Clues for Polygenic Inheritance of Pituitary Stalk Interruption Syndrome From Exome Sequencing in 20 Patients

Clues for Polygenic Inheritance of Pituitary Stalk Interruption Syndrome From Exome Sequencing in... Abstract Context Pituitary stalk interruption syndrome (PSIS) consists of a small/absent anterior pituitary lobe, an interrupted/absent pituitary stalk, and an ectopic posterior pituitary lobe. Mendelian forms of PSIS are detected infrequently (<5%), and a polygenic etiology has been suggested. GLI2 variants have been reported at a relatively high frequency in PSIS. Objective To provide further evidence for a non-Mendelian, polygenic etiology of PSIS. Methods Exome sequencing (trio approach) in 20 patients with isolated PSIS. In addition to searching for (potentially) pathogenic de novo and biallelic variants, a targeted search was performed in a panel of genes associated with midline brain development (223 genes). For GLI2 variants, both (potentially) pathogenic and relatively rare variants (<5% in the general population) were studied. The frequency of GLI2 variants was compared with that of a reference population. Results We found four additional candidate genes for isolated PSIS (DCHS1, ROBO2, CCDC88C, and KIF14) and one for syndromic PSIS (KAT6A). Eleven GLI2 variants were present in six patients. A higher frequency of a combination of two GLI2 variants (M1352V + D1520N) was found in the study group compared with a reference population (10% vs 0.68%). (Potentially) pathogenic variants were identified in genes associated with midline brain anomalies, including holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum and in genes involved in ciliopathies. Conclusion Combinations of variants in genes associated with midline brain anomalies are frequently present in PSIS and sustain the hypothesis of a polygenic cause of PSIS. Pituitary stalk interruption syndrome (PSIS) is a congenital anomaly of the pituitary gland consisting of a classic triad of magnetic resonance imaging (MRI) findings: (1) small or absent anterior pituitary lobe, (2) interrupted or absent pituitary stalk, and (3) ectopic posterior pituitary lobe. PSIS may be isolated or may occur in association with other midline brain malformations (syndromic PSIS) (1, 2). The clinical consequences of PSIS vary from isolated growth hormone (GH) deficiency to combined pituitary hormone deficiencies (CPHD) in which two or more anterior pituitary lobe functions [production of GH, thyrotropin, corticotropin, and gonadotropins (luteinizing hormone + follicle-stimulating hormone] may be insufficient. Prolactin level is usually elevated because of lack of hypothalamic dopaminergic inhibition. In isolated PSIS, typically only anterior pituitary hormone deficiencies occur, whereas in syndromic PSIS, posterior pituitary hormone deficiency (antidiuretic hormone) also may be present, with central diabetes insipidus as a clinical consequence (1, 2). The pituitary lobes and stalk have different embryological origins. The anterior lobe derives from the oral ectoderm, whereas the posterior lobe and pituitary stalk derive from the neural ectoderm. Various transcription factors are involved in the initial formation of the pituitary gland and the following cellular differentiation (3). Important signaling pathways involved in pituitary development are Wnt, Notch, Sonic Hedgehog (Shh), bone morphogenetic proteins (BMPs), and fibroblast growth factor (FGF). Mutations in genes encoding transcription factors in these pathways are found in <5% of PSIS cases and most often in association with other extrapituitary anomalies (4–8). In a recent review, Fang et al. (9) reported 30 candidate genes for PSIS and CPHD. Various genetic studies showed an association between PSIS and genes involved in holoprosencephaly, which is characterized by severely disturbed midline brain development, suggesting that PSIS may represent a mild form of this disorder (7). In particular, GLI2 variants, which are associated with holoprosencephaly, have been reported at a relatively high frequency in isolated PSIS (10–14). Furthermore, genes involved in hypogonadotropic hypogonadism (PROKR2, FGF8, FGFR1) have been associated with CPHD (6, 15–17). However, clinical expression is variable, including unaffected mutation-carrying relatives, suggesting involvement of additional genetic or environmental factors. Most studies searching for genetic causes of CPHD have been done in heterogeneous patient populations, including patients with other midline brain malformations, and most studies have focused on pathogenic mutations in specific genes, assuming a Mendelian inheritance (5). Because Mendelian forms of PSIS are detected only rarely, a polygenic and multifactorial etiology for PSIS should be considered as well (9). A polygenic inheritance has also been suggested for holoprosencephaly (18). In a very recent study using exome sequencing in 24 Chinese patients with isolated PSIS, heterozygous mutations, mostly in genes associated with Notch, Shh, and Wnt signaling pathways, were identified in 22 patients. Most patients (20 of 24) had more than one mutation, suggesting polygenic involvement. No mutations were found in genes known to be associated with pituitary malformation. Because parents were not included in this Chinese study, the authors were unable to determine whether mutations were de novo (19). To provide further evidence for a non-Mendelian, polygenic etiology of PSIS, we performed whole exome sequencing in 20 patients with isolated PSIS and their unaffected parents. In addition to searching for (potentially) pathogenic de novo and biallelic variants, we performed a targeted search in a large panel of genes associated with midline brain development. We included not only genes with a known association with pituitary formation (37 genes) but also genes associated with other midline brain malformations: holoprosencephaly (18 genes), hypogonadotropic hypogonadism (34 genes), and absent corpus callosum (134 genes). For each patient, all pathogenic variants and variants of unknown clinical significance (potentially pathogenic) were listed. Because rare GLI2 variants have been frequently reported in PSIS, we searched for (potentially) pathogenic GLI2 and for all relatively rare GLI2 variants (reported frequency <5% in the general population). Although rare GLI2 variants have been reported in association with PSIS, the frequency of (combinations of) these rare variants in the general population has not been investigated. To verify whether these rare GLI2 variants are indeed more common in PSIS, we compared the frequency of these variants in our study group with the frequency in a reference population. Patients and Methods Patients A database containing patients with congenital central hypothyroidism, located at the Department of Pediatric Endocrinology of the Academic Medical Center, Amsterdam, was searched for all patients with isolated PSIS. Most patients had been diagnosed after an abnormal result in the Dutch thyroxine-based neonatal screening program for congenital hypothyroidism (20). We selected 20 patients who had two or more pituitary hormone deficiencies and were still being treated at the Academic Medical Center. The diagnosis of PSIS was based on the three classic MRI findings. Patients with other (midline) brain abnormalities or with dysmorphic features or other pathologies suggestive of syndromic PSIS were excluded. The study was approved by the local medical ethics committee (NL47088.018.13). After informed consent was obtained, a venous blood sample was collected from all patients and their unaffected parents. Molecular studies Whole exome sequencing and variant calling were performed by the Beijing Genomic Institute. The capture used to enrich for exome sequences was the Agilent SureSelect Human All Exon V5 (50M) kit, and sequencing was done on either an Illumina or Complete Genomics platform. Variant prioritization Variant prioritization was done using the Cartagenia Bench Laboratory NGS (Agilent). Variants were considered potentially pathogenic when either resulting in truncation of the protein, predicted to result in a possibly/probably pathogenic variant by Polyphen2, or predicted to affect splicing and present at a frequency of <1% in the general population [based on the dbSNP database (dbSNP build 141 GRCh37.p13), ESP6500 (http://evs.gs.washington.edu/EVS/), 1000 Genomes Project (1000 Genomes phase 3 release, version 5.20130502), GoNL (http://www.nlgenome.nl/), and >900 in-house reference samples]. The validity of variants was judged by visual inspection of read data in Integrative Genomics Viewer or was confirmed by Sanger sequencing. A target gene panel for CPHD, holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum was made on the basis of results of a literature search (Table 1) (3, 8, 9, 18, 21, 22). Table 1. Gene Panels Used in the Targeted Search Genes Evaluated as Part of the Pituitary Panel (n = 37)  ARNT2, BMP2, BMP4, CDON,aFGF8,aFGF10, FGF18, FGFR1,aGATA2, GLI 1, GLI2,aGLI3, GLI4, GLI5, GLI6, GPR161, HESX1, IGSF1, LHX3, LHX4, NR5A1, OTX2, PAX6, PITX1, PITX2, POU1F1, PROP1, SHH, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, SOX1, SOX2, SOX3, TBX19, TGIF, WNT5a  Genes Evaluated as Part of the Pituitary Panel (n = 37)  ARNT2, BMP2, BMP4, CDON,aFGF8,aFGF10, FGF18, FGFR1,aGATA2, GLI 1, GLI2,aGLI3, GLI4, GLI5, GLI6, GPR161, HESX1, IGSF1, LHX3, LHX4, NR5A1, OTX2, PAX6, PITX1, PITX2, POU1F1, PROP1, SHH, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, SOX1, SOX2, SOX3, TBX19, TGIF, WNT5a  Genes Evaluated as Part of the Holoprosencephaly Panel (n = 18)  CDON,aDISP1, DLL1, FGF8,aFGFR1,aFOXH1, GAS1, GLI2,aHHAT, NODAL, PTCH1, SHH, SIX3, STIL, SUFU, TDGF1, TGIF1, ZIC2  Genes Evaluated as Part of the Holoprosencephaly Panel (n = 18)  CDON,aDISP1, DLL1, FGF8,aFGFR1,aFOXH1, GAS1, GLI2,aHHAT, NODAL, PTCH1, SHH, SIX3, STIL, SUFU, TDGF1, TGIF1, ZIC2  Genes Evaluated as Part of the Hypogonadotropic Hypogonadism Panel (n = 34)  AXL, CCDC141, CHD7, DMXL2, FEZF1, FGF8,aFGF17, FGFR1,aGNRH1, GNRHR, HESX1, HS6ST1, KAL1, KISS1, KISS1R, LEP, LEPR, NR0B1, NSMF, OL14RD, OTUD4, PCSK1, PNPLA6, PROK2, PROKR2, RNF216, SEMA3A, SEMA3E, SEMA7A, SOX10, STS, TAC3, TACR3, WDR11  Genes Evaluated as Part of the Hypogonadotropic Hypogonadism Panel (n = 34)  AXL, CCDC141, CHD7, DMXL2, FEZF1, FGF8,aFGF17, FGFR1,aGNRH1, GNRHR, HESX1, HS6ST1, KAL1, KISS1, KISS1R, LEP, LEPR, NR0B1, NSMF, OL14RD, OTUD4, PCSK1, PNPLA6, PROK2, PROKR2, RNF216, SEMA3A, SEMA3E, SEMA7A, SOX10, STS, TAC3, TACR3, WDR11  Genes Evaluated as Part of the Corpus Callosum Agenesis Panel (n = 134)  AHI1, AKT3, AMPD2, ANOP1, ARID1B, ARL13B, ARX, ASPM, ATR, ATRX, B9D1, B9D2, BCOR, BMP4, C12ORF57, CASK, CC2D2A, CENPJ, CEP152, CEP290, CEP41, CEP63, CREBBP, CTBP1, AS1, DCX, DHCR7, DHCR24, DIS3L2, DISC1, EFNB1, EOMES, EP300, EPG5, FGF8, FGFR1, FGFR2, FH, FKRP, FKTN, FLNA, GLI3, GPSM2, GTDC2, HCCS, HESX1, HS6ST1, HYLS1, IGBP1, IGF1, INPP5E, ISPD, KAT6B, KCC3, KIF7, L1CAM, LARGE, LRP2, MED12, MID1, MKS1, NDE1, NFIX, NIN, NPHP1, NPHP3, NSD1, OFD1, OTX1, PAX6, PDHA1, PDHB, POMGNT1, POMT1, POMT2, PYCR1, RAB18, RAB3GAP1, RAB3GAP2, RBBP8, RBM10, RELN, RNU4ATAC, RPGRIP1L, RPS6KA3, SCKL3, SOX2, SPG11, STRA6, TCF4, TCTN1, TCTN2, TCTN3, TMEM138, TMEM216, TMEM237, TMEM67, TUBA1A, TUBB2B, TUBB3, VAX1, WDR62, ZEB2  Genes Evaluated as Part of the Corpus Callosum Agenesis Panel (n = 134)  AHI1, AKT3, AMPD2, ANOP1, ARID1B, ARL13B, ARX, ASPM, ATR, ATRX, B9D1, B9D2, BCOR, BMP4, C12ORF57, CASK, CC2D2A, CENPJ, CEP152, CEP290, CEP41, CEP63, CREBBP, CTBP1, AS1, DCX, DHCR7, DHCR24, DIS3L2, DISC1, EFNB1, EOMES, EP300, EPG5, FGF8, FGFR1, FGFR2, FH, FKRP, FKTN, FLNA, GLI3, GPSM2, GTDC2, HCCS, HESX1, HS6ST1, HYLS1, IGBP1, IGF1, INPP5E, ISPD, KAT6B, KCC3, KIF7, L1CAM, LARGE, LRP2, MED12, MID1, MKS1, NDE1, NFIX, NIN, NPHP1, NPHP3, NSD1, OFD1, OTX1, PAX6, PDHA1, PDHB, POMGNT1, POMT1, POMT2, PYCR1, RAB18, RAB3GAP1, RAB3GAP2, RBBP8, RBM10, RELN, RNU4ATAC, RPGRIP1L, RPS6KA3, SCKL3, SOX2, SPG11, STRA6, TCF4, TCTN1, TCTN2, TCTN3, TMEM138, TMEM216, TMEM237, TMEM67, TUBA1A, TUBB2B, TUBB3, VAX1, WDR62, ZEB2  a Genes included in more than one list. View Large The exome data set was analyzed stepwise: First, we searched for potentially pathogenic variants in 37 genes known to be associated with pituitary development or function. Next, we searched for new candidate genes by identifying any potentially pathogenic de novo, homozygous, or compound heterozygous variants, including X-linked mutations in males. For each gene, a PubMed search was performed to determine function and/or brain expression pattern. Thereafter, we searched for potentially pathogenic variants in the panel of genes involved in midline brain pathologies: holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum. Lastly, we identified all GLI2 variants with a reported minor allele frequency (MAF) <0.05. The frequency of GLI2 variants with a MAF <0.05 was compared with the frequency of GLI2 variants with a MAF <0.05 in the 1000 Genomes database. Specific combinations of GLI2 variants, detected in the current study, were also searched for in the 1000 Genomes database. Because of the ethnic diversity of our study population, we used the 1000 Genomes database as an international reference population. Modeling and molecular dynamic study Three-dimensional models were constructed for the wild-type DCHS1 and DCHS1 I780V, P1519S, L2132P, H2729L mutants and for wild-type roundabout guidance receptor 2 (ROBO2; isoform 2a) and R314Q mutants. Full-length secondary structure predictions and the identification of most suitable templates were performed using the ROSETTA-based Robetta Web server (http://robetta.bakerlab.org). Using the homology modeling program Modeler 9v14 (www.salilab.org/modeller/), four pairs of models of the three extracellular domains (wild-type and mutants; residue range: 680 to 886, 1318 to 1641, 2062 to 2270, and 2536 to 2867) were generated considering previously published structural DCHS1 and ROBO2 data and Robetta outputs (23–26). Modeling was performed with the default parameters using the allHmodel protocol to include hydrogen atoms and the HETATM protocol to include Ca2+ with restricting Ca2+-binding residues side-chain distance and with thorough molecular dynamics optimization and refinement protocol. To study the mutational effects on structure, the molecular dynamics of each model pair was simulated using a NAMD 2.11 (http://www.ks.uiuc.edu/Research/namd/) plug-in in the VMD v1.9.2.27 program (http://www.ks.uiuc.edu/Research/vmd/) for 100 ns. Intermediate and final structures were evaluated in PyMol. Results We studied 20 unrelated patients (14 males) with an age range of 3 to 28 years (Table 2). Age at diagnosis ranged from 2 weeks to 5 years. Eleven children had been diagnosed with congenital central hypothyroidism after detection by neonatal screening, seven children after referral for growth retardation, one child for prolonged jaundice, and another child for neonatal hypotonia. Twelve patients had deficiency of four anterior pituitary hormones, seven patients had three hormone deficiencies, and one patient had two hormone deficiencies. All patients had GH deficiency. Central adrenal insufficiency was present in 19 patients, and hypogonadotropic hypogonadism was present in three patients of prepubertal age and in nine patients of postpubertal age. Three children had normal pubertal development, and in five prepubertal children, information on the status of the hypothalamic-pituitary-gonadal axis was not available. No patient had central diabetes insipidus. The medical history of parents was negative for endocrine deficiencies, and all parents had normal development, growth, and fertility. Table 2. Clinical Characteristics of Studied Patients With Isolated PSIS Case  Sex  Age (y)  Age at Diagnosis  Presentation  Pituitary Deficiencies  MRI Findings  1  M  8  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  2  F  25  5 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  3  M  28  3 mo  Prolonged jaundice  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  4  M  3  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, interrupted stalk, small AP  5  M  26  2 wk  Hypotonia  GH; TSH; ACTH  EPP, small stalk, small AP  6  F  10  2 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  7  F  9  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, absent AP  8  F  16  1 mo  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  9  M  23  6 mo  Abnormal NS  GH; TSH; ACTH  EPP, absent stalk, small AP  10  F  9  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  11  M  18  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  12  M  13  2 y  Growth retardation  GH; TSH  EPP, absent stalk, normal AP  13  M  23  6 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, normal AP  14  M  18  4 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  15  M  19  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  16  M  17  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, small stalk, small AP  17  F  5  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  18  M  5  1.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  19  M  8  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, small stalk, small AP  20  M  20  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  Case  Sex  Age (y)  Age at Diagnosis  Presentation  Pituitary Deficiencies  MRI Findings  1  M  8  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  2  F  25  5 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  3  M  28  3 mo  Prolonged jaundice  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  4  M  3  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, interrupted stalk, small AP  5  M  26  2 wk  Hypotonia  GH; TSH; ACTH  EPP, small stalk, small AP  6  F  10  2 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  7  F  9  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, absent AP  8  F  16  1 mo  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  9  M  23  6 mo  Abnormal NS  GH; TSH; ACTH  EPP, absent stalk, small AP  10  F  9  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  11  M  18  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  12  M  13  2 y  Growth retardation  GH; TSH  EPP, absent stalk, normal AP  13  M  23  6 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, normal AP  14  M  18  4 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  15  M  19  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  16  M  17  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, small stalk, small AP  17  F  5  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  18  M  5  1.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  19  M  8  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, small stalk, small AP  20  M  20  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  Abbreviations: ACTH, adrenocorticotropic hormone; AP, anterior pituitary; EPP, ectopic posterior pituitary; FSH, follicle-stimulating hormone; LH, luteinizing hormone; NS, neonatal screening result; TSH, thyrotropin. View Large For each patient, all pathogenic and potentially pathogenic variants and all GLI2 variants with an MAF <0.05 are shown in Tables 3 and 4 . Table 3. Exome Sequencing Results of Studied Patients With PSIS Including Variants, Reported Allele Frequency, Classification According to ACMG Guidelines, In Silico Prediction (Polyphen 2), and Phenotypes Known To Be Caused by Variants in Each Gene Case  Gene  Variant  dbSNP  Inheritance  Variant Classification  MAF (GnomAD)  In Silico Prediction  Gene Panel  Known Phenotype (MIM Number)  Information on Specific Variant  3  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Mother  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same two GLI2 variants in case 14 
Combination of these two variants previously described in patients with PSIS, including functional study showing reduced transcriptional activity and reduced luciferase activity (10, 13, 27)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Mother  VUS  0.0101  Probably damaging  4  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 18. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  RELN  c.9646G>A, p.E3216K (missense)  —  Mother  VUS  N  Benign  CCA  Lissencephaly 2 (MIM 257320)    IGSF1  c.498G>C, p.E166D  rs201255931  Mother  VUS  0.0005  Probably damaging  PIT  XL central hypothyroidism (MIM 300888)  5  INPP5E  c.902T>C, p.L301P (missense)  —  Mother  VUS  N  Probably damaging  CCA  Joubert syndrome 1 (MIM 213300)    6  KAT6A  c.235C>T, p.A79* (nonsense)  —  De novo  Pathogenic  N  Damaging  Not from panel  KAT6A neurodevelopmental syndrome (MIM 616268)  (28)  GLI3  c.539G>A, p.R180Q (missense)  rs140772904  Father  VUS  0.00004  Possibly damaging  PIT  Greig cephalopolysyndactyly syndrome (MIM 175700), Pallister-Hall syndrome (MIM 146510), postaxial polydactyly (MIM 174200), preaxial polydactyly (MIM 174700)  BMP4  c.804_815delCCGGCCCCTCCT, p.R269_L272del (deletion)  —  Father  VUS  N    PIT  Syndromic microphthalmia type 6 (MIM 607932)  GLI2  c.1761G>A, p.T587T (synonymous)  rs61732852  Mother  Likely benign  0.0119    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  7  DCHS1  c.6395T>C, p.L2132P (missense)  —  Father  VUS  N  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.2338A>G, p.I780V (missense)  rs145735483  Mother  VUS  0.0003  Benign        GLI2  c.1294G>A, p.V432M (missense)  rs142296407  Mother  VUS  0.0015  Possibly damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same variant described in patients with MPHD/PSIS (10, 11, 13)  8  NDE  c.488T>A, p.L163Q (missense)  —  Father  VUS  N  Probably damaging  CCA  Lissencephaly 4 (MIM 614019)    OTUD4  c.823G>T, p.V275L (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotropism (MIM 611744)  9  ROBO2  c.914G>A, p.R314Q (missense)  —  De novo  VUS  N  Probably damaging  Not from panel  Vesicoureteral reflux 2 (MIM 610878)    10  GLI2  c.963C>G, p.P321P (synonymous)  rs149894186  Mother  Benign  0.0046    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    GLI2  c.2088A>G, p.A696A (synonymous)  rs146059306  Mother  Benign  0.0009  GLI2  c.2262G>T, p.R754R (synonymous)  rs142856393  Father  Benign  0.0011  11  ASPM  c.10161+5G>C (insertion)  —  Mother  VUS/likely pathogenic (altered splicing?)  N    CCA  Primary autosomal recessive microcephaly (MIM 608716)    FGF8  c.77C>T, p.P26L (missense)  rs137852660  Mother  VUS  0.0019  Benign  PIT + HH  Hypogonadotropic hypogonadism 6 (MIM 612702)  Heterozygosity described in a patient with HH and partial empty sella [Falardeau et al. (29)]  CHD4  c.2374C>T, p.R792W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    12  CC2D2A  c.3055C>T, p.R1019* (nonsense)  rs370880399  Mother  Pathogenic  0.0001  Damaging  CCA  Joubert syndrome 9 (MIM 612285), Meckel syndrome 6 (MIM 612284)  Variant reported in compound heterozygosity with another variant in a number of patients with Joubert syndrome  NR0B1  c.315G>C, p.W105C (missense)  rs132630327  Mother  Pathogenic  0.00001  Damaging  HH  Congenital adrenal hypoplasia (MIM 300200), 46XY reversal 2 (MIM 300018)  Same mutation reported in patient with isolated mineralocorticoid deficiency (30)  ARNT2  c.1707G>T, p.Q569H (missense)  rs145379118  Father  VUS  0.0033  Possibly damaging  PIT  Webb-Dattani syndrome (MIM 615926)    13  PROK2  c.163delA p.I55* (nonsense)  rs554675432  Father  Pathogenic  0.0001  Damaging  HH  Hypogonadotropic hypogonadism 4 (MIM 610628)  Homozygosity reported in patients with anosmic hypogonadotropic hypogonadism (31, 32)  B9D1  c.151T>C, p.S51P (missense)  rs546359789  Father  VUS  0.00006  Probably damaging  CCA  Joubert syndrome 27 (MIM 617120), Meckel syndrome 9 (MIM 614209).  Variant reported in compound heterozygosity with another variant in a patient with Joubert syndrome [Srour et al. (33)]  14  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Father  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  See case 3 (same combination of GLI2 variants)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Father  VUS  0.0101  Probably damaging        GLI2  c.1944C>T, p.T648T (synonymous)  rs13008360  Both parents  Benign  0.0253          CHD4  c.5149C>T, p.R1717W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/ macula dystrophy (MIM 212550)  Same variant in patient 19. Variant previously described with reduced function [Carnes et al. (34)]  15  KIF14  c.3728A>G, p.K1243R  —  De novo  VUS  N  Probably damaging  Not from panel  Meckel syndrome 12 (MIM 616258)    16  GLI2  c.4145G>A, p.R1382H (missense)  rs200080112  Mother  VUS  0.0002  Probably damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    17  CCDC88C  c.3895C>T, p.R1299C (missense)  rs142539336  Father  VUS  0.0049  Possibly damaging  Not from panel  Nonsyndromic hydrocephalus (MIM 616053)    CCDC88C  c.1984G>A, p.E662K (missense)  Not in dbSNP  Mother  VUS  0.0001  Benign      TACR3  c.659T>C, p.L220P (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotrophic hypogonadism (MIM 614840)  18  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 4. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  CHD7  c.1324G>A, p.A442T (missense)  rs368086966  Mother  VUS  0.0002  Benign  HH  CHARGE syndrome (MIM 214800), Hypogonadotropic hypogonadism (MIM 612370)    19  DCHS1  c.8186A>T, p.H2729L (missense)  rs148148252  Father  VUS/likely pathogenic  0.0003  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.4555C>T, p.P1519S (missense)  rs199544459  Mother  VUS  0.0017  Benign        SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/macula dystrophy (MIM 212550)  Same variant in patient 14. Variant previously described with reduced function [Carnes et al. (34)]  BMP4  c.1001C>A, p.A334D (missense)  rs550409227  Mother  VUS  0.000008  Probably damaging  PIT  Syndromic microphthalmia type 6 (MIM 607932)    20  SLC12A6  c.1787C>T, p.P596L (missense)  —  Mother  VUS/likely pathogenic  N  Possibly damaging  CCA  AR corpus callosum agenesis + peripheral neuropathy (MIM 218000)  CC2D2A  c.3865A>G, p.T1289A (missense)  —  Mother  VUS  N  Possibly damaging  CCA  Joubert syndrome (MIM 612285), Meckel syndrome (MIM 612284)  Case  Gene  Variant  dbSNP  Inheritance  Variant Classification  MAF (GnomAD)  In Silico Prediction  Gene Panel  Known Phenotype (MIM Number)  Information on Specific Variant  3  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Mother  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same two GLI2 variants in case 14 
Combination of these two variants previously described in patients with PSIS, including functional study showing reduced transcriptional activity and reduced luciferase activity (10, 13, 27)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Mother  VUS  0.0101  Probably damaging  4  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 18. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  RELN  c.9646G>A, p.E3216K (missense)  —  Mother  VUS  N  Benign  CCA  Lissencephaly 2 (MIM 257320)    IGSF1  c.498G>C, p.E166D  rs201255931  Mother  VUS  0.0005  Probably damaging  PIT  XL central hypothyroidism (MIM 300888)  5  INPP5E  c.902T>C, p.L301P (missense)  —  Mother  VUS  N  Probably damaging  CCA  Joubert syndrome 1 (MIM 213300)    6  KAT6A  c.235C>T, p.A79* (nonsense)  —  De novo  Pathogenic  N  Damaging  Not from panel  KAT6A neurodevelopmental syndrome (MIM 616268)  (28)  GLI3  c.539G>A, p.R180Q (missense)  rs140772904  Father  VUS  0.00004  Possibly damaging  PIT  Greig cephalopolysyndactyly syndrome (MIM 175700), Pallister-Hall syndrome (MIM 146510), postaxial polydactyly (MIM 174200), preaxial polydactyly (MIM 174700)  BMP4  c.804_815delCCGGCCCCTCCT, p.R269_L272del (deletion)  —  Father  VUS  N    PIT  Syndromic microphthalmia type 6 (MIM 607932)  GLI2  c.1761G>A, p.T587T (synonymous)  rs61732852  Mother  Likely benign  0.0119    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  7  DCHS1  c.6395T>C, p.L2132P (missense)  —  Father  VUS  N  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.2338A>G, p.I780V (missense)  rs145735483  Mother  VUS  0.0003  Benign        GLI2  c.1294G>A, p.V432M (missense)  rs142296407  Mother  VUS  0.0015  Possibly damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same variant described in patients with MPHD/PSIS (10, 11, 13)  8  NDE  c.488T>A, p.L163Q (missense)  —  Father  VUS  N  Probably damaging  CCA  Lissencephaly 4 (MIM 614019)    OTUD4  c.823G>T, p.V275L (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotropism (MIM 611744)  9  ROBO2  c.914G>A, p.R314Q (missense)  —  De novo  VUS  N  Probably damaging  Not from panel  Vesicoureteral reflux 2 (MIM 610878)    10  GLI2  c.963C>G, p.P321P (synonymous)  rs149894186  Mother  Benign  0.0046    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    GLI2  c.2088A>G, p.A696A (synonymous)  rs146059306  Mother  Benign  0.0009  GLI2  c.2262G>T, p.R754R (synonymous)  rs142856393  Father  Benign  0.0011  11  ASPM  c.10161+5G>C (insertion)  —  Mother  VUS/likely pathogenic (altered splicing?)  N    CCA  Primary autosomal recessive microcephaly (MIM 608716)    FGF8  c.77C>T, p.P26L (missense)  rs137852660  Mother  VUS  0.0019  Benign  PIT + HH  Hypogonadotropic hypogonadism 6 (MIM 612702)  Heterozygosity described in a patient with HH and partial empty sella [Falardeau et al. (29)]  CHD4  c.2374C>T, p.R792W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    12  CC2D2A  c.3055C>T, p.R1019* (nonsense)  rs370880399  Mother  Pathogenic  0.0001  Damaging  CCA  Joubert syndrome 9 (MIM 612285), Meckel syndrome 6 (MIM 612284)  Variant reported in compound heterozygosity with another variant in a number of patients with Joubert syndrome  NR0B1  c.315G>C, p.W105C (missense)  rs132630327  Mother  Pathogenic  0.00001  Damaging  HH  Congenital adrenal hypoplasia (MIM 300200), 46XY reversal 2 (MIM 300018)  Same mutation reported in patient with isolated mineralocorticoid deficiency (30)  ARNT2  c.1707G>T, p.Q569H (missense)  rs145379118  Father  VUS  0.0033  Possibly damaging  PIT  Webb-Dattani syndrome (MIM 615926)    13  PROK2  c.163delA p.I55* (nonsense)  rs554675432  Father  Pathogenic  0.0001  Damaging  HH  Hypogonadotropic hypogonadism 4 (MIM 610628)  Homozygosity reported in patients with anosmic hypogonadotropic hypogonadism (31, 32)  B9D1  c.151T>C, p.S51P (missense)  rs546359789  Father  VUS  0.00006  Probably damaging  CCA  Joubert syndrome 27 (MIM 617120), Meckel syndrome 9 (MIM 614209).  Variant reported in compound heterozygosity with another variant in a patient with Joubert syndrome [Srour et al. (33)]  14  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Father  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  See case 3 (same combination of GLI2 variants)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Father  VUS  0.0101  Probably damaging        GLI2  c.1944C>T, p.T648T (synonymous)  rs13008360  Both parents  Benign  0.0253          CHD4  c.5149C>T, p.R1717W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/ macula dystrophy (MIM 212550)  Same variant in patient 19. Variant previously described with reduced function [Carnes et al. (34)]  15  KIF14  c.3728A>G, p.K1243R  —  De novo  VUS  N  Probably damaging  Not from panel  Meckel syndrome 12 (MIM 616258)    16  GLI2  c.4145G>A, p.R1382H (missense)  rs200080112  Mother  VUS  0.0002  Probably damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    17  CCDC88C  c.3895C>T, p.R1299C (missense)  rs142539336  Father  VUS  0.0049  Possibly damaging  Not from panel  Nonsyndromic hydrocephalus (MIM 616053)    CCDC88C  c.1984G>A, p.E662K (missense)  Not in dbSNP  Mother  VUS  0.0001  Benign      TACR3  c.659T>C, p.L220P (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotrophic hypogonadism (MIM 614840)  18  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 4. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  CHD7  c.1324G>A, p.A442T (missense)  rs368086966  Mother  VUS  0.0002  Benign  HH  CHARGE syndrome (MIM 214800), Hypogonadotropic hypogonadism (MIM 612370)    19  DCHS1  c.8186A>T, p.H2729L (missense)  rs148148252  Father  VUS/likely pathogenic  0.0003  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.4555C>T, p.P1519S (missense)  rs199544459  Mother  VUS  0.0017  Benign        SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/macula dystrophy (MIM 212550)  Same variant in patient 14. Variant previously described with reduced function [Carnes et al. (34)]  BMP4  c.1001C>A, p.A334D (missense)  rs550409227  Mother  VUS  0.000008  Probably damaging  PIT  Syndromic microphthalmia type 6 (MIM 607932)    20  SLC12A6  c.1787C>T, p.P596L (missense)  —  Mother  VUS/likely pathogenic  N  Possibly damaging  CCA  AR corpus callosum agenesis + peripheral neuropathy (MIM 218000)  CC2D2A  c.3865A>G, p.T1289A (missense)  —  Mother  VUS  N  Possibly damaging  CCA  Joubert syndrome (MIM 612285), Meckel syndrome (MIM 612284)  Abbreviations: ACMG, The American College of Medical Genetics and Genomics; AD, autosomal dominant; AR, autosomal recessive; CCA, corpus callosum agenesis; dbSNP, Single Nucleotide Polymorphism Database; HH, hypogonadotropic hypogonadism; HPE, holoprosencephaly; MIM, numerical assignment in Mendelian Inheritance in Man catalog; MPHD, multiple pituitary hormone deficiency; N, variant not previously reported; PIT, pituitary; VUS, variant of unknown significance. View Large Table 4. Exome Sequencing Results per Panel of Targeted Genes Including Number of Variants for Each Gene Case    1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  Pituitary panel   ARNT2                        1                   BMP4            1                          1     CHD4                      1      1               GLI3            1                               SIX6                            1          1     IGSF1        1                                  Holoprosencephaly panel   GLI2      2      1  1      3        3    1          Hypogonadotropic hypogonadism panel   B9D1                          1                 CHD7                                    1       FGF8                      1                     NR0B1                        1                   OTUD4                1                           PROK2                          1                 TACR3                                  1        Corpus callosum agenesis panel   ASPM                      1                     CC2D2A                        1                1   DHCR7        1                            1       INPP5E          1                                 NDE                1                           RELN        1                                   SLC12A6                                        1  New candidate genes   CCDC88C                                  2         DCHS1              2                        2     KAT6A          1                               KIF14                              1             ROBO2                  1                        Case    1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  Pituitary panel   ARNT2                        1                   BMP4            1                          1     CHD4                      1      1               GLI3            1                               SIX6                            1          1     IGSF1        1                                  Holoprosencephaly panel   GLI2      2      1  1      3        3    1          Hypogonadotropic hypogonadism panel   B9D1                          1                 CHD7                                    1       FGF8                      1                     NR0B1                        1                   OTUD4                1                           PROK2                          1                 TACR3                                  1        Corpus callosum agenesis panel   ASPM                      1                     CC2D2A                        1                1   DHCR7        1                            1       INPP5E          1                                 NDE                1                           RELN        1                                   SLC12A6                                        1  New candidate genes   CCDC88C                                  2         DCHS1              2                        2     KAT6A          1                               KIF14                              1             ROBO2                  1                        View Large Variants in pituitary genes In the panel of 37 genes associated with pituitary development, no pathogenic variants were found. However, several rare, potentially pathogenic variants were present in GLI2, GLI3, BMP4, IGSF1, CHD4, ARNT2, SIX6 (two patients, same variant), and FGF8. New candidate genes De novo or heterozygous mutations were found in five genes (in six patients) with a known expression/function in the brain: ROBO2, KAT6A, KIF14, compound heterozygosity for DCHS1 (two patients), and compound heterozygosity for CCDC88C. Protein modeling for both cases of the compound heterozygous DCHS1 mutations indicated disrupted interaction with the ligand FAT4, making functional consequences likely (Fig. 1). Protein modeling for the ROBO2 mutation indicated that the mutation was in the most mobile region of the ROBO2 protein. The mutation causes an extra H-bond, which solidifies the ROBO2 protein, probably leading to less adaptability to environmental influences and in interactions with the ligand SLIT (Fig. 2). Figure 1. View largeDownload slide (A) Schematic overview of the structure and interaction of DCHS1 and FAT4, including the sites of the presently reported mutations. (B‒E) Extracellular (EC) domains shown in cartoon presentation with mutation position marked and zoomed in on. The dashed arrows present orientation of extracellular domain in comparison with the neighboring domains, and the numbers present the corner of orientation. Affected residues are shown in sticks or line presentation. Calcium is in green spheres. (B, C) Mutations in case 7 are shown. (B) c.2338A>G, p.Ile780Val: wild-type = gray, mutation = red. Mutation is in EC7 and has a slight effect on the structural stability of the EC7 domain. EC6 and EC7 interact with FAT4, which may loosen the interaction. (C) c.6395T>C, p.Leu2132Pro: wild-type = gray, mutation = red. Mutation is in EC20 and has a structural effect on EC20 and even more so on EC21. Both mutant extracellular domains lose structural stability because of effects on Ca binding ability. Mutation also affects orientation of the EC20, which may cause less favorable DCHS1-FAT4 interaction. (D, E) Mutations in case 19 are shown. (D) c.4555C>T, p.Pro1519Ser: wild- type = gray, mutation = blue. Mutation is in EC14 and has a structural effect on EC13, EC14, and even more so on EC15. EC15 almost unfolds; only Ca2+ still holds the structure together. Immediately after Ca concentration went down, EC15 completely unfolded. Pro1519Ser disturbed beta structure, which affected Ca-binding sites between EC14 and EC15, potentiating the structural instability of EC15. Mutation also affected orientation of all mentioned extracellular domains, consequently leading to distorted interaction between DCHS1 and FAT4. (E) c.8186A>T, p.His2729Leu: wild-type = gray, mutation = blue. Mutation is in EC26. No significant structural effect was observed. Figure 1. View largeDownload slide (A) Schematic overview of the structure and interaction of DCHS1 and FAT4, including the sites of the presently reported mutations. (B‒E) Extracellular (EC) domains shown in cartoon presentation with mutation position marked and zoomed in on. The dashed arrows present orientation of extracellular domain in comparison with the neighboring domains, and the numbers present the corner of orientation. Affected residues are shown in sticks or line presentation. Calcium is in green spheres. (B, C) Mutations in case 7 are shown. (B) c.2338A>G, p.Ile780Val: wild-type = gray, mutation = red. Mutation is in EC7 and has a slight effect on the structural stability of the EC7 domain. EC6 and EC7 interact with FAT4, which may loosen the interaction. (C) c.6395T>C, p.Leu2132Pro: wild-type = gray, mutation = red. Mutation is in EC20 and has a structural effect on EC20 and even more so on EC21. Both mutant extracellular domains lose structural stability because of effects on Ca binding ability. Mutation also affects orientation of the EC20, which may cause less favorable DCHS1-FAT4 interaction. (D, E) Mutations in case 19 are shown. (D) c.4555C>T, p.Pro1519Ser: wild- type = gray, mutation = blue. Mutation is in EC14 and has a structural effect on EC13, EC14, and even more so on EC15. EC15 almost unfolds; only Ca2+ still holds the structure together. Immediately after Ca concentration went down, EC15 completely unfolded. Pro1519Ser disturbed beta structure, which affected Ca-binding sites between EC14 and EC15, potentiating the structural instability of EC15. Mutation also affected orientation of all mentioned extracellular domains, consequently leading to distorted interaction between DCHS1 and FAT4. (E) c.8186A>T, p.His2729Leu: wild-type = gray, mutation = blue. Mutation is in EC26. No significant structural effect was observed. Figure 2. View largeDownload slide Schematic overview of the structure and interaction of ROBO2 and SLIT. Right panel: SLIT (green), wild-type ROBO2 = gray, mutation (c.941G>A, p.Arg314Gln) = red. The mutation leads to an extra H-bond between residues 245 and 314, causing ROBO2 to become more rigid. Left panel: superposition of wild-type ROBO2-SLIT and mutated ROBO2 (c.941G>A, p.Arg314Gln)‒SLIT complex. Mutations are marked by arrows. Yellow lines indicate distance between their backbone atoms. Figure 2. View largeDownload slide Schematic overview of the structure and interaction of ROBO2 and SLIT. Right panel: SLIT (green), wild-type ROBO2 = gray, mutation (c.941G>A, p.Arg314Gln) = red. The mutation leads to an extra H-bond between residues 245 and 314, causing ROBO2 to become more rigid. Left panel: superposition of wild-type ROBO2-SLIT and mutated ROBO2 (c.941G>A, p.Arg314Gln)‒SLIT complex. Mutations are marked by arrows. Yellow lines indicate distance between their backbone atoms. Variants in genes associated with midline brain pathologies In the panel of genes associated with holoprosencephaly, 11 variants in GLI2 were found but no potential pathogenic variants in any of the other candidate genes. In the panel for hypogonadotropic hypogonadism, pathogenic mutations were found in NR0B1 and PROK2, and potentially pathogenic variants were found in OTUD4, FGF8, TACR3, and CHD7. In the panel for absent corpus callosum, pathogenic mutations were found in DHCR7 (two patients, identical mutation) and CC2D2A, and variants of unknown significance, predicted to potentially be damaging, were found in RELN, INPP5E, NDE, ASPM, B9D1, CC2D2A (two patients), and SLC12A6. All mutations found in the target panels were inherited from an unaffected parent (Table 3). We detected 11 GLI2 variants (six missense, five synonymous) with a MAF <0.05 in six patients; all were inherited from an unaffected parent. The total prevalence of GLI2 variants with a MAF <0.05 was 55% in our study population (11 variants in 20 individuals), which was comparable to the 58.6% prevalence of GLI2 variants with a MAF <0.05 in the 1000 Genomes database (1468 variants in 2504 individuals). Two of the 20 study patients had a combination of the missense mutations M1352V + D1520N. In the 1000 Genomes database, this specific combination was found in only 17 of 2504 individuals (0.68%). Discussion In this exome sequencing study of 20 unrelated patients with isolated PSIS and their unaffected parents, we identified five additional candidate genes for PSIS: DCHS1, ROBO2, CCDC88C, KIF14, and KAT6A. In addition, by using a target gene panel, we found potentially pathogenic variants in genes involved in midline brain formation in a panel of genes known to be associated with pituitary formation (in 8 of 37 genes) and in a panel of genes associated with holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum (11 of 186 genes); and also in the latter group, 5 pathogenic variants. Thirteen of 20 patients carried more than one variant, and all variants from the target panels were inherited from an unaffected parent. This diversity of (potentially) pathogenic variants and inheritance from unaffected parents are suggestive of a polygenic etiology of isolated PSIS. New candidate genes DCHS1 Two patients (nos. 7 and 19) were compound heterozygous for variants in DCHS1. Protein modeling indicated disrupted interaction with the ligand FAT4A, making functional consequences likely (Fig. 1). DCHS1 and FAT4 play a role in neuronal migration (35, 36). Pathogenic mutations in DCHS1 and FAT4 can cause Van Maldergem syndrome, which goes along with periventricular neuronal heterotopia, indicative of altered neuronal migration, and absent corpus callosum (24). Pituitary abnormalities have not been described. DCHS1 is expressed in the developing pituitary gland in mice (37). The present two patients had isolated PSIS without other brain abnormalities at MRI. We examined the patients carefully for signs suggestive of Van Maldergem syndrome, but except for broad hands and feet, present in both patients and absent in parents, there were no specific physical characteristics. One of the patients also had a GLI2 variant (c.1294G>A, maternally inherited), previously reported in patients with CPHD, and an ectopic posterior pituitary lobe (10, 11, 13). Because DCHS1 is involved in neuronal migration and is expressed in the pituitary gland and because we found two patients in this series of 20 patients with isolated PSIS, the DCHS1 gene may be considered a candidate gene for PSIS, but possibly only when variants are present in one or more other genes. ROBO2 Patient 9 had a de novo ROBO2 mutation (c.914G>A), which was not previously reported and was predicted to be probably damaging. Protein modeling indicated that the mutation is not in the ROBO2-SLIT interface but is in the most mobile region of the ROBO2 protein. The mutation leads to an extra H-bond and solidifies the ROBO2 protein, making functional consequences likely (Fig. 2). ROBO2 belongs to the ROBO family, part of the immunoglobulin superfamily of proteins that are highly conserved from fly to human. The encoded protein is a transmembrane receptor for the slit homolog 2 protein and functions in axon guidance across the midline of the mammalian central nervous system (38). ROBO2 mutations are associated with vesicoureteral reflux (39). ROBO2 isoform a is highly expressed in the developing human brain but not in the adult brain (40). The present patient did not have vesicoureteral reflux. After we had finished our study, Bashamboo et al. (41) identified ROBO1 mutations in five cases of PSIS. Four of the five patients had ocular anomalies, which the present patient did not have. The function of ROBO2 in axon guidance across the midline, the results of protein modeling, and the recent finding of ROBO1 mutations in PSIS make ROBO2 an excellent candidate gene for pituitary malformations, but functional studies are needed to prove pathogenicity. CCDC88C Patient 17 was compound heterozygous for CCDC88C variants. The R1299C was predicted to be possibly damaging by Polyphen2; the E662K mutation is very rare (MAF, 0.0001), predicted to be benign by Polyphen2. This gene encodes a ubiquitously expressed coiled‒coil domain‒containing protein that interacts with the disheveled protein and is a negative regulator of the Wnt signaling pathway. Autosomal recessive mutations in CCDC88C cause nonsyndromic hydrocephalus, associated with midline brain malformation (42). The present patient had no hydrocephalus and no midline brain malformation other than PSIS. Besides the CCDC88C variants, the patient also carried a TACR3 mutation, which was not described before and was predicted to be probably damaging. Homozygous TACR3 mutations are associated with hypogonadotropic hypogonadism. The present patient has complete anterior pituitary insufficiency, including hypogonadism. In this case, it is feasible that TACR3 contributed to the phenotype in a polygenic model. Functional studies will have to be performed to provide more evidence for the pathogenicity of the CCDC88C mutations, but the phenotype, including midline brain abnormalities, and the importance of the Wnt pathway in pituitary development make CCDC88C a promising candidate gene for PSIS. KIF14 Patient 15 had a de novoKIF14 mutation (c.3728A>G) that has not been described before and is predicted to be possibly damaging. Autosomal recessive KIF14 mutations have been linked to a lethal fetal ciliopathy, resembling Joubert syndrome (43). Primary cilia are involved in central nervous system development and are involved in signaling pathways, such as Shh and Wnt pathways. Ciliopathies are disorders caused by defects in the primary ciliary structure and include Joubert syndrome and Bardet-Biedl syndrome. In KIF14 mutant mice, the development of laminated structures in the central nervous system is affected, and the olfactory bulb was shown to be cytoarchitecturally disorganized (44). The olfactory placode is involved in hypothalamic-pituitary development with gonadotrophin-releasing hormone neurons migrating from the olfactory placode into the hypothalamus. Disruption of this migration is a key feature of Kallmann syndrome, consisting of hypogonadotropic hypogonadism and anosmia. Given this, mutations in KIF14 may well influence pituitary development. Because we found other heterozygous pathogenic mutations in genes linked to ciliopathies (INPP5E, CC2D2A, and B9D1), genes involved in primary ciliary structure/function may be involved in the PSIS phenotype and should be studied in more detail. KAT6A A de novo KAT6A mutation (c.235C>T) leading to a premature stop codon was found in patient 6. This patient’s case has been published in detail elsewhere (28). This patient had a developmental delay, with severely delayed speech, which was initially attributed to hypoglycemic brain damage due to untreated central adrenal insufficiency and GH deficiency in the first 2 years of life. In retrospect, she fulfills the criteria for KAT6A neurodevelopmental disorder and should not have been included in this study on isolated PSIS. Because subtle midline brain abnormalities were reported in KAT6A patients (cavum septum pellucidum, absent bulbus olfactorius), PSIS may be part of the phenotypic spectrum. However, because this patient also had potentially pathogenic BMP4 and GLI3 variants and a rare GLI2 variant (all present in the unaffected father), a polygenic cause of the pituitary malformation is also possible. Variants in genes associated with midline brain malformation Hypogonadotropic hypogonadism genes: PROK2, NR0B1 A pathogenic PROK2 frameshift mutation leading to a premature stop codon (c.163delA) was found in patient 13. The mutation was inherited from the unaffected father. Homozygous and heterozygous PROK2 and PROKR2 variants are associated with Kallmann syndrome and hypogonadotropic hypogonadism. Recently, PROKR2 variants were reported in patients with CPHD. In a UK series of 422 patients with CPHD (89% with septo-optic dysplasia and 11% with holoprosencephaly or midline clefts), PROKR2 mutations were found in 11 cases, but no mutations were found in PROK2 (16). In a Brazilian series of 156 patients with CPHD, two patients with a PROKR2 mutation were identified (PROK2 was not investigated) (17). In a French series of 72 patients with PSIS, two PROKR2 mutations were found (6). The presently reported PROK2 mutation was reported in a Portuguese family in which homozygous family members had hypogonadotropic hypogonadism with and without anosmia and heterozygous family members were unaffected (31). A Swiss patient with the same homozygous PROK2 mutation had hypogonadotropic hypogonadism with anosmia; data on heterozygous carriers were not available (32). The presently reported patient and his unaffected father both also had a rare, possibly damaging mutation in B9D1 associated with Joubert syndrome. The incomplete penetrance in the father may be due to the presence of variants in other genes or to epigenetic influences, which may fit a complex polygenic background for PSIS. A very rare missense mutation (MAF, 0.0001%) in the X-linked NR0B1 (c.315G>C) was found in patient 12. The mutation was inherited from the mother and predicted to be damaging. It has previously been linked to isolated mineralocorticoid deficiency and a mild form of congenital adrenal underdevelopment (30). Midline brain anomalies have not been reported before in individuals with NR0B1 variants. The same patient also had a rare pathogenic CC2D2A mutation. Absent corpus callosum: DCHR7, CCD2DA In three patients, pathogenic mutations in genes associated with absent corpus callosum were found: DCHR7 (c.452G>A; patients 4 + 18) and CCD2DA (c.3055C>T; patient 12). These genes are known to cause autosomal recessive disorders; DCHR7 mutations can cause Smith-Lemli-Opitz syndrome, and CCD2DA mutations can cause Meckel syndrome and Joubert syndrome. In each patient, the mutation was inherited from an unaffected parent, indicating that being a carrier in itself is insufficient to develop PSIS. CCD2DA is involved in ciliopathies, as is KIF14 (described previously). In the series of genes with variants classified as having unknown clinical significance, there are three genes (INPP5E, CC2D2A, and B9D1) that function as ciliopathy genes. This suggests that ciliopathy genes may be involved in pituitary development. Holoprosencephaly: GLI2 variants GLI2 is associated with holoprosencephaly and acts in the Wnt pathway. In a review, 25 patients (16 families) were reported to have heterozygous mutations in GLI2 and disturbed pituitary development (14). Most patients had an ectopic posterior pituitary. The suggested pattern of inheritance was autosomal dominant with incomplete penetrance and variable expression (14). In the current study, six patients had GLI2 variants with a MAF <0.05. Two patients had the combination of GLI2 variants M1352V + D1520N (patients 3 + 14). This combination has previously been reported in patients with PSIS, and functional studies have demonstrated reduced transcription activity and reduced luciferase activity (10, 13, 27). Another presently reported patient had a V432M variant that has also been described in patients with PSIS (10, 11, 13). The R1382H variant found in another patient has not been described before, was inherited from an unaffected mother, is very rare (MAF, 0.0002), and is potentially pathogenic. GLI2 variants with a MAF <0.05 were not more common in PSIS than in the general population (1000 Genomes); however, despite the small numbers, the specific combination of M1352V + D1520N variants seems to be important in PSIS because we found the combination in two of 20 patients (10%) compared with only 17 of 2504 individuals (0.68%) in the reference population (1000 Genomes). GLI2 variants have been reported at a relatively high frequency in patients with CPHD/PSIS, and GLI2 is likely an important factor in the polygenic background of pituitary development. Strengths and limitations Strengths of this study are the homogeneous group of study participants. A limitation is the inclusion of the KAT6A patient who had syndromic PSIS and not the isolated form. It emphasizes the importance of proper phenotyping in such studies (45). All studied patients had congenital central hypothyroidism, and therefore the results may not be representative for PSIS patients without central hypothyroidism. We presumed all parents were unaffected on the basis of their medical history. However, we did not perform endocrine studies or MRIs in parents. Exome sequencing by itself is limited by incomplete coverage of the exome and easily missed copy number variations, although the presently used targeted approach decreased the likelihood of missing deletions and duplications. Absolute proof for causality of combinations of variants in candidate genes can be obtained only by functional studies (46). It was beyond the aims and possibilities of the current study to perform such functional studies for each (combination of) candidate genes. Because we searched only for variants genes in lymphocytes, mosaicisms confined to affected tissue(s) cannot be excluded. Exome sequencing and genome sequencing can detect variants in genes, but epigenetic mechanisms such as DNA methylation, histone modification, and microRNAs may play a role, warranting additional studies. Systematically investigating large series of carefully phenotyped patients and collaboration between research groups are essential in the search for genes and pathways underlying disorders with a polygenic etiology (9). In conclusion, searching for causes and pathogenesis of a potentially polygenic disorder is complex. Here, we added four candidate genes for isolated PSIS (DCHS1, ROBO2, CCDC88C, and KIF14) and one for syndromic PSIS (KAT6A). In addition, we found 11 GLI2 variants in six patients and verified the higher frequency of a combination of two GLI2 variants in the study group compared with a reference population. We detected various (potentially) pathogenic variants in genes associated with midline brain anomalies and in genes involved in ciliary structure and function and suggest that these genes be included in future searches for a polygenic cause of PSIS and CPHD. Abbreviations: BMP bone morphogenetic protein CPHD combined pituitary hormone deficiencies FGF fibroblast growth factor GH growth hormone MAF minor allele frequency MRI magnetic resonance imaging PSIS pituitary stalk interruption syndrome ROBO2 roundabout guidance receptor 2. Acknowledgments Financial Support: N.Z.-S.: Work was partly funded by the Emma Foundation, Emma Children’s Hospital, Academic Medical Centre, Amsterdam, The Netherlands. Disclosure Summary: The authors have nothing to disclose. References 1. Bar C, Zadro C, Diene G, Oliver I, Pienkowski C, Jouret B, Cartault A, Ajaltouni Z, Salles JP, Sevely A, Tauber M, Edouard T. 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Nature . 2014; 508( 7497): 469– 476. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Clues for Polygenic Inheritance of Pituitary Stalk Interruption Syndrome From Exome Sequencing in 20 Patients

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Endocrine Society
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Copyright © 2018 Endocrine Society
ISSN
0021-972X
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1945-7197
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10.1210/jc.2017-01660
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Abstract

Abstract Context Pituitary stalk interruption syndrome (PSIS) consists of a small/absent anterior pituitary lobe, an interrupted/absent pituitary stalk, and an ectopic posterior pituitary lobe. Mendelian forms of PSIS are detected infrequently (<5%), and a polygenic etiology has been suggested. GLI2 variants have been reported at a relatively high frequency in PSIS. Objective To provide further evidence for a non-Mendelian, polygenic etiology of PSIS. Methods Exome sequencing (trio approach) in 20 patients with isolated PSIS. In addition to searching for (potentially) pathogenic de novo and biallelic variants, a targeted search was performed in a panel of genes associated with midline brain development (223 genes). For GLI2 variants, both (potentially) pathogenic and relatively rare variants (<5% in the general population) were studied. The frequency of GLI2 variants was compared with that of a reference population. Results We found four additional candidate genes for isolated PSIS (DCHS1, ROBO2, CCDC88C, and KIF14) and one for syndromic PSIS (KAT6A). Eleven GLI2 variants were present in six patients. A higher frequency of a combination of two GLI2 variants (M1352V + D1520N) was found in the study group compared with a reference population (10% vs 0.68%). (Potentially) pathogenic variants were identified in genes associated with midline brain anomalies, including holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum and in genes involved in ciliopathies. Conclusion Combinations of variants in genes associated with midline brain anomalies are frequently present in PSIS and sustain the hypothesis of a polygenic cause of PSIS. Pituitary stalk interruption syndrome (PSIS) is a congenital anomaly of the pituitary gland consisting of a classic triad of magnetic resonance imaging (MRI) findings: (1) small or absent anterior pituitary lobe, (2) interrupted or absent pituitary stalk, and (3) ectopic posterior pituitary lobe. PSIS may be isolated or may occur in association with other midline brain malformations (syndromic PSIS) (1, 2). The clinical consequences of PSIS vary from isolated growth hormone (GH) deficiency to combined pituitary hormone deficiencies (CPHD) in which two or more anterior pituitary lobe functions [production of GH, thyrotropin, corticotropin, and gonadotropins (luteinizing hormone + follicle-stimulating hormone] may be insufficient. Prolactin level is usually elevated because of lack of hypothalamic dopaminergic inhibition. In isolated PSIS, typically only anterior pituitary hormone deficiencies occur, whereas in syndromic PSIS, posterior pituitary hormone deficiency (antidiuretic hormone) also may be present, with central diabetes insipidus as a clinical consequence (1, 2). The pituitary lobes and stalk have different embryological origins. The anterior lobe derives from the oral ectoderm, whereas the posterior lobe and pituitary stalk derive from the neural ectoderm. Various transcription factors are involved in the initial formation of the pituitary gland and the following cellular differentiation (3). Important signaling pathways involved in pituitary development are Wnt, Notch, Sonic Hedgehog (Shh), bone morphogenetic proteins (BMPs), and fibroblast growth factor (FGF). Mutations in genes encoding transcription factors in these pathways are found in <5% of PSIS cases and most often in association with other extrapituitary anomalies (4–8). In a recent review, Fang et al. (9) reported 30 candidate genes for PSIS and CPHD. Various genetic studies showed an association between PSIS and genes involved in holoprosencephaly, which is characterized by severely disturbed midline brain development, suggesting that PSIS may represent a mild form of this disorder (7). In particular, GLI2 variants, which are associated with holoprosencephaly, have been reported at a relatively high frequency in isolated PSIS (10–14). Furthermore, genes involved in hypogonadotropic hypogonadism (PROKR2, FGF8, FGFR1) have been associated with CPHD (6, 15–17). However, clinical expression is variable, including unaffected mutation-carrying relatives, suggesting involvement of additional genetic or environmental factors. Most studies searching for genetic causes of CPHD have been done in heterogeneous patient populations, including patients with other midline brain malformations, and most studies have focused on pathogenic mutations in specific genes, assuming a Mendelian inheritance (5). Because Mendelian forms of PSIS are detected only rarely, a polygenic and multifactorial etiology for PSIS should be considered as well (9). A polygenic inheritance has also been suggested for holoprosencephaly (18). In a very recent study using exome sequencing in 24 Chinese patients with isolated PSIS, heterozygous mutations, mostly in genes associated with Notch, Shh, and Wnt signaling pathways, were identified in 22 patients. Most patients (20 of 24) had more than one mutation, suggesting polygenic involvement. No mutations were found in genes known to be associated with pituitary malformation. Because parents were not included in this Chinese study, the authors were unable to determine whether mutations were de novo (19). To provide further evidence for a non-Mendelian, polygenic etiology of PSIS, we performed whole exome sequencing in 20 patients with isolated PSIS and their unaffected parents. In addition to searching for (potentially) pathogenic de novo and biallelic variants, we performed a targeted search in a large panel of genes associated with midline brain development. We included not only genes with a known association with pituitary formation (37 genes) but also genes associated with other midline brain malformations: holoprosencephaly (18 genes), hypogonadotropic hypogonadism (34 genes), and absent corpus callosum (134 genes). For each patient, all pathogenic variants and variants of unknown clinical significance (potentially pathogenic) were listed. Because rare GLI2 variants have been frequently reported in PSIS, we searched for (potentially) pathogenic GLI2 and for all relatively rare GLI2 variants (reported frequency <5% in the general population). Although rare GLI2 variants have been reported in association with PSIS, the frequency of (combinations of) these rare variants in the general population has not been investigated. To verify whether these rare GLI2 variants are indeed more common in PSIS, we compared the frequency of these variants in our study group with the frequency in a reference population. Patients and Methods Patients A database containing patients with congenital central hypothyroidism, located at the Department of Pediatric Endocrinology of the Academic Medical Center, Amsterdam, was searched for all patients with isolated PSIS. Most patients had been diagnosed after an abnormal result in the Dutch thyroxine-based neonatal screening program for congenital hypothyroidism (20). We selected 20 patients who had two or more pituitary hormone deficiencies and were still being treated at the Academic Medical Center. The diagnosis of PSIS was based on the three classic MRI findings. Patients with other (midline) brain abnormalities or with dysmorphic features or other pathologies suggestive of syndromic PSIS were excluded. The study was approved by the local medical ethics committee (NL47088.018.13). After informed consent was obtained, a venous blood sample was collected from all patients and their unaffected parents. Molecular studies Whole exome sequencing and variant calling were performed by the Beijing Genomic Institute. The capture used to enrich for exome sequences was the Agilent SureSelect Human All Exon V5 (50M) kit, and sequencing was done on either an Illumina or Complete Genomics platform. Variant prioritization Variant prioritization was done using the Cartagenia Bench Laboratory NGS (Agilent). Variants were considered potentially pathogenic when either resulting in truncation of the protein, predicted to result in a possibly/probably pathogenic variant by Polyphen2, or predicted to affect splicing and present at a frequency of <1% in the general population [based on the dbSNP database (dbSNP build 141 GRCh37.p13), ESP6500 (http://evs.gs.washington.edu/EVS/), 1000 Genomes Project (1000 Genomes phase 3 release, version 5.20130502), GoNL (http://www.nlgenome.nl/), and >900 in-house reference samples]. The validity of variants was judged by visual inspection of read data in Integrative Genomics Viewer or was confirmed by Sanger sequencing. A target gene panel for CPHD, holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum was made on the basis of results of a literature search (Table 1) (3, 8, 9, 18, 21, 22). Table 1. Gene Panels Used in the Targeted Search Genes Evaluated as Part of the Pituitary Panel (n = 37)  ARNT2, BMP2, BMP4, CDON,aFGF8,aFGF10, FGF18, FGFR1,aGATA2, GLI 1, GLI2,aGLI3, GLI4, GLI5, GLI6, GPR161, HESX1, IGSF1, LHX3, LHX4, NR5A1, OTX2, PAX6, PITX1, PITX2, POU1F1, PROP1, SHH, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, SOX1, SOX2, SOX3, TBX19, TGIF, WNT5a  Genes Evaluated as Part of the Pituitary Panel (n = 37)  ARNT2, BMP2, BMP4, CDON,aFGF8,aFGF10, FGF18, FGFR1,aGATA2, GLI 1, GLI2,aGLI3, GLI4, GLI5, GLI6, GPR161, HESX1, IGSF1, LHX3, LHX4, NR5A1, OTX2, PAX6, PITX1, PITX2, POU1F1, PROP1, SHH, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, SOX1, SOX2, SOX3, TBX19, TGIF, WNT5a  Genes Evaluated as Part of the Holoprosencephaly Panel (n = 18)  CDON,aDISP1, DLL1, FGF8,aFGFR1,aFOXH1, GAS1, GLI2,aHHAT, NODAL, PTCH1, SHH, SIX3, STIL, SUFU, TDGF1, TGIF1, ZIC2  Genes Evaluated as Part of the Holoprosencephaly Panel (n = 18)  CDON,aDISP1, DLL1, FGF8,aFGFR1,aFOXH1, GAS1, GLI2,aHHAT, NODAL, PTCH1, SHH, SIX3, STIL, SUFU, TDGF1, TGIF1, ZIC2  Genes Evaluated as Part of the Hypogonadotropic Hypogonadism Panel (n = 34)  AXL, CCDC141, CHD7, DMXL2, FEZF1, FGF8,aFGF17, FGFR1,aGNRH1, GNRHR, HESX1, HS6ST1, KAL1, KISS1, KISS1R, LEP, LEPR, NR0B1, NSMF, OL14RD, OTUD4, PCSK1, PNPLA6, PROK2, PROKR2, RNF216, SEMA3A, SEMA3E, SEMA7A, SOX10, STS, TAC3, TACR3, WDR11  Genes Evaluated as Part of the Hypogonadotropic Hypogonadism Panel (n = 34)  AXL, CCDC141, CHD7, DMXL2, FEZF1, FGF8,aFGF17, FGFR1,aGNRH1, GNRHR, HESX1, HS6ST1, KAL1, KISS1, KISS1R, LEP, LEPR, NR0B1, NSMF, OL14RD, OTUD4, PCSK1, PNPLA6, PROK2, PROKR2, RNF216, SEMA3A, SEMA3E, SEMA7A, SOX10, STS, TAC3, TACR3, WDR11  Genes Evaluated as Part of the Corpus Callosum Agenesis Panel (n = 134)  AHI1, AKT3, AMPD2, ANOP1, ARID1B, ARL13B, ARX, ASPM, ATR, ATRX, B9D1, B9D2, BCOR, BMP4, C12ORF57, CASK, CC2D2A, CENPJ, CEP152, CEP290, CEP41, CEP63, CREBBP, CTBP1, AS1, DCX, DHCR7, DHCR24, DIS3L2, DISC1, EFNB1, EOMES, EP300, EPG5, FGF8, FGFR1, FGFR2, FH, FKRP, FKTN, FLNA, GLI3, GPSM2, GTDC2, HCCS, HESX1, HS6ST1, HYLS1, IGBP1, IGF1, INPP5E, ISPD, KAT6B, KCC3, KIF7, L1CAM, LARGE, LRP2, MED12, MID1, MKS1, NDE1, NFIX, NIN, NPHP1, NPHP3, NSD1, OFD1, OTX1, PAX6, PDHA1, PDHB, POMGNT1, POMT1, POMT2, PYCR1, RAB18, RAB3GAP1, RAB3GAP2, RBBP8, RBM10, RELN, RNU4ATAC, RPGRIP1L, RPS6KA3, SCKL3, SOX2, SPG11, STRA6, TCF4, TCTN1, TCTN2, TCTN3, TMEM138, TMEM216, TMEM237, TMEM67, TUBA1A, TUBB2B, TUBB3, VAX1, WDR62, ZEB2  Genes Evaluated as Part of the Corpus Callosum Agenesis Panel (n = 134)  AHI1, AKT3, AMPD2, ANOP1, ARID1B, ARL13B, ARX, ASPM, ATR, ATRX, B9D1, B9D2, BCOR, BMP4, C12ORF57, CASK, CC2D2A, CENPJ, CEP152, CEP290, CEP41, CEP63, CREBBP, CTBP1, AS1, DCX, DHCR7, DHCR24, DIS3L2, DISC1, EFNB1, EOMES, EP300, EPG5, FGF8, FGFR1, FGFR2, FH, FKRP, FKTN, FLNA, GLI3, GPSM2, GTDC2, HCCS, HESX1, HS6ST1, HYLS1, IGBP1, IGF1, INPP5E, ISPD, KAT6B, KCC3, KIF7, L1CAM, LARGE, LRP2, MED12, MID1, MKS1, NDE1, NFIX, NIN, NPHP1, NPHP3, NSD1, OFD1, OTX1, PAX6, PDHA1, PDHB, POMGNT1, POMT1, POMT2, PYCR1, RAB18, RAB3GAP1, RAB3GAP2, RBBP8, RBM10, RELN, RNU4ATAC, RPGRIP1L, RPS6KA3, SCKL3, SOX2, SPG11, STRA6, TCF4, TCTN1, TCTN2, TCTN3, TMEM138, TMEM216, TMEM237, TMEM67, TUBA1A, TUBB2B, TUBB3, VAX1, WDR62, ZEB2  a Genes included in more than one list. View Large The exome data set was analyzed stepwise: First, we searched for potentially pathogenic variants in 37 genes known to be associated with pituitary development or function. Next, we searched for new candidate genes by identifying any potentially pathogenic de novo, homozygous, or compound heterozygous variants, including X-linked mutations in males. For each gene, a PubMed search was performed to determine function and/or brain expression pattern. Thereafter, we searched for potentially pathogenic variants in the panel of genes involved in midline brain pathologies: holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum. Lastly, we identified all GLI2 variants with a reported minor allele frequency (MAF) <0.05. The frequency of GLI2 variants with a MAF <0.05 was compared with the frequency of GLI2 variants with a MAF <0.05 in the 1000 Genomes database. Specific combinations of GLI2 variants, detected in the current study, were also searched for in the 1000 Genomes database. Because of the ethnic diversity of our study population, we used the 1000 Genomes database as an international reference population. Modeling and molecular dynamic study Three-dimensional models were constructed for the wild-type DCHS1 and DCHS1 I780V, P1519S, L2132P, H2729L mutants and for wild-type roundabout guidance receptor 2 (ROBO2; isoform 2a) and R314Q mutants. Full-length secondary structure predictions and the identification of most suitable templates were performed using the ROSETTA-based Robetta Web server (http://robetta.bakerlab.org). Using the homology modeling program Modeler 9v14 (www.salilab.org/modeller/), four pairs of models of the three extracellular domains (wild-type and mutants; residue range: 680 to 886, 1318 to 1641, 2062 to 2270, and 2536 to 2867) were generated considering previously published structural DCHS1 and ROBO2 data and Robetta outputs (23–26). Modeling was performed with the default parameters using the allHmodel protocol to include hydrogen atoms and the HETATM protocol to include Ca2+ with restricting Ca2+-binding residues side-chain distance and with thorough molecular dynamics optimization and refinement protocol. To study the mutational effects on structure, the molecular dynamics of each model pair was simulated using a NAMD 2.11 (http://www.ks.uiuc.edu/Research/namd/) plug-in in the VMD v1.9.2.27 program (http://www.ks.uiuc.edu/Research/vmd/) for 100 ns. Intermediate and final structures were evaluated in PyMol. Results We studied 20 unrelated patients (14 males) with an age range of 3 to 28 years (Table 2). Age at diagnosis ranged from 2 weeks to 5 years. Eleven children had been diagnosed with congenital central hypothyroidism after detection by neonatal screening, seven children after referral for growth retardation, one child for prolonged jaundice, and another child for neonatal hypotonia. Twelve patients had deficiency of four anterior pituitary hormones, seven patients had three hormone deficiencies, and one patient had two hormone deficiencies. All patients had GH deficiency. Central adrenal insufficiency was present in 19 patients, and hypogonadotropic hypogonadism was present in three patients of prepubertal age and in nine patients of postpubertal age. Three children had normal pubertal development, and in five prepubertal children, information on the status of the hypothalamic-pituitary-gonadal axis was not available. No patient had central diabetes insipidus. The medical history of parents was negative for endocrine deficiencies, and all parents had normal development, growth, and fertility. Table 2. Clinical Characteristics of Studied Patients With Isolated PSIS Case  Sex  Age (y)  Age at Diagnosis  Presentation  Pituitary Deficiencies  MRI Findings  1  M  8  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  2  F  25  5 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  3  M  28  3 mo  Prolonged jaundice  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  4  M  3  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, interrupted stalk, small AP  5  M  26  2 wk  Hypotonia  GH; TSH; ACTH  EPP, small stalk, small AP  6  F  10  2 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  7  F  9  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, absent AP  8  F  16  1 mo  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  9  M  23  6 mo  Abnormal NS  GH; TSH; ACTH  EPP, absent stalk, small AP  10  F  9  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  11  M  18  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  12  M  13  2 y  Growth retardation  GH; TSH  EPP, absent stalk, normal AP  13  M  23  6 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, normal AP  14  M  18  4 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  15  M  19  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  16  M  17  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, small stalk, small AP  17  F  5  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  18  M  5  1.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  19  M  8  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, small stalk, small AP  20  M  20  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  Case  Sex  Age (y)  Age at Diagnosis  Presentation  Pituitary Deficiencies  MRI Findings  1  M  8  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  2  F  25  5 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  3  M  28  3 mo  Prolonged jaundice  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  4  M  3  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, interrupted stalk, small AP  5  M  26  2 wk  Hypotonia  GH; TSH; ACTH  EPP, small stalk, small AP  6  F  10  2 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  7  F  9  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, absent AP  8  F  16  1 mo  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  9  M  23  6 mo  Abnormal NS  GH; TSH; ACTH  EPP, absent stalk, small AP  10  F  9  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  11  M  18  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  12  M  13  2 y  Growth retardation  GH; TSH  EPP, absent stalk, normal AP  13  M  23  6 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, normal AP  14  M  18  4 y  Growth retardation  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, absent AP  15  M  19  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  16  M  17  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, small stalk, small AP  17  F  5  3 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH (tested at 3 mo)  EPP, absent stalk, small AP  18  M  5  1.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, absent stalk, small AP  19  M  8  2.5 y  Growth retardation  GH; TSH; ACTH; LH/FSH unknown  EPP, small stalk, small AP  20  M  20  2 wk  Abnormal NS  GH; TSH; ACTH; LH/FSH  EPP, absent stalk, small AP  Abbreviations: ACTH, adrenocorticotropic hormone; AP, anterior pituitary; EPP, ectopic posterior pituitary; FSH, follicle-stimulating hormone; LH, luteinizing hormone; NS, neonatal screening result; TSH, thyrotropin. View Large For each patient, all pathogenic and potentially pathogenic variants and all GLI2 variants with an MAF <0.05 are shown in Tables 3 and 4 . Table 3. Exome Sequencing Results of Studied Patients With PSIS Including Variants, Reported Allele Frequency, Classification According to ACMG Guidelines, In Silico Prediction (Polyphen 2), and Phenotypes Known To Be Caused by Variants in Each Gene Case  Gene  Variant  dbSNP  Inheritance  Variant Classification  MAF (GnomAD)  In Silico Prediction  Gene Panel  Known Phenotype (MIM Number)  Information on Specific Variant  3  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Mother  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same two GLI2 variants in case 14 
Combination of these two variants previously described in patients with PSIS, including functional study showing reduced transcriptional activity and reduced luciferase activity (10, 13, 27)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Mother  VUS  0.0101  Probably damaging  4  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 18. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  RELN  c.9646G>A, p.E3216K (missense)  —  Mother  VUS  N  Benign  CCA  Lissencephaly 2 (MIM 257320)    IGSF1  c.498G>C, p.E166D  rs201255931  Mother  VUS  0.0005  Probably damaging  PIT  XL central hypothyroidism (MIM 300888)  5  INPP5E  c.902T>C, p.L301P (missense)  —  Mother  VUS  N  Probably damaging  CCA  Joubert syndrome 1 (MIM 213300)    6  KAT6A  c.235C>T, p.A79* (nonsense)  —  De novo  Pathogenic  N  Damaging  Not from panel  KAT6A neurodevelopmental syndrome (MIM 616268)  (28)  GLI3  c.539G>A, p.R180Q (missense)  rs140772904  Father  VUS  0.00004  Possibly damaging  PIT  Greig cephalopolysyndactyly syndrome (MIM 175700), Pallister-Hall syndrome (MIM 146510), postaxial polydactyly (MIM 174200), preaxial polydactyly (MIM 174700)  BMP4  c.804_815delCCGGCCCCTCCT, p.R269_L272del (deletion)  —  Father  VUS  N    PIT  Syndromic microphthalmia type 6 (MIM 607932)  GLI2  c.1761G>A, p.T587T (synonymous)  rs61732852  Mother  Likely benign  0.0119    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  7  DCHS1  c.6395T>C, p.L2132P (missense)  —  Father  VUS  N  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.2338A>G, p.I780V (missense)  rs145735483  Mother  VUS  0.0003  Benign        GLI2  c.1294G>A, p.V432M (missense)  rs142296407  Mother  VUS  0.0015  Possibly damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same variant described in patients with MPHD/PSIS (10, 11, 13)  8  NDE  c.488T>A, p.L163Q (missense)  —  Father  VUS  N  Probably damaging  CCA  Lissencephaly 4 (MIM 614019)    OTUD4  c.823G>T, p.V275L (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotropism (MIM 611744)  9  ROBO2  c.914G>A, p.R314Q (missense)  —  De novo  VUS  N  Probably damaging  Not from panel  Vesicoureteral reflux 2 (MIM 610878)    10  GLI2  c.963C>G, p.P321P (synonymous)  rs149894186  Mother  Benign  0.0046    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    GLI2  c.2088A>G, p.A696A (synonymous)  rs146059306  Mother  Benign  0.0009  GLI2  c.2262G>T, p.R754R (synonymous)  rs142856393  Father  Benign  0.0011  11  ASPM  c.10161+5G>C (insertion)  —  Mother  VUS/likely pathogenic (altered splicing?)  N    CCA  Primary autosomal recessive microcephaly (MIM 608716)    FGF8  c.77C>T, p.P26L (missense)  rs137852660  Mother  VUS  0.0019  Benign  PIT + HH  Hypogonadotropic hypogonadism 6 (MIM 612702)  Heterozygosity described in a patient with HH and partial empty sella [Falardeau et al. (29)]  CHD4  c.2374C>T, p.R792W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    12  CC2D2A  c.3055C>T, p.R1019* (nonsense)  rs370880399  Mother  Pathogenic  0.0001  Damaging  CCA  Joubert syndrome 9 (MIM 612285), Meckel syndrome 6 (MIM 612284)  Variant reported in compound heterozygosity with another variant in a number of patients with Joubert syndrome  NR0B1  c.315G>C, p.W105C (missense)  rs132630327  Mother  Pathogenic  0.00001  Damaging  HH  Congenital adrenal hypoplasia (MIM 300200), 46XY reversal 2 (MIM 300018)  Same mutation reported in patient with isolated mineralocorticoid deficiency (30)  ARNT2  c.1707G>T, p.Q569H (missense)  rs145379118  Father  VUS  0.0033  Possibly damaging  PIT  Webb-Dattani syndrome (MIM 615926)    13  PROK2  c.163delA p.I55* (nonsense)  rs554675432  Father  Pathogenic  0.0001  Damaging  HH  Hypogonadotropic hypogonadism 4 (MIM 610628)  Homozygosity reported in patients with anosmic hypogonadotropic hypogonadism (31, 32)  B9D1  c.151T>C, p.S51P (missense)  rs546359789  Father  VUS  0.00006  Probably damaging  CCA  Joubert syndrome 27 (MIM 617120), Meckel syndrome 9 (MIM 614209).  Variant reported in compound heterozygosity with another variant in a patient with Joubert syndrome [Srour et al. (33)]  14  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Father  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  See case 3 (same combination of GLI2 variants)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Father  VUS  0.0101  Probably damaging        GLI2  c.1944C>T, p.T648T (synonymous)  rs13008360  Both parents  Benign  0.0253          CHD4  c.5149C>T, p.R1717W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/ macula dystrophy (MIM 212550)  Same variant in patient 19. Variant previously described with reduced function [Carnes et al. (34)]  15  KIF14  c.3728A>G, p.K1243R  —  De novo  VUS  N  Probably damaging  Not from panel  Meckel syndrome 12 (MIM 616258)    16  GLI2  c.4145G>A, p.R1382H (missense)  rs200080112  Mother  VUS  0.0002  Probably damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    17  CCDC88C  c.3895C>T, p.R1299C (missense)  rs142539336  Father  VUS  0.0049  Possibly damaging  Not from panel  Nonsyndromic hydrocephalus (MIM 616053)    CCDC88C  c.1984G>A, p.E662K (missense)  Not in dbSNP  Mother  VUS  0.0001  Benign      TACR3  c.659T>C, p.L220P (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotrophic hypogonadism (MIM 614840)  18  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 4. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  CHD7  c.1324G>A, p.A442T (missense)  rs368086966  Mother  VUS  0.0002  Benign  HH  CHARGE syndrome (MIM 214800), Hypogonadotropic hypogonadism (MIM 612370)    19  DCHS1  c.8186A>T, p.H2729L (missense)  rs148148252  Father  VUS/likely pathogenic  0.0003  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.4555C>T, p.P1519S (missense)  rs199544459  Mother  VUS  0.0017  Benign        SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/macula dystrophy (MIM 212550)  Same variant in patient 14. Variant previously described with reduced function [Carnes et al. (34)]  BMP4  c.1001C>A, p.A334D (missense)  rs550409227  Mother  VUS  0.000008  Probably damaging  PIT  Syndromic microphthalmia type 6 (MIM 607932)    20  SLC12A6  c.1787C>T, p.P596L (missense)  —  Mother  VUS/likely pathogenic  N  Possibly damaging  CCA  AR corpus callosum agenesis + peripheral neuropathy (MIM 218000)  CC2D2A  c.3865A>G, p.T1289A (missense)  —  Mother  VUS  N  Possibly damaging  CCA  Joubert syndrome (MIM 612285), Meckel syndrome (MIM 612284)  Case  Gene  Variant  dbSNP  Inheritance  Variant Classification  MAF (GnomAD)  In Silico Prediction  Gene Panel  Known Phenotype (MIM Number)  Information on Specific Variant  3  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Mother  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same two GLI2 variants in case 14 
Combination of these two variants previously described in patients with PSIS, including functional study showing reduced transcriptional activity and reduced luciferase activity (10, 13, 27)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Mother  VUS  0.0101  Probably damaging  4  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 18. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  RELN  c.9646G>A, p.E3216K (missense)  —  Mother  VUS  N  Benign  CCA  Lissencephaly 2 (MIM 257320)    IGSF1  c.498G>C, p.E166D  rs201255931  Mother  VUS  0.0005  Probably damaging  PIT  XL central hypothyroidism (MIM 300888)  5  INPP5E  c.902T>C, p.L301P (missense)  —  Mother  VUS  N  Probably damaging  CCA  Joubert syndrome 1 (MIM 213300)    6  KAT6A  c.235C>T, p.A79* (nonsense)  —  De novo  Pathogenic  N  Damaging  Not from panel  KAT6A neurodevelopmental syndrome (MIM 616268)  (28)  GLI3  c.539G>A, p.R180Q (missense)  rs140772904  Father  VUS  0.00004  Possibly damaging  PIT  Greig cephalopolysyndactyly syndrome (MIM 175700), Pallister-Hall syndrome (MIM 146510), postaxial polydactyly (MIM 174200), preaxial polydactyly (MIM 174700)  BMP4  c.804_815delCCGGCCCCTCCT, p.R269_L272del (deletion)  —  Father  VUS  N    PIT  Syndromic microphthalmia type 6 (MIM 607932)  GLI2  c.1761G>A, p.T587T (synonymous)  rs61732852  Mother  Likely benign  0.0119    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  7  DCHS1  c.6395T>C, p.L2132P (missense)  —  Father  VUS  N  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.2338A>G, p.I780V (missense)  rs145735483  Mother  VUS  0.0003  Benign        GLI2  c.1294G>A, p.V432M (missense)  rs142296407  Mother  VUS  0.0015  Possibly damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  Same variant described in patients with MPHD/PSIS (10, 11, 13)  8  NDE  c.488T>A, p.L163Q (missense)  —  Father  VUS  N  Probably damaging  CCA  Lissencephaly 4 (MIM 614019)    OTUD4  c.823G>T, p.V275L (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotropism (MIM 611744)  9  ROBO2  c.914G>A, p.R314Q (missense)  —  De novo  VUS  N  Probably damaging  Not from panel  Vesicoureteral reflux 2 (MIM 610878)    10  GLI2  c.963C>G, p.P321P (synonymous)  rs149894186  Mother  Benign  0.0046    PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    GLI2  c.2088A>G, p.A696A (synonymous)  rs146059306  Mother  Benign  0.0009  GLI2  c.2262G>T, p.R754R (synonymous)  rs142856393  Father  Benign  0.0011  11  ASPM  c.10161+5G>C (insertion)  —  Mother  VUS/likely pathogenic (altered splicing?)  N    CCA  Primary autosomal recessive microcephaly (MIM 608716)    FGF8  c.77C>T, p.P26L (missense)  rs137852660  Mother  VUS  0.0019  Benign  PIT + HH  Hypogonadotropic hypogonadism 6 (MIM 612702)  Heterozygosity described in a patient with HH and partial empty sella [Falardeau et al. (29)]  CHD4  c.2374C>T, p.R792W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    12  CC2D2A  c.3055C>T, p.R1019* (nonsense)  rs370880399  Mother  Pathogenic  0.0001  Damaging  CCA  Joubert syndrome 9 (MIM 612285), Meckel syndrome 6 (MIM 612284)  Variant reported in compound heterozygosity with another variant in a number of patients with Joubert syndrome  NR0B1  c.315G>C, p.W105C (missense)  rs132630327  Mother  Pathogenic  0.00001  Damaging  HH  Congenital adrenal hypoplasia (MIM 300200), 46XY reversal 2 (MIM 300018)  Same mutation reported in patient with isolated mineralocorticoid deficiency (30)  ARNT2  c.1707G>T, p.Q569H (missense)  rs145379118  Father  VUS  0.0033  Possibly damaging  PIT  Webb-Dattani syndrome (MIM 615926)    13  PROK2  c.163delA p.I55* (nonsense)  rs554675432  Father  Pathogenic  0.0001  Damaging  HH  Hypogonadotropic hypogonadism 4 (MIM 610628)  Homozygosity reported in patients with anosmic hypogonadotropic hypogonadism (31, 32)  B9D1  c.151T>C, p.S51P (missense)  rs546359789  Father  VUS  0.00006  Probably damaging  CCA  Joubert syndrome 27 (MIM 617120), Meckel syndrome 9 (MIM 614209).  Variant reported in compound heterozygosity with another variant in a patient with Joubert syndrome [Srour et al. (33)]  14  GLI2  c.4054A>G, p.M1352V (missense)  rs149140724  Father  Benign  0.0099  Benign  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)  See case 3 (same combination of GLI2 variants)  GLI2  c.4558G>A, p.D1520N (missense)  rs114814747  Father  VUS  0.0101  Probably damaging        GLI2  c.1944C>T, p.T648T (synonymous)  rs13008360  Both parents  Benign  0.0253          CHD4  c.5149C>T, p.R1717W (missense)  —  Father  VUS  N  Probably damaging  PIT  Sifrim-Hitz-Weiss syndrome (MIM 617159)    SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/ macula dystrophy (MIM 212550)  Same variant in patient 19. Variant previously described with reduced function [Carnes et al. (34)]  15  KIF14  c.3728A>G, p.K1243R  —  De novo  VUS  N  Probably damaging  Not from panel  Meckel syndrome 12 (MIM 616258)    16  GLI2  c.4145G>A, p.R1382H (missense)  rs200080112  Mother  VUS  0.0002  Probably damaging  PIT + HPE  Holoprosencephaly 9 (MIM 610829), Culler-Jones syndrome (MIM 615849)    17  CCDC88C  c.3895C>T, p.R1299C (missense)  rs142539336  Father  VUS  0.0049  Possibly damaging  Not from panel  Nonsyndromic hydrocephalus (MIM 616053)    CCDC88C  c.1984G>A, p.E662K (missense)  Not in dbSNP  Mother  VUS  0.0001  Benign      TACR3  c.659T>C, p.L220P (missense)  —  Mother  VUS  N  Probably damaging  HH  Hypogonadotrophic hypogonadism (MIM 614840)  18  DHCR7  c.452G>A, p.W151* (nonsense)  rs11555217  Mother  Pathogenic  0.0007  Damaging  CCA  Smith-Lemli-Opitz syndrome (MIM 270400)  Same mutation in patient 4. Homozygosity described in patients with Smith-Lemli-Opitz syndrome  CHD7  c.1324G>A, p.A442T (missense)  rs368086966  Mother  VUS  0.0002  Benign  HH  CHARGE syndrome (MIM 214800), Hypogonadotropic hypogonadism (MIM 612370)    19  DCHS1  c.8186A>T, p.H2729L (missense)  rs148148252  Father  VUS/likely pathogenic  0.0003  Probably damaging  Not from panel  Van Maldergem syndrome (MIM 601390), AD mitral valve prolapse (MIM 607829)    DCHS1  c.4555C>T, p.P1519S (missense)  rs199544459  Mother  VUS  0.0017  Benign        SIX6  c.385G>A, p.E129K (missense)  rs146737847  Mother  VUS  0.0040  Probably damaging  PIT  Optic disk anomalies + retina/macula dystrophy (MIM 212550)  Same variant in patient 14. Variant previously described with reduced function [Carnes et al. (34)]  BMP4  c.1001C>A, p.A334D (missense)  rs550409227  Mother  VUS  0.000008  Probably damaging  PIT  Syndromic microphthalmia type 6 (MIM 607932)    20  SLC12A6  c.1787C>T, p.P596L (missense)  —  Mother  VUS/likely pathogenic  N  Possibly damaging  CCA  AR corpus callosum agenesis + peripheral neuropathy (MIM 218000)  CC2D2A  c.3865A>G, p.T1289A (missense)  —  Mother  VUS  N  Possibly damaging  CCA  Joubert syndrome (MIM 612285), Meckel syndrome (MIM 612284)  Abbreviations: ACMG, The American College of Medical Genetics and Genomics; AD, autosomal dominant; AR, autosomal recessive; CCA, corpus callosum agenesis; dbSNP, Single Nucleotide Polymorphism Database; HH, hypogonadotropic hypogonadism; HPE, holoprosencephaly; MIM, numerical assignment in Mendelian Inheritance in Man catalog; MPHD, multiple pituitary hormone deficiency; N, variant not previously reported; PIT, pituitary; VUS, variant of unknown significance. View Large Table 4. Exome Sequencing Results per Panel of Targeted Genes Including Number of Variants for Each Gene Case    1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  Pituitary panel   ARNT2                        1                   BMP4            1                          1     CHD4                      1      1               GLI3            1                               SIX6                            1          1     IGSF1        1                                  Holoprosencephaly panel   GLI2      2      1  1      3        3    1          Hypogonadotropic hypogonadism panel   B9D1                          1                 CHD7                                    1       FGF8                      1                     NR0B1                        1                   OTUD4                1                           PROK2                          1                 TACR3                                  1        Corpus callosum agenesis panel   ASPM                      1                     CC2D2A                        1                1   DHCR7        1                            1       INPP5E          1                                 NDE                1                           RELN        1                                   SLC12A6                                        1  New candidate genes   CCDC88C                                  2         DCHS1              2                        2     KAT6A          1                               KIF14                              1             ROBO2                  1                        Case    1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  Pituitary panel   ARNT2                        1                   BMP4            1                          1     CHD4                      1      1               GLI3            1                               SIX6                            1          1     IGSF1        1                                  Holoprosencephaly panel   GLI2      2      1  1      3        3    1          Hypogonadotropic hypogonadism panel   B9D1                          1                 CHD7                                    1       FGF8                      1                     NR0B1                        1                   OTUD4                1                           PROK2                          1                 TACR3                                  1        Corpus callosum agenesis panel   ASPM                      1                     CC2D2A                        1                1   DHCR7        1                            1       INPP5E          1                                 NDE                1                           RELN        1                                   SLC12A6                                        1  New candidate genes   CCDC88C                                  2         DCHS1              2                        2     KAT6A          1                               KIF14                              1             ROBO2                  1                        View Large Variants in pituitary genes In the panel of 37 genes associated with pituitary development, no pathogenic variants were found. However, several rare, potentially pathogenic variants were present in GLI2, GLI3, BMP4, IGSF1, CHD4, ARNT2, SIX6 (two patients, same variant), and FGF8. New candidate genes De novo or heterozygous mutations were found in five genes (in six patients) with a known expression/function in the brain: ROBO2, KAT6A, KIF14, compound heterozygosity for DCHS1 (two patients), and compound heterozygosity for CCDC88C. Protein modeling for both cases of the compound heterozygous DCHS1 mutations indicated disrupted interaction with the ligand FAT4, making functional consequences likely (Fig. 1). Protein modeling for the ROBO2 mutation indicated that the mutation was in the most mobile region of the ROBO2 protein. The mutation causes an extra H-bond, which solidifies the ROBO2 protein, probably leading to less adaptability to environmental influences and in interactions with the ligand SLIT (Fig. 2). Figure 1. View largeDownload slide (A) Schematic overview of the structure and interaction of DCHS1 and FAT4, including the sites of the presently reported mutations. (B‒E) Extracellular (EC) domains shown in cartoon presentation with mutation position marked and zoomed in on. The dashed arrows present orientation of extracellular domain in comparison with the neighboring domains, and the numbers present the corner of orientation. Affected residues are shown in sticks or line presentation. Calcium is in green spheres. (B, C) Mutations in case 7 are shown. (B) c.2338A>G, p.Ile780Val: wild-type = gray, mutation = red. Mutation is in EC7 and has a slight effect on the structural stability of the EC7 domain. EC6 and EC7 interact with FAT4, which may loosen the interaction. (C) c.6395T>C, p.Leu2132Pro: wild-type = gray, mutation = red. Mutation is in EC20 and has a structural effect on EC20 and even more so on EC21. Both mutant extracellular domains lose structural stability because of effects on Ca binding ability. Mutation also affects orientation of the EC20, which may cause less favorable DCHS1-FAT4 interaction. (D, E) Mutations in case 19 are shown. (D) c.4555C>T, p.Pro1519Ser: wild- type = gray, mutation = blue. Mutation is in EC14 and has a structural effect on EC13, EC14, and even more so on EC15. EC15 almost unfolds; only Ca2+ still holds the structure together. Immediately after Ca concentration went down, EC15 completely unfolded. Pro1519Ser disturbed beta structure, which affected Ca-binding sites between EC14 and EC15, potentiating the structural instability of EC15. Mutation also affected orientation of all mentioned extracellular domains, consequently leading to distorted interaction between DCHS1 and FAT4. (E) c.8186A>T, p.His2729Leu: wild-type = gray, mutation = blue. Mutation is in EC26. No significant structural effect was observed. Figure 1. View largeDownload slide (A) Schematic overview of the structure and interaction of DCHS1 and FAT4, including the sites of the presently reported mutations. (B‒E) Extracellular (EC) domains shown in cartoon presentation with mutation position marked and zoomed in on. The dashed arrows present orientation of extracellular domain in comparison with the neighboring domains, and the numbers present the corner of orientation. Affected residues are shown in sticks or line presentation. Calcium is in green spheres. (B, C) Mutations in case 7 are shown. (B) c.2338A>G, p.Ile780Val: wild-type = gray, mutation = red. Mutation is in EC7 and has a slight effect on the structural stability of the EC7 domain. EC6 and EC7 interact with FAT4, which may loosen the interaction. (C) c.6395T>C, p.Leu2132Pro: wild-type = gray, mutation = red. Mutation is in EC20 and has a structural effect on EC20 and even more so on EC21. Both mutant extracellular domains lose structural stability because of effects on Ca binding ability. Mutation also affects orientation of the EC20, which may cause less favorable DCHS1-FAT4 interaction. (D, E) Mutations in case 19 are shown. (D) c.4555C>T, p.Pro1519Ser: wild- type = gray, mutation = blue. Mutation is in EC14 and has a structural effect on EC13, EC14, and even more so on EC15. EC15 almost unfolds; only Ca2+ still holds the structure together. Immediately after Ca concentration went down, EC15 completely unfolded. Pro1519Ser disturbed beta structure, which affected Ca-binding sites between EC14 and EC15, potentiating the structural instability of EC15. Mutation also affected orientation of all mentioned extracellular domains, consequently leading to distorted interaction between DCHS1 and FAT4. (E) c.8186A>T, p.His2729Leu: wild-type = gray, mutation = blue. Mutation is in EC26. No significant structural effect was observed. Figure 2. View largeDownload slide Schematic overview of the structure and interaction of ROBO2 and SLIT. Right panel: SLIT (green), wild-type ROBO2 = gray, mutation (c.941G>A, p.Arg314Gln) = red. The mutation leads to an extra H-bond between residues 245 and 314, causing ROBO2 to become more rigid. Left panel: superposition of wild-type ROBO2-SLIT and mutated ROBO2 (c.941G>A, p.Arg314Gln)‒SLIT complex. Mutations are marked by arrows. Yellow lines indicate distance between their backbone atoms. Figure 2. View largeDownload slide Schematic overview of the structure and interaction of ROBO2 and SLIT. Right panel: SLIT (green), wild-type ROBO2 = gray, mutation (c.941G>A, p.Arg314Gln) = red. The mutation leads to an extra H-bond between residues 245 and 314, causing ROBO2 to become more rigid. Left panel: superposition of wild-type ROBO2-SLIT and mutated ROBO2 (c.941G>A, p.Arg314Gln)‒SLIT complex. Mutations are marked by arrows. Yellow lines indicate distance between their backbone atoms. Variants in genes associated with midline brain pathologies In the panel of genes associated with holoprosencephaly, 11 variants in GLI2 were found but no potential pathogenic variants in any of the other candidate genes. In the panel for hypogonadotropic hypogonadism, pathogenic mutations were found in NR0B1 and PROK2, and potentially pathogenic variants were found in OTUD4, FGF8, TACR3, and CHD7. In the panel for absent corpus callosum, pathogenic mutations were found in DHCR7 (two patients, identical mutation) and CC2D2A, and variants of unknown significance, predicted to potentially be damaging, were found in RELN, INPP5E, NDE, ASPM, B9D1, CC2D2A (two patients), and SLC12A6. All mutations found in the target panels were inherited from an unaffected parent (Table 3). We detected 11 GLI2 variants (six missense, five synonymous) with a MAF <0.05 in six patients; all were inherited from an unaffected parent. The total prevalence of GLI2 variants with a MAF <0.05 was 55% in our study population (11 variants in 20 individuals), which was comparable to the 58.6% prevalence of GLI2 variants with a MAF <0.05 in the 1000 Genomes database (1468 variants in 2504 individuals). Two of the 20 study patients had a combination of the missense mutations M1352V + D1520N. In the 1000 Genomes database, this specific combination was found in only 17 of 2504 individuals (0.68%). Discussion In this exome sequencing study of 20 unrelated patients with isolated PSIS and their unaffected parents, we identified five additional candidate genes for PSIS: DCHS1, ROBO2, CCDC88C, KIF14, and KAT6A. In addition, by using a target gene panel, we found potentially pathogenic variants in genes involved in midline brain formation in a panel of genes known to be associated with pituitary formation (in 8 of 37 genes) and in a panel of genes associated with holoprosencephaly, hypogonadotropic hypogonadism, and absent corpus callosum (11 of 186 genes); and also in the latter group, 5 pathogenic variants. Thirteen of 20 patients carried more than one variant, and all variants from the target panels were inherited from an unaffected parent. This diversity of (potentially) pathogenic variants and inheritance from unaffected parents are suggestive of a polygenic etiology of isolated PSIS. New candidate genes DCHS1 Two patients (nos. 7 and 19) were compound heterozygous for variants in DCHS1. Protein modeling indicated disrupted interaction with the ligand FAT4A, making functional consequences likely (Fig. 1). DCHS1 and FAT4 play a role in neuronal migration (35, 36). Pathogenic mutations in DCHS1 and FAT4 can cause Van Maldergem syndrome, which goes along with periventricular neuronal heterotopia, indicative of altered neuronal migration, and absent corpus callosum (24). Pituitary abnormalities have not been described. DCHS1 is expressed in the developing pituitary gland in mice (37). The present two patients had isolated PSIS without other brain abnormalities at MRI. We examined the patients carefully for signs suggestive of Van Maldergem syndrome, but except for broad hands and feet, present in both patients and absent in parents, there were no specific physical characteristics. One of the patients also had a GLI2 variant (c.1294G>A, maternally inherited), previously reported in patients with CPHD, and an ectopic posterior pituitary lobe (10, 11, 13). Because DCHS1 is involved in neuronal migration and is expressed in the pituitary gland and because we found two patients in this series of 20 patients with isolated PSIS, the DCHS1 gene may be considered a candidate gene for PSIS, but possibly only when variants are present in one or more other genes. ROBO2 Patient 9 had a de novo ROBO2 mutation (c.914G>A), which was not previously reported and was predicted to be probably damaging. Protein modeling indicated that the mutation is not in the ROBO2-SLIT interface but is in the most mobile region of the ROBO2 protein. The mutation leads to an extra H-bond and solidifies the ROBO2 protein, making functional consequences likely (Fig. 2). ROBO2 belongs to the ROBO family, part of the immunoglobulin superfamily of proteins that are highly conserved from fly to human. The encoded protein is a transmembrane receptor for the slit homolog 2 protein and functions in axon guidance across the midline of the mammalian central nervous system (38). ROBO2 mutations are associated with vesicoureteral reflux (39). ROBO2 isoform a is highly expressed in the developing human brain but not in the adult brain (40). The present patient did not have vesicoureteral reflux. After we had finished our study, Bashamboo et al. (41) identified ROBO1 mutations in five cases of PSIS. Four of the five patients had ocular anomalies, which the present patient did not have. The function of ROBO2 in axon guidance across the midline, the results of protein modeling, and the recent finding of ROBO1 mutations in PSIS make ROBO2 an excellent candidate gene for pituitary malformations, but functional studies are needed to prove pathogenicity. CCDC88C Patient 17 was compound heterozygous for CCDC88C variants. The R1299C was predicted to be possibly damaging by Polyphen2; the E662K mutation is very rare (MAF, 0.0001), predicted to be benign by Polyphen2. This gene encodes a ubiquitously expressed coiled‒coil domain‒containing protein that interacts with the disheveled protein and is a negative regulator of the Wnt signaling pathway. Autosomal recessive mutations in CCDC88C cause nonsyndromic hydrocephalus, associated with midline brain malformation (42). The present patient had no hydrocephalus and no midline brain malformation other than PSIS. Besides the CCDC88C variants, the patient also carried a TACR3 mutation, which was not described before and was predicted to be probably damaging. Homozygous TACR3 mutations are associated with hypogonadotropic hypogonadism. The present patient has complete anterior pituitary insufficiency, including hypogonadism. In this case, it is feasible that TACR3 contributed to the phenotype in a polygenic model. Functional studies will have to be performed to provide more evidence for the pathogenicity of the CCDC88C mutations, but the phenotype, including midline brain abnormalities, and the importance of the Wnt pathway in pituitary development make CCDC88C a promising candidate gene for PSIS. KIF14 Patient 15 had a de novoKIF14 mutation (c.3728A>G) that has not been described before and is predicted to be possibly damaging. Autosomal recessive KIF14 mutations have been linked to a lethal fetal ciliopathy, resembling Joubert syndrome (43). Primary cilia are involved in central nervous system development and are involved in signaling pathways, such as Shh and Wnt pathways. Ciliopathies are disorders caused by defects in the primary ciliary structure and include Joubert syndrome and Bardet-Biedl syndrome. In KIF14 mutant mice, the development of laminated structures in the central nervous system is affected, and the olfactory bulb was shown to be cytoarchitecturally disorganized (44). The olfactory placode is involved in hypothalamic-pituitary development with gonadotrophin-releasing hormone neurons migrating from the olfactory placode into the hypothalamus. Disruption of this migration is a key feature of Kallmann syndrome, consisting of hypogonadotropic hypogonadism and anosmia. Given this, mutations in KIF14 may well influence pituitary development. Because we found other heterozygous pathogenic mutations in genes linked to ciliopathies (INPP5E, CC2D2A, and B9D1), genes involved in primary ciliary structure/function may be involved in the PSIS phenotype and should be studied in more detail. KAT6A A de novo KAT6A mutation (c.235C>T) leading to a premature stop codon was found in patient 6. This patient’s case has been published in detail elsewhere (28). This patient had a developmental delay, with severely delayed speech, which was initially attributed to hypoglycemic brain damage due to untreated central adrenal insufficiency and GH deficiency in the first 2 years of life. In retrospect, she fulfills the criteria for KAT6A neurodevelopmental disorder and should not have been included in this study on isolated PSIS. Because subtle midline brain abnormalities were reported in KAT6A patients (cavum septum pellucidum, absent bulbus olfactorius), PSIS may be part of the phenotypic spectrum. However, because this patient also had potentially pathogenic BMP4 and GLI3 variants and a rare GLI2 variant (all present in the unaffected father), a polygenic cause of the pituitary malformation is also possible. Variants in genes associated with midline brain malformation Hypogonadotropic hypogonadism genes: PROK2, NR0B1 A pathogenic PROK2 frameshift mutation leading to a premature stop codon (c.163delA) was found in patient 13. The mutation was inherited from the unaffected father. Homozygous and heterozygous PROK2 and PROKR2 variants are associated with Kallmann syndrome and hypogonadotropic hypogonadism. Recently, PROKR2 variants were reported in patients with CPHD. In a UK series of 422 patients with CPHD (89% with septo-optic dysplasia and 11% with holoprosencephaly or midline clefts), PROKR2 mutations were found in 11 cases, but no mutations were found in PROK2 (16). In a Brazilian series of 156 patients with CPHD, two patients with a PROKR2 mutation were identified (PROK2 was not investigated) (17). In a French series of 72 patients with PSIS, two PROKR2 mutations were found (6). The presently reported PROK2 mutation was reported in a Portuguese family in which homozygous family members had hypogonadotropic hypogonadism with and without anosmia and heterozygous family members were unaffected (31). A Swiss patient with the same homozygous PROK2 mutation had hypogonadotropic hypogonadism with anosmia; data on heterozygous carriers were not available (32). The presently reported patient and his unaffected father both also had a rare, possibly damaging mutation in B9D1 associated with Joubert syndrome. The incomplete penetrance in the father may be due to the presence of variants in other genes or to epigenetic influences, which may fit a complex polygenic background for PSIS. A very rare missense mutation (MAF, 0.0001%) in the X-linked NR0B1 (c.315G>C) was found in patient 12. The mutation was inherited from the mother and predicted to be damaging. It has previously been linked to isolated mineralocorticoid deficiency and a mild form of congenital adrenal underdevelopment (30). Midline brain anomalies have not been reported before in individuals with NR0B1 variants. The same patient also had a rare pathogenic CC2D2A mutation. Absent corpus callosum: DCHR7, CCD2DA In three patients, pathogenic mutations in genes associated with absent corpus callosum were found: DCHR7 (c.452G>A; patients 4 + 18) and CCD2DA (c.3055C>T; patient 12). These genes are known to cause autosomal recessive disorders; DCHR7 mutations can cause Smith-Lemli-Opitz syndrome, and CCD2DA mutations can cause Meckel syndrome and Joubert syndrome. In each patient, the mutation was inherited from an unaffected parent, indicating that being a carrier in itself is insufficient to develop PSIS. CCD2DA is involved in ciliopathies, as is KIF14 (described previously). In the series of genes with variants classified as having unknown clinical significance, there are three genes (INPP5E, CC2D2A, and B9D1) that function as ciliopathy genes. This suggests that ciliopathy genes may be involved in pituitary development. Holoprosencephaly: GLI2 variants GLI2 is associated with holoprosencephaly and acts in the Wnt pathway. In a review, 25 patients (16 families) were reported to have heterozygous mutations in GLI2 and disturbed pituitary development (14). Most patients had an ectopic posterior pituitary. The suggested pattern of inheritance was autosomal dominant with incomplete penetrance and variable expression (14). In the current study, six patients had GLI2 variants with a MAF <0.05. Two patients had the combination of GLI2 variants M1352V + D1520N (patients 3 + 14). This combination has previously been reported in patients with PSIS, and functional studies have demonstrated reduced transcription activity and reduced luciferase activity (10, 13, 27). Another presently reported patient had a V432M variant that has also been described in patients with PSIS (10, 11, 13). The R1382H variant found in another patient has not been described before, was inherited from an unaffected mother, is very rare (MAF, 0.0002), and is potentially pathogenic. GLI2 variants with a MAF <0.05 were not more common in PSIS than in the general population (1000 Genomes); however, despite the small numbers, the specific combination of M1352V + D1520N variants seems to be important in PSIS because we found the combination in two of 20 patients (10%) compared with only 17 of 2504 individuals (0.68%) in the reference population (1000 Genomes). GLI2 variants have been reported at a relatively high frequency in patients with CPHD/PSIS, and GLI2 is likely an important factor in the polygenic background of pituitary development. Strengths and limitations Strengths of this study are the homogeneous group of study participants. A limitation is the inclusion of the KAT6A patient who had syndromic PSIS and not the isolated form. It emphasizes the importance of proper phenotyping in such studies (45). All studied patients had congenital central hypothyroidism, and therefore the results may not be representative for PSIS patients without central hypothyroidism. We presumed all parents were unaffected on the basis of their medical history. However, we did not perform endocrine studies or MRIs in parents. Exome sequencing by itself is limited by incomplete coverage of the exome and easily missed copy number variations, although the presently used targeted approach decreased the likelihood of missing deletions and duplications. Absolute proof for causality of combinations of variants in candidate genes can be obtained only by functional studies (46). It was beyond the aims and possibilities of the current study to perform such functional studies for each (combination of) candidate genes. Because we searched only for variants genes in lymphocytes, mosaicisms confined to affected tissue(s) cannot be excluded. Exome sequencing and genome sequencing can detect variants in genes, but epigenetic mechanisms such as DNA methylation, histone modification, and microRNAs may play a role, warranting additional studies. Systematically investigating large series of carefully phenotyped patients and collaboration between research groups are essential in the search for genes and pathways underlying disorders with a polygenic etiology (9). In conclusion, searching for causes and pathogenesis of a potentially polygenic disorder is complex. Here, we added four candidate genes for isolated PSIS (DCHS1, ROBO2, CCDC88C, and KIF14) and one for syndromic PSIS (KAT6A). In addition, we found 11 GLI2 variants in six patients and verified the higher frequency of a combination of two GLI2 variants in the study group compared with a reference population. We detected various (potentially) pathogenic variants in genes associated with midline brain anomalies and in genes involved in ciliary structure and function and suggest that these genes be included in future searches for a polygenic cause of PSIS and CPHD. Abbreviations: BMP bone morphogenetic protein CPHD combined pituitary hormone deficiencies FGF fibroblast growth factor GH growth hormone MAF minor allele frequency MRI magnetic resonance imaging PSIS pituitary stalk interruption syndrome ROBO2 roundabout guidance receptor 2. Acknowledgments Financial Support: N.Z.-S.: Work was partly funded by the Emma Foundation, Emma Children’s Hospital, Academic Medical Centre, Amsterdam, The Netherlands. Disclosure Summary: The authors have nothing to disclose. References 1. Bar C, Zadro C, Diene G, Oliver I, Pienkowski C, Jouret B, Cartault A, Ajaltouni Z, Salles JP, Sevely A, Tauber M, Edouard T. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 1, 2018

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