Genomic analysis of ERVWE2 locus in patients with multiple sclerosis: absence of genetic association but potential role of human endogenous retrovirus type W elements in molecular mimicry with myelin antigen

Human endogenous retroviruses (HERVs) arise from ancient infections of the host germline cells by exogenous retroviruses, constituting 8% of the human genome. Elevated level of envelope transcripts from HERVs-W has been detected in CSF, plasma and brain tissues from patients with Multiple Sclerosis (MS), most of them from Xq22.3, 15q21.3, and 6q21 chromosomes. However, since the locus Xq22.3 (ERVWE2) lack the 5' LTR promoter and the putative protein should be truncated due to a stop codon, we investigated the ERVWE2 genomic loci from 84 individuals, including MS patients with active HERV-W expression detected in PBMC. In addition, an automated search for promoter sequences in 20 kb nearby region of ERVWE2 reference sequence was performed. Several putative binding sites for cellular cofactors and enhancers were found, suggesting that transcription may occur via alternative promoters. However, ERVWE2 DNA sequencing of MS and healthy individuals revealed that all of them harbor a stop codon at site 39, undermining the expression of a full-length protein. Finally, since plaque formation in central nervous system (CNS) of MS patients is attributed to immunological mechanisms triggered by autoimmune attack against myelin, we also investigated the level of similarity between envelope protein and myelin oligodendrocyte glycoprotein (MOG). Comparison of the MOG to the envelope identified five retroviral regions similar to the Ig-like domain of MOG. Interestingly, one of them includes T and B cell epitopes, capable to induce T effector functions and circulating Abs in rats. In sum, although no DNA substitutions that would link ERVWE2 to the MS pathogeny was found, the similarity between the envelope protein to MOG extends the idea that ERVEW2 may be involved on the immunopathogenesis of MS, maybe facilitating the MOG recognizing by the immune system. Although awaiting experimental evidences, the data presented here may expand the scope of the endogenous retroviruses involvement on MS pathogenesis

Local cohomology with supports in the non-free locus

[EN] The groups of local cohomology with supports in the non-free locus of a module are used in order to obtain three classifications and one characterization of four classes of modules

Heterozygous deletion of the PU.1 locus in human AML

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to acute myeloid leukemia (AML) in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (single nucleotide polymorphisms [SNPs]) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal acute myeloid leukemia M2 was identified with heterozygosity in 3 of the SNPs in the URE at remission. Loss of heterozygosity was further found in this patient at 2 polymorphic sites in the 5' promoter region and in 2 intronic sites flanking exon 4, thus suggesting loss of heterozygosity covering at least 40 kb of the PU.1 locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

A locus on chromosome 5 is associated with dilated cardiomyopathy in Doberman Pinschers

Dilated cardiomyopathy (DCM) is a heterogeneous group of heart diseases with a strong genetic background. Currently, many human DCM cases exist where no causative mutation can be identified. DCM also occurs with high prevalence in several large dog breeds. In the Doberman Pinscher a specific DCM form characterized by arrhythmias and/or echocardiographic changes has been intensively studied by veterinary cardiologists. We performed a genome-wide association study in Doberman Pinschers. Using 71 cases and 70 controls collected in Germany we identified a genome-wide significant association to DCM on chromosome 5. We validated the association in an independent cohort collected in the United Kingdom. There is no known DCM candidate gene under the association signal. Therefore, DCM in Doberman Pinschers offers the chance of identifying a novel DCM gene that might also be relevant for human health.

Sequential Quantitative Trait Locus Mapping in Experimental Crosses

The etiology of complex diseases is heterogeneous. The presence of risk alleles in one or more genetic loci affects the function of a variety of intermediate biological pathways, resulting in the overt expression of disease. Hence, there is an increasing focus on identifying the genetic basis of disease by sytematically studying phenotypic traits pertaining to the underlying biological functions. In this paper we focus on identifying genetic loci linked to quantitative phenotypic traits in experimental crosses. Such genetic mapping methods often use a one stage design by genotyping all the markers of interest on the available subjects. A genome scan based on single locus or multi-locus models is used to identify the putative loci. Since the number of quantitative trait loci (QTLs) is very likely to be small relative to the number of markers genotyped, a one-stage selective genotyping approach is commonly used to reduce the genotyping burden, whereby markers are genotyped solely on individuals with extreme trait values. This approach is powerful in the presence of a single quantitative trait locus (QTL) but may result in substantial loss of information in the presence of multiple QTLs. Here we investigate the efficiency of sequential two stage designs to identify QTLs in experimental populations. Our investigations for backcross and F2 crosses suggest that genotyping all the markers on 60% of the subjects in Stage 1 and genotyping the chromosomes significant at 20% level using additional subjects in Stage 2 and testing using all the subjects provides an efficient approach to identify the QTLs and utilizes only 70% of the genotyping burden relative to a one stage design, regardless of the heritability and genotyping density. Complex traits are a consequence of multiple QTLs conferring main effects as well as epistatic interactions. We propose a two-stage analytic approach where a single-locus genome scan is conducted in Stage 1 to identify promising chromosomes, and interactions are examined using the loci on these chromosomes in Stage 2. We examine settings under which the two-stage analytic approach provides sufficient power to detect the putative QTLs.

Allelic Heterogeneity at the Equine KIT Locus in Dominant White (W) Horses

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from approximately 50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the approximately 82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations.

DNA methylation patterns at the IGF2-H19 locus in sperm of Swiss Landrace and Swiss Large White boars

DNA methylation patterns at the IGF2-H19 locus were investigated in sperm DNA from Swiss Landrace (SL) and Swiss Large White (LW) boars. The putative IGF2 differentially methylated regions (DMR) 0, 1 and 2, a quantitative trait nucleotide (QTN) region in the intron 3 and a CpG island in the intron 4 of the IGF2 gene as well as three regions around porcine CTCF binding sites within the H19 differentially methylated domain (DMD) were selected for the DNA methylation analysis. In both breeds putative IGF2 DMR0, 1, 2 and H19 DMD were hypermethylated. Significant differences in DNA methylation content were found between the two breeds in the two DMD regions proximal to the H19 gene. The IGF2 QTN region and the CpG island in the IGF2 intron 4 were hypomethylated in sperm DNA of both breeds. The methylation analysis revealed significantly more methylated CpG sites in the intron 4 of sperm from the LW breed than in that from SL. No difference was found in global DNA methylation between the two breeds. These results indicate differences in DNA methylation patterns between breeds and it remains to be established whether variation in DNA methylation patterns impacts on phenotypic traits.

Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for 'tracking' the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.

Genome-wide analysis in German shepherd dogs reveals association of a locus on CFA 27 with atopic dermatitis

Humans and dogs are both affected by the allergic skin disease atopic dermatitis (AD), caused by an interaction between genetic and environmental factors. The German shepherd dog (GSD) is a high-risk breed for canine AD (CAD). In this study, we used a Swedish cohort of GSDs as a model for human AD. Serum IgA levels are known to be lower in GSDs compared to other breeds. We detected significantly lower IgA levels in the CAD cases compared to controls (p = 1.1 × 10(-5)) in our study population. We also detected a separation within the GSD cohort, where dogs could be grouped into two different subpopulations. Disease prevalence differed significantly between the subpopulations contributing to population stratification (λ = 1.3), which was successfully corrected for using a mixed model approach. A genome-wide association analysis of CAD was performed (n cases = 91, n controls = 88). IgA levels were included in the model, due to the high correlation between CAD and low IgA levels. In addition, we detected a correlation between IgA levels and the age at the time of sampling (corr = 0.42, p = 3.0 × 10(-9)), thus age was included in the model. A genome-wide significant association was detected on chromosome 27 (praw = 3.1 × 10(-7), pgenome = 0.03). The total associated region was defined as a ~1.5-Mb-long haplotype including eight genes. Through targeted re-sequencing and additional genotyping of a subset of identified SNPs, we defined 11 smaller haplotype blocks within the associated region. Two blocks showed the strongest association to CAD. The ~209-kb region, defined by the two blocks, harbors only the PKP2 gene, encoding Plakophilin 2 expressed in the desmosomes and important for skin structure. Our results may yield further insight into the genetics behind both canine and human AD.

A non-coding genomic duplication at the HMX1 locus is associated with crop ears in highland cattle.

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.

Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis.

BACKGROUND: We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus. RESULTS: In this study, we examined the effect of CO2/HCO3(-), pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3(-) increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2(-) bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested. CONCLUSIONS: This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

Importance of the ebp (endocarditis- and biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection.

BACKGROUND: We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis- and biofilm-associated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475). METHODS: OG1RF and TX5475 were administered transurethrally either at an ~1 : 1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model. RESULTS: No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P=.0033) and bladders (P< or =.0001). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P=.015 to P=.049), and 50% infective doses of OG1RF for kidneys and bladder (9.1x10(1) and 3.5x10(3) cfu, respectively) were 2-3 log(10) lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475. CONCLUSION: The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

A molecular study of the rhodopsin locus in patients with retinitis pigmentosa

DNA for this study was collected from a sample of 133 retinitis pigmentosa (RP) patients and the rhodopsin locus molecularly analyzed by linkage and for disease specific mutations. The cohort of patients consisted of 85 individuals diagnosed with autosomal dominant RP (adRP), and 48 patients representing other forms of retinitis pigmentosa or retinal dystrophy related disease. In three large families with adRP rhodopsin was excluded from linkage to the disease locus. A search for subtle mutations in the rhodopsin coding region using single strand conformational polymorphisms (SSCP) and sequencing detected a total of 14 unique sequence variants in 24 unrelated patients. These variants included one splicing variant, 5168 -1G-A, one deletion variant of 17 base pairs causing a frame shift at codon 332, and 12 misense variants: Pro23His, Leu46Arg, Gly106Trp, Arg135Pro, Pro171Glu, Pro180Ala, Glu181Lys, Asp190Asn, His211Arg, Ser270Arg, Leu328Pro and Pro347Thr. All but three of the missense variants change amino acids that are evolutionarily conserved. The Pro23His mutation was found in 10 unrelated individuals with family histories of adRP and not in any normal controls (over 80 chromosomes tested). The Pro180Ala mutation was present in a patient with simplex RP and probably represents a new mutation. Three normal polymorphic nucleotide substitutions, A-269-G, T-3982-C, and G-5145-A, were also identified. We conclude, based on this study, that 25% of adRP cases are attributable to rhodopsin mutations.^ Clinical data, including ERG results and visual field testing, was available for patients with eleven different mutations. The eleven patients were all diagnosed with RP, however the severity of the disease varied with five patients mildly affected and diagnosed with type II adRP and 5 patients severely affected and diagnosed with type I adRP. The patient with simplex RP was mildly affected. The location of the mutations within the rhodopsin protein was randomly associated with the severity of the disease in those patients evaluated. However, four mutations, Pro23His, Leu46Arg, Pro347Thr, and 5168 -1G-A, are particularly interesting. The Pro23His mutation appears to have radiated from a recent common ancestor of the affected patients as all of them share a common haplotype at the rhodopsin locus. The Leu46Arg mutation causes an unusually severe form of RP. Hydropathy analysis of the mutated sequence revealed a marked change in the hydrophobicity of this first transmembrane spanning region. Codon 347 has been the target of multiple mutations with at least six documented changes at the position, significantly more than expected by a random distribution of mutations. Finally the splice-site variant is extremely variable in its expression in the family studied. Similar mutations have been reported in other cases of adRP and postulated to be involved in autosomal recessive RP (arRP). Mechanisms to account for the variable expression of rhodopsin mutations in relation to RP heterogeneity are discussed. (Abstract shortened by UMI.) ^

Mitotic mapping of a novel tumor suppressor locus on human chromosome 3q important in osteosarcoma tumorigenesis

Tumor-specific loss of constitutional heterozygosity by deletion, mitotic recombination or nondisjunction is a common mechanism for tumor suppressor allele inactivation. When loss of heterozygosity is the result of mitotic recombination, or a segmental deletion event, only a portion of the chromosome is lost. This information can be used to map the location of new tumor suppressor genes. In osteosarcoma, the highest frequencies of loss of heterozygosity have been reported for chromosomes 3q, 13q, 17p. On chromosomes 13q and 17p, allelic losses are associated with loss of function at the retinoblastoma susceptibility locus (RB1) and the p53 locus, respectively. Chromosome 3q is also of particular interest because the high percent of loss of heterozygosity (62%-75%) suggests the presence of another tumor suppressor important for osteosarcoma tumorigenesis. To localize this putative tumor suppressor gene, we used polymorphic markers on chromosome 3q to find the smallest common region of allele loss. This putative tumor suppressor was localized to a 700 kb region on chromosome 3q26.2 between the polymorphic loci D3S1282 and D3S1246. ^