Expanded Newborn Screening Using Genome Sequencing for Early Actionable Conditions.

Importance: The feasibility of implementing genome sequencing as an adjunct to traditional newborn screening (NBS) in newborns of different racial and ethnic groups is not well understood. Objective: To report interim results of acceptability, feasibility, and outcomes of an ongoing genomic NBS study in a diverse population in New York City within the context of the New York State Department of Health Newborn Screening Program. Design, Setting, and Participants: The Genomic Uniform-screening Against Rare Disease in All Newborns (GUARDIAN) study was a multisite, single-group, prospective, observational investigation of supplemental newborn genome screening with a planned enrollment of 100 000 participants. Parent-reported race and ethnicity were recorded at the time of recruitment. Results of the first 4000 newborns enrolled in 6 New York City hospitals between September 2022 and July 2023 are reported here as part of a prespecified interim analysis. Exposure: Sequencing of 156 early-onset genetic conditions with established interventions selected by the investigators were screened in all participants and 99 neurodevelopmental disorders associated with seizures were optional. Main Outcomes and Measures: The primary outcome was screen-positive rate. Additional outcomes included enrollment rate and successful completion of sequencing. Results: Over 11 months, 5555 families were approached and 4000 (72.0%) consented to participate. Enrolled participants reflected a diverse group by parent-reported race (American Indian or Alaska Native, 0.5%; Asian, 16.5%; Black, 25.1%; Native Hawaiian or Other Pacific Islander, 0.1%; White, 44.7%; 2 or more races, 13.0%) and ethnicity (Hispanic, 44.0%; not Hispanic, 56.0%). The majority of families consented to screening of both groups of conditions (both groups, 90.6%; disorders with established interventions only, 9.4%). Testing was successfully completed for 99.6% of cases. The screen-positive rate was 3.7%, including treatable conditions that are not currently included in NBS. Conclusions and Relevance: These interim findings demonstrate the feasibility of targeted interpretation of a predefined set of genes from genome sequencing in a population of different racial and ethnic groups. DNA sequencing offers an additional method to improve screening for conditions already included in NBS and to add those that cannot be readily screened because there is no biomarker currently detectable in dried blood spots. Additional studies are required to understand if these findings are generalizable to populations of different racial and ethnic groups and whether introduction of sequencing leads to changes in management and improved health outcomes. Trial Registration: ClinicalTrials.gov Identifier: NCT05990179.

CLN2 disease resulting from a novel homozygous deep intronic splice variant in TPP1 discovered using long-read sequencing.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative disorder with enzyme replacement therapy available. We present two siblings with a clinical diagnosis of CLN2 disease, but no identifiable TPP1 variants after standard clinical testing. Long-read sequencing identified a homozygous deep intronic variant predicted to affect splicing, confirmed by clinical DNA and RNA sequencing. This case demonstrates how traditional laboratory assays can complement emerging molecular technologies to provide a precise molecular diagnosis.

Interference of nuclear mitochondrial DNA segments in mitochondrial DNA testing resembles biparental transmission of mitochondrial DNA in humans.

PURPOSE: Reports have questioned the dogma of exclusive maternal transmission of human mitochondrial DNA (mtDNA), including the recent report of an admixture of two mtDNA haplogroups in individuals from three multigeneration families. This was interpreted as being consistent with biparental transmission of mtDNA in an autosomal dominant-like mode. The authenticity and frequency of these findings are debated. METHODS: We retrospectively analyzed individuals with two mtDNA haplogroups from 2017 to 2019 and selected four families for further study. RESULTS: We identified this phenomenon in 104/27,388 (approximately 1/263) unrelated individuals. Further study revealed (1) a male with two mitochondrial haplogroups transmits only one haplogroup to some of his offspring, consistent with nuclear transmission; (2) the heteroplasmy level of paternally transmitted variants is highest in blood, lower in buccal, and absent in muscle or urine of the same individual, indicating it is inversely correlated with mtDNA content; and (3) paternally transmitted apparent large-scale mtDNA deletions/duplications are not associated with a disease phenotype. CONCLUSION: These findings strongly suggest that the observed mitochondrial haplogroup of paternal origin resulted from coamplification of rare, concatenated nuclear mtDNA segments with genuine mtDNA during testing. Evaluation of additional specimen types can help clarify the clinical significance of the observed results.

Correction to: Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.

The original version of this article unfortunately contained a mistake.

Hereditary fructose intolerance mimicking a biochemical phenotype of mucolipidosis: A review of the literature of secondary causes of lysosomal enzyme activity elevation in serum.

We describe a patient with failure to thrive, hepatomegaly, liver dysfunction, and elevation of multiple plasma lysosomal enzyme activities mimicking mucolipidosis II or III, in whom a diagnosis of hereditary fructose intolerance (HFI) was ultimately obtained. She presented before introduction of solid foods, given her consumption of a fructose-containing infant formula. We present the most extensive panel of lysosomal enzyme activities reported to date in a patient with HFI, and propose that multiple enzyme elevations in plasma, especially when in conjunction with a normal plasma α-mannosidase activity, should elicit a differential diagnosis of HFI. We also performed a review of the literature on the different etiologies of elevated lysosomal enzyme activities in serum or plasma. © 2016 Wiley Periodicals, Inc.

A de novo mutation in the ß-tubulin gene TUBB4A results in the leukoencephalopathy hypomyelination with atrophy of the basal ganglia and cerebellum.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and ß-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC's clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.

Resting-state striato-frontal functional connectivity is sensitive to DAT1 genotype and predicts executive function.

Individual differences in striatal dopamine (DA) signaling have been associated both with individual differences in executive function in healthy individuals and with risk for psychiatric disorders defined by executive dysfunction. We used resting-state functional connectivity in 50 healthy adults to examine whether a polymorphism of the dopamine transporter gene (DAT1), which regulates striatal DA function, affects striatal functional connectivity in healthy adults, and whether that connectivity predicts executive function. We found that 9/10 heterozygotes, who are believed to have higher striatal DA signaling, demonstrated stronger connectivity between dorsal caudate (DC) and insular, dorsal anterior cingulate, and dorsolateral prefrontal regions, as well as between ventral striatum and ventrolateral prefrontal cortex, than 10/10 homozygotes. Across subjects, stronger DC-seeded connectivity predicted superior N-back working memory performance, while stronger ventral striatum-seeded connectivity predicted reduced impulsivity in everyday life. Further, mediation analysis suggested that connectivity strength mediated relationships between DAT1 genotype and behavior. These findings suggest that resting-state striato-frontal connectivity may be an endophenotype for executive function in healthy individuals.

A clinicopathologic study of diencephalic pediatric low-grade gliomas with BRAF V600 mutation.

Among brain tumors, the BRAF (V600E) mutation is frequently associated with pleomorphic xanthoastrocytomas (PXAs) and gangliogliomas (GGs). This oncogenic mutation is also detected in ~5 % of other pediatric low-grade gliomas (LGGs) including pilocytic astrocytomas (PAs) and diffuse astrocytomas. In the current multi-institutional study of 56 non-PXA/non-GG diencephalic pediatric LGGs, the BRAF (V600) mutation rate is 36 %. V600-mutant tumors demonstrate a predilection for infants and young children (

Circulating and urinary microRNA profile in focal segmental glomerulosclerosis: a pilot study.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNA molecules that play important roles in the pathogenesis of various kidney diseases. We investigated whether patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) have distinct circulating and urinary miRNA expression profiles that could lead to potential development of noninvasive biomarkers of the disease. MATERIALS AND METHODS: Exosome miRNAs were extracted from plasma and urine samples of patients with primary FSGS (n = 16) or MCD (n = 5) and healthy controls (n = 5). Differences in miRNA abundance were examined using Affymetrix GeneChip miRNA 3.0 arrays. QRT-PCR was used to validate the findings from the array. RESULTS: Comparison analysis of FSGS versus MCD revealed 126 and 155 differentially expressed miRNAs in plasma and in urine, respectively. Only 38 of these miRNAs were previously cited, whereas the remaining miRNAs have not been described. Comparison analysis showed that a significant number of miRNAs were downregulated in both plasma and urine samples of patients with FSGS compared to those with MCD. Plasma levels of miR-30b, miR-30c, miR-34b, miR-34c and miR-342 and urine levels of mir-1225-5p were upregulated in patients with MCD compared to patients with FSGS and controls (P < 0.001). Urinary levels of mir-1915 and miR-663 were downregulated in patients with FSGS compared to MCD and controls (P < 0.001), whereas the urinary levels of miR-155 were upregulated in patients with FSGS when compared to patients with MCD and controls (P < 0.005). CONCLUSIONS: Patients with FSGS and MCD have a unique circulating and urinary miRNA profile. The diagnostic and prognostic potential of miRNAs in FSGS and MCD warrants further studies.

Multi-omic integrated networks connect DNA methylation and miRNA with skeletal muscle plasticity to chronic exercise in Type 2 diabetic obesity.

Epigenomic regulation of the transcriptome by DNA methylation and posttranscriptional gene silencing by miRNAs are potential environmental modulators of skeletal muscle plasticity to chronic exercise in healthy and diseased populations. We utilized transcriptome networks to connect exercise-induced differential methylation and miRNA with functional skeletal muscle plasticity. Biopsies of the vastus lateralis were collected from middle-aged Polynesian men and women with morbid obesity (44 kg/m(2) ± 10) and Type 2 diabetes before and following 16 wk of resistance (n = 9) or endurance training (n = 8). Longitudinal transcriptome, methylome, and microRNA (miRNA) responses were obtained via microarray, filtered by novel effect-size based false discovery rate probe selection preceding bioinformatic interrogation. Metabolic and microvascular transcriptome topology dominated the network landscape following endurance exercise. Lipid and glucose metabolism modules were connected to: microRNA (miR)-29a; promoter region hypomethylation of nuclear receptor factor (NRF1) and fatty acid transporter (SLC27A4), and hypermethylation of fatty acid synthase, and to exon hypomethylation of 6-phosphofructo-2-kinase and Ser/Thr protein kinase. Directional change in the endurance networks was validated by lower intramyocellular lipid, increased capillarity, GLUT4, hexokinase, and mitochondrial enzyme activity and proteome. Resistance training also lowered lipid and increased enzyme activity and caused GLUT4 promoter hypomethylation; however, training was inconsequential to GLUT4, capillarity, and metabolic transcriptome. miR-195 connected to negative regulation of vascular development. To conclude, integrated molecular network modelling revealed differential DNA methylation and miRNA expression changes occur in skeletal muscle in response to chronic exercise training that are most pronounced with endurance training and topographically associated with functional metabolic and microvascular plasticity relevant to diabetes rehabilitation.

Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.

Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild-type DIPG samples. This study presents the first comprehensive multidimensional protein, mRNA, and methylation profiling of pediatric brain tumor specimens, detecting the presence of two subgroups within our DIPG cohort. This multidimensional analysis of DIPG provides increased analytical power to more fully explore molecular signatures of DIPGs, with implications for evaluating potential molecular subtypes and biomarker discovery for assessing response to therapy.

DNA methylation profile associated with rapid decline in kidney function: findings from the CRIC study.

BACKGROUND: Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD). METHODS: We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate. RESULTS: The mean eGFR slope was 2.2 (1.4) and -5.1 (1.2) mL/min/1.73 m(2) in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E-05 to 9.5E-05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E-03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD. CONCLUSIONS: Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD.

Racial disparity with on-treatment platelet reactivity in patients undergoing percutaneous coronary intervention.

BACKGROUND: On-treatment platelet reactivity to clopidogrel is variable and in part genetic dependent. In African American (AA) patients, the relation between on-treatment platelet reactivity to clopidogrel and the factors that influence this interaction is unknown. The present study aims to evaluate on-treatment platelet reactivity to clopidogrel in AA patients and its interaction to race and CYP2C19*2 loss of function mutation. METHODS: The study cohort included 289 consecutive patients presenting for percutaneous coronary intervention who were entered into a prospective observational registry. High on-treatment platelet reactivity (HTPR) was defined as P2Y12 reaction units (PRU) ≥208 with VerifyNow P2Y12 assay and >50% by vasodilator-stimulated phosphoprotein phosphorylation assay platelet reactivity index (VASP PRI) measured 6 to 24 hours postprocedure. CYP2C19*2 (rs4244285) genotype was analyzed by real-time polymerase chain reaction. RESULTS: The prevalence of HTPR by both PRU (56% vs 35%, P = .003) and VASP PRI (67% vs 45%, P = .002) is more common in AAs compared with whites, respectively. African American patients had higher on-treatment mean PRU (207 ± 110 vs 160 ± 102, P = .002) and VASP PRI (49 ± 26 vs 38 ± 26, P = .004). African Americans also had a higher prevalence of CYP2C19*2 allele carrier status compared with whites (43% vs 29%, P = .04). African American race (P = .008) and CYP2C19*2 allele status (P = .02) independently had significant effects on PRU and VASP. Multivariable logistic regression analysis has shown that both CYP2C19*2 allele carrier status and AA race were independent correlates of HTPR for PRU ≥208. CONCLUSIONS: African American patients undergoing percutaneous coronary intervention not only have a higher prevalence of HTPR to clopidogrel but also have higher CYP2C19*2 allele carrier status compared with whites. Careful selection of antiplatelet agents should be considered in an AA population at higher risk for ischemic complications.

Effect of dopamine transporter genotype on intrinsic functional connectivity depends on cognitive state.

Functional connectivity between brain regions can define large-scale neural networks and provide information about relationships between those networks. We examined how relationships within and across intrinsic connectivity networks were 1) sensitive to individual differences in dopaminergic function, 2) modulated by cognitive state, and 3) associated with executive behavioral traits. We found that regardless of cognitive state, connections between frontal, parietal, and striatal nodes of Task-Positive networks (TPNs) and Task-Negative networks (TNNs) showed higher functional connectivity in 10/10 homozygotes of the dopamine transporter gene, a polymorphism influencing synaptic dopamine, than in 9/10 heterozygotes. However, performance of a working memory task (a state requiring dopamine release) modulated genotype differences selectively, such that cross-network connectivity between TPNs and TNNs was higher in 10/10 than 9/10 subjects during working memory but not during rest. This increased cross-network connectivity was associated with increased self-reported measures of impulsivity and inattention traits. By linking a gene regulating synaptic dopamine to a phenotype characterized by inefficient executive function, these findings validate cross-network connectivity as an endophenotype of executive dysfunction.

Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies.

BACKGROUND: We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis. METHODS: We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12,393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644). FINDINGS: In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10(-13)). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10(-9)). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction. INTERPRETATION: Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD. FUNDING: The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.

Variants of the ankyrin repeat domain 6 gene (ANKRD6) and muscle and physical activity phenotypes among European-derived American adults.

Ankyrin repeat domain 6 (ANKRD6) is a ubiquitous protein that associates with early development in mammals and is highly expressed in the brain, spinal cord, and heart of humans. We examined the role of 8 ANKRD6 single-nucleotide polymorphisms (SNPs) on muscle performance and habitual physical activity (PA). Single-nucleotide polymorphisms were 545 T>A (rs9362667), 485 M>L (rs61736690), 233 T>M (rs2273238), 128 I>L (rs3748085), 631 P>L (rs61739327), 122 Q>E (rs16881983), 197805 G>A (rs9344950), and 710 L>X (NOVEL). This study consisted of 922 healthy, untrained, European-derived American men (n = 376, 23.6 ± 0.3 years, 25.0 ± 0.2 kg·m(-2)) and women (n = 546, 23.2 ± 0.2 years, 24.0 ± 0.2 kg·m(-2)). Muscle strength (maximum voluntary contraction [MVC] and 1 repetition maximum [1RM]) and size (cross-sectional area [CSA]) were assessed before and after 12 weeks of unilateral resistance training (RT). A subsample (n = 536, 23.4 ± 0.2 years, 24.6 ± 0.2 kg·m(-2)) completed the Paffenbarger Physical Activity Questionnaire. Associations among ANKRD6 genotypes and muscle phenotypes were tested with repeated measure analysis of covariance (ANCOVA) and PA phenotypes with multivariate ANCOVA, with age and body mass index as covariates. ANKRD6 122 Q>E was associated with increased baseline biceps CSA. ANKRD6 545 A>T and ANKRD6 710 L>X were associated with increased 1RM and MVC in response to RT, respectively. ANKRD6 631 P>L was associated with increased biceps CSA response to RT and time spent in moderate-intensity PA among the total sample and women. ANKRD6 genetic variants were associated with the muscle size and strength response to RT and habitual PA levels. Further research is needed to validate our results and explore mechanisms for the associations we observed.

Characterization of the ZBTB42 gene in humans and mice.

A 12 kb haplotype upstream of the key signaling protein gene, AKT1, has been associated with insulin resistance and metabolic syndrome (Devaney et al. 2010). The region contains the first exon and promoter sequences of AKT1, but also includes the complete transcript unit for a highly conserved yet uncharacterized zinc finger-containing protein (ZBTB42). One of the component SNPs of the 12 kb haplotype metabolic syndrome haplotype changes a conserved amino acid in the predicted ZBTB42 protein, increasing the potential significance of the ZBTB42 transcript unit for contributing to disease risk. Using RT-PCR of human and mouse cells, we verified that the two exon ZBTB42 was expressed and correctly spliced in human skeletal muscle, and murine C2C12 cells. Production of peptide antibodies showed the expected protein in human (47 kD) and mouse (49 kD) immunoblots, and murine tissue distribution showed strongest expression in muscle and ovary. Immunostaining showed nuclear localization of the ZBTB42 protein in human muscle. Confocal imaging analyses of murine muscle showed ZBTB42 distributed in the nucleoplasm, with particular enrichment in nuclei underlying the neuromuscular junctions. The genetic association data of metabolic syndrome, coupled with the molecular characterization of the ZBTB42 transcript unit and encoded protein presented here, suggests that ZBTB42 may be involved in metabolic syndrome phenotypes.

AKT1 polymorphisms are associated with risk for metabolic syndrome.

Converging lines of evidence suggest that AKT1 is a major mediator of the responses to insulin,insulin-like growth factor 1 (IGF1), and glucose. AKT1 also plays a key role in the regulation of both muscle cell hypertrophy and atrophy. We hypothesized that AKT1 variants may play a role in the endophenotypes that makeup metabolic syndrome. We studied a 12-kb region including the first exon of the AKT1 gene for association with metabolic syndrome-related phenotypes in four study populations [FAMUSS cohort (n = 574; age 23.7 ± 5.7 years), Strong Heart Study (SHS) (n = 2,134; age 55.5 ± 7.9 years), Dynamics of Health, Aging and Body Composition (Health ABC) (n = 3,075; age 73.6 ± 2.9 years), and Studies of a Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE)(n = 175; age 40­65 years)]. We identified a three SNP haplotype that we call H1, which represents the ancestral alleles eles at the three loci and H2, which represents the derived alleles at the three loci. In young adult European Americans (FAMUSS), H1 was associated with higher fasting glucose levels in females. In middle age Native Americans (SHS), H1 carriers showed higher fasting insulin and HOMA in males, and higher BMI in females. Inolder African-American and European American subjects(Health ABC) H1 carriers showed a higher incidence of metabolic syndrome. Homozygotes for the H1 haplotype showed about twice the risk of metabolic syndrome in both males and females (p < 0.001). In middle-aged European Americans with insulin resistance (STRRIDE) studied by intravenous glucose tolerance test (IVGTT), H1 carriers showed increased insulin resistance due to the Sg component (p = 0.021). The 12-kb haplotype is a risk factor for metabolic syndrome and insulin resistance that needs to be explored in further populations.

The 1p13.3 LDL (C)-associated locus shows large effect sizes in young populations.

Genome-wide association studies (GWASs) have identified polymorphic loci associated with coronary artery disease (CAD) risk factors (i.e. serum lipids) in adult populations (42-69 y). We hypothesized that younger populations would show a greater relative genetic component due to fewer confounding variables. We examined the influence of 20 GWAS loci associated with serum lipids and insulin metabolism, in a university student cohort (n = 548; mean age = 24 y), and replicated statistically associated results in a second study cohort of primary school students (n = 810, mean age = 11.5 y). Nineteen loci showed no relationship with studied risk factors in young adults. However, the ancestral allele of the rs646776 (SORT1) locus was strongly associated with increased LDL (C) in young adults [TT: 97.6 ± 1.0 mg/dL (n = 345) versus CT/CC: 87.3 ± 1.0 mg/dL (n = 203); p = 3 × 10(x6)] and children [TT: 94.0 ± 1.3 mg/dL (n = 551) versus CT/CC: 84.7 ± 1.4 mg/dL (n = 259); p = 4 × 10(x6)]. This locus is responsible for 3.6% of population variance in young adults and 2.5% of population variance in children. The effect size of the SORT1 locus is considerably higher in young populations (2.5-4.1%) compared with older subjects (1%).

Functional characterization of a haplotype in the AKT1 gene associated with glucose homeostasis and metabolic syndrome.

A small 12-kb haplotype upstream of the AKT1 gene has been found to be associated with insulin resistance phenotypes. We sought to define the functional consequences of the three component polymorphic loci (rs1130214, rs10141867, rs33925946) on AKT1 and the upstream ZBTB42 gene. 5' RACE analysis of AKT1 transcripts in human skeletal muscle biopsies showed the predominant promoter to be 2.5 kb upstream of exon 2, and distinct from those promoters previously reported in rat. We then studied the effect of each of the three haplotype polymorphisms in transcriptional reporter assays in muscle, bone, and fat cell culture models, and found that each modulated enhancer and repressor activity are in a cell-specific and differentiation-specific manner. Our results in promoter assays are consistent with the human phenotype data; we found an anabolic effect on muscle and bone with increased mRNA expression of AKT1, and catabolic effect on fat with decreased expression. To test the hypothesis that rs10141867 affects transcription levels of the novel zinc finger protein ZBTB42 in vivo, we developed the allele-specific expression assay using Taqman technology to test for allelic differences within heterozygotes. The allele containing the derived polymorphism (haplotype H2) showed a 1.75-fold increase in expression in human skeletal muscle. Our data show a particularly complex effect of the component polymorphisms of a single haplotype on cells and tissues, suggesting that the coordination of different tissue-specific effects may have driven selection for the H2 haplotype. In light of the recent abundance of SNP association studies, our approach can serve as a method for exploring the biological function of polymorphisms that show significant genotype/phenotype associations.