De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder

De novo mutations in MED13, a component of the Mediator complex, are associated with a novel... Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non- truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiqui- tination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes. Introduction The introduction of next-generation sequencing techniques has rapidly improved the identification of genes that associ- ate with rare disease. Although developmental delay (DD) Lot Snijders Blok and Susan M. Hiatt contributed equally as first and intellectual disability (ID) are relatively common (Boat authors, and Tjitske Kleefstra and Gregory M. Cooper contribued and Wu 2015; Boyle et al. 2011), there is extreme genetic equally as last authors. heterogeneity among affected patients and a large fraction of patients with DD/ID remain refractory to diagnosis (Vis- Electronic supplementary material The online version of this sers et al. 2016). In unsolved cases, the understanding of article (https ://doi.org/10.1007/s0043 9-018-1887-y) contains supplementary material, which is available to authorized users. gene–disease relationships has greatly benefited from col- laboration between clinical genetics teams (Sobreira et al. * Tjitske Kleefstra 2015). In fact, many recently discovered DD/ID genes have tjitske.kleefstra@radboudumc.nl come from “matchmaking” (Au et al. 2015; Harms et al. * Gregory M. Cooper 2017; Kernohan et al. 2017), where websites such as Gen- gcooper@hudsonalpha.org eMatcher (Sobreira et al. 2015) facilitate the comparison Extended author information available on the last page of the article Vol.:(0123456789) 1 3 376 Human Genetics (2018) 137:375–388 of patients with rare genotypes and phenotypes across the described (Bowling et  al. 2017). In each patient, the world. observed MED13 mutation was considered to be the most Here we present the results of a collaboration facilitated likely contributor to the phenotype, and no additional patho- by GeneMatcher (Sobreira et  al. 2015) in which multi- genic or likely pathogenic variants were found. ple clinical and research groups independently identified individuals with DD/ID and related phenotypes with rare Three‑dimensional modeling protein-altering variation in MED13. This genotype-driven approach enabled us to characterize the phenotypes and Protein modeling was performed as previously described mutational spectrum of a cohort of 13 patients, each with a (Prokop et al. 2017). Modeling of MED13 interacting with likely pathogenic variant in MED13. FBXW7 was performed using PDB 2OVQ, replacing mol- Although MED13 has not been previously linked to a dis- ecule C with the MED13 amino acids 321–330. Binding order, it is a paralog of MED13L, mutations of which have energy was calculated following each patient variant inser- been found to cause ID, speech impairment and heart defects tion and energy minimization using AMBER14 force field (Adegbola et al. 2015; Muncke et al. 2003; van Haelst et al. (http://amber md.org/) in YASARA. 2015). The gene products MED13 and MED13L are mutu- ally exclusive components of the reversible CDK8-module RNA isolation of the Mediator complex, a multi-protein complex that is required for the expression of all protein-coding genes (Con- 2.5  mL of blood was collected in PAXgene RNA tubes away et al. 2005; Malik and Roeder 2005). In this study, we (PreAnalytiX #762165) according to the manufacturer’s show that variants in MED13 are also associated with a neu- instructions and stored short-term at − 20 °C. RNA was iso- rodevelopmental disorder, and delineate the corresponding lated using a PAX gene Blood RNA Kit (Qiagen #762164) phenotypic features and mutational spectrum. according to the manufacturer’s instructions. Isolated RNA was quantified by Qubit® (Thermo Fisher #Q32855). Materials and methods cDNA synthesis Informed consent First strand synthesis of cDNA was performed from 150 ng Informed consent to publish de-identified data was obtained of RNA isolated from blood using Superscript™ III (Thermo from all patients, either as part of the diagnostic workflow or Fisher #18080044) according to manufacturer’s instructions as part of a research study (Bowling et al. 2017). Informed using random primers (Invitrogen #48190011) for +/− RT consent to publish clinical photographs was also obtained reactions. The products were diluted 1:10 in water before when applicable. Informed consent matched the local ethi- use in qPCR reactions. cal guidelines. qPCR Exome/genome sequencing qPCR was performed according to manufacturer’s protocols In patients A, B, D, E, F, G, I, K, L and M, whole exome using Taqman gene expression master mix (ThermoFisher sequencing and variant filtering were performed as previ- #4369016) and FAM-MGB Taqman probes directed against ously published (de Ligt et al. 2012; Deciphering Develop- MED13 (ThermoFisher Hs01080701_m1 catalog #4331182) mental Disorders 2015; Neveling et al. 2013; Sollis et al. and GAPDH (ThermoFisher #4352934E). qPCR reactions 2017; Tanaka et  al. 2015). In patient C, targeted Sanger were carried out in a QuantStudio 6 Flex Real-Time PCR sequencing was performed to confirm the presence of the system (Applied Biosystems) using 40 cycles of amplifi- MED13 variant (L131*) that was first identified in patient cation. Raw C values were obtained, normalized first to B. For patient H, whole genome sequencing was performed the GAPDH loading control, and then to the proband. We using Illumina’s HiSeq X ten platform. Sequencing reads tested an additional loading control [AGPAT-data not shown were mapped against the hs37d5 reference using GATK. (ThermoFisher Hs00965850_g1; catalog #4331182)], but Variants were called using GATK’s Haplotype Caller. Vari- the data were like those normalized to GAPDH. ants were filtered using frequencies from the ExAC and gnomAD databases (mean allele frequency < 0.003) and for conservation using PhastCons (> 0.5) and PhyloP (> 4). For patient J, whole genome sequencing, variant prioritiza- tion, and Sanger validation were performed as previously 1 3 Human Genetics (2018) 137:375–388 377 MED13 in blood despite detecting MED13 in neural pre- Sanger sequencing cursor nuclear lysates: Bethyl #278A, Abcam #ab49468, and Abcam #ab76923 (data not shown). cDNA template was amplified using primers to the region of interest: 5′ -CG A GGC T CT TAT GG A A CT G AT G AA Statistical enrichment of MED13 variants in DD/ID TC-3′ (forward) and 5′-GATCC AT CGT GCTTT C AGA CA CAT C-3′ (reverse). No amplification was observed in the cohorts no RT condition. PCR conditions were: 500  nM prim- ers, 3% DMSO, 1x Phusion HF (NEB #M0531L), 0.5 µL We compared the frequency of observed de novo MED13 variation identified in two large sequencing cohorts to the cDNA template, and cycling at (98 °C, 30 s), (98 °C, 10 s; 60 °C, 30 s; 72 °C, 45 s)x35, (72 °C, 7 m), (4 °C, ∞). The expected frequency of variation in MED13 based on its gene specific mutation rate (Samocha et al. 2014) using an Exact additional reverse primer 5′-AAAT GCTT CA TTG TTA CC GTC AGC T-3′ and the additional forward primers 5′-TCC Poisson Test in R (R Core Team. R: A language and envi- ronment for statistical computing (http://www.r-project.or g). AAA AGA AAC GAT GTG AGT ATG CAG-3′, 5′-CTC TCT TCA GCC AGT TCT TCA GGA T-3′, 5′-ACA ATT TCA TAA Vienna). AATGGC T GGCCG A -3′, 5′-CGAGGC T CTT ATGG AA CT GATG AAT C-3′, 5′-GTGCTT T CTCC ATTT GCT CTT CCT Results T-3′ were used for sequencing, along with the primers used for amplification from cDNA. Chromatograms were quan- Phenotypes tified using ab1PeakReporter (Thermo Fisher). We collected detailed clinical information of 13 patients Western Blot with rare, protein-altering MED13 variants. Eleven variants were confirmed to be de novo, and one patient (patient B) Whole blood was collected using cell processing tubes inherited the variant from her mother who is also affected (patient C). Phenotypic data summarizing the spectrum of (BD #362760), isolated according to the manufacturer’s instructions, and stored in liquid nitrogen in CTS™ features of this cohort of 13 patients are shown in Table 1. All patients had developmental delays with varying sever- Synth-a-Freeze Medium (Thermo Fisher # A13713-01) until use. As a control for antibody specificity, MED13 ity and course. In the patients that underwent formal intel- ligence testing, total IQ levels varied from 85 (lower range was knocked down in neural precursor cells (clone BC1, MTI-GlobalStem #GSC-4311) by generating stable lines of normal IQ) to an IQ between 35 and 50 (moderate ID). Five patients had an Autism Spectrum Disorder (ASD), and using puromycin selection expressing shRNA against MED13 (Sigma Aldrich # SHCLNG-NM_005121; three patients were diagnosed with Attention Deficit Hyper - activity Disorder (ADHD). All patients had speech delays TRCN0000234904) compared to a GFP shRNA control in the same vector (Addgene # 30323). Cell pellets were and/or disorders, with delayed milestones in speech and lan- guage development. While several patients had expressive processed using the NE-PER™ (Thermo Fisher #78833) nuclear and cytoplasmic extraction kit according to the and receptive language problems, in the majority of patients, speech production was significantly more impaired than lan- manufacturer’s instructions, and nuclear extracts were used for the blot shown (whole cell extracts, even at very guage comprehension. Three patients (patient A, K and M) showed characteristics of speech apraxia, a developmental high concentrations, did not produce sufficient signal). 60 µg of protein was loaded for patient blood samples, speech disorder in which affected individuals have difficul- ties accurately programming the motor sequences required and 15 µg of protein was loaded for neural precursor cell samples. Blots were blocked for 1 h at room temperature in to produce fluent speech. Patient A had a mild ID, but showed speech apraxia with a mixed receptive and expres- LICOR blocking buffer (LICOR #927-40000), then blots were probed (with washes in PBS-T (0.05% Tween-20) sive language disorder, and limited verbal expression at the age of 8 years. Patient M had a non-verbal IQ of 70 with a and a secondary probe for 1 h after each primary probe) with 1:250 rabbit anti-MED13 (Bethyl #A301-277A) for severe speech/language disorder. Her expressive speech was severely affected, with signs of speech apraxia. At the age of 3 days at 4 °C, 1:1,000 mouse anti-HDAC2 (clone 3F3, SCBT #sc-81599) overnight at 4 °C as a loading control, 8 years she only used single words and very short sentences. Patient K developed some speech capabilities, but showed and 1:1000 rabbit anti-HSP90 (abcam #ab115660) over- night at 4 °C as an additional loading control. Secondary regression at the age of 13 months and has since remained non-verbal. probes were used at 1:20,000 (LICOR #926-32211 and #926-68070). Three other primary antibodies were tested Seven of 13 patients showed delays in motor develop- ment, most of which affected at least the gross motor skills for MED13, but did not show sufficient signal to detect 1 3 378 Human Genetics (2018) 137:375–388 1 3 Table 1 Clinical features of patients with MED13 mutations and molecular characterization Patient A Patient B Patient C Patient D Patient E Patient F Patient G Patient H Patient I Patient J Patient K Patient L Patient M Molecular characterization  cDNA variant c.125del c.392T>G c.392T>G c.977C>T c.975_977 c.979C>T c.980C>A c.1618C>A c.1745T>A c.4198C>T c.4487delC c.6178C>A c.6191C>T (NM_005121.2) delTAC  Predicted protein P42Lfs*6 L131* L131* T326I T326del P327S P327Q P540T L582* R1400* T1496Mfs Q2060K A2064V effect  CADD v.1.3 31.0 37.0 37.0 25.0 20.5 23.4 25.2 26.3 40 41 35 24.1 25.7  GERP + + RS 5.67 5.32 5.32 5.5 5.5 5.5 5.5 6.16 6.02 4.58 5.86 6.04 6.04  Inheritance De novo Maternal Unknown De novo De novo De novo De novo De novo De novo De novo De novo De novo De novo (daughter of pt. C) Clinical characterization  Gender M F F M F M F M M F M F F  Age at last visit 8 5 32 19 9 11 3 10 6 13 5 10 6 (years)  Height + 0.6 SD + 0.5 SD average − 0.2 SD + 2.2 SD + 0.5 SD + 0.3 SD − 0.9 SD 0 SD − 0.7 SD − 2 SD + 0.5 SD − 2 SD  Weight (for + 1.8 SD + 1.9 SD average − 0.8 SD + 1.2 SD − 0.9 SD − 1.1 SD + 0.6 SD + 0.5 SD 0 SD − 1.6 SD + 0.4 SD + 0.7 SD height)  Head circumfer- − 1 SD + 0.2 SD NA − 0.5 SD + 1.1 SD − 0.9 SD − 0.3 SD − 1.5 SD − 0.5 SD NA 0 SD − 2 SD + 1 SD ence  Intellectual Dis- Mild ID Mild/border- Borderline Mild ID Mild ID Mild ID DD Borderline ID Mild ID Moderate ID DD DD Mild/border- ability (ID) / line ID ID (IQ 65) (IQ 85 with (IQ 61) line ID Developmental (IQ 80–85) working (IQ 70) Delay (DD) memory score 68 on WISC-IV)  Speech delay/ + + + + + + + +/− + + + + + disorder Speech Delayed Delayed Delayed Moderate mixed Delayed Mainly Borderline At age 6y Moderate Severe speech Severe speech Severe apraxia speech speech speech receptive and expres- expressive (verbal com- expres- expressive disorder delay, 5–10 speech/ with develop- develop- develop- expressive sive and speech prehension sive and language with words language mixed ment, mild ment, ment, mild language disor- receptive problems score = 87 receptive disorder regression, disorder, recep- articulation expressive articula- der, decreased language on WISC- speech at speech expressive tive and problems language tion vocabulary IV) age equiva- apraxia, language expres- problems problems, and language lent < 2y receptive most sive in child- normal formulation language is affected, language hood. At language difficulties fine signs of disorder, adult age compre- speech limited only spo- hension apraxia verbal radic and expres- mild word- sion and finding lan- problems guage- based learning disorder Human Genetics (2018) 137:375–388 379 1 3 Table 1 (continued) Patient A Patient B Patient C Patient D Patient E Patient F Patient G Patient H Patient I Patient J Patient K Patient L Patient M  Delayed motor + − − + + + + − + NR − − + development (Only fine (walked at (walked on (walked at (walked at 25 m) (walked at (walked at (walked before (walked at 2 (walked at (walked at motor 14 m) time) 20 m) 22 m) 26 m) 12 m) years; early 12 m) 20 m) skills delays, now delayed) mostly on target with peers)  Autism spectrum ADHD NA − − − ASD ASD − − ASD, ADHD ASD ASD, ADHD − disorder (ASD) /ADHD  Brain MRI Normal NA NA Normal Bulbous splenium Normal Normal Small area of Normal NA NA Normal Mild frontal of corpus cal- abnormal atrophy, losum (likely signal in left otherwise normal variant) occipital normal lobe  Eye/vision abnor- Astigma- probably − Visual Congenital nys- − Strabismus, − Astigmatism NR NR Duane Duane malities tism amblyopia impair- tagmus, outer papil anomaly anomaly ment, retinal atrophy edema pale optic temporal to nerves both optic discs, optic nerves low normal in size on MRI  Heart abnormali- NR − NR − History of Dilated − NR NR Subaortic NR NR NR ties murmur, normal aortic stenosis echo and ECG root and pulmonary artery  Chronic obstipa- NR NR + NR + NR + NR NR NR NR NR + tion  Other features Sloping Small and Kyphosis, Hypotonia, mild Hypotonia, Hypotonia Epilepsy Chronic sleep Chronic sleep Conductive (features shoulders, laterally pes cavus proximal weak- Conduc- (drug-resist- disturbances distur- hearing loss, reported in small and deviated ness, fatigues tive ant with bances Precocious two unrelated laterally halluces easily, clumsy hearing myoclonic- puberty patients in bold) deviated gait, transient loss, Mild atonic halluces lactic acidosis scoliosis, seizures) with illness, pes cavus congenital left hip dysplasia NR not reported, NA not assessed 380 Human Genetics (2018) 137:375–388 (6 of 7), although one patient was reported to have only v1.3 (Kircher et al. 2014), with scores ranging from 20.5 fine motor delays. Three patients had hypotonia (patient E, to 41 (Table 1). Six patients had five unique variants that F and G). One patient (patient H) developed severe drug- are predicted to be truncating: three nonsense mutations resistant myoclonic-atonic epilepsy at 4 years of age with (p.Leu131* in Patients B and C, p.Leu582* in Patient I and generalized clonic, myoclonic, atonic, tonic and atypical p.Arg1400* in Patient J) and two frameshift variants lead- absence seizures. MRI screening of this patient showed a ing to a premature stop codon (p.Pro42Leufs*6 in patient A small abnormality in the left occipital lobe of his brain that and p.Thr1496Metfs*11 in Patient K). The remaining vari- did not correspond to the electrophysiological onset or the ants include six missense variants and a single amino acid semiology of his seizures. In other patients, MRI scans were deletion. These seven variants form two apparent clusters: not performed or showed no clear abnormalities, except for one in the N-terminal conserved phosphodegron domain mild frontal atrophy in patient M. and the other in the C-terminal domain (Fig.  2a). These Eight patients (62%) presented with eye or vision abnor- seven variants were all found to lie within motifs that are malities. Two patients (patients L and M) presented with highly conserved between MED13 and MED13L (Fig. 2b) Duane anomaly, a congenital type of strabismus that is char- and affect sites under high codon selection (Fig.  2c). These acterized by non-progressive horizontal ophtalmoplegia and missense variants and the in-frame deletion are each located retraction of the globe with attempted adduction, together on surface-exposed sites within a three-dimensional model with narrowing of the palpebral fissure (Andrews et  al. of the MED13 protein (Fig.  3). The four mutations that 1993). One patient (patient G) had strabismus, two patients cluster in the N-terminal domain affect two adjacent amino had astigmatism (patient A and I), and one patient (patient acids (p.Thr326 and p.Pro327) that are known to be part E) had congenital nystagmus. While only one patient (patient of a conserved phosphodegron that is required for binding D) had a visual impairment, three patients had optic nerve with SCF-Fbw7 ubiquitin ligase for degradation (Davis et al. abnormalities: pale optic nerves in patient D, papilledema 2013). Using interaction data from Davis et al. and PDB in patient G, and in patient E outer retinal atrophy temporal structure 2OVQ, which has Fbw7 interacting with a similar to both optic discs was reported with relatively small optic motif as MED13, we modeled this interaction for MED13 nerves on a MRI-scan. followed by insertion of each variant and calculation of Several other interesting phenotypes were observed in at binding energy. All four variants (p.Thr326Ile, p.Thr326del, least two patients in the cohort. Four patients presented with p.Pro327Ser, p.Pro327Gln) are predicted to alter the phos- chronic obstipation (patients C, E, G and M). Two patients phorylation and Fbw7 interaction with drastic decreases in had conductive hearing loss (patients F and L). Two patients binding energy to Fbw7 (Supplementary Fig. 1). The two had congenital heart abnormalities: a mildly dilated aortic missense changes clustering in the C-terminal portion of root and pulmonary artery (both improving over time) in the protein (p.Gln2060Lys and p.Ala2064Val; in patients L patient F, and a subaortic stenosis in patient J. Two patients and M, respectively) were also studied in more detail. One were reported to have chronic sleep issues (patient J and K). of the changes (p.Ala2064Val) is predicted to be structure- Overlapping facial characteristics were reported, includ- altering through increasing hydrophobic collapse, second- ing widely spaced eyes with narrow palpebral fissures and ary structure formation, and increasing aliphatic index of a peri-orbital fullness, a broad and high nasal bridge, full nasal surface exposed linear motif. This results in a decrease of tip, synophrys, a flat philtrum and a wide mouth with thin the regions linear interacting peptide potential that is highly upper lip (Fig. 1). conserved and likely functional (Supplementary Fig. 2). The remaining missense variant (p.Pro540Thr in Patient H) lies Variants and predicted consequences within a highly conserved linear motif centered near amino acid 538 (Fig. 2b); it results in the formation of a high prob- The MED13 transcript (NM_005121.2) encodes a large pro- ability Casein Kinase 1 phosphorylation motif, which could tein consisting of 2174 amino acids (NP_005112.2). The lead to additional interaction with proteins containing fork- Pfam database characterizes two domains within the MED13 head-associated domains when analyzed through the ELM protein: an N-terminal domain (aa 11–383) and a C-terminal database (Dinkel et al. 2016) (Fig. 3). domain (aa 1640–2163), as shown in Fig. 2a. Analysis of conservation across the length of the protein indicates sev- Eec ff ts of truncating MED13 mutation on transcript eral highly conserved residues that lie between these two and protein levels domains (Fig. 2b). All 12 unique variants found in our patients are absent As truncating mutations often lead to nonsense-medi- from the gnomAD database (Lek et al. 2016) and TOPMED ated decay and haploinsufficiency, we aimed to examine Bravo database ( https ://br avo .sph.umic h .edu/fr eez e3a/ the effects of a truncating MED13 mutation on levels of hg19/) and are predicted to be highly deleterious by CADD MED13 transcript and MED13 protein. We performed 1 3 Human Genetics (2018) 137:375–388 381 Fig. 1 Facial phenotypes of seven individuals with a MED13 variant. Overlapping facial characteristics include peri-orbital fullness, narrow pal- pebral fissures, a broad and high nasal bridge, full nasal tip, synophrys, flat philtrum, wide mouth and a thin upper lip RT-PCR on cDNA transcribed from RNA of patient J, who Enrichment of de novo MED13 variants in DD/ID was heterozygous for a nonsense mutation (c.4198C > T; cohorts p.Arg1400*). We compared the MED13 transcript level of the patient to her biological parents and two healthy con- We quantified the extent of enrichment of de novo variants trols (Fig. 4a). No differences in MED13 transcript levels in MED13 within DD/ID-affected probands. We used only were detectable between the affected patient and the unaf- the two largest cohorts considered within this study, each fected parents or controls (One-way ANOVA p = 0.5913). of which yielded at least two de novo MED13 variants. Sanger sequencing of cDNA amplicons from the child Five patients described here (A, E, F, I, and K) come from demonstrated the presence of the aberrant transcript in a cohort of 11,149 affected individuals, and two patients, the child (Fig. 4b), at ~ 70% levels relative to the normal one of which is described here (patient L), were identified transcript (Fig. 4c). To assess the effect of the nonsense within the Deciphering Developmental Disorders (DDD) mutation on protein levels, a western blot was performed study of 4293 trios (Deciphering Developmental Disor- on nuclear extracts from mononuclear blood cells of the ders 2017). Both studies suggest a rate close to 1 de novo patient and controls (Fig. 4d). While full-length MED13 variant affecting MED13 per ~ 2200 DD/ID-affected indi- protein was present in the patient (and in the controls), no viduals. When comparing the number of observed de novo truncated MED13 protein product could be detected. The mutations in MED13 to the expected number based on the MED13 protein level of the patient was not clearly differ - gene specific mutation rate of MED13 for missense, splice- −5 ent compared with the MED13 protein level of the father. site, nonsense and frameshift mutations [6.237 × 10 per chromosome (Samocha et al. 2014)], we find evidence for a significant enrichment among DD/ID-affected individu - als (7 variants in 30,884 alleles; p = 0.00371). 1 3 382 Human Genetics (2018) 137:375–388 Fig. 2 Analysis of mutations: location, conservation and codon usage such that the most selected motifs of a protein are identified as peaks. of variant sites. a Identified mutations are shown within a linear rep- The center of each highly conserved linear motif is labeled and those resentation of the MED13 protein, consisting of 2174 amino acids. containing variants described in this paper are boxed. c Codon usage Missense mutations and the in-frame deletion are shown in blue, throughout evolution for the locations of all missense mutations and and nonsense and frameshift mutations in green. Six of the seven the in-frame deletion. All five sites are under high selection with non-truncating mutations in our MED13 cohort cluster in two small multiple synonymous (Syn, gray) amino acids in 352 open reading regions within the N-terminal and C-terminal domains of the MED13 frames (ORFs) of MED13 and MED13L with only a single nonsyn- protein. Affected amino acids p.Thr326 and p.Pro327 and are part of onymous (Nonsyn, red) change. Numbers indicate instances where a conserved phosphodegron (CPD), which is shown in orange. Two ORFs in other species deviate from the conserved codon usage. Of LxxLL nuclear receptor-binding motifs are also noted. b Analysis of note, for three locations (326, 327 and 540) the codon used differs conservation throughout the protein was performed using amino acid between MED13 and MED13L with the amino acid conserved. In selection scores as previously published (Prokop et  al. 2017), using these cases, numbers indicate where ORFs in other species deviate a 21 codon sliding window for both MED13 and MED13L aligned from conserved codon usage in their respective ortholog ID with speech delay and/or speech disorders. Addition- Discussion ally a broad spectrum of other common features is seen, including ASD, ADHD, various eye abnormalities and By molecular and clinical characterization of a cohort mild facial dysmorphisms. Based on the phenotypes of of 13 patients with variants in MED13, we here provide patients presented here, we do not yet see a clear geno- evidence for a new neurodevelopmental disorder. This type-phenotype correlation between type and location of MED13-associated syndrome is characterized by DD/ 1 3 Human Genetics (2018) 137:375–388 383 Fig. 3 Location of missense mutations and in-frame deletion in three- basic, magenta = conserved human variants of interest. A zoomed dimensional structure of MED13 and conservation of affected amino in view of the three different affected regions are shown, along with acids. A full model of MED13 protein created with I-TASSER mod- amino acid alignments from MED13 and MED13L. An asterisk (*) eling was combined with 152 species sequences for MED13 using indicates 100% conservation in all sequences and a colon (:) indicates ConSurf mapping. Amino acid coloring is as followed: gray = not functional conservation. Linear motifs mapped with the Eukaryotic conserved, yellow = conserved hydrophobic, green = conserved Linear Motif tool are shown below sites for 326–327 and 540 hydrophilic, red = conserved polar acidic, blue = conserved polar the mutations and severity of clinical features. However, it to RNA polymerase II (Pol II) (Allen and Taatjes 2015). is notable that the two patients with Duane anomaly have a Binding of the CDK8-module to Mediator has been reported missense mutation in a similar location in the C-terminal to prevent the association of Mediator with the Pol II pre- domain of the MED13 protein, and that the optic nerve initiation complex, thus preventing transcription initiation abnormalities are reported in patients with mutations and/or re-initiation. In this way, the CDK8-module is consid- affecting residues p.Thr326 or p.Pro327 only. ered a key molecular switch in Pol II mediated transcription MED13 is a component of the CDK8-kinase module, (Knuesel et al. 2009). MED13, as well as the other subunits which can reversibly bind the Mediator complex. Media- of the CDK8-module, are known to be critical regulators tor is a multi-protein complex that is required for assem- of developmental gene expression programs in Drosophila, bly and stabilization of the pre-initiation complex, which zebrafish and C. elegans (Carrera et al. 2008; Poss et al. is essential for transcription initiation (Chen et al. 2012; 2013). MED13, or its paralog MED13L, forms a direct Hantsche and Cramer 2017). The core function of Media- connection of the CDK8 module with the core Mediator tor is to transmit signals from various transcription factors 1 3 384 Human Genetics (2018) 137:375–388 Fig. 4 Analysis of transcript and protein levels in patient with non- base signal by trace). The father and mother do not have any signal at sense mutation. a Level of MED13 transcript was measured by qPCR the mutant base above the level of noise. d Western blot for MED13 and normalized to GAPDH and proband (patient J). No differences (and HSP90 and HDAC2 as loading controls) from nuclear extracts of were detectable between groups (One-way ANOVA p = 0.5913). An patient peripheral blood mononuclear cells or a neural precursor cell additional loading control (AGPAT) produced very similar results line (present to demonstrate antibody specificity with a knockdown (data not shown). b Representative Sanger traces from cDNA ampli- (KD) control). If the nonsense mutation resulted in a stable protein, cons demonstrating the presence of the variant in the proband, and a product at approximately 150 kDa would be expected, which is not absence in the father and mother. c Quantification of the chromato- present. No protein was recoverable from the blood sample from the grams of all Sanger sequences reveals less signal from the base on the mother mutant allele (p < 0.0001 by paired t-test compared to the wildtype complex (Daniels 2013), and protein turnover of MED13 which targets the MED13 protein for ubiquitination and (or MED13L) may be critical in modulating the pools of degradation (Davis et al. 2013). In fact, it has already been Mediator-CDK8 kinase complex in cells (Davis et al. 2013; shown that a specific amino acid substitution at position 326 Knuesel et al. 2009; Tsai et al. 2013). in MED13 (p.Thr326Ala) leads to impaired binding of Fbw7 Three missense mutations (p.Thr326Ile, p.Pro327Ser and to the phosphodegron of MED13/MED13L, thus prevent- p.Pro327Gln) and one in-frame-deletion (p.Thr326del) in ing MED13/MED13L ubiquitination and degradation (Davis our cohort are likely to affect MED13 protein turnover due to et al. 2013). Therefore, a variant at this position may lead to their location within a conserved phosphodegron. This phos- increased levels of MED13 protein in the cell. As Fbw7 is phodegron is recognized by the SCF-Fbw7 ubiquitin ligase, proposed to target only MED13 or MED13L proteins that are bound to the core Mediator complex (Davis et al. 2013), 1 3 Human Genetics (2018) 137:375–388 385 these mutations may have an effect on the CDK8 module- Mutations in MED12 have also been associated with intel- Mediator association and subsequently on transcription reg- lectual disability. In addition to ID and speech delays both ulation. The potential effects of the p.Pro540Thr missense MED12 patients and several MED13 probands described variant are also intriguing. Protein modeling suggests that here present with eye abnormalities (eye movement dis- this variant could introduce an additional Casein Kinase 1 orders, and abnormalities of the retina and optic nerves) phosphorylation site, thus potentially increasing interactions (Clark et al. 2009; Donnio et al. 2017) and chronic obstipa- with forkhead-associated domains involved in protein–pro- tion (Donnio et al. 2017; Lyons 1993). In addition to the tein interactions. MED12 subunit, a disruption of CDK19 was reported in a We also observed five unique mutations predicted to trun- patient with ID, microcephaly and congenital retinal folds cate MED13. In assessments of RNA and protein levels in (Mukhopadhyay et al. 2010). Patient J and her unaffected parents, the variant transcript It is of particular relevance to this study that variation was detected in the proband but no truncated protein could in the MED13-paralog MED13L has been shown to cause be observed. While these results are inconclusive with a neurodevelopmental disorder as well (Asadollahi et al. regards to the molecular mechanism of pathogenicity in 2013). Given the similar molecular roles for MED13 and this particular proband, loss-of-function mechanisms remain MED13L, we aimed to compare and contrast phenotypes an attractive possibility. Patterns of variation in MED13 in presented by both groups of individuals using information human population databases indicate that MED13 is rela- provided in the literature. The main phenotypic character- tively intolerant to loss-of-function variation; MED13 has a istics of MED13L-associated syndrome are (borderline) ID Rare Variant Intolerance Score (RVIS) that ranks among the with delayed speech and language development, and a vari- top 1.66% of all genes (Petrovski et al. 2013) and an ExAC able spectrum of other features including autism, hypotonia, pLI score of 1.00 (Lek et al. 2016). characteristic facial features and heart defects (Adegbola We show an enrichment of de novo MED13 muta- et al. 2015; Caro-Llopis et al. 2016; Martinez et al. 2017; tions compared to what is expected under a null model Muncke et al. 2003; van Haelst et al. 2015). Many of these (p = 0.00371) in two large ID/DD patient cohorts. We features clearly overlap with the phenotypes in our MED13 acknowledge that this p value does not exceed a genome- cohort. However, similar to the heterogeneity observed wide evidence threshold and by itself proves association. here in patients with MED13 variation, the spectrum of However, the enrichment p value does not account for five phenotypes observed among MED13L mutation carriers is de novo variants described here from smaller cohorts that quite broad. The identification and detailed phenotyping of were discovered independent of, and prior to, assessment additional patients with MED13 and MED13L mutations is of the statistical evidence from the larger cohorts. We also needed to elucidate the complete spectrum of associated fea- observed clustering of missense mutations in our cohort, tures, and to reveal the similarities and differences between which by itself is an argument for pathogenicity (Lelieveld the two syndromes. et al. 2017). Additionally, independent genetic studies also We believe that the data presented in this study cou- support the disease relevance of variation in MED13. There pled to the additional evidence available from other studies is one report of an 800-kb microdeletion including MED13 strongly support the conclusion that rare protein-altering and five other genes in a patient with moderate ID, short variation in MED13 underlie a new neurodevelopmental stature, mild dysmorphisms, and hearing loss (Boutry-Kryza disorder. Key results from this study include: a significant et al. 2012); the authors proposed MED13 as the most likely enrichment of de novo mutations in MED13 within ID/DD causal candidate gene. Additionally, a de novo frameshift cohorts (p = 0.00371); the clustering and conservation levels (p.Pro286Leufs*86) and a de novo variant that likely affects of the positions affected by the observed missense variation splicing (D + 3; c.814+3A>G) were observed in a cohort of (Fig. 2a, b); the computationally predicted deleteriousness 2508 probands with ASD (Iossifov et al. 2014), and three of the observed mutations (Table 1; Fig. 3, Supplementary rare protein-altering variants in MED13 (p.Ala418Thr, Fig. 1); and the overlap of phenotypic features among the 13 p.Arg512*, p.Tyr1649*) were also found in a separate ASD patients presented here, including speech difficulties (13/13), cohort (Yuen et al. 2017). intellectual disability (at least 9/13), and eye or vision prob- Other Mediator subunits, including other CDK8-kinase lems (8/13). Supporting evidence from other studies include: module-associated disease genes, have been associated with the existence of mutations affecting MED13 in at least six various neurodevelopmental disorders. Variants in MED12 independent families affected by pediatric neurodevelop- have been associated with ID syndromes with congenital mental disorders; the intolerance of MED13 to mutations abnormalities, including Opitz-Kaveggia syndrome (MIM in the general human population (pLI = 1.00, RVIS score of 305,450) (Risheg et al. 2007), Lujan-Fryns syndrome (MIM 1.66%); and the previously established disease-associations 309,520) (Schwartz et al. 2007) and X-linked Ohdo syn- of several other Mediator subunits, including MED13L, a drome (MIM 300,896) (Vulto-van Silfhout et  al. 2013). functionally related paralog of MED13. While the precise 1 3 386 Human Genetics (2018) 137:375–388 Allen BL, Taatjes DJ (2015) The Mediator complex: a central integra- pathogenic mechanisms have yet to be elucidated—some tor of transcription. Nat Rev Mol Cell Biol 16:155–166. https :// of the mutations observed here are predicted to stabilize doi.org/10.1038/nrm39 51 MED13 protein while others are predicted to lead to loss-of- Andrews CV, Hunter DG, Engle EC (1993) Duane Syndrome. In: function—we find it highly likely that mutational disruption Pagon RA et al (eds) GeneReviews(R). Seattle (WA) Asadollahi R et al (2013) Dosage changes of MED13L further delineate of normal MED13 function leads to disease, adding MED13 its role in congenital heart defects and intellectual disability. Eur J to the list of Mediator-associated, in particular CDK8-kinase Hum Genet 21:1100–1104. https ://doi.org/10.1038/ejhg.2013.17 module-associated, neurodevelopmental disorders. Au PYB et al (2015) GeneMatcher aids in the identification of a new malformation syndrome with intellectual disability, unique facial Acknowledgements We thank all patients and families for their contri- dysmorphisms, and skeletal and connective tissue abnormalities butions. This work was supported by the Max Planck Society (S.E.F.), caused by de novo variants in. HNRNPK Hum Mut 36:1009– a grant from the US National Human Genome Research Institute 1014. https ://doi.org/10.1002/humu.22837 (NHGRI; UM1HG007301) (S.M.H., K.M.B., J.N.C., K.L.E., E.M.B., Boat TF, Wu JT (eds) (2015) mental disorders and disabilities among G.M.C.), and the Netherlands Organisation for Scientific Research low-income children. Washington (DC). https://doi.or g/10.17226 (NWO) Gravitation Grant 24.001.006 to the Language in Interaction /21780 Consortium (L.S.B., H.G.B., S.E.F.). Patient L was part of the DDD Boutry-Kryza N et al (2012) An 800 kb deletion at 17q23.2 includ- study cohort (DECIPHER ID: DDD263479). The DDD study presents ing the MED13 (THRAP1) gene, revealed by aCGH in a patient independent research commissioned by the Health Innovation Chal- with a SMC 17. Am J Med Genet A 158A:400–405. https ://doi. lenge Fund [grant number HICF-1009-003], a parallel funding partner- org/10.1002/ajmg.a.34222 p ship between the Wellcome Trust and the Department of Health, and Bowling KM et al (2017) Genomic diagnosis for children with intel- the Wellcome Trust Sanger Institute [grant number WT098051]. The lectual disability and/or developmental delay. Genome Med 9:43. views expressed in this publication are those of the author(s) and not https ://doi.org/10.1186/s1307 3-017-0433-1 necessarily those of the Wellcome Trust or the Department of Health. Boyle CA et al (2011) Trends in the prevalence of developmental dis- The study has UK Research Ethics Committee approval (10/H0305/83, abilities in US children 1997–2008. Pediatrics 127:1034–1042. granted by the Cambridge South REC, and GEN/284/12 granted by the https ://doi.org/10.1542/peds.2010-2989 Republic of Ireland REC). The research team acknowledges the support Caro-Llopis A, Rosello M, Orellana C, Oltra S, Monfort S, Mayo of the National Institute for Health Research, through the Comprehen- S, Martinez F (2016) De novo mutations in genes of mediator sive Clinical Research Network. This study makes use of DECIPHER complex causing syndromic intellectual disability: mediatorpa- (http://decipher .sanger .ac.uk), which is funded by the Wellcome Trust. thy or transcriptomopathy? Pediatr Res 80:809–815. https ://doi. org/10.1038/pr.2016.162 Carrera I, Janody F, Leeds N, Duveau F, Treisman JE (2008) Pygopus Compliance with ethical standards activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. 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Circulation https ://doi.org/10.1016/j.ajhg.2013.01.007 1 3 388 Human Genetics (2018) 137:375–388 Affiliations 1,2,3 4 4 4 4 Lot Snijders Blok  · Susan M. Hiatt  · Kevin M. Bowling  · Jeremy W. Prokop  · Krysta L. Engel  · 4 5 6 6 7 J. Nicholas Cochran  · E. Martina Bebin  · Emilia K. Bijlsma  · Claudia A. L. Ruivenkamp  · Paulien Terhal  · 7 8 9 10 11 Marleen E. H. Simon  · Rosemarie Smith  · Jane A. Hurst  · The DDD study  · Heather McLaughlin  · 11 11 12 12 13 13 Richard Person  · Amy Crunk  · Michael F. Wangler  · Haley Streff  · Joseph D. Symonds  · Sameer M. Zuberi  · 14 15 16 16,17 18 Katherine S. Elliott  · Victoria R. Sanders  · Abigail Masunga  · Robert J. Hopkin  · Holly A. Dubbs  · 18 1 1,3,19 2,3 1,3 Xilma R. Ortiz‑Gonzalez  · Rolph Pfundt  · Han G. Brunner  · Simon E. Fisher  · Tjitske Kleefstra  · Gregory M. Cooper 1 12 Human Genetics Department, Radboud University Medical Department of Molecular and Human Genetics, Baylor Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands College of Medicine, Houston, TX, USA 2 13 Language and Genetics Department, Max Planck Institute Paediatric Neurosciences Research Group, University for Psycholinguistics, Nijmegen, The Netherlands of Glasgow and Royal Hospital for Children, Glasgow G51 4TF, UK Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA University of Alabama at Birmingham, Birmingham, AL, USA Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA Division of Genetics, Department of Pediatrics, Maine Medical Center, Portland, ME, USA Department of Clinical Genetics, GROW School for Oncology and Developmental Biology, Maastricht UMC, Great Ormond Street Hospital for Children, London, UK Maastricht, The Netherlands Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK GeneDx, 207 Perry Parkway, Gaithersburg, MD 20877, USA 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Genetics Springer Journals

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Abstract

Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non- truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiqui- tination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes. Introduction The introduction of next-generation sequencing techniques has rapidly improved the identification of genes that associ- ate with rare disease. Although developmental delay (DD) Lot Snijders Blok and Susan M. Hiatt contributed equally as first and intellectual disability (ID) are relatively common (Boat authors, and Tjitske Kleefstra and Gregory M. Cooper contribued and Wu 2015; Boyle et al. 2011), there is extreme genetic equally as last authors. heterogeneity among affected patients and a large fraction of patients with DD/ID remain refractory to diagnosis (Vis- Electronic supplementary material The online version of this sers et al. 2016). In unsolved cases, the understanding of article (https ://doi.org/10.1007/s0043 9-018-1887-y) contains supplementary material, which is available to authorized users. gene–disease relationships has greatly benefited from col- laboration between clinical genetics teams (Sobreira et al. * Tjitske Kleefstra 2015). In fact, many recently discovered DD/ID genes have tjitske.kleefstra@radboudumc.nl come from “matchmaking” (Au et al. 2015; Harms et al. * Gregory M. Cooper 2017; Kernohan et al. 2017), where websites such as Gen- gcooper@hudsonalpha.org eMatcher (Sobreira et al. 2015) facilitate the comparison Extended author information available on the last page of the article Vol.:(0123456789) 1 3 376 Human Genetics (2018) 137:375–388 of patients with rare genotypes and phenotypes across the described (Bowling et  al. 2017). In each patient, the world. observed MED13 mutation was considered to be the most Here we present the results of a collaboration facilitated likely contributor to the phenotype, and no additional patho- by GeneMatcher (Sobreira et  al. 2015) in which multi- genic or likely pathogenic variants were found. ple clinical and research groups independently identified individuals with DD/ID and related phenotypes with rare Three‑dimensional modeling protein-altering variation in MED13. This genotype-driven approach enabled us to characterize the phenotypes and Protein modeling was performed as previously described mutational spectrum of a cohort of 13 patients, each with a (Prokop et al. 2017). Modeling of MED13 interacting with likely pathogenic variant in MED13. FBXW7 was performed using PDB 2OVQ, replacing mol- Although MED13 has not been previously linked to a dis- ecule C with the MED13 amino acids 321–330. Binding order, it is a paralog of MED13L, mutations of which have energy was calculated following each patient variant inser- been found to cause ID, speech impairment and heart defects tion and energy minimization using AMBER14 force field (Adegbola et al. 2015; Muncke et al. 2003; van Haelst et al. (http://amber md.org/) in YASARA. 2015). The gene products MED13 and MED13L are mutu- ally exclusive components of the reversible CDK8-module RNA isolation of the Mediator complex, a multi-protein complex that is required for the expression of all protein-coding genes (Con- 2.5  mL of blood was collected in PAXgene RNA tubes away et al. 2005; Malik and Roeder 2005). In this study, we (PreAnalytiX #762165) according to the manufacturer’s show that variants in MED13 are also associated with a neu- instructions and stored short-term at − 20 °C. RNA was iso- rodevelopmental disorder, and delineate the corresponding lated using a PAX gene Blood RNA Kit (Qiagen #762164) phenotypic features and mutational spectrum. according to the manufacturer’s instructions. Isolated RNA was quantified by Qubit® (Thermo Fisher #Q32855). Materials and methods cDNA synthesis Informed consent First strand synthesis of cDNA was performed from 150 ng Informed consent to publish de-identified data was obtained of RNA isolated from blood using Superscript™ III (Thermo from all patients, either as part of the diagnostic workflow or Fisher #18080044) according to manufacturer’s instructions as part of a research study (Bowling et al. 2017). Informed using random primers (Invitrogen #48190011) for +/− RT consent to publish clinical photographs was also obtained reactions. The products were diluted 1:10 in water before when applicable. Informed consent matched the local ethi- use in qPCR reactions. cal guidelines. qPCR Exome/genome sequencing qPCR was performed according to manufacturer’s protocols In patients A, B, D, E, F, G, I, K, L and M, whole exome using Taqman gene expression master mix (ThermoFisher sequencing and variant filtering were performed as previ- #4369016) and FAM-MGB Taqman probes directed against ously published (de Ligt et al. 2012; Deciphering Develop- MED13 (ThermoFisher Hs01080701_m1 catalog #4331182) mental Disorders 2015; Neveling et al. 2013; Sollis et al. and GAPDH (ThermoFisher #4352934E). qPCR reactions 2017; Tanaka et  al. 2015). In patient C, targeted Sanger were carried out in a QuantStudio 6 Flex Real-Time PCR sequencing was performed to confirm the presence of the system (Applied Biosystems) using 40 cycles of amplifi- MED13 variant (L131*) that was first identified in patient cation. Raw C values were obtained, normalized first to B. For patient H, whole genome sequencing was performed the GAPDH loading control, and then to the proband. We using Illumina’s HiSeq X ten platform. Sequencing reads tested an additional loading control [AGPAT-data not shown were mapped against the hs37d5 reference using GATK. (ThermoFisher Hs00965850_g1; catalog #4331182)], but Variants were called using GATK’s Haplotype Caller. Vari- the data were like those normalized to GAPDH. ants were filtered using frequencies from the ExAC and gnomAD databases (mean allele frequency < 0.003) and for conservation using PhastCons (> 0.5) and PhyloP (> 4). For patient J, whole genome sequencing, variant prioritiza- tion, and Sanger validation were performed as previously 1 3 Human Genetics (2018) 137:375–388 377 MED13 in blood despite detecting MED13 in neural pre- Sanger sequencing cursor nuclear lysates: Bethyl #278A, Abcam #ab49468, and Abcam #ab76923 (data not shown). cDNA template was amplified using primers to the region of interest: 5′ -CG A GGC T CT TAT GG A A CT G AT G AA Statistical enrichment of MED13 variants in DD/ID TC-3′ (forward) and 5′-GATCC AT CGT GCTTT C AGA CA CAT C-3′ (reverse). No amplification was observed in the cohorts no RT condition. PCR conditions were: 500  nM prim- ers, 3% DMSO, 1x Phusion HF (NEB #M0531L), 0.5 µL We compared the frequency of observed de novo MED13 variation identified in two large sequencing cohorts to the cDNA template, and cycling at (98 °C, 30 s), (98 °C, 10 s; 60 °C, 30 s; 72 °C, 45 s)x35, (72 °C, 7 m), (4 °C, ∞). The expected frequency of variation in MED13 based on its gene specific mutation rate (Samocha et al. 2014) using an Exact additional reverse primer 5′-AAAT GCTT CA TTG TTA CC GTC AGC T-3′ and the additional forward primers 5′-TCC Poisson Test in R (R Core Team. R: A language and envi- ronment for statistical computing (http://www.r-project.or g). AAA AGA AAC GAT GTG AGT ATG CAG-3′, 5′-CTC TCT TCA GCC AGT TCT TCA GGA T-3′, 5′-ACA ATT TCA TAA Vienna). AATGGC T GGCCG A -3′, 5′-CGAGGC T CTT ATGG AA CT GATG AAT C-3′, 5′-GTGCTT T CTCC ATTT GCT CTT CCT Results T-3′ were used for sequencing, along with the primers used for amplification from cDNA. Chromatograms were quan- Phenotypes tified using ab1PeakReporter (Thermo Fisher). We collected detailed clinical information of 13 patients Western Blot with rare, protein-altering MED13 variants. Eleven variants were confirmed to be de novo, and one patient (patient B) Whole blood was collected using cell processing tubes inherited the variant from her mother who is also affected (patient C). Phenotypic data summarizing the spectrum of (BD #362760), isolated according to the manufacturer’s instructions, and stored in liquid nitrogen in CTS™ features of this cohort of 13 patients are shown in Table 1. All patients had developmental delays with varying sever- Synth-a-Freeze Medium (Thermo Fisher # A13713-01) until use. As a control for antibody specificity, MED13 ity and course. In the patients that underwent formal intel- ligence testing, total IQ levels varied from 85 (lower range was knocked down in neural precursor cells (clone BC1, MTI-GlobalStem #GSC-4311) by generating stable lines of normal IQ) to an IQ between 35 and 50 (moderate ID). Five patients had an Autism Spectrum Disorder (ASD), and using puromycin selection expressing shRNA against MED13 (Sigma Aldrich # SHCLNG-NM_005121; three patients were diagnosed with Attention Deficit Hyper - activity Disorder (ADHD). All patients had speech delays TRCN0000234904) compared to a GFP shRNA control in the same vector (Addgene # 30323). Cell pellets were and/or disorders, with delayed milestones in speech and lan- guage development. While several patients had expressive processed using the NE-PER™ (Thermo Fisher #78833) nuclear and cytoplasmic extraction kit according to the and receptive language problems, in the majority of patients, speech production was significantly more impaired than lan- manufacturer’s instructions, and nuclear extracts were used for the blot shown (whole cell extracts, even at very guage comprehension. Three patients (patient A, K and M) showed characteristics of speech apraxia, a developmental high concentrations, did not produce sufficient signal). 60 µg of protein was loaded for patient blood samples, speech disorder in which affected individuals have difficul- ties accurately programming the motor sequences required and 15 µg of protein was loaded for neural precursor cell samples. Blots were blocked for 1 h at room temperature in to produce fluent speech. Patient A had a mild ID, but showed speech apraxia with a mixed receptive and expres- LICOR blocking buffer (LICOR #927-40000), then blots were probed (with washes in PBS-T (0.05% Tween-20) sive language disorder, and limited verbal expression at the age of 8 years. Patient M had a non-verbal IQ of 70 with a and a secondary probe for 1 h after each primary probe) with 1:250 rabbit anti-MED13 (Bethyl #A301-277A) for severe speech/language disorder. Her expressive speech was severely affected, with signs of speech apraxia. At the age of 3 days at 4 °C, 1:1,000 mouse anti-HDAC2 (clone 3F3, SCBT #sc-81599) overnight at 4 °C as a loading control, 8 years she only used single words and very short sentences. Patient K developed some speech capabilities, but showed and 1:1000 rabbit anti-HSP90 (abcam #ab115660) over- night at 4 °C as an additional loading control. Secondary regression at the age of 13 months and has since remained non-verbal. probes were used at 1:20,000 (LICOR #926-32211 and #926-68070). Three other primary antibodies were tested Seven of 13 patients showed delays in motor develop- ment, most of which affected at least the gross motor skills for MED13, but did not show sufficient signal to detect 1 3 378 Human Genetics (2018) 137:375–388 1 3 Table 1 Clinical features of patients with MED13 mutations and molecular characterization Patient A Patient B Patient C Patient D Patient E Patient F Patient G Patient H Patient I Patient J Patient K Patient L Patient M Molecular characterization  cDNA variant c.125del c.392T>G c.392T>G c.977C>T c.975_977 c.979C>T c.980C>A c.1618C>A c.1745T>A c.4198C>T c.4487delC c.6178C>A c.6191C>T (NM_005121.2) delTAC  Predicted protein P42Lfs*6 L131* L131* T326I T326del P327S P327Q P540T L582* R1400* T1496Mfs Q2060K A2064V effect  CADD v.1.3 31.0 37.0 37.0 25.0 20.5 23.4 25.2 26.3 40 41 35 24.1 25.7  GERP + + RS 5.67 5.32 5.32 5.5 5.5 5.5 5.5 6.16 6.02 4.58 5.86 6.04 6.04  Inheritance De novo Maternal Unknown De novo De novo De novo De novo De novo De novo De novo De novo De novo De novo (daughter of pt. C) Clinical characterization  Gender M F F M F M F M M F M F F  Age at last visit 8 5 32 19 9 11 3 10 6 13 5 10 6 (years)  Height + 0.6 SD + 0.5 SD average − 0.2 SD + 2.2 SD + 0.5 SD + 0.3 SD − 0.9 SD 0 SD − 0.7 SD − 2 SD + 0.5 SD − 2 SD  Weight (for + 1.8 SD + 1.9 SD average − 0.8 SD + 1.2 SD − 0.9 SD − 1.1 SD + 0.6 SD + 0.5 SD 0 SD − 1.6 SD + 0.4 SD + 0.7 SD height)  Head circumfer- − 1 SD + 0.2 SD NA − 0.5 SD + 1.1 SD − 0.9 SD − 0.3 SD − 1.5 SD − 0.5 SD NA 0 SD − 2 SD + 1 SD ence  Intellectual Dis- Mild ID Mild/border- Borderline Mild ID Mild ID Mild ID DD Borderline ID Mild ID Moderate ID DD DD Mild/border- ability (ID) / line ID ID (IQ 65) (IQ 85 with (IQ 61) line ID Developmental (IQ 80–85) working (IQ 70) Delay (DD) memory score 68 on WISC-IV)  Speech delay/ + + + + + + + +/− + + + + + disorder Speech Delayed Delayed Delayed Moderate mixed Delayed Mainly Borderline At age 6y Moderate Severe speech Severe speech Severe apraxia speech speech speech receptive and expres- expressive (verbal com- expres- expressive disorder delay, 5–10 speech/ with develop- develop- develop- expressive sive and speech prehension sive and language with words language mixed ment, mild ment, ment, mild language disor- receptive problems score = 87 receptive disorder regression, disorder, recep- articulation expressive articula- der, decreased language on WISC- speech at speech expressive tive and problems language tion vocabulary IV) age equiva- apraxia, language expres- problems problems, and language lent < 2y receptive most sive in child- normal formulation language is affected, language hood. At language difficulties fine signs of disorder, adult age compre- speech limited only spo- hension apraxia verbal radic and expres- mild word- sion and finding lan- problems guage- based learning disorder Human Genetics (2018) 137:375–388 379 1 3 Table 1 (continued) Patient A Patient B Patient C Patient D Patient E Patient F Patient G Patient H Patient I Patient J Patient K Patient L Patient M  Delayed motor + − − + + + + − + NR − − + development (Only fine (walked at (walked on (walked at (walked at 25 m) (walked at (walked at (walked before (walked at 2 (walked at (walked at motor 14 m) time) 20 m) 22 m) 26 m) 12 m) years; early 12 m) 20 m) skills delays, now delayed) mostly on target with peers)  Autism spectrum ADHD NA − − − ASD ASD − − ASD, ADHD ASD ASD, ADHD − disorder (ASD) /ADHD  Brain MRI Normal NA NA Normal Bulbous splenium Normal Normal Small area of Normal NA NA Normal Mild frontal of corpus cal- abnormal atrophy, losum (likely signal in left otherwise normal variant) occipital normal lobe  Eye/vision abnor- Astigma- probably − Visual Congenital nys- − Strabismus, − Astigmatism NR NR Duane Duane malities tism amblyopia impair- tagmus, outer papil anomaly anomaly ment, retinal atrophy edema pale optic temporal to nerves both optic discs, optic nerves low normal in size on MRI  Heart abnormali- NR − NR − History of Dilated − NR NR Subaortic NR NR NR ties murmur, normal aortic stenosis echo and ECG root and pulmonary artery  Chronic obstipa- NR NR + NR + NR + NR NR NR NR NR + tion  Other features Sloping Small and Kyphosis, Hypotonia, mild Hypotonia, Hypotonia Epilepsy Chronic sleep Chronic sleep Conductive (features shoulders, laterally pes cavus proximal weak- Conduc- (drug-resist- disturbances distur- hearing loss, reported in small and deviated ness, fatigues tive ant with bances Precocious two unrelated laterally halluces easily, clumsy hearing myoclonic- puberty patients in bold) deviated gait, transient loss, Mild atonic halluces lactic acidosis scoliosis, seizures) with illness, pes cavus congenital left hip dysplasia NR not reported, NA not assessed 380 Human Genetics (2018) 137:375–388 (6 of 7), although one patient was reported to have only v1.3 (Kircher et al. 2014), with scores ranging from 20.5 fine motor delays. Three patients had hypotonia (patient E, to 41 (Table 1). Six patients had five unique variants that F and G). One patient (patient H) developed severe drug- are predicted to be truncating: three nonsense mutations resistant myoclonic-atonic epilepsy at 4 years of age with (p.Leu131* in Patients B and C, p.Leu582* in Patient I and generalized clonic, myoclonic, atonic, tonic and atypical p.Arg1400* in Patient J) and two frameshift variants lead- absence seizures. MRI screening of this patient showed a ing to a premature stop codon (p.Pro42Leufs*6 in patient A small abnormality in the left occipital lobe of his brain that and p.Thr1496Metfs*11 in Patient K). The remaining vari- did not correspond to the electrophysiological onset or the ants include six missense variants and a single amino acid semiology of his seizures. In other patients, MRI scans were deletion. These seven variants form two apparent clusters: not performed or showed no clear abnormalities, except for one in the N-terminal conserved phosphodegron domain mild frontal atrophy in patient M. and the other in the C-terminal domain (Fig.  2a). These Eight patients (62%) presented with eye or vision abnor- seven variants were all found to lie within motifs that are malities. Two patients (patients L and M) presented with highly conserved between MED13 and MED13L (Fig. 2b) Duane anomaly, a congenital type of strabismus that is char- and affect sites under high codon selection (Fig.  2c). These acterized by non-progressive horizontal ophtalmoplegia and missense variants and the in-frame deletion are each located retraction of the globe with attempted adduction, together on surface-exposed sites within a three-dimensional model with narrowing of the palpebral fissure (Andrews et  al. of the MED13 protein (Fig.  3). The four mutations that 1993). One patient (patient G) had strabismus, two patients cluster in the N-terminal domain affect two adjacent amino had astigmatism (patient A and I), and one patient (patient acids (p.Thr326 and p.Pro327) that are known to be part E) had congenital nystagmus. While only one patient (patient of a conserved phosphodegron that is required for binding D) had a visual impairment, three patients had optic nerve with SCF-Fbw7 ubiquitin ligase for degradation (Davis et al. abnormalities: pale optic nerves in patient D, papilledema 2013). Using interaction data from Davis et al. and PDB in patient G, and in patient E outer retinal atrophy temporal structure 2OVQ, which has Fbw7 interacting with a similar to both optic discs was reported with relatively small optic motif as MED13, we modeled this interaction for MED13 nerves on a MRI-scan. followed by insertion of each variant and calculation of Several other interesting phenotypes were observed in at binding energy. All four variants (p.Thr326Ile, p.Thr326del, least two patients in the cohort. Four patients presented with p.Pro327Ser, p.Pro327Gln) are predicted to alter the phos- chronic obstipation (patients C, E, G and M). Two patients phorylation and Fbw7 interaction with drastic decreases in had conductive hearing loss (patients F and L). Two patients binding energy to Fbw7 (Supplementary Fig. 1). The two had congenital heart abnormalities: a mildly dilated aortic missense changes clustering in the C-terminal portion of root and pulmonary artery (both improving over time) in the protein (p.Gln2060Lys and p.Ala2064Val; in patients L patient F, and a subaortic stenosis in patient J. Two patients and M, respectively) were also studied in more detail. One were reported to have chronic sleep issues (patient J and K). of the changes (p.Ala2064Val) is predicted to be structure- Overlapping facial characteristics were reported, includ- altering through increasing hydrophobic collapse, second- ing widely spaced eyes with narrow palpebral fissures and ary structure formation, and increasing aliphatic index of a peri-orbital fullness, a broad and high nasal bridge, full nasal surface exposed linear motif. This results in a decrease of tip, synophrys, a flat philtrum and a wide mouth with thin the regions linear interacting peptide potential that is highly upper lip (Fig. 1). conserved and likely functional (Supplementary Fig. 2). The remaining missense variant (p.Pro540Thr in Patient H) lies Variants and predicted consequences within a highly conserved linear motif centered near amino acid 538 (Fig. 2b); it results in the formation of a high prob- The MED13 transcript (NM_005121.2) encodes a large pro- ability Casein Kinase 1 phosphorylation motif, which could tein consisting of 2174 amino acids (NP_005112.2). The lead to additional interaction with proteins containing fork- Pfam database characterizes two domains within the MED13 head-associated domains when analyzed through the ELM protein: an N-terminal domain (aa 11–383) and a C-terminal database (Dinkel et al. 2016) (Fig. 3). domain (aa 1640–2163), as shown in Fig. 2a. Analysis of conservation across the length of the protein indicates sev- Eec ff ts of truncating MED13 mutation on transcript eral highly conserved residues that lie between these two and protein levels domains (Fig. 2b). All 12 unique variants found in our patients are absent As truncating mutations often lead to nonsense-medi- from the gnomAD database (Lek et al. 2016) and TOPMED ated decay and haploinsufficiency, we aimed to examine Bravo database ( https ://br avo .sph.umic h .edu/fr eez e3a/ the effects of a truncating MED13 mutation on levels of hg19/) and are predicted to be highly deleterious by CADD MED13 transcript and MED13 protein. We performed 1 3 Human Genetics (2018) 137:375–388 381 Fig. 1 Facial phenotypes of seven individuals with a MED13 variant. Overlapping facial characteristics include peri-orbital fullness, narrow pal- pebral fissures, a broad and high nasal bridge, full nasal tip, synophrys, flat philtrum, wide mouth and a thin upper lip RT-PCR on cDNA transcribed from RNA of patient J, who Enrichment of de novo MED13 variants in DD/ID was heterozygous for a nonsense mutation (c.4198C > T; cohorts p.Arg1400*). We compared the MED13 transcript level of the patient to her biological parents and two healthy con- We quantified the extent of enrichment of de novo variants trols (Fig. 4a). No differences in MED13 transcript levels in MED13 within DD/ID-affected probands. We used only were detectable between the affected patient and the unaf- the two largest cohorts considered within this study, each fected parents or controls (One-way ANOVA p = 0.5913). of which yielded at least two de novo MED13 variants. Sanger sequencing of cDNA amplicons from the child Five patients described here (A, E, F, I, and K) come from demonstrated the presence of the aberrant transcript in a cohort of 11,149 affected individuals, and two patients, the child (Fig. 4b), at ~ 70% levels relative to the normal one of which is described here (patient L), were identified transcript (Fig. 4c). To assess the effect of the nonsense within the Deciphering Developmental Disorders (DDD) mutation on protein levels, a western blot was performed study of 4293 trios (Deciphering Developmental Disor- on nuclear extracts from mononuclear blood cells of the ders 2017). Both studies suggest a rate close to 1 de novo patient and controls (Fig. 4d). While full-length MED13 variant affecting MED13 per ~ 2200 DD/ID-affected indi- protein was present in the patient (and in the controls), no viduals. When comparing the number of observed de novo truncated MED13 protein product could be detected. The mutations in MED13 to the expected number based on the MED13 protein level of the patient was not clearly differ - gene specific mutation rate of MED13 for missense, splice- −5 ent compared with the MED13 protein level of the father. site, nonsense and frameshift mutations [6.237 × 10 per chromosome (Samocha et al. 2014)], we find evidence for a significant enrichment among DD/ID-affected individu - als (7 variants in 30,884 alleles; p = 0.00371). 1 3 382 Human Genetics (2018) 137:375–388 Fig. 2 Analysis of mutations: location, conservation and codon usage such that the most selected motifs of a protein are identified as peaks. of variant sites. a Identified mutations are shown within a linear rep- The center of each highly conserved linear motif is labeled and those resentation of the MED13 protein, consisting of 2174 amino acids. containing variants described in this paper are boxed. c Codon usage Missense mutations and the in-frame deletion are shown in blue, throughout evolution for the locations of all missense mutations and and nonsense and frameshift mutations in green. Six of the seven the in-frame deletion. All five sites are under high selection with non-truncating mutations in our MED13 cohort cluster in two small multiple synonymous (Syn, gray) amino acids in 352 open reading regions within the N-terminal and C-terminal domains of the MED13 frames (ORFs) of MED13 and MED13L with only a single nonsyn- protein. Affected amino acids p.Thr326 and p.Pro327 and are part of onymous (Nonsyn, red) change. Numbers indicate instances where a conserved phosphodegron (CPD), which is shown in orange. Two ORFs in other species deviate from the conserved codon usage. Of LxxLL nuclear receptor-binding motifs are also noted. b Analysis of note, for three locations (326, 327 and 540) the codon used differs conservation throughout the protein was performed using amino acid between MED13 and MED13L with the amino acid conserved. In selection scores as previously published (Prokop et  al. 2017), using these cases, numbers indicate where ORFs in other species deviate a 21 codon sliding window for both MED13 and MED13L aligned from conserved codon usage in their respective ortholog ID with speech delay and/or speech disorders. Addition- Discussion ally a broad spectrum of other common features is seen, including ASD, ADHD, various eye abnormalities and By molecular and clinical characterization of a cohort mild facial dysmorphisms. Based on the phenotypes of of 13 patients with variants in MED13, we here provide patients presented here, we do not yet see a clear geno- evidence for a new neurodevelopmental disorder. This type-phenotype correlation between type and location of MED13-associated syndrome is characterized by DD/ 1 3 Human Genetics (2018) 137:375–388 383 Fig. 3 Location of missense mutations and in-frame deletion in three- basic, magenta = conserved human variants of interest. A zoomed dimensional structure of MED13 and conservation of affected amino in view of the three different affected regions are shown, along with acids. A full model of MED13 protein created with I-TASSER mod- amino acid alignments from MED13 and MED13L. An asterisk (*) eling was combined with 152 species sequences for MED13 using indicates 100% conservation in all sequences and a colon (:) indicates ConSurf mapping. Amino acid coloring is as followed: gray = not functional conservation. Linear motifs mapped with the Eukaryotic conserved, yellow = conserved hydrophobic, green = conserved Linear Motif tool are shown below sites for 326–327 and 540 hydrophilic, red = conserved polar acidic, blue = conserved polar the mutations and severity of clinical features. However, it to RNA polymerase II (Pol II) (Allen and Taatjes 2015). is notable that the two patients with Duane anomaly have a Binding of the CDK8-module to Mediator has been reported missense mutation in a similar location in the C-terminal to prevent the association of Mediator with the Pol II pre- domain of the MED13 protein, and that the optic nerve initiation complex, thus preventing transcription initiation abnormalities are reported in patients with mutations and/or re-initiation. In this way, the CDK8-module is consid- affecting residues p.Thr326 or p.Pro327 only. ered a key molecular switch in Pol II mediated transcription MED13 is a component of the CDK8-kinase module, (Knuesel et al. 2009). MED13, as well as the other subunits which can reversibly bind the Mediator complex. Media- of the CDK8-module, are known to be critical regulators tor is a multi-protein complex that is required for assem- of developmental gene expression programs in Drosophila, bly and stabilization of the pre-initiation complex, which zebrafish and C. elegans (Carrera et al. 2008; Poss et al. is essential for transcription initiation (Chen et al. 2012; 2013). MED13, or its paralog MED13L, forms a direct Hantsche and Cramer 2017). The core function of Media- connection of the CDK8 module with the core Mediator tor is to transmit signals from various transcription factors 1 3 384 Human Genetics (2018) 137:375–388 Fig. 4 Analysis of transcript and protein levels in patient with non- base signal by trace). The father and mother do not have any signal at sense mutation. a Level of MED13 transcript was measured by qPCR the mutant base above the level of noise. d Western blot for MED13 and normalized to GAPDH and proband (patient J). No differences (and HSP90 and HDAC2 as loading controls) from nuclear extracts of were detectable between groups (One-way ANOVA p = 0.5913). An patient peripheral blood mononuclear cells or a neural precursor cell additional loading control (AGPAT) produced very similar results line (present to demonstrate antibody specificity with a knockdown (data not shown). b Representative Sanger traces from cDNA ampli- (KD) control). If the nonsense mutation resulted in a stable protein, cons demonstrating the presence of the variant in the proband, and a product at approximately 150 kDa would be expected, which is not absence in the father and mother. c Quantification of the chromato- present. No protein was recoverable from the blood sample from the grams of all Sanger sequences reveals less signal from the base on the mother mutant allele (p < 0.0001 by paired t-test compared to the wildtype complex (Daniels 2013), and protein turnover of MED13 which targets the MED13 protein for ubiquitination and (or MED13L) may be critical in modulating the pools of degradation (Davis et al. 2013). In fact, it has already been Mediator-CDK8 kinase complex in cells (Davis et al. 2013; shown that a specific amino acid substitution at position 326 Knuesel et al. 2009; Tsai et al. 2013). in MED13 (p.Thr326Ala) leads to impaired binding of Fbw7 Three missense mutations (p.Thr326Ile, p.Pro327Ser and to the phosphodegron of MED13/MED13L, thus prevent- p.Pro327Gln) and one in-frame-deletion (p.Thr326del) in ing MED13/MED13L ubiquitination and degradation (Davis our cohort are likely to affect MED13 protein turnover due to et al. 2013). Therefore, a variant at this position may lead to their location within a conserved phosphodegron. This phos- increased levels of MED13 protein in the cell. As Fbw7 is phodegron is recognized by the SCF-Fbw7 ubiquitin ligase, proposed to target only MED13 or MED13L proteins that are bound to the core Mediator complex (Davis et al. 2013), 1 3 Human Genetics (2018) 137:375–388 385 these mutations may have an effect on the CDK8 module- Mutations in MED12 have also been associated with intel- Mediator association and subsequently on transcription reg- lectual disability. In addition to ID and speech delays both ulation. The potential effects of the p.Pro540Thr missense MED12 patients and several MED13 probands described variant are also intriguing. Protein modeling suggests that here present with eye abnormalities (eye movement dis- this variant could introduce an additional Casein Kinase 1 orders, and abnormalities of the retina and optic nerves) phosphorylation site, thus potentially increasing interactions (Clark et al. 2009; Donnio et al. 2017) and chronic obstipa- with forkhead-associated domains involved in protein–pro- tion (Donnio et al. 2017; Lyons 1993). In addition to the tein interactions. MED12 subunit, a disruption of CDK19 was reported in a We also observed five unique mutations predicted to trun- patient with ID, microcephaly and congenital retinal folds cate MED13. In assessments of RNA and protein levels in (Mukhopadhyay et al. 2010). Patient J and her unaffected parents, the variant transcript It is of particular relevance to this study that variation was detected in the proband but no truncated protein could in the MED13-paralog MED13L has been shown to cause be observed. While these results are inconclusive with a neurodevelopmental disorder as well (Asadollahi et al. regards to the molecular mechanism of pathogenicity in 2013). Given the similar molecular roles for MED13 and this particular proband, loss-of-function mechanisms remain MED13L, we aimed to compare and contrast phenotypes an attractive possibility. Patterns of variation in MED13 in presented by both groups of individuals using information human population databases indicate that MED13 is rela- provided in the literature. The main phenotypic character- tively intolerant to loss-of-function variation; MED13 has a istics of MED13L-associated syndrome are (borderline) ID Rare Variant Intolerance Score (RVIS) that ranks among the with delayed speech and language development, and a vari- top 1.66% of all genes (Petrovski et al. 2013) and an ExAC able spectrum of other features including autism, hypotonia, pLI score of 1.00 (Lek et al. 2016). characteristic facial features and heart defects (Adegbola We show an enrichment of de novo MED13 muta- et al. 2015; Caro-Llopis et al. 2016; Martinez et al. 2017; tions compared to what is expected under a null model Muncke et al. 2003; van Haelst et al. 2015). Many of these (p = 0.00371) in two large ID/DD patient cohorts. We features clearly overlap with the phenotypes in our MED13 acknowledge that this p value does not exceed a genome- cohort. However, similar to the heterogeneity observed wide evidence threshold and by itself proves association. here in patients with MED13 variation, the spectrum of However, the enrichment p value does not account for five phenotypes observed among MED13L mutation carriers is de novo variants described here from smaller cohorts that quite broad. The identification and detailed phenotyping of were discovered independent of, and prior to, assessment additional patients with MED13 and MED13L mutations is of the statistical evidence from the larger cohorts. We also needed to elucidate the complete spectrum of associated fea- observed clustering of missense mutations in our cohort, tures, and to reveal the similarities and differences between which by itself is an argument for pathogenicity (Lelieveld the two syndromes. et al. 2017). Additionally, independent genetic studies also We believe that the data presented in this study cou- support the disease relevance of variation in MED13. There pled to the additional evidence available from other studies is one report of an 800-kb microdeletion including MED13 strongly support the conclusion that rare protein-altering and five other genes in a patient with moderate ID, short variation in MED13 underlie a new neurodevelopmental stature, mild dysmorphisms, and hearing loss (Boutry-Kryza disorder. Key results from this study include: a significant et al. 2012); the authors proposed MED13 as the most likely enrichment of de novo mutations in MED13 within ID/DD causal candidate gene. Additionally, a de novo frameshift cohorts (p = 0.00371); the clustering and conservation levels (p.Pro286Leufs*86) and a de novo variant that likely affects of the positions affected by the observed missense variation splicing (D + 3; c.814+3A>G) were observed in a cohort of (Fig. 2a, b); the computationally predicted deleteriousness 2508 probands with ASD (Iossifov et al. 2014), and three of the observed mutations (Table 1; Fig. 3, Supplementary rare protein-altering variants in MED13 (p.Ala418Thr, Fig. 1); and the overlap of phenotypic features among the 13 p.Arg512*, p.Tyr1649*) were also found in a separate ASD patients presented here, including speech difficulties (13/13), cohort (Yuen et al. 2017). intellectual disability (at least 9/13), and eye or vision prob- Other Mediator subunits, including other CDK8-kinase lems (8/13). Supporting evidence from other studies include: module-associated disease genes, have been associated with the existence of mutations affecting MED13 in at least six various neurodevelopmental disorders. Variants in MED12 independent families affected by pediatric neurodevelop- have been associated with ID syndromes with congenital mental disorders; the intolerance of MED13 to mutations abnormalities, including Opitz-Kaveggia syndrome (MIM in the general human population (pLI = 1.00, RVIS score of 305,450) (Risheg et al. 2007), Lujan-Fryns syndrome (MIM 1.66%); and the previously established disease-associations 309,520) (Schwartz et al. 2007) and X-linked Ohdo syn- of several other Mediator subunits, including MED13L, a drome (MIM 300,896) (Vulto-van Silfhout et  al. 2013). functionally related paralog of MED13. While the precise 1 3 386 Human Genetics (2018) 137:375–388 Allen BL, Taatjes DJ (2015) The Mediator complex: a central integra- pathogenic mechanisms have yet to be elucidated—some tor of transcription. Nat Rev Mol Cell Biol 16:155–166. https :// of the mutations observed here are predicted to stabilize doi.org/10.1038/nrm39 51 MED13 protein while others are predicted to lead to loss-of- Andrews CV, Hunter DG, Engle EC (1993) Duane Syndrome. In: function—we find it highly likely that mutational disruption Pagon RA et al (eds) GeneReviews(R). Seattle (WA) Asadollahi R et al (2013) Dosage changes of MED13L further delineate of normal MED13 function leads to disease, adding MED13 its role in congenital heart defects and intellectual disability. Eur J to the list of Mediator-associated, in particular CDK8-kinase Hum Genet 21:1100–1104. https ://doi.org/10.1038/ejhg.2013.17 module-associated, neurodevelopmental disorders. Au PYB et al (2015) GeneMatcher aids in the identification of a new malformation syndrome with intellectual disability, unique facial Acknowledgements We thank all patients and families for their contri- dysmorphisms, and skeletal and connective tissue abnormalities butions. This work was supported by the Max Planck Society (S.E.F.), caused by de novo variants in. HNRNPK Hum Mut 36:1009– a grant from the US National Human Genome Research Institute 1014. https ://doi.org/10.1002/humu.22837 (NHGRI; UM1HG007301) (S.M.H., K.M.B., J.N.C., K.L.E., E.M.B., Boat TF, Wu JT (eds) (2015) mental disorders and disabilities among G.M.C.), and the Netherlands Organisation for Scientific Research low-income children. Washington (DC). https://doi.or g/10.17226 (NWO) Gravitation Grant 24.001.006 to the Language in Interaction /21780 Consortium (L.S.B., H.G.B., S.E.F.). Patient L was part of the DDD Boutry-Kryza N et al (2012) An 800 kb deletion at 17q23.2 includ- study cohort (DECIPHER ID: DDD263479). The DDD study presents ing the MED13 (THRAP1) gene, revealed by aCGH in a patient independent research commissioned by the Health Innovation Chal- with a SMC 17. Am J Med Genet A 158A:400–405. https ://doi. lenge Fund [grant number HICF-1009-003], a parallel funding partner- org/10.1002/ajmg.a.34222 p ship between the Wellcome Trust and the Department of Health, and Bowling KM et al (2017) Genomic diagnosis for children with intel- the Wellcome Trust Sanger Institute [grant number WT098051]. The lectual disability and/or developmental delay. Genome Med 9:43. views expressed in this publication are those of the author(s) and not https ://doi.org/10.1186/s1307 3-017-0433-1 necessarily those of the Wellcome Trust or the Department of Health. Boyle CA et al (2011) Trends in the prevalence of developmental dis- The study has UK Research Ethics Committee approval (10/H0305/83, abilities in US children 1997–2008. Pediatrics 127:1034–1042. granted by the Cambridge South REC, and GEN/284/12 granted by the https ://doi.org/10.1542/peds.2010-2989 Republic of Ireland REC). The research team acknowledges the support Caro-Llopis A, Rosello M, Orellana C, Oltra S, Monfort S, Mayo of the National Institute for Health Research, through the Comprehen- S, Martinez F (2016) De novo mutations in genes of mediator sive Clinical Research Network. This study makes use of DECIPHER complex causing syndromic intellectual disability: mediatorpa- (http://decipher .sanger .ac.uk), which is funded by the Wellcome Trust. thy or transcriptomopathy? Pediatr Res 80:809–815. https ://doi. org/10.1038/pr.2016.162 Carrera I, Janody F, Leeds N, Duveau F, Treisman JE (2008) Pygopus Compliance with ethical standards activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. 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Circulation https ://doi.org/10.1016/j.ajhg.2013.01.007 1 3 388 Human Genetics (2018) 137:375–388 Affiliations 1,2,3 4 4 4 4 Lot Snijders Blok  · Susan M. Hiatt  · Kevin M. Bowling  · Jeremy W. Prokop  · Krysta L. Engel  · 4 5 6 6 7 J. Nicholas Cochran  · E. Martina Bebin  · Emilia K. Bijlsma  · Claudia A. L. Ruivenkamp  · Paulien Terhal  · 7 8 9 10 11 Marleen E. H. Simon  · Rosemarie Smith  · Jane A. Hurst  · The DDD study  · Heather McLaughlin  · 11 11 12 12 13 13 Richard Person  · Amy Crunk  · Michael F. Wangler  · Haley Streff  · Joseph D. Symonds  · Sameer M. Zuberi  · 14 15 16 16,17 18 Katherine S. Elliott  · Victoria R. Sanders  · Abigail Masunga  · Robert J. Hopkin  · Holly A. Dubbs  · 18 1 1,3,19 2,3 1,3 Xilma R. Ortiz‑Gonzalez  · Rolph Pfundt  · Han G. Brunner  · Simon E. Fisher  · Tjitske Kleefstra  · Gregory M. Cooper 1 12 Human Genetics Department, Radboud University Medical Department of Molecular and Human Genetics, Baylor Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands College of Medicine, Houston, TX, USA 2 13 Language and Genetics Department, Max Planck Institute Paediatric Neurosciences Research Group, University for Psycholinguistics, Nijmegen, The Netherlands of Glasgow and Royal Hospital for Children, Glasgow G51 4TF, UK Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA University of Alabama at Birmingham, Birmingham, AL, USA Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA Division of Genetics, Department of Pediatrics, Maine Medical Center, Portland, ME, USA Department of Clinical Genetics, GROW School for Oncology and Developmental Biology, Maastricht UMC, Great Ormond Street Hospital for Children, London, UK Maastricht, The Netherlands Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK GeneDx, 207 Perry Parkway, Gaithersburg, MD 20877, USA 1 3

Journal

Human GeneticsSpringer Journals

Published: May 8, 2018

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