Genetic Variation and Mendelian Randomization Approaches.

While genome-wide association studies (GWAS) on levels of nuclear receptors are sparse, the genetics of ligands of these receptors (steroid hormones, thyroid hormones, and liposoluble vitamins) have been extensively studied in GWAS of predominantly European populations. Hundreds of genetic variants across the genome have been associated with serum levels of nuclear receptor ligands, shedding light on the physiology of hormone metabolism. These GWAS findings have been used to explore causal associations of these hormones with complex human traits and diseases in Mendelian randomization (MR) studies, and in studies using polygenic risk scores to quantify the genetic predisposition to higher/lower hormone levels. As such, besides providing insights into hormonal pathophysiology and its causal relationship with clinical complications, GWAS-identified genetic markers could ultimately play an important role in the daily clinical management of patients. As large trans-ethnic GWAS on levels of nuclear receptor ligands emerge, and with the fast advances in genotyping techniques and constant decrease of the genotyping costs, studying an individual's genetically predicted hormonal profile could be the next step in personalizing the management of patients with pathologies related to nuclear receptors and their ligands.

Role of interleukin 6 signaling pathway in the anti-inflammatory effects of statins on coronary artery disease: Evidence from Mendelian randomization analysis.

BACKGROUND: Statins are currently widely used in the prevention of coronary artery disease (CAD) primarily for lipid-lowering with a potential anti-inflammatory effect. However, it is not clear if their potential anti-inflammatory effects are mediated through the interleukin 6 (IL-6) signaling pathway. METHODS: Using the Mendelian randomization (MR) approach followed by multivariable MR analyses, we examined the extent to which the effects of statins on CAD might be mediated through the IL-6 signaling pathway. RESULTS: Our observations showed that HMG-CoA reductase, using LDL levels as a proxy, had a significant effect on upstream IL-6 (ßMR = 0.47, P-IVW = 0.01) and nominally significant effects on IL-6RA (ßMR = 0.22, P-IVW = 0.047) and APOB (ßMR = 0.82, P-IVW = 1.8 × 10-33). While the IL-6 signaling cascade (IL-6RA ßMR = -0.06, P-IVW = 3.45 × 10-20 and IL-6 ßMR = -0.03, P-IVW = 0.09) and the anti-inflammatory effect of HMG-CoA reductase (ßMR = -0.31, P-IVW = 0.01) was found to influence the risk of CAD, the multivariable MR (MVMR) model indicated that the anti-inflammatory effect of HMG-CoA reductase is not likely to be mediated through the IL-6 signaling cascade, including APOB and IL-6RA (MVMRß = 0.23, P = 0.688). CONCLUSIONS: Our findings suggest that statins may use inflammatory mechanisms independent of the IL-6 signaling pathway to prevent CAD. This result could potentially affect the definition of the target population for statin use.

Metabolome-wide Mendelian randomization for age at menarche and age at natural menopause.

BACKGROUND: The role of metabolism in the variation of age at menarche (AAM) and age at natural menopause (ANM) in the female population is not entirely known. We aimed to investigate the causal role of circulating metabolites in AAM and ANM using Mendelian randomization (MR). METHODS: We combined MR with genetic colocalization to investigate potential causal associations between 658 metabolites and AAM and between 684 metabolites and ANM. We extracted genetic instruments for our exposures from four genome-wide association studies (GWAS) on circulating metabolites and queried the effects of these variants on the outcomes in two large GWAS from the ReproGen consortium. Additionally, we assessed the mediating role of the body mass index (BMI) in these associations, identified metabolic pathways implicated in AAM and ANM, and sought validation for selected metabolites in the Avon Longitudinal Study of Parents and Children (ALSPAC). RESULTS: Our analysis identified 10 candidate metabolites for AAM, but none of them colocalized with AAM. For ANM, 76 metabolites were prioritized (FDR-adjusted MR P-value ≤ 0.05), with 17 colocalizing, primarily in the glycerophosphocholines class, including the omega-3 fatty acid and phosphatidylcholine (PC) categories. Pathway analyses and validation in ALSPAC mothers also highlighted the role of omega and polyunsaturated fatty acids levels in delaying age at menopause. CONCLUSIONS: Our study suggests that metabolites from the glycerophosphocholine and fatty acid families play a causal role in the timing of both menarche and menopause. This underscores the significance of specific metabolic pathways in the biology of female reproductive longevity.

Mendelian randomization identifies circulating proteins as biomarkers for age at menarche and age at natural menopause.

Age at menarche (AAM) and age at natural menopause (ANM) are highly heritable traits and have been linked to various health outcomes. We aimed to identify circulating proteins associated with altered ANM and AAM using an unbiased two-sample Mendelian randomization (MR) and colocalization approach. By testing causal effects of 1,271 proteins on AAM, we identified 22 proteins causally associated with AAM in MR, among which 13 proteins (GCKR, FOXO3, SEMA3G, PATE4, AZGP1, NEGR1, LHB, DLK1, ANXA2, YWHAB, DNAJB12, RMDN1 and HPGDS) colocalized. Among 1,349 proteins tested for causal association with ANM using MR, we identified 19 causal proteins among which 7 proteins (CPNE1, TYMP, DNER, ADAMTS13, LCT, ARL and PLXNA1) colocalized. Follow-up pathway and gene enrichment analyses demonstrated links between AAM-related proteins and obesity and diabetes, and between AAM and ANM-related proteins and various types of cancer. In conclusion, we identified proteomic signatures of reproductive ageing in women, highlighting biological processes at both ends of the reproductive lifespan.

Understanding the genetic complexity of puberty timing across the allele frequency spectrum.

Pubertal timing varies considerably and is associated with later health outcomes. We performed multi-ancestry genetic analyses on ~800,000 women, identifying 1,080 signals for age at menarche. Collectively, these explained 11% of trait variance in an independent sample. Women at the top and bottom 1% of polygenic risk exhibited ~11 and ~14-fold higher risks of delayed and precocious puberty, respectively. We identified several genes harboring rare loss-of-function variants in ~200,000 women, including variants in ZNF483, which abolished the impact of polygenic risk. Variant-to-gene mapping approaches and mouse gonadotropin-releasing hormone neuron RNA sequencing implicated 665 genes, including an uncharacterized G-protein-coupled receptor, GPR83, which amplified the signaling of MC3R, a key nutritional sensor. Shared signals with menopause timing at genes involved in DNA damage response suggest that the ovarian reserve might signal centrally to trigger puberty. We also highlight body size-dependent and independent mechanisms that potentially link reproductive timing to later life disease.

Understanding the genetic complexity of puberty timing across the allele frequency spectrum.

Pubertal timing varies considerably and has been associated with a range of health outcomes in later life. To elucidate the underlying biological mechanisms, we performed multi-ancestry genetic analyses in ~800,000 women, identifying 1,080 independent signals associated with age at menarche. Collectively these loci explained 11% of the trait variance in an independent sample, with women at the top and bottom 1% of polygenic risk exhibiting a ~11 and ~14-fold higher risk of delayed and precocious pubertal development, respectively. These common variant analyses were supported by exome sequence analysis of ~220,000 women, identifying several genes, including rare loss of function variants in ZNF483 which abolished the impact of polygenic risk. Next, we implicated 660 genes in pubertal development using a combination of in silico variant-to-gene mapping approaches and integration with dynamic gene expression data from mouse embryonic GnRH neurons. This included an uncharacterized G-protein coupled receptor GPR83, which we demonstrate amplifies signaling of MC3R, a key sensor of nutritional status. Finally, we identified several genes, including ovary-expressed genes involved in DNA damage response that co-localize with signals associated with menopause timing, leading us to hypothesize that the ovarian reserve might signal centrally to trigger puberty. Collectively these findings extend our understanding of the biological complexity of puberty timing and highlight body size dependent and independent mechanisms that potentially link reproductive timing to later life disease.

Connecting Genomics and Proteomics to Identify Protein Biomarkers for Adult and Youth-Onset Type 2 Diabetes: A Two-Sample Mendelian Randomization Study.

Type 2 diabetes shows an increasing prevalence in both adults and children. Identification of biomarkers for both youth and adult-onset type 2 diabetes is crucial for development of screening tools or drug targets. In this study, using two-sample Mendelian randomization (MR), we identified 22 circulating proteins causally linked to adult type 2 diabetes and 11 proteins with suggestive evidence for association with youth-onset type 2 diabetes. Among these, colocalization analysis further supported a role in type 2 diabetes for C-type mannose receptor 2 (MR odds ratio [OR] 0.85 [95% CI 0.79-0.92] per genetically predicted SD increase in protein level), MANS domain containing 4 (MR OR 0.90 [95% CI 0.88-0.92]), sodium/potassium-transporting ATPase subunit ß2 (MR OR 1.10 [95% CI 1.06-1.15]), endoplasmic reticulum oxidoreductase 1ß (MR OR 1.09 [95% CI 1.05-1.14]), spermatogenesis-associated protein 20 (MR OR 1.12 [95% CI 1.06-1.18]), haptoglobin (MR OR 0.96 [95% CI 0.94-0.98]), and α1-3-N-acetylgalactosaminyltransferase and α1-3-galactosyltransferase (MR OR 1.04 [95% CI 1.03-1.05]). Our findings support a causal role in type 2 diabetes for a set of circulating proteins, which represent promising type 2 diabetes drug targets.

Clinically Relevant Circulating Protein Biomarkers for Type 1 Diabetes: Evidence From a Two-Sample Mendelian Randomization Study.

OBJECTIVE: To identify circulating proteins influencing type 1 diabetes susceptibility using Mendelian randomization (MR). RESEARCH DESIGN AND METHODS: We used a large-scale two-sample MR study, using cis genetic determinants (protein quantitative trait loci [pQTL]) of up to 1,611 circulating proteins from five large genome-wide association studies, to screen for causal associations of these proteins with type 1 diabetes risk in 9,684 case subjects with type 1 diabetes and 15,743 control subjects. Further, pleiotropy-robust MR methods were used in sensitivity analyses using both cis and trans-pQTL. RESULTS: We found that a genetically predicted SD increase in signal regulatory protein gamma (SIRPG) level was associated with increased risk of type 1 diabetes risk (MR odds ratio [OR] 1.66 [95% 1.36-2.03]; P = 7.1 × 10-7). The risk of type 1 diabetes increased almost twofold per genetically predicted standard deviation (SD) increase in interleukin-27 Epstein-Barr virus-induced 3 (IL27-EBI3) protein levels (MR OR 1.97 [95% CI 1.48-2.62]; P = 3.7 × 10-6). However, an SD increase in chymotrypsinogen B1 (CTRB1) was associated with decreased risk of type 1 diabetes (MR OR 0.84 [95% CI 0.77-0.90]; P = 6.1 × 10-6). Sensitivity analyses using MR methods testing for pleiotropy while including trans-pQTL showed similar results. While the MR-Egger suggested no pleotropic effect (P value MR-Egger intercept = 0.31), there was evidence of pleiotropy in MR-PRESSO (P value global test = 0.006). CONCLUSIONS: We identified three novel circulating protein biomarkers associated with type 1 diabetes risk using an MR approach. These biomarkers are promising targets for development of drugs and/or of screening tools for early prediction of type 1 diabetes.

Morquio-like dysostosis multiplex presenting with neuronopathic features is a distinct GLB1-related phenotype.

BACKGROUND: Morquio B disease (MBD) is a distinct GLB1-related dysostosis multiplex presenting a mild phenocopy of GALNS-related Morquio A disease. Previously reported cases from European countries carry the W273L variant on at least one GLB1 allele and exhibit a pure skeletal phenotype (pure MBD). Only a minority of MBD cases have been described with additional neuronopathic findings (MBD plus). OBJECTIVES AND METHODS: With the aim to further describe patterns of MBD-related dysostosis multiplex, we analyzed clinical, biochemical, and genetic features in 17 cases with GLB1-related dysostosis multiplex living and diagnosed in Brazil. RESULTS: About 14 of the 17 individuals had three or more skeletal findings characteristic of Morquio syndrome. Two had no additional neuronopathic features (pure MBD) and 12 exhibited additional neuronopathic features (MBD plus). Three of the 17 cases had mild dysostosis without distinct features of MBD. Seven of the 12 MBD plus patients had signs of spinal cord compression (SCC), as a result of progressive spinal vertebral dysostosis. There was an age-dependent increase in the number of skeletal findings and in the severity of growth impairment. GLB1 mutation analysis was completed in 10 of the 14 MBD patients. T500A occurred in compound heterozygosity in 8 of the 19 alleles. CONCLUSION: Our study extends the phenotypic spectrum of GLB1-related conditions by describing a cohort of patients with MBD and GM1-gangliosidosis (MBD plus). Targeting the progressive nature of the skeletal manifestations in the development of new therapies for GLB1-related conditions is warranted.

Low dietary riboflavin but not folate predicts increased fracture risk in postmenopausal women homozygous for the MTHFR 677 T allele.

UNLABELLED: The MTHFR C677T polymorphism is associated with mildly elevated homocysteine levels when folate and/or riboflavin status is low. Furthermore, a mildly elevated homocysteine level is a risk factor for osteoporotic fractures. We studied whether dietary intake of riboflavin and folate modifies the effects of the MTHFR C677T variant on fracture risk in 5,035 men and women from the Rotterdam Study. We found that the MTHFR C677T variant interacts with dietary riboflavin intake to influence fracture risk in women. INTRODUCTION: The MTHFR C677T polymorphism is associated with mildly elevated homocysteine (Hcy) levels in the presence of low folate and/or riboflavin status. A mildly elevated Hcy level was recently identified as a modifiable risk factor for osteoporotic fracture. We studied whether dietary intake of riboflavin and folate modifies the effects of the MTHFR C677T polymorphism on BMD and fracture risk. MATERIALS AND METHODS: We studied 5,035 individuals from the Rotterdam Study, >or=55 yr of age, who had data available on MTHFR, nutrient intake, and fracture risk. We performed analysis on Hcy levels in a total of 666 individuals, whereas BMD data were present for 4,646 individuals (2,692 women). RESULTS: In the total population, neither the MTHFR C677T polymorphism nor low riboflavin intake was associated with fracture risk and BMD. However, in the lowest quartile of riboflavin intake, female 677-T homozygotes had a 1.8 (95% CI: 1.1-2.9, p = 0.01) times higher risk for incident osteoporotic fractures and a 2.6 (95% CI: 1.3-5.1, p = 0.01) times higher risk for fragility fractures compared with the 677-CC genotype (interaction, p = 0.0002). This effect was not seen for baseline BMD in both men and women. No significant influence was found for dietary folate intake on the association between the MTHFR C677T genotype and fracture risk or BMD. In the lowest quartile of dietary riboflavin intake, T-homozygous individuals (men and women combined) had higher (22.5%) Hcy levels compared with C-homozygotes (mean difference = 3.44 microM, p = 0. 01; trend, p = 0.02). CONCLUSIONS: In this cohort of elderly whites, the MTHFR C677T variant interacts with dietary riboflavin intake to influence fracture risk in women.

Effect of dietary B vitamins on BMD and risk of fracture in elderly men and women: the Rotterdam study.

A mildly elevated homocysteine (Hcy) level is a novel and potentially modifiable risk factor for age-related osteoporotic fractures. Elevated Hcy levels can have a nutritional cause, such as inadequate intake of folate, riboflavin, pyridoxine or cobalamin, which serve as cofactors or substrates for the enzymes involved in the Hcy metabolism. We examined the association between intake of Hcy-related B vitamin (riboflavin, pyridoxine, folate and cobalamin) and femoral neck bone mineral density BMD (FN-BMD) and the risk of fracture in a large population-based cohort of elderly Caucasians. We studied 5304 individuals aged 55 years and over from the Rotterdam Study. Dietary intake of nutrients was obtained from food frequency questionnaires. Incident non-vertebral fractures were recorded during a mean follow-up period of 7.4 years, and vertebral fractures were assessed by X-rays during a mean follow-up period of 6.4 years. We observed a small but significant positive association between dietary pyridoxine (beta = 0.09, p = 1 x 10(-8)) and riboflavin intake (beta = 0.06, p = 0.002) and baseline FN-BMD. In addition, after controlling for gender, age and BMI, pyridoxine intake was inversely correlated to fracture risk. As compared to the three lowest quartiles, individuals in the highest quartile of age- and energy-adjusted dietary pyridoxine intake had a decreased risk of non-vertebral fractures (HR = 0.77, 95% CI = 0.65-0.92, p = 0.005) and of fragility fractures (HR = 0.55, 95% CI = 0.40-0.77, p = 0.0004). Further adjustments for other dietary B vitamins (riboflavin, folate and cobalamin), dietary intake of calcium, vitamin D, vitamin A and vitamin K, protein and energy intake, smoking and BMD did not essentially modify these results. We conclude that increased dietary riboflavin and pyridoxine intake was associated with higher FN-BMD. Furthermore, we found a reduction in risk of fracture in relation to dietary pyridoxine intake independent of BMD. These findings highlight the importance of considering nutritional factors in epidemiological studies of osteoporosis and fractures.

Maternal Western dietary patterns and the risk of developing a cleft lip with or without a cleft palate.

OBJECTIVE: To identify maternal dietary patterns in association with a cleft lip or cleft palate or both in the offspring. METHODS: In a case-control study of 203 mothers of a child with a cleft lip or cleft palate and 178 mothers with non-malformed offspring, maternal nutritional intakes were assessed 14 months after the birth of the index child to estimate the preconception intake. We measured serum and red blood cell folate, serum vitamin B12, whole blood vitamin B6, and total plasma homocysteine as biomarkers. Dietary patterns were analyzed by factor analysis. Univariate and multivariate analyses were performed and odds ratios with 95% confidence intervals calculated. RESULTS: Two major dietary patterns were identified. The Western dietary pattern, eg, high in meat, pizza, legumes, and potatoes, and low in fruits, was associated with a higher risk of a cleft lip or cleft palate (odds ratio 1.9; 95% confidence interval 1.2-3.1). This risk remained significant after adjustment for potential confounders of maternal education and smoking at the time of the study, and periconception use of folic acid or multivitamins. This dietary pattern was associated with lower red blood cell folate (P=.02), vitamin B6 (P=.001), vitamin B12 (P=.02), and higher homocysteine (P=.05) concentrations. The use of the Prudent pattern, eg, high intakes of fish, garlic, nuts, vegetables, increased vitamin B12 (P<.001) and serum folate (P=.05) levels, was not associated with cleft lip or cleft palate risk compared with the Western diet. CONCLUSION: The use of the maternal Western diet increases the risk of offspring with a cleft lip or cleft palate approximately two fold. Therefore, dietary and lifestyle profiles should be included in preconception screening programs. LEVEL OF EVIDENCE: II.

The -1997 G/T and Sp1 polymorphisms in the collagen type I alpha1 (COLIA1) gene in relation to changes in femoral neck bone mineral density and the risk of fracture in the elderly: the Rotterdam study.

The COLIA1 Sp1 polymorphism has been associated with bone mineral density (BMD) and fracture. A promoter polymorphism, -1997 G/T, also has been associated with BMD. In this study, we examined whether these polymorphisms alone and in the form of haplotypes influence bone parameters and fracture risk in a large population-based cohort of elderly Caucasians. We determined the COLIA1 -1997 G/T (promoter) and Sp1 G/T (intron) polymorphisms in 6,280 individuals and inferred haplotypes. Femoral neck BMD and BMD change were compared across COLIA1 genotypes at baseline and follow-up (mean 6.5 years). We also investigated the relationship between the COLIA1 polymorphisms and incident nonvertebral fractures, which were recorded during a mean follow-up period of 7.4 years. Vertebral fractures were assessed by radiographs on 3,456 genotyped individuals. Femoral neck BMD measured at baseline was 3.8% lower in women carrying two copies of the T-Sp1 allele (P for trend = 0.03). No genotype dependent differences in BMD loss were observed. In women homozygous for the T allele of the Sp1 polymorphism, the risk of fragility fracture increased 2.3 times (95% confidence interval 1.4-3.9, P = 0.001). No such association was observed with the promoter polymorphism. In men, no association with either the Sp1 or the -1997 G/T promoter polymorphism was seen with BMD or fracture. High linkage disequilibrium (LD; D' = 0.99, r (2 )= 0.03) exists between the two studied polymorphisms. We observed three haplotypes in our population: haplotype 1 (G(promoter)-G(intron)) frequency (f) = 69%, haplotype 2 (G(promoter)-T(intron)) f = 17.6%, and haplotype 3 (T(promoter)-G(intron)) f = 13.4%. Haplotype 2 was associated with a 2.1-fold increased risk of fragility fracture in women (95% confidence interval 1.2-3.7, P = 0.001). We confirm that the COLIA1 Sp1 polymorphism influences BMD and the risk of fracture in postmenopausal Caucasian women. In contrast, we found no independent effect of the -1997 G/T promoter polymorphism on BMD or fracture.