Biallelic variant in DACH1, encoding Dachshund Homolog 1, defines a novel candidate locus for recessive postaxial polydactyly type A

Polydactyly or hexadactyly is characterized by an extra digit/toe with or without a bone. Currently, variants in ten genes have been implicated in the non-syndromic form of polydactyly.DNA from a single affected individual having bilateral postaxial polydactyly was subjected to whole exome sequencing (WES), followed by Sanger sequencing. Homology modeling was performed for the identified variant and advance microscopy imaging approaches were used to reveal the localization of the DACH1 protein at the base of primary cilia.A disease-causing biallelic missense variant (c.563G > A; p.Cys188Tyr; NM_080760.5) was identified in the DACH1 gene segregating perfectly within the family. Structural analysis using homology modeling of the DACH1 protein revealed secondary structure change that might result in loss of function or influence downstream interactions. Moreover, siRNA-mediated depletion of DACH1 showed a key role of DACH1 in ciliogenesis and cilia function.This study provides the first evidence of involvement of the DACH1 gene in digits development in humans and its role in primary cilia. This signifies the importance and yet unexplored role of DACH1.     

Genetic analysis of <Emphasis Type="Italic">COL11A2</Emphasis> in Korean patients with autosomal dominant non-syndromic hearing loss

The collagen type XI alpha 2 gene (COL11A2) is associated with autosomal dominant non-syndromic hearing loss (ADNSHL), and all mutations of this gene in ADNSHL are missense mutations. To evaluate its potential as a major causative gene of ADNSHL in the Korean population, we performed genetic analysis of COL11A2 in 75 unrelated Korean patients with ADNSHL. Consequently, 5 non-synonymous variants, 7 synonymous variants, and 6 intronic variants were identified in COL11A2. Among them, a novel variant, p.G829R (c.2485G>C) was found in a patient as a heterozygote. However, pedigree analysis showed this variation was not co-segregated with hearing loss. Previously reported variants p.G230W (c.688G>T) and p.P1422L (c.4265C>T) were discovered in Korean patients. However, these variants were also detected in normal individuals. These results suggest that COL11A2 is not a major causative gene of ADNSHL in the Korean population.     

Exome Sequencing Identifies a Novel Gene,WNK1, for Susceptibility to Pelvic Organ Prolapse (POP)

Pelvic organ prolapse (POP) is a common gynecological disorder; however, the genetic components remain largely unidentified. Exome sequencing has been widely used to identify pathogenic gene mutations of several diseases because of its high chromosomal coverage and accuracy. In this study, we performed whole exome sequencing (WES), for the first time, on 8 peripheral blood DNA samples from representative POP cases. After filtering the sequencing data from the dbSNP database (build 138) and the 1000 Genomes Project, 2 missense variants in WNK1, c.2668G > A (p.G890R) and c.6761C> T (p.P2254L), were identified and further validated via Sanger sequencing. In validation stage, the c.2668G > A (p.G890R) variant and 8 additional variants were detected in 11 out of 161 POP patients. All these variants were absent in 231 healthy controls. Functional experiments showed that fibroblasts from the utero-sacral ligaments of POP with WNK1 mutations exhibited loose and irregular alignment compared with fibroblasts from healthy controls. In sum, our study identified a novel gene, WNK1, for POP susceptibility, expanded the causal mutation spectrums of POP, and provided evidence for the genetic diagnosis and medical management of POP in the future.     

A Mutation in VPS35, Encoding a Subunit of the Retromer Complex, Causes Late-Onset Parkinson Disease

To identify rare causal variants in late-onset Parkinson disease (PD), we investigated an Austrian family with 16 affected individuals by exome sequencing. We found a missense mutation, c.1858G>A (p.Asp620Asn), in the VPS35 gene in all seven affected family members who are alive. By screening additional PD cases, we saw the same variant cosegregating with the disease in an autosomal-dominant mode with high but incomplete penetrance in two further families with five and ten affected members, respectively. The mean age of onset in the affected individuals was 53 years. Genotyping showed that the shared haplotype extends across 65 kilobases around VPS35. Screening the entire VPS35 coding sequence in an additional 860 cases and 1014 controls revealed six further nonsynonymous missense variants. Three were only present in cases, two were only present in controls, and one was present in cases and controls. The familial mutation p.Asp620Asn and a further variant, c.1570C>T (p.Arg524Trp), detected in a sporadic PD case were predicted to be damaging by sequence-based and molecular-dynamics analyses. VPS35 is a component of the retromer complex and mediates retrograde transport between endosomes and the trans-Golgi network, and it has recently been found to be involved in Alzheimer disease.     

A de novo germline mutation of APC for inheritable colon cancer in a Chinese family using multigene next generation sequencing

Inheritable colorectal cancers (CRC) accounted for about 20% of the CRC cases, such as hereditary nonpolyposis colorectal cancer (HNPCC), Gardner syndrome and familial adenomatous polyposis (FAP). A four-generation Han Chinese family was found affected with polyposis in colons. Inferred from the pedigree structure, the disease in this family showed an autosomal dominant inheritance model. To locate the causal mutations in this family, genomic DNAs were extracted and the next generation sequencing for 5 genes relating to colon cancer performed by Ion Torrent Personal Genome Machine with a 314 chip. The reads were aligned with human reference genome hg19 to call variants in the 5 genes. After analysis, 14 variants were detected in the sequenced sample and 13 been collected in dbSNP database and assigned with a rs identification number. In these variants, 9 were synonymous, 4 missense and 1 non-sense. In them, 2 rare variants (c.694C>T in APC and c.1690A>G in MSH2) might be the putative causal mutations for familial adenomatous polyposis (FAP) since the rarity of the mutated allele in normal controls. c.694C>T was detected in only affected members and generated a premature stop codon in APC. It should be a de novo germline mutation making APC containing this stop codon as targets for nonsense-mediated mRNA decay (NMD). c.1690A>G in MSH2 was not only detected in affected members, but also in normal ones in the family. Functional prediction revealed that the amino acid affected by this variant had no effect on the function of MSH2. Here, we report a de novo germline mutation of APC as the causal variant in a Chinese family with inheritable colon cancer by the next generation sequencing.     

Functional Analysis Helps to Define KCNC3 Mutational Spectrum in Dutch Ataxia Cases

Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominantly inherited neurodegenerative disorder of the cerebellum caused by mutations in the voltage gated potassium channel KCNC3. To identify novel pathogenic SCA13 mutations in KCNC3 and to gain insights into the disease prevalence in the Netherlands, we sequenced the entire coding region of KCNC3 in 848 Dutch cerebellar ataxia patients with familial or sporadic origin. We evaluated the pathogenicity of the identified variants by co-segregation analysis and in silico prediction followed by biochemical and electrophysiological studies. We identified 19 variants in KCNC3 including 2 non-coding, 11 missense and 6 synonymous variants. Two missense variants did not co-segregate with the disease and were excluded as potentially disease-causing mutations. We also identified the previously reported p.R420H and p.R423H mutations in our cohort. Of the remaining 7 missense variants, functional analysis revealed that 2 missense variants shifted Kv3.3 channel activation to more negative voltages. These variations were associated with early disease onset and mild intellectual disability. Additionally, one other missense variant shifted channel activation to more positive voltages and was associated with spastic ataxic gait. Whereas, the remaining missense variants did not change any of the channel characteristics. Of these three functional variants, only one variant was in silico predicted to be damaging and segregated with disease. The other two variants were in silico predicted to be benign and co-segregation analysis was not optimal or could only be partially confirmed. Therefore, we conclude that we have identified at least one novel pathogenic mutation in KCNC3 that cause SCA13 and two additionally potential SCA13 mutations. This leads to an estimate of SCA13 prevalence in the Netherlands to be between 0.6% and 1.3%.     

Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF,and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.     

Genetic Testing of Non-familial Deaf Patients for <Emphasis Type="Italic">CIB2</Emphasis> and <Emphasis Type="Italic">GJB2</Emphasis> Mutations: Phenotype and Genetic Counselling

CIB2 and GJB2 genes variants contribute significantly in familial cases of prelingual recessive hearing loss (HL). This study was aimed to determine the CIB2 and GJB2 variants and associated phenotype in 150 non-familial individuals with HL. After getting informed consent, 150 non-familial deaf patients were enrolled and blood samples were obtained for DNA extraction. Pure tone air conduction audiometry was performed. Coding exons of CIB2 and GJB2 genes were Sanger sequenced. A tetra primer ARMS assay was developed for recurrent CIB2 variant. Four bi-allelic GJB2 variants, c.71G>A p.(Trp24*), c.231G>A p.(Trp77*), c.235delC p.(Leu79Cysfs3*) and c.35delG p.(Gly11Leufs24*), were found in nine hearing impaired individuals. We also found four homozygotes and five carriers of c.380G>A p. (Arg127His) variant of controversial clinical significance. CIB2 sequencing revealed single recurrent variant c.272T>C p. (Phe91Ser) segregating with HL in ten individuals. Among our patients, c.71G>A (p.Trp24*) was the most common variant, accounted for 45% of GJB2 variants. Two known GJB2 variants, c.235delC p. (Leu79Cysfs3*) and c.310del14 p. (Lys105Argfs2*), are reported here for the first time in Pakistani population. Our data further support the benign nature of c.380G>A p. (Arg127His) variant. For CIB2, c.272T>C p. (Phe91Ser) is the second common cause of HL among our sporadic cases. Phenotypically, in our patients, individuals homozygous for GJB2 variants had profound HL, whereas CIB2 homozygotes had severe to profound prelingual HL. Our results suggest that GJB2 and CIB2 are common cause of HL in different Pakistani ethnicities.     

Defective pre-mRNA splicing in PKD1 due to presumed missense and synonymous mutations causing autosomal dominant polycystic disease

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G > A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117 bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A > T (p.G109G) and c.11257C > A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level.     

Imputation of Exome Sequence Variants into Population- Based Samples and Blood-Cell-Trait-Associated Loci in African Americans: NHLBI GO Exome Sequencing Project

Researchers have successfully applied exome sequencing to discover causal variants in selected individuals with familial, highly penetrant disorders. We demonstrate the utility of exome sequencing followed by imputation for discovering low-frequency variants associated with complex quantitative traits. We performed exome sequencing in a reference panel of 761 African Americans and then imputed newly discovered variants into a larger sample of more than 13,000 African Americans for association testing with the blood cell traits hemoglobin, hematocrit, white blood count, and platelet count. First, we illustrate the feasibility of our approach by demonstrating genome-wide-significant associations for variants that are not covered by conventional genotyping arrays; for example, one such association is that between higher platelet count and an MPL c.117G>T (p.Lys39Asn) variant encoding a p.Lys39Asn amino acid substitution of the thrombpoietin receptor gene (p = 1.5 × 10⁻¹¹). Second, we identified an association between missense variants of LCT and higher white blood count (p = 4 × 10⁻¹³). Third, we identified low-frequency coding variants that might account for allelic heterogeneity at several known blood cell-associated loci: MPL c.754T>C (p.Tyr252His) was associated with higher platelet count; CD36 c.975T>G (p.Tyr325^∗) was associated with lower platelet count; and several missense variants at the α-globin gene locus were associated with lower hemoglobin. By identifying low-frequency missense variants associated with blood cell traits not previously reported by genome-wide association studies, we establish that exome sequencing followed by imputation is a powerful approach to dissecting complex, genetically heterogeneous traits in large population-based studies.     

Trans-activation-based risk assessment of <Emphasis Type="Italic">BRCA1</Emphasis> BRCT variants with unknown clinical significance

Background

Deleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants.

Results

Twelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3).

Conclusions

The modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.

     

The rare intracellular RET mutation p.S891A in a Chinese Han family with familial medullary thyroid carcinoma

We report intracellular RET mutation in a Han Chinese pedigree with familial medullary thyroid carcinoma (FMTC). Direct sequencing of RET proto-oncogene identified a missense c.2671T>G (p.S891A) mutation in 6 of 14 family members. The single nucleotide polymorphisms c. 135A>G (p.A45A), IVS4+48A>G, c. 1296A>G (p.A432A), c. 2071G>A (p.G691S), c. 2307T>G (p.L769L) and a variant c. 833C>A (p.T278N) were also found in 6 carriers. Among 5 of the 6 carriers presented medullary thyroid carcinoma (MTC) as an isolated clinical phenotype, with elevated basal serum calcitonin (Ct). Two underwent non-normative thyroidectomy either two or four times without physician awareness or diagnosis of this disease at initial treatment, but with elevated Ct. One with elevated pre-Ct accepted total thyroidectomy (TT) with modified bilateral neck dissection (MBiND), and whose seventh posterior rib MTC metastases was confirmed 5 months after surgery. Moreover, results of two affected individuals with elevated Ct were reduced to normal after TT with MBiND or prophylactic VI compartmental dissection. However, only another carrier with the variant p.T278N had slightly elevated Ct rejected surgery and was strictly monitored. Given these case results, we suggest that screening of RET and pre-surgical Ct levels in the management of MTC patients is essential for earlier diagnosis and more normative initial treatment, that FMTC patients with cervical lymph nodes metastases may be cured by TT with MBiND, and that prophylactic VI compartmental dissection should be avoided when Ct levels are low.     

Missense mutations in the BMP15 gene are associated with ovarian failure

Premature ovarian failure (POF) is an unexplained amenorrhoea (>6 months) with raised levels of gonadotropins (FSH>40 U/L) occurring before the age of 40 years. Recent studies have elucidated the role of oocyte derived growth factors (BMP15 and GDF9) in maintenance of folliculogenesis, granulosa cell (GC) proliferation and overall fertility. Our recently published work showed presence of two rare missense variants in the GDF9 gene associated with ovarian failure (Dixit et al. 2005, Menopause 12:749–754). The present case-control study has been structured to establish the role of BMP15 germline status associated with ovarian failure. Sequence analysis of the coding region of the BMP15 gene was carried out in a cohort of women with POF (n=133), primary amenorrhoea (n=60), and secondary amenorrhoea (n=9) compared with control females (n=197). This study revealed a total of 18 germline variants in the coding region of BMP15 gene, including 16 novel variants. These novel variants include one intronic variant, one 3’ flanking variant, one silent variant, and 13 missense variants. Eleven missense variants were present only in cases with complete absence in the control females. The remaining two missense variants viz. c.308A>G (p.Asn103Ser) and c.788_789insTCT (p.Leu263_Arg264insLeu) were present both in the cases and in the controls. The c.788_789insTCT variant was significantly higher in primary amenorrhoea cases than in the controls (Fisher’s exact test, P=0.034). Three frequent variants c.-9C>G, c.308A>G, and c.852C>T were chosen for haplotyping. The haplotype G-G-C was found to be significantly associated with ovarian failure (P=0.0075). In a nutshell, the BMP15 gene is highly associated with etiology of ovarian failure.     

Functional consequences of Genetics variant in TMC1 and TMC2 within a United Arab Emirates family with Pre-lingual hearing loss

Hearing loss (HL) is the most prevalent sensory disorder whose etiology comes from environmental and/or genetic factors. Approximately 60 % of HL cases are due to mutations in genes responsible for maintaining a normal hearing function. Despite the monogenic inheritance of hereditary hearing loss (HHL), its diagnosis is challenging as both clinical and genetic heterogeneity characterizes it. Through the development of next-generation sequencing (NGS) techniques, the number of identified mutations responsible for HHL has increased exponentially during the last decade. Mutations in the TMC1 have been reported in several patients with nonsyndromic hereditary hearing loss (NSHHL), more precisely in cases with an autosomal recessive inheritance pattern. In this study, we conducted whole-exome sequencing (WES) analysis of a United Arabs Emirates (UAE) family with autosomal recessive nonsyndromic hearing loss (ARNSHL). This analysis revealed segregation of the TMC1 missense mutation c.596A > T (p.Asn199Ile) with the disease. Bioinformatics analysis supported the pathogenic effect of this mutation and predicted its impact at the proteomics level. Molecular docking analysis of TMC2WT, TMC2R123K, TMC2Q205R, and TMC2R123K + Q205R. Finally, protein docking results suggest a role for TMC2 variants in the phenotypic variability observed within the investigated family.     

Identification and characterization of the compound heterozygous variants of TYR gene in a northern Chinese family with Oculocutaneous albinism type 1

Oculocutaneous albinism (OCA) is a genetically heterogeneous disease and is most inherited in an autosomal recessive manner. The characteristic manifestation of OCA is due to disfunction of melanin synthesis. OCA1 is the most severe subtype of OCA and is caused by homozygous or compound heterozygous variants in tyrosinase (TYR) gene, which is the key gene for melanin synthesis. This study aimed to identify the genetic variants of a northern Chinese family with OCA1. Clinical information and peripheral blood samples were collected. PCR amplification and Sanger sequencing were used to detect the entire exons and adjacent flanking sequences of TYR gene. Functional prediction of variants was performed by various bioinformatic analyses, while the pathogenicity classification of variants was evaluated according to ACMG standards and guidelines. A missense variant NM_000372.5:c.107G > C;NP_000363.1:p.C36S was discovered in TYR gene which converted cysteine to serine. Another variant in intron, NM_000372.5:c.1037–7 T > A, also affected the function of TYR gene. We verified the pathogenicity of the intron variant with a pCAS2 mini-gene based splicing assay and found that c.1037–7 T > A led to an insertion of 5 bp upstream from the common acceptor site of exon 3, which caused a frameshift TYR:c.1037–7 T > A:p.G346Efs*11. The results showed that the compound heterozygous variants c.107G > C:p.C36S and c.1037–7 T > A:p.G346Efs*11 of TYR gene were the pathogenic variants for this OCA1 family.     

Functional Properties of Rare Missense Variants of Human CDH13 Found in Adult Attention Deficit/Hyperactivity Disorder (ADHD) Patients

The CDH13 gene codes for T-cadherin, a GPI-anchored protein with cell adhesion properties that is highly expressed in the brain and cardiovascular system. Previous studies have suggested that CDH13 may be a promising candidate gene for Attention Deficit/Hyperactivity Disorder (ADHD). The aims of this study were to identify, functionally characterize, and estimate the frequency of coding CDH13 variants in adult ADHD patients and controls. We performed sequencing of the CDH13 gene in 169 Norwegian adult ADHD patients and 63 controls and genotyping of the identified variants in 641 patients and 668 controls. Native and green fluorescent protein tagged wild type and variant CDH13 proteins were expressed and studied in CHO and HEK293 cells, respectively. Sequencing identified seven rare missense CDH13 variants, one of which was novel. By genotyping, we found a cumulative frequency of these rare variants of 2.9% in controls and 3.2% in ADHD patients, implying that much larger samples are needed to obtain adequate power to study the genetic association between ADHD and rare CDH13 variants. Protein expression and localization studies in CHO cells and HEK293 cells showed that the wild type and mutant proteins were processed according to the canonical processing of GPI-anchored proteins. Although some of the mutations were predicted to severely affect protein secondary structure and stability, no significant differences were observed between the expression levels and distribution of the wild type and mutant proteins in either HEK293 or CHO cells. This is the first study where the frequency of coding CDH13 variants in patients and controls is reported and also where the functional properties of these variants are examined. Further investigations are needed to conclude whether CDH13 is involved in the pathogenesis of ADHD or other conditions.     

Germline Mutations in MAP3K6 Are Associated with Familial Gastric Cancer

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.