A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

Copy number variation in patients with cervical artery dissection.

Cervical artery dissection (CeAD) occurs in healthy young individuals and often entails ischemic stroke. Skin biopsies from most CeAD-patients show minor connective tissue alterations. We search for rare genetic deletions and duplication that may predispose to CeAD. Forty-nine non-traumatic CeAD-patients with electron microscopic (EM) alterations of their dermal connective tissue (EM+ patients) and 21 patients with normal connective tissue in skin biopsies (EM- patients) were analyzed. Affymetrix 6.0 microarrays (Affymetrix) from all patients were screened for copy number variants (CNVs). CNVs absent from 403 control subjects and from 2402 published disease-free individuals were considered as CeAD-associated. The genetic content of undentified CNVs was analyzed by means of the Gene Ontology (GO) Term Mapper to detect associations with biological processes. In 49 EM+ patients we identified 13 CeAD-associated CNVs harboring 83 protein-coding genes. In 21 EM- patients we found five CeAD-associated CNVs containing only nine genes (comparison of CNV gene density between the groups: Mann-Whitney P=0.039). Patients' CNVs were enriched for genes involved in extracellular matrix organization (COL5A2, COL3A1, SNTA1, P=0.035), collagen fibril organization COL5A2, COL3A1, (P=0.0001) and possibly for genes involved in transforming growth factor beta (TGF)-beta receptor signaling pathway (COL3A1, DUPS22, P=0.068). We conclude that rare genetic variants may contribute to the pathogenesis of CeAD, in particular in patients with a microscopic connective tissue phenotype.

Increased expression of cell-cell signaling genes by stimulated mononuclear leukocytes in patients with previous atherothrombotic stroke. A whole genome expression profile study.

BACKGROUND/AIMS: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke. METHODS: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests. RESULTS: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell-cell signaling. CONCLUSION: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell-cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result.

Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro.

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.

Genetic analysis of familial connective tissue alterations associated with cervical artery dissections suggests locus heterogeneity.

BACKGROUND AND PURPOSE: Cervical artery dissections (CAD) can be associated with connective tissue aberrations in skin biopsies. The analysis of healthy relatives of patients suggested that the connective tissue phenotype is familial with an autosomal dominant inheritance. METHODS: We performed genetic linkage studies in 3 families of patients with CAD. Connective tissue phenotypes for the patients and all family members were assessed by electron microscopic study of skin biopsies. A genome-wide linkage analysis of 1 family (1 patient with 8 healthy relatives) indicated 2 candidate loci. Three genes were subsequently studied by sequence analysis. Part of the genome was also studied by linkage analysis in 2 further families. RESULTS: The genome-wide scan in a single family suggested linkage between the hypothetical mutation causing the connective tissue phenotype and informative genetic markers on chromosome 15q24 (logarithm of the odds score: Z= +2.1). A second possible candidate locus (Z=+1.9) was found on chromosome 10q26. Sequence analysis of 3 candidate genes in the suggestive locus (chondroitin sulfate proteoglycan4 [CSPG4], lysyl oxidase-like1 [LOXL1] and fibroblast growth factor receptor2 [FGFR2]) did not lead to the identification of a mutation responsible for connective tissue alterations. In 2 additional smaller families the loci on chromosome 15q24 and 10q26 were excluded by linkage analysis. CONCLUSIONS: Linkage analysis of a large family with CAD-associated connective tissue alterations suggested the presence of a candidate locus on chromosome 15q2 or on chromosome 10q26. Sequence analysis did not lead to the identification of a mutated candidate gene in 1 of these loci. The study of 2 additional pedigrees indicated locus heterogeneity for the connective tissue phenotype of CAD patients.

Multiple levels of regulation of the interleukin-6 system in stroke.

BACKGROUND AND PURPOSE: Serum levels of the cytokine interleukin-6 (IL-6) rise markedly in stroke. IL-6 is a key regulator of inflammatory mechanisms that play an important part in stroke pathophysiology. The action of IL-6 is modified by its soluble receptor subunits sgp130 and sIL-6R. The purpose of this study was to investigate whether serum levels of the receptor subunits are changed after ischemic stroke and to define the role of genetic influences on IL-6 expression in acute stroke. METHODS: In 48 patients with acute stroke and 48 age- and sex-matched control subjects, serum concentrations of IL-6, sgp130, and sIL-6R were measured by enzyme-linked immunosorbent assay. Furthermore, IL-6 promoter haplotypes comprising 4 different polymorphisms (-597G-->A, -572G-->C, -373A(n)T(n), -174G-->C) were determined by DNA sequencing and allele-specific oligonucleotide polymerase chain reaction. The effect of the common haplotypes on IL-6 gene transcription was tested by transfecting reporter fusion genes in the astrocytelike cell line U373. RESULTS: Whereas serum concentrations of IL-6 significantly rose (P<0.001), sgp130 levels were transiently reduced after stroke (P<0.05), and sIL-6R levels remained unchanged. IL-6 levels depended on the infarct size and the haplotype of the promoter region. The common haplotype A-G-8/12-C was associated with low IL-6 levels after stroke and a reduced induction of IL-6 transcription on stimulation with an adenosine analog in vitro. CONCLUSIONS: The data demonstrate genetic variation in the expression of IL-6 in stroke. Induction of the inflammatory response by IL-6 might be enhanced by a transient downregulation of the potential IL-6 antagonist sgp130.

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control.

We have identified parameters which define a causal role of HPV16 in head and neck cancer. Twenty-eight tumours which were typed positive for HPV16 DNA, were comprehensively analysed for expression of the viral oncogenes E6 and E7, the status of the p53 gene, and the protein status of pRb and p16(INK4a). In a subset of cases, we have searched for integrated viral DNA, and have determined the genomic status of the E6 gene. Expression of E6/E7 was found in 12 tumours most of which were derived from the oropharynx, whereas p53 mutations were present in 13 tumours from various sites. The tumours either carried p53 mutations but did not express E6/E7, or they did express E6/E7 but were p53-wild-type. Coexistence of E6/E7 expression with a mutated p53 was found in only one case. Strikingly, in most p53-mutated tumours without E6/E7 expression, we found the E6 gene to be disrupted. E6/E7 expression was associated with reduced pRb and overexpressed p16(INK4a). Viral-cellular fusion transcripts were found in two cases. Our data demonstrate that HPV16 DNA-positivity in head and neck cancers is not indicative of a causal role. A causal role of HPV16 in head and neck cancer is defined by: E6/E7 expression, viral integration with an intact E6 gene, and perturbation of pRb cell cycle control. Mostly, the p53 gene is wild-type.