Leveraging pleiotropy to discover and interpret GWAS results for sleep-associated traits.

Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required phenotyping. This reduces statistical power and limits discovery of multiple genetic associations. We present a strategy to leverage pleiotropy between traits to both discover new loci and to provide mechanistic hypotheses of the underlying pathophysiology. Specifically, we combine a colocalization test with a locus-level test of pleiotropy. In simulations, we show that this approach is highly selective for identifying true pleiotropy driven by the same causative variant, thereby improves the chance to replicate the associations in underpowered validation cohorts and leads to higher interpretability. Here, as an exemplar, we use Obstructive Sleep Apnea (OSA), a common disorder diagnosed using overnight multi-channel physiological testing. We leverage pleiotropy with relevant cellular and cardio-metabolic phenotypes and gene expression traits to map new risk loci in an underpowered OSA GWAS. We identify several pleiotropic loci harboring suggestive associations to OSA and genome-wide significant associations to other traits, and show that their OSA association replicates in independent cohorts of diverse ancestries. By investigating pleiotropic loci, our strategy allows proposing new hypotheses about OSA pathobiology across many physiological layers. For example, we identify and replicate the pleiotropy across the plateletcrit, OSA and an eQTL of DNA primase subunit 1 (PRIM1) in immune cells. We find suggestive links between OSA, a measure of lung function (FEV1/FVC), and an eQTL of matrix metallopeptidase 15 (MMP15) in lung tissue. We also link a previously known genome-wide significant peak for OSA in the hexokinase 1 (HK1) locus to hematocrit and other red blood cell related traits. Thus, the analysis of pleiotropic associations has the potential to assemble diverse phenotypes into a chain of mechanistic hypotheses that provide insight into the pathogenesis of complex human diseases.

C-Reactive Protein Levels Correlate with Measures of Dysglycemia and Gut Microbiome Profiles.

C-reactive protein (CRP) is a commonly used marker of low-grade inflammation as well as a marker of acute infection. CRP levels are elevated in those with diabetes and increased CRP concentrations are a risk factor for developing type 2 diabetes. Gut microbiome effects on metabolism and immune responses can impact chronic inflammation, including affecting CRP levels, that in turn can lead to the development and maintenance of dysglycemia. Using a high-sensitivity C-reactive protein (hsCRP) assay capable of detecting subtle changes in C-reactive protein, we show that higher hsCRP levels specifically correlate with worsening glycemia, reduced microbial richness and evenness, and with a reduction in the Firmicutes/Bacteroidota ratio. These data demonstrate a pivotal role for CRP not only in the context of worsening glycemia and changes to the gut microbiota, but also highlight CRP as a potential target for mitigating type 2 diabetes progression or as a therapeutic target that could be manipulated through the microbiome. Understanding these processes will provide insights into the etiology of type 2 diabetes in addition to opening doors leading to possible novel diagnostic strategies and therapeutics.

Associations of variants In the hexokinase 1 and interleukin 18 receptor regions with oxyhemoglobin saturation during sleep.

Sleep disordered breathing (SDB)-related overnight hypoxemia is associated with cardiometabolic disease and other comorbidities. Understanding the genetic bases for variations in nocturnal hypoxemia may help understand mechanisms influencing oxygenation and SDB-related mortality. We conducted genome-wide association tests across 10 cohorts and 4 populations to identify genetic variants associated with three correlated measures of overnight oxyhemoglobin saturation: average and minimum oxyhemoglobin saturation during sleep and the percent of sleep with oxyhemoglobin saturation under 90%. The discovery sample consisted of 8,326 individuals. Variants with p < 1 × 10(-6) were analyzed in a replication group of 14,410 individuals. We identified 3 significantly associated regions, including 2 regions in multi-ethnic analyses (2q12, 10q22). SNPs in the 2q12 region associated with minimum SpO2 (rs78136548 p = 2.70 × 10(-10)). SNPs at 10q22 were associated with all three traits including average SpO2 (rs72805692 p = 4.58 × 10(-8)). SNPs in both regions were associated in over 20,000 individuals and are supported by prior associations or functional evidence. Four additional significant regions were detected in secondary sex-stratified and combined discovery and replication analyses, including a region overlapping Reelin, a known marker of respiratory complex neurons.These are the first genome-wide significant findings reported for oxyhemoglobin saturation during sleep, a phenotype of high clinical interest. Our replicated associations with HK1 and IL18R1 suggest that variants in inflammatory pathways, such as the biologically-plausible NLRP3 inflammasome, may contribute to nocturnal hypoxemia.

Functionally oriented analysis of cardiometabolic traits in a trans-ethnic sample.

Interpretation of genetic association results is difficult because signals often lack biological context. To generate hypotheses of the functional genetic etiology of complex cardiometabolic traits, we estimated the genetically determined component of gene expression from common variants using PrediXcan (1) and determined genes with differential predicted expression by trait. PrediXcan imputes tissue-specific expression levels from genetic variation using variant-level effect on gene expression in transcriptome data. To explore the value of imputed genetically regulated gene expression (GReX) models across different ancestral populations, we evaluated imputed expression levels for predictive accuracy genome-wide in RNA sequence data in samples drawn from European-ancestry and African-ancestry populations and identified substantial predictive power using European-derived models in a non-European target population. We then tested the association of GReX on 15 cardiometabolic traits including blood lipid levels, body mass index, height, blood pressure, fasting glucose and insulin, RR interval, fibrinogen level, factor VII level and white blood cell and platelet counts in 15 755 individuals across three ancestry groups, resulting in 20 novel gene-phenotype associations reaching experiment-wide significance across ancestries. In addition, we identified 18 significant novel gene-phenotype associations in our ancestry-specific analyses. Top associations were assessed for additional support via query of S-PrediXcan (2) results derived from publicly available genome-wide association studies summary data. Collectively, these findings illustrate the utility of transcriptome-based imputation models for discovery of cardiometabolic effect genes in a diverse dataset.

Admixture mapping identifies novel loci for obstructive sleep apnea in Hispanic/Latino Americans.

Obstructive sleep apnea (OSA) is a common disorder associated with increased risk of cardiovascular disease and mortality. Its prevalence and severity vary across ancestral background. Although OSA traits are heritable, few genetic associations have been identified. To identify genetic regions associated with OSA and improve statistical power, we applied admixture mapping on three primary OSA traits [the apnea hypopnea index (AHI), overnight average oxyhemoglobin saturation (SaO2) and percentage time SaO2 < 90%] and a secondary trait (respiratory event duration) in a Hispanic/Latino American population study of 11 575 individuals with significant variation in ancestral background. Linear mixed models were performed using previously inferred African, European and Amerindian local genetic ancestry markers. Global African ancestry was associated with a lower AHI, higher SaO2 and shorter event duration. Admixture mapping analysis of the primary OSA traits identified local African ancestry at the chromosomal region 2q37 as genome-wide significantly associated with AHI (P < 5.7 × 10-5), and European and Amerindian ancestries at 18q21 suggestively associated with both AHI and percentage time SaO2 < 90% (P < 10-3). Follow-up joint ancestry-SNP association analyses identified novel variants in ferrochelatase (FECH), significantly associated with AHI and percentage time SaO2 < 90% after adjusting for multiple tests (P < 8 × 10-6). These signals contributed to the admixture mapping associations and were replicated in independent cohorts. In this first admixture mapping study of OSA, novel associations with variants in the iron/heme metabolism pathway suggest a role for iron in influencing respiratory traits underlying OSA.

Metabolomic profiles associated with subtypes of prediabetes among Mexican Americans in Starr County, Texas, USA.

AIMS/HYPOTHESIS: To understand the complex metabolic changes that occur long before the diagnosis of type 2 diabetes, we investigated differences in metabolomic profiles in plasma between prediabetic and normoglycaemic individuals for subtypes of prediabetes defined by fasting glucose, 2 h glucose and HbA1c measures. METHODS: Untargeted metabolomics data were obtained from 155 plasma samples from 127 Mexican American individuals from Starr County, TX, USA. None had type 2 diabetes at the time of sample collection and 69 had prediabetes by at least one criterion. We tested statistical associations of amino acids and other metabolites with each subtype of prediabetes. RESULTS: We identified distinctive differences in amino acid profiles between prediabetic and normoglycaemic individuals, with further differences in amino acid levels among subtypes of prediabetes. When testing all named metabolites, several fatty acids were also significantly associated with 2 h glucose levels. Multivariate discriminative analyses show that untargeted metabolomic data have considerable potential for identifying metabolic differences among subtypes of prediabetes. CONCLUSIONS/INTERPRETATION: People with each subtype of prediabetes have a distinctive metabolomic signature, beyond the well-known differences in branched-chain amino acids. DATA AVAILABILITY: Metabolomics data are available through the NCBI database of Genotypes and Phenotypes (dbGaP, accession number phs001166; www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001166.v1.p1).

Impact of Diabetes on the Gut and Salivary IgA Microbiomes.

Mucosal surfaces like those present in the lung, gut, and mouth interface with distinct external environments. These mucosal gateways are not only portals of entry for potential pathogens but also homes to microbial communities that impact host health. Secretory immunoglobulin A (SIgA) is the single most abundant acquired immune component secreted onto mucosal surfaces and, via the process of immune exclusion, shapes the architecture of these microbiomes. Not all microorganisms at mucosal surfaces are targeted by SIgA; therefore, a better understanding of the SIgA-coated fraction may identify the microbial constituents that stimulate host immune responses in the context of health and disease. Chronic diseases like type 2 diabetes are associated with altered microbial communities (dysbiosis) that in turn affect immune-mediated homeostasis. 16S rRNA gene sequencing of SIgA-coated/uncoated bacteria (IgA-Biome) was conducted on stool and saliva samples of normoglycemic participants and individuals with prediabetes or diabetes (n = 8/group). These analyses demonstrated shifts in relative abundance in the IgA-Biome profiles between normoglycemic, prediabetic, or diabetic samples distinct from that of the overall microbiome. Differences in IgA-Biome alpha diversity were apparent for both stool and saliva, while overarching bacterial community differences (beta diversity) were also observed in saliva. These data suggest that IgA-Biome analyses can be used to identify novel microbial signatures associated with diabetes and support the need for further studies exploring these communities. Ultimately, an understanding of the IgA-Biome may promote the development of novel strategies to restructure the microbiome as a means of preventing or treating diseases associated with dysbiosis at mucosal surfaces.

Sleep apnea and galectin-3: possible sex-specific relationship.

PURPOSE: Sleep apnea is associated with increased risk of cardiovascular disease. Elevated plasma galectin-3 levels, a biomarker associated with myocardial fibrosis, are also associated with adverse cardiovascular events, including heart failure. Our objective was to determine the relationship between severity of sleep apnea and plasma levels of galectin-3 and to determine whether this relationship was modified by sex. METHODS: We performed a cross-sectional study of 471 Mexican Americans from Starr County, TX who underwent an overnight, in-home sleep evaluation, and plasma measurement of galectin-3. Severity of sleep apnea was based on apnea hypopnea index (AHI). Multivariable linear regression modeling was used to determine the association between categories of sleep apnea and galectin-3. We also tested for interactions by sex. RESULTS: The mean age was 53 years, and 74% of the cohort was female. The prevalence of moderate to severe sleep apnea (AHI > 15 apnea-hypopnea events per hour) was 36.7%. Moderate to severe sleep apnea was associated with increased levels of galectin-3 in the entire population, but we identified a statistically significant interaction between galectin-3 levels and category of sleep apnea by sex (p for interaction = 0.02). Plasma galectin levels were significantly higher in women with moderate or severe sleep apnea than women with no/mild sleep apnea (multivariable adjusted p < 0.001), but not in men (p = 0.5). CONCLUSIONS: Sleep apnea is associated elevated galectin-3 levels in women but not men. Our findings highlight a possible sex-specific relationship between sleep apnea and galectin-3, a biomarker of potential myocardial fibrosis that has been associated with increased cardiovascular risk.

Variants in angiopoietin-2 (ANGPT2) contribute to variation in nocturnal oxyhaemoglobin saturation level.

Genetic determinants of sleep-disordered breathing (SDB), a common set of disorders that contribute to significant cardiovascular and neuropsychiatric morbidity, are not clear. Overnight nocturnal oxygen saturation (SaO2) is a clinically relevant and easily measured indicator of SDB severity but its genetic contribution has never been studied. Our recent study suggests nocturnal SaO2 is heritable. We performed linkage analysis, association analysis and haplotype analysis of average nocturnal oxyhaemoglobin saturation in participants in the Cleveland Family Study (CFS), followed by gene-based association and additional tests in four independent samples. Linkage analysis identified a peak (LOD = 4.29) on chromosome 8p23. Follow-up association analysis identified two haplotypes in angiopoietin-2 (ANGPT2) that significantly contributed to the variation of SaO2 (P = 8 × 10-5) and accounted for a portion of the linkage evidence. Gene-based association analysis replicated the association of ANGPT2 and nocturnal SaO2. A rare missense SNP rs200291021 in ANGPT2 was associated with serum angiopoietin-2 level (P = 1.29 × 10-4), which was associated with SaO2 (P = 0.002). Our study provides the first evidence for the association of ANGPT2, a gene previously implicated in acute lung injury syndromes, with nocturnal SaO2, suggesting that this gene has a broad range of effects on gas exchange, including influencing oxygenation during sleep.

Transancestral fine-mapping of four type 2 diabetes susceptibility loci highlights potential causal regulatory mechanisms.

To gain insight into potential regulatory mechanisms through which the effects of variants at four established type 2 diabetes (T2D) susceptibility loci (CDKAL1, CDKN2A-B, IGF2BP2 and KCNQ1) are mediated, we undertook transancestral fine-mapping in 22 086 cases and 42 539 controls of East Asian, European, South Asian, African American and Mexican American descent. Through high-density imputation and conditional analyses, we identified seven distinct association signals at these four loci, each with allelic effects on T2D susceptibility that were homogenous across ancestry groups. By leveraging differences in the structure of linkage disequilibrium between diverse populations, and increased sample size, we localised the variants most likely to drive each distinct association signal. We demonstrated that integration of these genetic fine-mapping data with genomic annotation can highlight potential causal regulatory elements in T2D-relevant tissues. These analyses provide insight into the mechanisms through which T2D association signals are mediated, and suggest future routes to understanding the biology of specific disease susceptibility loci.

FOXP3 mutations causing early-onset insulin-requiring diabetes but without other features of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.

Diabetes occurs in 1/90 000 to 1/160 000 births and when diagnosed under 6 months of age is very likely to have a primary genetic cause. FOXP3 encodes a transcription factor critical for T regulatory cell function and mutations are known to cause "immune dysregulation, polyendocrinopathy (including insulin-requiring diabetes), enteropathy, X-linked" (IPEX) syndrome. This condition is often fatal unless patients receive a bone-marrow transplant. Here we describe the phenotype of male neonates and infants who had insulin-requiring diabetes without other features of IPEX syndrome and were found to have mutations in FOXP3. Whole-exome or next generation sequencing of genes of interest was carried out in subjects with isolated neonatal diabetes without a known genetic cause. RT-PCR was carried out to investigate the effects on RNA splicing of a novel intronic splice-site variant. Four male subjects were found to have FOXP3 variants in the hemizygous state: p.Arg114Trp, p.Arg347His, p.Lys393Met, and c.1044+5G>A which was detected in 2 unrelated probands and in a brother diagnosed with diabetes at 2.1 years of age. Of these, p.Arg114Trp is likely a benign rare variant found in individuals of Ashkenazi Jewish ancestry and p.Arg347His has been previously described in patients with classic IPEX syndrome. The p.Lys393Met and c.1044+5G>A variants are novel to this study. RT-PCR studies of the c.1044+5G>A splice variant confirmed it affected RNA splicing by generating both a wild type and truncated transcript. We conclude that FOXP3 mutations can cause early-onset insulin-requiring diabetes with or without other features of IPEX syndrome.

Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus.

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.

Contribution of common non-synonymous variants in PCSK1 to body mass index variation and risk of obesity: a systematic review and meta-analysis with evidence from up to 331 175 individuals.

Polymorphisms rs6232 and rs6234/rs6235 in PCSK1 have been associated with extreme obesity [e.g. body mass index (BMI) ≥ 40 kg/m(2)], but their contribution to common obesity (BMI ≥ 30 kg/m(2)) and BMI variation in a multi-ethnic context is unclear. To fill this gap, we collected phenotypic and genetic data in up to 331 175 individuals from diverse ethnic groups. This process involved a systematic review of the literature in PubMed, Web of Science, Embase and the NIH GWAS catalog complemented by data extraction from pre-existing GWAS or custom-arrays in consortia and single studies. We employed recently developed global meta-analytic random-effects methods to calculate summary odds ratios (OR) and 95% confidence intervals (CIs) or beta estimates and standard errors (SE) for the obesity status and BMI analyses, respectively. Significant associations were found with binary obesity status for rs6232 (OR = 1.15, 95% CI 1.06-1.24, P = 6.08 × 10(-6)) and rs6234/rs6235 (OR = 1.07, 95% CI 1.04-1.10, P = 3.00 × 10(-7)). Similarly, significant associations were found with continuous BMI for rs6232 (ß = 0.03, 95% CI 0.00-0.07; P = 0.047) and rs6234/rs6235 (ß = 0.02, 95% CI 0.00-0.03; P = 5.57 × 10(-4)). Ethnicity, age and study ascertainment significantly modulated the association of PCSK1 polymorphisms with obesity. In summary, we demonstrate evidence that common gene variation in PCSK1 contributes to BMI variation and susceptibility to common obesity in the largest known meta-analysis published to date in genetic epidemiology.

Cross-tissue and tissue-specific eQTLs: partitioning the heritability of a complex trait.

Top signals from genome-wide association studies (GWASs) of type 2 diabetes (T2D) are enriched with expression quantitative trait loci (eQTLs) identified in skeletal muscle and adipose tissue. We therefore hypothesized that such eQTLs might account for a disproportionate share of the heritability estimated from all SNPs interrogated through GWASs. To test this hypothesis, we applied linear mixed models to the Wellcome Trust Case Control Consortium (WTCCC) T2D data set and to data sets representing Mexican Americans from Starr County, TX, and Mexicans from Mexico City. We estimated the proportion of phenotypic variance attributable to the additive effect of all variants interrogated in these GWASs, as well as a much smaller set of variants identified as eQTLs in human adipose tissue, skeletal muscle, and lymphoblastoid cell lines. The narrow-sense heritability explained by all interrogated SNPs in each of these data sets was substantially greater than the heritability accounted for by genome-wide-significant SNPs (∼10%); GWAS SNPs explained over 50% of phenotypic variance in the WTCCC, Starr County, and Mexico City data sets. The estimate of heritability attributable to cross-tissue eQTLs was greater in the WTCCC data set and among lean Hispanics, whereas adipose eQTLs significantly explained heritability among Hispanics with a body mass index ≥ 30. These results support an important role for regulatory variants in the genetic component of T2D susceptibility, particularly for eQTLs that elicit effects across insulin-responsive peripheral tissues.

Genetic Associations with Obstructive Sleep Apnea Traits in Hispanic/Latino Americans.

RATIONALE: Obstructive sleep apnea is a common disorder associated with increased risk for cardiovascular disease, diabetes, and premature mortality. Although there is strong clinical and epidemiologic evidence supporting the importance of genetic factors in influencing obstructive sleep apnea, its genetic basis is still largely unknown. Prior genetic studies focused on traits defined using the apnea-hypopnea index, which contains limited information on potentially important genetically determined physiologic factors, such as propensity for hypoxemia and respiratory arousability. OBJECTIVES: To define novel obstructive sleep apnea genetic risk loci for obstructive sleep apnea, we conducted genome-wide association studies of quantitative traits in Hispanic/Latino Americans from three cohorts. METHODS: Genome-wide data from as many as 12,558 participants in the Hispanic Community Health Study/Study of Latinos, Multi-Ethnic Study of Atherosclerosis, and Starr County Health Studies population-based cohorts were metaanalyzed for association with the apnea-hypopnea index, average oxygen saturation during sleep, and average respiratory event duration. MEASUREMENTS AND MAIN RESULTS: Two novel loci were identified at genome-level significance (rs11691765, GPR83, P = 1.90 × 10-8 for the apnea-hypopnea index, and rs35424364; C6ORF183/CCDC162P, P = 4.88 × 10-8 for respiratory event duration) and seven additional loci were identified with suggestive significance (P < 5 × 10-7). Secondary sex-stratified analyses also identified one significant and several suggestive associations. Multiple loci overlapped genes with biologic plausibility. CONCLUSIONS: These are the first genome-level significant findings reported for obstructive sleep apnea-related physiologic traits in any population. These findings identify novel associations in inflammatory, hypoxia signaling, and sleep pathways.

Hypertension in Pregnancy and Future Cardiovascular Event Risk in Siblings.

Hypertension in pregnancy is a risk factor for future hypertension and cardiovascular disease. This may reflect an underlying familial predisposition or persistent damage caused by the hypertensive pregnancy. We sought to isolate the effect of hypertension in pregnancy by comparing the risk of hypertension and cardiovascular disease in women who had hypertension in pregnancy and their sisters who did not using the dataset from the Genetic Epidemiology Network of Arteriopathy study, which examined the genetics of hypertension in white, black, and Hispanic siblings. This analysis included all sibships with at least one parous woman and at least one other sibling. After gathering demographic and pregnancy data, BP and serum analytes were measured. Disease-free survival was examined using Kaplan-Meier curves and Cox proportional hazards regression. Compared with their sisters who did not have hypertension in pregnancy, women who had hypertension in pregnancy were more likely to develop new onset hypertension later in life, after adjusting for body mass index and diabetes (hazard ratio 1.75, 95% confidence interval 1.27-2.42). A sibling history of hypertension in pregnancy was also associated with an increased risk of hypertension in brothers and unaffected sisters, whereas an increased risk of cardiovascular events was observed in brothers only. These results suggest familial factors contribute to the increased risk of future hypertension in women who had hypertension in pregnancy. Further studies are needed to clarify the potential role of nonfamilial factors. Furthermore, a sibling history of hypertension in pregnancy may be a novel familial risk factor for future hypertension.

Beyond type 2 diabetes, obesity and hypertension: an axis including sleep apnea, left ventricular hypertrophy, endothelial dysfunction, and aortic stiffness among Mexican Americans in Starr County, Texas.

BACKGROUND: There is an increasing appreciation for a series of less traditional risk factors that should not be ignored when considering type 2 diabetes, obesity, hypertension, and cardiovascular disease. These include aortic stiffness, cardiac structure, impaired endothelial function and obstructive sleep apnea. They are associated to varying degrees with each disease categorization and with each other. It is not clear whether they represent additional complications, concomitants or antecedents of disease. Starr County, Texas, with its predominantly Mexican American population has been shown previously to bear a disproportionate burden of the major disease categories, but little is known about the distribution of these less traditional factors. METHODS: Type 2 diabetes, obesity and hypertension frequencies were determined through a systematic survey of Starr County conducted from 2002 to 2006. Individuals from this examination and an enriched set with type 2 diabetes were re-examined from 2010 to 2014 including assessment of cardiac structure, sleep apnea, endothelial function and aortic stiffness. Individual and combined frequencies of these inter-related (i.e., axis) conditions were estimated and associations evaluated. RESULTS: Household screening of 5230 individuals aged 20 years and above followed by direct physical assessment of 1610 identified 23.7 % of men and 26.7 % of women with type 2 diabetes, 46.2 and 49.5 % of men and women, respectively with obesity and 32.1 and 32.4 % with hypertension. Evaluation of pulse wave velocity, left ventricular mass, endothelial function and sleep apnea identified 22.3, 12.7, 48.6 and 45.2 % of men as having "at risk" values for each condition, respectively. Corresponding numbers in women were 16.0, 17.9, 23.6 and 28.8 %. Cumulatively, 88 % of the population has one or more of these while 50 % have three or more. CONCLUSIONS: The full axis of conditions is high among Mexican Americans in Starr County, Texas. Individual and joint patterns suggest a genesis well before overt disease. Whether they are all mediated by common underlying factors or whether there exist multiple mechanisms remains to be seen.

Continued lessons from the INS gene: an intronic mutation causing diabetes through a novel mechanism.

BACKGROUND: Diabetes in neonates usually has a monogenic aetiology; however, the cause remains unknown in 20-30%. Heterozygous INS mutations represent one of the most common gene causes of neonatal diabetes mellitus. METHODS: Clinical and functional characterisation of a novel homozygous intronic mutation (c.187+241G>A) in the insulin gene in a child identified through the Monogenic Diabetes Registry (http://monogenicdiabetes.uchicago.edu). RESULTS: The proband had insulin-requiring diabetes from birth. Ultrasonography revealed a structurally normal pancreas and C-peptide was undetectable despite readily detectable amylin, suggesting the presence of dysfunctional ß cells. Whole-exome sequencing revealed the novel mutation. In silico analysis predicted a mutant mRNA product resulting from preferential recognition of a newly created splice site. Wild-type and mutant human insulin gene constructs were derived and transiently expressed in INS-1 cells. We confirmed the predicted transcript and found an additional transcript created via an ectopic splice acceptor site. CONCLUSIONS: Dominant INS mutations cause diabetes via a mutated translational product causing endoplasmic reticulum stress. We describe a novel mechanism of diabetes, without ß cell death, due to creation of two unstable mutant transcripts predicted to undergo nonsense and non-stop-mediated decay, respectively. Our discovery may have broader implications for those with insulin deficiency later in life.

Neonatal diabetes, gallbladder agenesis, duodenal atresia, and intestinal malrotation caused by a novel homozygous mutation in RFX6.

Recently, bi-allelic mutations in the transcription factor RFX6 were described as the cause of a rare condition characterized by neonatal diabetes with pancreatic and biliary hypoplasia and duodenal/jejunal atresia. A male infant developed severe hyperglycemia (446 mg/dL) within 24 h of birth. Acute abdominal concerns by day five necessitated exploratory surgery that revealed duodenal atresia, gallbladder agenesis, annular pancreas and intestinal malrotation. He also exhibited chronic diarrhea and feeding intolerance, cholestatic jaundice, and subsequent liver failure. He died of sepsis at four months old while awaiting liver transplantation. The phenotype of neonatal diabetes with intestinal atresia and biliary agenesis clearly pointed to RFX6 as the causative gene; indeed, whole exome sequencing revealed a novel homozygous RFX6 mutation c.779A>C; p.Lys260Thr (K260T). This missense mutation also changes the consensus 5' splice donor site before intron 7 and is thus predicted to cause disruption in splicing. Both parents, who were not known to be related, were heterozygous carriers. Targeted genetic testing based on consideration of phenotypic features may reveal a cause among the many genes now associated with heterogeneous forms of monogenic neonatal diabetes. Our study demonstrates the feasibility of using modern sequencing technology to identify one such rare cause. Continued research is needed to determine the possible cost-effectiveness of this approach, especially when clear phenotypic clues are absent. Further study of patients with RFX6 mutations should clarify its role in pancreatic, intestinal and enteroendocrine cellular development and explain features such as the diarrhea exhibited in our case.

Microcephaly, epilepsy, and neonatal diabetes due to compound heterozygous mutations in IER3IP1: insights into the natural history of a rare disorder.

Neonatal diabetes mellitus is known to have over 20 different monogenic causes. A syndrome of permanent neonatal diabetes along with primary microcephaly with simplified gyral pattern associated with severe infantile epileptic encephalopathy was recently described in two independent reports in which disease-causing homozygous mutations were identified in the immediate early response-3 interacting protein-1 (IER3IP1) gene. We report here an affected male born to a non-consanguineous couple who was noted to have insulin-requiring permanent neonatal diabetes, microcephaly, and generalized seizures. He was also found to have cortical blindness, severe developmental delay and numerous dysmorphic features. He experienced a slow improvement but not abrogation of seizure frequency and severity on numerous anti-epileptic agents. His clinical course was further complicated by recurrent respiratory tract infections and he died at 8 years of age. Whole exome sequencing was performed on DNA from the proband and parents. He was found to be a compound heterozygote with two different mutations in IER3IP1: p.Val21Gly (V21G) and a novel frameshift mutation p.Phe27fsSer*25. IER3IP1 is a highly conserved protein with marked expression in the cerebral cortex and in beta cells. This is the first reported case of compound heterozygous mutations within IER3IP1 resulting in neonatal diabetes. The triad of microcephaly, generalized seizures, and permanent neonatal diabetes should prompt screening for mutations in IER3IP1. As mutations in genes such as NEUROD1 and PTF1A could cause a similar phenotype, next-generation sequencing approaches-such as exome sequencing reported here-may be an efficient means of uncovering a diagnosis in future cases.