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Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects. more...

Revertant mosaicism, or "natural gene therapy", is the phenomenon in which germline mutations are corrected by somatic events. In recent years, revertant mosaicism has been identified in all major types of epidermolysis bullosa, the group of heritable blistering disorders caused by mutations in the genes encoding epidermal adhesion proteins. Moreover, revertant mosaicism appears to be present in all patients with a specific subtype of recessive epidermolysis bullosa. We therefore hypothesized that revertant mosaicism should be expected at least in all patients with recessive forms of epidermolysis bullosa. Naturally corrected, patient-own cells are of extreme interest for their promising therapeutic potential, and their presence in all patients would open exciting, new treatment perspectives to those patients. To test our hypothesis, we determined the probability that single nucleotide reversions occur in patients' skin using a mathematical developmental model. According to our model, reverse mutations are expected to occur frequently (estimated 216x) in each patient's skin. Reverse mutations should, however, occur early in embryogenesis to be able to drive the emergence of recognizable revertant patches, which is expected to occur in only one per ~10,000 patients. This underestimate, compared to our clinical observations, can be explained by the "late-but-fitter revertant cell" hypothesis: reverse mutations arise at later stages of development, but provide revertant cells with a selective growth advantage in vivo that drives the development of recognizable healthy skin patches. Our results can be extrapolated to any other organ with stem cell division numbers comparable to skin, which may offer novel future therapeutic options for other genetic conditions if these revertant cells can be identified and isolated. more...

Background: The clinical outcomes of noncompaction cardiomyopathy (NCCM) range from asymptomatic to heart failure, arrhythmias, and sudden cardiac death. Genetics play an important role in NCCM., Objectives: This study investigated the correlations among genetics, clinical features, and outcomes in adults and children diagnosed with NCCM., Methods: A retrospective multicenter study from 4 cardiogenetic centers in the Netherlands classified 327 unrelated NCCM patients into 3 categories: 1) genetic, with a mutation in 32% (81 adults; 23 children) of patients; 2) probably genetic, familial cardiomyopathy without a mutation in 16% (45 adults; 8 children) of patients; or 3) sporadic, no family history, without mutation in 52% (149 adults; 21 children) of patients. Clinical features and major adverse cardiac events (MACE) during follow-up were compared across the children and adults., Results: MYH7, MYBPC3, and TTN mutations were the most common mutations (71%) found in genetic NCCM. The risk of having reduced left ventricular (LV) systolic dysfunction was higher for genetic patients compared with the probably genetic and sporadic cases (p = 0.024), with the highest risk in patients with multiple mutations and TTN mutations. Mutations were more frequent in children (p = 0.04) and were associated with MACE (p = 0.025). Adults were more likely to have sporadic NCCM. High risk for cardiac events in children and adults was related to LV systolic dysfunction in mutation carriers, but not in sporadic cases. Patients with MYH7 mutations had low risk for MACE (p = 0.03)., Conclusions: NCCM is a heterogeneous condition, and genetic stratification has a role in clinical care. Distinguishing genetic from nongenetic NCCM complements prediction of outcome and may lead to management and follow-up tailored to genetic status., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.) more...

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs ∗ 51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.) more...

Congenital short-bowel syndrome (CSBS) is a rare neonatal pathology associated with poor prognosis and high mortality rate. We describe a newborn presenting CSBS intestinal malrotation and chronic intestinal pseudo-obstruction syndrome (CIPS), compound heterozygous for two previously unreported heterozygous mutations in Coxsackie and adenovirus receptor-like membrane protein (CLMP) gene, one in intron 1 (c.28+1G>C), the other on exon 4 (c502C>T, p.R168X). Both mutations are predicted to be pathogenic, leading to impaired splicing and the appearance of a premature stop codon, respectively. Our case is remarkable in that it concerns two heterozygous truncating mutations associated with a good clinical prognosis with a favorable cerebral and gastrointestinal outcome and a substantial enteral input at 8 months of age, despite a small intestine measuring only 35cm., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.) more...

Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder., (Copyright © 2016. Published by Elsevier B.V.) more...

Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite-unstable endometrial tumors. These tumors are particularly prone to mutations in repeats due to mismatch repair deficiency. We screened this coding repeat in our collection of microsatellite-unstable endometrial tumors (EC) and colorectal tumors (CRC). We found 50% mutation frequency for EC and 77% mutation frequency for CRC. These results confirm the previous study on the involvement of RPL22 in EC and, more importantly, reports for the first time such high mutation frequency in this gene in colorectal cancer. Furthermore, considering the high mutation frequency found, our data point toward an important role for RPL22 in microsatellite instability carcinogenesis., (© 2014 WILEY PERIODICALS, INC.) more...

Background: Childhood-onset pulmonary arterial hypertension (PAH) is rare and differs from adult-onset disease in clinical presentation, with often unexplained mental retardation and dysmorphic features (MR/DF). Mutations in the major PAH gene, BMPR2, were reported to cause PAH in only 10-16% of childhood-onset patients. We aimed to identify more genes associated with childhood-onset PAH., Methods: We studied 20 consecutive cases with idiopathic or heritable PAH. In patients with accompanying MR/DF (n=6) array-comparative genomic hybridisation analysis was performed, with the aim of finding common deletion regions containing candidate genes for PAH. Three patients had overlapping deletions of 17q23.2. TBX2 and TBX4 were selected from this area as candidate genes and sequenced in all 20 children. After identifying TBX4 mutations in these children, we subsequently sequenced TBX4 in a cohort of 49 adults with PAH. Because TBX4 mutations are known to cause small patella syndrome (SPS), all patients with newly detected TBX4 mutations were screened for features of SPS. We also screened a third cohort of 23 patients with SPS for PAH., Results: TBX4 mutations (n=3) or TBX4-containing deletions (n=3) were detected in 6 out of 20 children with PAH (30%). All living patients and two parents with TBX4 mutations appeared to have previously unrecognised SPS. In the adult PAH-cohort, one TBX4 mutation (2%) was detected. Screening in the cohort of (predominantly adult) SPS patients revealed no PAH., Conclusions: These data indicate that TBX4 mutations are associated with childhood-onset PAH, but that the prevalence of PAH in adult TBX4 mutation carriers is low. more...

Purpose: Autosomal recessive congenital short bowel syndrome is caused by mutations in CLMP. No mutations were found in the affected males of a family with presumed X-linked congenital short bowel syndrome or in an isolated male patient. Our aim was to identify the disease-causing mutation in these patients., Methods: We performed mutation analysis of the second exon of FLNA in the two surviving affected males of the presumed X-linked family and in the isolated patient., Results: We identified a novel 2-base-pair deletion in the second exon of FLNA in all these male patients. The deletion is located between two nearby methionines at the N-terminus of filamin A. Previous studies showed that translation of FLNA occurs from both methionines, resulting in two isoforms of the protein. We hypothesized that the longer isoform is no longer translated due to the mutation and that this mutation is therefore not lethal for males in utero., Conclusion: Our findings emphasize that congenital short bowel syndrome can be the presenting symptom in male patients with mutations in FLNA. more...

Background: Hirschsprung disease (HSCR) is a congenital disease characterized by the absence of ganglion cells in various length of distal digestive tract. The rearranged during transfection gene (RET) is considered the major gene in HSCR. Although an increasing number of HSCR-associated RET coding sequence (CDS) mutations have been identified in recent years, not many have been investigated for functional consequence on the RET protein., Methods and Results: We examined the functional implications of the de novo RET-CDS mutations V145G, Y483X, V636fsX1, and F961L that we first identified in sporadic Chinese patients with HSCR. The V145G disrupted RET glycosylation and F961L RET phosphorylation. Presumably, the truncation mutations would affect the translocation or the anchoring of the RET protein onto the cellular membrane., Conclusion: The study of RET-CDS mutations that appear de novo is essential not only for understanding the mechanistic of the disease but also for penetrance and recurrence risk estimations, being the ultimate goal for the improvement in disease management and counseling., (Copyright © 2011 Wiley Periodicals, Inc.) more...

Dystrophic epidermolysis bullosa (DEB) is a heritable blistering disorder that can be inherited autosomal dominantly (DDEB) or recessively (RDEB) and covers a group of several distinctive phenotypes. A large number of unique COL7A1 mutations have been shown to underlie DEB. Although general genotype-phenotype correlation rules have emerged, many exceptions to these rules exist, compromising disease diagnosing and genetic counseling. We therefore constructed the International DEB Patient Registry (http://www.deb-central.org), aimed at worldwide collection and sharing of phenotypic and genotypic information on DEB. As of May 2011, this MOLGENIS-based registry contains detailed information on 508 published and 71 unpublished patients and their 388 unique COL7A1 mutations, and includes all combinations of mutations. The current registry RDEB versus DDEB ratio of 4:1, if compared to prevalence figures, suggests underreporting of DDEB in the literature. Thirty-eight percent of mutations stored introduce a premature termination codon (PTC) and 43% an amino acid change. Submission wizards allow users to quickly and easily share novel information. This registry will be of great help in disease diagnosing and genetic counseling and will lead to novel insights, especially in the rare phenotypes of which there is often lack of understanding. Altogether, this registry will greatly benefit the DEB patients., (© 2011 Wiley-Liss, Inc.) more...

Background: Bi-allelic germline mutations of one of the DNA mismatch repair genes, so far predominantly found in PMS2, cause constitutional MMR-deficiency syndrome. This rare disorder is characterised by paediatric intestinal cancer and other malignancies. We report the clinical, immunohistochemical and genetic characterisation of four families with bi-allelic germline PMS2 mutations. We present an overview of the published gastrointestinal manifestations of CMMR-D syndrome and propose recommendations for gastro-intestinal screening., Methods and Results: The first proband developed a cerebral angiosarcoma at age 2 and two colorectal adenomas at age 7. Genetic testing identified a complete PMS2 gene deletion and a frameshift c.736_741delinsTGTGTGTGAAG (p.Pro246CysfsX3) mutation. In the second family, both the proband and her brother had multiple intestinal adenomas, initially wrongly diagnosed as familial adenomatous polyposis. A splice site c.2174+1G>A, and a missense c.137G>T (p.Ser46Ile) mutation in PMS2 were identified. The third patient was diagnosed with multiple colorectal adenomas at age 11; he developed a high-grade dysplastic colorectal adenocarcinoma at age 21. Two intragenic PMS2 deletions were found. The fourth proband developed a cerebral anaplastic ganglioma at age 9 and a high-grade colerectal dysplastic adenoma at age 10 and carries a homozygous c.2174+1G>A mutation. Tumours of all patients showed microsatellite instability and/or loss of PMS2 expression., Conclusions: Our findings show the association between bi-allelic germline PMS2 mutations and severe childhood-onset gastrointestinal manifestations, and support the notion that patients with early-onset gastrointestinal adenomas and cancer should be investigated for CMMR-D syndrome. We recommend yearly follow-up with colonoscopy from age 6 and simultaneous video-capsule small bowel enteroscopy from age 8., (Copyright © 2011 Elsevier Ltd. All rights reserved.) more...

Hirschsprung disease (HSCR) is a congenital malformation characterized by the absence of enteric neurons in the distal part of the colon. Several genes have been implicated in the development of this disease that together account for 20% of all cases, implying that other genes are involved. Since HSCR is frequently associated with other congenital malformations, the functional characterization of the proteins encoded by the genes involved in these syndromes can provide insights into the protein-network involved in HSCR development. Recently, we found that KBP, encoded by the gene involved in a HSCR- associated syndrome called Goldberg-Shprintzen syndrome, interacts with SCG10, a stathmin-like protein. To determine if SCG10 is involved in the etiology of HSCR, we determined SCG10 expression levels during development and screened 85 HSCR patients for SCG10 mutations. We showed that SCG10 expression increases during development but no germline mutation was found in any of these patients. In conclusion, this study shows that SCG10 is not directly implicated in HSCR development. However, an indirect involvement of SCG10 cannot be ruled out as this can be due to a secondary effect caused by its direct interactors. more...

The L1 syndrome is an X-linked recessive disease caused by mutations in the L1CAM gene. To date more than 200 different mutations have been reported, scattered over the entire gene, about 35% being missense mutations. Although it is tempting to consider these missense mutations as being disease-causing, one should be careful in drawing any firm conclusions, unless there is additional supporting information. This is in contrast to truncating mutations, which are always considered to be disease-causing, unless they involve truncations close to the gene stop codon. In order to allow conclusions to be drawn on the disease-causing nature of L1CAM (missense) mutations, we have updated and upgraded our LICAM mutation database with more pathogenicity data and clinical information collected from the literature or generated by our own research. As a result, the renewed database offers condensed scientific information, allowing conclusions to be drawn on the pathogenicity and severity of LICAM mutations based on multiple factors. The L1CAM Mutation Database is at: www.l1cammutationdatabase.info. more...

Dystrophic epidermolysis bullosa is a heritable skin disease in which blisters occur because of a defect in type VII collagen resulting from mutations in the COL7A1 gene that is composed of 118 exons. Although a few mutations are specific to certain populations owing to founder effects, and although a few mutational hotspots exist, most mutations are unique to families and can be found scattered throughout the entire COL7A1 gene. This emphasizes the need for a sensitive, reliable, and efficient mutation scanning technique. Therefore, we developed a conformation-sensitive capillary electrophoresis (CSCE) system for COL7A1 mutation scanning. Here we report on the design and validation of this system. The CSCE technique is based on the principle of heteroduplex formation when polymerase chain reaction-amplified DNA fragments containing heterozygous sequence changes are slowly reannealed. These fluorescently labeled fragments have different migration characteristics and can be detected on a multi-capillary automated sequencer. Validation was performed by analysis of 29 known COL7A1 sequence changes, covering 33% of amplicons. After optimization of the conditions, all 29 sequence changes were detected by the CSCE system, irrespective of length or CG-content of amplicons and position of sequence changes, reflecting an analytical sensitivity of 90.2-100% (95% confidence interval). We conclude that this CSCE system is a rapid, reliable, cost-effective, and highly sensitive way of mutation scanning for COL7A1 in a molecular genetics service laboratory. more...

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation, although somatic mutations in MMR genes have been described. We aimed to investigate which MMR defects are present in SGC. Twenty-nine MSI-H SGC investigated previously for MLH1 promoter hypermethylation were screened for somatic mutations in MLH1, MSH2, MSH6, MLH3, and MBD4 by denaturing gradient gel electrophoresis and sequencing. Five truncating mutations (three in MSH6, one in MLH3, and one in MBD4) and one missense mutation (MLH1) were identified. Of these, three truncating mutations were in MSI-H cases that lack MLH1 hypermethylation. As all truncating mutations were found in the coding poly-A tracts, it seems likely that they result from the MSI phenotype rather than cause it. In summary, somatic mutations in MMR genes are rare in SGC and do not explain the development of these tumors reflecting, rather than causing, the mutator phenotype. Other MMR genes are probably involved in MSI-H gastric cancer without MLH1 hypermethylation. more...

Background: Among the most frequently encountered mutations in dilated cardiomyopathy (DCM) are those in the lamin A/C (LMNA) gene. Our goal was to analyze the LMNA gene in patients with DCM and/or conduction disease referred to the cardiogenetics outpatient clinic and to evaluate the prevalence of LMNA mutations and their clinical expression., Methods and Results: The LMNA gene was screened in 61 index patients. Eleven mutations (including 6 novel) were identified, mainly in the subgroup of familial DCM with cardiac conduction disease (3/10 index patients) and in patients with DCM and Emery-Dreifuss, Limb-Girdle, or unclassified forms of muscular dystrophy (7/8 index patients). In addition, a mutation was identified in 1 of 4 families with only cardiac conduction disease. We did not identify any large deletions or duplications. Genotype-phenotype relationships revealed a high rate of sudden death and cardiac transplants in carriers of the p.N195K mutation. Our study confirmed that the p.R225X mutation leads to cardiac conduction disease with late or no development of DCM, underscoring the importance of this mutation in putative familial "lone conduction disease." Nearly one third of LMNA mutation carriers had experienced a thromboembolic event., Conclusions: This study highlights the role of LMNA mutations in DCM and related disorders. A severe phenotype in p.N195K mutation carriers and preferential cardiac conduction disease in p.R225X carriers was encountered. Because of the clinical variability, including the development of associated symptoms in time, LMNA screening should be considered in patients with DCM or familial lone conduction disease. more...

Objectives: The goal of this study was to identify the underlying gene defect in a family with inherited myocardial fibrosis., Background: A large family with an autosomal dominantly inherited form of myocardial fibrosis with a highly malignant clinical outcome has been investigated. Because myocardial fibrosis preceded the clinical and echocardiographic signs, we consider the disease to be a hereditary form of cardiac fibrosis., Methods: Twenty-five family members were clinically evaluated, and 5 unaffected and 8 affected family members were included in a genome-wide linkage study., Results: The highest logarithm of the odds (LOD) score (LOD = 2.6) was found in the region of the lamin AC (LMNA) gene. The LMNA mutation analysis, both by denaturing gradient gel electrophoresis and sequencing, failed to show a mutation. Subsequent Southern blotting, complementary deoxyribonucleic acid sequencing, and multiplex ligation-dependent probe amplification analysis, however, revealed a deletion of the start codon-containing exon and an adjacent noncoding exon. In vitro studies demonstrated that the deletion results in the formation of nuclear aggregates of lamin, suggesting that the mutant allele is being transcribed., Conclusions: This novel LMNA deletion causes a distinct, highly malignant cardiomyopathy with early-onset primary cardiac fibrosis likely due to an effect of the shortened mutant protein, which secondarily leads to arrhythmias and end-stage cardiac failure. more...

Background: Mutations in the plakophilin-2 gene (PKP2) have been found in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC). Hence, genetic screening can potentially be a valuable tool in the diagnostic workup of patients with ARVC., Methods and Results: To establish the prevalence and character of PKP2 mutations and to study potential differences in the associated phenotype, we evaluated 96 index patients, including 56 who fulfilled the published task force criteria. In addition, 114 family members from 34 of these 56 ARVC index patients were phenotyped. In 24 of these 56 ARVC patients (43%), 14 different (11 novel) PKP2 mutations were identified. Four different mutations were found more than once; haplotype analyses revealed identical haplotypes in the different mutation carriers, suggesting founder mutations. No specific genotype-phenotype correlations could be identified, except that negative T waves in V(2) and V(3) occurred more often in PKP2 mutation carriers (P<0.05). Of the 34 index patients whose family members were phenotyped, 23 familial cases were identified. PKP2 mutations were identified in 16 of these 23 ARVC index patients (70%) with familial ARVC. On the other hand, no PKP2 mutations at all were found in 11 probands without additional affected family members (P<0.001)., Conclusions: PKP2 mutations can be identified in nearly half of the Dutch patients fulfilling the ARVC criteria. In familial ARVC, even the vast majority (70%) is caused by PKP2 mutations. However, nonfamilial ARVC is not related to PKP2. The high yield of mutational analysis in familial ARVC is unique in inherited cardiomyopathies. more...

Biallelic germline mutations of MUTYH-a gene encoding a base excision repair protein-are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P = 0.002) and the published controls (P = 0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk. more...

Chromosomal instability in colon tumors implies the presence of numerical and structural chromosome aberrations and is further characterized by the absence of microsatellite instability and the occurrence of KRAS and/or TP53 mutations. In a previous screening of 194 colon tumors for both microsatellite instability and TP53 mutation, we found 25 microsatellite-unstable tumors, in 9 (36%) of which, presumed to be chromosomally stable, there were TP53 mutations. This prompted us to investigate whether a TP53 mutation in these microsatellite-unstable tumors would be an indicator of chromosomal instability, that is, whether this would be a category of tumors showing both microsatellite and chromosomal instability. For chromosomal instability assessment, we performed array-comparative genomic hybridization analysis of tumor and control DNA extracted from formalin-fixed, paraffin-embedded stage III colon tumor specimens. The array consisted of 435 subtelomere-specific BACs. We compared all but one (whose DNA was of bad quality) of the microsatellite-unstable TP53 mutation-containing tumors (8) with a similarly sized group of microsatellite-unstable tumors without TP53 mutation (11). Microsatellite-unstable tumors with a TP53 mutation showed on average 0.9 aberrations (range 0-3) when assessed with this array system. Those without a TP53 mutation showed on average 0.7 aberrations (range 0-2). Thus, microsatellite-unstable tumors showed few chromosomal abnormalities regardless of TP53 mutation status. Because, in our study, the microsatellite-stable tumors had on average 3.4chromosomal abnormalities (range 0-7), a clear difference exists between microsatellite-unstable and -stable tumors. Because a substantial proportion of microsatellite-unstable colon tumors carry a TP53 mutation while showing relativelyfewchromosomal aberrations, a TP53 mutation in these tumors cannot be considered to be an indicator of chromosomal instability., (Copyright 2005 Wiley-Liss, Inc.) more...

The RET proto-oncogene encodes a receptor tyrosine kinase whose dysfunction plays a crucial role in the development of several neural crest disorders. Distinct activating RET mutations cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Despite clear correlations between the mutations found in these cancer syndromes and their phenotypes, the molecular mechanisms connecting the mutated receptor to the different disease phenotypes are far from completely understood. Luciferase reporter assays in combination with immunoprecipitations, and Western and immunohistochemistry analyses were done in order to characterize the signaling properties of two FMTC-associated RET mutations, Y791F and S891A, respectively, both affecting the tyrosine kinase domain of the receptor. We show that these RET-FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line-derived neurotrophic factor. Moreover, we show that the dysfunctional signaling properties of these mutants, when compared with wild-type RET, involve constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, we show that STAT3 activation is mediated by a signaling pathway involving Src, JAK1, and JAK2, differing from STAT3 activation promoted by RET(C634R) which was previously found to be independent of Src and JAKs. Three-dimensional modeling of the RET catalytic domain suggested that the structural changes promoted by the respective amino acids substitutions lead to a more accessible substrate and ATP-binding monomeric conformation. Finally, immunohistochemical analysis of FMTC tumor samples support the in vitro data, because nuclear localized, Y705-phosphorylated STAT3, as well as a high degree of RET expression at the plasma membrane was observed. more...

Hirschsprung disease (HSCR), a congenital disorder characterized by intestinal obstruction due to absence of enteric ganglia along variable lengths of the intestinal tract, occurs both in familial and sporadic cases. RET mutations have been found in approximately 50% of the families, but explains only a minority of sporadic cases. This study aims at investigating a possible role of RET in sporadic HSCR patients. Haplotypes of 13 DNA markers, within and flanking RET, have been determined for 117 sporadic HSCR patients and their parents. Strong association was observed for six markers in the 5' region of RET. The largest distortions in allele transmission were found at the same markers. One single haplotype composed of these six markers was present in 55.6% of patients versus 16.2% of controls. Odds ratios (ORs) revealed a highly increased risk of homozygotes for this haplotype to develop HSCR (OR>20). These results allowed us to conclude that RET plays a crucial role in HSCR even when no RET mutations are found. An unknown functional disease variant(s) with a dosage-dependent effect in HSCR is likely located between the promoter region and exon 2 of RET. more...

Recently we identified a new variant, S845G, in the MLH3 gene in 7 out of 327 patients suspected of hereditary nonpolyposis colorectal cancer but not fulfilling the Amsterdam criteria and in 1 out of 188 control subjects. As this variant might play a role in causing sporadic colorectal cancer, we analyzed its prevalence in sporadic colorectal cancer patients. We analyzed a small part of exon 1 of the MLH3 gene, including the S845G variant, in germline DNA of 467 white sporadic colorectal cancer patients and 497 white controls. The S845G variant was detected in five patients and eight controls; the results thus indicate that this variant does not confer an increased colorectal cancer risk. Another variant (P844L) was clearly a polymorphism. Three other missense variants were rare and the sample size of the study was too small to conclude whether they are pathogenic. In conclusion, no association was observed between two MLH3 variants (P844L and S845G) and colorectal cancer risk. more...

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles. [ABSTRACT FROM AUTHOR] more...