Mutation Frequency in Main Susceptibility Genes Among Patients With Head and Neck Paragangliomas

Head and neck paragangliomas (HNPGLs) are rare neuroendocrine tumors that have a high degree of heritability and are predominantly associated with mutations in ten genes, such as SDHx, SDHAF2, VHL, RET, NF1, TMEM127, MAX, FH, MEN2, and SLC25A11. Elucidating the mutation prevalence is crucial for the development of genetic testing. In this study, we identified pathogenic/likely pathogenic variants in the main susceptibility genes in 102 Russian patients with HNPGLs (82 carotid and 23 vagal paragangliomas) using whole exome sequencing. Pathogenic/likely pathogenic variants were detected in 43% (44/102) of patients. We identified the following variant distribution of the tested genes: SDHA (1%), SDHB (10%), SDHC (5%), SDHD (24.5%), and RET (5%). SDHD variants were observed in the majority of the patients with bilateral/multiple paragangliomas. Thus, among Russian patients with HNPGLs the most frequently mutated gene was SDHD followed by SDHB, SDHC, RET, and SDHA.

In this study, we aimed to assess the frequency of variants in the main susceptibility genes for HNPGLs, such as SDHx, SDHAF2, VHL, RET, NF1, TMEM127, MAX, FH, MEN2, and SLC25A11, among a representative set of Russian patients with CPGLs and VPGLs.

Sanger Sequencing
To validate the whole exome sequencing data, Sanger sequencing was performed in Evrogen. Primer sequences are available on request.

RESULTS
A representative set of HNPGL samples were collected from 102 Russian patients diagnosed with CPGLs (n = 82) and VPGLs (n = 23), including 76 patients with single CPGL, 20 patients with single VPGL, and 6 patients with bilateral/multiple PGLs (three of them had both carotid and vagal paragangliomas) ( Table 1). These tumor samples were analyzed for the presence of pathogenic/likely pathogenic variants in the main susceptibility genes for HNPGLs: SDHx, SDHAF2, VHL, RET, NF1, TMEM127, MAX, FH, MEN2, and SLC25A11. Variants were classified as pathogenic or likely pathogenic according to the annotations in the ClinVar database or by using the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) (Richards et al., 2015).
Next, we analyzed the age at diagnosis and sex ratio for SDHB, SDHC, SDHD, and RET variants within the cohort of patients with HNPGLs (Table 3). Variants in the SDHC and SDHD genes were diagnosed with approximately equal frequency in males and females taking into account 1:3 male to female ratio among the studied cohort. SDHB variants were found third-fold more frequent in females when RET variants were detected about two-fold more frequent in males. Variants in SDHB, SDHC, and SDHD were observed in all age groups and were more often detected in patients aged between 19-40 and 41-60 years. RET variants were identified in two groups of patients aged 19-40 and 41-60 years. Notably, frequency of variants in all the genes was the lowest in patients aged 61-80 years.

DISCUSSION
In HNPGLs, the occurrence and frequency of mutations in the SDHx genes are extensively studied and are of importance for the diagnosis and management of the disease. The prevalence of mutations in other susceptibility genes has been poorly investigated. Notably, most studies have been focused on germline variants. In this work, we cannot establish germline and somatic mutation status for identified variants, since we used the archival collection of tumors for which blood or other normal tissues were not available. We obtained data on the overall frequency of pathogenic/likely pathogenic variants in the main susceptibility genes that allows to better understand molecular basis of the tumor development in Russian patients. A more similar study was performed for 24 Spanish patients with HNPGLs, who were subjected to genetic testing for germline and somatic mutations in the SDHx genes (Curras-Freixes et al., 2015). In contrast to our data, the majority of mutations were detected in SDHB (33%, 8/24) followed by SDHD (21%, 5/24) and SDHC (4%, 1/24). Two patients with SDHB mutations and one patient with SDHD mutation were characterized by metastatic tumors but no SDHA variants were detected.
In Russian patients, we revealed the likely pathogenic variant in RET in 5% of cases (5/102), including four patients with CPGLs and one patient with VPGL. Activation mutations in the proto-oncogene RET lead to the development of an autosomal dominant syndrome called multiple endocrine neoplasia type 2 (MEN2). Up to 50% of patients with MEN2 develop PCCs (Pedulla et al., 2014). HNPGLs have been rarely described in patients with MEN2 syndrome (Boedeker et al., 2009).  However, several studies have reported RET mutations in HNPGLs without any association with MEN2 syndrome. A RET mutation was previously detected in a patient with multiple paragangliomas (Ding et al., 2019). Moreover, the likely pathogenic germline variant NM_020975: c.A2372T, p.Y791F (chr10: 43613908, rs77724903) in RET was identified in two out of four members of a family with multiple and malignant paragangliomas (Choi Jdo et al., 2014). All four members carried pathogenic SDHD mutations. In addition, in this family, the RET mutation was observed in the male adult with bilateral carotid body and jugulotympanic paragangliomas and his son with unilateral CPGLs. Here, we also detected this RET variant in all RET-mutated tumors. Moreover, this variant co-occurred with the pathogenic start-loss variant in SDHA and the pathogenic splice site variant in SDHB. Collectively, data from our study and previous studies suggest that this RET variant can occur both alone and together with SDHD, SDHA, and SDHB pathogenic variants. However, according to the ClinVar database, this RET mutation was annotated as pathogenic variant associated with the MEN2 syndrome and its pathogenicity has not been proved by any functional studies. Thus, the role of the RET variant in the development of HNPGLs is controversial taking into account that we detected this variant in patients, who were not diagnosed with the MEN2 syndrome.
In the study, we also analyzed male to female ratio and age groups for identified variants in the SDHx and RET genes. Currently, clinic-genetic correlations in association with mutations in these genes in HNPGLs have been poorly investigated. Moreover, reported data are related to germline mutations and can be compared only conditionally with our results. Thus, Mario Hermsen with colleagues studied 23 males and 51 females and with HNPGLs and found 8:6 and 5:9 ratios for SDHB and SDHD mutations, respectively (Hermsen et al., 2010). Taking into account the initial male to female ratio approximately 1:2, we can see that SDHD mutations were detected about equally in males and females that is concordance with our data. SDHB mutations were more frequently revealed in males compared with females, while we obtained opposite results. In addition, it was observed that SDHB and SDHD mutations were diagnosed predominantly in patients aged <50 years. We showed that patients aged <61 years carried variants in these genes more frequently. Also, age-related penetrance for SDHB and SDHD mutations was shown to be increased by 50 years in HNPGLs (Neumann et al., 2004). In our study, we also observed higher number of patients aged 41-60 years with SDHB and SDHD variants.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found here: https://www.ncbi.nlm. nih.gov/bioproject/PRJNA639937.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Vishnevsky Institute of Surgery. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
AS and AVK: conceptualization and writing-review and editing. GK, VP, AD, AS, and NM: formal analysis. MF, EP, and ZG: investigation. GR, DK, and AG: resources. AP, GA, and ADK: writing-review and editing. All authors contributed to the article and approved the submitted version. Center for Precision Genome Editing and Genetic Technologies for Biomedicine, EIMB RAS for providing the computing power and techniques for the data analysis. This work was performed using the equipment of EIMB RAS ''Genome'' center (http:// www.eimb.ru/rus/ckp/ccu_genome_c.php).