Expression of the peptide C4b-binding protein beta in the arthritic joint.

BACKGROUND: C4b-binding protein (C4BP) is a plasma oligomeric glycoprotein that participates in the regulation of complement and haemostasis. Complement-regulatory activity depends on the C4BPalpha-polypeptide, whereas the C4BPbeta-polypeptide inactivates protein S, interfering with the anti-coagulatory protein C-dependent pathway. OBJECTIVE: To investigate the expression of C4BPbeta in the rheumatoid joint. METHODS: Expression of C4BP was studied in synovial explants from patients with rheumatoid arthritis, osteoarthritis and healthy controls, using immunohistochemistry and in situ hybridisation. C4BP isoforms and free C4BPbeta were studied in synovial effusions from patients with rheumatoid arthritis, osteoarthritis and microcrystalline arthritis (MCA) by immunoblotting; total and free protein S levels were studied by enzyme immunoassay. RESULTS: C4BPbeta was overexpressed in the synovial membranes of patients with rheumatoid arthritis, in close association with the severity of synovitis and the extension of interstitial fibrin deposits. As many as 85% fluids from patients with rheumatoid arthritis contained free C4BPbeta, whereas this unusual polypeptide was present in 50% fluids from patients with MCA and 40% fluids from patients with osteoarthritis. Free protein S at the effusions was pathologically reduced in patients with rheumatoid arthritis and MCA, and remained normal in patients with osteoarthritis. CONCLUSION: C4BPbeta is expressed by the inflamed synovial tissue, where it can participate in processes of tissue remodelling associated with invasive growth.

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

High frequency of alkaptonuria in Slovakia: evidence for the appearance of multiple mutations in HGO involving different mutational hot spots.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups. One notable exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our laboratory for >80 AKU chromosomes from different European and non-European countries. The results show that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool. Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations, most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a small geographical region.

Familial syndromic esophageal atresia maps to 2p23-p24.

Esophageal atresia (EA) is a common life-threatening congenital anomaly that occurs in 1/3,000 newborns. Little is known of the genetic factors that underlie EA. Oculodigitoesophageoduodenal (ODED) syndrome (also known as "Feingold syndrome") is a rare autosomal dominant disorder with digital abnormalities, microcephaly, short palpebral fissures, mild learning disability, and esophageal/duodenal atresia. We studied four pedigrees, including a three-generation Dutch family with 11 affected members. Linkage analysis was initially aimed at chromosomal regions harboring candidate genes for this disorder. Twelve different genomic regions covering 15 candidate genes (approximately 15% of the genome) were excluded from involvement in the ODED syndrome. A subsequent nondirective mapping approach revealed evidence for linkage between the syndrome and marker D2S390 (maximum LOD score 4.51 at recombination fraction 0). A submicroscopic deletion in a fourth family with ODED provided independent confirmation of this genetic localization and narrowed the critical region to 7.3 cM in the 2p23-p24 region. These results show that haploinsufficiency for a gene or genes in 2p23-p24 is associated with syndromic EA.

Mutational spectrum of the EPM2A gene in progressive myoclonus epilepsy of Lafora: high degree of allelic heterogeneity and prevalence of deletions.

Progressive myoclonus epilepsy of the Lafora type (Lafora disease) is an autosomal recessive disease characterised by epilepsy, myoclonus, progressive neurological deterioration and the presence of glycogen-like intracellular inclusion bodies (Lafora bodies). We recently cloned the major gene for Lafora disease (EPM2A) and characterised the corresponding product, a putative protein tyrosine phosphatase (LAFPTPase). Here we report the complete coding sequence of the EPM2A gene and the analysis of this gene in 68 Lafora disease chromosomes. We describe 11 novel mutations: three missense (F84L, G240S and P301L), one nonsense (Y86stop), three < 40 bp microdeletions (K90fs, Ex1-32bpdel, Ex1-33bpdel), and two deletions affecting the entire exon 1 (Ex1-del1 and Ex1-del2). In addition, we have identified three patients with a null allele in non-exonic microsatellites EPM2A-3 or EPM2A-4, suggesting the presence of two distinct > 3 kb deletions affecting exon 2 (Ex2-del1 and Ex2-del2). Considering these mutations, a total of 25 mutations, 60% of them generating truncations, have been described thus far in the EPM2A gene. In spite of this remarkable allelic heterogeneity, the R241stop EPM2A mutation was found in approximately 40% of the Lafora disease patients. We also report the characterisation of five new microsatellite markers and one SNP in the EPM2A gene and describe the haplotypic associations of alleles at these sites in normal and EPM2A chromosomes. This analysis suggests that both founder effect and recurrence have contributed to the relatively high prevalence of R241stop mutation in Spain. The data reported here represent the first systematic analysis of the mutational events in the EPM2A gene in Lafora disease patients and provide insight into the origin and evolution of the different EPM2A alleles.

A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2).

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Genetic polymorphism of human C4-binding protein.

Two different forms of human C4-bp, C4-bp A and C4-bp B, have been identified by isoelectric focusing (IEF) of neuraminidase-treated EDTA-plasma samples. Family studies demonstrate Mendelian segregation of these forms, indicating that they are under gentic control. This conclusion is supported by IEF analysis of the two variants purified by affinity chromatography. Under completely denaturing conditions, C4-bp B was found to be composed of two subunits that focused at different pH, whereas C4-bp A contains only the more basic one. These results suggest that a single autosomal locus with at least two codominant alleles coding for the subunits controls the IEF variation of C4-bp in humans. The allele designated C4BP*1 codes for a subunit that, after neuraminidase treatment, focuses at pH = 6.65. The allele C4BP*2 codes for a different subunit that focuses at pH = 6.60. The C4-bp A phenotype corresponds to the genotype C4BP*1,C4BP*1 and the phenotype C4-bp B to the genotype C4BP*1,C4BP*2. The phenotype corresponding to the C4BP*2,C4BP*2 homozygous genotype has not been encountered thus far. Initial linkage data indicate that the C4BP locus is not closely linked to either the HLA or to the C3 loci.