A genomewide screen for autism: strong evidence for linkage to chromosomes 2q, 7q, and 16p

Autism is characterized by impairments in reciprocal communication and social interaction and by repetitive and stereotyped patterns of activities and interests. Evidence for a strong underlying genetic predisposition comes from twin and family studies, although susceptibility genes have not yet been identified. A whole-genome screen for linkage, using 83 sib pairs with autism, has been completed, and 119 markers have been genotyped in 13 candidate regions in a further 69 sib pairs. The addition of new families and markers provides further support for previous reports of linkages on chromosomes 7q and 16p. Two new regions of linkage have also been identified on chromosomes 2q and 17q. The most significant finding was a multipoint maximum LOD score (MLS) of 3.74 at marker D2S2188 on chromosome 2; this MLS increased to 4.80 when only sib pairs fulfilling strict diagnostic criteria were included. The susceptibility region on chromosome 7 was the next most significant, generating a multipoint MLS of 3.20 at marker D7S477. Chromosome 16 generated a multipoint MLS of 2.93 at D16S3102, whereas chromosome 17 generated a multipoint MLS of 2.34 at HTTINT2. With the addition of new families, there was no increased allele sharing at a number of other loci originally showing some evidence of linkage. These results support the continuing collection of multiplex sib-pair families to identify autism-susceptibility genes.     

Use of monozygotic twins in search for breast cancer susceptibility loci.

We have used Swedish monozygotic twins concordant for breast cancer to study genetic changes associated with the development of breast cancer. Because loss of heterozygosity (LOH) at a specific genomic region may reflect the presence of a tumour suppressor gene, loss of the same allele in both of the twins concordant for breast cancer may pinpoint a tumour suppressor gene that confers a strong predisposition to breast cancer. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a set of microsatellite markers on chromosomes 1, 13, 16 and 17, containing loci with known tumour suppressor genes. The two main regions, where more twin pairs than expected had lost the same allele, were located at 16qtel', including markers D16S393, D16S305 and D16S413, and at 17p13, distal to the p53 locus. Our results show that the monozygotic twin model can be used to suggest candidate regions of potential tumour suppressor genes, even with a limited number of twin pairs.     

Genes in the vicinity of <Emphasis Type="Italic">CFTR </Emphasis>modulate the cystic fibrosis phenotype in highly concordant or discordant F508del homozygous sib pairs

Cystic fibrosis (CF) is the most common severe autosomal recessive disease among Caucasians and is caused by lesions within the cystic fibrosis transmembrane conductance regulator ( CFTR) gene. The variability of CF disease severity suggests the effect of modifying factors. Thirty-four highly concordant and highly discordant F508del homozygous sib pairs, who have been selected out of a group of 114 pairs for extreme disease phenotypes by nutritional and pulmonary status, were typed at single nucleotide polymorphisms (SNPs) and short tandem repeat polymorphisms (STRPs) in the 24-cM CFTR-spanning region between D7S525 and D7S495. Allele frequencies differed significantly at D7S495, located within a 21-cM distance 3' of CFTR, comparing concordant mildly affected, concordant severely affected and discordant sib pairs, as judged by hypothesis-free permutation analysis by Monte Carlo simulation. A rare haplotype of two SNPs within the leptin gene promotor was found exclusively among the concordant mildly affected pairs. All concordant sib pairs shared the paternal F508del chromosome between CFTR and D7S495, whilst the cohort of discordant sib pairs inherited equal proportions of recombined and non-recombined parental chromosomes. We conclude that disease manifestation in CF is modulated by loci in the partially imprinted region 3' of CFTR that determine stature, food intake and energy homeostasis, such as the Silver-Russel-Syndrome candidate gene region and LEP.     

A genomewide scan for loci involved in attention-deficit/hyperactivity disorder

Attention deficit/hyperactivity disorder (ADHD) is a common heritable disorder with a childhood onset. Molecular genetic studies of ADHD have previously focused on examining the roles of specific candidate genes, primarily those involved in dopaminergic pathways. We have performed the first systematic genomewide linkage scan for loci influencing ADHD in 126 affected sib pairs, using a approximately 10-cM grid of microsatellite markers. Allele-sharing linkage methods enabled us to exclude any loci with a lambda(s) of > or =3 from 96% of the genome and those with a lambda(s) of > or =2.5 from 91%, indicating that there is unlikely to be a major gene involved in ADHD susceptibility in our sample. Under a strict diagnostic scheme we could exclude all screened regions of the X chromosome for a locus-specific lambda(s) of >/=2 in brother-brother pairs, demonstrating that the excess of affected males with ADHD is probably not attributable to a major X-linked effect. Qualitative trait maximum LOD score analyses pointed to a number of chromosomal sites that may contain genetic risk factors of moderate effect. None exceeded genomewide significance thresholds, but LOD scores were >1.5 for regions on 5p12, 10q26, 12q23, and 16p13. Quantitative-trait analysis of ADHD symptom counts implicated a region on 12p13 (maximum LOD 2.6) that also yielded a LOD >1 when qualitative methods were used. A survey of regions containing 36 genes that have been proposed as candidates for ADHD indicated that 29 of these genes, including DRD4 and DAT1, could be excluded for a lambda(s) of 2. Only three of the candidates-DRD5, 5HTT, and CALCYON-coincided with sites of positive linkage identified by our screen. Two of the regions highlighted in the present study, 2q24 and 16p13, coincided with the top linkage peaks reported by a recent genome-scan study of autistic sib pairs.     

Genomewide scan of hoarding in sib pairs in which both sibs have Gilles de la Tourette syndrome

A genome scan of the hoarding phenotype (a component of obsessive-compulsive disorder) was conducted on 77 sib pairs collected by the Tourette Syndrome Association International Consortium for Genetics (TSAICG). All sib pairs were concordant for a diagnosis of Gilles de la Tourette syndrome (GTS). However, the analyses reported here were conducted for hoarding as both a dichotomous trait and a quantitative trait. Not all sib pairs in the sample were concordant for hoarding. Standard linkage analyses were performed using GENEHUNTER and Haseman-Elston methods. In addition, novel analyses with a recursive-partitioning technique were employed. Significant allele sharing was observed for both the dichotomous and the quantitative hoarding phenotypes for markers at 4q34-35 (P=.0007), by use of GENEHUNTER, and at 5q35.2-35.3 (P=.000002) and 17q25 (P=.00002), by use of the revisited Haseman-Elston method. The 4q site is in proximity to D4S1625, which was identified by the TSAICG as a region linked to the GTS phenotype. The recursive-partitioning technique examined multiple markers simultaneously. Results suggest joint effects of specific loci on 5q and 4q, with an overall P value of.000003. Although P values were not adjusted for multiple comparison, nearly all were much smaller than the customary significance level of.0001 for genomewide scans.     

Pleiotropic effects of a chromosome 3 locus on speech-sound disorder and reading

Speech-sound disorder (SSD) is a complex behavioral disorder characterized by speech-sound production errors associated with deficits in articulation, phonological processes, and cognitive linguistic processes. SSD is prevalent in childhood and is comorbid with disorders of language, spelling, and reading disability, or dyslexia. Previous research suggests that developmental problems in domains associated with speech and language acquisition place a child at risk for dyslexia. Recent genetic studies have identified several candidate regions for dyslexia, including one on chromosome 3 segregating in a large Finnish pedigree. To explore common genetic influences on SSD and reading, we examined linkage for several quantitative traits to markers in the pericentrometric region of chromosome 3 in 77 families ascertained through a child with SSD. The quantitative scores measured several processes underlying speech-sound production, including phonological memory, phonological representation, articulation, receptive and expressive vocabulary, and reading decoding and comprehension skills. Model-free linkage analysis was followed by identification of sib pairs with linkage and construction of core shared haplotypes. In our multipoint analyses, measures of phonological memory demonstrated the strongest linkage (marker D3S2465, P=5.6 x 10(-5), and marker D3S3716, P=6.8 x 10(-4)). Tests for single-word decoding also demonstrated linkage (real word reading: marker D3S2465, P=.004; nonsense word reading: marker D3S1595, P=.005). The minimum shared haplotype in sib pairs with similar trait values spans 4.9 cM and is bounded by markers D3S3049 and D3S3045. Our results suggest that domains common to SSD and dyslexia are pleiotropically influenced by a putative quantitative trait locus on chromosome 3.     

Association of the chromosomal region 2q35 with type 1 diabetes mellitus in the Russian patients from Moscow

To map human chromosome 2 region associated with type 1 diabetes mellitus, 89 families with concordant and discordant sib pairs were analyzed. Linkage and association with type 1 diabetes were examined using polymorphic microsatellite markers spanning the region of about 4 Mb. The linkage plot was constructed, and association of the five microsatellite markers within the chromosomal region 2q35 was examined. Polymorphic marker D2S137 (Z′ = 3.225, p _c = 0.0048) demonstrated maximum linkage and association with type 1 diabetes.     

A genomewide linkage scan for quantitative-trait loci for obesity phenotypes

Obesity is an increasingly serious health problem in the world. Body mass index (BMI), percentage fat mass, and body fat mass are important indices of obesity. For a sample of pedigrees that contains >10,000 relative pairs (including 1,249 sib pairs) that are useful for linkage analyses, we performed a whole-genome linkage scan, using 380 microsatellite markers to identify genomic regions that may contain quantitative-trait loci (QTLs) for obesity. Each pedigree was ascertained through a proband who has extremely low bone mass, which translates into a low BMI. A major QTL for BMI was identified on 2q14 near the marker D2S347 with a LOD score of 4.04 in two-point analysis and a maximum LOD score (MLS) of 4.44 in multipoint analysis. The genomic region near 2q14 also achieved an MLS >2.0 for percentage of fat mass and body fat mass. For the putative QTL on 2q14, as much as 28.2% of BMI variation (after adjustment for age and sex) may be attributable to this locus. In addition, several other genomic regions that may contain obesity-related QTLs are suggested. For example, 1p36 near the marker D1S468 may contain a QTL for BMI variation, with a LOD score of 2.75 in two-point analysis and an MLS of 2.09 in multipoint analysis. The genomic regions identified in this and earlier reports are compared for further exploration in extension studies that use larger samples and/or denser markers for confirmation and fine-mapping studies, to eventually identify major functional genes involved in obesity.     

Evidence for Linkage of Bipolar Disorder to Chromosome 18 with a Parent-of-Origin Effect

A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sib-pair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = .0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father's sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; θ = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study.     

Genome search in celiac disease.

Celiac disease (CD), a malabsorption disorder of the small intestine, results from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the entire genetic susceptibility. To determine the localization of other genetic risk factors, a systematic screening of the genome has been undertaken. The typing information of 281 markers on 110 affected sib pairs and their parents was used to test linkage. Systematic linkage analysis was first performed on 39 pairs in which both sibs had a symptomatic form of CD. Replication of the regions of interest was then carried out on 71 pairs in which one sib had a symptomatic form and the other a silent form of CD. In addition to the HLA loci, our study suggests that a risk factor in 5qter is involved in both forms of CD (symptomatic and silent). Furthermore, a factor on 11qter possibly differentiates the two forms. In contrast, none of the regions recently published was confirmed by the present screening.     

A Clinically Orientated Approach for Combining Discordant and Concordant Sib Pairs

The use of extreme discordant sib pairs (EDSP) or extreme concordant sib pairs (ECSP) has recently been proposed to increase power for mapping quantitative traits in humans (RISCH and ZHANG, 1995, 1996). In this paper we propose a test statistic to jointly analyze EDSP and ECSP based on a clinical sampling procedure. This test statistic does not fulfill any optimality criteria. However, this approach is useful for quantitative traits of clinical significance for which EDSP are rare and/or expensive to ascertain. We show how sample size calculations can be adjusted for recombination using single markers, multipoint analysis, incompletely polymorphic markers and varying proportions of ECSP. If the true genetic model is unknown, the combined approach appears to be more robust than sampling based on only EDSP or only ECSP. We discuss how to find the optimal proportion of EDSP and ECSP to be included in an analysis under power considerations.     

Association of the chromosomal region 2q35 with type 1 diabetes mellitus in the Russian patients from Moscow

To map human chromosome 2 region associated with type 1 diabetes mellitus, 89 families with concordant and discordant sib pairs were analyzed. Linkage and association with type 1 diabetes were examined using polymorphic microsatellite markers spanning the region of about 4 Mb. The linkage plot was constructed, and association of the five microsatellite markers within the chromosomal region 2q35 was examined. Polymorphic marker D2S137 (Z' = 3.225, p(c) = 0.0048) demonstrated maximum linkage and association with type 1 diabetes.     

A unified sampling approach for multipoint analysis of qualitative and quantitative traits in sib pairs

Recent advances in molecular biology have enhanced the opportunity to conduct multipoint mapping for complex diseases. Concurrently, one sees a growing interest in the use of quantitative traits in linkage studies. Here, we present a multipoint sib-pair approach to locate the map position (tau) of a trait locus that controls the observed phenotype (qualitative or quantitative), along with a measure of statistical uncertainty. This method builds on a parametric representation for the expected identical-by-descent statistic at an arbitrary locus, conditional on an event reflecting the sampling scheme, such as affected sib pairs, for qualitative traits, or extreme discordant (ED) sib pairs, for quantitative traits. Our results suggest that the variance about tau&d4;, the estimator of tau, can be reduced by as much as 60%-70% by reducing the length of intervals between markers by one half. For quantitative traits, we examine the precision gain (measured by the variance reduction in tau&d4;) by genotyping extremely concordant (EC) sib pairs and including them along with ED sib pairs in the statistical analysis. The precision gain depends heavily on the residual correlation of the quantitative trait for sib pairs but considerably less on the allele frequency and exact genetic mechanism. Since complex traits involve multiple loci and, hence, the residual correlation cannot be ignored, our finding strongly suggests that one should incorporate EC sib pairs along with ED sib pairs, in both design and analysis. Finally, we empirically establish a simple linear relationship between the magnitude of precision gain and the ratio of the number of ED pairs to the number of EC pairs. This relationship allows investigators to address issues of cost effectiveness that are due to the need for phenotyping and genotyping subjects.     

Replication study supports evidence for linkage to 9p24 in obsessive-compulsive disorder

Obsessive-compulsive disorder (OCD) is a severe psychiatric illness that is characterized by intrusive and senseless thoughts and impulses (obsessions) and by repetitive behaviors (compulsions). Family, twin, and segregation studies support the presence of both genetic and environmental susceptibility factors, and the only published genome scan for OCD identified a candidate region on 9p24 at marker D9S288 that met criteria for suggestive significance (Hanna et al. 2002). In an attempt to replicate this finding, we genotyped 50 pedigrees with OCD, using microsatellite markers spanning the 9p24 candidate region, and analyzed the data, using parametric and nonparametric linkage analyses under both a narrow phenotype model (DSM-IV OCD definite; 41 affected sib pairs) and a broad phenotype model (DSM-IV OCD definite and probable; 50 affected sib pairs). Similar to what was described by Hanna et al. (2002), our strongest findings came with the dominant parameters and the narrow phenotype model: the parametric signal peaked at marker D9S1792 with an HLOD of 2.26 ( alpha =0.59), and the nonparametric linkage signal (NPL) peaked at marker D9S1813 with an NPL of 2.52 (P=.006). These findings are striking in that D9S1813 and D9S1792 lie within 0.5 cM (<350 kb) of the original 9p24 linkage signal at D9S288; furthermore, pedigree-based association analyses also implicated the 9p24 candidate region by identifying two markers (D9S288 and GATA62F03) with modest evidence (P=.046 and .02, respectively) for association.     

A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder with a complex genetic component. We report the first major genomewide screen of multiplex families with RA gathered in the United States. The North American Rheumatoid Arthritis Consortium, using well-defined clinical criteria, has collected 257 families containing 301 affected sibling pairs with RA. A genome screen for allele sharing was performed, using 379 microsatellite markers. A nonparametric analysis using SIBPAL confirmed linkage of the HLA locus to RA (P < .00005), with lambdaHLA = 1.79. However, the analysis also revealed a number of non-HLA loci on chromosomes 1 (D1S235), 4 (D4S1647), 12 (D12S373), 16 (D16S403), and 17 (D17S1301), with evidence for linkage at a significance level of P<.005. Analysis of X-linked markers using the MLOD method from ASPEX also suggests linkage to the telomeric marker DXS6807. Stratifying the families into white or seropositive subgroups revealed some additional markers that showed improvement in significance over the full data set. Several of the regions that showed evidence for nominal significance (P < .05) in our data set had previously been implicated in RA (D16S516 and D17S1301) or in other diseases of an autoimmune nature, including systemic lupus erythematosus (D1S235), inflammatory bowel disease (D4S1647, D5S1462, and D16S516), multiple sclerosis (D12S1052), and ankylosing spondylitis (D16S516). Therefore, genes in the HLA complex play a major role in RA susceptibility, but several other regions also contribute significantly to overall genetic risk.     

Genome-wide searching of rare genetic variants in WTCCC data

Although they have demonstrated success in searching for common variants for complex diseases, genome-wide association (GWA) studies are less successful in detecting rare genetic variants because of the poor statistical power of most of current methods. We developed a two-stage method that can apply to GWA studies for detecting rare variants. Here we report the results of applying this two-stage method to the Wellcome Trust Case Control Consortium (WTCCC) dataset that include seven complex diseases: bipolar disorder, cardiovascular disease, hypertension (HT), rheumatoid arthritis, Crohn’s disease, type 1 diabetes and type 2 diabetes (T2D). We identified 24 genes or regions that reach genome wide significance. Eight of them are novel and were not reported in the WTCCC study. The cumulative risk (or protective) haplotype frequency for each of the 8 genes or regions is small, being at most 11%. For each of the novel genes, the risk (or protective) haplotype set cannot be tagged by the common SNPs available in chips (r ² < 0.32). The gene identified in HT was further replicated in the Framingham Heart Study, and is also significantly associated with T2D. Our analysis suggests that searching for rare genetic variants is feasible in current GWA studies and candidate gene studies, and the results can severe as guides to future resequencing studies to identify the underlying rare functional variants.     

Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21-q24

Despite recent advances in the molecular genetics of type 2 diabetes, the majority of susceptibility genes in humans remain to be identified. We therefore conducted a 10-cM genomewide search (401 microsatellite markers) for type 2 diabetes-related traits in 637 members of 143 French pedigrees ascertained through multiple diabetic siblings, to map such genes in the white population. Nonparametric two-point and multipoint linkage analyzes-using the MAPMAKER-SIBS (MLS) and MAXIMUM-BINOMIAL-LIKELIHOOD (MLB) programs for autosomal markers and the ASPEX program for chromosome X markers-were performed with six diabetic phenotypes: diabetes and diabetes or glucose intolerance (GI), as well as with each of the two phenotypes associated with normal body weight (body-mass index<27 kg/m(2)) or early age at diagnosis (<45 years). In a second step, high-resolution genetic mapping ( approximately 2 cM) was performed in regions on chromosomes 1 and 3 loci showing the strongest linkage to diabetic traits. We found evidence for linkage with diabetes or GI diagnosed at age <45 years in 92 affected sib pairs from 55 families at the D3S1580 locus on chromosome 3q27-qter using MAPMAKER-SIBS (MLS = 4.67, P=.000004), supported by the MLB statistic (MLB-LOD=3.43, P=.00003). We also found suggestive linkage between the lean diabetic status and markers APOA2-D1S484 (MLS = 3. 04, P=.00018; MLB-LOD=2.99, P=.00010) on chromosome 1q21-q24. Several other chromosomal regions showed indication of linkage with diabetic traits, including markers on chromosome 2p21-p16, 10q26, 20p, and 20q. These results (a) showed evidence for a novel susceptibility locus for type 2 diabetes in French whites on chromosome 3q27-qter and (b) confirmed the previously reported diabetes-susceptibility locus on chromosome 1q21-q24. Saturation on both chromosomes narrowed the regions of interest down to an interval of <7 cM.     

Concordance of murine quantitative trait loci for salt-induced hypertension with rat and human loci

To investigate the genetic control of salt-induced hypertension, we performed a quantitative trait locus analysis on male mice from a reciprocal backcross between the salt-sensitive C57BL/6J and the normotensive A/J inbred mouse strains after they were provided with water containing 1% salt for 2 weeks. Genome-wide scans performed on these mice and analyzed with a combination of conventional marker-based regressions and a novel simultaneous search for pairs revealed six significant quantitative trait loci associated with salt-induced blood pressure, two of which were interacting loci. These six loci, named Bpq1-6 for blood pressure quantitative trait loci, mapped to D1Mit334, D1Mit14, D4Mit164, D5Mit31, D6Mit15, and D15Mit13. Furthermore, five of these six loci were concordant with hypertension loci in rats, and four were concordant with hypertension loci in humans, suggesting that quantitative trait loci mapping in model organisms can be used to guide the search for human blood pressure genes.     

Whole-Exome Sequencing of 2,000 Danish Individuals and the Role of Rare Coding Variants in Type 2 Diabetes

It has been hypothesized that, in aggregate, rare variants in coding regions of genes explain a substantial fraction of the heritability of common diseases. We sequenced the exomes of 1,000 Danish cases with common forms of type 2 diabetes (including body mass index > 27.5 kg/m² and hypertension) and 1,000 healthy controls to an average depth of 56×. Our simulations suggest that our study had the statistical power to detect at least one causal gene (a gene containing causal mutations) if the heritability of these common diseases was explained by rare variants in the coding regions of a limited number of genes. We applied a series of gene-based tests to detect such susceptibility genes. However, no gene showed a significant association with disease risk after we corrected for the number of genes analyzed. Thus, we could reject a model for the genetic architecture of type 2 diabetes where rare nonsynonymous variants clustered in a modest number of genes (fewer than 20) are responsible for the majority of disease risk.     

Linkage analysis with candidate genes: the Taiwan young-onset hypertension genetic study

A genetic linkage study of young-onset hypertension was performed on data from 59 nucleus families of Han Chinese residing in Taiwan. Thirty seven microsatellite markers near 18 hypertension candidate genes were genotyped. In a nonparametric identity-by-descent sibpair analysis, a positive linkage signal (defined as P<0.05) was found for four microsatellite markers, viz., D1S1612 (P=0.0162), D1S547 (P=0.0263), D8S 1145 (P= 0.0284), and D17S2193 (P=0.0256), which were located near genes for atrial natriuretic peptide (NPPA)/glucose transporter 5 (SLC2A5), angiotensinogen (AGT), lipoprotein lipase (LPL), and angiotensin-conveting enzyme (DCP1), respectively. Marker D5S1480 located near beta-2-adrenergic receptor (ADRB2) had a borderline P value (P=0.0785) for the positive signal. Comprehensive genotyping with further markers in these regions is underway to confirm whether these genes are linked to young-onset hypertension.