Association of rare missense variants in the second intracellular loop of NaV1.7 sodium channels with familial autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder often accompanied by intellectual disability, language impairment and medical co-morbidities. The heritability of autism is high and multiple genes have been implicated as causal. However, most of these genes have been identified in de novo cases. To further the understanding of familial autism, we performed whole-exome sequencing on five families in which second- and third-degree relatives were affected. By focusing on novel and protein-altering variants, we identified a small set of candidate genes. Among these, a novel private missense C1143F variant in the second intracellular loop of the voltage-gated sodium channel NaV1.7, encoded by the SCN9A gene, was identified in one family. Through electrophysiological analysis, we show that NaV1.7C1143F exhibits partial loss-of-function effects, resulting in slower recovery from inactivation and decreased excitability in cultured cortical neurons. Furthermore, for the same intracellular loop of NaV1.7, we found an excess of rare variants in a case-control variant-burden study. Functional analysis of one of these variants, M932L/V991L, also demonstrated reduced firing in cortical neurons. However, although this variant is rare in Caucasians, it is frequent in Latino population, suggesting that genetic background can alter its effects on phenotype. Although the involvement of the SCN1A and SCN2A genes encoding NaV1.1 and NaV1.2 channels in de novo ASD has previously been demonstrated, our study indicates the involvement of inherited SCN9A variants and partial loss-of-function of NaV1.7 channels in the etiology of rare familial ASD.

Variants regulating ZBTB4 are associated with age-at-onset of Alzheimer's disease.

The identification of novel genetic modifiers of age-at-onset (AAO) of Alzheimer's disease (AD) could advance our understanding of AD and provide novel therapeutic targets. A previous genome scan for modifiers of AAO among families affected by early-onset AD caused by the PSEN2 N141I variant identified 2 loci with significant evidence for linkage: 1q23.3 and 17p13.2. Here, we describe the fine-mapping of these 2 linkage regions, and test for replication in 6 independent datasets. By fine-mapping these linkage signals in a single large family, we reduced the linkage regions to 11% their original size and nominated 54 candidate variants. Among the 11 variants associated with AAO of AD in a larger sample of Germans from Russia, the strongest evidence implicated promoter variants influencing NCSTN on 1q23.3 and ZBTB4 on 17p13.2. The association between ZBTB4 and AAO of AD was replicated by multiple variants in independent, trans-ethnic datasets. Our results show association between AAO of AD and both ZBTB4 and NCSTN. ZBTB4 is a transcriptional repressor that regulates the cell cycle, including the apoptotic response to amyloid beta, while NCSTN is part of the gamma secretase complex, known to influence amyloid beta production. These genes therefore suggest important roles for amyloid beta and cell cycle pathways in AAO of AD.

Multipoint genome-wide linkage scan for nonword repetition in a multigenerational family further supports chromosome 13q as a locus for verbal trait disorders.

Verbal trait disorders encompass a wide range of conditions and are marked by deficits in five domains that impair a person's ability to communicate: speech, language, reading, spelling, and writing. Nonword repetition is a robust endophenotype for verbal trait disorders that is sensitive to cognitive processes critical to verbal development, including auditory processing, phonological working memory, and motor planning and programming. In the present study, we present a six-generation extended pedigree with a history of verbal trait disorders. Using genome-wide multipoint variance component linkage analysis of nonword repetition, we identified a region spanning chromosome 13q14-q21 with LOD = 4.45 between 52 and 55 cM, spanning approximately 5.5 Mb on chromosome 13. This region overlaps with SLI3, a locus implicated in reading disability in families with a history of specific language impairment. Our study of a large multigenerational family with verbal trait disorders further implicates the SLI3 region in verbal trait disorders. Future studies will further refine the specific causal genetic factors in this locus on chromosome 13q that contribute to language traits.

Family-based genome scan for age at onset of late-onset Alzheimer's disease in whole exome sequencing data.

Alzheimer's disease (AD) is a common and complex neurodegenerative disease. Age at onset (AAO) of AD is an important component phenotype with a genetic basis, and identification of genes in which variation affects AAO would contribute to identification of factors that affect timing of onset. Increase in AAO through prevention or therapeutic measures would have enormous benefits by delaying AD and its associated morbidities. In this paper, we performed a family-based genome-wide association study for AAO of late-onset AD in whole exome sequence data generated in multigenerational families with multiple AD cases. We conducted single marker and gene-based burden tests for common and rare variants, respectively. We combined association analyses with variance component linkage analysis, and with reference to prior studies, in order to enhance evidence of the identified genes. For variants and genes implicated by the association study, we performed a gene-set enrichment analysis to identify potential novel pathways associated with AAO of AD. We found statistically significant association with AAO for three genes (WRN, NTN4 and LAMC3) with common associated variants, and for four genes (SLC8A3, SLC19A3, MADD and LRRK2) with multiple rare-associated variants that have a plausible biological function related to AD. The genes we have identified are in pathways that are strong candidates for involvement in the development of AD pathology and may lead to a better understanding of AD pathogenesis.

Identification of rare variants from exome sequence in a large pedigree with autism.

We carried out analyses with the goal of identifying rare variants in exome sequence data that contribute to disease risk for a complex trait. We analyzed a large, 47-member, multigenerational pedigree with 11 cases of autism spectrum disorder, using genotypes from 3 technologies representing increasing resolution: a multiallelic linkage marker panel, a dense diallelic marker panel, and variants from exome sequencing. Genome-scan marker genotypes were available on most subjects, and exome sequence data was available on 5 subjects. We used genome-scan linkage analysis to identify and prioritize the chromosome 22 region of interest, and to select subjects for exome sequencing. Inheritance vectors (IVs) generated by Markov chain Monte Carlo analysis of multilocus marker data were the foundation of most analyses. Genotype imputation used IVs to determine which sequence variants reside on the haplotype that co-segregates with the autism diagnosis. Together with a rare-allele frequency filter, we identified only one rare variant on the risk haplotype, illustrating the potential of this approach to prioritize variants. The associated gene, MYH9, is biologically unlikely, and we speculate that for this complex trait, the key variants may lie outside the exome.

Meta-analysis of 32 genome-wide linkage studies of schizophrenia.

A genome scan meta-analysis (GSMA) was carried out on 32 independent genome-wide linkage scan analyses that included 3255 pedigrees with 7413 genotyped cases affected with schizophrenia (SCZ) or related disorders. The primary GSMA divided the autosomes into 120 bins, rank-ordered the bins within each study according to the most positive linkage result in each bin, summed these ranks (weighted for study size) for each bin across studies and determined the empirical probability of a given summed rank (P(SR)) by simulation. Suggestive evidence for linkage was observed in two single bins, on chromosomes 5q (142-168 Mb) and 2q (103-134 Mb). Genome-wide evidence for linkage was detected on chromosome 2q (119-152 Mb) when bin boundaries were shifted to the middle of the previous bins. The primary analysis met empirical criteria for 'aggregate' genome-wide significance, indicating that some or all of 10 bins are likely to contain loci linked to SCZ, including regions of chromosomes 1, 2q, 3q, 4q, 5q, 8p and 10q. In a secondary analysis of 22 studies of European-ancestry samples, suggestive evidence for linkage was observed on chromosome 8p (16-33 Mb). Although the newer genome-wide association methodology has greater power to detect weak associations to single common DNA sequence variants, linkage analysis can detect diverse genetic effects that segregate in families, including multiple rare variants within one locus or several weakly associated loci in the same region. Therefore, the regions supported by this meta-analysis deserve close attention in future studies.

Evidence for multiple loci from a genome scan of autism kindreds.

We performed a genome-wide linkage scan using highly polymorphic microsatellite markers. To minimize genetic heterogeneity, we focused on sibpairs meeting the strict diagnosis of autism. In our primary analyses, we observed a strong linkage signal (P=0.0006, 133.16 cM) on chromosome 7q at a location coincident with other linkage studies. When a more relaxed diagnostic criteria was used, linkage evidence at this location was weaker (P=0.01). The sample was stratified into families with only male affected subjects (MO) and families with at least one female affected subject (FC). The strongest signal unique to the MO group was on chromosome 11 (P=0.0009, 83.82 cM), and for the FC group on chromosome 4 (P=0.002, 111.41 cM). We also divided the sample into regression positive and regression negative families. The regression-positive group showed modest linkage signals on chromosomes 10 (P=0.003, 0 cM) and 14 (P=0.005, 104.2 cM). More significant peaks were seen in the regression negative group on chromosomes 3 (P=0.0002, 140.06 cM) and 4 (P=0.0005, 111.41 cM). Finally, we used language acquisition data as a quantitative trait in our linkage analysis and observed a chromosome 9 signal (149.01 cM) of P=0.00006 and an empirical P-value of 0.0008 at the same location. Our work provides strong conformation for an autism locus on 7q and suggestive evidence for several other chromosomal locations. Diagnostic specificity and detailed analysis of the autism phenotype is critical for identifying autism loci.

A genome scan in multigenerational families with dyslexia: Identification of a novel locus on chromosome 2q that contributes to phonological decoding efficiency.

Dyslexia is a common and complex developmental disorder manifested by unexpected difficulty in learning to read. Multiple different measures are used for diagnosis, and may reflect different biological pathways related to the disorder. Impaired phonological decoding (translation of written words without meaning cues into spoken words) is thought to be a core deficit. We present a genome scan of two continuous measures of phonological decoding ability: phonemic decoding efficiency (PDE) and word attack (WA). PDE measures both accuracy and speed of phonological decoding, whereas WA measures accuracy alone. Multipoint variance component linkage analyses (VC) and Markov chain Monte-Carlo (MCMC) multipoint joint linkage and segregation analyses were performed on 108 families. A strong signal was observed on chromosome 2 for PDE using both VC (LOD=2.65) and MCMC methods (intensity ratio (IR)=32.1). The IR is an estimate of the ratio of the posterior to prior probability of linkage in MCMC analysis. The chromosome 2 signal was not seen for WA. More detailed mapping with additional markers provided statistically significant evidence for linkage of PDE to chromosome 2, with VC-LOD=3.0 and IR=59.6 at D2S1399. Parametric analyses of PDE, using a model obtained by complex segregation analysis, provided a multipoint maximum LOD=2.89. The consistency of results from three analytic approaches provides strong evidence for a locus on chromosome 2 that influences speed but not accuracy of phonological decoding.

Genome-wide scan in a large complex pedigree with predominantly male schizophrenics from the island of Kosrae: evidence for linkage to chromosome 2q.

It is widely accepted that founder populations hold promise for mapping loci for complex traits. However, the outcome of these mapping efforts will most likely depend on the individual demographic characteristics and historical circumstances surrounding the founding of a given genetic isolate. The 'ideal' features of a founder population are currently unknown. The Micronesian islandic population of Kosrae, one of the four islands comprising the Federated States of Micronesia (FSM), was founded by a small number of settlers and went through a secondary genetic 'bottleneck' in the mid-19th century. The potential for reduced etiological (genetic and environmental) heterogeneity, as well as the opportunity to ascertain extended and statistically powerful pedigrees makes the Kosraen population attractive for mapping schizophrenia susceptibility genes. Our exhaustive case ascertainment from this islandic population identified 32 patients who met DSM-IV criteria for schizophrenia or schizoaffective disorder. Three of these were siblings in one nuclear family, and 27 were from a single large and complex schizophrenia kindred that includes a total of 251 individuals. One of the most startling findings in our ascertained sample was the great difference in male and female disease rates. A genome-wide scan provided initial suggestive evidence for linkage to markers on chromosomes 1, 2, 3, 7, 13, 15, 19, and X. Follow-up multipoint analyses gave additional support for a region on 2q37 that includes a schizophrenia locus previously identified in another small genetic isolate, with a well-established recent genealogical history and a small number of founders, located on the eastern border of Finland. In addition to providing further support for a schizophrenia susceptibility locus at 2q37, our results highlight the analytic challenges associated with extremely large and complex pedigrees, as well as the limitations associated with genetic studies of complex traits in small islandic populations.

TAU as a susceptibility gene for amyotropic lateral sclerosis-parkinsonism dementia complex of Guam.

BACKGROUND: A Guam variant of amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia complex (PDC-G) are found in the Chamorro people of Guam. Both disorders have overlapping neuropathologic findings, with neurofibrillary tangles in spinal cord and brain. The cause of ALS-G-PDC-G is unknown, although inheritance and environment appear important. Because neurofibrillary tangles containing tau protein are present in ALS-G-PDC-G, and because mutations in the tau gene (TAU) cause autosomal dominant frontotemporal dementia, TAU was examined as a candidate gene for ALS-G-PDC-G. METHODS: TAU was evaluated by DNA sequence analysis in subjects with ALS-G-PDC-G, by linkage analysis of TAU polymorphisms in an extended pedigree from the village of Umatac, and by evaluation of linkage disequilibrium with polymorphic markers flanking and within TAU. RESULTS: Linkage disequilibrium between ALS-G-PDC-G and the TAU polymorphism CA3662 was observed. For this 2-allele system, PDC and ALS cases were significantly less likely than Guamanian controls to have the 1 allele (4.9% and 2% vs 11.5%, respectively; Fisher exact P =.007). DNA sequence analysis of TAU coding regions did not demonstrate a mutation responsible for ALS-G-PDC-G. Analysis of TAU genotypes in an extended pedigree of subjects from Umatac showed obligate recombinants between TAU and ALS-G-PDC-G. Linkage analysis of the Umatac pedigree indicates that TAU is not the major gene for ALS-G-PDC-G. CONCLUSIONS: The genetic association between ALS-G-PDC-G implicates TAU in the genetic susceptibility to ALS-G-PDC-G. TAU may be a modifying gene increasing risk for ALS-G-PDC-G in the presence of another, as yet, unidentified gene.

On estimating the proportion of variance in a phenotypic trait attributable to a measured locus.

The measured genotype approach can be used to estimate the variance contributions of specific candidate loci to quantitative traits of interest. We show here that both the naive estimate of measured-locus heritability, obtained by invoking infinite-sample theory, and an estimate obtained from a bias-corrected variance estimate based on finite-sample theory, produce biased estimates of heritability. We identify the sources of bias, and quantify their effects. The two sources of bias are: (1) the estimation of heritability from population samples as the ratio of two variances, and (2) the existence of sampling error. We show that neither heritability estimator is less biased (in absolute value) than the other in all situations, and the choice of an ideal estimator is therefore a function of the sample size and magnitude of the locus-specific contribution to the overall phenotypic variance. In most cases the bias is small, so that the practical implications of using either estimator are expected to be minimal.

The importance of connections: joining components of the Hutterite pedigree.

We analyzed a quantitative trait (serum IgE levels), and a binary trait (asthma), in four Hutterite sub-pedigrees. A genome screen for asthma was performed using GENEHUNTER, and interesting regions were followed up using extended pedigrees and the FASTLINK package. Markov chain Monte Carlo (MCMC) methods were used to assess haplotype sharing among affected individuals (MORGAN/AUTOZYG), and to perform a combined oligogenic segregation and linkage analysis (LOKI) for log10(IgE). We found evidence for at least two susceptibility loci for asthma on chromosome 5, and a QTL for log10(IgE) on chromosome 1. Our analyses demonstrate that using the most complete pedigree structure possible is advisable, with attention to the possibility of heterogeneity among subunits of a very large pedigree.

Bias in multipoint linkage analysis arising from map misspecification.

Multipoint linkage analysis methods are often used in human genetic studies. Although multipoint methods increase power for a linkage analysis and will become essential if use of diallelic markers becomes widespread, the methods in use assume an accurate meiotic marker map. Unfortunately, uncertainties in estimates of between-marker meiotic distances are large. Also, sex-averaged maps are generally used, but recombination rates differ in males and females. Both these types of map misspecification can lead to lod score bias, but such bias has not previously been systematically quantified. We examine multipoint lod score bias arising from these map misspecifications, in both the presence and absence of actual linkage. We define bias as the expected difference between the lod score computed under the misspecified map and that computed under the true map. With actual linkage, any map misspecification causes negative bias in lod scores, resulting in loss of power to detect linkage. In most cases, bias is modest, only reaching clearly detectable levels when both types of misspecification are substantial. In the absence of linkage, map misspecification can cause positive or negative bias: falsely assuming a 1:1 female:male ratio always causes positive bias; using too large a distance gives a positive bias; using too small a distance gives a negative bias. This bias can inflate the false-positive rate, especially when the sample size is modest. We conclude that although current sex-averaged maps are suitable for a first-pass multipoint screen, the potential for bias from map misspecification should be evaluated in following up results from such an analysis.

Segregation analysis of phenotypic components of learning disabilities. I. Nonword memory and digit span.

Dyslexia is a common and complex disorder with evidence for a genetic component. Multiple loci (i.e., quantitative-trait loci [QTLs]) are likely to be involved, but the number is unknown. Diagnosis is complicated by the lack of a standard protocol, and many diagnostic measures have been proposed as understanding of the component processes has evolved. One or more genes may, in turn, influence these measures. To date, little work has been done to evaluate the mode of inheritance of individual component-as opposed to composite-phenotypes, beyond family or twin correlation studies that initially demonstrate evidence for a genetic basis of such components. Here we use two approaches to segregation analysis in 102 nuclear families to estimate genetic models for component phenotypes associated with dyslexia: digit span and a nonword-repetition task. Both measures are related to phonological skills, one of the key component processes in dyslexia. We use oligogenic-trait segregation analysis to estimate the number of QTLs contributing to each phenotype, and we use complex segregation analysis to identify the most parsimonious inheritance models. We provide evidence in support of both a major-gene mode of inheritance for the nonword-repetition task, with approximately 2.4 contributing QTLs, and for a genetic basis of digit span, with approximately 1.9 contributing QTLs. Results obtained by reciprocal adjustment of measures suggest that genes contributing to digit span may contribute to the nonword-repetition score but that there are additional QTLs involved in nonword repetition. Our study adds to existing studies of the genetic basis of composite phenotypes related to dyslexia, by providing evidence for major-gene modes of inheritance of these single-measure component phenotypes.

Linkage of hereditary distal myopathy with desmin accumulation to 2q.

We are investigating the genetics of a large family with an autosomal dominant form of hereditary distal myopathy. This slowly progressive myopathy begins during early adulthood in the distal leg muscles, producing a gait disturbance. Cardiomyopathy is also present in most affected family members, manifesting itself as conduction block or congestive heart failure. Histologically, an accumulation of the protein, desmin, occurs in the subsarcolemmal spaces of myofibers. We have performed linkage analyses of this family, and have mapped the location of the gene causing the myopathy to human chromosome 2q33. The gene is within a 17-cM segment of chromosome 2q bounded by the DNA markers D2S2248 and D2S401. The best candidate gene for this myopathy is desmin.

The number of trait loci in late-onset Alzheimer disease.

Although it is clear that apoE plays an important role in the genetics of late-onset Alzheimer disease (AD), evidence exists that additional genes may play a role in AD, and estimates of the total contribution of apoE to the variance in onset of AD vary widely. Unfortunately, little information is available on the number and contribution of additional genes. We estimated the number of additional quantitative-trait loci and their contribution to the variance in age at onset of AD, as well as the contribution of apoE and sex, in an oligogenic segregation analysis of 75 families (742 individuals) ascertained for members with late-onset AD. We found evidence that four additional loci make a contribution to the variance in age at onset of late-onset AD that is similar to or greater in magnitude than that made by apoE, with one locus making a contribution several times greater than that of apoE. Additionally, we confirmed previous findings of a dose effect for the apoE varepsilon4 allele, a protective effect for the varepsilon2 allele, evidence for allelic interactions at the apoE locus, and a small protective effect for males. Furthermore, although we estimate that the apoE genotype can make a difference of

Familial aggregation of dyslexia phenotypes.

There is evidence for genetic contributions to reading disability, but the phenotypic heterogeneity associated with the clinical diagnosis may make identification of the underlying genetic basis difficult. In order to elucidate distinct phenotypic features that may be contributing to the genotypic heterogeneity, we assessed the familial aggregation patterns of Verbal IQ and 24 phenotypic measures associated with dyslexia in 102 nuclear families ascertained through probands in grades 1 through 6 who met the criteria for this disorder. Correlations between relatives were computed for all diagnostic phenotypes, using a generalized estimating equation (GEE) approach. GEE is a recently developed semiparametric method for handling correlated data. The method is robust to model misspecification and flexible in adjusting for the subjects' characteristics and pedigree sizes as well as for the ascertainment process, while estimating the correlations between related subjects. The Nonword Memory (NWM) subtest of a prepublication version of the Comprehensive Test of Phonological Processing (CTOPP) and Phonemic Decoding Efficiency (PDE) subtest of a prepublication version of the Test of Word Reading Efficiency (TOWRE) showed correlation patterns in relatives that are strongly supportive of a genetic basis. The Wechsler Scale Digit Span, the Word Attack subtest of the Woodcock Reading Mastery Test--Revised, and the Spelling subtest of the Wide Range Achievement Test--Third Edition had slightly weaker evidence of a genetic basis. Five additional phenotypes (the Spelling subtest of the Wechsler Individual Achievement Test, the Accuracy, Rate, and Comprehension subtests of the Gray Oral Reading Test--Third Edition, and Rapid Automatized Naming of Letters and Numbers) gave suggestive evidence of such a pattern. The results cross-validate in that evidence for a pattern consistent with a genetic basis was obtained for two measures of phonological short-term memory (CTOPP Nonword Memory and WISCIII or WAIS-R Digit Span), for two measures of phonological decoding (WRMT-R Word Attack and TOWRE Phonemic Decoding Efficiency), and for two measures of spelling from dictation (WRAT-3 Spelling and, to a lesser extent, WIAT Spelling). These measures are thus good candidates for more sophisticated segregation analyses that can formulate models for incorporation into linkage analyses.