Germline variants in POLE are associated with early onset mismatch repair deficient colorectal cancer

Germline variants affecting the exonuclease domains of POLE and POLD1 predispose to multiple colorectal adenomas and/or colorectal cancer (CRC). The aim of this study was to estimate the prevalence of previously described heterozygous germline variants POLE c.1270C>G, p.(Leu424Val) and POLD1 c.1433G>A, p.(Ser478Asn) in a Dutch series of unexplained familial, early onset CRC and polyposis index cases. We examined 1188 familial CRC and polyposis index patients for POLE p.(Leu424Val) and POLD1 p.(Ser478Asn) variants using competitive allele-specific PCR. In addition, protein expression of the POLE and DNA mismatch repair genes was studied by immunohistochemistry in tumours from POLE carriers. Somatic mutations were screened using semiconductor sequencing. We detected three index patients (0.25%) with a POLE p.(Leu424Val) variant. In one patient, the variant was found to be de-novo. Tumours from three patients from two families were microsatellite instable, and immunohistochemistry showed MSH6/MSH2 deficiency suggestive of Lynch syndrome. Somatic mutations but no germline MSH6 and MSH2 variants were subsequently found, and one tumour displayed a hypermutator phenotype. None of the 1188 patients carried the POLD1 p.(Ser478Asn) variant. POLE germline variant carriers are also associated with a microsatellite instable CRC. POLE DNA analysis now seems warranted in microsatellite instable CRC, especially in the absence of a causative DNA mismatch repair gene germline variant.     

Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome‐wide and targeted DNA copy number profiling and resequencing in early‐onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Paternal thrombophilic gene mutations are not associated with recurrent miscarriage

Problem Recurrent miscarriage (RM) affects 1–3% of couples. So far, diagnostic procedures are performed only in female patients. However, the main part of the placentary perfusion is encoded by both the maternal and the paternal genome. Method of study In this case–control study, German couples with two (n = 49) or three and more RM (n = 102) and 157 German control couples were analyzed for the factor V‐Leiden 1691G>A mutation (FVL), the prothrombin (PT) 20210G>A substitution, and the 677C>T replacement in the 5,10‐methylenetetrahydrofolate reductase (MTHFR) gene. Results No significant differences in the prevalence of the FVL, PT or MTHFR mutation were observed in male partners of RM patients and in control men [RM/control: FVL heterozygous 13/151 (8.6%): 14/157 (8.9%) (P = 0.9); PT heterozygous 2/151 (1.3%): 7/157 (4.5%) (P = 0.097); PT homozygous 0/151: 2/157 (1.3%); MTHFR homozygous 19/151 (12.6%): 18/157 (11.5%) (P = 0.12)]. This was also true for female RM patients. However, miscarriage during the embryonal period (5–10 weeks of gestation) was significantly associated with a maternal heterozygous FVL mutation (P = 0.014). Conclusion Recurrent miscarriage was not associated with paternal thrombophilia. Men of the control group showed an even higher incidence of the PT and MTHFR mutations. Abortions in the embryonic phase of fetal development were associated with a significantly higher incidence of maternal heterozygosity for FVL.     

CARD15 gene mutations are not associated with ankylosing spondylitis

An insertion mutation at nucleotide 3020 (3020insC) and a missense mutation G2722C in the CARD15 gene on chromosome 16p have been reported to be associated with Crohn's disease (CD). The protein encoded by the CARD15 gene is expressed in peripheral monocytes and regulates apoptosis and NF-kappaB activation, factors which play an important role in inflammation. Since CD and ankylosing spondylitis (AS) are interrelated disorders, we have investigated whether these mutations in the CARD15 gene are also associated with AS. We studied 113 unrelated AS patients and 152 unrelated healthy controls. No significant differences were found between patients and controls in the prevalence of the insertion 3020insC mutation and the G2722C missense mutation, OR = 1.36, 95% CI: 0.27-6.84, P = 0.70 and OR = 0.58; 95% CI: 0.18-1.94; P = 0.38, respectively. We conclude that the insertion 3020insC mutation and the G2722C missense mutation in the CARD15 gene are not involved in the susceptibility to AS.     

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer

The Partner and Localizer of BRCA2 (PALB2) protein has been linked to Fanconi anemia and breast cancer predisposition. Here we present data of a comprehensive mutation screening of the PALB2 gene in 818 familial cases of breast cancer from Germany. By analyzing the entire coding region of PALB2, we found seven truncating mutations (six of them novel) in families tested negative for BRCA1/2-mutations. In addition, two novel potentially disease causing missense mutations were found. Remarkably, only one mutation reported previously in other populations, was also identified in the German population. No PALB2 mutation carriers were identified in 450 unaffected controls. Thus, our observations indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the German population. As PALB2-deficient tumors were shown to be sensitive to Poly(ADP-ribose) Polymerase (PARP) inhibitors, our study has implications for newly developed, favorable treatment options in familial breast cancer.     

Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer

Inherited mutations in the E-cadherin gene ( CDH1 ) were described recently in three Maori kindreds with familial gastric cancer. Familial gastric cancer is genetically heterogeneous and it is not clear what proportion of gastric cancer susceptibility in non-Maori populations is due to germline CDH1 mutations. Therefore, we screened eight familial gastric cancer kindreds of British and Irish origin for germline CDH1 mutations, by SSCP analysis of all 16 exons and flanking sequences. Each family contained: (i) two cases of gastric cancer in first degree relatives with one affected before age 50 years; or (ii) three or more cases of gastric cancer. Novel germline CDH1 mutations (a nonsense and a splice site) were detected in two families (25%). Both mutations were predicted to truncate the E-cadherin protein in the signal peptide domain. In one family there was evidence of non-penetrance and susceptibility to both gastric and colorectal cancer; thus, in addition to six cases of gastric cancer, a CDH1 mutation carrier developed colorectal cancer at age 30 years. We have confirmed that germline mutations in the CDH1 gene cause familial gastric cancer in non-Maori populations. However, only a minority of familial gastric cancers can be accounted for by CDH1 mutations. Loss of E-cadherin function has been implicated in the pathogenesis of sporadic colorectal and other cancers, and our findings provide evidence that germline CDH1 mutations predispose to early onset colorectal cancer. Thus, CDH1 should be investigated as a cause of inherited susceptibility to both gastric and colorectal cancers.     

Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC), the most common hereditary colon cancer syndrome, is a dominant disorder caused by germline defects in mismatch repair (MMR) genes. Identification of MMR gene mutations can have direct clinical implications in counseling and management of HNPCC families. We screened 44 HNPCC and 97 suspected HNPCC Korean families for germline mutations in three MMR genes: MLH1, MSH2 and MSH6. We identified twelve novel mutations: nine in MLH1(c.632_633insT, c.808_811delACTT, c.845C>G, c.1625A>C, c.1730+1delG, c.1907T>C, c.1918C>T, c.2104-2A>G and c.2170T>A), two in MSH2 (c.1886A>G, c.1316_1318delCCT) and one in MSH6 (c.3488A>T). In addition, two statically significant cSNPs in MLH1: c.1128T>C ( p=0.008 in HNPCC and p=0.037 in early-onset CRC) and c.2168C>A ( p<0.001 in HNPCC). Interestingly, the most frequent mutation, c.1757_1758insC in MLH1, was a founder mutation inherited from a common Korean ancestor.     

Dominant genes for colorectal cancer are not rare

The genetic basis for colorectal cancer was investigated by complex segregation analysis of a published series of consecutive pedigrees ascertained through patients undergoing treatment for colorectal cancer. Analysis favoured a dominant gene or genes with a frequency of 0·006 with a lifetime penetrance of 0·63. These genes account for 81% of colorectal cancer in patients under 35, however, by 65 about 85% are phenocopies.     

Germline variation in the oxidative DNA repair genes NUDT1 and OGG1 is not associated with hereditary colorectal cancer or polyposis

The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next‐generation sequencing in 529 families (441 uncharacterized MMR‐proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case–control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss‐of‐function, and splice‐site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer‐free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.     

CDH1 mutations in gastric cancers are not associated with family history

CDH1 mutation is the most frequent genetic alteration in hereditary diffuse gastric cancer (GC) and early onset diffuse GC patients. However, the incidence of CDH1 mutations in sporadic GC with or without family history has not been studied. This retrospective study includes a total of 993 Korean patients with primary advanced GC who underwent surgery and received palliative chemotherapy. Targeted deep sequencing was performed in all cases and family history of GC was searched with survival analysis. We found CDH1 alterations in 146 of 993 patients (14.7 %) and 8 were germline (0.8 %). Out of 146 patients with CDH1 mutations, 25 (17.1 %) had a family history of GC in one of their first relatives, and 12 patients (8.2 %) were diagnosed with familial GC (FGC). All cases with FGC were diffuse type by Lauren classification, and only one harbored a previously reported germline mutation of CDH1 (c.2638 G > A) and the remaining 11 harbored known somatic CDH1 mutations. Among all patients with CDH1 mutation, there was no significant survival difference between patients with family history or FGC. In the 847 patients without CDH1 mutation, 189 (22.3 %) had a family history of GC and 92 patients (10.9 %) were FGC. CDH1 mutations were more frequent in patients with early onset (<45 years) GC (45.5 %) compared with patients with late onset GC (10.9 %) (p = 0.001), but were not significantly associated with the family history of GC (p > 0.05). CDH1 mutations are mostly somatic and typically are not associated with family history.     

Isolated oligodontia associated with mutations in EDARADD, AXIN2, MSX1, and PAX9 genes

Oligodontia is defined as the congenital lack of six or more permanent teeth, excluding third molars. Oligodontia as well as hypodontia (lack of one or more permanent teeth) are highly heritable conditions associated with mutations in the AXIN2, MSX1, PAX9, EDA, and EDAR genes. Here we define the prevalence of mutations in the AXIN2, MSX1, PAX9, EDA, and EDAR genes, and the novel candidate gene EDARADD in a cohort of 93 Swedish probands with non-syndromic, isolated oligodontia. Mutation screening was performed using denaturing gradient gel electrophoresis and DNA sequence analysis. Analyses of the coding sequences of the six genes showed sequence alterations predicted to be damaging or potentially damaging in ten of 93 probands (10.8%). Mutations were identified in the EDARADD (n = 1), AXIN2 (n = 3), MSX1 (n = 2), and PAX9 (n = 4) genes, respectively. None of the 10 probands with mutations had other self-reported symptoms from ectodermal tissues. The oral parameters were similar when comparing individuals with and without mutations but a family history of oligodontia was three times more frequent for probands with mutations. EDARADD mutations have previously been reported in a few families segregating hypohidrotic ectodermal dysplasia and this is, to our knowledge, the first report of an EDARADD mutation associated with isolated oligodontia.     

Germline MUTYH (MYH) mutations in Portuguese individuals with multiple colorectal adenomas

Germinal mutations in the base excision repair (BER) gene MUTYH (MYH) have recently been described in association with predisposition to multiple colorectal adenomas and cancer. In contrast to the classic dominant condition of familial adenomatous polyposis (FAP) due to germinal mutations in the APC gene, the MYH polyposis is an autosomal recessive disease. The identification of individuals affected by MYH polyposis brings new and important implications for the diagnostic, screening, genetic counseling, follow up and therapeutic options in these patients. In this study, screening for germinal mutations in the MYH gene was performed in 53 Portuguese individuals with multiple colorectal adenomas or classic adenomatous polyposis, in whom no mutation had been identified in the APC gene. The results revealed the presence of biallelic germline MYH mutations in 21 patients. In addition, we here report 3 mutations (c.340T>C [p.Y114H]; c.503G>A [p.R168H]; and c.1186_1187insGG [p.E396fsX437]) which, to our knowledge, have not been previously described.     

Germline LEMD3 mutations are rare in sporadic patients with isolated melorheostosis

To further explore the allelic heterogeneity within the group of LEMD3-related disorders, we have screened a larger series of patients including 5 probands with osteopoikilosis or Buschke-Ollendorff syndrome (BOS), 2 families with the co-occurrence of melorheostosis and BOS, and 12 unrelated patients with isolated melorheostosis. Seven novel LEMD3 mutations were identified, all predicted to result in loss-of-function of the protein. We confirm that loss-of-function mutations in the LEMD3 gene can result in either osteopoikilosis or BOS. However, LEMD3 germline mutations were only found in two melorheostosis patients belonging to a different BOS family and one sporadic patient with melorheostosis. The additional presence of osteopoikilosis lesions in these patients seemed to distinguish them from the group of sporadic melorheostosis patients where no germline LEMD3 mutation was identified. Somatic mosaicism for a LEMD3 mutation in the latter group was also not observed, and therefore we must conclude that the genetic defect in the majority of sporadic and isolated melorheostosis remains unknown.     

Germline mutations but not somatic changes at the MYH locus contribute to the pathogenesis of unselected colorectal cancers

MYH-associated polyposis is a recently described, autosomal recessive condition comprising multiple colorectal adenomas and cancer. This disease is caused by germline mutations in the base excision repair (BER) gene MYH. Genes involved in the BER pathway are thus good candidates for involvement in the pathogenesis of sporadic tumors of the large bowel. We have screened a set of 75 sporadic colorectal cancers for mutations in MYH, MTH1, and OGG1. Allelic loss at MYH was also assessed. Selected samples were screened for mutations and allele loss at APC and mutations in p53, K-ras, and beta-catenin. A panel of 35 colorectal cancer cell lines was screened for MYH mRNA and protein expression. One of 75 cancers had bi-allelic germline mutations in MYH and on retrospective analysis of medical records this patient was found to have synchronous multiple small adenomas in addition to carcinoma. No somatic MYH mutations were found and mRNA and protein were expressed in all of our cell lines. There were no clearly pathogenic mutations in MTH1 or OGG1 in any tumor. Bi-allelic germline MYH mutations cause approximately 1 to 3% of unselected colorectal cancers, but appear always to be associated with multiple adenomas. Somatic inactivation of the DNA glycosylases involved in the BER pathway however does not appear to be involved in colorectal tumorigenesis.     

Mutations of P4HA2 encoding prolyl 4-hydroxylase 2 are associated with nonsyndromic high myopia

PurposeHigh myopia is one of the leading causes of blindness worldwide, with high heritability. However, only a few causative genes have been identified, and the pathogenesis is still unclear. Our aim was to identify a novel causative gene in a family with autosomal-dominant, nonsyndromic high myopia.MethodsWhole-genome linkage and whole-exome sequencing were conducted on the family. Real-time quantitative polymerase chain reaction and immunoblotting were applied to test the functional consequence of the candidate mutation. Sanger sequencing was performed to screen for the candidate gene in other families or sporadic cases.ResultsA heterozygous missense mutation, c.871G>A (p.Glu291Lys), within P4HA2 was cosegregating with the phenotype in the family. The segregating mutation caused premature degradation of unstable messenger RNA. Subsequent screening for P4HA2 in 186 cases identified an additional four mutations in 5 cases, including the frameshift mutation c.1349_1350delGT (p.Arg451Glyfs*8), the nonsense mutation c.1327A>G (p.Lys443*), and two deleterious missense mutations, c.419A>G (p.Gln140Arg) and c.448A>G (p.Ile150Val). The missense mutation c.419A>G (p.Gln140Arg) was recurrently identified in a sporadic case and was segregating in a three-generation family.ConclusionP4HA2 was identified as a novel causative gene for nonsyndromic high myopia. This study also indicated that the disruption of posttranslational modifications of collagen is an important factor in the pathogenesis of high myopia.Genet Med 17 4, 300–306.     

Pedigree analysis supports a correlation between an AXIN2 variant and polyposis/colorectal cancer

We present a patient with a history of colonic polyposis and family history significant for colon polyps and colorectal cancer (CRC). The patient and the family also had a history of bone loss of the jaw and early tooth loss, consistent with oligodontia. Genetic testing revealed the patient to have a previously unpublished variant of unknown significance (VUS) in the AXIN2 gene. These clinical findings have been demonstrated previously in only two other families, both of which exhibited oligodontia, colorectal neoplasia (polyps and cancer) and a heterozygous mutation in AXIN2. The AXIN2 protein is component of the Wnt pathway, which is known to be vital for organism development and cellular homeostasis. Alterations of the Wnt pathway lead to cell proliferation and neoplasm, in addition to agenesis of physical structures (such as teeth). The analysis of our pedigree further supports an association between colonic neoplasm (polyposis and CRC), the AXIN2 gene in general, and this particular VUS. It also highlights the importance of analyzing and disseminating information on pedigrees with less commonly encountered genomic abnormalities so that genotypic-phenotypic correlations can be solidified.