Veronica J. Vieland

An autosomal genomic screen for autism

Autism is a severe neurodevelopmental disorder defined by social and communication deficits and ritualistic-repetitive behaviors that are detectable in early childhood. The etiology of idiopathic autism is strongly genetic, and oligogenic transmission is likely. The first stage of a two-stage genomic screen for autism was carried out by the Collaborative Linkage Study of Autism on individuals affected with autism from 75 families ascertained through an affected sib-pair. The strongest multipoint results were for regions on chromosomes 13 and 7. The highest maximum multipoint heterogeneity LOD (MMLS/het) score is 3.0 at D13S800 (approximately 55 cM from the telomere) under the recessive model, with an estimated 35% of families linked to this locus. The next highest peak is an MMLS/het score of 2.3 at 19 cM, between D13S217 and D13S1229. Our third highest MMLS/het score of 2.2 is on chromosome 7 and is consistent with the International Molecular Genetic Study of Autism Consortium report of a possible susceptibility locus somewhere within 7q31-33. These regions and others will be followed up in the second stage of our study by typing additional markers in both the original and a second set of identically ascertained autism families, which are currently being collected. By comparing results across a number of studies, we expect to be able to narrow our search for autism susceptibility genes to a small number of genomic regions.

A Major Susceptibility Locus for Specific Language Impairment Is Located on 13q21

Children who fail to develop language normally-in the absence of explanatory factors such as neurological disorders, hearing impairment, or lack of adequate opportunity-are clinically described as having specific language impairment (SLI). SLI has a prevalence of approximately 7% in children entering school and is associated with later difficulties in learning to read. Research indicates that genetic factors are important in the etiology of SLI. Studies have consistently demonstrated that SLI aggregates in families. Increased monozygotic versus dizygotic twin concordance rates indicate that heredity, not just shared environment, is the cause of the familial clustering. We have collected five pedigrees of Celtic ancestry that segregate SLI, and we have conducted genomewide categorical linkage analysis, using model-based LOD score techniques. Analysis was conducted under both dominant and recessive models by use of three phenotypic classifications: clinical diagnosis, language impairment (spoken language quotient <85) and reading discrepancy (nonverbal IQ minus non-word reading >15). Chromosome 13 yielded a maximum multipoint LOD score of 3.92 under the recessive reading discrepancy model. Simulation to correct for multiple models and multiple phenotypes indicated that the genomewide empirical P value is <.01. As an alternative measure, we also computed the posterior probability of linkage (PPL), obtaining a PPL of 53% in the same region. One other genomic region yielded suggestive results on chromosome 2 (multipoint LOD score 2.86, genomic P value <.06 under the recessive language impairment model). Our findings underscore the utility of traditional LOD-score-based methods in finding genes for complex diseases, specifically, SLI.

Examination of AVPR1a as an autism susceptibility gene.

Impaired reciprocal social interaction is one of the core features of autism. While its determinants are complex, one biomolecular pathway that clearly influences social behavior is the arginine–vasopressin (AVP) system. The behavioral effects of AVP are mediated through the AVP receptor 1a (AVPR1a), making the AVPR1a gene a reasonable candidate for autism susceptibility. We tested the genes contribution to autism by screening its exons in 125 independent autistic probands and genotyping two promoter polymorphisms in 65 autism affected sibling pair (ASP) families. While we found no nonconservative coding sequence changes, we did identify evidence of linkage and of linkage disequilibrium. These results were most pronounced in a subset of the ASP families with relatively less severe impairment of language. Thus, though we did not demonstrate a disease-causing variant in the coding sequence, numerous nontraditional disease-causing genetic abnormalities are known to exist that would escape detection by traditional gene screening methods. Given the emerging biological, animal model, and now genetic data, AVPR1a and genes in the AVP system remain strong candidates for involvement in autism susceptibility and deserve continued scrutiny.

Results of a genome-wide genetic screen for panic disorder.

Panic disorder is characterized by spontaneous and recurrent panic attacks, often accompanied by agoraphobia. The results of family, twin, and segregation studies suggest a genetic role in the etiology of the illness. We have genotyped up to 23 families that have a high density of panic disorder with 540 microsatellite DNA markers in a first-pass genomic screen. The thirteen best families (ELOD > 6.0 under the dominant genetic model) have been genotyped with an ordered set of markers encompassing all the autosomes, at an average marker density of 11 cM. Over 110,000 genotypes have been generated on the whole set of families, and the data have been analyzed under both a dominant and a recessive model, and with the program SIBPAIR. No lod scores exceed 2.0 for either parametric model. Two markers give lod scores over 1.0 under the dominant model (chromosomes 1p and 20p), and four do under the recessive model (7p, 17p, 20q, and X/Y). One of these (20p) may be particularly promising. Analysis with SIBPAIR yielded P values equivalent to a lod score of 1.0 or greater (i.e., P < .016, one-sided, uncorrected for multiple tests) for 11 marker loci (2, 7p, 8p, 8q, 9p, 11q, 12q, 16p, 20p and 20q).

The replication requirement.

Human geneticists withhold judgment on any report of linkage or allelic association until it is independently replicated. But the independent replication criterion has become harder to meet. Does this signal the demise of the reverse genetics paradigm or a problem with the replication requirement itself?

Examination of Potential Overlap in Autism and Language Loci on Chromosomes 2, 7, and 13 in Two Independent Samples Ascertained for Specific Language Impairment

Specific language impairment is a neurodevelopmental disorder characterized by impairments essentially restricted to the domain of language and language learning skills. This contrasts with autism, which is a pervasive developmental disorder defined by multiple impairments in language, social reciprocity, narrow interests and/or repetitive behaviors. Genetic linkage studies and family data suggest that the two disorders may have genetic components in common. Two samples, from Canada and the US, selected for specific language impairment were genotyped at loci where such common genes are likely to reside. Significant evidence for linkage was previously observed at chromosome 13q21 in our Canadian sample (HLOD 3.56) and was confirmed in our US sample (HLOD 2.61). Using the posterior probability of linkage (PPL) to combine evidence for linkage across the two samples yielded a PPL over 92%. Two additional loci on chromosome 2 and 7 showed weak evidence for linkage. However, a marker in the cystic fibrosis transmembrane conductance regulator (7q31) showed evidence for association to SLI, confirming results from another group (O’Brien et al. 2003). Our results indicate that using samples selected for components of the autism phenotype may be a useful adjunct to autism genetics.

Effect of allelic heterogeneity on the power of the transmission disequilibrium test.

Due in part to an influential paper by Risch and Merikangas [(1996) Science 273:1516–1517], which suggested that disequilibrium tests would have greater power to detect genes of small effect than would linkage tests, interest in the use of the Transmission Disequilibrium Test (TDT) as an analysis tool for genomewide studies is steadily growing. However, the paper by Risch and Merikangas made several simplifying assumptions. One such assumption was that the underlying gene showed allelic homogeneity, and another was that the allele being measured was the actual susceptibility allele. Here we investigate the effect of allelic heterogeneity on the power of the TDT using multiplicative, additive, dominant, and recessive modes of inheritances in the context of a genomewide study. We further distinguish two cases: first, that the marker alleles are the actual susceptibility alleles, and second, that alleles are measured at a marker linked to the disease gene with zero recombination. We consider two family structures, either a single affected offspring (SAO) and two parents, or an affected sib‐pair (ASP) and two parents. We find that, as expected, the power of the TDT declines as the number of susceptibility alleles at the locus being tested increases and the effect on power can be substantial. When a linked marker is measured rather than a susceptibility allele itself, sample sizes reach unattainable levels when as few as two susceptibility alleles are present. Across all the models we consider, the required number of families for a TDT with ASP sampling varies from 19 to over a million families. Thus, the TDT may not be an optimal test in the context of genomic screens under more biologically realistic assumptions. Genet. Epidemiol. 18:143–156, 2000.

Rare familial 16q21 microdeletions under a linkage peak implicate cadherin 8 ( CDH8 ) in susceptibility to autism and learning disability

Background Autism spectrum disorder (ASD) is characterised by impairments in social communication and by a pattern of repetitive behaviours, with learning disability (LD) typically seen in up to 70% of cases. A recent study using the PPL statistical framework identified a novel region of genetic linkage on chromosome 16q21 that is limited to ASD families with LD. Methods In this study, two families with autism and/or LD are described which harbour rare >1.6 Mb microdeletions located within this linkage region. The deletion breakpoints are mapped at base-pair resolution and segregation analysis is performed using a combination of 1M single nucleotide polymorphism (SNP) technology, array comparative genomic hybridisation (CGH), long-range PCR, and Sanger sequencing. The frequency of similar genomic variants in control subjects is determined through analysis of published SNP array data. Expression of CDH8, the only gene disrupted by these microdeletions, is assessed using reverse transcriptase PCR and in situ hybridisation analysis of 9 week human embryos. Results The deletion of chr16: 60 025 584–61 667 839 was transmitted to three of three boys with autism and LD and none of four unaffected siblings, from their unaffected mother. In a second family, an overlapping deletion of chr16: 58 724 527–60 547 472 was transmitted to an individual with severe LD from his father with moderate LD. No copy number variations (CNVs) disrupting CDH8 were observed in 5023 controls. Expression analysis indicates that the two CDH8 isoforms are present in the developing human cortex. Conclusion Rare familial 16q21 microdeletions and expression analysis implicate CDH8 in susceptibility to autism and LD.

Power to Detect Linkage Based on Multiple Sets of Data in the Presence of Locus Heterogeneity: Comparative Evaluation of Model-Based Linkage Methods for Affected Sib Pair Data

The development of rigorous methods for evaluating the overall strength of evidence for genetic linkage based on multiple sets of data is becoming increasingly important in connection with genomic screens for complex disorders. We consider here what happens when we attempt to increase power to detect linkage by pooling multiple independently collected sets of families under conditions of variable levels of locus heterogeneity across samples. We show that power can be substantially reduced in pooled samples when compared to the most informative constituent subsamples considered alone, in spite of the increased sample size afforded by pooling. We demonstrate that for affected sib pair data, a simple adaptation of the lod score (which we call the compound lod), which allows for intersample admixture differences can afford appreciably higher power than the ordinary heterogeneity lod; and also, that a statistic we have proposed elsewhere, the posterior probability of linkage, performs at least as well as the compound lod while having considerable computational advantages. The companion paper (this issue, pp 217–225) shows further that in application to multiple data sets, familiar model-free methods are in some sense equivalent to ordinary lod scores based on data pooling, and that they therefore will also suffer dramatic losses in power for pooled data in the presence of locus heterogeneity and other complicating factors.

Bayesian analysis of a previously published genome screen for panic disorder reveals new and compelling evidence for linkage to chromosome 7

This article presents a Bayesian re‐analysis of a linkage study of panic disorder Crowe et al. [2001: Am J Med Genet (Neuropsychiatr Genet) 105:105–109]. In the initial analysis Crowe et al. failed to find compelling evidence for linkage based on either LOD scores or NPL scores anywhere in the genome. The maximum LOD score was 2.23 on chromosome 7 at marker D7S2846 (57.79 cM according to Marshfield). Over the past several years we have been developing a Bayesian alternative approach to linkage analysis, based on direct measurement of the posterior probability of linkage (PPL), and have shown elsewhere that this approach has several advantages over the available alternatives for mapping complex‐disease genes Vieland [1998: Am J Med Genet 63:947–954]; Wang et al. [1999: Genet Epidemiol 17(Suppl 1):S749–S754]; Wang et al. [2000: Ann Hum Genet 64:533–553]; and Vieland et al. [2001: Hum Hered 51:199–208]. One limitation of this approach in previous applications has been that it required the investigator to specify a fixed genetic model for the trait. We employ a new implementation of the PPL that treats the unknown trait model as a vector of nuisance parameters, which is integrated out of the PPL equation. When we apply this new model‐integrated version of the PPL to the data of Crowe et al. [2001: Am J Med Genet (Neuropsychiatr Genet) 105:105–109] we obtain much clearer evidence than previously reported for a locus on chromosome 7, with an 80% probability of linkage to marker D7S521. A second location is also identified on chromosome 16 near marker D16S749 (PPL = 24%). The results for the remainder of the genome are consistently low. The two loci identified here are also supported by independent evidence from other studies.