Analysis of rare APC variants at the mRNA level: six pathogenic mutations and literature review.

In monogenic disorders, the functional evaluation of rare, unclassified variants helps to assess their pathogenic relevance and can improve differential diagnosis and predictive testing. We characterized six rare APC variants in patients with familial adenomatous polyposis at the mRNA level. APC variants c.531 + 5G>C and c.532-8G>A in intron 4, c.1409-2_1409delAGG in intron 10, c.1548G>A in exon 11, and a large duplication of exons 10 and 11 result in a premature stop codon attributable to aberrant transcripts whereas the variant c.1742A>G leads to the in-frame deletion of exon 13 and results in the removal of a functional motif. Mutation c.1548G>A was detected in the index patient but not in his affected father, suggesting mutational mosaicism. A literature review shows that most of the rare APC variants detected by routine diagnostics and further analyzed at the transcript level were evaluated as pathogenic. The majority of rare APC variants, particularly those located close to exon-intron boundaries, could be classified as pathogenic because of aberrant splicing. Our study shows that the characterization of rare variants at the mRNA level is crucial for the evaluation of pathogenicity and underlying mutational mechanisms, and could lead to better treatment modalities.

Compound heterozygosity for two MSH6 mutations in a patient with early onset colorectal cancer, vitiligo and systemic lupus erythematosus.

Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC) is an autosomal dominant condition caused by heterozygous germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. Rare cases have been reported of an inherited bi-allelic deficiency of MMR genes, associated with multiple café-au-lait spots, early onset CNS tumors, hematological malignancies, and early onset gastrointestinal neoplasia. We report on a patient with vitiligo in segments of the integument who developed systemic lupus erythematosus (SLE) at the age of 16, and four synchronous colorectal cancers at age 17 years. Examination of the colorectal cancer tissue showed high microsatellite instability (MSI-H) and an exclusive loss of expression of the MSH6 protein. Immunohistochemical analysis of normal colon tissue also showed loss of MSH6, pointing to a bi-allelic MSH6 mutation. Sequencing of the MSH6 gene showed the two germline mutations; c.1806_1809delAAAG;p.Glu604LeufsX5 and c.3226C > T;p.Arg1076Cys. We confirmed that the two mutations are on two different alleles by allele-specific PCR. To our knowledge, neither parent is clinically affected. They did not wish to be tested for the mutations identified in their daughter. These data suggest that bi-allelic mutations of one of the MMR genes should be considered in patients who develop early-onset multiple HNPCC-associated tumors and autoimmune disorders, even in absence of either hematological malignancies or brain tumors.

Somatic APC mosaicism: a frequent cause of familial adenomatous polyposis (FAP).

Somatic mutational mosaicism presents a challenge for both molecular and clinical diagnostics and may contribute to deviations from predicted genotype-phenotype correlations. During APC mutation screening in 1,248 unrelated patients with familial adenomatous polyposis (FAP), we identified 75 cases with an assumed or confirmed de novo mutation. Prescreening methods (protein truncation test [PTT], DHPLC) indicated the presence of somatic mosaicism in eight cases (11%). Sequencing of the corresponding fragments revealed very weak mutation signals, pointing to the presence of either nonsense or frameshift mutations at low level. All mutations were confirmed and quantified by SNaPshot analysis: in leukocyte DNA from the eight patients, the percentage of mosaicism varied between 5.5% and 77%, while the proportion of the mutation in DNA extracted from adenomas of the respective patient was consistently higher. The eight mutations identified as mosaic are localized within codons 216-1464 of the APC gene. According to the known genotype-phenotype correlation, patients with mutations in this region exhibit typical or severe FAP. However, six of the eight patients presented with an attenuated or atypical polyposis phenotype. Our data demonstrate that in a fraction of FAP patients the causative APC mutation may not be detected due to weak signals or somatic mosaicism that is restricted to tissues other than blood. SNaPshot analysis was proven to be an easy, rapid, and reliable method of confirming low-level mutations and evaluating the degree of mosaicism. Some of the deviations from the expected phenotype in FAP can be explained by the presence of somatic mosaicism.

A complex rearrangement in the APC gene uncovered by multiplex ligation-dependent probe amplification.

Germline mutations in the tumor suppressor gene APC are the underlying cause of familial adenomatous polyposis, an autosomal-dominant cancer predisposition syndrome of the colorectum. Here, we describe a complex pathogenic rearrangement in the APC gene that was detected during deletion screening and transmitted throughout at least three generations. The rearrangement consists of a deletion of 604 bp in intron 4 that impairs the binding site of the reverse primer for exon 4 and of an insertion of 119 bp in exon 4 that interferes with the binding site of the multiplex ligation-dependent probe amplification (MLPA) probes for exon 4. The insertion is composed of three duplicated sequences derived from exon 4, intron 3, and intron 4, all in inverse direction. By transcript analysis, we found that the mutation results in complete skipping of exon 4 and that it leads to a frameshift. The rearrangement would not have been identified had it occurred outside the MLPA hybridization site. Our findings demonstrate that part of the pathogenic mutations remain undetected by routine methods. Moreover, MLPA and RNA analysis alone would have led to an incorrect interpretation of a genomic deletion of exon 4.

Absence of association between cyclin D1 (CCND1) G870A polymorphism and age of onset in hereditary nonpolyposis colorectal cancer.

CCND1 encodes cyclin D1, which plays an important role in the G1 to S phase transition of the cell cycle. A common polymorphism (c.G870A) increases alternate splicing. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mismatch repair (MMR) genes, mainly MSH2 and MLH1, and shows a wide range in the age of its onset (AO), suggesting the existence of other modifying genetic factors. To date, two studies have investigated the association between CCND1 G/A variation and AO in HNPCC with contradictory results in 86 and 146 MMR mutation carriers, respectively. To clarify the role of the CCND1 G/A variation in HNPCC, we performed a study in 406 individuals carrying exclusively clear cut pathogenic mutations in MSH2 or MLH1. We did not observe a significant difference in genotype frequencies of affected and unaffected mutation carriers and healthy controls. A significant association between CCND1 genotypes and AO was found neither in the global comparison (log-rank, P = 0.2981; Wilcoxon, P = 0.2567) nor in a multivariate Cox regression analysis (hazard ratios 1.111, 95%CI 0.950-1.299, P = 0.188 and 1.090, 95%CI 0.868-1.369, P = 0.459 for the additive and dominant effect, respectively). We conclude, that the CCND1 G870A sequence variation is not a genetic modifier of the phenotype of HNPCC.

High proportion of large genomic STK11 deletions in Peutz-Jeghers syndrome.

Germline mutations in the STK11 gene have been identified in 10-70% of patients with Peutz-Jeghers syndrome (PJS), an autosomal-dominant hamartomatous polyposis syndrome. A second locus was assumed in a large proportion of PJS patients. To date, STK11 alterations comprise mainly point mutations; only a small number of large deletions have been reported. We performed a mutation analysis for the STK11 gene in 71 patients. Of these, 56 met the clinical criteria for PJS and 12 were presumed to have PJS because of mucocutaneous pigmentation only or bowel problems due to isolated PJS polyps. No clinical information was available for the remaining three patients. By direct sequencing of the coding region of the STK11 gene, we identified point mutations in 37 of 71 patients (52%). We examined the remaining 34 patients by means of the multiplex ligation-dependent probe amplification (MLPA) method, and detected deletions in 17 patients. In four patients the deletion extended over all 10 exons, and in eight patients only the promoter region and exon 1 were deleted. The remaining deletions encompassed exons 2-10 (in two patients), exons 2-3, exons 4-5, or exon 8. When only patients who met the clinical criteria for PJS are considered, the overall mutation detection rate increases to 94% (64% point mutations and 30% large deletions). No mutation was identified in any of the 12 presumed cases. In conclusion, we found that approximately one-third of the patients who met the clinical PJS criteria exhibited large genomic deletions that were readily detectable by MLPA. Screening for point mutations and large deletions by direct sequencing or MLPA, respectively, increased the mutation detection rate in the STK11 gene up to 94%. There may be still other mutations in the STK11 gene that are not detectable by the methods applied here. Therefore, it is questionable whether a second PJS locus exists at all.

Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a deficiency in DNA mismatch repair in consequence of germline mutations mainly in the genes MSH2 and MLH1. Around 10% of patients suspected of HNPCC are identified with large genomic deletions that cannot be detected by conventional methods of mutation screening. The recently developed multiplex ligation-dependent probe amplification (MLPA) proved to be an easy to perform method for deletion detection and is reliable when more than one exon is deleted. We show that, in some cases, apparent deletions of single exons may actually result from single base substitutions or small insertions/deletions in the hybridisation sequence of MLPA probes. We conclude that single exon deletions, detected by MLPA or multiplex PCR, should be validated with additional methods.

Large genomic aberrations in MSH2 and MLH1 genes are frequent in Chinese colorectal cancer.

Hereditary nonpolyposis colorectal cancer is caused by inactivating mutations in the genes of the DNA mismatch repair (MMR) system. Studies have shown that large-fragment aberrations in MMR genes are responsible for a considerable proportion of hereditary colorectal cancer (CRC), but it has been rarely reported in Chinese patients. Here we used multiplex ligation-dependent probe amplification to analyze the genomic rearrangements of 45 Chinese hereditary CRC families, 20 young-age CRC patients (onset of CRC at younger than 50 years and no family history), and 13 patients with sporadic CRC diagnosed at age 50 years or older. Overall, we found 9 (13.8%) large genomic deletions or duplications: 7 out of 45 CRC patients with family history and 2 out of 20 young CRC patients. In all alterations, five genomic deletions were uncovered in the MSH2 gene, as well as one deletion and three duplications in the MLH1 gene. Furthermore, two of the duplications unveiled in this study may have more than a four-copy increase of the exon showing duplication in MLH1. The results indicate that genomic aberrations, large-fragment deletions and duplications, in both MSH2 and MLH1 genes play a role in the pathogenesis of Chinese CRC patients with a family history, as reported in western populations. Moreover, the genomic aberrations in these genes might also be a frequent cause of CRC at a young age in China.

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1.

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.

Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, the relevance of rare exonic single-base substitutions at nucleotide positions close to splice sites that are predicted to result in missense or silent (SNP) variants or substitutions in introns at splice-site positions that are not highly conserved has not been systematically examined in FAP patients. In 34 index patients, we identified 26 different heterozygous single-base substitutions at or close to the splice sites. We characterized five exonic mutations in exon 4 (c.423G>T), exon 14 (c.1956C>T, c.1957A>G, and c.1957A>C), and exon 15 (c.1959G>A) by transcript analysis and by splice-prediction programs (BDGP, SpliceSiteFinder, and ESEfinder). The splicing patterns of these variants were compared to those of 16 different substitutions at highly or less-conserved intronic splice-site positions, and to normal controls. In addition, we analyzed cosegregation of the variants with affected family members and examined the genotype-phenotype correlation. We could demonstrate that the four unclear variants in exon 4 and 14 that are predicted to result in missense or silent mutations in fact lead to complete exon skipping due to aberrant splicing; one possible explanation for this observed effect might be the disruption of exonic splicing enhancer (ESE) motifs. In contrast, the substitution at the first position of exon 15 seems to actually be a silent variant. We present the first systematic evaluation of different single-base substitutions in APC at or close to splice sites at transcript level. We show that the consequence of exonic mutations cannot be evaluated only by the predicted change in amino acid sequence but rather by the change at DNA level. The functional analysis of variants with unknown pathogenic effect plays an important role in increasing the mutation detection rate and achieving validation of predictive testing.

Arylamine N-acetyltransferase type 2 and glutathione S-transferases M1 and T1 polymorphisms in familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) exhibits a variable phenotype even in carriers of the same adenomatous polyposis coli germline mutation. Xenobiotic-metabolizing enzymes such as N-acetyltransferases (NATs) and glutathione S-transferases (GSTs) were reported to modify the individual risk for colorectal cancer. We examined whether the polymorphisms of the NAT2, GSTM1, and GSTT1 enzymes affect age at diagnosis of first colorectal adenomas or extracolonic manifestations in 411 FAP patients. Neither age at diagnosis of colorectal adenomas nor occurrence of extra-intestinal tumors differed significantly between genotypes at the NAT2 and GSTM1 loci, whereas GSTT1 polymorphism showed an uncertain association with extra-intestinal manifestations. Combinations of supposed at risk genotypes of the three enzymes showed no significant differences either. Thus, NAT2, GSTM1, or GSTT1 are unlikely to modify the disease phenotype in FAP patients.

Frequency and parental origin of de novo APC mutations in familial adenomatous polyposis.

A predominance of de novo mutations in the paternal germ line has been reported for several disorders; however, in familial adenomatous polyposis (FAP), the parental origin of APC mutations has been scarcely analysed so far. Among 563 unrelated FAP families with known family history, we identified 58 patients with a suspected de novo mutation in the APC gene. A germline mutation was detected in 52 of them; in 38 patients, the mutation could be excluded in both parents. The five base pair deletion at codon 1309 (c.3927_3931delAAAGA) was over-represented in the group of patients with suspected de novo mutations (17/58=29%), when compared to the group of familial cases (26/505=5%); thus, the high frequency of this mutation is not due to a founder effect but rather due to de novo mutation events. Parental origin of de novo mutations could be traced in 16 families, including three families with large chromosomal deletions. Four mutations were of maternal and 12 of paternal origin, pointing to a moderate preponderance towards paternal origin. Sex-related differences of mutation types could be observed: large deletions and single-base substitutions were exclusively of paternal origin, whereas the small deletions were equally distributed (maternal/paternal ratio 4:4).

Loss of DNA mismatch repair proteins in skin tumors from patients with Muir-Torre syndrome and MSH2 or MLH1 germline mutations: establishment of immunohistochemical analysis as a screening test.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous skin tumors (or multiple keratoacanthomas) and internal malignancies. A subtype of MTS is allelic to hereditary nonpolyposis colorectal cancer and is caused by germline mutations in the DNA mismatch repair genes MSH2 or MLH1. In these cases both internal and skin tumors show characteristic microsatellite instability (MSI). The aim of the present study was to determine whether immunohistochemical examination of MSH2 or MLH1 protein expression in MTS-associated skin tumors can be used as a diagnostic screening tool to identify patients with germline mutations in MSH2 or MLH1. In the present study 28 skin lesions from 17 patients (20 sebaceous gland tumors, 4 sebaceous hyperplasias, 3 keratoacanthomas, and 1 squamous cell carcinoma) were tested immunohistochemically with antibodies against MSH2 and MLH1. Eighteen of these tumors were from eight patients with known MSH2 germline mutations, two tumors were from a patient with a germline mutation in MLH1, and eight microsatellite stable sporadic skin tumors served as controls. One sample had to be excluded because of a lack of immunoreactivity. All eight microsatellite stable tumors expressed both DNA repair proteins. In 15 of the tumors from MSH2 germline mutation carriers, loss of MSH2 expression was observed, one tumor showed reduced MSH2 expression, and one tumor displayed positive immunoreactivity to MSH2. Both tumors of the MLH1 germline mutation carrier showed loss of the MLH1 protein. In conclusion, our findings demonstrate that immunohistochemical testing of MTS-related skin tumors is a reliable screening method with high predictive value for the diagnosis of the DNA mismatch repair-deficient MTS.

MUTYH-associated polyposis (MAP): evidence for the origin of the common European mutations p.Tyr179Cys and p.Gly396Asp by founder events.

MUTYH-associated polyposis (MAP) is an autosomal recessive adenomatous polyposis caused by biallelic germline mutations of the base-excision-repair gene MUTYH. In MAP patients of European origin, the combined allele frequency of the mutations p.Tyr179Cys and p.Gly396Asp ranges between 50 and 82%, while these mutations have not been identified in Far Eastern Asian populations, supporting the hypothesis that a founder effect has occurred at some point in European history. To investigate the natural history of the two common European MUTYH alleles, we genotyped six gene-flanking microsatellite markers in 80 unrelated Italian and German MAP patients segregating one or both mutations and calculated their age in generations (g) by using DMLE+2.2 software. Three distinct common haplotypes, one for p.Tyr179Cys and two for p.Gly396Asp, were identified. Estimated mutation ages were 305 g (95% CS: 271-418) for p.Tyr179Cys and 350 g (95% CS: 313-435) for p.Gly396Asp. These results provide evidence for strong founder effects and suggest that the p.Tyr179Cys and p.Gly396Asp mutations derive from ancestors who lived between 5-8 thousand years and 6-9 thousand years B.C., respectively.

Ten recently identified associations between nsSNPs and colorectal cancer could not be replicated in German families.

Ten non-synonymous single nucleotide polymorphisms (nsSNPs), which were recently associated with colorectal cancer risk in a comprehensive, array based study (AKAP9 M463I, DKK3 G335R, AMPD1 Q12X, LIPC L356F, PSMB9 V32I, THBS1 N700S, CA6 S90G, ASCC3 C1995S, DHX36 S416C and CPA4 G303C) were re-evaluated in the present study based on 626 German familial non-HNPCC colorectal cancer patients and 736 healthy controls. No associations of any of the 10 nsSNPs with colorectal cancer could be replicated. The combined analyses indicated that further research based on additional independent samples is required.

May the APC gene somatic mutations in tumor tissues influence the clinical features of Chinese sporadic colorectal cancers?

The APC gene plays an important role in colorectal carcinogenesis. The impact of APC mutations on the clinical features in sporadic CRC remains to be uncovered. The APC gene was screened for mutations with systematic analysis techniques including DHPLC, PTT, MLPA and DNA sequencing in 43 Chinese sporadic CRC patients. Twenty nine somatic mutations (in 17 different types) in APC gene were found in 18 of 43 sporadic CRC patients. Of those, nine were novel mutations. Higher frequency of somatic APC mutations was found in younger CRC patients than that in elder ones. The biallelic somatic mutations of APC gene were identified in four CRC patients whose tumors had more invasive clinical features. The nonsense mutation Arg1114X in APC gene was found in five of 43 CRC tumor tissues. A higher cancer metastasis rate was uncovered in CRC patients with this mutation. The somatic mutations of APC gene may influence the clinical features of sporadic CRC. Arg1114X in APC gene, as a hot spot mutation in Chinese CRC, may predispose to the cancer metastasis of sporadic CRC.

Nine novel pathogenic germline mutations in MLH1, MSH2, MSH6 and PMS2 in families with Lynch syndrome.

Many germline mutations in the DNA mismatch repair genes have been described so far leading to the clinical phenotype of Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC). Most mutations are private mutations. We report on nine novel pathogenic germline mutations that have been found in families meeting either the Amsterdam or the Bethesda criteria. These findings include the mutations MLH1,c.884+4A>G, MLH1,c.1377_1378insA;p.Glu460ArgfsX19, MLH1,c.1415_1416delGA;p.Arg472ThrfsX5, MSH2,c.301G>T;p.Glu101X, MSH2,c.638_639delTG;p.Leu213GlnfsX18, MSH2,c.842C>A;p.Ser281X, MSH2,c.859G>T;p.Gly287X, MSH6,c.2503C>T;p.Gln835X and a large genomic deletion of exons 1-10 of the PMS2 gene. The mutation MLH1,c.884+4A>G detected in two families results in a complete skipping of exon 10 on mRNA level and thus has been considered as pathogenic. In all cases the tumor tissue of the index patient revealed high microsatellite instability (MSI-H) and showed a complete loss of expression of the affected protein in the tumor cells by immunohistochemistry (IHC). The findings underline the importance of a pre-screening of tumor tissue for an efficient definition of conspicuous cases.

Should children at risk for familial adenomatous polyposis be screened for hepatoblastoma and children with apparently sporadic hepatoblastoma be screened for APC germline mutations?

BACKGROUND: Hepatoblastoma (HB) is the most frequent liver tumor in childhood, occurring in the first few years of life. Surgery combined with chemotherapy has resulted in dramatic improvements in prognosis. However, even today, about one quarter of affected children do not survive the disease. Compared to the general population, the risk of HB is 750-7,500 times higher in children predisposed to familial adenomatous polyposis (FAP), an autosomal-dominant cancer predispostion syndrome caused by germline mutations in the tumor suppressor gene APC. Only limited data exist about the frequency of APC germline mutations in cases of apparently sporadic HB without a family history of FAP. PROCEDURE: In our sample of 1,166 German FAP families, all known cases of HB were registered. In addition, 50 patients with apparently sporadic HB were examined for APC germline mutations. RESULTS: In the FAP families, seven unrelated cases of HB are documented; three had been detected at an advanced stage. In patients with apparently sporadic HB, germline mutations in the APC gene were identified in 10%. CONCLUSIONS: These data raise the issue of the appropriate screening for HB in children of FAP patients. To date, the efficiency of surveillance for HB is unclear. In Beckwith-Wiedemann syndrome (BWS), recent studies suggest an earlier detection of both Wilms tumor and HB by frequent screening. We discuss the rationale and implications of a screening program; besides the examination procedure itself, screening for HB in children of FAP patients would have important consequences for the policy of predictive testing in FAP. In a substantial fraction of sporadic HB, the disease is obviously the first manifestation of a de novo FAP. These patients should be identified by routine APC mutation screening and undergo colorectal surveillance thereafter.

MUTYH-associated polyposis: 70 of 71 patients with biallelic mutations present with an attenuated or atypical phenotype.

To determine the frequency, mutation spectrum and phenotype of the recently described autosomal recessive MUTYH-associated polyposis (MAP), we performed a systematic search for MUTYH (MYH) mutations by sequencing the complete coding region of the gene in 329 unselected APC mutation-negative index patients with the clinical diagnosis of familial adenomatous polyposis (FAP) or attenuated FAP (AFAP). Biallelic germline mutations in MUTYH were identified in 55 of the 329 unselected patients (17%) and in another 9 selected index cases. About one-fifth (20%) of the 64 unrelated MAP patients harboured none of the 2 hot-spot missense mutations Y165C and/or G382D. Including 7 affected relatives, almost all MAP patients presented with either an attenuated (80%) or with an atypical phenotype (18%). Fifty percentage of the MAP patients had colorectal cancer at diagnosis. Duodenal polyposis was found in 18%, thyroid and stomach cancer in 1 case, other extraintestinal manifestations associated with FAP were not observed. In 8 families, vertical segregation was suspected; in 2 of these families, biallelic mutations were identified in 2 generations. Monoallelic changes with predicted functional relevance were found in 0.9% of the 329 patients, which is in accordance with the carrier frequency in the general population. In conclusion, biallelic MUTYH mutations are the underlying genetic basis in a substantial fraction of patients with adenomatous polyposis. The phenotype of MAP is best characterised as attenuated or atypical, respectively. Colorectal surveillance starting at about 18 years of age is recommended for biallelic mutation carriers and siblings of MAP patients, who refuse predictive testing.