James A. Perry

From Genotype to Phenotype: Nonsense Variants in SLC13A1 Are Associated with Decreased Serum Sulfate and Increased Serum Aminotransferases

Using genomic applications to glean insights into human biology, we systematically searched for nonsense single nucleotide variants (SNVs) that are rare in the general population but enriched in the Old Order Amish (Amish) due to founder effect. We identified two nonlinked, nonsense SNVs (R12X and W48X) in SLC13A1 (allele frequencies 0.29% and 0.74% in the Amish; enriched 1.2-fold and 3.7-fold, compared to the outbred Caucasian population, respectively). SLC13A1 encodes the apical sodium-sulfate cotransporter (NaS1) responsible for sulfate (re)absorption in the kidneys and intestine. SLC13A1 R12X and W48X were independently associated with a 27.6% (P = 2.7 × 10−8) and 27.3% (P = 6.9 × 10−14) decrease in serum sulfate, respectively (P = 8.8 × 10-20 for carriers of either SLC13A1 nonsense SNV). We further performed the first exome- and genome-wide association study (ExWAS/GWAS) of serum sulfate and identified a missense variant (L348P) in SLC26A1, which encodes the basolateral sulfate-anion transporter (Sat1), that was associated with decreased serum sulfate (P = 4.4 × 10−12). Consistent with sulfate’s role in xenobiotic detoxification and protection against acetaminophen-induced hepatotoxicity, SLC13A1 nonsense SNV carriers had higher aminotransferase levels compared to noncarriers. Furthermore, SLC26A1 L348P was associated with lower whole-body bone mineral density (BMD) and higher serum calcium, consistent with the osteochondrodysplasia exhibited by dogs and sheep with naturally occurring, homozygous, loss-of-function mutations in Slc13a1. This study demonstrates the power and translational potential of systematic identification and characterization of rare, loss-of-function variants and warrants additional studies to better understand the importance of sulfate in human physiology, disease, and drug toxicity.

Clopidogrel Improves Skin Microcirculatory Endothelial Function in Persons With Heightened Platelet Aggregation

Background Platelet activation can lead to enhanced oxidative stress, inflammatory response, and endothelial dysfunction. To quantify the effects of platelet inhibition on endothelial function, we assessed platelet activity of healthy persons before and after clopidogrel administration and evaluated its effects on endothelial function. We hypothesized that clopidogrel, by attenuating platelet activity, would result in enhanced endothelial function. Methods and Results Microcirculatory endothelial function was quantified by laser Doppler flowmetry (LDF) mediated by thermal hyperemia (TH) and postocclusive reactive hyperemia, respectively, in 287 and 241 relatively healthy and homogenous Old Order Amish persons. LDF and platelet aggregation measures were obtained at baseline and after 7 days of clopidogrel administration. Our primary outcome was percentage change in post‐ versus preclopidogrel LDF measures. Preclopidogrel TH‐LDF and platelet aggregation were higher in women than in men (P<0.001). Clopidogrel administration was associated with ≈2‐fold higher percentage change in TH‐LDF in participants with high versus low baseline platelet aggregation (39.4±10.1% versus 17.4±5.6%, P=0.03). Clopidogrel also increased absolute TH‐LDF measures in persons with high platelet aggregation (1757±766 to 2154±1055, P=0.03), with a more prominent effect in women (1909±846 to 2518±1048, P=0.001). There was no evidence that clopidogrel influenced postocclusive reactive hyperemia LDF measures. Conclusions The administration of clopidogrel in healthy persons with high baseline platelet aggregation results in improved TH‐induced microcirculatory endothelial function. These data suggest that clopidogrel may have a beneficial effect on microcirculatory endothelial function, presumably through antiplatelet activity, and may confer additional vascular benefits. Clinical Trial Registration URL: https://www.clinicaltrials.gov. Unique identifier: NCT00799396.

Deep-coverage whole genome sequences and blood lipids among 16,324 individuals

Large-scale deep-coverage whole-genome sequencing (WGS) is now feasible and offers potential advantages for locus discovery. We perform WGS in 16,324 participants from four ancestries at mean depth >29X and analyze genotypes with four quantitative traits—plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, and triglycerides. Common variant association yields known loci except for few variants previously poorly imputed. Rare coding variant association yields known Mendelian dyslipidemia genes but rare non-coding variant association detects no signals. A high 2M-SNP LDL-C polygenic score (top 5th percentile) confers similar effect size to a monogenic mutation (~30 mg/dl higher for each); however, among those with severe hypercholesterolemia, 23% have a high polygenic score and only 2% carry a monogenic mutation. At these sample sizes and for these phenotypes, the incremental value of WGS for discovery is limited but WGS permits simultaneous assessment of monogenic and polygenic models to severe hypercholesterolemia.Common genetic variants associated with plasma lipids have been extensively studied for a better understanding of common diseases. Here, the authors use whole-genome sequencing of 16,324 individuals to analyze rare variant associations and to determine their monogenic and polygenic contribution to lipid traits.

An APOO Pseudogene on Chromosome 5q is Associated with LDL-C Levels

Background: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels. Methods: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association. Results: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1, increased LDL-C and vascular plaque formation. Conclusions: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.

Assignment of Functional Relevance to Genes at Type 2 Diabetes-Associated Loci Through Investigation of β-Cell Mass Deficits

Type 2 diabetes (T2D) has been associated with a large number of genomic loci, many of which encompass multiple genes without a definitive causal gene. This complexity has hindered efforts to clearly identify functional candidate genes and interpret their role in mediating susceptibility to disease. Here we examined the relevance of individual genes found at T2D-associated loci by assessing their potential contribution to a phenotype relevant to the disease state: production and maintenance of β-cell mass. Using transgenic zebrafish in which β-cell mass could be rapidly visualized in vivo, we systematically suppressed the expression of orthologs of genes found at T2D-associated genomic loci. Overall, we tested 67 orthologs, many of which had no known relevance to β-cell mass, at 62 human T2D-associated loci, including eight loci with multiple candidate genes. In total we identified 25 genes that were necessary for proper β-cell mass, providing functional evidence for their role in a physiological phenotype directly related to T2D. Of these, 16 had not previously been implicated in the regulation of β-cell mass. Strikingly, we identified single functional candidate genes at the majority of the loci for which multiple genes were analyzed. Further investigation into the contribution of the 25 genes to the adaptive capacity of β-cells suggested that the majority of genes were not required for glucose-induced expansion of β-cell mass but were significantly necessary for the regeneration of β-cells. These findings suggest that genetically programmed deficiencies in β-cell mass may be related to impaired maintenance. Finally, we investigated the relevance of our findings to human T2D onset in diabetic individuals from the Old Order Amish and found that risk alleles in β-cell mass genes were associated with significantly younger age of onset and lower body mass index. Taken together, our study offers a functional approach to assign relevance to genes at T2D-associated loci and offers experimental evidence for the defining role of β-cell mass maintenance in genetic susceptibility to T2D onset.

Evaluation of WISP1 as a candidate gene for bone mineral density in the Old Order Amish

Wnt1-inducible signaling pathway protein-1 (WISP1) is a novel target of the Wnt pathway for modulating osteogenesis and improving bone strength. However, it is not clear if genetic variants in the WISP1 region are associated with bone mineral density (BMD) in human. The aim of this study is to investigate the role of genetic variation in WISP1 gene as a determinant of BMD in 1,510 Old Order Amish (OOA). We performed regional association analysis of 58 tag variants within 5 kb upstream and downstream to WISP1 with BMD and found 5 variants that were associated with BMD at multiple skeletal sites (P values from 2.89 × 10−6 to 1.62 × 10−2), with some significant associations even after adjustment for multiple comparisons. To replicate these results in an independent dataset, we performed a look-up of BMD associations with these variants in European ancestry subjects from the large GEFOS Consortium and observed the nominal associations of two of these variants with BMD (P values: 0.031 to 0.048). In conclusion, we have demonstrated that genetic variants surrounding WISP1 are associated with BMD at multiple skeletal sites in the OOA, thus influencing osteoporosis risk. These results support a role for the WISP1 gene on influencing variation in BMD.

Sex-specific effects of serum sulfate level and SLC13A1 nonsense variants on DHEA homeostasis

Context Sulfate is critical in the biotransformation of multiple compounds via sulfation. These compounds include neurotransmitters, proteoglycans, xenobiotics, and hormones such as dehydroepiandrosterone (DHEA). Sulfation reactions are thought to be rate-limited by endogenous sulfate concentrations. The gene, SLC13A1, encodes the sodium-sulfate cotransporter NaS1, responsible for sulfate (re)absorption in the intestines and kidneys. We previously reported two rare, non-linked, nonsense variants in SLC13A1 (R12X and W48X) associated with hyposulfatemia (P = 9 × 10− 20). Objective To examine the effect of serum sulfate concentration and sulfate-lowering genotype on DHEA homeostasis. Design Retrospective cohort study. Setting Academic research. Patients Participants of the Amish Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and the Amish Hereditary and Phenotype Intervention (HAPI) Study. Main outcome measures DHEA, DHEA-S, and DHEA-S/DHEA ratio. Results Increased serum sulfate was associated with decreased DHEA-S (P = 0.03) and DHEA-S/DHEA ratio (P = 0.06) in males but not females. Female SLC13A1 nonsense variant carriers, who had lower serum sulfate (P = 9 × 10− 13), exhibited 14% lower DHEA levels (P = 0.01) and 7% higher DHEA-S/DHEA ratios compared to female non-carriers (P = 0.002). Consistent with this finding, female SLC13A1 nonsense variant carriers also had lower total testosterone levels compared to non-carrier females (P = 0.03). Conclusions Our results demonstrate an inverse relationship between serum sulfate, and DHEA-S and DHEA-S/DHEA ratio in men, while also suggesting that the sulfate-lowering variants, SLC13A1 R12X and W48X, decrease DHEA and testosterone levels, and increase DHEA-S/DHEA ratio in women. While paradoxical, these results illustrate the complexity of the mechanisms involved in DHEA homeostasis and warrant additional studies to better understand sulfates role in hormone physiology.