A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups.

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the approximately 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (approximately 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome.

Sanfilippo A syndrome is one of four recognised Sanfilippo sub-types (A, B, C and D) that result from deficiencies of different enzymes involved in the lysosomal degradation of heparan sulphate; patients suffer from severe neurological disorders. The Sanfilippo syndrome sub-types are also known as mucopolysaccharidosis (MPS) type III (MPS-IIIA, B, C and D), and are part of the large group of lysosomal storage disorders. Each of the MPS-III types is inherited as an autosomal recessive disorder with considerable variation in severity of clinical phenotype. The incidence of Sanfilippo syndrome has been estimated at 1:24,000 in The Netherlands with MPS IIIA (MIM #252900) the most common. MPS-IIIA is the predominant MPS-III in the United Kingdom, and has a similar high incidence to that found in The Netherlands (E. Wraith, personal communication). There is a particularly high incidence of a clinically severe form of MPS-IIIA in the Cayman Islands with a carrier frequency of 0.1 (ref. 4). Due to the mild somatic disease compared to other MPS disorders there is difficulty in diagnosing mild cases of MPS-III, hence Sanfilippo syndrome may be underdiagnosed, especially in patients with mild mental retardation. Here, we report the isolation, sequence and expression of cDNA clones encoding the enzyme sulphamidase (EC 3.10.1.1). In addition, we report the chromosomal localisation of the sulphamidase gene as being 17q25.3. An 11-bp deletion, present in sulphamidase cDNA from two unrelated Sanfilippo A patients, is described.

The morquio A syndrome (mucopolysaccharidosis IVA) gene maps to 16q24.3.

The gene for N-acetylgalactosamine-6-sulfatase, the deficiency of which results in Morquio A syndrome (mucopolysaccharidosis type IVA), was assigned to chromosome 16 at band q24.3 by fluorescence in situ hybridization. Localization of this band was confirmed by PCR analysis of a somatic cell hybrid panel used for fine mapping of chromosome 16.

Human alpha-L-iduronidase: cDNA isolation and expression.

alpha-L-Iduronidase (IDUA; EC 3.2.1.76) is a lysosomal hydrolase in the metabolic pathway responsible for the degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. A deficiency of IDUA in humans leads to the accumulation of these glycosaminoglycans and results in the lysosomal storage disorder mucopolysaccharidosis type I. We have isolated and sequenced cDNA clones containing part of the human IDUA coding region and used PCR from reverse-transcribed RNA to obtain the full IDUA sequence. Analysis of the predicted 653-amino acid precursor protein shows that IDUA has a 26-amino acid signal peptide that is cleaved immediately prior to the amino terminus of the 74-kDa polypeptide present in human liver IDUA. The protein sequence contains six potential N-glycosylation sites. Northern blot analysis with IDUA cDNA detected only a single 2.3-kilobase mRNA species in human placental RNA; however, PCR analysis of fibroblast, liver, kidney, and placental RNA showed the existence of alternatively spliced mRNA from the IDUA gene. Southern blot analysis failed to detect major deletions or gene rearrangements in any of the 40 mucopolysaccharidosis type I patients studied. Expression of a full-length IDUA cDNA construct in Chinese hamster ovary cells produced human IDUA protein at a level 13-fold higher than, and with a specific activity comparable to, IDUA present in normal human fibroblasts.

Development of a highly sensitive assay, based on the polymerase chain reaction, for rare B-lymphocyte clones in a polyclonal population.

A method has been developed to use the polymerase chain reaction to amplify and sequence the chain determining region 3 (CDR 3) of the human immunoglobulin heavy-chain gene, and to use the sequence as a marker for rare neoplastic B lymphocytes. Consensus primers for the Variable and Joining regions of the gene were constructed and shown to enable efficient amplification, directed cloning, and sequencing of CDR 3. Using leukaemic cell line PFMC as a test system, CDR 3 was sequenced, specific primers synthesized, and PFMC DNA was detected down to a dilution of 1:1300 in DNA from normal lymphocytes. This strategy should be useful for monitoring therapy and detecting early disease relapse in B lymphoproliferative disease.