Estrogen receptor α regulates area-adjusted bone mineral content in late pubertal girls

Context: Whether the action of estrogen in skeletal development depends on estrogen receptor α as encoded by the ESR1 gene is unknown. Objectives: The aim of this study was to establish whether the gain in area-adjusted bone mineral content (ABMC) in girls occurs in late puberty and to examine whether the magnitude of this gain is related to ESR1 polymorphisms. Design: We conducted a cross-sectional analysis. Setting: The study involved the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based prospective study. Participants: Participants included 3097 11-yr-olds with DNA samples, dual x-ray absorptiometry measurements, and pubertal stage information. Outcomes: Outcome measures included separate prespecified analyses in boys and girls of the relationship between ABMC derived from total body dual x-ray absorptiometry scans and Tanner stage and of the interaction between ABMC, Tanner stage, and ESR1 polymorphisms. Results: Total body less head and spinal ABMC were higher in girls in Tanner stages 4 and 5, compared with those in Tanner stages 1, 2, and 3. In contrast, height increased throughout puberty. No differences were observed in ABMC according to Tanner stage in boys. For rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms, differences in spinal ABMC in late puberty were 2-fold greater in girls who were homozygous for the C and G alleles, respectively (P = 0.001). For rs7757956, the difference in total body less head ABMC in late puberty was 50% less in individuals homozygous or heterozygous for the A allele (P = 0.006). Conclusions: Gains in ABMC in late pubertal girls are strongly associated with ESR1 polymorphisms, suggesting that estrogen contributes to this process via an estrogen receptor α-dependent pathway.

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

A review of the MHC genetics of rheumatoid arthritis

Rheumatoid arthritis is a common complex genetic disease, and, despite a significant genetic element, no gene other than HLA-DRB1 has been clearly demonstrated to be involved in the disease. However, this association accounts for less than half the overall genetic susceptibility. Investigation of other candidate genes, in particular those that reside within the major histocompatibility complex, are hampered by the presence of strong linkage disequilibrium and problems with study design. © 2004 Nature Publishing Group All rights reserved.

The effect of transforming growth factor β1 gene polymorphisms in ankylosing spondylitis

Objectives. To determine whether genetic polymorphisms in or near the transforming growth factor β1 (TGFB1) locus were associated d with susceptibility to or severity of ankylosing spondylitis (AS). Methods. Five intragenic single-nucleotide polymorphisms (SNP) and three microsatellite markers flanking the TGFB1 locus were genotyped. Seven hundred and sixty-two individuals from 184 multiplex families were genotyped for the microsatellite markers and two of the promoter SNPs. One thousand and two individuals from 212 English and 170 Finnish families with AS were genotyped for all five intragenic SNPs. A structured questionnaire was used to assess the age of symptom onset, disease duration and disease severity scores, including the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) and BASFI (Bath Ankylosing Spondylitis Functional Index). Results. A weak association was noted between the rare TGFB1 + 1632 T allele and AS in the Finnish population (P = 0.04) and in the combined data set (P = 0.03). No association was noted between any other SNPs or SNP haplotype and AS, even among those families with positive non-parametric linkage scores. The TGFB1 +1632 polymorphism was also associated with a younger age of symptom onset (English population, allele 2 associated with age of onset greater by 4.2 yr, P = 0.05; combined data set, allele 2 associated with age of onset greater by 3.2 yr, P = 0.02). A haplotype of coding region SNPs (TGFB1 +869/ +915+1632 alleles 2/1/2) was associated with age of symptom onset in both the English parent-case trios and the combined data set (English data set, haplotype 2/1/2 associated with age of onset greater by 4.9 yr, P = 0.03; combined data set, haplotype 2/1/2 associated with greater age of onset by 4.2 yr, P = 0.006). Weak linkage with AS susceptibility was noted and the peak LOD score was 1.3 at distance 2 cM centromeric to the TGFB1 gene. No other linkage or association was found between quantitative traits and the markers. Conclusion. This study suggests that the polymorphisms within the TGFB1 gene play at most a small role in AS and that other genes encoded on chromosome 19 are involved in susceptibility to the disease.

Interethnic studies of TNF polymorphisms confirm the likely presence of a second MHC susceptibility locus in ankylosing spondylitis

The objective of this study was to investigate TNF promoter region polymorphisms for association with susceptibility to ankylosing spondylitis (AS). The TNF -238 and -308 polymorphisms were genotyped in 306 English AS cases and 204 ethnically matched healthy B27-positive controls, and 96 southern German AS cases, 58 B27-positive and 251 B27-negative ethnically matched controls. Additionally, the TNF -376 polymorphism was genotyped in the southern German cases and controls. In the southern German AS patients a significant reduction in TNF -308.2 alleles was seen, compared with B27 positive controls (odds ratio 0.4, P= 0.03, 95% confidence interval 0.2-0.9), but no difference in allele frequencies was observed at TNF -238. Significant association between AS and both TNF -238 and TNF -308 was excluded in the English cases. These results confirm previous observations in the southern German population of association between TNF promoter region polymorphisms and AS, but the lack of association in the English population suggests that these polymorphisms themselves are unlikely to be directly involved. More likely, a second, non-HLA-B, MHC locus is involved in susceptibility to AS in these two populations.

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same region.

Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation

Population substructure and hybridization, among other factors, have the potential to cause erroneous associations in linkage disequilibrium (LD) mapping. Two closely related spotted gum eucalypts, Corymbia variegata and C. henryi (Myrtaceae) occur in sympatry in the east coast of Australia and potentially interbreed. They are morphologically similar but are distinguished as separate species based on capsule and foliage size. To determine whether they hybridize in nature and its implications for LD mapping, we investigated the level of molecular divergence between the two species at two sympatric locations separated by 300 kilometres. Very few individuals of intermediate morphology were identified, despite the two species occurring only metres apart. Analysis of genetic structure using 12 microsatellite loci showed that genetic differentiation between populations of the same species at different locations (FST = 0.07 for both species; p = 0.0001) was significantly higher than that observed between species at each location (mean FST = 0.02 and 0.04 for Cherry tree and Bunyaville respectively; p = 0.0001; all Mann-Whitney U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, suggesting recurrent local gene flow between the two species. The lack of significant allele frequency differences implies no population stratification along taxonomic lines. This suggested that there is little concern for cryptic hybridization when sampling from sites of sympatry for LD mapping.

Quantitative and molecular genetics of juvenile wood traits in radiata and slash/Caribbean pines.

The Juvenile Wood Initiative (JWI) project has been running successfully since July 2003 under a Research Agreement with FWPA and Letters of Association with the consortium partners STBA (Southern Tree Breeding Association), ArborGen and FPQ (Forestry Plantations Queensland). Over the last five and half years, JWI scientists in CSIRO, FPQ, and STBA have completed all 12 major milestones and 28 component milestones according to the project schedule. We have made benchmark progress in understanding the genetic control of wood formation and interrelationships among wood traits. The project has made 15 primary scientific findings and several results have been adopted by industry as summarized below. This progress was detailed in 10 technical reports to funding organizations and industry clients. Team scientists produced 16 scientific manuscripts (8 published, 1 in press, 2 submitted, and several others in the process of submission) and 15 conference papers or presentations. Primary Scientific Findings. The 15 major scientific findings related to wood science, inheritance and the genetic basis of juvenile wood traits are: 1. An optimal method to predict stiffness of standing trees in slash/Caribbean pine is to combine gravimetric basic density from 12 mm increment cores with a standing tree prediction of MoE using a time of flight acoustic tool. This was the most accurate and cheapest way to rank trees for breeding selection for slash/Caribbean hybrid pine. This method was also recommended for radiata pine. 2. Wood density breeding values were predicted for the first time in the STBA breeding population using a large sample of 7,078 trees (increment cores) and it was estimated that selection of the best 250 trees for deployment will produce wood density gains of 12.4%. 3. Large genetic variation for a suite of wood quality traits including density, MFA, spiral grain, shrinkage, acoustic and non-acoustic stiffness (MoE) for clear wood and standing trees were observed. Genetic gains of between 8 and 49% were predicted for these wood quality traits with selection intensity between 1 to 10% for radiata pine. 4. Site had a major effect on juvenile-mature wood transition age and the effect of selective breeding for a shorter juvenile wood formation phase was only moderate (about 10% genetic gain with 10% selection intensity, equivalent to about 2 years reduction of juvenile wood). 5. The study found no usable site by genotype interactions for the wood quality traits of density, MFA and MoE for both radiata and slash/Caribbean pines, suggesting that assessment of wood properties on one or two sites will provide reliable estimates of the genetic worth of individuals for use in future breeding. 6. There were significant and sizable genotype by environment interactions between the mainland and Tasmanian regions and within Tasmania for DBH and branch size. 7. Strong genetic correlations between rings for density, MFA and MoE for both radiata and slash/Caribbean pines were observed. This suggests that selection for improved wood properties in the innermost rings would also result in improvement of wood properties in the subsequent rings, as well as improved average performance of the entire core. 8. Strong genetic correlations between pure species and hybrid performance for each of the wood quality traits were observed in the hybrid pines. Parental performance can be used to identify the hybrid families which are most likely to have superior juvenile wood properties of the slash/Caribbean F1 hybrid in southeast Queensland. 9. Large unfavourable genetic correlations between growth and wood quality traits were a prominent feature in radiata pine, indicating that overcoming this unfavourable genetic correlation will be a major technical issue in progressing radiata pine breeding. 10. The project created the first radiata pine 18 k cDNA microarray and generated 5,952 radiata pine xylogenesis expressed sequence tags (ESTs) which assembled into 3,304 unigenes. 11. A total of 348 genes were identified as preferentially expressed genes in earlywood or latewood while a total of 168 genes were identified as preferentially expressed genes in either juvenile or mature wood. 12. Juvenile earlywood has a distinct transcriptome relative to other stages of wood development. 13. Discovered rapid decay of linkage disequilibrium (LD) in radiata pine with LD decaying to approximately 50% within 1,700 base pairs (within a typical gene). A total of 913 SNPS from sequencing 177,380 base pairs were identified for association genetic studies. 14. 149 SNPs from 44 genes and 255 SNPs from a further 51 genes (total 95 genes) were selected for association analysis with 62 wood traits, and 30 SNPs were shortlisted for their significant association with variation of wood quality traits (density, MFA and MoE) with individual significant SNPs accounting for between 1.9 and 9.7% of the total genetic variation in traits. 15. Index selection using breeding objectives was the most profitable selection method for radiata pine, but in the long term it may not be the most effective in dealing with negative genetic correlations between wood volume and quality traits. A combination of economic and biological approaches may be needed to deal with the strong adverse correlation.

Fifteen microsatellite loci for the jungle perch, Kuhlia rupestris

We developed and optimized 15 polymorphic microsatellite loci in the jungle perch, Kuhlia rupestris. Loci were screened in a single population (n = 24) from Fraser Island, Queensland, Australia. Number of alleles per locus ranged from 3 to 19 and observed heterozygosity from 0.25 to 1. No significant linkage disequilibrium was detected between any pair of loci. Genotype proportions for these loci in the population sampled were in Hardy–Weinberg equilibrium.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

Genomic inflation factors under polygenic inheritance

Population structure, including population stratification and cryptic relatedness, can cause spurious associations in genome-wide association studies (GWAS). Usually, the scaled median or mean test statistic for association calculated from multiple single-nucleotide-polymorphisms across the genome is used to assess such effects, and 'genomic control' can be applied subsequently to adjust test statistics at individual loci by a genomic inflation factor. Published GWAS have clearly shown that there are many loci underlying genetic variation for a wide range of complex diseases and traits, implying that a substantial proportion of the genome should show inflation of the test statistic. Here, we show by theory, simulation and analysis of data that in the absence of population structure and other technical artefacts, but in the presence of polygenic inheritance, substantial genomic inflation is expected. Its magnitude depends on sample size, heritability, linkage disequilibrium structure and the number of causal variants. Our predictions are consistent with empirical observations on height in independent samples of ~4000 and ~133,000 individuals.

Function of psoriasis susceptibility gene CCHCR1

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, neoangiogenesis and inflammation. Its etiology is multifactorial, as both the environmental and genetic factors have an important role in the pathogenesis of psoriasis. The exact disease mechanism behind psoriasis still remains unknown. The most important genetic susceptibility region for psoriasis has been located to PSORS1 locus in chromosome 6. The area includes multiply good candidate genes but the strong linkage disequilibrium between them has made genetic studies difficult. One of the candidate genes in PSORS1 is CCHCR1, which has a psoriasis-associated gene form CCHCR1*WWCC. The aim of the study was to elucidate the function of CCHCR1 and its potential role in the pathogenesis of psoriasis. In this study, transgenic mice expressing either the healthy or psoriasis-associated gene form of CCHCR1 were engineered and characterized. Mice were phenotypically normal but their gene expression profiles revealed many similarities to that observed in human psoriatic skin. In addition, the psoriasis-associated gene form had specific impacts on the expression of many genes relevant to the pathogenesis of psoriasis. We also challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoylphorbol-13-acetate (TPA). The experiments revealed that CCHCR1 impacts on keratinocyte proliferation by limiting it. In addition, we demonstrated that CCHCR1 has a role in steroidogenesis and showed that both CCHCR1 forms promote synthesis of steroids. Also many agents relevant either for steroidogenesis or cell proliferation were shown to regulate the expression level of CCHCR1. The present study showed that CCHCR1 has functional properties relevant in the context of psoriasis. Firstly, CCHCR1 affects proliferation of keratinocytes as it may function as a negative regulator of keratinocyte proliferation. Secondly, CCHCR1 also has a role in steroidogenesis, a function relevant both in the pathogenesis of psoriasis and regulation of cell proliferation. This study suggests that aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.

Educational attainment: A genome wide association study in 9538 Australians

BACKGROUND Correlations between Educational Attainment (EA) and measures of cognitive performance are as high as 0.8. This makes EA an attractive alternative phenotype for studies wishing to map genes affecting cognition due to the ease of collecting EA data compared to other cognitive phenotypes such as IQ. METHODOLOGY In an Australian family sample of 9538 individuals we performed a genome-wide association scan (GWAS) using the imputed genotypes of approximately 2.4 million single nucleotide polymorphisms (SNP) for a 6-point scale measure of EA. Top hits were checked for replication in an independent sample of 968 individuals. A gene-based test of association was then applied to the GWAS results. Additionally we performed prediction analyses using the GWAS results from our discovery sample to assess the percentage of EA and full scale IQ variance explained by the predicted scores. RESULTS The best SNP fell short of having a genome-wide significant p-value (p = 9.77x10(-7)). In our independent replication sample six SNPs among the top 50 hits pruned for linkage disequilibrium (r(2)<0.8) had a p-value<0.05 but only one of these SNPs survived correction for multiple testing--rs7106258 (p = 9.7*10(-4)) located in an intergenic region of chromosome 11q14.1. The gene based test results were non-significant and our prediction analyses show that the predicted scores explained little variance in EA in our replication sample. CONCLUSION While we have identified a polymorphism chromosome 11q14.1 associated with EA, further replication is warranted. Overall, the absence of genome-wide significant p-values in our large discovery sample confirmed the high polygenic architecture of EA. Only the assembly of large samples or meta-analytic efforts will be able to assess the implication of common DNA polymorphisms in the etiology of EA.

Association between ORMDL3, IL1RL1 and a deletion on chromosome 17q21 with asthma risk in Australia

Genome-wide association studies followed by replication provide a powerful approach to map genetic risk factors for asthma. We sought to search for new variants associated with asthma and attempt to replicate the association with four loci reported previously (ORMDL3, PDE4D, DENND1B and IL1RL1). Genome-wide association analyses of individual single nucleotide polymorphisms (SNPs), rare copy number variants (CNVs) and overall CNV burden were carried out in 986 asthma cases and 1846 asthma-free controls from Australia. The most-associated locus in the SNP analysis was ORMDL3 (rs6503525, P = 4.8 x 10(-)(7)). Five other loci were associated with P < 10(-)(5), most notably the chemokine CXC motif ligand 14 (CXCL14) gene (rs31263, P = 7.8 x 10(-)(6)). We found no evidence for association with the specific risk variants reported recently for PDE4D, DENND1B and ILR1L1. However, a variant in IL1RL1 that is in low linkage disequilibrium with that reported previously was associated with asthma risk after accounting for all variants tested (rs10197862, gene wide P = 0.01). This association replicated convincingly in an independent cohort (P = 2.4 x 10(-)(4)). A 300-kb deletion on chromosome 17q21 was associated with asthma risk, but this did not reach experiment-wide significance. Asthma cases and controls had comparable CNV rates, length and number of genes affected by deletions or duplications. In conclusion, we confirm the association between asthma risk and variants in ORMDL3 and identify a novel risk variant in IL1RL1. Follow-up of the 17q21 deletion in larger cohorts is warranted.

Common SNPs explain a large proportion of the heritability for human height

SNPs discovered by genome-wide association studies (GWASs) account for only a small fraction of the genetic variation of complex traits in human populations. Where is the remaining heritability? We estimated the proportion of variance for human height explained by 294,831 SNPs genotyped on 3,925 unrelated individuals using a linear model analysis, and validated the estimation method with simulations based on the observed genotype data. We show that 45% of variance can be explained by considering all SNPs simultaneously. Thus, most of the heritability is not missing but has not previously been detected because the individual effects are too small to pass stringent significance tests. We provide evidence that the remaining heritability is due to incomplete linkage disequilibrium between causal variants and genotyped SNPs, exacerbated by causal variants having lower minor allele frequency than the SNPs explored to date.