Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome.

The inherited osteolyses or 'vanishing bone' syndromes are a group of rare disorders of unknown etiology characterized by destruction and resorption of affected bones. The multicentric osteolyses are notable for interphalangeal joint erosions that mimic severe juvenile rheumatoid arthritis (OMIMs 166300, 259600, 259610 and 277950). We recently described an autosomal recessive form of multicentric osteolysis with carpal and tarsal resorption, crippling arthritic changes, marked osteoporosis, palmar and plantar subcutaneous nodules and distinctive facies in a number of consanguineous Saudi Arabian families. We localized the disease gene to 16q12-21 by using members of these families for a genome-wide search for homozygous-by-descent microsatellite markers. Haplotype analysis narrowed the critical region to a 1.2-cM region that spans the gene encoding MMP-2 (gelatinase A, collagenase type IV; (ref. 3). We detected no MMP2 enzymatic activity in the serum or fibroblasts of affected family members. We identified two family-specific homoallelic MMP2 mutations: R101H and Y244X. The nonsense mutation effects a deletion of the substrate-binding and catalytic sites and the fibronectin type II-like and hemopexin/TIMP2 binding domains. Based on molecular modeling, the missense mutation disrupts hydrogen bond formation within the highly conserved prodomain adjacent to the catalytic zinc ion.     

DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression.     

Molecular analysis of the muscle pathology associated with mitochondrial DNA deletions.

Large-scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns-Sayre syndrome or isolated ocular myopathy who harboured a sub-population of partially-deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially-encoded polypeptides were found in affected muscle fibres, but while many COX-deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild-type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.     

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.     

Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.     

PTPRC (CD45) is not associated with the development of multiple sclerosis in U.S. patients

A C-->G nucleotide transition in exon 4 of PTPRC (encoding protein-tyrosine phosphatase receptor-type C, also known as CD45) was recently reported to be genetically associated with the development of multiple sclerosis (MS). We performed an extensive evaluation of this polymorphism using large family-based and case-control comparisons. Overall, we observed no evidence of genetic association between the PTPRC polymorphism and MS susceptibility or disease course.     

Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter.

Deafness can result from a variety of gene defects. Some genes involved in the physiology of hearing encode membrane transporters that regulate the ionic composition of the fluid bathing the inner ear. The endolymph is an extracellular fluid with an atypical composition that resembles the intracellular milieu, high in K+ and low in Na+. Recent studies have emphasized the prominent role of K+ channels in endolymph secretion and mechanical transduction. Coupled electroneutral transport of Na+, K+ and Cl- is mediated by two isoforms of the Na-K-2Cl co-transporter: the absorptive isoform BSC1 (also called NKCC2, encoded by Slc12a1 in mouse) that is exclusively expressed in kidney; and BSC2/NKCC1 (encoded by Slc12a2 in mouse), the secretory isoform which has a wider pattern of expression including epithelia, muscle cells, neurons and red blood cells. These co-transporters share 57% homology at the amino acid level and are pharmacologically inhibited by loop diuretics. There is functional and histochemical evidence for the presence of the secretory isoform of the Na-K-2Cl co-transporter in gerbil, rat and rabbit inner ear. We disrupted mouse Slc12a2 and report here that Slc12a2-/- mice are deaf and exhibit classic shaker/waltzer behaviour, indicative of inner-ear defects. We localized the co-transporter to key secreting epithelia of the mouse inner ear and show that absence of functional co-transporter leads to structural damages in the inner ear consistent with a decrease in endolymph secretion.     

Genetic mapping with SNP markers in Drosophila.

Map-based positional cloning of Drosophila melanogaster genes is hampered by both the time-consuming, error-prone nature of traditional methods for genetic mapping and the difficulties in aligning the genetic and cytological maps with the genome sequence. The identification of sequence polymorphisms in the Drosophila genome will make it possible to map mutations directly to the genome sequence with high accuracy and resolution. Here we report the identification of 7,223 single-nucleotide polymorphisms (SNPs) and 1,392 insertions/deletions (InDels) in common laboratory strains of Drosophila. These sequence polymorphisms define a map of 787 autosomal marker loci with a resolution of 114 kb. We have established PCR product-length polymorphism (PLP) or restriction fragment-length polymorphism (RFLP) assays for 215 of these markers. We demonstrate the use of this map by delimiting two mutations to intervals of 169 kb and 307 kb, respectively. Using a local high-density SNP map, we also mapped a third mutation to a resolution of approximately 2 kb, sufficient to localize the mutation within a single gene. These methods should accelerate the rate of positional cloning in Drosophila.     

Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2'-5'-oligoadenylate(2-5A)-dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24-25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.     

Mutation in codon 713 of the beta amyloid precursor protein gene presenting with schizophrenia.

Following reports of mutations of codon 717 in exon 17 of the amyloid precursor protein (APP) gene in early-onset familial Alzheimer's disease, we screened exon 17 for new mutations in presenile dementia. The majority of the 105 patients screened had definite or probable Alzheimer's disease, but we also included atypical cases and some chronic schizophrenics. We identified a single abnormal case--a chronic schizophrenic with cognitive defects. Sequencing revealed a C to T nucleotide substitution which produces an alanine to valine change at codon 713. We were unable to detect the mutation in the remaining members of the original cohort nor in a further 100 chronic schizophrenics and 100 non-demented controls. Nonetheless, the position of the mutation in a critical portion of the APP gene suggests that it may well prove to be pathogenic.     

Maternal imprinting of the mouse Snrpn gene and conserved linkage homology with the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.     

Mutations in the V2 vasopressin receptor gene are associated with X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disorder in which the kidney is insensitive to the antidiuretic hormone, vasopressin. It has been proposed that the kidney-specific V2 vasopressin receptor, a G protein-coupled receptor, is defective in this disorder as both the disease and the receptor map to Xq28. We report six unique mutations in the V2 receptor gene of five unrelated NDI patients, with one patient having two mutations. The most severely affected patient has a nonsense mutation which would terminate the protein in transmembrane domain III. Other mutations include three missense mutations, a frameshift and one small in-frame deletion. These results represent one of the first examples of recessive mutations affecting a G protein-coupled receptor.     

Novel mutations in families with unusual and variable disorders of the skeletal muscle sodium channel.

Mutations in the skeletal muscle sodium channel gene (SCN4A) have been described in paramyotonia congenita (PMC) and hyperkalaemic periodic paralysis (HPP). We have found two mutations in SCN4A which affect regions of the sodium channel not previously associated with a disease phenotype. Furthermore, affected family members display an unusual mixture of clinical features reminiscent of PMC, HPP and of a third disorder, myotonia congenita (MC). The highly variable individual expression of these symptoms, including in some cases apparent non-penetrance, implies the existence of modifying factors. Mutations in SCN4A can produce a broad range of phenotypes in muscle diseases characterized by episodic abnormalities of membrane excitability.     

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.     

Defective colour vision associated with a missense mutation in the human green visual pigment gene.

All red/green colour vision defects described so far have been associated with gross rearrangements within the red/green opsin gene array (Xq28). We now describe a male with severe deuteranomaly without such a rearrangement. A substitution of a highly conserved cysteine by arginine at position 203 in the green opsins presumably accounted for his colour vision defect. Surprisingly, this mutation was fairly common (2%) in the population but apparently was not always expressed. In analogy with nonexpression of some 5'green-red hybrid genes in persons with normal colour vision, we suggest that failure of manifestation occurs when the mutant gene is located at a distal (3') position among several green opsin genes. This mutation might also predispose to certain X-linked retinal dystrophies.     

A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease-like 2.

We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.