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Hirschsprung's disease (HSCR) is a congenital disorder characterised by the absence of ganglia along variable lengths of the intestine. The RET gene is the major HSCR gene. Reduced penetrance of RET mutations and phenotypic variability suggest the involvement of additional modifying genes in the disease. A RET-dependent modifier locus was mapped to 9q31 in families bearing no coding sequence (CDS) RET mutations. Yet, the 9q31 causative locus is to be identified. To fine-map the 9q31 region, we genotyped 301 tag-SNPs spanning 7 Mb on 137 HSCR Dutch trios. This revealed two HSCR-associated regions that were further investigated in 173 Chinese HSCR patients and 436 controls using the genotype data obtained from a genome-wide association study recently conducted. Within one of the two identified regions SVEP1 SNPs were found associated with Dutch HSCR patients in the absence of RET mutations. This ratifies the reported linkage to the 9q31 region in HSCR families with no RET CDS mutations. However, this finding could not be replicated. In Chinese, HSCR was found associated with IKBKAP. In contrast, this association was stronger in patients carrying RET CDS mutations with p = 5.10 x 10(-6) [OR = 3.32 (1.99, 5.59)] after replication. The HSCR-association found for IKBKAP in Chinese suggests population specificity and implies that RET mutation carriers may have an additional risk. Our finding is supported by the role of IKBKAP in the development of the nervous system.

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.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome. Germline mutations in five different mismatch repair (MMR) genes, MSH2, MSH6, MLH1, MLH3, and PMS2 are linked to HNPCC. Here, we describe two colon cancer families in which the index patients carry missense mutations in both MSH2 and MSH6. The MSH2 mutation, I145M, is the same in both families, whereas the MSH6 mutations are different (R1095H and L1354Q). The families do not fulfil the international criteria for HNPCC, one family comprising two and the other family four colon cancer patients, all in one generation, resembling a recessive rather than dominant inheritance characteristic of HNPCC. The tumors of the index patients showed microsatellite instability. Functional analysis was performed to determine which one of the mutations could primarily underlie the cancer susceptibility in the families. MSH2 and MSH6 are known to form a heterodimeric complex (MutSalpha) responsible for mismatch recognition. The interaction of each mutated protein with its wild-type partner and with its mutated partner present in the colon cancer patient, and the MMR function of the mutated MutSalpha complexes were determined. Since none of the three mutations affected the MSH2-MSH6 interaction or the function of MutSalpha in an in-vitro MMR assay, our results suggest that alone the mutations do not cause MMR deficiency typical of HNPCC. However, our results do not exclude the possible compound pathogenicity of the two mutations.

Hereditary paroxysmal ataxia, or episodic ataxia (EA), is a rare, genetically heterogeneous neurological disorder characterized by attacks of generalized ataxia. By direct sequence analysis, a different missense mutation of the potassium channel gene (KCNA1) has been identified in three families with EA.

Expression of the tyrosine kinase receptor RET has previously been detected in normal hematopoietic cells, and especially in cells of the myeloid lineage. Furthermore, RET was shown to be differentially expressed in acute myeloid leukemia (AML), a disease characterized by excessive cell growth and aberrant maturation of cells, with the highest levels of expression in leukemias with monocytic differentiation. RET is known to be expressed in cells from the excretory system and from the developing central and peripheral nervous system. Both activating and inactivating aberrations in the RET gene have been detected in disorders derived from these tissues. To investigate whether the differential expression is a primary defect in AML, the presence of RET alterations was scanned by Southern blot analysis on DNA of blasts obtained from 17 AML patients. However, no RET gene aberrations were found. Subsequently, denaturing gradient gel electrophoresis (DGGE) analysis was performed on the DNA of blasts from ten selected cases. All five variants detected turned out to represent neutral DNA polymorphisms, including a novel polymorphism in exon 14. Since we were unable to detect mutations of RET in AML, it is unlikely that it plays an important role in leukemogenesis.

Neuroblastoma occasionally occurs in diseases associated with abnormal neurocrest differentiation, e.g. Hirschsprung disease. Expression studies in developing mice suggest that the proto-oncogene RET plays a role in neurocrest differentiation. In humans expression of RET is limited to certain tumor types, including neuroblastoma, that derive from migrating neural crest cells. Mutations of RET are found associated with Hirschsprung disease. These data prompted us to investigate expression of RET and to search for gene mutations in neuroblastoma. Out of 16 neuroblastoma cell lines analyzed, 9 show clear expression of RET in a Northern blot analysis. In a single strandt conformation polymorphism (SSCP) analysis of all exons, no mutations were detected other than neutral polymorphisms. In a patient with neuroblastoma, from a family in which different neurocrestopathies, including neuroblastoma and Hirschsprung disease, had occurred, we also failed to detect RET mutations. Possibly, expression of RET in neuroblastoma merely reflects the differentiation status of the tumor cells. The absence of mutations suggests that RET does not play a crucial role in the tumorigenesis of neuroblastoma.

Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.

Seven polymorphic cosmids previously assigned to 10cen-q11.2 were mapped between D10S34 and RBP3, and ordered by interphase in situ hybridization and yeast artificial chromosome analysis. Some of the presumed unique sequences from the centromeric region have homologies either within the same region or within the centromeric region of other chromosomes.