A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment.

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.

Common alleles at 6q25.1 and 1p11.2 are associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers.

Two single nucleotide polymorphisms (SNPs) at 6q25.1, near the ESR1 gene, have been implicated in the susceptibility to breast cancer for Asian (rs2046210) and European women (rs9397435). A genome-wide association study in Europeans identified two further breast cancer susceptibility variants: rs11249433 at 1p11.2 and rs999737 in RAD51L1 at 14q24.1. Although previously identified breast cancer susceptibility variants have been shown to be associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers, the involvement of these SNPs to breast cancer susceptibility in mutation carriers is currently unknown. To address this, we genotyped these SNPs in BRCA1 and BRCA2 mutation carriers from 42 studies from the Consortium of Investigators of Modifiers of BRCA1/2. In the analysis of 14 123 BRCA1 and 8053 BRCA2 mutation carriers of European ancestry, the 6q25.1 SNPs (r(2) = 0.14) were independently associated with the risk of breast cancer for BRCA1 mutation carriers [hazard ratio (HR) = 1.17, 95% confidence interval (CI): 1.11-1.23, P-trend = 4.5 × 10(-9) for rs2046210; HR = 1.28, 95% CI: 1.18-1.40, P-trend = 1.3 × 10(-8) for rs9397435], but only rs9397435 was associated with the risk for BRCA2 carriers (HR = 1.14, 95% CI: 1.01-1.28, P-trend = 0.031). SNP rs11249433 (1p11.2) was associated with the risk of breast cancer for BRCA2 mutation carriers (HR = 1.09, 95% CI: 1.02-1.17, P-trend = 0.015), but was not associated with breast cancer risk for BRCA1 mutation carriers (HR = 0.97, 95% CI: 0.92-1.02, P-trend = 0.20). SNP rs999737 (RAD51L1) was not associated with breast cancer risk for either BRCA1 or BRCA2 mutation carriers (P-trend = 0.27 and 0.30, respectively). The identification of SNPs at 6q25.1 associated with breast cancer risk for BRCA1 mutation carriers will lead to a better understanding of the biology of tumour development in these women.

Population-based survey of cancer risks in chromosome 3 translocation carriers.

Familial renal cell carcinoma (RCC) is genetically heterogeneous and may be associated with germline mutations in a number of genes. Twelve different constitutional translocations involving chromosome 3 have also been described in association with inherited RCC. In some families the lifetime risk of RCC in chromosome 3 translocation carriers has been estimated to be more than 80%; however the cancer risks in patients with chromosome 3 translocations not ascertained because of a family history of RCC are not well defined. We report a retrospective population-based study using Danish national cytogenetic and cancer registries to clarify tumor risks associated with constitutional translocations involving chromosome 3. We identified 222 (105 females, 117 males) individuals with a constitutional chromosome 3 translocation and compared their cancer risks to those of the Danish population. None of the chromosome 3 translocation carriers had developed RCC at the time of study (female 95% CIs 0.000-0.042, male 95% CIs 0.000-0.038) (P = 1.0 and P = 0.498 for females and males compared to Danish population). Fourteen translocation carriers had developed cancer but there was no evidence of an excess of early onset disease and lifetime cancer risks in chromosome 3 translocation carriers were similar that in the Danish population. There was no association between cancer risk and location of the chromosome 3 breakpoint (HR = 1.322, P = 0.673). These findings suggest that, in the absence of a family history of RCC or evidence of disruption of a specific tumor suppressor gene, chromosome 3 translocations carriers are not at high risk of developing RCC.

Laser microdissection and microsatellite analysis of colorectal adenocarcinomas.

BACKGROUND: Microsatellite instability (MSI) is an important marker in colorectal cancer. The analysis may be difficult if the tumour is heterogeneous or only scarce material is available. The aim of this study was to apply laser microdissection (LMD) to MSI analysis in an attempt to allow diagnosis in these situations. MATERIALS AND METHODS: Twenty-two primary tumours and eight lymph node metastases from twenty patients were laser microdissected and MSI analysis was performed with an optimised multiplex PCR. Differences in allelic size between tumour and blood were evaluated to determine the MSI status. RESULTS: The method proved efficient in as little as 4,000 microm3 formalin-treated and paraffin-embedded tumour tissue. The result of microsatellite analysis was independent of sample location in the primary tumour and its metastasis. CONCLUSION: LMD followed by a multiplex PCR is a useful method for MSI analysis in cases of tumour heterogeneity and scarce tumour material.

Identification of chromosome aberrations in sporadic microsatellite stable and unstable colorectal cancers using array comparative genomic hybridization.

Colorectal cancer (CRC) is one of the most common cancers in Denmark and in the western world in general, and the prognosis is generally poor. According to the traditional molecular classification of sporadic colorectal cancer, microsatellite stable (MSS)/chromosome unstable (CIN) colorectal cancers constitute approximately 85% of sporadic cases, whereas microsatellite unstable (MSI) cases constitute the remaining 15%. In this study, we used array comparative genomic hybridization (aCGH) to identify genomic hotspot regions that harbor recurrent copy number changes. The study material comprised fresh samples from 40 MSS tumors and 20 MSI tumors obtained from 60 Danish CRC patients. We identified five small genomic regions (<15 megabases) exhibiting recurrent copy number loss, which, to our knowledge, have not been reported in previously published aCGH studies of CRC: 3p25.3, 3p21.2-p21.31, 5q13.2, 12q24.23-q24.31, and 12q24.23-q24.31. These regions contain several potentially important tumor suppressor genes that may play a role in a significant proportion of both sporadic MSS CRC and MSI CRC. Furthermore, the generated aCGH data are in support of the recently proposed classification of sporadic CRC into MSS CIN+, MSI CIN-, MSI CIN+, and MSS CIN- cancers.

Resumption of mitosis in frozen-thawed embryos is not related to the chromosomal constitution.

OBJECTIVE: To study the relation between the resumption of mitosis after thaw and chromosomal constitution in frozen-thawed embryos. In addition, to evaluate the correlation among the three parameters of resumption of mitosis after thaw, postthaw blastomere loss, and multinucleation. DESIGN: Frozen-thawed embryos were morphologically evaluated at thaw and after 24 hours of culture. Then, fluorescence in situ hybridization (FISH) analysis, including enumeration of 13 chromosomes, was performed by using a combination of peptide nucleic acid and DNA probes. SETTING: In vitro fertilization laboratory. PATIENT(S): Forty IVF and/or intracytoplasmic sperm injection patients. INTERVENTION(S): Embryo thawing, morphological evaluation, and fluorescence in situ hybridization analysis for aneuploidy screening. MAIN OUTCOME MEASURE(S): Resumption of mitosis, blastomere loss, multinucleation, and chromosome enumeration. RESULT(S): No difference was observed in the chromosomal constitution of embryos with and without resumption of mitosis. Neither was the postthaw blastomere loss connected to the chromosomal constitution. The resumption of mitosis was not associated with postthaw loss of blastomeres or with multinuclearity. CONCLUSION(S): Resumption of mitosis and blastomere loss of frozen-thawed embryos is not related to chromosome aberrations in the embryo. Further, the resumption of mitosis is not correlated with multinucleation. However, the high incidence of multinucleated embryos after thawing indicates that the freezing and thawing procedure may affect this condition.

Mapping of a novel ocular and cutaneous malignant melanoma susceptibility locus to chromosome 9q21.32.

An estimated 10% of all cutaneous malignant melanoma (CMM) cases are inherited, but the genetics of familial CMM are largely unknown. Ocular malignant melanoma (OMM), which is rare, may be associated with familial CMM. We performed a genome-wide scan of two Danish pedigrees with multiple cases of OMM (N = 10) and CMM (N = 3) and other malignancies (with no germline mutations in CDKN2A, CDK4, BRCA1, and BRCA2) to identify melanoma susceptibility genes. Results of parametric linkage analysis are presented as logarithm of the odds (LOD) scores, and all P values are two-sided. Peak two-point parametric LOD score of 2.2 (P = .0007) at D9S167 on chromosome 9q21 was observed. Targeted analysis of a third Danish family with OMM and CMM patients confirmed 9q21 linkage, providing a combined four-point parametric LOD score of 3.02 (nominal P = .00003 and genome-wide P = .086). Two families shared a common haplotype comprising three adjacent and highly polymorphic markers, limiting the region to less than 5 cM and 3 Mbp at 9q21.32. Expression of RASEF, a known gene in this region, was examined in tumor tissue from 10 sporadic CMM lesions and was found to be decreased in 70% of these tumors compared with RASEF expression in a human reference RNA pool from 10 different cell types and in 10 breast tumors.