A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT‐PCR analyses of patient RNA or functional splicing reporter minigene assays, are ...required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch‐repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.
We compared minigene splicing assays with patient RNA analyses for 28 intronic, exonic and deep‐intronic variants in the Mismatch repair genes associated with Lynch syndrome. 100% concordance was found between both tests in assessment of aberrant splicing, although we found differences in the exact splice pattern for four variants. We discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.
Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome (LS). In this study, we identified and characterized a novel SINE‐VNTR‐Alu (SVA) insertion in exon 12 of MSH2 in an ...individual with early‐onset colorectal cancer and a very strong LS family history. RT‐PCR analysis indicated a larger aberrant MSH2 transcript in one of the family members. MSK‐IMPACT next‐generation sequencing and long‐range PCR analyses revealed an insertion in MSH2 exon 12 at the c.1972 position in an antisense orientation. The insertion was further characterized as an SVA element approximately 3 kb in length, belonging to the SVA_F1 family of retrotransposons. This variant also segregated with LS related cancers in four affected family members in this family. Based on this evidence, this MSH2 SVA insertion is considered pathogenic.
Autosomal dominant variants in LDB3 (also known as ZASP), encoding the PDZ-LIM domain-binding factor, have been linked to a late onset phenotype of cardiomyopathy and myofibrillar myopathy in humans. ...However, despite knockout mice displaying a much more severe phenotype with premature death, bi-allelic variants in LDB3 have not yet been reported. Here we identify biallelic loss-of-function variants in five unrelated cardiomyopathy families by next-generation sequencing. In the first family, we identified compound heterozygous LOF variants in LDB3 in a fetus with bilateral talipes and mild left cardiac ventricular enlargement. Ultra-structural examination revealed highly irregular Z-disc formation, and RNA analysis demonstrated little/no expression of LDB3 protein with a functional C-terminal LIM domain in muscle tissue from the affected fetus. In a second family, a homozygous LDB3 nonsense variant was identified in a young girl with severe early-onset dilated cardiomyopathy with left ventricular non-compaction; the same homozygous nonsense variant was identified in a third unrelated female infant with dilated cardiomyopathy. We further identified homozygous LDB3 frameshift variants in two unrelated probands diagnosed with cardiomegaly and severely reduced left ventricular ejection fraction. Our findings demonstrate that recessive LDB3 variants can lead to an early-onset severe human phenotype of cardiomyopathy and myopathy, reminiscent of the knockout mouse phenotype, and supporting a loss of function mechanism.
High-throughput sequencing efforts in molecular tumour diagnostics detect increasing numbers of novel variants, including variants predicted to affect splicing. In silico prediction tools can ...reliably predict the effect of variant disrupting canonical splice sites; however, experimental validation is required to confirm aberrant splicing. Here, we present RNA analysis performed for 13 canonical splice site variants predicted or known to result in splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Total nucleic acid was successfully isolated for 10 variants from eight formalin-fixed paraffin-embedded (FFPE) tumour tissues and two B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known variant in the B-cell line, aberrant splicing could only be detected after formalin fixation, which indicated that formalin fixation could possibly inhibit RNA degradation. Aberrant splicing was concluded in three of four predicted splice variants of uncertain significance, supporting their pathogenic effect. With this assay, somatic splice variants can be easily and rapidly analysed, enabling retrospective analysis to support the pathogenicity of variants predicted to result in splicing when only FFPE material is available.
Biallelic pathogenic variants in the mismatch repair (MMR) genes cause a recessive childhood cancer predisposition syndrome known as constitutional mismatch repair deficiency (CMMRD). Family members ...with a heterozygous MMR variant have Lynch syndrome. We aimed at estimating cancer risk in these heterozygous carriers as a novel approach to avoid complicated statistical methods to correct for ascertainment bias.
Cumulative colorectal cancer incidence was estimated in a cohort of PMS2- and MSH6-associated families, ascertained by the CMMRD phenotype of the index, by using mutation probabilities based on kinship coefficients as analytical weights in a proportional hazard regression on the cause-specific hazards. Confidence intervals (CIs) were obtained by bootstrapping at the family level.
The estimated cumulative colorectal cancer risk at age 70 years for heterozygous PMS2 variant carriers was 8.7% (95% CI 4.3-12.7%) for both sexes combined, and 9.9% (95% CI 4.9-15.3%) for men and 5.9% (95% CI 1.6-11.1%) for women separately. For heterozygous MSH6 variant carriers these estimates are 11.8% (95% CI 4.5-22.7%) for both sexes combined, 10.0% (95% CI 1.83-24.5%) for men and 11.7% (95% CI 2.10-26.5%) for women.
Our findings are consistent with previous reports that used more complex statistical methods to correct for ascertainment bias. These results underline the need for MMR gene-specific surveillance protocols for Lynch syndrome.
ABSTRACT
Monoallelic PMS2 germline mutations cause 5%–15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch ...repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA‐ and RNA‐based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety‐one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.
We present a comprehensive overview of PMS2 mutations found in 121 Lynch syndrome and nine CMMRD patients from The Netherlands. PMS2 mutation analysis was performed with recently improved protocols circumventing interference of pseudogene sequences. Eight PMS2 mutation carriers (6%) showed discordant IHC whereas ten patients with isolated PMS2 loss in their tumors harbored an MLH1 mutation.
Background #x0026; Aims: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating ...PMS2 mutations have been reported in HNPCC-suspected families. Our aim was to further assess the role of PMS2 in HNPCC. Methods: We performed Southern blot analysis in 112 patients from MLH1-, MSH2-, and MSH6-negative HNPCC-like families. A subgroup (n = 38) of these patients was analyzed by denaturing gradient gel electrophoresis (DGGE). In a second study group consisting of 775 index patients with familial colorectal cancer, we performed immunohistochemistry using antibodies against MLH1, MSH2, MSH6, and PMS2 proteins. In 8 of 775 tumors, only loss of PMS2 expression was found. In these cases, we performed Southern blot analysis and DGGE. Segregation analysis was performed in the families with a (possibly) deleterious mutation. Results: Seven novel mutations were identified: 4 genomic rearrangements and 3 truncating point mutations. Three of these 7 families fulfill the Amsterdam II criteria. The pattern of inheritance is autosomal dominant with a milder phenotype compared with families with pathogenic MLH1 or MSH2 mutations. Microsatellite instability and immunohistochemical analysis performed in HNPCC-related tumors from proven carriers showed a microsatellite instability high phenotype and loss of PMS2 protein expression in all tumors. Conclusions: We show that heterozygous truncating mutations in PMS2 do play a role in a small subset of HNPCC-like families. PMS2 mutation analysis is indicated in patients diagnosed with a colorectal tumor with absent staining for the PMS2 protein.
A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include ...variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis. Hum Mutat 0,1-8, 2008.
The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies,
MSH2 and
MLH1 mutations were found in approximately ...two-thirds of the Amsterdam-criteria–positive families and in much lower percentages of the Amsterdam-criteria–negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for
MSH2, MLH1, and
MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria–positive families and in 7 (70%) of the 10 Amsterdam-criteria–negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in
MSH2 or
MLH1, and only three were in
MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in
MSH2 and 2 in
MLH1). Notably, a deletion encompassing exons 1–6 of
MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in
MSH2, accounting for ∼10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.
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