A nonsense loss-of-function mutation in PCSK1 contributes to dominantly inherited human obesity.

BACKGROUND: A significant proportion of severe familial forms of obesity remain genetically elusive. Taking advantage of our unique cohort of multigenerational obese families, we aimed to assess the contribution of rare mutations in 29 common obesity-associated genes to familial obesity, and to evaluate in these families the putative presence of nine known monogenic forms of obesity. METHODS: Through next-generation sequencing, we sequenced the coding regions of 34 genes involved in polygenic and/or monogenic forms of obesity in 201 participants (75 normal weight individuals, 54 overweight individuals and 72 individuals with obesity class I, II or III) from 13 French families. In vitro functional analyses were performed to investigate the mutation PCSK1-p.Arg80* which was identified in a family. RESULTS: A novel heterozygous nonsense variant in PCSK1 (p.Arg80*), encoding a propeptide truncated to less than two exons (out of 14), was found to co-segregate with obesity in a three-generation family. We demonstrated that this mutation inhibits PCSK1 enzyme activity and that this inhibition most likely does not involve a strong physical interaction. Furthermore, both mutations PCSK1-p.Asn180Ser and POMC-p.Phe144Leu, which had previously been reported to be associated with severe obesity, were also identified in this study, but did not co-segregate with obesity. Finally, we did not identify any rare mutations co-segregating with obesity in common obesity susceptibility genes, except for CADM2 and QPCTL, where we found two novel variants (p.Arg81His and p.Leu98Pro, respectively) in three obese individuals. CONCLUSIONS: We showed for the first time that a nonsense mutation in PCSK1 was likely to cause dominantly inherited human obesity, due to the inhibiting properties of the propeptide fragment encoded by the null allele. Furthermore, the present family sequencing design challenged the contribution of previously reported mutations to monogenic or at least severe obesity.

The callipyge mutation enhances the expression of coregulated imprinted genes in cis without affecting their imprinting status.

The callipyge (CLPG) phenotype (from kappa(alpha)lambda(iota), "beautiful," and pi(iota)gamma(epsilon), "buttocks") described in sheep is an inherited muscular hypertrophy that is subject to an unusual parent-of-origin effect referred to as polar overdominance: only heterozygous individuals having inherited the CLPG mutation from their sire exhibit the muscular hypertrophy. The callipyge (clpg) locus was mapped to a chromosome segment of approximately 400 kb (refs. 2-4), which was shown to contain four genes (DLK1, GTL2, PEG11 and MEG8) that are preferentially expressed in skeletal muscle and subject to parental imprinting in this tissue. Here we describe the effect of the CLPG mutation on the expression of these four genes, and demonstrate that callipyge individuals have a unique expression profile that may account for the observed polar overdominance.

The 1993-94 Généthon human genetic linkage map.

In 1992, we described a second-generation genetic linkage map of the human genome. Using 1,267 new microsatellite markers, we now present a new genetic linkage map containing a total of 2,066 (AC)n short tandem repeats, 60% of which show a heterozygosity of over 0.7. Statistical linkage analysis based on the genotyping of eight large CEPH families placed these markers in the 23 linkage groups. The map includes 1,266 intervals and spans a total distance of 3690 centiMorgans (cM). A total of 1,041 markers could be ordered with odds ratios greater than 1000:1. About 56% of this map is at a distance of 1 cM or less from one of its markers.

Localization of Friedreich ataxia phenotype with selective vitamin E deficiency to chromosome 8q by homozygosity mapping.

Friedreich ataxia and ataxia with selective vitamin E deficiency (AVED) share very similar clinical phenotypes. We have mapped the AVED locus to proximal 8q with only three large consanguinous Tunisian families, representing to our knowledge the first use of homozygosity mapping for primary linkage analysis. Subsequently, three additional families showed linkage with the same markers. A maximum lod score of 17.9 was obtained at theta = 0 for the haplotype D8S260-D8S510, consisting of the two closest markers. With only 6 families, the AVED locus is therefore mapped precisely as illustrated by the lod-1 confidence interval of 2.4 cM on either side of D8S260-D8S510. Isolation of a yeast artificial chromosome contig > 800 kilobases (kb) showed that D8S260 and D8S510 are less than 400 kb apart.

A complete genomic screen for multiple sclerosis underscores a role for the major histocompatability complex. The Multiple Sclerosis Genetics Group.

Multiple sclerosis (MS), an inflammatory autoimmune demyelinating disorder of the central nervous system, is the most common cause of acquired neurological dysfunction arising in the second to fourth decades of life. A genetic component to MS is indicated by an increased relative risk of 20-40 to siblings compared to the general population (lambda s), and an increased concordance rate in monozygotic compared to dizygotic twins. Association and/or linkage studies to candidate genes have produced many reports of significant genetic effects including those for the major histocompatability complex (MHC; particularly the HLA-DR2 allele), immunoglobulin heavy chain (IgH), T-cell receptor (TCR) and myelin basic protein (MBP) loci. With the exception of the MHC, however, these results have been difficult to replicate and/or apply beyond isolated populations. We have therefore conducted a two-stage, multi-analytical genomic screen to identify genomic regions potentially harbouring MS susceptibility genes. We genotyped 443 markers and 19 such regions were identified. These included the MHC region on 6p, the only region with a consistently reported genetic effect. However, no single locus generated overwhelming evidence of linkage. Our results suggest that a multifactorial aetiology, including both environmental and multiple genetic factors of moderate effect, is more likely than an aetiology consisting of simple mendelian disease gene(s).

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies.

BACKGROUND: Meiotic recombination events do not occur randomly along a chromosome, but appear to be restricted to specific regions. In addition, some regions in the genome undergo recombination more frequently in the germ cells of one sex than the other. Genomic imprinting, the process by which the two parental alleles of a gene are differentially marked, is another genetic phenomenon associated with inheritance from only one parent or the other. The mechanisms that control meiotic recombination and genomic imprinting are unknown, but both phenomena necessarily depend on the presence of some DNA signal sequences and/or on the structure of the surrounding chromatin domain. RESULTS: In the present study, we compared the frequencies of sex-specific recombination events in three chromosomal regions of the human genome that contain clustered imprinted genes. Alignment of the genetic and physical maps of the ZNF127-SNRPN-IPW-PAR-5-PAR-1 region on chromosome 15q11-q13 (associated with Prader-Willi and Angelman syndromes) and the IGF2-H19 region on chromosome 11p15.5 (associated with Beckwith-Wiedemann syndrome) shows that both regions recombine with very high frequency during male meiosis, and with very low frequency during female meiosis. A third region around the WT-1 gene on chromosome 11p13 also recombines with higher frequency during male meiosis. CONCLUSIONS: The results show that the two best-known imprinted regions in the human genome are characterized by significant differences in recombination frequency during male and female meioses. A third, less well-characterized, imprinted region shows a similar sex-specific bias. On the basis of these observations, we propose a model suggesting that the region-specific differential accessibility of DNA that leads to differential recombination rates during male and female meioses also leads to the male- and female-specific modification of the signal sequences that control genomic imprinting.

Alterations of the DNA repair gene OGG1 in human clear cell carcinomas of the kidney.

The OGG1 gene, which codes for a DNA repair protein with antimutator activity, is located on chromosome 3p25, a frequent site of allelic deletions in many types of human tumors, including renal clear cell cancers. We present the analysis of 99 renal tumors for alterations in the OGG1 gene to determine its association with tumorigenesis. Loss of heterozygosity in the 3p25 region was found for 85% of the informative cases. We detected somatic missense mutations of the OGG1 gene in 4 of the 99 tumor samples. Biochemical analysis of the mutant proteins revealed that a substitution at codon 46 impairs the enzymatic activity. We also describe the occurrence of several polymorphisms as well as aberrantly spliced OGG1 transcripts.

Identification of three distinct regions of allelic deletions on the short arm of chromosome 8 in hepatocellular carcinoma.

The chromosome 8p is associated with a large number of allelic imbalances in epithelial tumors including hepatocellular carcinoma (HCC). However, no tumor suppressor gene has been identified so far in this particular region of the genome. To further clarify the pattern of allelic deletions on chromosome 8p in HCC, we have undertaken high-density polymorphic marker analysis of 109 paired normal and primary tumor samples using 40 microsatellites positioned every 2 cm in average throughout 8p. We found that 60% of the tumors exhibited loss of heterozygosity (LOH) at one or more loci at 8p with three distinct minimal deleted areas: a 13 cm region in the distal part of 8p21, a 9 cm area in the more proximal portion of 8p22 and a 5 cm area in 8p23. These data strongly suggest the presence of at least three novel tumor suppressor loci on 8p in hepatocellular carcinoma.

Genotyping Procedures in Linkage Mapping

Genotyping methods based on nonradioactive detection of PCR products and suitable for large-scale mapping projects are described. Two alternative techniques are proposed for the genotyping of polymorphic short tandem repeats or microsatellite markers. The first is designed for investigators who do not have access to automatic sequencing machines. This technique uses multiplex analysis of PCR products that are separated on sequencing gels, transferred to nylon membranes, and detected by hybridization with nonradioactive probes. The second technique uses automatic sequencing machines for the detection of fluorescently labeled PCR products. Another method describes the analysis of nonpolymorphic markers in whole-genome radiation hybrids. This method uses separation and detection of PCR products on agarose gels.

Chromosomal localization of the adrenoleukodystrophy-related gene in man and mice.

We report here on the chromosomal mapping of the adrenoleukodystrophy-related (ALDR) gene on both the human and the mouse genomes. This gene encodes a peroxisomal ATP binding cassette transporter, closely related to the transporter identified as responsible for the adrenoleukodystrophy phenotype. ALDR maps on the syntenic region on murine and human autosomes. In addition, we could determine its position in relation to known microsatellite framework markers; this will allow to test its role in Zellweger syndrome and/ or related peroxisomal disorders.

A new human nm23 homologue (nm23-H5) specifically expressed in testis germinal cells.

We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.

Effect of interferon-alpha (IFN alpha) on various human tumor xenografts.

The growth of some human tumor xenografts (3 out of 8, melanoma, non-Hodgkin's lymphoma, squamous cell carcinoma) was successfully--but moderately and temporarily--inhibited, when interferon-alpha (EGIS, Hungary) was given for 10 days. The route of administration (intratumoral or intraperitoneal) was usually not a decisive factor. An attempt to potentiate IFNa action with Zymozan or Cyclophosphamide did not succeed.

Drug action and chromatin structure. I. Adriamycin binding to core particle of nucleosomes and subsequent enhanced DNA fragmentation induced by micrococcal nuclease.

The relevance of the subunit structure of chromatin to the mode of action of Adriamycin in Novikoff hepatoma had been investigated. To elucidate whether drug binding takes place at random along the chromatin fiber or not Novikoff hepatoma nuclei treated with Adriamycin in vivo were digested with Micrococcal nuclease and were subsequently separated into three fractions, S1, S2 and P2. In these chromatin fraction Adriamycin and the DNA content was determined. DNA were isolated from the above fractions and were analyzed by polyacrylamide gel electrophoresis, furthermore the S2 fraction was sedimented on sucrose density gradient. The results indicate that binding of Adriamycin takes place preferentially on the core particle inducing structural alterations. Adriamycin binding not only enhanced DNA nuclease digestion but altered the size of the fragments.

Further studies on the biochemical characterization of the MC-29 virus derived transplantable hepatoma (VTH). II. Modification of cyclic adenosine-3',5'-monophosphate levels by catecholamines, glucagon and Vinca alkaloids in normal chicken liver and VTH.

Basic and stimulated intracellular cAMP concentrations were measured in normal chicken liver and MC-29-virus-derived transplantable hepatoma (VTH) slices after in vitro incubation. Data indicated the preservation of catecholamine receptor but a loss of glucagon receptor in VTH. Comparing the relative stimulatory action of various catecholamines on cAMP concentration it was concluded that as in normal liver a predominantly beta 2-adrenergic receptor exists in the VTH, but its response to adrenaline is greater. Vinca alkaloids induced higher cAMP concentration in VTH than in normal liver. This stimulation was abolished by glucagon, while catecholamines and Vincristine acted in a synergistic manner on cAMP concentration.

Differentiating Alström from Bardet-Biedl syndrome (BBS) using systematic ciliopathy genes sequencing.

INTRODUCTION: Early onset retinal degeneration associated with obesity can present a diagnostic challenge in paediatric ophthalmology practice. Clinical overlap between Bardet-Biedl syndrome (BBS) and Alström syndrome has been described, although the two entities are genetically distinct. To date, 16 genes are known to be associated with BBS (BBS1-16) and only one gene has been identified for Alström syndrome (ALMS1). MATERIALS AND METHODS: In collaboration with the French National Center for Sequencing (CNS, Evry), all coding exons and flanking introns were sequenced for 27 ciliopathy genes (BBS1-12, MGC1203, TTC21b, AHI1, NPHP2-8 (NPHP6=BBS14), MKS1(BBS13), MKS3, C2ORF86, SDCCAG8, ALMS1) in 96 patients referred with a clinical diagnosis of BBS. ALMS1 gene analysis included sequencing of all coding exons. RESULTS: BBS known gene mutations were found in 44 patients (36 with two mutations and 8 heterozygous). ALMS1 mutations were found in four cases. The rate of ALMS1 mutations among patients suspected of having BBS was 4.2%. DISCUSSION: Clinically, all four patients presented early-onset severe retinal degeneration with congenital nystagmus associated with obesity. The difficult early differential diagnosis between the two syndromes is outlined. One mutation had already been reported (c.11310delAGAG/p.R3770fsX) and three were novel (c.2293C > T/p.Q765X, c.6823insA/p.R2275fsX, c.9046delA/p.N3016fsX). CONCLUSIONS: Ciliopathy genes sequencing can be very helpful in providing a timely diagnosis in this group of patients, hence appropriate genetic counselling for families and adequate medical follow-up for affected children.