Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer

Epimutations in the germline, such as methylation of the MLH1 gene, may contribute to hereditary cancer syndrome in human, but their transmission to offspring has never been documented. Here we report a family with inheritance, in three successive generations, of germline allele-specific and mosaic hypermethylation of the MSH2 gene, without evidence of DNA mismatch repair gene mutation. Three siblings carrying the germline methylation developed early-onset colorectal or endometrial cancers, all with microsatellite instability and MSH2 protein loss. Clonal bisulfite sequencing and pyrosequencing showed different methylation levels in different somatic tissues, with the highest level recorded in rectal mucosa and colon cancer tissue, and the lowest in blood leukocytes. This mosaic state of germline methylation with different tissue distribution could act as the first hit and provide a mechanism for genetic disease inheritance that may deviate from the mendelian pattern and be overlooked in conventional leukocyte-based genetic diagnosis strategy.     

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.     

Deletions and epimutations affecting the human 14q32.2 imprinted region in individuals with paternal and maternal upd(14)-like phenotypes

Human chromosome 14q32.2 carries a cluster of imprinted genes including paternally expressed genes (PEGs) such as DLK1 and RTL1 and maternally expressed genes (MEGs) such as MEG3 (also known as GTL2), RTL1as (RTL1 antisense) and MEG8 (refs. 1,2), together with the intergenic differentially methylated region (IG-DMR) and the MEG3-DMR. Consistent with this, paternal and maternal uniparental disomy for chromosome 14 (upd(14)pat and upd(14)mat) cause distinct phenotypes. We studied eight individuals (cases 1-8) with a upd(14)pat-like phenotype and three individuals (cases 9-11) with a upd(14)mat-like phenotype in the absence of upd(14) and identified various deletions and epimutations affecting the imprinted region. The results, together with recent mouse data, imply that the IG-DMR has an important cis-acting regulatory function on the maternally inherited chromosome and that excessive RTL1 expression and decreased DLK1 and RTL1 expression are relevant to upd(14)pat-like and upd(14)mat-like phenotypes, respectively.     

A germline variant in the TP53 polyadenylation signal confers cancer susceptibility

To identify new risk variants for cutaneous basal cell carcinoma, we performed a genome-wide association study of 16 million SNPs identified through whole-genome sequencing of 457 Icelanders. We imputed genotypes for 41,675 Illumina SNP chip-typed Icelanders and their relatives. In the discovery phase, the strongest signal came from rs78378222[C] (odds ratio (OR) = 2.36, P = 5.2 × 10(-17)), which has a frequency of 0.0192 in the Icelandic population. We then confirmed this association in non-Icelandic samples (OR = 1.75, P = 0.0060; overall OR = 2.16, P = 2.2 × 10(-20)). rs78378222 is in the 3' untranslated region of TP53 and changes the AATAAA polyadenylation signal to AATACA, resulting in impaired 3'-end processing of TP53 mRNA. Investigation of other tumor types identified associations of this SNP with prostate cancer (OR = 1.44, P = 2.4 × 10(-6)), glioma (OR = 2.35, P = 1.0 × 10(-5)) and colorectal adenoma (OR = 1.39, P = 1.6 × 10(-4)). However, we observed no effect for breast cancer, a common Li-Fraumeni syndrome tumor (OR = 1.06, P = 0.57, 95% confidence interval 0.88-1.27).     

Mitotic recombination effects homozygosity for NF1 germline mutations in neurofibromas.

Pure populations of neurofibroma-derived Schwann cells bearing both NF1 mutated alleles (NF1-/-) have been isolated from different neurofibromas showing loss of heterozygosity of nearly the entire 17q chromosome. By comparing molecular and fluorescent in situ hybridization analysis of these cells, we demonstrate mitotic recombination is the mechanism underlying this type of loss of heterozygosity leading to reduction to homozygosity of NF1 germline mutation.     

Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes

The nature of mendelian inheritance assumes that all tissues in which a phenotype of interest is expressed have a uniform diploid karyotype, which is often not the case in cancer cells. Owing to nonrandom gains of chromosomes, trisomies are present in many cases of leukemia and other malignances. We used polymorphisms in the genes encoding thiopurine S-methyltransferase (TPMT), gamma-glutamyl hydrolase (GGH) and the reduced folate carrier (SLC19A1) to assess the nature of chromosomal acquisition and its influence on genotype-phenotype concordance in cancer cells. TPMT and GGH activities in somatic cells were concordant with germline genotypes, whereas activities in leukemia cells were determined by chromosomal number and whether the acquired chromosomes contained a wild-type or variant allele. Leukemia cells that had acquired an additional chromosome containing a wild-type TPMT or GGH allele had significantly lower accumulation of thioguanine nucleotides or methotrexate polyglutamates, respectively. Among these genes, there was a comparable number of acquired chromosomes with wild-type and variant alleles. Therefore, chromosomal gain can alter the concordance of germline genotype and cancer cell phenotypes, indicating that allele-specific quantitative genotyping may be required to define cancer pharmacogenomics unequivocally.     

Recombination rate and reproductive success in humans

Intergenerational mixing of DNA through meiotic recombinations of homologous chromosomes during gametogenesis is a major event that generates diversity in the eukaryotic genome. We examined genome-wide microsatellite data for 23,066 individuals, providing information on recombination events of 14,140 maternal and paternal meioses each, and found a positive correlation between maternal recombination counts of an offspring and maternal age. We postulated that the recombination rate of eggs does not increase with maternal age, but that the apparent increase is the consequence of selection. Specifically, a high recombination count increased the chance of a gamete becoming a live birth, and this effect became more pronounced with advancing maternal age. Further support for this hypothesis came from our observation that mothers with high oocyte recombination rate tend to have more children. Hence, not only do recombinations have a role in evolution by yielding diverse combinations of gene variants for natural selection, but they are also under selection themselves.     

The sex-specific genetic architecture of quantitative traits in humans

Mapping genetically complex traits remains one of the greatest challenges in human genetics today. In particular, gene-environment and gene-gene interactions, genetic heterogeneity and incomplete penetrance make thorough genetic dissection of complex traits difficult, if not impossible. Sex could be considered an environmental factor that can modify both penetrance and expressivity of a wide variety of traits. Sex is easily determined and has measurable effects on recognizable morphology; neurobiological circuits; susceptibility to autoimmune disease, diabetes, asthma, cardiovascular and psychiatric disease; and quantitative traits like blood pressure, obesity and lipid levels, among others. In this study, we evaluated sex-specific heritability and genome-wide linkages for 17 quantitative traits in the Hutterites. The results of this study could have important implications for mapping complex trait genes.     

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.     

A germline mutation in the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome.

Breast cancer in men is rare--among the risk factors that have been identified are a family history of breast cancer and evidence of androgen insufficiency. We report a family in which two brothers who both developed breast cancer had clinical and endocrinological evidence of androgen resistance. Sequence analysis revealed a mutation in the androgen receptor gene on the X chromosome, within the region encoding the DNA binding domain. This is the first report of a germline mutation in a member of the steroid/thyroid hormone receptor superfamily associated with the development of cancer.