Exclusion of candidate genes and chromosomal regions in familial neuroblastoma

Two families with recurrence of neuroblastoma one Italian and one British with three and two affected children respectively were genotyped using polymorphic markers on chromosome 1 spanning the p32-p36 region frequently deleted in neuroblastoma tumor cells. Linkage to this region was excluded by haplotype inspection and negative lod scores. Furthermore, the exclusion of genes involved in neurocristopathies sometimes associated with neuroblastoma was carried out by typing the Italian family with polymorphic markers located in or near the corresponding genes. Finally, linkage analysis in the two families showed negative lod scores for markers spanning the 16p12-13 chromosomal region where a locus for familial neuroblastoma has been recently mapped. Our findings indicate that different genes are involved in the pathogenesis of familial neuroblastoma.     

Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization

Childhood neuroblastoma, an embryonal neoplasm of sympathetic nervous system progenitors, occurs in a familial form with an autosomal dominant mode of inheritance. Genetic susceptibility to this disorder is thought to arise via a germline mutation affecting a tumor suppressor gene, in accord with the two-hit model established for familial and sporadic retinoblastoma. Surprisingly, the familial neuroblastoma predisposition locus does not map to chromosome band 1p36, a genomic region likely to contain one or more neuroblastoma suppressor genes. We reasoned that inherited point mutations affecting one allele would be unmasked in many cases by somatically acquired deletions of the second allele that included the target gene in the tumor cells from these patients. Thus, to identify chromosomal regions that might contain suppressor genes important in hereditary neuroblastoma, we analyzed six familial tumors by comparative genomic hybridization. Recurrent losses of genetic material were detected on chromosome arms 3p (consensus region, 3p24-pter), 10p (consensus, 10p12-p13), 10q (consensus, 10q25-qter), 16q (consensus, 16q12-q22), and 20q (consensus, 20q13.3-qter), in addition to the regions commonly deleted in sporadic neuroblastomas (1p36 and 11q). These chromosomal sites may harbor novel tumor suppressor genes that could aid in our understanding of the predisposition to and pathogenesis of familial neuroblastoma and potentially sporadic tumors as well. Genes Chromosom. Cancer 19:176–184, 1997. © 1997 Wiley-Liss Inc.     

Nonsyndromic cleft lip and palate: four chromosomal regions of interest

Nonsyndromic cleft lip with or without cleft palate (NSCLP), a common birth defect affecting 1/700 live births and 4,000 newborns/year in the United States, is associated with short and long-term morbidity. As such, it has significant impact on the health care system. NSCLP is a complex disorder that results from the interaction of genetic and environmental factors that are slowly being defined. Genomic scans have suggested a number of regions that may contain NSCLP loci. In this study, we have evaluated regions identified by a previously published genomic scan of affected sib-pairs and have found evidence of association in sixty-five multiplex families for eight chromosomal regions. Four of these regions, 2q37, 11p12-14, 12q13, and 16p13, have also been identified in second genomic scan of multiplex families from China. One region, defined by D11S1392, gave evidence suggesting the presence of an NSCLP locus in all three studies. Altogether, these results suggest chromosomal regions that should be targeted in order to identify NSCLP loci.     

Meta-analysis of four new genome scans for lipid parameters and analysis of positional candidates in positive linkage regions

Lipid levels in plasma strongly influence the risk for coronary heart disease. To localise and subsequently identify genes affecting lipid levels, we performed four genome-wide linkage scans followed by combined linkage/association analysis. Genome-scans were performed in 701 dizygotic twin pairs from four samples with data on plasma levels of HDL- and LDL-cholesterol and their major protein constituents, apolipoprotein AI (ApoAI) and Apolipoprotein B (ApoB). To maximise power, the genome scans were analysed simultaneously using a well-established meta-analysis method that was newly applied to linkage analysis. Overall LOD scores were estimated using the means of the sample-specific quantitative trait locus (QTL) effects inversely weighted by the standard errors obtained using an inverse regression method. Possible heterogeneity was accounted for with a random effects model. Suggestive linkage for HDL-C was observed on 8p23.1 and 12q21.2 and for ApoAI on 1q21.3. For LDL-C and ApoB, linkage regions frequently coincided (2p24.1, 2q32.1, 19p13.2 and 19q13.31). Six of the putative QTLs replicated previous findings. After fine mapping, three maximum LOD scores mapped within 1 cM of major candidate genes, namely APOB (LOD=2.1), LDLR (LOD=1.9) and APOE (LOD=1.7). APOB haplotypes explained 27% of the QTL effect observed for LDL-C on 2p24.1 and reduced the LOD-score by 0.82. Accounting for the effect of the LDLR and APOE haplotypes did not change the LOD score close to the LDLR gene but abolished the linkage signal at the APOE gene. In conclusion, application of a new meta-analysis approach maximised the power to detect QTLs for lipid levels and improved the precision of their location estimate.     

Genome-wide analysis of extended pedigrees confirms IL2-IL21 linkage and shows additional regions of interest potentially influencing coeliac disease risk

Coeliac disease is a chronic inflammatory condition of the small intestine, triggered by dietary exposure to gluten in genetically susceptible individuals. Risk alleles at HLA-DQA1 and HLA-DQB1 are necessary for disease development, but are alone not sufficient for disease onset. We aimed to identify novel loci underlying susceptibility to coeliac disease through the use of extended Finnish and Hungarian families with multiple affected individuals. An initial whole-genome linkage approach yielded several loci that were followed up further using the Immunochip custom array. Loci with a parametric logarithm of odds (LOD) score of >1.3 were identified at 4q, 6p [human leukocyte antigen (HLA) region], 6q, 7p, 17p, 17q and at 22p. The 4q and 6q loci have been identified previously in coeliac disease risk, whereas follow-up analyses indicate that the 17p and 22p loci may be novel risk loci for coeliac disease. These loci harbour previously described risk variants for other autoimmune diseases, but their segregation patterns do not explain the linkage to coeliac disease. We followed up the linkage to the 4q region, containing the previously described interleukin (IL)2 and IL21 genes. The risk variants at 4q in the studied pedigrees are most likely distinct from previously described risk variants, indicating that the observed linkage may be due to rare high-risk variants of still unknown nature. The importance of this locus to coeliac disease risk was further shown by the finding that serum levels of IL21 were elevated in both untreated and treated coeliac patients compared to controls.     

Cosmid mapping and locus linkage within the human chromosomal region 22q13.1

Many human meningiomas show loss of heterozygosity at distal loci but retain constitutional heterozygosity at one or more proximal loci of 22q. Molecular analysis indicted deletions involving at least the region 22q12.3-qter. In this region, distal to myoglobin, the putative meningioma locus ought to be expected. Long-range mapping was performed around two loci from 22q12.3–q13.1 (D22S16 and PDGFB, the most proximal locus to be lost in meningioma). D22S16, originally assigned to 22q13-qter by isotopic in situ hybridization, was placed in the vicinity of PDGFB by utilizing a set of somatic cell hybrids, an assignment confirmed by fluorescence in situ hybridization (FISH) of a cosmid clone containing the D22S16 locus. Moreover, pulsed field gel electrophoresis suggests a close linkage of both markers within 630 kb.     

Combined segregation and linkage analysis of HLA markers in familial psoriasis

Marker-based segregation analysis (MBSA) is a modification of a published method of combined linkage and segregation analysis (Am J Hum Genet 51: 1111-1126, 1992), to determine whether a candidate gene known to be associated with the disease of interest is truly segregating with the disease in families. Here we outline the conceptual basis of MBSA and present a Monte Carlo method for significance testing. The method is applied to PSORS1, a locus within the major histocompatibility complex (MHC) for which linkage and linkage disequilibrium with psoriasis has already been demonstrated. The results are very consistent with our current knowledge of PSORS1, and suggest that MBSA can provide useful information on genotype-phenotype relationships such as penetrance and allelic heterogeneity.     

Bayesian analysis of haplotypes for linkage disequilibrium mapping

Haplotype analysis of disease chromosomes can help identify probable historical recombination events and localize disease mutations. Most available analyses use only marginal and pairwise allele frequency information. We have developed a Bayesian framework that utilizes full haplotype information to overcome various complications such as multiple founders, unphased chromosomes, data contamination, and incomplete marker data. A stochastic model is used to describe the dependence structure among several variables characterizing the observed haplotypes, for example, the ancestral haplotypes and their ages, mutation rate, recombination events, and the location of the disease mutation. An efficient Markov chain Monte Carlo algorithm was developed for computing the estimates of the quantities of interest. The method is shown to perform well in both real data sets (cystic fibrosis data and Friedreich ataxia data) and simulated data sets. The program that implements the proposed method, BLADE, as well as the two real datasets, can be obtained from http://www.fas.harvard.edu/~junliu/TechRept/01folder/diseq_prog.tar.gz.     

小儿尿道下裂与染色体异常的关系探讨

目的了解男性儿童以尿道下裂为主要症状的外生殖器畸形与染色体异常的关系。方法收集2002年~2008年9月间来我院就诊尿道下裂的患儿资料,每份病例都有完整的染色体检查和B超记录,有些需要有激素水平检测,必要时要有性腺活检。符合条件的共237例,根据临床表现分为两组：A组为单纯性尿道下裂共185例;B组为伴有其他外生殖器畸形组52例。结果 A组,发现6例为纯合或嵌合体（≥3%）的异常核型,发生率为3.2%;9例为低比例（≤3%）嵌合体,发生率4.9%。其中仅1例为性染色体异常,余为常染色体异常。B组,发现9例为纯合或嵌合体且全部为性染色体异常;1例常染色体异常的低比例嵌合体,发生率分别为17.3%、1.9%。10例染色体异常者中6例伴有双（或单）侧隐睾和（或）其他外生殖器畸形,4例只伴有其他畸形。结论本研究中,单纯性尿道下裂以常染色体异常为主;尿道下裂合并其他外生殖器畸形如隐睾、阴囊分裂、阴茎阴囊转位、小睾丸、小阴茎等要特别注意,性染色体异常率高（17.3%）。上述畸形要常规执行染色体核型分析。早期诊断、合理治疗、尽早进行遗传咨询,对于优生优育具有重要的指导意义。     

Association and linkage analysis of candidate chromosomal regions in multiple sclerosis: indication of disease genes in 12q23 and 7ptr-15

Four recent genome-wide screen studies in multiple sclerosis (MS) identified a number of candidate regions for susceptibility genes in addition to the HLA complex in 6p21. However, none of these regions provided formally significant evidence for genome-wide linkage. We have investigated such regions in 46 Swedish multiplex MS families, 28 singleton families, 190 sporadic MS patients and 148 normal controls by parametric and nonparametric linkage and association analysis. One microsatellite marker, in 12q23, provided evidence for association in addition to suggestive transmission distortion and slightly positive linkage. In addition, a marker in 7ptr-15 showed a significant transmission distortion as well as a highly significant score in affected pedigree member analysis, but not quite significant deviations in association analysis. One of three markers in 5p, a region implicated in all four previous studies, showed a weakly positive lod score, but no other evidence of importance. Markers in 2p23, 5q11-13, 6q25, 7q21-22, 11q21-23, 13q33-34, 16p13.2, 18p11.32-23, Xp21.3 provided little or no evidence of importance for MS. In summary, these data support the importance of genome-wide screens in the identification of new candidate loci in polygenic disorders.     

Genomewide linkage analysis of familial prostate cancer in the Japanese population

Prostate cancer (PC) is one of the most common causes of cancer mortality in Western countries, and familial aggregation of PC is well known. Multiple PC susceptibility loci have been reported in Western countries, but attempts to confirm the loci in independent data sets have proven to be inconsistent. We performed a genomewide linkage analysis with 53 affected sib pairs to identify genetic loci related to PC in a Japanese population. Two linkage analyses, GENEHUNTER-PLUS and SIBPAL, were applied and detected nominal statistical significance of linkage to PC at chromosome 1p and 8p, which were reported as being loci for PC in Caucasians. The best evidence of linkage was detected near D8S550 on 8p23 (maximum Z_lr=2.25, P=0.037), and the second-best evidence of linkage was observed near D1S2667 on 1p36 (maximum Z_lr=2.24, P=0.034). This is the first genetic mapping of PC in Japanese, and the results suggest that susceptibilities to PC lie close to D8S550 on 8p23 and D1S2667 on 1p36.     

Fine mapping of a schizophrenia susceptibility locus at chromosome 6q23: increased evidence for linkage and reduced linkage interval

We previously reported an autosomal scan for schizophrenia susceptibility loci in a systematically recruited sample of Arab Israeli families. The scan detected significant evidence for linkage at chromosome 6q23 with a nonparametric LOD score (NPL) of 4.60 (P=0.000004) and a multipoint parametric LOD score of 4.16. In order to refine this finding we typed 42 additional microsatellite markers on chromosome 6q between D6S1570 (99.01 cM from the pter) and D6S281 (190.14 from the pter) in the same sample (average intermarker distance approximately 1.7 cM). In the 23 cM region between D6S1715 and D6S311, markers were more closely spaced ( approximately 1.1 cM). Multipoint nonparametric and parametric and single point linkage analyses were performed. The peak NPL rose to 4.98 (P=0.00000058) at D6S1626 (136.97 cM), immediately adjacent to D6S292 (NPL 4.98, P=0.00000068), the marker that gave the highest NPL in the original genome scan, under the broad diagnostic category. The putative susceptibility region (NPL-1) was reduced from 12.0 to 4.96 cM. The peak multipoint parametric LOD score was 4.63 at D6S1626 under a dominant genetic model, core diagnostic category and the LOD-1 interval was 2.10 cM. The maximum single point LOD score (3.55, theta=0.01) was also at D6S1626 (dominant model, core diagnostic category). Increased evidence for linkage in the same sample as in the original genome scan and consistent localization of the linkage peak add further support for the presence of a schizophrenia susceptibility locus at chromosome 6q23. Moreover, the markedly reduced linkage interval greatly improves prospects for identifying a schizophrenia susceptibility gene within the implicated region.     

Fine deletion mapping on the long arm of chromosome 9 in sporadic and familial basal cell carcinomas

Basal cell carcinomas (BCCs) are the most common sporadic cancersworldwide. They are also a cardinal manifestation of a familialcancer predisposition syndrome, naevoid BCC syndrome (NBCCS).The gene responsible for NBCCS is likely to be a tumour suppressorgene and has been genetically mapped to a 2cM region betweenmicrosatellite markers, D9S196 and 38 famillal) were examinedfor loss of heterozygosity (LOH) in the candidate region ofthe NBCCS gene. Deletions were found in 46% and all LOH is consistentwith genetic mapping of the NBCC locus. These findings stronglysupport the hypothesis that inactivation of the putative tumoursuppressor, the NBCCS gene, is important in the formation ofsporadic BCCs. One sporadic tumour indicates that the smallestregion of overlap of these deletions is within the intervalbetween D9S287 and D9S180. If this is confirmed in additionaltumours, it would further narrow down the NBCCS region and excludeone candidate gene, that for the C complementation group ofFanconl anaemia, which maps proximally to D9S287. However, itwould not exclude another candidate, the gene for the A complementationgroup of xeroderma pigmentosum (XPAC). Evidence of imprintingwas also sought but preliminary data indicate that it is unlikelyto occur at the NBCCS locus.     

Family-based association studies of alpha-adrenergic receptor genes in chromosomal regions with linkage to bipolar affective disorder.

Several lines of evidence suggest an involvement of the noradrenergic neurotransmitter system in the pathogenesis of bipolar affective disorder (BPAD). Three genes for alpha-adrenergic receptors (ADRA) are located in chromosomal regions that showed evidence for linkage: The alpha(1c)-adrenergic (ADRA-1C) receptor gene on 8p21, the alpha(2a)-adrenergic (ADRA-2A) receptor gene on 10q25, and the alpha(2c)-adrenergic (ADRA-2C) receptor on 4p16. In a BPAD sample of 120 parent-offspring triads, we genotyped a 492 Cys/Arg variant in exon 2 of the ADRA-1C gene, a -1291 G/C variant in the 5'UTR of the ADRA-2A gene, and a STR marker (adra2c1) in the 5'UTR of the ADRA-2C gene. Using the Transmission Disequilibrium Test (TDT), no significant differences in transmissions were observed for any of the three ADRA genes.     

Integrated mapping,chromosomal sequencing and sequence analysis of Cryptosporidium parvum

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.     

Fine mapping and association studies of a high-density lipoprotein cholesterol linkage region on chromosome 16 in French-Canadian subjects

Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for cardiovascular disease. To identify novel genetic variants that contribute to HDL-C, we performed genome-wide scans and quantitative association studies in two study samples: a Quebec-wide study consisting of 11 multigenerational families and a study of 61 families from the Saguenay–Lac St-Jean (SLSJ) region of Quebec. The heritability of HDL-C in these study samples was 0.73 and 0.49, respectively. Variance components linkage methods identified a LOD score of 2.61 at 98 cM near the marker D16S515 in Quebec-wide families and an LOD score of 2.96 at 86 cM near the marker D16S2624 in SLSJ families. In the Quebec-wide sample, four families showed segregation over a 25.5-cM (18 Mb) region, which was further reduced to 6.6 Mb with additional markers. The coding regions of all genes within this region were sequenced. A missense variant in CHST6 segregated in four families and, with additional families, we observed a P value of 0.015 for this variant. However, an association study of this single-nucleotide polymorphism (SNP) in unrelated Quebec-wide samples was not significant. We also identified an SNP (rs11646677) in the same region, which was significantly associated with a low HDL-C (P=0.016) in the SLSJ study sample. In addition, RT-PCR results from cultured cells showed a significant difference in the expression of CHST6 and KIAA1576, another gene in the region. Our data constitute additional evidence for a locus on chromosome 16q23-24 that affects HDL-C levels in two independent French-Canadian studies.     

Combined segregation and linkage analysis of nonsyndromic orofacial cleft in two candidate regions

We applied a complex segregation analysis to 46 pedigrees with a total of 121 nuclear families and 660 individuals, to verify hypotheses regarding the inheritance of OFC and linkage with markers on chromosomes 6 and 2. The pointer program for segregation analysis strongly rejected the hypothesis of no familial transmission of OFC in these families. When the hypothesis of a two-locus model was tested with comds , the analysis showed the presence of at least two loci and the model assuming a dominant major gene and a recessive modifier locus was statistically accepted. Given the fitted two-locus model, we tested for a possible linkage between the major OFC locus and the two markers studied. For D6S259, the estimate of the recombination fraction was θ=0.098, corresponding to a LOD score around 2.1. On the contrary, the data analysis concerning the D2S378 marker showed an estimate of the recombination fraction not significantly different from the independence hypothesis.     

Fine mapping of whole RB1 gene deletions in retinoblastoma patients confirms PCDH8 as a candidate gene for psychomotor delay

Retinoblastoma (Rb) results from inactivation of both alleles of the RB1 gene located in 13q14.2. Whole-germline monoallelic deletions of the RB1 gene (6% of RB1 mutational spectrum) sometimes cause a variable degree of psychomotor delay and several dysmorphic abnormalities. Breakpoints in 12 Rb patients with or without psychomotor delay were mapped to specifically define the role of chromosomal regions adjacent to RB1 in psychomotor delay. A high-resolution CGH array focusing on RB1 and its flanking region was designed to precisely map the deletion. Comparative analysis detected a 4-Mb critical interval, including a candidate gene protocadherin 8 (PCDH8). PCDH8 is thought to function in signalling pathways and cell adhesion in a central nervous system-specific manner, making loss of PCDH8 one of the probable causes of psychomotor delay in RB1-deleted patients. Consequently, we propose to systematically use high-resolution CGH in cases of partial or complete RB1 deletion encompassing the telomeric flanking region to characterize the putative loss of PCDH8 and to better define genotype/phenotype correlations, eventually leading to optimized genetic counselling and psychomotor follow-up.