Rare DNA variants in the brain-derived neurotrophic factor gene increase risk for attention-deficit hyperactivity disorder: a next-generation sequencing study.

Attention-deficit hyperactivity disorder (ADHD) is a prevalent and highly heritable disorder of childhood with negative lifetime outcomes. Although candidate gene and genome-wide association studies have identified promising common variant signals, these explain only a fraction of the heritability of ADHD. The observation that rare structural variants confer substantial risk to psychiatric disorders suggests that rare variants might explain a portion of the missing heritability for ADHD. Here we believe we performed the first large-scale next-generation targeted sequencing study of ADHD in 152 child and adolescent cases and 188 controls across an a priori set of 117 genes. A multi-marker gene-level analysis of rare (<1% frequency) single-nucleotide variants (SNVs) revealed that the gene encoding brain-derived neurotrophic factor (BDNF) was associated with ADHD at Bonferroni corrected levels. Sanger sequencing confirmed the existence of all novel rare BDNF variants. Our results implicate BDNF as a genetic risk factor for ADHD, potentially by virtue of its critical role in neurodevelopment and synaptic plasticity.

Linkage and association analysis of ADHD endophenotypes in extended and multigenerational pedigrees from a genetic isolate.

Attention-deficit/hyperactivity disorder (ADHD) is a heritable, chronic, neurodevelopmental disorder with serious long-term repercussions. Despite being one of the most common cognitive disorders, the clinical diagnosis of ADHD is based on subjective assessments of perceived behaviors. Endophenotypes (neurobiological markers that cosegregate and are associated with an illness) are thought to provide a more powerful and objective framework for revealing the underlying neurobiology than syndromic psychiatric classification. Here, we present the results of applying genetic linkage and association analyses to neuropsychological endophenotypes using microsatellite and single nucleotide polymorphisms. We found several new genetic regions linked and/or associated with these endophenotypes, and others previously associated to ADHD, for example, loci harbored in the LPHN3, FGF1, POLR2A, CHRNA4 and ANKFY1 genes. These findings, when compared with those linked and/or associated to ADHD, suggest that these endophenotypes lie on shared pathways. The genetic information provided by this study offers a novel and complementary method of assessing the genetic causes underpinning the susceptibility to behavioral conditions and may offer new insights on the neurobiology of the disorder.

APOE*E2 allele delays age of onset in PSEN1 E280A Alzheimer's disease.

Alzheimer's disease (AD) age of onset (ADAOO) varies greatly between individuals, with unique causal mutations suggesting the role of modifying genetic and environmental interactions. We analyzed ~50 000 common and rare functional genomic variants from 71 individuals of the 'Paisa' pedigree, the world's largest pedigree segregating a severe form of early-onset AD, who were affected carriers of the fully penetrant E280A mutation in the presenilin-1 (PSEN1) gene. Affected carriers with ages at the extremes of the ADAOO distribution (30s-70s age range), and linear mixed-effects models were used to build single-locus regression models outlining the ADAOO. We identified the rs7412 (APOE*E2 allele) as a whole exome-wide ADAOO modifier that delays ADAOO by ~12 years (ß=11.74, 95% confidence interval (CI): 8.07-15.41, P=6.31 × 10(-8), PFDR=2.48 × 10(-3)). Subsequently, to evaluate comprehensively the APOE (apolipoprotein E) haplotype variants (E1/E2/E3/E4), the markers rs7412 and rs429358 were genotyped in 93 AD affected carriers of the E280A mutation. We found that the APOE*E2 allele, and not APOE*E4, modifies ADAOO in carriers of the E280A mutation (ß=8.24, 95% CI: 4.45-12.01, P=3.84 × 10(-5)). Exploratory linear mixed-effects multilocus analysis suggested that other functional variants harbored in genes involved in cell proliferation, protein degradation, apoptotic and immune dysregulation processes (i.e., GPR20, TRIM22, FCRL5, AOAH, PINLYP, IFI16, RC3H1 and DFNA5) might interact with the APOE*E2 allele. Interestingly, suggestive evidence as an ADAOO modifier was found for one of these variants (GPR20) in a set of patients with sporadic AD from the Paisa genetic isolate. This is the first study demonstrating that the APOE*E2 allele modifies the natural history of AD typified by the age of onset in E280A mutation carriers. To the best of our knowledge, this is the largest analyzed sample of patients with a unique mutation sharing uniform environment. Formal replication of our results in other populations and in other forms of AD will be crucial for prediction, follow-up and presumably developing new therapeutic strategies for patients either at risk or affected by AD.

Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees. Australian Breast Cancer Family Study.

Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.

Association of AKAP6 and MIR2113 with cognitive performance in a population-based sample of older adults.

Genetic factors make a substantial contribution to inter-individual variability in cognitive function. A recent meta-analysis of genome-wide association studies identified two loci, AKAP6 and MIR2113, that are associated with general cognitive function. Here, we extend this previous research by investigating the association of MIR2113 and AKAP6 with baseline and longitudinal non-linear change across a broad spectrum of cognitive domains in a community-based cohort of older adults without dementia. Two single nucleotide polymorphisms (SNPs), MIR211-rs10457441 and AKAP6-rs17522122 were genotyped in 1570 non-demented older Australians of European ancestry, who were examined up to 4 times over 12 years. Linear mixed effects models were used to examine the association between AKAP6 and MIR2113 with cognitive performance in episodic memory, working memory, vocabulary, perceptual speed and reaction time at baseline and with linear and quadratic rates of change. AKAP6-rs17522122*T was associated with worse baseline performance in episodic memory, working memory, vocabulary and perceptual speed, but it was not associated with cognitive change in any domain. MIR2113-rs10457441*T was associated with accelerated decline in episodic memory. No other associations with baseline cognitive performance or with linear or quadratic rate or cognitive changes were observed for this SNP. These results confirm the previous finding that AKAP6 is associated with performance across multiple cognitive domains at baseline but not with cognitive decline, while MIR2113 primarily affects the rate at which memory declines over time.

HLA-DP variation as additional risk factor in IDDM.

A significant increase in the frequency of DPw3/6 alleles defined by restriction-fragment-length polymorphism is observed in insulin-dependent diabetes mellitus (IDDM) patients relative to healthy control subjects (34.6 vs. 10.5%, P less than 0.009). Log-linear modeling demonstrates that this association is independent of HLA-DR3 and -DR4 and IDDM association and cannot be attributed to linkage disequilibrium between HLA-DP and -DR. The analysis also demonstrates an absence of interactive effects among the antigens in conferring IDDM susceptibility. The strength of the DPw3/6 association is not significantly less than that of either DR3 or DR4.

The pattern of mammalian evolution and the relative rate of molecular evolution.

The rates of nucleotide substitution at four genes in four orders of eutherian mammals are compared in relative rate tests using marsupial orthologs for reference. There is no evidence of systematic variation in evolutionary rate among the orders. The sequences are used to reconstruct the phylogeny of the orders using maximum likelihood, parsimony and compatibility methods. A branching order of rodent then ungulate then primate and lagomorph is overwhelmingly indicated. The nodes of the nucleotide based cladograms are widely separated in relation to the total lengths of the branches. The assumption of a star phylogeny that underlies Kimura's test for molecular evolutionary rate variation is shown to be invalid for eutherian mammals. Excess variance in nucleotide or amino acid differences between mammalian orders, above that predicted by neutral theory is explained better by variation in divergence time than by variation in evolutionary rate.

The ecological genetics of introduced populations of the giant toad Bufo marinus. II. Effective population size.

The allele frequencies are described at ten polymorphic enzyme loci (of a total of 22 loci sampled) in 15 populations of the neotropical giant toad, Bufo marinus, introduced to Hawaii and Australia in the 1930s. The history of establishment of the ten populations is described and used as a framework for the analysis of allele frequency variances. The variances are used to determine the effective sizes of the populations. The estimates obtained (390 and 346) are reasonably precise, homogeneous between localities and much smaller than estimates of neighborhood size obtained previously using ecological methods. This discrepancy is discussed, and it is concluded that the estimates obtained here using genetic methods are the more reliable.

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations.

Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution.

The complete sequence of the MC1R locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF); however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MC1R coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought.

RFLP-defined HLA-DP polymorphism in four ethnic groups.

Patterns of BglII, MspI, and TaqI DP alpha and DP beta hybridizing restriction fragment length polymorphisms are compared with primed lymphocyte typing--determined specificities in 28 10th International Histocompatibility Workshop core cell lines. Correlation of specific RFLP patterns with most recognized DP types is confirmed, although the RFLPs do not distinguish DPw3 from DPw6. Analysis of DP-region nucleotide sequence data shows that the observed RFLPs are those expected to hybridize to the DP alpha and beta probes and are thus not due to cross-hybridization to other genes. The distribution of RFLPs in Chinese, Micronesian, South Indian, and white Australian populations is described. The most frequent DP specificity in Chinese is DPw5. In the other populations DPw4 is the most common specificity, although DPw5 is also relatively common in Micronesians. Four new DP alpha RFLP patterns and three new DP beta patterns are described. There are also numerous unusual combinations of DP alpha and DP beta alleles particularly in the South Indian population.

Association of a polymorphism of the dopamine transporter gene with externalizing behavior problems and associated temperament traits: a longitudinal study from infancy to the mid-teens.

There have been reports that a variable number of tandem repeats (VNTR) polymorphism situated in the 3' untranslated region of the dopamine transporter gene is associated with attention-deficit hyperactivity disorder. On the basis of these findings, we predicted an association of this polymorphism with hyperactivity, other externalizing behavior problems, and related temperament traits in a general population sample. The association was investigated using children participating in a longitudinal study of childhood temperament and development. DNA was taken from 660 children who had been assessed for temperament from 4-8 months to 15-16 years, and for behavior problems from 3-4 to 15-16 years. No significant associations were found at any age. There are a number of methodological differences from earlier studies that might explain the lack of associations with hyperactivity. It is also possible that the earlier findings are not replicable.

Association of smoking and personality with a polymorphism of the dopamine transporter gene: results from a community survey.

In studies that used mixed volunteer samples, Lerman et al. [1999: Health Psychol 18:14-20] and Sabol et al. [1999: Health Psychol 18:7-13] reported on an association of smoking with a polymorphism of the dopamine transporter gene. We attempted to replicate this association in a nonvolunteer community sample of 861 Caucasians. No associations were found with either smoking initiation or smoking cessation. Sabol et al. [1999] also reported on an association of the dopamine transporter polymorphism with the personality trait of novelty seeking. However, we failed to find any associations with a range of personality traits, including a scale of fun seeking that correlates with novelty seeking. These negative findings suggest that either the original associations are not replicable or that any association is very small.

COMT and DRD3 polymorphisms, environmental exposures, and personality traits related to common mental disorders.

In a community sample of 2,327 Caucasians, we tested the hypotheses that polymorphisms in the COMT and DRD3 genes are associated with personality traits conferring vulnerability to anxiety, depression, or alcohol misuse, or with current symptoms of these; and that the association is stronger in persons who also have been exposed to stressor experiences. To conserve resources and to allow replication, the genetic analysis was undertaken in two stages. For the COMT polymorphism, no statistically significant associations were found in the first sample of 862 persons. The remainder of the sample was therefore not analysed for that gene. For the DRD3 polymorphism, those in the first sample with at least one of the Ser(9) alleles had significantly higher scores in neuroticism (p=0.006) and behavioral inhibition (p=0.003). There was a trend, failing to meet the 1% significance criterion, for those with this genotype also to have higher depression and anxiety. The groups did not differ in alcohol use. In persons with the Ser(9) allele who were also exposed to stressors, there was a higher level of depression at the 5% level; and the depression level was higher in homozygotes. But when the remainder of the sample (1,465) was analysed, none of the associations reached statistical significance. We conclude that neither the COMT nor DRD3 polymorphisms are associated with anxiety, depression, or alcohol abuse. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:102-107, 2000

Association of a functional polymorphism of the monoamine oxidase A gene promoter with personality and psychiatric symptoms.

A functional polymorphism in the promoter of the monoamine oxidase gene has recently been described by Sabol et al. This polymorphism is a strong candidate for associations with personality traits and psychiatric symptoms. We report relevant data from a general population sample of 850 Caucasian Australians. We found no associations with anxiety and depression symptoms, with personality traits that predispose to anxiety (neuroticism, behavioral inhibition, negative affect) or to a personality trait related to antisocial behavior (psychoticism).

Measuring environmental factors can enhance the search for disease causing genes?

The value of the concurrent measurement of environmental factors in studies aimed at the discovery of disease causing genes has been questioned on the grounds that such an approach fails to increase study power. This report discusses the issue and shows with examples from the recent literature that the examination of a gene disease association within an environmental subgroup can provide enhanced opportunities for detecting gene effects. The concurrent collection of environmental as well as genetic factors in studies of disease aetiology may enhance study informativeness and validity in several ways, including an increase in the power of the study to detect gene disease associations.

Attitudes and practices of doctors toward spouse assault victims: an Australian study.

The attitudes and practices of 96 doctors toward spousal assault victims in the Australian Capital Territory, Australia, were investigated by questionnaire surveys distributed to general practitioners. The results indicate that although most doctors believe that members of their profession should play a role in prevention and treatment, many doctors may be failing to do so. In general, the sample was fairly homogeneous attitudinally, although long-serving practitioners tend to have more conservative attitudes about the causes of spousal assault and about doctors' role in prevention; and female doctors have more liberal beliefs than males about some aspects of causation and intervention. However, the latters' responses are less sympathetic when questioned about their own feelings and practices. Very few of the doctors had received any training on domestic violence issues, and for those that had, such training was minimal. There is nevertheless an indication that training has influenced some beliefs. The findings are not surprising in the context of the historical and current Australian ethos which perpetuates traditional gender stratification and wife battering.