Obesity-associated variants within FTO form long-range functional connections with IRX3.

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

An 8q24 gene desert variant associated with prostate cancer risk confers differential in vivo activity to a MYC enhancer.

Genome-wide association studies (GWAS) routinely identify risk variants in noncoding DNA, as exemplified by reports of multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer in five independent regions in a gene desert on 8q24. Two of these regions also have been associated with breast and colorectal cancer. These findings implicate functional variation within long-range cis-regulatory elements in disease etiology. We used an in vivo bacterial artificial chromosome (BAC) enhancer-trapping strategy in mice to scan a half-megabase of the 8q24 gene desert encompassing the prostate cancer-associated regions for long-range cis-regulatory elements. These BAC assays identified both prostate and mammary gland enhancer activities within the region. We demonstrate that the 8q24 cancer-associated variant rs6983267 lies within an in vivo prostate enhancer whose expression mimics that of the nearby MYC proto-oncogene. Additionally, we show that the cancer risk allele increases prostate enhancer activity in vivo relative to the non-risk allele. This allele-specific enhancer activity is detectable during early prostate development and throughout prostate maturation, raising the possibility that this SNP could assert its influence on prostate cancer risk before tumorigenesis occurs. Our study represents an efficient strategy to build experimentally on GWAS findings with an in vivo method for rapidly scanning large regions of noncoding DNA for functional cis-regulatory sequences harboring variation implicated in complex diseases.

Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression.

Neuroblastoma is a heterogeneous neoplasm that has served as a paradigm for the clinical utility of somatically acquired genomic aberrations. DNA copy number alterations (CNA) are currently used to predict prognosis, including MYCN amplification and deletions at chromosome bands 1p36 and 11q23. We predicted that genome-wide assessment of DNA aberrations in neuroblastoma tumors would provide a more precise estimation of clinical phenotype, and could be used to predict outcome. We measured CNAs in a representative set of 82 diagnostic tumors on a customized high-resolution BAC array-based CGH platform supplemented with additional clones across 1p36, 2p24, 3p21-22, 11q14-24, and 16p12-13, and integrated these data with RNA expression data. We used an unbiased statistical method to define a set of minimal common regions (MCRs) of aberration. Unsupervised hierarchical clustering identified four distinct genomic subclasses. First, a subset of tumors with a clinically benign phenotype showed predominantly whole chromosome gains and losses. Second, tumors with MYCN amplification had a unique genomic signature of 1p deletion and 17q gain, but few other rearrangements. Third, tumors with an aggressive clinical phenotype without MYCN amplification, showed multiple structural rearrangements. Most notable were deletions of 3p, 4p, and 11q and gain of 1q, 2p, 12q, and 17q. Lastly, there was a subset of tumors with an aggressive clinical phenotype and no detectable DNA CNAs. The genomic subsets were highly correlated with patient outcome, and individual MCRs remained prognostic in a multivariable model. DNA signature patterns embed important prognostic information in diagnostic neuroblastoma samples, and can identify candidate cancer-related genes.

Genetic linkage study of high-grade myopia in a Hutterite population from South Dakota.

PURPOSE: Myopia is a common, complex disorder, and severe forms have implications for blindness due to increased risk of premature cataracts, glaucoma, retinal detachment, and macular degeneration. Autosomal dominant (AD) non-syndromic high-grade myopia has been mapped to chromosomes 18p11.31, 12q21-23, 17q21-23, 7q36, 2q37.1, 7p15.3, 15q12-13, 3q26, 4q12, 8p23, 4q22-q27, 1p36, and Xq23-q25. Here, we demonstrate evidence of linkage for AD non-syndromic high-grade myopia in a large Hutterite family to a locus on chromosome 10q21.1. METHODS: After clinical evaluation, genomic DNA was genotyped from 29 members of a Hutterite family from South Dakota (7 affected). The average refractive error of affected individuals was -7.04 diopters. Microsatellite markers were used to exclude linkage to the known AD nonsyndromic high-grade myopia loci as well as to syndromic high-grade myopia loci. A genome screen was then performed using 382 markers with an average inter-marker distance of 10 cM followed by fine-point mapping in all regions of the genome that gave positive LOD scores. SimWalk2 software was used for multipoint linkage based on AD and autosomal recessive (AR) models with a penetrance of 90% and a disease allele frequency of 0.001. RESULTS: A maximum multipoint LOD score of 3.22 was achieved under an AD model at microsatellite marker D10S1643. Fine point mapping and haplotype analysis defined a critical region of 2.67 cM on chromosome 10q21.1. Haplotype analysis demonstrated two distinct haplotypes segregating with high-grade myopia, indicative of two distinct mutations occurring in the same gene. CONCLUSIONS: We have identified a presumptive myopia locus for high-grade myopia based on linkage and haplotype analysis.

Subtelomeric deletions of chromosome 6p: molecular and cytogenetic characterization of three new cases with phenotypic overlap with Ritscher-Schinzel (3C) syndrome.

We have identified six children in three families with subtelomeric deletions of 6p25 and a recognizable phenotype consisting of ptosis, posterior embryotoxon, optic nerve abnormalities, mild glaucoma, Dandy-Walker malformation, hydrocephalus, atrial septal defect, patent ductus arteriosus, and mild mental retardation. There is considerable clinical overlap between these children and individuals with the Ritscher-Schinzel (or cranio-cerebello-cardiac (3C)) syndrome (OMIM #220210). Clinical features of 3C syndrome include craniofacial anomalies (macrocephaly, prominent forehead and occiput, foramina parietalia, hypertelorism, down-slanting palpebral fissures, ocular colobomas, depressed nasal bridge, narrow or cleft palate, and low-set ears), cerebellar malformations (variable manifestations of a Dandy-Walker malformation with moderate mental retardation), and cardiac defects (primarily septal defects). Since the original report, over 25 patients with 3C syndrome have been reported. Recessive inheritance has been postulated based on recurrence in siblings born to unaffected parents and parental consanguinity in two familial cases. Molecular and cytogenetic mapping of the 6p deletions in these three families with subtelomeric deletions of chromosome 6p have defined a 1.3 Mb minimally deleted critical region. To determine if 6p deletions are common in 3C syndrome, we analyzed seven unrelated individuals with 3C syndrome for deletions of this region. Three forkhead genes (FOXF1 and FOXQ1 from within the critical region, and FOXC1 proximal to this region) were evaluated as potential candidate disease genes for this disorder. No deletions or disease-causing mutations were identified.

Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.