DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe

STUDY OBJECTIVE Prior research has identified five common genetic variants associated with narcolepsy with cataplexy in Caucasian patients. To replicate and/or extend these findings, we have tested HLA-DQB1, the previously identified 5 variants, and 10 other potential variants in a large European sample of narcolepsy with cataplexy subjects. DESIGN Retrospective case-control study. SETTING A recent study showed that over 76% of significant genome-wide association variants lie within DNase I hypersensitive sites (DHSs). From our previous GWAS, we identified 30 single nucleotide polymorphisms (SNPs) with P < 10(-4) mapping to DHSs. Ten SNPs tagging these sites, HLADQB1, and all previously reported SNPs significantly associated with narcolepsy were tested for replication. PATIENTS AND PARTICIPANTS For GWAS, 1,261 narcolepsy patients and 1,422 HLA-DQB1*06:02-matched controls were included. For HLA study, 1,218 patients and 3,541 controls were included. MEASUREMENTS AND RESULTS None of the top variants within DHSs were replicated. Out of the five previously reported SNPs, only rs2858884 within the HLA region (P < 2x10(-9)) and rs1154155 within the TRA locus (P < 2x10(-8)) replicated. DQB1 typing confirmed that DQB1*06:02 confers an extraordinary risk (odds ratio 251). Four protective alleles (DQB1*06:03, odds ratio 0.17, DQB1*05:01, odds ratio 0.56, DQB1*06:09 odds ratio 0.21, DQB1*02 odds ratio 0.76) were also identified. CONCLUSION An overwhelming portion of genetic risk for narcolepsy with cataplexy is found at DQB1 locus. Since DQB1*06:02 positive subjects are at 251-fold increase in risk for narcolepsy, and all recent cases of narcolepsy after H1N1 vaccination are positive for this allele, DQB1 genotyping may be relevant to public health policy.

Comparative genetic analyses point to HCP5 as susceptibility locus for HCV-associated hepatocellular carcinoma

BACKGROUND & AIMS: Recently, genetic variations in MICA (lead single nucleotide polymorphism [SNP] rs2596542) were identified by a genome-wide association study (GWAS) to be associated with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) in Japanese patients. In the present study, we sought to determine whether this SNP is predictive of HCC development in the Caucasian population as well. METHODS: An extended region around rs2596542 was genotyped in 1924 HCV-infected patients from the Swiss Hepatitis C Cohort Study (SCCS). Pair-wise correlation between key SNPs was calculated both in the Japanese and European populations (HapMap3: CEU and JPT). RESULTS: To our surprise, the minor allele A of rs2596542 in proximity of MICA appeared to have a protective impact on HCC development in Caucasians, which represents an inverse association as compared to the one observed in the Japanese population. Detailed fine-mapping analyses revealed a new SNP in HCP5 (rs2244546) upstream of MICA as strong predictor of HCV-related HCC in the SCCS (univariable p=0.027; multivariable p=0.0002, odds ratio=3.96, 95% confidence interval=1.90-8.27). This newly identified SNP had a similarly directed effect on HCC in both Caucasian and Japanese populations, suggesting that rs2244546 may better tag a putative true variant than the originally identified SNPs. CONCLUSIONS: Our data confirms the MICA/HCP5 region as susceptibility locus for HCV-related HCC and identifies rs2244546 in HCP5 as a novel tagging SNP. In addition, our data exemplify the need for conducting meta-analyses of cohorts of different ethnicities in order to fine map GWAS signals.

The brown coat colour of Coppernecked goats is associated with a non-synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome-wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome-wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non-synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss-of-function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the (496) Asp allele might possibly act in a dominant manner. The (496) Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.

A novel myeloid leukemia suppressor locus at human chromosome 5q13.3

Molecular mechanisms that underlie preleukemic myelodysplasia (MDS) and acute myelogenous leukemia (AML) are poorly understood. In MDS or AML with a refractory clinical course, more than 30% of patients have acquired interstitial or complete deletions of chromosome 5. The 5q13.3 chromosomal segment is commonly lost as the result of 5q deletion. Reciprocal and unbalanced translocations of 5q13.3 can also occur as sole anomalies associated with refractory AML or MDS. This study addresses the hypothesis that a critical gene at 5q13.3 functions either as a classical tumor suppressor or as a chromosomal translocation partner and contributes to leukemogenesis. ^ Previous studies from our laboratory delineated a critical region of loss to a 2.5–3.0Mb interval at 5q13.3 between microsatellite markers D5S672 and GATA-P18104. The critical region of loss was later resolved to an interval of approximately 2Mb between the markers D5S672 and D5S2029. I, then generated a long range physical map of yeast artificial chromosomes (YACs) and developed novel sequence tagged sites (STS). To enhance the resolution of this map, bacterial artificial chromosomes (BACs) were used to construct a triply linked contig across a 1 Mb interval. These BACs were used as probes for fluorescent in situ hybridization (FISH) on an AML cell line to define the 5q13.3 critical region. A 200kb BAC, 484a9, spans the translocation breakpoint in this cell line. A novel gene, SSDP2 (single stranded DNA binding protein), is disrupted at the breakpoint because its first four exons are encoded within 140kb of BAC 484a9. This finding suggests that SSDP2 is the critical gene at 5q13.3. ^ In addition, I made an observation that deletions of chromosome 5q13 co-segregate with loss of the chromosome 17p. In some cases the deletions result from unbalanced translocations between 5q13 and 17p13. It was confirmed that the TP53 gene is deleted in patients with 17p loss, and the remaining allele harbors somatic mutation. Thus, the genetic basis for the aggressive clinical course in AML and MDS may be caused by functional cooperation between deletion or disruption of the 5q13.3 critical gene and inactivation of TP53. ^

Altered tumorigenic phenotype in mice with a hypomorphic p53 mutant allele

Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Evolution of foraging behavior in Drosophila by density-dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind SC10-RNase in SI N. alata SC10SC10 and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia × SI N. alata SC10SC10) hybrids with reduced levels of HT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both SC10-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

Molecular characterization of four induced alleles at the Ednrb locus

The piebald locus on mouse chromosome 14 encodes the endothelin-B receptor (EDNRB), a G protein-coupled, seven-transmembrane domain protein, which is required for neural crest-derived melanocyte and enteric neuron development. A spontaneous null allele of Ednrb results in homozygous mice that are predominantly white and die as juveniles from megacolon. To identify the important domains for EDNRB function, four recessive juvenile lethal alleles created by either radiation or chemical mutagens (Ednrb27Pub, Ednrb17FrS, Ednrb1Chlc, and Ednrb3Chlo) were examined at the molecular level. Ednrb27Pub mice harbor a mutation at a critical proline residue in the fifth transmembrane domain of the EDNRB protein. A gross genomic alteration within the Ednrb gene in Ednrb3Chlo results in the production of aberrantly sized transcripts and no authentic Ednrb mRNA. Ednrb17FrS mice exhibited a decreased level of Ednrb mRNA, supporting previous observations that the degree of spotting in piebald mice is dependent on the amount of EDNRB expressed. Finally, no molecular defect was detected in Ednrb1Chlc mice, which produce normal levels of Ednrb mRNA in adult brain, suggesting that the mutation affects important regulatory elements that mediate the expression of the gene during development.

Identification of nitric oxide synthase as a protective locus against tuberculosis

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2−/− mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-l-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.

The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.

A normal β-globin allele as a modifier gene ameliorating the severity of α-thalassemia in mice

Thalassemia is a heritable human anemia caused by a variety of mutations that affect expression of the α- or the β-chain of hemoglobin. The expressivity of the phenotype is likely to be influenced by unlinked modifying genes. Indeed, by using a mouse model of α-thalassemia, we find that its phenotype is strongly influenced by the genetic background in which the α-thalassemia mutation resides [129sv/ev/129sv/ev (severe) or 129sv/ev/C57BL/6 (mild)]. Linkage mapping indicates that the modifying gene is very tightly linked to the β-globin locus (Lod score = 13.3). Furthermore, the severity of the phenotype correlates with the size of β-chain-containing inclusion bodies that accumulate in red blood cells and likely accelerate their destruction. The β-major globin chains encoded by the two strains differ by three amino acids, one of which is a glycine-to-cysteine substitution at position 13. The Cys-13 should be available for interchain disulfide bridging and consequent aggregation between excess β-chains. This normal polymorphic variation between murine β-globin chains could account for the modifying action of the unlinked β-globin locus. Here, the variation in severity of the phenotype would not depend on a change in the ratio between α- and β-chains but on the chemical nature of the normal β-chain, which is in excess. This work also indicates that modifying genes can be normal variants that—absent an apparent physiologic rationale—may be difficult to identify on the basis of structure alone.