Construction of YAC Contigs at Human Chromosome 11q22.3-q23.1 Region Covering the Ataxia Telangiectasia Locus

Ataxia telangiectasia (AT) is an autosomal recessive diseaseof unknown etiology associated with cerebellar ataxia, telangiectasia,immune dysfunction, higher cancer risk, genomic instabilityand hypersensitivity to ionizing radiation. The major AT loci,AT-A and AT-C, are shown to be closely linked at chromosome11q22–q23. The most recent genetic linkage mapping andlinkage disequilibrium analysis have localized the major ATloci to a sequence of approximately 850 kb between the markersD11S1819 and D11S1818. The isolation of yeast artificial chromosomesspanning the AT region is an essential step to identify thegene or genes responsible for the mutation(s). We isolated atotal of 20 YAC clones from three independent YAC libraries,using sequence tagged sites mapped in the AT region as primersfor PCR-based YAC screening. The PCR assay for the presenceor absence of 16 different DNA markers allowed us to constructand to order four YAC contigs at the AT region. One of the contigswhich consists of the 10 YAC clones, covers about 2 Mb of DNAat the boundary between Giemsa-positive band 11q22.3 and Giemsa-negativeband 11q23.1 and includes the entire region of the major ATlocus between D11S1819 and D11S1818. Thus, the YAC contigs willfacilitate the positional cloning approach for searching transcribedsequences from the defined genomic region.     

A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy

We recently described mutations of the neuronal sodium-channel alpha-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.     

Assessment of 2q23.1 microdeletion syndrome implicates MBD5 as a single causal locus of intellectual disability, epilepsy, and autism spectrum disorder

Persons with neurodevelopmental disorders or autism spectrum disorder (ASD) often harbor chromosomal microdeletions, yet the individual genetic contributors within these regions have not been systematically evaluated. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosomal region 2q23.1 and acquired 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints; aligned microdeletions to determine the critical region; assessed effects on mRNA expression; and examined medical records, photos, and clinical evaluations. We identified a single gene, methyl-CpG-binding domain 5 (MBD5), as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency of mRNA expression, intellectual disability, epilepsy, and autistic features. Fourteen alterations, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening, disrupted MBD5 alone. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. No copy-number alterations of MBD5 were observed in 7878 controls, suggesting MBD5 alterations are highly penetrant. We surveyed MBD5 coding variations among 747 ASD subjects compared to 2043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG-binding domain missense variant, p.79Gly>Glu (c.236G>A) (p = 0.012). These results suggest that genetic alterations of MBD5 cause features of 2q23.1 microdeletion syndrome and that this epigenetic regulator significantly contributes to ASD risk, warranting further consideration in research and clinical diagnostic screening and highlighting the importance of chromatin remodeling in the etiology of these complex disorders.     

A novel linkage to generalized vitiligo on 4q13-q21 identified in a genomewide linkage analysis of Chinese families

Generalized vitiligo is a common, autoimmune, familial-clustering depigmentary disorder of the skin and hair that results from selective destruction of melanocytes. Generalized vitiligo is likely a heterogeneous disease, with five susceptibility loci reported so far--on chromosomes 1p31, 6p21, 7q, 8p, and 17p13--in white populations. To investigate vitiligo susceptibility loci in the Chinese population, we performed a genomewide linkage analysis in 57 multiplex Chinese families, each with at least two affected siblings, and we identified interesting linkage evidence on 1p36, 4q13-q21, 6p21-p22, 6q24-q25, 14q12-q13, and 22q12. Subsequently, to extract more linkage information, we investigated our initial genomewide linkage findings in a follow-up analysis of 49 new families and additional markers. Our initial genomewide linkage analysis and our subsequent follow-up analysis have identified a novel linkage to vitiligo on 4q13-q21, with highly significant linkage evidence (a nonparametic LOD score of 4.62 [P=.000003] and a heterogeneity LOD score of 4.01, under a recessive inheritance model), suggesting that 4q13-q21 likely harbors a major susceptibility locus for vitiligo in the Chinese population. We observed a minimal overlap between the linkage results of our current genomewide analysis in the Chinese population and the results of previous analyses in white populations, and we thus hypothesize that, as a polygenic disorder, vitiligo may be associated with great genetic heterogeneity and a substantial difference in its genetic basis between ethnic populations.     

Genome-wide scan and fine-mapping linkage study of androgenetic alopecia reveals a locus on chromosome 3q26

Androgenetic alopecia (AGA, male pattern baldness) is the most common form of hair loss. The origin of AGA is genetic, with the X chromosome located androgen receptor gene (AR) being the only risk gene identified to date. We present the results of a genome-wide linkage study of 95 families and linkage fine mapping of the 3q21-q29, 11q14-q25, 18p11-q23, and 19p13-q13 regions in an extended sample of 125 families of German descent. The locus with strongest evidence for linkage was mapped to 3q26 with a nonparametric linkage (NPL) score of 3.97 (empirical p value = 0.00055). This is the first step toward the identification of new susceptibility genes in AGA, a process which will provide important insights into the molecular and cellular basis of scalp hair loss.     

Autozygosity mapping of a seckel syndrome locus to chromosome 3q22. 1-q24

Seckel syndrome (MIM 210600) is an autosomal recessive disorder of low birth weight, severe microcephaly, and dysmorphic facial appearance with receding forehead, prominent nose, and micrognathia. We have performed a genomic screen in two consanguineous families of Pakistani origin and found that the disorder segregates with markers between loci D3S1316 and D3S3710, which map to chromosome 3q22.1-q24. Analysis using HOMOZ/MAPMAKER gave a maximum LOD score of 8.72. All five affected individuals were homozygous for the same allele, for two adjacent polymorphic markers within the region segregating with the disease, narrowing the region to 12 cM.     

Genetic mapping of the Camurati-Engelmann disease locus to chromosome 19q13.1-q13.3

Camurati-Engelmann disease (CED [MIM 131300]), or progressive diaphyseal dysplasia, is an autosomal dominant sclerosing bone dysplasia characterized by progressive bone formation along the periosteal and endosteal surfaces at the diaphyseal and metaphyseal regions of long bones and cranial hyperostosis, particularly at the skull base. The gene for CED, or its chromosomal localization, has not yet been identified. We performed a genomewide linkage analysis of two unrelated Japanese families with CED, in which a total of 27 members were available for this study; 16 of them were affected with the disease. Two-point linkage analysis revealed a maximum LOD score of 7.41 (recombination fraction.00; penetrance 1.00) for the D19S918 microsatellite marker locus. Haplotype analysis revealed that all the affected individuals shared a common haplotype observed, in each family, between D19S881 and D19S606, at chromosome 19q13.1-q13.3. These findings, together with a genetic distance among the marker loci, indicate that the CED locus can be assigned to a 15.1-cM segment between D19S881 and D19S606.     

SCN1A rs3812718 polymorphism and susceptibility to epilepsy with febrile seizures: A meta-analysis

Background

Evidence showed that the SCN1A IVS5N+5G>A polymorphism might be associated with susceptibility to epilepsy with febrile seizures (EFS), however, the published data were inconclusive. Therefore, a meta-analysis was performed to estimate the overall EFS risk with the polymorphism.

Methods

The PubMed and Medline were searched up to March, 2013 for studies on the association between SCN1A IVS5N+5G>A polymorphism and EFS risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by means of a genetic model free approach. The heterogeneity and sensitivity of each report and the publication bias were also performed. All the statistical analyses were done using the STATA 11.0 software.

Result

A total of 6 studies with 2719 cases and 2317 controls met the selection criteria. We found significant association between SCN1A polymorphism and EFS (A vs. G: OR = 1.498, 95%CI = 1.138–1.972; AA vs. GG: OR = 2.292, 95%CI = 1.620–3.243; AG vs. GG: OR = 1.414, 95%CI = 1.010–1.978; recessive model: OR = 1.747, 95%CI = 1.119–2.728 and dominant model: OR = 1.730, 95%CI = 1.259–2.376). When compared with the epilepsy without febrile seizure (EWFS), the subgroup analysis stratified by ethnicity showed that the SNP was significantly associated with EFS in Caucasian (A vs. G: OR = 1.505, 95%CI = 1.218–1.861; AA vs. GG: OR = 2.081, 95%CI = 1.358–3.189; recessive model: OR = 1.715, 95%CI = 1.273–2.310 and dominant model: OR = 1.625, 95%CI = 1.096–2.410), but not in Indian and Chinese. When applying Bonferroni correction (significance was set at 0.05/20), the Caucasian still has robust association with EFS and epilepsy.

Conclusion

The present meta-analysis suggests that SCN1A IVS5N+5G>A polymorphism is a risk factor of EFS and epilepsy, especially in Caucasian.     

Significant evidence for linkage of mite-sensitive childhood asthma to chromosome 5q31-q33 near the interleukin 12 B locus by a genome-wide search in Japanese families

Childhood-onset asthma is frequently found in association with atopy. Although asthmatic children may develop IgE antibodies against variety of allergens, asthma is associated primarily with allergy to house-dust mites, molds, or other allergens. In this study, we conducted a genome-wide linkage search in 47 Japanese families (197 members) with more than two mite-sensitive atopic asthmatics (65 affected sib-pairs) using 398 markers. Multipoint linkage analysis was carried out for atopic asthma as a qualitative trait using the MAPMAKER/SIB program. We observed significant evidence for linkage with maximum lod scores (MLS) of 4.8 near the interleukin 12 B gene locus on chromosome 5q31-q33. In addition, suggestive evidence on 4q35 with MLS = 2.7 and on 13q11 with MLS = 2.4 was obtained. The other possible linkage regions included 6p22-p21.3 (MLS = 2.1), 12q21-q23 (MLS = 1.9), and 13q14.1-q14.3 (MLS = 2.0). Many of the linkage loci suggested in this study were at or close to those suggested by genome-wide studies for asthma in Caucasian populations. The present study suggests the contribution of the interleukin 12 B gene or nearby gene(s) to mite-sensitive atopic asthma and a considerable number of genetic variants common across Caucasians and Japanese populations contributing to asthma, although the relative importance of various variants may differ between the groups.     

Childhood absence epilepsy with tonic-clonic seizures and electroencephalogram 3-4-Hz spike and multispike-slow wave complexes: linkage to chromosome 8q24.

Childhood absence epilepsy (CAE), a common form of idiopathic generalized epilepsy, accounts for 5%-15% of childhood epilepsies. To map the chromosomal locus of persisting CAE, we studied the clinical and electroencephalographic traits of 78 members of a five-generation family from Bombay, India. The model-free affected-pedigree member method was used during initial screening with chromosome 6p, 8q, and 1p microsatellites, and only individuals with absence seizures and/or electroencephalogram 3-4-Hz spike- and multispike-slow wave complexes were considered to be affected. Significant P values of .00000-.02 for several markers on 8q were obtained. Two-point linkage analysis, assuming autosomal dominant inheritance with 50% penetrance, yielded a maximum LOD score (Zmax) of 3.6 for D8S502. No other locus in the genome achieved a significant Zmax. For five smaller multiplex families, summed Zmax was 2.4 for D8S537 and 1.7 for D8S1761. Haplotypes composed of the same 8q24 microsatellites segregated with affected members of the large family from India and with all five smaller families. Recombinations positioned the CAE gene in a 3.2-cM interval.     

Sex- and age-dependent DNA methylation at the 17q12-q21 locus associated with childhood asthma

Chromosomal region 17q12-q21 is one of the best-replicated genome-wide association study (GWAS) hits and associated with childhood-onset asthma. However, the mechanism by which the genetic association is restricted to childhood-onset disease is unclear. During childhood, more boys than girls develop asthma. Therefore, we tested the hypothesis that the 17q12-q21 genetic association was sex-specific. Indeed, a TDT test showed that in the Saguenay-Lac-Saint-Jean familial collection, the 17q12-q21 association was significant among male, but not among female asthmatic subjects. We next hypothesized that the bias in the genetic association resulted from sex-specific and/or age-dependent DNA methylation at regulatory regions and determined the methylation profiles of five 17q12-q21 gene promoters using the bisulfite sequencing methylation assay. We identified a single regulatory region within the zona pellucida binding protein 2 (ZPBP2) gene, which showed statistically significant differences between males and females with respect to DNA methylation. DNA methylation also varied with age and was higher in adult males compared to boys. We have recently identified two functionally important polymorphisms, both within the ZPBP2 gene that influence expression levels of neighboring genes. Combined with the results of the present work, these data converge pointing to the same 5 kb region within the ZPBP2 gene as a critical region for both gene expression regulation and predisposition to asthma. Our data show that sex- and age-dependent DNA methylation may act as a modifier of genetic effects and influence the results of genetic association studies.     

Genome-wide high-density SNP-based linkage analysis of infantile hypertrophic pyloric stenosis identifies loci on chromosomes 11q14-q22 and Xq23

Infantile hypertrophic pyloric stenosis (IHPS) has an incidence of 1-8 per 1000 live births and is inherited as a complex sex-modified multifactorial trait with a striking male preponderance. Syndromic and monogenic forms exist, and two loci have been identified. Infants present with vomiting due to gastric-outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. A genome-wide SNP-based high-density linkage scan was carried out on 81 IHPS pedigrees. Nonparametric and parametric linkage analysis identified loci on chromosomes 11q14-q22 (Z(max) = 3.9, p < 0.0001; HLOD(max) = 3.4, alpha = 0.34) and Xq23 (Z(max) = 4.3, p < 0.00001; HLOD(max) = 4.8, alpha = 0.56). The two linked chromosomal regions each harbor functional candidate genes that are members of the canonical transient receptor potential (TRPC) family of ion channels and have a potential role in smooth-muscle control and hypertrophy.     

Refined genetic and physical localization of the Wagner disease (WGN1) locus and the genes CRTL1 and CSPG2 to a 2- to 2.5-cM region of chromosome 5q14.3

Wagner syndrome (WGN1; MIM 143200), an autosomal dominant vitreoretinopathy characterized by chorioretinal atrophy, cataract, and retinal detachment, is linked to 5q14.3. Other vitreoretinopathies without systemic stigmata, including erosive vitreoretinopathy, are also linked to this region and are likely to be allelic. Within the critical region lie genes encoding two extracellular macromolecules, link protein (CRTL1) and versican (CSPG2), which are important in binding hyaluronan, a significant component of the mammalian vitreous gel, and which therefore represent excellent candidates for Wagner syndrome. Genetic mapping presented here in two further families reduces the critical region to approximately 2 cM. Subsequent refinement of the physical map allows ordering of known polymorphic microsatellites and excludes CRTL1 as a likely candidate for the disorder. CSPG2 is shown to lie within the critical region; however, analysis of the complete coding region of the mature peptide reveals no clear evidence that it is the gene underlying WGN1.     

Linkage analysis of 5 novel van der Woude syndrome kindreds to 1q32-q41 markers further supports locus homogeneity of the disease trait.

van der Woude syndrome (vWS, MIM 119300) is a rare autosomal dominant clefting condition with cardinal features of mucous cysts (lower-lip pits) and clefts to the lip and/or palate. The vWS gene has been assigned to a locus in 1q32-q41 by linkage analysis and physical mapping. We have investigated 5 novel vWS families through probands attended for cleft lip and/or palate repair at the Department of Maxillofacial Surgery of H?pital Trousseau, Paris, in order to tentatively refine the genetic map of the vWS region in 1q32-q41 and possibly identify unlinked pedigrees. Linkage analysis was carried out to 6 microsatellite markers (D1S249, D1S425, D1S491, D1S205, D1S414, D1S425), yielding a maximum cumulative LOD score of Z = 3.27 at theta = 0.00 for D1S245. The innermost four markers were found to be tightly linked to one another, with no evidence for recombination. Our results support linkage of vWS within a region of tightly linked markers and do not favour locus heterogeneity of the disease trait.     

Dinucleotide repeat polymorphism at the D4S2458 locus close to the PKD2 locus on human chromosome 4q

A new polymorphic CA repeat sequence was identified within the candidate region fot the autosomal dominant polycystic kidney disease type 2 (PKD2) locus. It should be a useful marker in the localization of this gene.     

Physical localisation of the chromosomal marker D13S31 places the Wilson disease locus at the junction of bands q14.3 and q21.1 of chromosome 13

D13S31 is the marker closest to the Wilson disease locus according to genetic analysis. Its physical localisation was refined by fluorescent in situ hybridisation to the junction to chromosomal bands 13q14.3 and 13q21.1. Using polymerase chain reaction analysis, D13S31 and D13S59 (the closest proximal and distal marker, respectively) were found to be located on the end of the der(13) consisting of 13pter-13q14.3: in the somatic cell hybrid ICD, and to be absent from the cell lines WC-H38B3B6 containing a del(13) (13pter-q13::13q21.1-qter) and KSF39 containing a del(13) (13pter-q14.1:).