Charlene J. Williams

Evidence against either a premature stop codon or the absence of obese gene mRNA in human obesity.

Obese (ob) gene expression in abdominal subcutaneous adipocytes from lean and obese humans was examined. The full coding region of the ob gene was isolated from a human adipocyte cDNA library. Translation of the insert confirmed the reported amino acid sequence. There was no difference in the sequence of an reverse transcription PCR product of the coding region from five lean and five obese subjects. The nonsense mutation in the ob mouse which results in the conversion of arginine 105 to a stop codon was not present in human obesity. In all 10 human cDNAs, arginine 105 was encoded by CGG, consequently two nucleotide substitutions would be required to result in a stop codon. To compare the amount of ob gene expression in lean and obese individuals, radiolabed primer was used in the PCR reaction with beta-actin as a control. There was 72% more ob gene expression (P < 0.01) in eight obese subjects (body mass index, BMI = 42.8 +/- 2.7) compared to eight lean controls (BMI = 22.4 +/- 0.8). Regression analysis indicated a positive correlation between BMI and the amount of ob message (P < 0.005). There was no difference in the amount of beta-actin expression in the two groups. These results provide evidence that ob gene expression is increased in human obesity; furthermore, the mutations present in the mouse ob gene were not detected in the human mRNA population.

Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition

Multiple genetic variants of CARD15/NOD2 have been associated with susceptibility to Crohns disease and Blau syndrome. NOD2 recognizes muramyl dipeptide (MDP) derived from bacterial peptidoglycan (PGN), but the molecular basis of recognition remains elusive. We performed systematic mutational analysis to gain insights into the function of NOD2 and molecular mechanisms of disease susceptibility. Using an archive of 519 mutations covering ∼50% of the amino‐acid residues of NOD2, the essential regulatory domains and specific residues of NOD2 involved in recognition of MDP were identified. The analysis revealed distinct roles for N‐terminal and C‐terminal leucine‐rich repeats (LRRs) in the modulation of NOD2 activation and bacterial recognition. Within the C‐terminal LRRs, variable residues predicted to form the β‐strand/βturn structure were found to be essential for the response to MDP. In addition, we analyzed NOD1, a NOD2‐related protein, revealing conserved and nonconserved amino‐acid residues involved in PGN recognition. These results provide new insights into the molecular function and regulation of NOD2 and related NOD family proteins.

First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q

Osteoporosis is characterized by low bone density, and osteopenia is responsible for 1.5 million fractures in the United States annually.1 In order to identify regions of the genome which are likely to contain genes predisposing to osteopenia, we genotyped 149 members of seven large pedigrees having recurrence of low bone mineral density (BMD) with 330 DNA markers spread throughout the autosomal genome. Linkage analysis for this quantitative trait was carried out using spine and hip BMD values by the classical lod-score method using a genetic model with parameters estimated from the seven families. In addition, non-parametric analysis was performed using the traditional Haseman-Elston approach in 74 independent sib pairs from the same pedigrees. The maximum lod score obtained by parametric analysis in all families combined was +2.08 (θ = 0.05) for the marker CD3D on chromosome 11q. All other combined lod scores from the parametric analysis were less than +1.90, the threshold for suggestive linkage. Non-parametric analysis suggested linkage of low BMD to chromosomes 1p36 (Zmax = +3.51 for D1S450) and 2p23-24 (Zmax = +2.07 for D2S149). Maximum multi-point lod scores for these regions were +2.29 and +2.25, respectively. A third region with associated lod scores above the threshold of suggestive linkage in both single-point and multi-point non-parametric analysis was on chromosome 4qter (Zmax = +2.95 for D4S1539 and Zmax = +2.48 for D4S1554). Our data suggest the existence of multiple genes involved in controlling spine and hip BMD, and indicate several candidate regions for further screening in this and other independent samples.

The hypothalamic leptin receptor in humans : Identification of incidental sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations

Leptin-receptor gene expression in hypothalamic tissue from lean and obese humans was examined. The full-length leptin receptor, that is believed to transmit the leptin signal, is expressed in human hypothalamus. There was no difference in the amount of leptin-receptor mRNA In seven lean (BMI 23.3 +/- 0.9 kg/m2) and eight obese (BMI 36.9 +/- 1.5) subjects as determined by reverse transcription-polymerase chain reaction. A sequence polymorphism (A-->G) was detected at position 668 of the leptin receptor cDNA. This second base substitution changed a glutamine to an arginine at position 223 of the leptin receptor protein. Of 15 subjects analyzed, 11 were heterozygous for this base change and 3 were homozygous. The occurrence [correction of occurance] of the polymorphic allele(s) did not correlate with BMI in the population studied. The mutation responsible for the defect in the leptin receptor in db/db mice was not detected in any obese human, nor was the fa/fa rat mutation. These results provide evidence that the leptin resistance observed in obese humans is not due to a defect in the leptin receptor.

Mutations in ANKH Cause Chondrocalcinosis

Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.

Identification of Incidental Sequence Polymorphisms and Absence of the db/db Mouse and fa/fa Rat Mutations

Leptin-receptor gene expression in hypothalamic tissue from lean and obese humans was examined. The fulllength leptin receptor, that is believed to transmit the leptin signal, is expressed in human hypothalamus. There was no difference in the amount of leptin-receptor mRNA in seven lean (BMI 23.3 ± 0.9 kg/m2) and eight obese (BMI 36.9 ± 1.5) subjects as determined by reverse transcription-polymerase chain reaction. A sequence polymorphism (A→G) was detected at position 668 of the leptin receptor cDNA. This second base substitution changed a glutamine to an arginine at position 223 of the leptin receptor protein. Of 15 subjects analyzed, 11 were heterozygous for this base change and 3 were homozygous. The occurance of the polymorphic allele(s) did not correlate with BMI in the population studied. The mutation responsible for the defect in the leptin receptor in db/db mice was not detected in any obese human, nor was the fa/fa rat mutation. These results provide evidence that the leptin resistance observed in obese humans is not due to a defect in the leptin receptor.

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH.

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Internal deletion in a collagen gene in a perinatal lethal form of osteogenesis imperfecta

Cloned probes specific for unique genes have proven to be powerful tools in defining the nature of genetic diseases such as the thalassaemias1 and growth hormone deficiencies2. A similar approach should be useful in defining heritable diseases of type I collagen, the heterotrimer of two α1(I) chains and one α2(I) chain, which is the most abundant member of the collagen family of proteins. Recently, cloned cDNAs and genomic DNAs for the two polypeptide chains of the type I collagen3–10 have become available and have been used to elucidate the chromosomal location of the corresponding genes11–14. Here, we have used several of these cloned DNAs to demonstrate the presence of an internal deletion of about 0.5 kilobases (kb) in one allele for the proα1(I) chain in a patient with osteogenesis imperfecta (OI), a group of heritable disorders which are characterized by brittle bones but which are highly heterogeneous both phenotypically and biochemically15,16.

Spondyloepiphyseal dysplasia and precocious osteoarthritis in a family with an Arg75→Cys mutation in the procollagen type II gene (COL2A1)

Direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA from a patient with spondyloepiphyseal dysplasia and precocious osteoarthritis revealed a single-base change in exon 11 of the type II procollagen gene (COL2A1), which produces an Arg→ Cys mutation in one allele. The proband is a member of a large Chilean kindred presenting with chondrodysplasia of the hips, knees, shoulders, elbows, and spine associated with severe, early-onset osteoarthritis. All affected individuals exhibit mildly short stature; in addition, five out of seven affected family members display shortened metacarpals or metatarsals. DNA from affected and unaffected family members was PCR-amplified and analysis of restriction digests of the products determined that the mutation segregated with the disease with a lod score of 2.2 at zero recombination. The mutation, which resides in the triple-helical region of type II procollagen at amino acid position 75, is the second example of an Arg→Cys mutation in the COL2A1 gene in heritable cartilaginous disease and is the first example of a point mutation in the amino terminal region of the α1(II) chain, that results in a spondyloepiphyseal dysplastic phenotype.

Refinement of the Chromosome 5p Locus for Familial Calcium Pyrophosphate Dihydrate Deposition Disease

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.