Arylsulphatase A Pseudodeficiency (ARSA-PD), hypertension and chronic renal disease in Aboriginal Australians.

Chronic renal disease (CRD) associated with cardiovascular disease (CVD) and/or type 2 diabetes (T2D) is a significant problem in Aboriginal Australians. Whole exome sequencing data (N = 72) showed enrichment for ClinVar pathogenic variants in gene sets/pathways linking lipoprotein, lipid and glucose metabolism. The top Ingenuity Pathway Analysis canonical pathways were Farsenoid X Receptor and Retinoid Receptor (FXR/RXR; (P = 1.86 × 10-7), Liver X Receptor and Retinoid Receptor (LXR/RXR; P = 2.88 × 10-6), and atherosclerosis signalling (P = 3.80 × 10-6). Top pathways/processes identified using Enrichr included: Reactome 2016 chylomicron-mediated lipid transport (P = 3.55 × 10-7); Wiki 2016 statin (P = 8.29 × 10-8); GO Biological Processes 2017 chylomicron remodelling (P = 1.92 × 10-8). ClinVar arylsulfatase A pseudodeficiency (ARSA-PD) pathogenic variants were common, including the missense variant c.511 G > A (p.Asp171Asn; rs74315466; frequency 0.44) only reported in Polynesians. This variant is in cis with known ARSA-PD 3' regulatory c.*96 A > G (rs6151429; frequency 0.47) and missense c.1055 A > G (p.Asn352Ser; rs2071421; frequency 0.47) variants. These latter two variants are associated with T2D (risk haplotype GG; odds ratio 2.67; 95% CI 2.32-3.08; P = 2.43 × 10-4) in genome-wide association data (N = 402), but are more strongly associated with quantitative traits (DBP, SBP, ACR, eGFR) for hypertension and renal function in non-diabetic than diabetic subgroups. Traits associated with CVD, CRD and T2D in Aboriginal Australians provide novel insight into function of ARSA-PD variants.

First genome-wide association study in an Australian aboriginal population provides insights into genetic risk factors for body mass index and type 2 diabetes.

A body mass index (BMI) >22kg/m2 is a risk factor for type 2 diabetes (T2D) in Aboriginal Australians. To identify loci associated with BMI and T2D we undertook a genome-wide association study using 1,075,436 quality-controlled single nucleotide polymorphisms (SNPs) genotyped (Illumina 2.5M Duo Beadchip) in 402 individuals in extended pedigrees from a Western Australian Aboriginal community. Imputation using the thousand genomes (1000G) reference panel extended the analysis to 6,724,284 post quality-control autosomal SNPs. No associations achieved genome-wide significance, commonly accepted as P<5x10-8. Nevertheless, genes/pathways in common with other ethnicities were identified despite the arrival of Aboriginal people in Australia >45,000 years ago. The top hit (rs10868204 Pgenotyped = 1.50x10-6; rs11140653 Pimputed_1000G = 2.90x10-7) for BMI lies 5' of NTRK2, the type 2 neurotrophic tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF) that regulates energy balance downstream of melanocortin-4 receptor (MC4R). PIK3C2G (rs12816270 Pgenotyped = 8.06x10-6; rs10841048 Pimputed_1000G = 6.28x10-7) was associated with BMI, but not with T2D as reported elsewhere. BMI also associated with CNTNAP2 (rs6960319 Pgenotyped = 4.65x10-5; rs13225016 Pimputed_1000G = 6.57x10-5), previously identified as the strongest gene-by-environment interaction for BMI in African-Americans. The top hit (rs11240074 Pgenotyped = 5.59x10-6, Pimputed_1000G = 5.73x10-6) for T2D lies 5' of BCL9 that, along with TCF7L2, promotes beta-catenin's transcriptional activity in the WNT signaling pathway. Additional hits occurred in genes affecting pancreatic (KCNJ6, KCNA1) and/or GABA (GABRR1, KCNA1) functions. Notable associations observed for genes previously identified at genome-wide significance in other populations included MC4R (Pgenotyped = 4.49x10-4) for BMI and IGF2BP2 Pimputed_1000G = 2.55x10-6) for T2D. Our results may provide novel functional leads in understanding disease pathogenesis in this Australian Aboriginal population.

Genetic and functional evidence for a locus controlling otitis media at chromosome 10q26.3.

BACKGROUND: Otitis media (OM) is a common childhood disease characterised by middle ear effusion and inflammation. Susceptibility to recurrent acute OM and chronic OM with effusion is 40-70% heritable. Linkage studies provide evidence for multiple putative OM susceptibility loci. This study attempts to replicate these linkages in a Western Australian (WA) population, and to identify the etiological gene(s) in a replicated region. METHODS: Microsatellites were genotyped in 468 individuals from 101 multicase families (208 OM cases) from the WA Family Study of OM (WAFSOM) and non-parametric linkage analysis carried out in ALLEGRO. Association mapping utilized dense single nucleotide polymorphism (SNP) data extracted from Illumina 660 W-Quad analysis of 256 OM cases and 575 controls from the WA Pregnancy Cohort (Raine) Study. Logistic regression analysis was undertaken in ProbABEL. RT-PCR was used to compare gene expression in paired adenoid and tonsil samples, and in epithelial and macrophage cell lines. Comparative genomics methods were used to identify putative regulatory elements and transcription factor binding sites potentially affected by associated SNPs. RESULTS: Evidence for linkage was observed at 10q26.3 (Zlr = 2.69; P = 0.0036; D10S1770) with borderline evidence for linkage at 10q22.3 (Zlr = 1.64; P = 0.05; D10S206). No evidence for linkage was seen at 3p25.3, 17q12, or 19q13.43. Peak association at 10q26.3 was in the intergenic region between TCERG1L and PPP2R2D (rs7922424; P = 9.47 × 10-6), immediately under the peak of linkage. Independent associations were observed at DOCK1 (rs9418832; P = 7.48 × 10-5) and ADAM12 (rs7902734; P = 8.04 × 10-4). RT-PCR analysis confirmed expression of all 4 genes in adenoid samples. ADAM12, DOCK1 and PPP2R2D, but not TCERG1L, were expressed in respiratory epithelial and macrophage cell lines. A significantly associated polymorphism (rs7087384) in strong LD with the top SNP (rs7922424; r2 = 0.97) alters a transcription factor binding site (CREB/CREBP) in the intergenic region between TCERG1L and PPP2R2D. CONCLUSIONS: OM linkage was replicated at 10q26.3. Whilst multiple genes could contribute to this linkage, the weight of evidence supports PPP2R2D, a TGF-ß/Activin/Nodal pathway modulator, as the more likely functional candidate lying immediately under the linkage peak for OM susceptibility at chromosome 10q26.3.

Genetic and functional evidence for a role for SLC11A1 in susceptibility to otitis media in early childhood in a Western Australian population.

Otitis media (OM) is a common disease in early childhood characterised by inflammation of the middle ear. Susceptibility to recurrent acute OM (rAOM; ≥3 episodes AOM in 6 months) and chronic OM with effusion (COME; middle ear effusion ≥3 months) is 40-70% heritable. Three bacterial pathogens commonly associated with OM, Streptococcus pneumoniae (Sp), non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mc), have been observed within adenoids and as facultative intracellular pathogens that invade and survive in mononuclear cells. Case/pseudo-control conditional logistic regression analysis of variants in the SLC11A1 gene, initially identified for its role in resistance to intra-macrophage pathogens in mice, revealed association with OM at four polymorphisms (Pbest=0.025) in 531 families (660 affected children) from the Western Australian Family Study of Otitis Media. This included association at the functional promoter GTn polymorphism (rs34448891) with alleles that regulate high (allele 3; odds ratio=1.2, 95% CI 1.00-1.44, P=0.04) versus low (allele 2; odds ratio=0.83, 95% CI 0.69-0.99, P=0.04) SLC11A1 expression. Haplotype and stepwise conditional logistic regression analyses support a single genetic effect in the proximal region of SLC11A1, with the haplotype 3_C_C_G across rs34448891_rs2276631_rs3731865_rs2695343 significantly (P=0.008) over-transmitted to affected offspring. Stratified analysis showed no association with OM in children who had undergone adenoidectomy (296 children), whereas children with adenoids intact (364 children) showed improved significance at the GTn polymorphism (allele 3: odds ratio=1.38, 95% CI=1.10-1.75, P=0.006). Quantitative RT/PCR demonstrated high expression of SLC11A1 in mononuclear cells isolated from adenoid tissue, with a trend for decreased expression with increasing copies of GTn allele 2. Expression of SLC11A1 was enhanced at 12 (P=1.2×10(-3)) and 24h (P<1.0×10(-4)) after infection of Mono-Mac-6 cells with NTHi. This study identifies SLC11A1 as a novel candidate for OM susceptibility, particularly in children with adenoids intact. Further analysis in other cohorts is required to validate these observations.

Alternative recruitment strategies influence saliva sample return rates in community-based genetic association studies.

Collection of saliva for DNA extraction has created new opportunities to recruit participants from the community for genetic association studies. However, sample return rates are variable. No prior study has specifically addressed how study design impacts sample return. Using data from three large-scale genetic association studies we compared recruitment strategy and sample return rates. We found highly significant differences in sample return rates between the studies. In studies that recruited retrospectively, overall returns were much lower from families with a self-limiting condition who provided samples at a research centre or home visit, than adult elderly individuals with a chronic disease who provided samples by post (59% vs. 84%). Prospective recruitment was associated with high agreement to participate (72%), but subsequent low return of actual saliva samples (42%). A telephone call had marginal effect on recruitment in a retrospective family study, but significantly improved returns in a prospective family study. We found no effect upon DNA yield comparing observed versus unobserved sample collection, or between male and female adult participants. Overall, study design significantly impacts upon response rates for genetic association studies recruiting from the community. Our findings will help researchers in constructing and costing a recruitment protocol.

Genome-wide association study to identify the genetic determinants of otitis media susceptibility in childhood.

BACKGROUND: Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥ 3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥ 3 months) is 40-70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported. METHODS AND FINDINGS: Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at CAPN14 (rs6755194: OR = 1.90; 95%CI 1.47-2.45; P(adj-PCA) = 8.3 × 10(-7)) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29-1.99; P(adj-PCA) = 2.2 × 10(-5)) observed at the adjacent GALNT14 gene. In a gene-based analysis in VEGAS, BPIFA3 (P(Gene) = 2 × 10(-5)) and BPIFA1 (P(Gene) = 1.07 × 10(-4)) in the BPIFA gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (P(adj-PCA)<10(-5)) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFß pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS. CONCLUSIONS: This first discovery GWAS for an OM phenotype has identified CAPN14 and GALNT14 on chromosome 2p23.1 and the BPIFA gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.