Clinical Aspects of SDHA-Related Pheochromocytoma and Paraganglioma: A Nationwide Study

Clinical Aspects of SDHA-Related Pheochromocytoma and Paraganglioma: A Nationwide Study Abstract Context Paraganglioma (PGL) has the highest degree of heritability among human neoplasms. Current clinical understanding of germline SDHA mutation carriers is limited. Objective To estimate the contribution of SDHA mutations in PGL and to assess clinical manifestations and age-related penetrance. Design Nationwide retrospective cohort study. Setting Tertiary referral centers in the Netherlands (multicenter). Patients Germline SDHA analysis was performed in 393 patients with genetically unexplained PGL. Subsequently, 30 index SDHA mutation carriers and 56 nonindex carriers were studied. Main Outcome Measures SDHA mutation detection yield, clinical manifestations, and SDHA-related disease penetrance. Results Pathogenic germline SDHA variants were identified in 30 of the 393 referred patients with PGL (7.6%), who had head and neck PGL (21 of 174 [12%]), pheochromocytoma (4 of 191 [2%]), or sympathetic PGL (5 of 28 [18%]). The median age at diagnosis was 43 years (range, 17 to 81 years) in index SDHA mutation carriers compared with 52 years (range, 7 to 90 years) in nonmutation carriers (P = 0.002). The estimated penetrance of any SDHA-related manifestation was 10% at age 70 years (95% confidence interval, 0% to 21%) in nonindex mutation carriers. Conclusion Germline SDHA mutations are relatively common (7.6%) in patients with genetically unexplained PGL. Most index patients presented with apparently sporadic PGL. In this SDHA series, the largest assembled so far, we found the lowest penetrance of all major PGL predisposition genes. This suggests that recommendations for genetic counseling of at-risk relatives and stringency of surveillance for SDHA mutation carriers might need to be reassessed. Paragangliomas (PGLs) are rare neuroendocrine tumors that carry the highest degree of heritability among human neoplasms (1, 2). PGLs are classified according to their anatomic location (intra-adrenal or extra-adrenal PGL) and whether they are of sympathetic or parasympathetic origin. Head and neck paragangliomas (HNPGLs) emerge from the parasympathetic nervous system and are usually benign, slow-growing tumors (3, 4). Common sites include the carotid body, the temporal bone, and the vagal body. Parasympathetic PGLs are most often nonsecreting, although about 30% are associated with elevated levels of the dopamine metabolite 3-methoxytyramine (3-MT) (5). Pheochromocytoma (PHEO) and sympathetic paraganglioma (SPGL) are catecholamine-secreting tumors (6). PHEOs are derived from the chromaffin cells of the adrenal medulla, and SPGLs are found in close relationship to the peripheral sympathetic nervous system from the level of the superior cervical ganglion down the trunk into the pelvis (7). Metastases are more often present in SPGL than are PHEO and HNPGL (3). About one third of patients with PGL have been reported to carry pathogenic germline variants in a growing list of susceptibility genes (8). The most described genes are NF1, RET, VHL, SDHD, SDHC, SDHB, SDHAF2, SDHA, TMEM127, and MAX. Germline variants in the succinate dehydrogenase (SDH) genes are the most common genetic cause of PGLs, occurring in up to 15% of all patients with PGL and half of all familiar cases (2, 9). In 2010, a direct association between germline SDHA variants and PGL was reported (10). SDH-associated syndromes are characterized by the development of PGLs, with an additional risk for developing other tumor types [e.g., clear cell renal cancer (RCC), gastrointestinal stromal tumors (GISTs), and, more rarely, neuroendocrine tumors and pituitary adenomas] (11–13). So far, information on prevalence, phenotype, penetrance, and pathogenicity of SDHA variants is limited to one large series (14) and a few small series (15, 16). In this study, we performed a nationwide evaluation of germline SDHA analyses undertaken in patients with PGL and characterized the clinical manifestations and disease penetrance in 30 index SDHA mutation carriers and their relatives. Patients and Methods Study population and design All patients with an established diagnosis of PGL who were referred for germline SDHA analysis in the Netherlands from February 2011 through July 2016 were included in this study. Referred patients with PGL were grouped into three clinical subgroups—HNPGL, PHEO, or SPGL—on the basis of clinical, biochemical, imaging, and/or histological characteristics. Data on sex, diagnosis, and age at diagnosis were retrieved from DNA request forms. In accordance with the Dutch national genetic testing strategy, all patients with PGL referred for SDHA analysis lacked pathogenic germline variants in SDHB, SDHC, SDHD, and SDHAF2. All patients with PHEO and SPGL furthermore lacked pathogenic germline variants in TMEM127, MAX, RET, and VHL and had no clinical symptoms suggesting neurofibromatosis type 1. Index patients with (likely) pathogenic SDHA variants or variants of uncertain significance (VUS) were evaluated and subsequently counseled by a clinical geneticist in their regional University Medical Center. Patients with pathogenic and likely pathogenic variants are annotated as SDHA mutation carriers in this manuscript. Clinical characteristics (e.g., sex, age at diagnosis, tumor location or locations, presence of metastases, biochemical phenotype, and additional non-PGL tumors) and pedigrees were collected. Genetic counseling and testing for the family-specific (likely) pathogenic SDHA variant were offered to relatives via cascade screening. All SDHA mutation carriers age ≥18 years were referred to departments of otorhinolaryngology and endocrinology for annual clinical surveillance aimed at detecting PGL. According to national guidelines (17), surveillance consisted of magnetic resonance imaging of the head and neck region once every 3 years and magnetic resonance imaging or computed tomography of the thorax, abdomen, and pelvis once every 2 years. Annual routine biochemical testing included the measurement of (nor)epinephrine, (nor)metanephrine, dopamine, and/or 3-MT in 24-hour urine samples and/or plasma, depending on the center. In cases with excessive catecholamine secretion (i.e., any value above the upper reference limit), radiological assessment of the thorax, abdomen, and pelvis was performed to identify potential sources of excessive catecholamine production. The current study was approved by the local medical ethical committee of Leiden University Medical Center (G16.063). DNA sequencing and data analysis Germline SDHA variant analysis was performed in the Department of Human Genetics at the Radboud University Medical Center and the Laboratory for Diagnostic Genome Analysis of the Department of Clinical Genetics at Leiden University Medical Center, the Netherlands. Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures. Germline SDHA analysis was performed with Sanger sequencing or next-generation (gene panel) sequencing (NGS) depending on the testing period. For the detailed NGS procedure, see the Supplemental Method. Coding variants were analyzed for their effect on function by using the Alamut software package, version (Interactive Biosoftware, Rouen, France), which incorporates Align GVGD (18), polymorphism phenotyping (PolyPhen2) (19), and sorting intolerant from tolerant (SIFT) (20). Variants were annotated to the Genbank reference sequence NM_004168.2. The Leiden Open Variation Database (http://www.lovd.nl/SDHA) was consulted to find variants previously described and classified. Variant interpretation was done in line with the recent consensus statement on NGS-based diagnostic testing of hereditary PHEO and PGLs (21). Variant nomenclature is in accordance with Human Genome Variation Society guidelines, version 2.0 (http://www.hgvs.org). To obtain further support for the pathogenicity of certain SDHA variants, SDHA immunohistochemistry and loss-of-heterozygosity (LOH) analysis were performed on formalin-fixed, paraffin-embedded samples, as described elsewhere (22). Statistical analysis Descriptive statistics were used to characterize the study population, to determine the age of PGL onset, and to examine the difference between patients with germline SDHA mutation and those without germline SDHA mutation. Continuous variables were analyzed by using an independent sample t test. Dichotomous variables were compared by using the χ2 test. Age-related penetrance of PGL was estimated by using the Kaplan-Meier method. Because most of the nonindex mutation carriers were recently identified, we used the age at 1 year after DNA analysis (or age at death) for the penetrance estimation. By completion of this manuscript, >80% of the nonindex mutation carriers had participated in surveillance at least once. Statistical significance was set at P < 0.05, and the analyses were conducted by using SPSS software, version 23.0 (IBM, Armonk, NY). Results SDHA case detection in the study population Pathogenic germline SDHA variants were identified in 30 of 393 (7.6%) patients with PGL who were referred for SDHA genetic testing. The clinical characteristics of the study population (SDHA vs non-SDHA) are listed in Table 1. Within the clinical PGL subgroups, pathogenic SDHA variants were identified in 21 of 174 patients with HNPGL (12%), 4 of 191 patients with PHEO (2%), and 5 of 28 patients with SPGL (18%). The median age at diagnosis of PGL was 43 years (range, 17 to 81 years) in SDHA mutation carriers and 52 years (range, 7 to 90 years) in those without a detectable mutation (P = 0.002). Half of the SDHA mutation carriers were males compared with 32% of patients without an SDHA mutation (P = 0.049). Table 1. Clinical Characteristics of Patients With Germline SDHA Mutation and Patients Without Germline SDHA Mutation Characteristic  SDHA Mutation (n = 30)  No SDHA Mutation (n = 363)  P Value  Mutation Yield (%)  All patients with PGL        7.6   Age at diagnosis (y)  43 (17–81)  52 (7–90)  0.002     Males, n (%)  15 (50)  117 (32)  0.049    Patients with HNPGL  21  153    12   Age at diagnosis (y)  43 (18–81)  54 (19–90)  0.008     Males, n (%)  10 (48)  33 (22)  0.010    Patients with PHEO  4  186    2   Age at diagnosis (y)  35 (17–70)  51 (7–81)  0.14     Males, n (%)  0  71 (38)  0.118    Patients with SPGL  5  24    18   Age at diagnosis (y)  36 (22–60)  53 (23–73)  0.118     Males, n (%)  5 (100)  13 (54)  0.055    Characteristic  SDHA Mutation (n = 30)  No SDHA Mutation (n = 363)  P Value  Mutation Yield (%)  All patients with PGL        7.6   Age at diagnosis (y)  43 (17–81)  52 (7–90)  0.002     Males, n (%)  15 (50)  117 (32)  0.049    Patients with HNPGL  21  153    12   Age at diagnosis (y)  43 (18–81)  54 (19–90)  0.008     Males, n (%)  10 (48)  33 (22)  0.010    Patients with PHEO  4  186    2   Age at diagnosis (y)  35 (17–70)  51 (7–81)  0.14     Males, n (%)  0  71 (38)  0.118    Patients with SPGL  5  24    18   Age at diagnosis (y)  36 (22–60)  53 (23–73)  0.118     Males, n (%)  5 (100)  13 (54)  0.055    Data are presented as median (range) or number and percentage. P values are derived from χ2 or independent sample t test. View Large SDHA variants Seven different (likely) pathogenic germline SDHA variants were identified in 30 patients with PGL (Table 2). Three variants had been reported previously: The common nonsense mutation c.91C>T, (p.Arg31*) (10) was observed in 23 patients, the c.1753C>T, (p.Arg585Trp) (16) missense mutation was observed in two patients, and the nonsense c.1534C>T, p.(Arg512*) (22) was observed in one patient. Moreover, four not previously reported and three previously reported SDHA VUS were identified (Supplemental Table 1). All VUS were identified in patients with apparently sporadic HNPGL, diagnosed between the ages of 28 and 52 years. Additional immunohistochemical staining and LOH analysis were performed in two of the seven VUS-related HNPGLs but showed no loss of SDHA or SDHB staining or LOH. The other five HNPGLs were not available for further analysis. Table 2. Clinical and Molecular Characteristics of 30 Index SDHA Mutation Carriers and Their Relatives Patient No.  Family   Sex  Tumors Observed (Age at Detection, y)  Biochemistry Results  Family History  Germline SDHA Variant  Tested Relatives (Carriersa )  Reference  1  A  Female  GCT-ri (43)  Normal  Negative  c.91C>T, p.(Arg31*)  5 (3)  10  2  B  Male  GVT-le (38)  Normal  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  3  C  Female  GVT-ri (81)  Normal  Negative  c.91C>T, p.(Arg31*)  3 (1)  10  4  D  Female  GCT-le 2x (35)  Normal  Negative  c.91C>T, p.(Arg31*)  1 (1)  10  5  E  Female  GCT-le, GCT-ri, GJT-le (48)  Normal  Negative  c.91C>T, p.(Arg31*)  6 (4)  10  6  F  Male  GCT-le (30)  NA  Negative  c.91C>T, p.(Arg31*)  10 (6)  10  7  G  Male  GCT-ri (56), Prolactinoma (58)  Normal  Negative  c.91C>T, p.(Arg31*)  4 (2)  10  8  H  Male  GJT-le (43)  Normal  Negative  c.1432_1432+1delGGb  3 (2)  Not previously reported  9  I  Female  GCT-ri, GCT-le (26, 49, 50), Prolactinoma (45), Multiple Meningioma (45,62)  Normal  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  10  J  Male  GCT-ri (23)  Normal  GISTc  c.91C>T, p.(Arg31*)  8 (6)  10  11  K  Male  GJTT-le (38)  3-MT  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  12  L  Female  GCT-le (40)  Normal  RCCc  c.985C>T, p.(Arg329*)  1 (1)  Not previously reported  13  M  Female  GCT-le (61)  NA  Negative  c.91C>T, p.(Arg31*)  8 (5)  10  14  M  Female  GJTT-ri (58)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  15  M  Female  GVT-le (53)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  16  M  Female  GVT-ri (42)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  17  M  Female  GVT-ri (53)  Normal  RCCd  c.91C>T, p.(Arg31*)  0  10  18  M  Male  GJT-ri (18)  NA  Negative  c.91C>T, p.(Arg31*)  0  10  19  M  Male  GCT-ri (48), uveal melanoma (48)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  20  M  Male  GVT-ri (60)  3-MT  Negative  c.1795-3C>Gb  0  Not previously reported  21  M  Male  GVT-ri, GVT-le, GCT-ri (49)  Normal  Negative  c.667delG, p.(Asp223fs)  0  Not previously reported  22  N  Female  PHEO-ri (17)  NM  Negative  c.1753C>T, p.(Arg585Trp)  3 (1)  16  23  N  Female  PHEO-ri (20), Wilms tumor (4)  NM  Negative  c.1753C>T, (p.Arg585Trp)  0  16  24  N  Female  PHEO-le (50), metastasis (60)  NM  Negative  c.91C>T, p.(Arg31*)  0  10  25  N  Female  PHEO-ri (70), RCC (70)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  26  O  Male  Para-aortal SPGL (60)  NA  RCCe  c.91C>T, p.(Arg31*)  10 (8)  10  27  P  Male  Testis SPGL (23), metastases (26)  NM/3-MT  HNPGLc  c.1534C>T, p.(Arg512*)  3 (4)  22  28  Q  Male  Retroperitoneal para-aortal SPGL (50)  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (3)  10  29  R  Male  Malignant retroperitoneal SPGL (36)f  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  30  S  Male  Para-aortal SPGL (22)  NM  Negative  c.91C>T, p.(Arg31*)  3 (3)  10  Patient No.  Family   Sex  Tumors Observed (Age at Detection, y)  Biochemistry Results  Family History  Germline SDHA Variant  Tested Relatives (Carriersa )  Reference  1  A  Female  GCT-ri (43)  Normal  Negative  c.91C>T, p.(Arg31*)  5 (3)  10  2  B  Male  GVT-le (38)  Normal  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  3  C  Female  GVT-ri (81)  Normal  Negative  c.91C>T, p.(Arg31*)  3 (1)  10  4  D  Female  GCT-le 2x (35)  Normal  Negative  c.91C>T, p.(Arg31*)  1 (1)  10  5  E  Female  GCT-le, GCT-ri, GJT-le (48)  Normal  Negative  c.91C>T, p.(Arg31*)  6 (4)  10  6  F  Male  GCT-le (30)  NA  Negative  c.91C>T, p.(Arg31*)  10 (6)  10  7  G  Male  GCT-ri (56), Prolactinoma (58)  Normal  Negative  c.91C>T, p.(Arg31*)  4 (2)  10  8  H  Male  GJT-le (43)  Normal  Negative  c.1432_1432+1delGGb  3 (2)  Not previously reported  9  I  Female  GCT-ri, GCT-le (26, 49, 50), Prolactinoma (45), Multiple Meningioma (45,62)  Normal  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  10  J  Male  GCT-ri (23)  Normal  GISTc  c.91C>T, p.(Arg31*)  8 (6)  10  11  K  Male  GJTT-le (38)  3-MT  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  12  L  Female  GCT-le (40)  Normal  RCCc  c.985C>T, p.(Arg329*)  1 (1)  Not previously reported  13  M  Female  GCT-le (61)  NA  Negative  c.91C>T, p.(Arg31*)  8 (5)  10  14  M  Female  GJTT-ri (58)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  15  M  Female  GVT-le (53)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  16  M  Female  GVT-ri (42)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  17  M  Female  GVT-ri (53)  Normal  RCCd  c.91C>T, p.(Arg31*)  0  10  18  M  Male  GJT-ri (18)  NA  Negative  c.91C>T, p.(Arg31*)  0  10  19  M  Male  GCT-ri (48), uveal melanoma (48)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  20  M  Male  GVT-ri (60)  3-MT  Negative  c.1795-3C>Gb  0  Not previously reported  21  M  Male  GVT-ri, GVT-le, GCT-ri (49)  Normal  Negative  c.667delG, p.(Asp223fs)  0  Not previously reported  22  N  Female  PHEO-ri (17)  NM  Negative  c.1753C>T, p.(Arg585Trp)  3 (1)  16  23  N  Female  PHEO-ri (20), Wilms tumor (4)  NM  Negative  c.1753C>T, (p.Arg585Trp)  0  16  24  N  Female  PHEO-le (50), metastasis (60)  NM  Negative  c.91C>T, p.(Arg31*)  0  10  25  N  Female  PHEO-ri (70), RCC (70)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  26  O  Male  Para-aortal SPGL (60)  NA  RCCe  c.91C>T, p.(Arg31*)  10 (8)  10  27  P  Male  Testis SPGL (23), metastases (26)  NM/3-MT  HNPGLc  c.1534C>T, p.(Arg512*)  3 (4)  22  28  Q  Male  Retroperitoneal para-aortal SPGL (50)  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (3)  10  29  R  Male  Malignant retroperitoneal SPGL (36)f  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  30  S  Male  Para-aortal SPGL (22)  NM  Negative  c.91C>T, p.(Arg31*)  3 (3)  10  See Supplemental Fig. 2 for the pedigrees of families A through S. Abbreviations: GCT, glomus carotid body tumor; GJT, glomus jugularis tumor; GJTT, glomus jugulotympanicum tumor; GTT, glomus tympanicum tumor; GVT, glomus vagal tumor; ri, right; le, left; NA, not available; PTC, papillary thyroid carcinoma. a Including obligate mutation carriers. Reference sequence SDHA NC000005.9, NM004168.3. b Likely pathogenic SDHA variant. c SDHA mutation carrier. d SDHA mutation status unknown. e No SDHA mutation carrier. f Dead of disease. View Large Clinical manifestations in index SDHA mutation carriers The clinical and molecular characteristics of the 30 index patients with germline pathogenic SDHA variants are listed in Table 2. Germline SDHA mutations were identified in 21 index patients with HNPGL, 4 with PHEO, and 5 with SPGL. Four patients were diagnosed with multiple HNPGLs. The HNPGL anatomic locations were distributed as follows: 15 carotid body, 8 vagal, 3 jugular, and 2 jugular tympanic. Two patients with HNPGL had elevated 3-MT levels. Three patients with PHEO had elevated normetanephrine levels. One patient had developed a metastatic PHEO, but no bilateral PHEOs were detected. Four SPGLs had a retroperitoneal para-aortal location and one SPGL was found in the testis. Four patients with SPGL had elevated normetanephrine levels, three in combination with elevated 3-MT. Two patients developed metastatic SPGL, and one of these patients (no. 27) died at age 27 years. Furthermore, three index mutation carriers (no. 7, no. 9, and no. 25) were diagnosed with one other possibly SDHA-related feature, including pituitary adenoma (at ages 58 and 45 years, respectively) and RCC (age 70 years), respectively. One pituitary adenoma was immunonegative for both SDHA and SDHB and contained an additional somatic pathogenic SDHA variant (p.Asp38Val), likely resulting in biallelic inactivation of SDHA (Supplemental Fig. 1) (23). The other pituitary adenoma was not resected and therefore not analyzed. Conversely, the RCC tissue showed no loss of SDHA immunohistochemical staining, suggesting that it was not SDHA-related. Three additional tumor types were reported in index SDHA mutation carriers: multiple meningioma (patient no. 9), uveal melanoma (no. 19, BAP1-mutation negative) and Wilms tumor (no. 23). However, it is not clear whether these tumors were related to the SDHA mutation. Immunohistochemical staining showed no loss of SDHA staining in both meningiomas. The uveal melanoma lesion and Wilms tumor were not available for analysis. Five SDHA mutation carriers had a positive family history for SDHA-related tumors, including HNPGL (patient no. 27), GIST (no. 10), and RCC (no. 12, no. 17, and no. 26). Clinical manifestations in SDHA families In total, 94 available relatives were tested via cascade screening for their familial pathogenic SDHA variant, revealing 51 nonindex carriers and 5 obligate carriers. Pedigrees of the 19 SDHA families with at least one nonindex mutation carrier are shown in Supplemental Fig. 2. Remarkably, we could confirm in all families, except one (index no. 3, diagnosed at age 81 years), that the mutation was inherited from an unaffected parent. The median age at DNA analysis in the nonindex SDHA mutation carriers was 58 years (range, 7 to 94 years). In total, 3 of 56 (5%) nonindex SDHA mutation carriers were diagnosed as having one (possible) SDHA-related tumor: HNPGL (n = 1), GIST (n = 1), and RCC (n = 1). Family history did not reveal any not-tested relatives with (possible) SDHA-related tumors. The estimated penetrance of any SDHA-related tumor is shown in Fig. 1. The age-related penetrance values for all 86 SDHA mutation carriers were 7% at age 25 years [95% confidence interval (CI), 2% to 12%], 26% at age 50 years (95% CI, 16% to 36%), and 50% at age 70 years (95% CI, 34% to 66%). The age-related penetrance values for the 56 nonindex SDHA mutation carriers were 0% at age 25 years, 2% at age 50 years (95% CI, 0% to 6%), and 10% at age 70 years (95% CI, 0% to 21%). By completion of this manuscript, 51 nonindex carriers were lacking any identified SDHA-related feature, indicating that they could be considered to be healthy mutation carriers. Figure 1. View largeDownload slide Age-related penetrance of any SDHA-related manifestations. Figure 1. View largeDownload slide Age-related penetrance of any SDHA-related manifestations. Discussion This nationwide retrospective SDHA survey investigated SDHA mutation detection yield and clinical phenotype in patients with genetically unexplained PGL. We identified pathogenic germline SDHA variants in 30 of 393 (7.6%) patients with PGL. Most of our index SDHA mutation carriers presented with an apparently sporadic HNPGL. Remarkably, most germline mutations were inherited from an unaffected parent. The clinical phenotype in our SDHA families is similar to that seen in previous studies (i.e., with few non-PGL tumors, such as GIST, RCC, and pituitary adenoma) (2, 14). This study highlights the low age-related penetrance: 10% at age 70 years in nonindex SDHA mutation carriers. However, some index mutation carriers presented at very young ages and/or with metastatic disease. These results may give cause to reconsider the current surveillance protocol for SDHA mutation carriers. The age at first examination and/or the interval between screenings could possibly be less stringent than for SDHB/C/D mutation carriers. SDHA mutation analysis On the basis of a detection yield of 7.6% in this nationwide cohort analysis, we recommend germline SDHA analysis for all individuals with PGL, preferably by using gene panels. To date, at least 15 genes have been associated with hereditary PGL, and it is likely that further rare and low-penetrant genes will be identified. Until recently, a stepwise mutation testing protocol was applied in those suspected of having familial PGL. Multiple algorithms were used, including age at presentation, location of tumor, multifocal or metastatic disease, presence of syndromic features, and family history (1). This type of testing protocol is expensive and time-consuming. Nowadays, gene panel testing using NGS is fast and cost-effective for germline genetic testing of patients with PGL (24). However, molecular analysis of SDHA in NGS panels could be challenging because of the presence of four pseudogenes that are highly homologous to both the coding regions of SDHA and the intronic regions of the gene. According to our data, additional SDHA Sanger sequencing should be considered in patients with HNPGL and SPGL without detectable mutations following NGS. The SDHA mutation detection yield in patients with apparently sporadic HNPGL in our study population (12%) was higher than in a previous study (6%), whereas the detection yield in patients with PHEO in our study population (2%) was similar to that of patients without HNPGL in that study (2%) (14). Although no specific data are available on SDHA mutation detection yields in SPGLs, our detection yield was high (18%); however, it was seen against a background of a small sample size. We identified eight SDHA variants not previously reported, including four pathogenic variants and four VUS. More than 60 unique SDHA nonsense and missense variants have been reported in the Leiden Open Variation Database, and they are evenly distributed across coding exons. No SDHA genotype-phenotype relationship has yet been established. Among Dutch SDHA index mutation carriers, the pathogenic variant c.91C>T (p.Arg31*) was most frequent (21 of 30), in contrast to the 5 of 29 in a previous study of non-Dutch index patients, a greater than fourfold difference in frequency (14). This variant has an allele frequency of 0.027% in the Exome Aggregation Consortium database, and 0.039% (6:16000) in our in-house whole-exome sequencing database (unpublished data). Together, these data suggest that SDHA p.Arg31* is a Dutch founder mutation, in the same vein as the very common SDHB and SDHD founder mutations reported in the Netherlands (25). Genetic counseling Exploring the genetic basis of hereditary PGL after appropriate counseling provides opportunities for early detection of PGL in patients and relatives. Early removal of tumors may prevent or minimize complications related to mass effects, catecholamine hypersecretion, and metastatic transformation. However, this is counterbalanced by the need for lifelong surveillance starting at an early age and the possible psychological burden of not knowing whether, when, and how (benign or metastatic) these tumors will develop. This is a particular challenge in the case of SDHA, for which penetrance appears to be much lower than for SDHD and somewhat lower than for SDHB (26). Prospective studies in SDHA mutation carriers—including genotype-phenotype relationships, genetic modifiers, and/or environmental factors—are required to determine the optimal age at which surveillance should be initiated and the best monitoring intervals to capture the different SDHA-related manifestations as they develop. Strengths and limitations of the study The current study has several strengths as well as some limitations. Its main strength is the size of the cohort investigated, representing the largest SDHA series to date (n = 84 carriers) This was possible because of the close collaboration of several Dutch university medical centers. A further strength is that all patients with PGL referred for germline SDHA analysis in the Netherlands within a defined period (2011 to 2016) were included in the study. Finally, the study was initiated at the Center for Endocrine Tumors Leiden and the Radboud Adrenal Center, both tertiary referral centers recognized as national and European centers of excellence for rare endocrine tumors, including PGL. The study also has limitations. First, the estimated mutation detection yield in this study was found in a retrospective diagnostic cohort and therefore might not be representative of the total patient population. However, a large proportion of the study population was systematically referred within a defined period, and these patients did not differ in age, sex, and diagnosis from other patients (unpublished data). Second, a possible explanation for the relatively low penetrance in our SDHA families could be inadequate surveillance and incomplete follow-up data. On the other hand, the over-representation of index patients (29 of 37) in a previous study leads to an overestimation of penetrance (14). Conclusion Germline SDHA mutations are relatively frequent (7.6%) in patients with genetically unexplained PGL, even in the absence of familial or clinical indications for inherited PGL. Mutation analysis of SDHA should therefore be included in the genetic testing of all patients with PGL, preferably by using gene panels. This study confirms the long-suspected low penetrance of disease in SDHA mutation carriers and suggests that recommended guidelines for genetic counseling of at-risk relatives and surveillance in mutation carriers might need to be revised. Abbreviations: 3-MT 3-methoxytyramine CI confidence interval GIST gastrointestinal stromal tumor HNPGL head and neck paragangliomas LOH loss of heterozygosity NGS next-generation sequencing PGL paraganglioma PHEO pheochromocytoma RCC clear cell renal cancer SPGL sympathetic paraganglioma VUS variants of uncertain significance. Acknowledgments Disclosure Summary: The authors have nothing to disclose. References 1. Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, Naruse M, Pacak K, Young WF, Jr; Endocrine Society. Pheochromocytoma and paraganglioma: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab . 2014; 99( 6): 1915– 1942. Google Scholar CrossRef Search ADS PubMed  2. 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SDHA mutations in adult and pediatric wild-type gastrointestinal stromal tumors. Modern Pathol . 2013; 26: 456– 463. Google Scholar CrossRef Search ADS   13. Dwight T, Mann K, Benn DE, Robinson BG, McKelvie P, Gill AJ, Winship I, Clifton-Bligh RJ. Familial SDHA mutation associated with pituitary adenoma and pheochromocytoma/paraganglioma. J Clin Endocrinol Metab . 2013; 98( 6): E1103– E1108. Google Scholar CrossRef Search ADS PubMed  14. Bausch B, Schiavi F, Ni Y, Welander J, Patocs A, Ngeow J, Wellner U, Malinoc A, Taschin E, Barbon G, Lanza V, Söderkvist P, Stenman A, Larsson C, Svahn F, Chen JL, Marquard J, Fraenkel M, Walter MA, Peczkowska M, Prejbisz A, Jarzab B, Hasse-Lazar K, Petersenn S, Moeller LC, Meyer A, Reisch N, Trupka A, Brase C, Galiano M, Preuss SF, Kwok P, Lendvai N, Berisha G, Makay Ö, Boedeker CC, Weryha G, Racz K, Januszewicz A, Walz MK, Gimm O, Opocher G, Eng C, Neumann HPH; European-American-Asian Pheochromocytoma-Paraganglioma Registry Study Group. 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Rijken JA, Niemeijer ND, Jonker MA, Eijkelenkamp K, Jansen JC, van Berkel A, HJL MT, Kunst HPM, Bisschop P, Kerstens MN, Dreijerink KMA, van Dooren MF, van der Horst-Schrivers ANA, Hes FJ, Leenmans CR, Corssmit EPM, Hensen EF. The penetrance of paraganglioma and pheochromocytoma in SDHB germline mutation carriers [published online before print May 14, 2017]. Clin Genet . 18. Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB, de Silva D, Zharkikh A, Thomas A. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet . 2006; 43( 4): 295– 305. Google Scholar CrossRef Search ADS PubMed  19. Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet . 2013; Chapter 7: Unit7. Google Scholar CrossRef Search ADS   20. Flanagan SE, Patch AM, Ellard S. Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genet Test Mol Biomarkers . 2010; 14( 4): 533– 537. Google Scholar CrossRef Search ADS PubMed  21. Toledo RA, Burnichon N, Cascon A, Benn DE, Bayley JP, Welander J, Tops CM, Firth H, Dwight T, Ercolino T, Mannelli M, Opocher G, Clifton-Bligh R, Gimm O, Maher ER, Robledo M, Gimenez-Roqueplo AP, Dahia PL; NGS in PPGL (NGSnPPGL) Study Group. Consensus statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas. Nat Rev Endocrinol . 2017; 13( 4): 233– 247. Google Scholar CrossRef Search ADS PubMed  22. Wagner AJ, Remillard SP, Zhang YX, Doyle LA, George S, Hornick JL. Loss of expression of SDHA predicts SDHA mutations in gastrointestinal stromal tumors. Modern Pathol . 2013; 26: 289– 294. Google Scholar CrossRef Search ADS   23. Niemeijer ND, Papathomas TG, Korpershoek E, de Krijger RR, Oudijk L, Morreau H, Bayley JP, Hes FJ, Jansen JC, Dinjens WN, Corssmit EP. Succinate dehydrogenase (SDH)-deficient pancreatic neuroendocrine tumor expands the SDH-related tumor spectrum. J Clin Endocrinol Metab . 2015; 100( 10): E1386– E1393. Google Scholar CrossRef Search ADS PubMed  24. Welander J, Andreasson A, Juhlin CC, Wiseman RW, Bäckdahl M, Höög A, Larsson C, Gimm O, Söderkvist P. Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma. J Clin Endocrinol Metab . 2014; 99( 7): E1352– E1360. Google Scholar CrossRef Search ADS PubMed  25. Hensen EF, van Duinen N, Jansen JC, Corssmit EP, Tops CM, Romijn JA, Vriends AH, van der Mey AG, Cornelisse CJ, Devilee P, Bayley JP. High prevalence of founder mutations of the succinate dehydrogenase genes in the Netherlands. Clin Genet . 2012; 81( 3): 284– 288. 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Endocrine Society
Copyright
Copyright © 2018 Endocrine Society
ISSN
0021-972X
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1945-7197
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10.1210/jc.2017-01762
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Abstract

Abstract Context Paraganglioma (PGL) has the highest degree of heritability among human neoplasms. Current clinical understanding of germline SDHA mutation carriers is limited. Objective To estimate the contribution of SDHA mutations in PGL and to assess clinical manifestations and age-related penetrance. Design Nationwide retrospective cohort study. Setting Tertiary referral centers in the Netherlands (multicenter). Patients Germline SDHA analysis was performed in 393 patients with genetically unexplained PGL. Subsequently, 30 index SDHA mutation carriers and 56 nonindex carriers were studied. Main Outcome Measures SDHA mutation detection yield, clinical manifestations, and SDHA-related disease penetrance. Results Pathogenic germline SDHA variants were identified in 30 of the 393 referred patients with PGL (7.6%), who had head and neck PGL (21 of 174 [12%]), pheochromocytoma (4 of 191 [2%]), or sympathetic PGL (5 of 28 [18%]). The median age at diagnosis was 43 years (range, 17 to 81 years) in index SDHA mutation carriers compared with 52 years (range, 7 to 90 years) in nonmutation carriers (P = 0.002). The estimated penetrance of any SDHA-related manifestation was 10% at age 70 years (95% confidence interval, 0% to 21%) in nonindex mutation carriers. Conclusion Germline SDHA mutations are relatively common (7.6%) in patients with genetically unexplained PGL. Most index patients presented with apparently sporadic PGL. In this SDHA series, the largest assembled so far, we found the lowest penetrance of all major PGL predisposition genes. This suggests that recommendations for genetic counseling of at-risk relatives and stringency of surveillance for SDHA mutation carriers might need to be reassessed. Paragangliomas (PGLs) are rare neuroendocrine tumors that carry the highest degree of heritability among human neoplasms (1, 2). PGLs are classified according to their anatomic location (intra-adrenal or extra-adrenal PGL) and whether they are of sympathetic or parasympathetic origin. Head and neck paragangliomas (HNPGLs) emerge from the parasympathetic nervous system and are usually benign, slow-growing tumors (3, 4). Common sites include the carotid body, the temporal bone, and the vagal body. Parasympathetic PGLs are most often nonsecreting, although about 30% are associated with elevated levels of the dopamine metabolite 3-methoxytyramine (3-MT) (5). Pheochromocytoma (PHEO) and sympathetic paraganglioma (SPGL) are catecholamine-secreting tumors (6). PHEOs are derived from the chromaffin cells of the adrenal medulla, and SPGLs are found in close relationship to the peripheral sympathetic nervous system from the level of the superior cervical ganglion down the trunk into the pelvis (7). Metastases are more often present in SPGL than are PHEO and HNPGL (3). About one third of patients with PGL have been reported to carry pathogenic germline variants in a growing list of susceptibility genes (8). The most described genes are NF1, RET, VHL, SDHD, SDHC, SDHB, SDHAF2, SDHA, TMEM127, and MAX. Germline variants in the succinate dehydrogenase (SDH) genes are the most common genetic cause of PGLs, occurring in up to 15% of all patients with PGL and half of all familiar cases (2, 9). In 2010, a direct association between germline SDHA variants and PGL was reported (10). SDH-associated syndromes are characterized by the development of PGLs, with an additional risk for developing other tumor types [e.g., clear cell renal cancer (RCC), gastrointestinal stromal tumors (GISTs), and, more rarely, neuroendocrine tumors and pituitary adenomas] (11–13). So far, information on prevalence, phenotype, penetrance, and pathogenicity of SDHA variants is limited to one large series (14) and a few small series (15, 16). In this study, we performed a nationwide evaluation of germline SDHA analyses undertaken in patients with PGL and characterized the clinical manifestations and disease penetrance in 30 index SDHA mutation carriers and their relatives. Patients and Methods Study population and design All patients with an established diagnosis of PGL who were referred for germline SDHA analysis in the Netherlands from February 2011 through July 2016 were included in this study. Referred patients with PGL were grouped into three clinical subgroups—HNPGL, PHEO, or SPGL—on the basis of clinical, biochemical, imaging, and/or histological characteristics. Data on sex, diagnosis, and age at diagnosis were retrieved from DNA request forms. In accordance with the Dutch national genetic testing strategy, all patients with PGL referred for SDHA analysis lacked pathogenic germline variants in SDHB, SDHC, SDHD, and SDHAF2. All patients with PHEO and SPGL furthermore lacked pathogenic germline variants in TMEM127, MAX, RET, and VHL and had no clinical symptoms suggesting neurofibromatosis type 1. Index patients with (likely) pathogenic SDHA variants or variants of uncertain significance (VUS) were evaluated and subsequently counseled by a clinical geneticist in their regional University Medical Center. Patients with pathogenic and likely pathogenic variants are annotated as SDHA mutation carriers in this manuscript. Clinical characteristics (e.g., sex, age at diagnosis, tumor location or locations, presence of metastases, biochemical phenotype, and additional non-PGL tumors) and pedigrees were collected. Genetic counseling and testing for the family-specific (likely) pathogenic SDHA variant were offered to relatives via cascade screening. All SDHA mutation carriers age ≥18 years were referred to departments of otorhinolaryngology and endocrinology for annual clinical surveillance aimed at detecting PGL. According to national guidelines (17), surveillance consisted of magnetic resonance imaging of the head and neck region once every 3 years and magnetic resonance imaging or computed tomography of the thorax, abdomen, and pelvis once every 2 years. Annual routine biochemical testing included the measurement of (nor)epinephrine, (nor)metanephrine, dopamine, and/or 3-MT in 24-hour urine samples and/or plasma, depending on the center. In cases with excessive catecholamine secretion (i.e., any value above the upper reference limit), radiological assessment of the thorax, abdomen, and pelvis was performed to identify potential sources of excessive catecholamine production. The current study was approved by the local medical ethical committee of Leiden University Medical Center (G16.063). DNA sequencing and data analysis Germline SDHA variant analysis was performed in the Department of Human Genetics at the Radboud University Medical Center and the Laboratory for Diagnostic Genome Analysis of the Department of Clinical Genetics at Leiden University Medical Center, the Netherlands. Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures. Germline SDHA analysis was performed with Sanger sequencing or next-generation (gene panel) sequencing (NGS) depending on the testing period. For the detailed NGS procedure, see the Supplemental Method. Coding variants were analyzed for their effect on function by using the Alamut software package, version (Interactive Biosoftware, Rouen, France), which incorporates Align GVGD (18), polymorphism phenotyping (PolyPhen2) (19), and sorting intolerant from tolerant (SIFT) (20). Variants were annotated to the Genbank reference sequence NM_004168.2. The Leiden Open Variation Database (http://www.lovd.nl/SDHA) was consulted to find variants previously described and classified. Variant interpretation was done in line with the recent consensus statement on NGS-based diagnostic testing of hereditary PHEO and PGLs (21). Variant nomenclature is in accordance with Human Genome Variation Society guidelines, version 2.0 (http://www.hgvs.org). To obtain further support for the pathogenicity of certain SDHA variants, SDHA immunohistochemistry and loss-of-heterozygosity (LOH) analysis were performed on formalin-fixed, paraffin-embedded samples, as described elsewhere (22). Statistical analysis Descriptive statistics were used to characterize the study population, to determine the age of PGL onset, and to examine the difference between patients with germline SDHA mutation and those without germline SDHA mutation. Continuous variables were analyzed by using an independent sample t test. Dichotomous variables were compared by using the χ2 test. Age-related penetrance of PGL was estimated by using the Kaplan-Meier method. Because most of the nonindex mutation carriers were recently identified, we used the age at 1 year after DNA analysis (or age at death) for the penetrance estimation. By completion of this manuscript, >80% of the nonindex mutation carriers had participated in surveillance at least once. Statistical significance was set at P < 0.05, and the analyses were conducted by using SPSS software, version 23.0 (IBM, Armonk, NY). Results SDHA case detection in the study population Pathogenic germline SDHA variants were identified in 30 of 393 (7.6%) patients with PGL who were referred for SDHA genetic testing. The clinical characteristics of the study population (SDHA vs non-SDHA) are listed in Table 1. Within the clinical PGL subgroups, pathogenic SDHA variants were identified in 21 of 174 patients with HNPGL (12%), 4 of 191 patients with PHEO (2%), and 5 of 28 patients with SPGL (18%). The median age at diagnosis of PGL was 43 years (range, 17 to 81 years) in SDHA mutation carriers and 52 years (range, 7 to 90 years) in those without a detectable mutation (P = 0.002). Half of the SDHA mutation carriers were males compared with 32% of patients without an SDHA mutation (P = 0.049). Table 1. Clinical Characteristics of Patients With Germline SDHA Mutation and Patients Without Germline SDHA Mutation Characteristic  SDHA Mutation (n = 30)  No SDHA Mutation (n = 363)  P Value  Mutation Yield (%)  All patients with PGL        7.6   Age at diagnosis (y)  43 (17–81)  52 (7–90)  0.002     Males, n (%)  15 (50)  117 (32)  0.049    Patients with HNPGL  21  153    12   Age at diagnosis (y)  43 (18–81)  54 (19–90)  0.008     Males, n (%)  10 (48)  33 (22)  0.010    Patients with PHEO  4  186    2   Age at diagnosis (y)  35 (17–70)  51 (7–81)  0.14     Males, n (%)  0  71 (38)  0.118    Patients with SPGL  5  24    18   Age at diagnosis (y)  36 (22–60)  53 (23–73)  0.118     Males, n (%)  5 (100)  13 (54)  0.055    Characteristic  SDHA Mutation (n = 30)  No SDHA Mutation (n = 363)  P Value  Mutation Yield (%)  All patients with PGL        7.6   Age at diagnosis (y)  43 (17–81)  52 (7–90)  0.002     Males, n (%)  15 (50)  117 (32)  0.049    Patients with HNPGL  21  153    12   Age at diagnosis (y)  43 (18–81)  54 (19–90)  0.008     Males, n (%)  10 (48)  33 (22)  0.010    Patients with PHEO  4  186    2   Age at diagnosis (y)  35 (17–70)  51 (7–81)  0.14     Males, n (%)  0  71 (38)  0.118    Patients with SPGL  5  24    18   Age at diagnosis (y)  36 (22–60)  53 (23–73)  0.118     Males, n (%)  5 (100)  13 (54)  0.055    Data are presented as median (range) or number and percentage. P values are derived from χ2 or independent sample t test. View Large SDHA variants Seven different (likely) pathogenic germline SDHA variants were identified in 30 patients with PGL (Table 2). Three variants had been reported previously: The common nonsense mutation c.91C>T, (p.Arg31*) (10) was observed in 23 patients, the c.1753C>T, (p.Arg585Trp) (16) missense mutation was observed in two patients, and the nonsense c.1534C>T, p.(Arg512*) (22) was observed in one patient. Moreover, four not previously reported and three previously reported SDHA VUS were identified (Supplemental Table 1). All VUS were identified in patients with apparently sporadic HNPGL, diagnosed between the ages of 28 and 52 years. Additional immunohistochemical staining and LOH analysis were performed in two of the seven VUS-related HNPGLs but showed no loss of SDHA or SDHB staining or LOH. The other five HNPGLs were not available for further analysis. Table 2. Clinical and Molecular Characteristics of 30 Index SDHA Mutation Carriers and Their Relatives Patient No.  Family   Sex  Tumors Observed (Age at Detection, y)  Biochemistry Results  Family History  Germline SDHA Variant  Tested Relatives (Carriersa )  Reference  1  A  Female  GCT-ri (43)  Normal  Negative  c.91C>T, p.(Arg31*)  5 (3)  10  2  B  Male  GVT-le (38)  Normal  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  3  C  Female  GVT-ri (81)  Normal  Negative  c.91C>T, p.(Arg31*)  3 (1)  10  4  D  Female  GCT-le 2x (35)  Normal  Negative  c.91C>T, p.(Arg31*)  1 (1)  10  5  E  Female  GCT-le, GCT-ri, GJT-le (48)  Normal  Negative  c.91C>T, p.(Arg31*)  6 (4)  10  6  F  Male  GCT-le (30)  NA  Negative  c.91C>T, p.(Arg31*)  10 (6)  10  7  G  Male  GCT-ri (56), Prolactinoma (58)  Normal  Negative  c.91C>T, p.(Arg31*)  4 (2)  10  8  H  Male  GJT-le (43)  Normal  Negative  c.1432_1432+1delGGb  3 (2)  Not previously reported  9  I  Female  GCT-ri, GCT-le (26, 49, 50), Prolactinoma (45), Multiple Meningioma (45,62)  Normal  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  10  J  Male  GCT-ri (23)  Normal  GISTc  c.91C>T, p.(Arg31*)  8 (6)  10  11  K  Male  GJTT-le (38)  3-MT  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  12  L  Female  GCT-le (40)  Normal  RCCc  c.985C>T, p.(Arg329*)  1 (1)  Not previously reported  13  M  Female  GCT-le (61)  NA  Negative  c.91C>T, p.(Arg31*)  8 (5)  10  14  M  Female  GJTT-ri (58)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  15  M  Female  GVT-le (53)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  16  M  Female  GVT-ri (42)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  17  M  Female  GVT-ri (53)  Normal  RCCd  c.91C>T, p.(Arg31*)  0  10  18  M  Male  GJT-ri (18)  NA  Negative  c.91C>T, p.(Arg31*)  0  10  19  M  Male  GCT-ri (48), uveal melanoma (48)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  20  M  Male  GVT-ri (60)  3-MT  Negative  c.1795-3C>Gb  0  Not previously reported  21  M  Male  GVT-ri, GVT-le, GCT-ri (49)  Normal  Negative  c.667delG, p.(Asp223fs)  0  Not previously reported  22  N  Female  PHEO-ri (17)  NM  Negative  c.1753C>T, p.(Arg585Trp)  3 (1)  16  23  N  Female  PHEO-ri (20), Wilms tumor (4)  NM  Negative  c.1753C>T, (p.Arg585Trp)  0  16  24  N  Female  PHEO-le (50), metastasis (60)  NM  Negative  c.91C>T, p.(Arg31*)  0  10  25  N  Female  PHEO-ri (70), RCC (70)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  26  O  Male  Para-aortal SPGL (60)  NA  RCCe  c.91C>T, p.(Arg31*)  10 (8)  10  27  P  Male  Testis SPGL (23), metastases (26)  NM/3-MT  HNPGLc  c.1534C>T, p.(Arg512*)  3 (4)  22  28  Q  Male  Retroperitoneal para-aortal SPGL (50)  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (3)  10  29  R  Male  Malignant retroperitoneal SPGL (36)f  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  30  S  Male  Para-aortal SPGL (22)  NM  Negative  c.91C>T, p.(Arg31*)  3 (3)  10  Patient No.  Family   Sex  Tumors Observed (Age at Detection, y)  Biochemistry Results  Family History  Germline SDHA Variant  Tested Relatives (Carriersa )  Reference  1  A  Female  GCT-ri (43)  Normal  Negative  c.91C>T, p.(Arg31*)  5 (3)  10  2  B  Male  GVT-le (38)  Normal  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  3  C  Female  GVT-ri (81)  Normal  Negative  c.91C>T, p.(Arg31*)  3 (1)  10  4  D  Female  GCT-le 2x (35)  Normal  Negative  c.91C>T, p.(Arg31*)  1 (1)  10  5  E  Female  GCT-le, GCT-ri, GJT-le (48)  Normal  Negative  c.91C>T, p.(Arg31*)  6 (4)  10  6  F  Male  GCT-le (30)  NA  Negative  c.91C>T, p.(Arg31*)  10 (6)  10  7  G  Male  GCT-ri (56), Prolactinoma (58)  Normal  Negative  c.91C>T, p.(Arg31*)  4 (2)  10  8  H  Male  GJT-le (43)  Normal  Negative  c.1432_1432+1delGGb  3 (2)  Not previously reported  9  I  Female  GCT-ri, GCT-le (26, 49, 50), Prolactinoma (45), Multiple Meningioma (45,62)  Normal  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  10  J  Male  GCT-ri (23)  Normal  GISTc  c.91C>T, p.(Arg31*)  8 (6)  10  11  K  Male  GJTT-le (38)  3-MT  Negative  c.91C>T, p.(Arg31*)  2 (1)  10  12  L  Female  GCT-le (40)  Normal  RCCc  c.985C>T, p.(Arg329*)  1 (1)  Not previously reported  13  M  Female  GCT-le (61)  NA  Negative  c.91C>T, p.(Arg31*)  8 (5)  10  14  M  Female  GJTT-ri (58)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  15  M  Female  GVT-le (53)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  16  M  Female  GVT-ri (42)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  17  M  Female  GVT-ri (53)  Normal  RCCd  c.91C>T, p.(Arg31*)  0  10  18  M  Male  GJT-ri (18)  NA  Negative  c.91C>T, p.(Arg31*)  0  10  19  M  Male  GCT-ri (48), uveal melanoma (48)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  20  M  Male  GVT-ri (60)  3-MT  Negative  c.1795-3C>Gb  0  Not previously reported  21  M  Male  GVT-ri, GVT-le, GCT-ri (49)  Normal  Negative  c.667delG, p.(Asp223fs)  0  Not previously reported  22  N  Female  PHEO-ri (17)  NM  Negative  c.1753C>T, p.(Arg585Trp)  3 (1)  16  23  N  Female  PHEO-ri (20), Wilms tumor (4)  NM  Negative  c.1753C>T, (p.Arg585Trp)  0  16  24  N  Female  PHEO-le (50), metastasis (60)  NM  Negative  c.91C>T, p.(Arg31*)  0  10  25  N  Female  PHEO-ri (70), RCC (70)  Normal  Negative  c.91C>T, p.(Arg31*)  0  10  26  O  Male  Para-aortal SPGL (60)  NA  RCCe  c.91C>T, p.(Arg31*)  10 (8)  10  27  P  Male  Testis SPGL (23), metastases (26)  NM/3-MT  HNPGLc  c.1534C>T, p.(Arg512*)  3 (4)  22  28  Q  Male  Retroperitoneal para-aortal SPGL (50)  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (3)  10  29  R  Male  Malignant retroperitoneal SPGL (36)f  NM/3-MT  Negative  c.91C>T, p.(Arg31*)  8 (2)  10  30  S  Male  Para-aortal SPGL (22)  NM  Negative  c.91C>T, p.(Arg31*)  3 (3)  10  See Supplemental Fig. 2 for the pedigrees of families A through S. Abbreviations: GCT, glomus carotid body tumor; GJT, glomus jugularis tumor; GJTT, glomus jugulotympanicum tumor; GTT, glomus tympanicum tumor; GVT, glomus vagal tumor; ri, right; le, left; NA, not available; PTC, papillary thyroid carcinoma. a Including obligate mutation carriers. Reference sequence SDHA NC000005.9, NM004168.3. b Likely pathogenic SDHA variant. c SDHA mutation carrier. d SDHA mutation status unknown. e No SDHA mutation carrier. f Dead of disease. View Large Clinical manifestations in index SDHA mutation carriers The clinical and molecular characteristics of the 30 index patients with germline pathogenic SDHA variants are listed in Table 2. Germline SDHA mutations were identified in 21 index patients with HNPGL, 4 with PHEO, and 5 with SPGL. Four patients were diagnosed with multiple HNPGLs. The HNPGL anatomic locations were distributed as follows: 15 carotid body, 8 vagal, 3 jugular, and 2 jugular tympanic. Two patients with HNPGL had elevated 3-MT levels. Three patients with PHEO had elevated normetanephrine levels. One patient had developed a metastatic PHEO, but no bilateral PHEOs were detected. Four SPGLs had a retroperitoneal para-aortal location and one SPGL was found in the testis. Four patients with SPGL had elevated normetanephrine levels, three in combination with elevated 3-MT. Two patients developed metastatic SPGL, and one of these patients (no. 27) died at age 27 years. Furthermore, three index mutation carriers (no. 7, no. 9, and no. 25) were diagnosed with one other possibly SDHA-related feature, including pituitary adenoma (at ages 58 and 45 years, respectively) and RCC (age 70 years), respectively. One pituitary adenoma was immunonegative for both SDHA and SDHB and contained an additional somatic pathogenic SDHA variant (p.Asp38Val), likely resulting in biallelic inactivation of SDHA (Supplemental Fig. 1) (23). The other pituitary adenoma was not resected and therefore not analyzed. Conversely, the RCC tissue showed no loss of SDHA immunohistochemical staining, suggesting that it was not SDHA-related. Three additional tumor types were reported in index SDHA mutation carriers: multiple meningioma (patient no. 9), uveal melanoma (no. 19, BAP1-mutation negative) and Wilms tumor (no. 23). However, it is not clear whether these tumors were related to the SDHA mutation. Immunohistochemical staining showed no loss of SDHA staining in both meningiomas. The uveal melanoma lesion and Wilms tumor were not available for analysis. Five SDHA mutation carriers had a positive family history for SDHA-related tumors, including HNPGL (patient no. 27), GIST (no. 10), and RCC (no. 12, no. 17, and no. 26). Clinical manifestations in SDHA families In total, 94 available relatives were tested via cascade screening for their familial pathogenic SDHA variant, revealing 51 nonindex carriers and 5 obligate carriers. Pedigrees of the 19 SDHA families with at least one nonindex mutation carrier are shown in Supplemental Fig. 2. Remarkably, we could confirm in all families, except one (index no. 3, diagnosed at age 81 years), that the mutation was inherited from an unaffected parent. The median age at DNA analysis in the nonindex SDHA mutation carriers was 58 years (range, 7 to 94 years). In total, 3 of 56 (5%) nonindex SDHA mutation carriers were diagnosed as having one (possible) SDHA-related tumor: HNPGL (n = 1), GIST (n = 1), and RCC (n = 1). Family history did not reveal any not-tested relatives with (possible) SDHA-related tumors. The estimated penetrance of any SDHA-related tumor is shown in Fig. 1. The age-related penetrance values for all 86 SDHA mutation carriers were 7% at age 25 years [95% confidence interval (CI), 2% to 12%], 26% at age 50 years (95% CI, 16% to 36%), and 50% at age 70 years (95% CI, 34% to 66%). The age-related penetrance values for the 56 nonindex SDHA mutation carriers were 0% at age 25 years, 2% at age 50 years (95% CI, 0% to 6%), and 10% at age 70 years (95% CI, 0% to 21%). By completion of this manuscript, 51 nonindex carriers were lacking any identified SDHA-related feature, indicating that they could be considered to be healthy mutation carriers. Figure 1. View largeDownload slide Age-related penetrance of any SDHA-related manifestations. Figure 1. View largeDownload slide Age-related penetrance of any SDHA-related manifestations. Discussion This nationwide retrospective SDHA survey investigated SDHA mutation detection yield and clinical phenotype in patients with genetically unexplained PGL. We identified pathogenic germline SDHA variants in 30 of 393 (7.6%) patients with PGL. Most of our index SDHA mutation carriers presented with an apparently sporadic HNPGL. Remarkably, most germline mutations were inherited from an unaffected parent. The clinical phenotype in our SDHA families is similar to that seen in previous studies (i.e., with few non-PGL tumors, such as GIST, RCC, and pituitary adenoma) (2, 14). This study highlights the low age-related penetrance: 10% at age 70 years in nonindex SDHA mutation carriers. However, some index mutation carriers presented at very young ages and/or with metastatic disease. These results may give cause to reconsider the current surveillance protocol for SDHA mutation carriers. The age at first examination and/or the interval between screenings could possibly be less stringent than for SDHB/C/D mutation carriers. SDHA mutation analysis On the basis of a detection yield of 7.6% in this nationwide cohort analysis, we recommend germline SDHA analysis for all individuals with PGL, preferably by using gene panels. To date, at least 15 genes have been associated with hereditary PGL, and it is likely that further rare and low-penetrant genes will be identified. Until recently, a stepwise mutation testing protocol was applied in those suspected of having familial PGL. Multiple algorithms were used, including age at presentation, location of tumor, multifocal or metastatic disease, presence of syndromic features, and family history (1). This type of testing protocol is expensive and time-consuming. Nowadays, gene panel testing using NGS is fast and cost-effective for germline genetic testing of patients with PGL (24). However, molecular analysis of SDHA in NGS panels could be challenging because of the presence of four pseudogenes that are highly homologous to both the coding regions of SDHA and the intronic regions of the gene. According to our data, additional SDHA Sanger sequencing should be considered in patients with HNPGL and SPGL without detectable mutations following NGS. The SDHA mutation detection yield in patients with apparently sporadic HNPGL in our study population (12%) was higher than in a previous study (6%), whereas the detection yield in patients with PHEO in our study population (2%) was similar to that of patients without HNPGL in that study (2%) (14). Although no specific data are available on SDHA mutation detection yields in SPGLs, our detection yield was high (18%); however, it was seen against a background of a small sample size. We identified eight SDHA variants not previously reported, including four pathogenic variants and four VUS. More than 60 unique SDHA nonsense and missense variants have been reported in the Leiden Open Variation Database, and they are evenly distributed across coding exons. No SDHA genotype-phenotype relationship has yet been established. Among Dutch SDHA index mutation carriers, the pathogenic variant c.91C>T (p.Arg31*) was most frequent (21 of 30), in contrast to the 5 of 29 in a previous study of non-Dutch index patients, a greater than fourfold difference in frequency (14). This variant has an allele frequency of 0.027% in the Exome Aggregation Consortium database, and 0.039% (6:16000) in our in-house whole-exome sequencing database (unpublished data). Together, these data suggest that SDHA p.Arg31* is a Dutch founder mutation, in the same vein as the very common SDHB and SDHD founder mutations reported in the Netherlands (25). Genetic counseling Exploring the genetic basis of hereditary PGL after appropriate counseling provides opportunities for early detection of PGL in patients and relatives. Early removal of tumors may prevent or minimize complications related to mass effects, catecholamine hypersecretion, and metastatic transformation. However, this is counterbalanced by the need for lifelong surveillance starting at an early age and the possible psychological burden of not knowing whether, when, and how (benign or metastatic) these tumors will develop. This is a particular challenge in the case of SDHA, for which penetrance appears to be much lower than for SDHD and somewhat lower than for SDHB (26). Prospective studies in SDHA mutation carriers—including genotype-phenotype relationships, genetic modifiers, and/or environmental factors—are required to determine the optimal age at which surveillance should be initiated and the best monitoring intervals to capture the different SDHA-related manifestations as they develop. Strengths and limitations of the study The current study has several strengths as well as some limitations. Its main strength is the size of the cohort investigated, representing the largest SDHA series to date (n = 84 carriers) This was possible because of the close collaboration of several Dutch university medical centers. A further strength is that all patients with PGL referred for germline SDHA analysis in the Netherlands within a defined period (2011 to 2016) were included in the study. Finally, the study was initiated at the Center for Endocrine Tumors Leiden and the Radboud Adrenal Center, both tertiary referral centers recognized as national and European centers of excellence for rare endocrine tumors, including PGL. The study also has limitations. First, the estimated mutation detection yield in this study was found in a retrospective diagnostic cohort and therefore might not be representative of the total patient population. However, a large proportion of the study population was systematically referred within a defined period, and these patients did not differ in age, sex, and diagnosis from other patients (unpublished data). Second, a possible explanation for the relatively low penetrance in our SDHA families could be inadequate surveillance and incomplete follow-up data. On the other hand, the over-representation of index patients (29 of 37) in a previous study leads to an overestimation of penetrance (14). Conclusion Germline SDHA mutations are relatively frequent (7.6%) in patients with genetically unexplained PGL, even in the absence of familial or clinical indications for inherited PGL. Mutation analysis of SDHA should therefore be included in the genetic testing of all patients with PGL, preferably by using gene panels. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 1, 2018

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