Biological, clinical and population relevance of 95 loci for blood lipids.

Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Defects in TRH signaling, not thyroid hormone transport, underlie IGSF1-deficiency syndrome

Pathogenic mutations in the carboxyl-terminal domain (CTD) of immunoglobulin superfamily member 1 (IGSF1, formerly known as InhBP/p120) cause IGSF1-deficiency syndrome, a novel form of central hypothyroidism. IGSF1-deficient (Igsf1ex1/y) mice have demonstrably upregulated levels of thyrotropin-releasing hormone (Trh) mRNA in the hypothalamus, which may reflect low circulating thyroid hormones (THs) and/or impaired TH transport into the hypothalamus. Delivery of THs to the hypothalamus requires their active transport across the blood-cerebrospinal fluid barrier (BCSFB) by TH transporters, the most notable of which is monocarboxylate transporter 8 (MCT8). Here we investigated the possibility that increased Trh expression in the hypothalamus of Igsf1ex1/y mice was reflective of impaired MCT8-mediated TH transport across the BCSFB. We demonstrate a novel interaction between IGSF1 and MCT8 that occurs at the plasma membrane, which is specific to MCT8 as IGSF1 does not interact with the closely related MCT10. With both IGSF1 and MCT8 expressed at the apical membrane of the choroid plexus (CP), which forms the BCSFB, of wild-type mice, we suspected that IGSF1 acted as an ancillary protein to MCT8, trafficking MCT8 to the apical membrane. However, MCT8 remains apically localized in the CP of Igsf1ex1/y mice, suggesting that IGSF1 is dispensable for MCT8 subcellular localization. Furthermore, we did not detect an effect of IGSF1 on TH influx in vitro, nor does impaired TH transport into the hypothalamus account for increased Trh expression in Igsf1ex1/y mice. However, Igsf1ex1/y mice had a significantly blunted thyroid-stimulating hormone (TSH) response to hypothyroidism and exogenous TRH administration, as well as low Trhr expression. These results suggest that TH transport is not affected by the loss of IGSF1 in the hypothalamus, and that downregulation of TRH receptor may directly impair TRH-mediated TSH secretion.Les mutations pathogéniques dans le domaine de la terminaison carboxyle (CTD) du membre de la superfamille des immunoglobulines 1 (IGSF1, auparavant connu sous le nom de InhBP/p120) cause un syndrome dû à une déficience en IGSF1, une nouvelle forme d'hypothyroïdie centrale. Les souris déficientes en IGSF1 (Igsf1ex1/y) démontrent un niveau élevé d'ARNm de l'hormone thyréotrope (Trh) au niveau de l'hypothalamus, ce qui pourrait signifier une baisse des hormones thyroïdiennes (THs) dans la circulation et/ou un problème de transport des THs vers l'hypothalamus. Pour accéder à l'hypothalamus, les THs doivent être activement transportées au travers de la barrière sang-liquide céphalo-rachidien (BCSFB) par des transporteurs de TH, dont l'un des plus important est le transporteur monocarboxylate 8 (MCT8). Cette thèse examine la possibilité qu'une augmentation de l'ARNm de Trh au niveau de l'hypothalamus des souris Igsf1ex1/y reflète un trouble de transport des THs au travers de la BCSFB, qui est dépendent de MCT8. Nous avons démontré une nouvelle interaction entre MCT8 et IGSF1 à la surface de la membrane cellulaire. Cette interaction est spécifique pour MCT8 car IGSF1 n'interagie pas avec MCT10 un membre rapproché de MCT8. De plus, parce que IGSF1 et MCT8 interagissent à la membrane apicale du plexus choroïde (CP), qui forme la BCSFB chez les souris de type sauvage, nous croyons que IGSF1 pourrait diriger MCT8 vers la membrane apicale des cellules. Par contre, la localisation de MCT8 à la membrane apicale demeure la même dans le CP des souris Igsf1ex1/y, ce qui suggère que IGSF1 n'est pas nécessaire à la localisation de MCT8 dans le CP. Selon nos expériences qui adressent la fonction de cette interaction in vitro, IGSF1 n'a aucun effet sur l'influx des THs. De plus, chez les souris Igsf1ex1/y un trouble de transport des THs vers l'hypothalamus ne semble pas être responsable pour l'augmentation de l'ARNm de Trh. Par contre, les souris Igsf1ex1/y ont une réponse de l'hormone thyréostimuline (TSH) diminuée lorsque que l'axe est provoqué par des niveaux bas de THs ou une administration de TRH exogène, ainsi qu'un niveau d'ARNm de Trhr bas. Les résultats présentés ici suggèrent que le transport des THs n'est pas affecté par la perte d'expression de IGSF1 au niveau de l'hypothalamus, et que la réduction des récepteurs de TRH pourrait directement affecter la sécrétion de TSH par TRH

Three Novel IGSF1 Mutations in Four Japanese Patients With X-Linked Congenital Central Hypothyroidism

Context: Congenital central hypothyroidism (C-CH) is a rare disease. We investigated the molecular basis of unexplained C-CH in 4 Japanese boys. Patients and Methods: C-CH was diagnosed by low free T-4 and/or T-3 and low basal TSH concentrations. We used whole-exome sequencing of one patient with C-CH to identify potential disease-causing mutations. Thereafter, PCR direct sequencing was performed to Identify genetic defects underlying C-CH in 3 more patients. We then assessed the effects of mutations identified in the Ig superfamily, member 1 (IGSF1), gene on protein expression and membrane trafficking. Results: All patients had congenital hypothyroidism, and 2 had definitive prolactin deficiency. Two patients were detected by neonatal screening. The other patients were diagnosed by short stature and failure to thrive. We identified a novel nonsense variant in IGSF1 by whole-exome sequencing in patient 1, which was confirmed by PCR direct sequencing (p.R1189X). PCR direct sequencing identified the identical nonsense mutation in patient 2. Patients 3 and 4 harbored distinct missense (p.V1082E) or nonsense (p.Q645X) mutations in IGSF1. The mothers of patients 1, 3, and 4 were heterozygous for these mutations. The R1189X mutant, which lacks the transmembrane domain, failed to traffic to the plasma membrane. V1082E could be observed at the cell surface, but at greatly diminished levels relative to the wild-type form of the protein. The severely truncated Q645X mutant could not be detected by Western blot. Conclusion: Our findings provide additional genetic evidence that loss-of-function mutations in IGSF1 cause an X-linked form of C-CH and variable prolactin deficiency

The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein

Mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1) cause an X-linked form of central hypothyroidism. The canonical form of IGSF1 is a transmembrane glycoprotein with 12 immunoglobulin (Ig) loops. The protein is co-translationally cleaved into two sub-domains. The carboxyl-terminal domain (CTD), which contains the last 7 Ig loops, is trafficked to the plasma membrane. Most pathogenic mutations in IGSF1 map to the portion of the gene encoding the CTD. IGSF1/Igsf1 encodes a variety of transcripts. A little studied, but abundant splice variant encodes a truncated form of the protein, predicted to contain the first 2 Ig loops of the full-length IGSF1. The protein (hereafter referred to as IGSF1 isoform 2 or IGSF1-2) is likely retained in most individuals with IGSF1 mutations. [...

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action

Primers used for cloning and mutagenesis.

<p>Primers used for cloning and mutagenesis.</p

Murine and rat IGSF1-2 proteins are not secreted from transfected cells.

<p>HEK293 cells were transfected with expression plasmids for wild-type (WT) or glycosylation mutant (NQ) forms of murine or rat IGSF1-2, murine transthyretin (TTR), or empty vector (pcDNA4). Note, in all cases, proteins were expressed with Myc/His tags at their C-termini. Media (top two panels) and whole cell protein lysates (bottom panel) were collected and analyzed by SDS-PAGE and immunoblotting for Myc. In the middle panel, proteins in culture medium were analyzed directly. In the top panel, proteins in the media were analyzed following Ni-NTA purification and enrichment.</p