Frequency and characterization of mutations in genes in a large cohort of patients referred to MODY registry.

OBJECTIVES: There have been few large-scale studies utilizing exome sequencing for genetically undiagnosed maturity onset diabetes of the young (MODY), a monogenic form of diabetes that is under-recognized. We describe a cohort of 160 individuals with suspected monogenic diabetes who were genetically assessed for mutations in genes known to cause MODY. METHODS: We used a tiered testing approach focusing initially on GCK and HNF1A and then expanding to exome sequencing for those individuals without identified mutations in GCK or HNF1A. The average age of onset of hyperglycemia or diabetes diagnosis was 19 years (median 14 years) with an average HbA1C of 7.1%. RESULTS: Sixty (37.5%) probands had heterozygous likely pathogenic/pathogenic variants in one of the MODY genes, 90% of which were in GCK or HNF1A. Less frequently, mutations were identified in PDX1, HNF4A, HNF1B, and KCNJ11. For those probands with available family members, 100% of the variants segregated with diabetes in the family. Cascade genetic testing in families identified 75 additional family members with a familial MODY mutation. CONCLUSIONS: Our study is one of the largest and most ethnically diverse studies using exome sequencing to assess MODY genes. Tiered testing is an effective strategy to genetically diagnose atypical diabetes, and familial cascade genetic testing identified on average one additional family member with monogenic diabetes for each mutation identified in a proband.

Corrigendum: MC4R-dependent suppression of appetite by bone-derived lipocalin 2.

This corrects the article DOI: 10.1038/nature21697.

MC4R-dependent suppression of appetite by bone-derived lipocalin 2.

Bone has recently emerged as a pleiotropic endocrine organ that secretes at least two hormones, FGF23 and osteocalcin, which regulate kidney function and glucose homeostasis, respectively. These findings have raised the question of whether other bone-derived hormones exist and what their potential functions are. Here we identify, through molecular and genetic analyses in mice, lipocalin 2 (LCN2) as an osteoblast-enriched, secreted protein. Loss- and gain-of-function experiments in mice demonstrate that osteoblast-derived LCN2 maintains glucose homeostasis by inducing insulin secretion and improves glucose tolerance and insulin sensitivity. In addition, osteoblast-derived LCN2 inhibits food intake. LCN2 crosses the blood-brain barrier, binds to the melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial neurons of the hypothalamus and activates an MC4R-dependent anorexigenic (appetite-suppressing) pathway. These results identify LCN2 as a bone-derived hormone with metabolic regulatory effects, which suppresses appetite in a MC4R-dependent manner, and show that the control of appetite is an endocrine function of bone.

Hypomorphism for RPGRIP1L, a ciliary gene vicinal to the FTO locus, causes increased adiposity in mice.

Common polymorphisms in the first intron of FTO are associated with increased body weight in adults. Previous studies have suggested that a CUX1-regulatory element within the implicated FTO region controls expression of FTO and the nearby ciliary gene, RPGRIP1L. Given the role of ciliary genes in energy homeostasis, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. We find that Rpgrip1l⁺/⁻ mice are hyperphagic and fatter, and display diminished suppression of food intake in response to leptin administration. In the hypothalamus of Rpgrip1l⁺/⁻ mice, and in human fibroblasts with hypomorphic mutations in RPGRIP1L, the number of AcIII-positive cilia is diminished, accompanied by impaired convening of the leptin receptor to the vicinity of the cilium, and diminished pStat3 in response to leptin. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO locus.

Bitter taste phenotype and body weight predict children's selection of sweet and savory foods at a palatable test-meal.

Previous studies show that children who are sensitive to the bitter taste of 6-n-propylthiouracil (PROP) report more frequent intake of sweets and less frequent intake of meats (savory fats) relative to children who are PROP insensitive. Laboratory studies are needed to confirm these findings. In this study, seventy-nine 4- to 6-year-olds from diverse ethnicities attended four laboratory sessions, the last of which included a palatable buffet consisting of savory-fats (e.g. pizza), sweet-fats (e.g. cookies, cakes), and sweets (e.g. juices, candies). PROP phenotype was classified by two methods: 1) a common screening procedure to divide children into tasters and nontasters, and 2) a three-concentration method used to approximate PROP thresholds. Height and weight were measured and saliva was collected for genotyping TAS2R38, a bitter taste receptor related to the PROP phenotype. Data were analyzed by General Linear Model ANOVA with intake from savory fats, sweet-fats, and sweets as dependent variables and PROP status as the independent variable. BMI z-score, sex, age, and ethnicity were included as covariates. Adjusted energy intake from the food group "sweets" at the test-meal was greater for tasters than for nontasters. PROP status did not influence children's adjusted intake of savory-fats, but BMI z-score did. The TAS2R38 genotype did not impact intake at the test-meal. At a palatable buffet, PROP taster children preferentially consumed more sweets than nontaster children, while heavier children consumed more savory fats. These findings may have implications for understanding differences in susceptibility to hyperphagia.

Whole-exome sequencing identifies novel LEPR mutations in individuals with severe early onset obesity.

OBJECTIVE: Obesity is a major public health problem that increases the risk for a broad spectrum of co-morbid conditions. Despite evidence for a strong genetic contribution to susceptibility to obesity, previous efforts to discover the relevant genes using positional cloning have failed to account for most of the apparent genetic risk variance. DESIGN AND METHODS: Deploying a strategy combining analysis of exome sequencing data in extremely obese members of four consanguineous families with segregation analysis, we screened for causal genetic variants. Filter-based analysis and homozygosity mapping were used to identify and prioritize putative functional variants. RESULTS: Two novel frameshift mutations in the leptin receptor in two of the families were identified. CONCLUSIONS: These results provide proof-of-principle that whole-exome sequencing of families segregating for extreme obesity can identify causal pathogenic mutations. The methods described here can be extended to additional families segregating for extreme obesity and should enable the identification of mutations in novel genes that predispose to obesity.

Association of candidate genes with nonsyndromic clefts in Honduran and Colombian populations.

OBJECTIVES/HYPOTHESIS: Orofacial clefts are the most common craniofacial birth defects in humans, with the majority of orofacial clefts occurring as nonsyndromic cleft lip with or without cleft palate (NSCLP). We previously demonstrated associations between single-nucleotide polymorphisms (SNPs) in the IRF6 gene and NSCLP in the Honduran population. Here we investigated other candidate genes and chromosomal regions associated with NSCLP identified from genome-wide association studies (GWAS), including MAFB, ABCA4, 8q24, 9q22, 10q25, and 17q22 in two independent Hispanic populations. STUDY DESIGN: Case-control and family-based association testing. METHODS: Honduran families with two or more members with NSCLP (multiplex) were identified. DNA was collected from affected and unaffected family members (488) and 99 gender-matched controls. NSCLP Colombian families were identified; DNA was collected from 26 proband-parent trios. All participants were genotyped for 17 SNPs in six chromosomal regions. Case-control association and family-based association testing (FBAT) analyses were conducted. RESULTS: Seven SNPs demonstrated association in at least one model in the Honduran population. In the Colombian families, five SNPs demonstrated significance in FBAT when patients with isolated cleft palate (CP) were included; four overlapped with SNPs demonstrating significance in the Honduran population, two with the same allele. One SNP retained significance with CP excluded. CONCLUSIONS: This study supports the previous GWAS findings and is the first to suggest a role for FOXE1, ABCA4, and MAFB in orofacial clefting in two separate Hispanic populations.

Common variants in the CD36 gene are associated with oral fat perception, fat preferences, and obesity in African Americans.

Animal studies show that CD36, a fatty acid translocase, is involved in fat detection and preference, but these findings have not been reported in humans. The objective of this study was to determine whether human genetic variation in 5 common CD36 polymorphisms is associated with oral fat perception of Italian salad dressings, self-reported acceptance of high-fat foods and obesity in African-American adults (n = 317). Ratings of perceived oiliness, fat content, and creaminess were assessed on a 170-mm visual analogue scale (VAS) in response to salad dressings that were 5%, 35%, and 55% fat-by-weight content. Acceptance of added fats and oils and high-fat foods was self-reported and anthropometric measures were taken in the laboratory. DNA was isolated from saliva and genotyped at 5 CD36 polymorphisms. Three polymorphisms, rs1761667, rs3840546, and rs1527483 were associated with the outcomes. Participants with the A/A genotype at rs1761667 reported greater perceived creaminess, regardless of the fat concentration of the salad dressings (P < 0.01) and higher mean acceptance of added fats and oils (P = 0.02) compared to those with other genotypes at this site. Individuals who had C/T or T/T genotypes at rs1527483 also perceived greater fat content in the salad dressings, independent of fat concentration (P = 0.03). BMI and waist circumference were higher in participants who were homozygous for a deletion (D/D) at rs3840546, compared to I/D or D/D individuals (P < 0.001), but only 2 D/D individuals were tested, so this finding needs replication. This is the first study to demonstrate an association between common variants in CD36 and fat ingestive behaviors in humans.

Mutations in ZIC3 and ACVR2B are a common cause of heterotaxy and associated cardiovascular anomalies.

BACKGROUND: Heterotaxy syndrome is caused by left-right asymmetry disturbances and is associated with abnormal lateralisation of the abdominal and thoracic organs. The heart is frequently involved and the severity of the abnormality usually determines the outcome. METHODS: We performed a direct sequence analysis of the coding sequence of genes including Zinc Finger Protein of the Cerebellum 3, Left-Right Determination Factor 2, Activin A Receptor Type IIB, and Cryptic in 47 patients with laterality defects and congenital cardiac disease. RESULTS: Of the 47 patients, 31 (66%) had atrioventricular septal defects, 34 (72%) had abnormal systemic venous return, 25 (53%) had transposed or malposed great arteries, and 20 (43%) had pulmonary venous abnormalities. We identified two novel genetic changes in Zinc Finger Protein of the Cerebellum 3, and these variants were not present in 100 ethnically matched control samples. One previously reported missense mutation in Activin A Receptor Type IIB was identified in two unrelated subjects. The genetic changes identified in this study are all located in conserved regions and are predicted to affect protein function in left-right axis formation and cardiovascular development. CONCLUSIONS: Mutations in Zinc Finger Protein of the Cerebellum 3 and Activin A Receptor Type IIB were identified in 4 of the 47 patients with heterotaxy syndrome for a yield of approximately 8.5%. Our results expand the mutation spectrum of monogenic heterotaxy syndrome with associated cardiac anomalies and suggest that there are other causes of heterotaxy yet to be identified.

Association of plastin 3 expression with disease severity in spinal muscular atrophy only in postpubertal females.

OBJECTIVE: To investigate the potential association of plastin 3 (PLS3) expression levels in the blood with disease severity in spinal muscular atrophy (SMA). DESIGN: Measurement of PLS3 messenger RNA levels in the blood of patients with types I, II, and III SMA. SETTING: Pediatric Neuromuscular Clinical Research Network SMA Natural History study. PARTICIPANTS: A cohort of 88 patients of both sexes who had SMA. MAIN OUTCOME MEASURES: Levels of PLS3 messenger RNA in relation to SMA type and SMN2 copy number. RESULTS: Prepubertal female and younger male (<11 years) patients had approximately 2-fold-higher levels of PLS3 expression than did postpubertal female and older male (≥11 years) patients, respectively (P ≤ .001). Expression of PLS3 in male patients did not correlate with SMA clinical type or SMN2 copy number in either age group (P > .10). In postpubertal female patients, PLS3 expression was greatest in patients with type III SMA, was intermediate in patients with type II SMA, and was lowest in patients with type I SMA. Expression of PLS3 correlated with SMA type, SMN2 copy number, and the gross motor function measure only in postpubertal female patients. CONCLUSION: The PLS3 gene may be an age- and/or puberty-specific and sex-specific modifier of SMA.

Sex differences in the effects of inherited bitter thiourea sensitivity on body weight in 4-6-year-old children.

Previous studies have shown that inherited taste blindness to bitter compounds like 6-n-propylthiouracil (PROP) may be a risk factor for obesity, but this literature has been highly controversial. The objectives of this study were (i) to confirm findings that show an interaction between PROP status and sex on BMI z-score, and (ii) to determine if sex also interacts with variations in TAS2R38 (phenylthiocarbamide (PTC) genotype) to influence weight status in 4-6 year olds. Also, we tested whether nontaster children consumed more fat and total energy at laboratory-based meals. Seventy-two ethnically diverse children who ranged in weight status were classified as tasters (N = 52) or nontasters (N = 20) using a standard PROP screening solution. Anthropometric measures were taken, and at the end of each visit, children ate ad libitum from test meals intended for exploratory purposes. Genomic DNA was extracted from saliva and alleles at TAS2R38 were genotyped for A49P polymorphisms. In 75.8% of children, PTC genotype predicted PROP phenotype, whereas in 24.4%, genotype did not predict phenotype. PROP nontaster males had higher BMI z-scores than taster-males and females in both groups (P < 0.05), but due to a three-way interaction between PROP phenotype, TAS2R38 genotype, and sex, this relationship was only true for children who were homozygous for the bitter-insensitive allele (P < 0.0005). There were no differences in test-meal intake as a function of PROP phenotype or TAS2R38 genotype. These results suggest that the TAS2R38 variation, PROP phenotype, and sex interact to impact obesity risk in children. Future studies should be done to determine how this trait influences energy balance.

Glucokinase mutations in young children with hyperglycemia.

BACKGROUND: The etiology of mild hyperglycemia without ketoacidosis in young children is often unknown. Maturity onset diabetes of youth (MODY) is a form of diabetes mellitus (DM) characterized by fasting hyperglycemia without evidence for autoimmune destruction of beta-cells. METHODS: We genetically analyzed four families of young children with fasting hyperglycemia with family histories of diabetes for mutations in the genes for hepatocyte nuclear factor 4 alpha (HNF4alpha), glucokinase (GCK), and hepatocyte nuclear factor 1 alpha (HNF1alpha), the genes responsible for MODY1, MODY2, and MODY3, respectively. RESULTS: We identified mutations in GCK (Gly258Asp, Arg303Trp, and Arg191Gln) in three of the four families. Molecular genetic characterization in these children clarified the etiology and prognosis of the hyperglycemia and allowed discontinuation of insulin therapy in one family. CONCLUSIONS: We conclude that molecular evaluation for MODY in children with mild fasting hyperglycemia without ketosis with family histories of diabetes can provide important prognostic information to guide therapy and exclude preclinical type 1 diabetes mellitus.

Prenatal diagnosis of factor X deficiency using a combination of direct mutation detection and linkage analysis with an intragenic single nucleotide polymorphism.

OBJECTIVE: To present a family in which it was possible to perform prenatal diagnosis for recessively inherited factor X deficiency using both direct mutation detection as well as linkage analysis. METHODS: In a family where both parents were known to be carriers of factor X deficiency, fetal DNA was obtained from an ongoing pregnancy by CVS in the first trimester. Direct DNA sequencing was used to detect a previously identified factor X mutation, and linkage analysis using a single nucleotide polymorphism (SNP) was used to follow the segregation of the other parent's factor X alleles. RESULTS: Our studies predicted the fetus in question to be a heterozygote carrier of factor X deficiency, and this was demonstrated to be correct at birth. CONCLUSIONS: This is the first reported case of prenatal diagnosis of factor X deficiency. In addition, this case demonstrates the remarkable utility of SNPs in linkage analysis of rare genetic disorders.

Rapid prenatal diagnosis of sickle cell diseases using oligonucleotide ligation assay coupled with laser-induced capillary fluorescence detection.

Prenatal diagnosis of sickle cell diseases has been available for several years, and our laboratory has performed over 1000 prenatal diagnoses. However, currently available techniques are labor-intensive and time-consuming, and thus the diagnosis is delayed, making the mother's decision difficult. We describe a rapid, high-throughput technique based on the ligation assay coupled with automated capillary fluorescence detection. This new approach allows the diagnosis of both Hgb S and Hgb C to be available in a few hours. We have utilized this technique in 30 prenatal diagnoses and found it to be in complete agreement with the standard diagnoses.