Gene-nutrient interactions that impact magnesium homeostasis increase risk for neural tube defects in mice exposed to dolutegravir.

In 2018, data from a surveillance study in Botswana evaluating adverse birth outcomes raised concerns that women on antiretroviral therapy (ART) containing dolutegravir (DTG) may be at increased risk for neural tube defects (NTDs). The mechanism of action for DTG involves chelation of Mg2+ ions in the active site of the viral integrase. Plasma Mg2+ homeostasis is maintained primarily through dietary intake and reabsorption in the kidneys. Inadequate dietary Mg2+ intake over several months results in slow depletion of plasma Mg2+ and chronic latent hypomagnesemia, a condition prevalent in women of reproductive age worldwide. Mg2+ is critical for normal embryonic development and neural tube closure. We hypothesized that DTG therapy might slowly deplete plasma Mg2+ and reduce the amount available to the embryo, and that mice with pre-existing hypomagnesemia due to genetic variation and/or dietary Mg2+ insufficiency at the time of conception and initiation of DTG treatment would be at increased risk for NTDs. We used two different approaches to test our hypothesis: 1) we selected mouse strains that had inherently different basal plasma Mg2+ levels and 2) placed mice on diets with different concentrations of Mg2+. Plasma and urine Mg2+ were determined prior to timed mating. Pregnant mice were treated daily with vehicle or DTG beginning on the day of conception and embryos examined for NTDs on gestational day 9.5. Plasma DTG was measured for pharmacokinetic analysis. Our results demonstrate that hypomagnesemia prior to conception, due to genetic variation and/or insufficient dietary Mg2+ intake, increases the risk for NTDs in mice exposed to DTG. We also analyzed whole-exome sequencing data from inbred mouse strains and identified 9 predicted deleterious missense variants in Fam111a that were unique to the LM/Bc strain. Human FAM111A variants are associated with hypomagnesemia and renal Mg2+ wasting. The LM/Bc strain exhibits this same phenotype and was the strain most susceptible to DTG-NTDs. Our results suggest that monitoring plasma Mg2+ levels in patients on ART regimens that include DTG, identifying other risk factors that impact Mg2+ homeostasis, and correcting deficiencies in this micronutrient might provide an effective strategy for mitigating NTD risk.

Genome-wide screen to identify genetic loci associated with cognitive decline in late-life depression.

OBJECTIVE: This study sought to conduct a comprehensive search for genetic risk of cognitive decline in the context of geriatric depression. DESIGN: A genome-wide association study (GWAS) analysis in the Neurocognitive Outcomes of Depression in the Elderly (NCODE) study. SETTING: Longitudinal, naturalistic follow-up study. PARTICIPANTS: Older depressed adults, both outpatients and inpatients, receiving care at an academic medical center. MEASUREMENTS: The Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropsychological battery was administered to the study participants at baseline and a minimum of twice within a subsequent 3-year period in order to measure cognitive decline. A GWAS analysis was conducted to identify genetic variation that is associated with baseline and change in the CERAD Total Score (CERAD-TS) in NCODE. RESULTS: The GWAS of baseline CERAD-TS revealed a significant association with an intergenic single-nucleotide polymorphism (SNP) on chromosome 6, rs17662598, that surpassed adjustment for multiple testing (p = 3.7 × 10-7; false discovery rate q = 0.0371). For each additional G allele, average baseline CERAD-TS decreased by 8.656 points. The most significant SNP that lies within a gene was rs11666579 in SLC27A1 (p = 1.1 × 10-5). Each additional copy of the G allele was associated with an average decrease of baseline CERAD-TS of 4.829 points. SLC27A1 is involved with processing docosahexaenoic acid (DHA), an endogenous neuroprotective compound in the brain. Decreased levels of DHA have been associated with the development of Alzheimer's disease. The most significant SNP associated with CERAD-TS decline over time was rs73240021 in GRXCR1 (p = 1.1 × 10-6), a gene previously linked with deafness. However, none of the associations within genes survived adjustment for multiple testing. CONCLUSIONS: Our GWAS of cognitive function and decline among individuals with late-life depression (LLD) has identified promising candidate genes that, upon replication in other cohorts of LLD, may be potential biomarkers for cognitive decline and suggests DHA supplementation as a possible therapy of interest.

Largest GWAS of PTSD (N=20 070) yields genetic overlap with schizophrenia and sex differences in heritability.

The Psychiatric Genomics Consortium-Posttraumatic Stress Disorder group (PGC-PTSD) combined genome-wide case-control molecular genetic data across 11 multiethnic studies to quantify PTSD heritability, to examine potential shared genetic risk with schizophrenia, bipolar disorder, and major depressive disorder and to identify risk loci for PTSD. Examining 20 730 individuals, we report a molecular genetics-based heritability estimate (h2SNP) for European-American females of 29% that is similar to h2SNP for schizophrenia and is substantially higher than h2SNP in European-American males (estimate not distinguishable from zero). We found strong evidence of overlapping genetic risk between PTSD and schizophrenia along with more modest evidence of overlap with bipolar and major depressive disorder. No single-nucleotide polymorphisms (SNPs) exceeded genome-wide significance in the transethnic (overall) meta-analysis and we do not replicate previously reported associations. Still, SNP-level summary statistics made available here afford the best-available molecular genetic index of PTSD-for both European- and African-American individuals-and can be used in polygenic risk prediction and genetic correlation studies of diverse phenotypes. Publication of summary statistics for ∼10 000 African Americans contributes to the broader goal of increased ancestral diversity in genomic data resources. In sum, the results demonstrate genetic influences on the development of PTSD, identify shared genetic risk between PTSD and other psychiatric disorders and highlight the importance of multiethnic/racial samples. As has been the case with schizophrenia and other complex genetic disorders, larger sample sizes are needed to identify specific risk loci.

SNPs in dopamine D2 receptor gene (DRD2) and norepinephrine transporter gene (NET) are associated with continuous performance task (CPT) phenotypes in ADHD children and their families.

Haplotype-tagging SNP analyses were conducted to identify molecular genetic substrates of quantitative phenotypes derived from performance on a Continuous Performance Task (CPT). Three hundred sixty-four individuals were sampled from 152 families ascertained on the basis of at least one child having ADHD. Probands, their affected and unaffected siblings, and parents were administered a CPT. Four different components of performance were analyzed and tested for association with SNPs from 10 candidate genes involved in monoaminergic function. After correcting for multiple comparisons and controlling for multiple individuals from the same family, significant associations were identified between commission errors and SNPs in the DRD2 gene (rs2075654, rs1079596), and between reaction time variability and a SNP in the NET gene (rs3785155). These findings suggest that commission errors and reaction time variability are excellent candidates as ADHD endophenotypes based on previously published criteria. Results also shed light on the molecular genetic basis of specific processes that may underlie the disorder.

An analysis paradigm for investigating multi-locus effects in complex disease: examination of three GABA receptor subunit genes on 15q11-q13 as risk factors for autistic disorder.

Gene-gene interactions are likely involved in many complex genetic disorders and new statistical approaches for detecting such interactions are needed. We propose a multi-analytic paradigm, relying on convergence of evidence across multiple analysis tools. Our paradigm tests for main and interactive effects, through allele, genotype and haplotype association. We applied our paradigm to genotype data from three GABAA receptor subunit genes (GABRB3, GABRA5, and GABRG3) on chromosome 15 in 470 Caucasian autism families. Previously implicated in autism, we hypothesized these genes interact to contribute to risk. We detected no evidence of main effects by allelic (PDT, FBAT) or genotypic (genotype-PDT) association at individual markers. However, three two-marker haplotypes in GABRG3 were significant (HBAT). We detected no significant multi-locus associations using genotype-PDT analysis or the EMDR data reduction program. However, consistent with the haplotype findings, the best single locus EMDR model selected a GABRG3 marker. Further, the best pairwise genotype-PDT result involved GABRB3 and GABRG3, and all multi-locus EMDR models also selected GABRB3 and GABRG3 markers. GABA receptor subunit genes do not significantly interact to contribute to autism risk in our overall data set. However, the consistency of results across analyses suggests that we have defined a useful framework for evaluating gene-gene interactions.

Identification of significant association and gene-gene interaction of GABA receptor subunit genes in autism.

Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis--with the pedigree disequilibrium test and the family-based association test--and for genotypic and haplotypic association analysis--with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a significant two-locus gene-gene effect model involving RS1912960 in GABRA4 and RS2351299 in GABRB1. Further support for this two-locus model came from both the multilocus geno-PDT and the APL test, which indicated a common genotype and haplotype combination positively associated with disease. Finally, these results were also consistent with the results from the conditional logistic regression, which confirmed the interaction between GABRA4 and GABRB1 (odds ratio = 2.9 for interaction term; P = .002). Through the convergence of all analyses, we conclude that GABRA4 is involved in the etiology of autism and potentially increases autism risk through interaction with GABRB1. These results support the hypothesis that GABA receptor subunit genes are involved in autism, most likely via complex gene-gene interactions.

An autosomal genomic screen for dementia in an extended Amish family.

Apolipoprotein E (APOE) is the only universally confirmed susceptibility gene for late-onset Alzheimer disease (LOAD), although many loci are believed to modulate LOAD risk. The genetic homogeneity of isolated populations, such as the Amish, potentially provide increased power to identify LOAD susceptibility genes. Population homogeneity in these special populations may reduce the total number of susceptibility genes contributing to the complex disorder, thereby increasing the ability to identify any one susceptibility gene. Dementia in the Amish is clinically indistinguishable from LOAD in the general population. Previous studies in the Amish demonstrated a significantly decreased frequency of the APOE-4 susceptibility allele, but significant familial clustering of dementia [M.A. Pericak-Vance, C.C. Johnson, J.B. Rimmler, A.M. Saunders, L.C. Robinson, E.G. D'Hondt, C.E. Jackson, J.L. Haines, Alzheimer's disease and apolipoprotein E-4 allele in an Amish population, Ann. Neurol. 39 (1996) 700-704]. These data suggested that a genetic etiology independent of APOE may underlie the dementia observed in this population. In the present analysis, we focused on a large, multiplex, inbred Amish family (24 sampled individuals; 10 of whom are affected). We completed a genomic screen to identify novel LOAD loci (n=316 genetic markers), using both model-dependent "affecteds-only" analysis (dominant and recessive) and model-independent affected relative pair analysis. Interesting results (lod>1.5 or p<0.01) were obtained for markers on eight chromosomes (2q, 5q, 6q, 7p, 8p, 8q, 11p, 18p, 18q, and 19q). The highest overall score was a multipoint lod score of 3.1 on chromosome 11p. Most regions we identified were not previously detected by genomic screens of outbred populations and may represent population-specific susceptibilities to LOAD. These loci are currently under further investigation in a study of LOAD including additional Amish families.

Analysis of the RELN gene as a genetic risk factor for autism.

Several genome-wide screens have indicated the presence of an autism susceptibility locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this region is the candidate gene reelin (RELN). RELN encodes a signaling protein that plays a pivotal role in the migration of several neuronal cell types and in the development of neural connections. Given these neurodevelopmental functions, recent reports that RELN influences genetic risk for autism are of significant interest. The total data set consists of 218 Caucasian families collected by our group, 85 Caucasian families collected by AGRE, and 68 Caucasian families collected at Tufts University were tested for genetic association of RELN variants to autism. Markers included five single-nucleotide polymorphisms (SNPs) and a repeat in the 5'-untranslated region (5'-UTR). Tests for association in Duke and AGRE families were also performed on four additional SNPs in the genes PSMC2 and ORC5L, which flank RELN. Family-based association analyses (PDT, Geno-PDT, and FBAT) were used to test for association of single-locus markers and multilocus haplotypes with autism. The most significant association identified from this combined data set was for the 5'-UTR repeat (PDT P-value=0.002). These analyses show the potential of RELN as an important contributor to genetic risk in autism.

Identification and expression analysis of spastin gene mutations in hereditary spastic paraplegia.

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

Testing for contributions of mitochondrial DNA mutations to complex diseases.

Several complex disorders are suspected of being associated with mitochondrial DNA (mtDNA) mutations. We studied the statistical properties of a test based on proband-relative pairs to identify potential mtDNA mutation involvement in a complex disorder. The test compares the recurrence risk of relatives of probands along the mitochondrial lineage with that of relatives along the nonmitochondrial lineage. If mtDNA mutations are involved, the recurrence risk will be higher among relatives in the mitochondrial lineage. The form of the test is independent of the assumed models of inheritance and interaction of the nuclear autosomal mutations with mtDNA mutations. The power of the test, however, differs among the different models and by the type of proband-relative pairs used in the test. We considered heterogeneity models with and without phenocopies, a three-state heteroplasmic mtDNA transmission model, and a multiplicative epistasis model. Under the heterogeneity model, the power of the test increases as the relationship between the proband and the relative becomes more distant. Under the multiplicative epistasis model, the power of the test decreases as the relationship between the proband and the relative becomes more distant.

Examination of factors associated with instability of the FMR1 CGG repeat.

We examined premutation-female transmissions and premutation-male transmissions of the FMR1 CGG repeat to carrier offspring, to identify factors associated with instability of the repeat. First we investigated associations between parental and offspring repeat size. Premutation-female repeat size was positively correlated with the risk of having full-mutation offspring, confirming previous reports. Similarly, premutation-male repeat size was positively correlated with the daughter's repeat size. However, increasing paternal repeat size was associated also with both increased risk of contraction and decreased magnitude of the repeat-size change passed to the daughter. We hypothesized that the difference between the female and male transmissions was due simply to selection against full-mutation sperm. To test this hypothesis, we simulated selection against full-mutation eggs, by only examining premutation-female transmissions to their premutation offspring. Among this subset of premutation-female transmissions, associations between maternal and offspring repeat size were similar to those observed in premutation-male transmissions. This suggests that the difference between female and male transmissions may be due to selection against full-mutation sperm. Increasing maternal age was associated with increasing risk of expansion to the full mutation, possibly because of selection for smaller alleles within the offspring's soma over time; a similar effect of increasing paternal age may be due to the same selection process. Last, we have evidence that the reported association between offspring sex and risk of expansion may be due to ascertainment bias. Thus, female and male offspring are equally likely to inherit the full mutation.