Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease with a world-wide distribution. It usually presents in the sixth decade with progressive swallowing difficulties (dysphagia), eyelid drooping (ptosis) and proximal limb weakness. Unique nuclear filament inclusions in skeletal muscle fibres are its pathological hallmark. We isolated the poly(A) binding protein 2 gene (PABP2) from a 217-kb candidate interval on chromosome 14q11 (B.B. et al., manuscript submitted). A (GCG)6 repeat encoding a polyalanine tract located at the N terminus of the protein was expanded to (GCG)8-13 in the 144 OPMD families screened. More severe phenotypes were observed in compound heterozygotes for the (GCG)9 mutation and a (GCG)7 allele that is found in 2% of the population, whereas homozygosity for the (GCG)7 allele leads to autosomal recessive OPMD. Thus the (GCG)7 allele is an example of a polymorphism which can act either as a modifier of a dominant phenotype or as a recessive mutation. Pathological expansions of the polyalanine tract may cause mutated PABP2 oligomers to accumulate as filament inclusions in nuclei.

Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPM1.

Progressive myoclonus epilepsy type 1 (EPM1, also known as Unverricht-Lundborg disease) is an autosomal recessive disorder characterized by progressively worsening myoclonic jerks, frequent generalized tonic-clonic seizures, and a slowly progressive decline in cognition. Recently, two mutations in the cystatin B gene (also known as stefin B, STFB) mapping to 21q22.3 have been implicated in the EPM1 phenotype: a G-->C substitution in the last nucleotide of intron 1 that was predicted to cause a splicing defect in one family, and a C-->T substitution that would change an Arg codon (CGA) to a stop codon (TGA) at amino acid position 68, resulting in a truncated cystatin B protein in two other families. A fourth family showed undetectable amounts of STFB mRNA by northern blot analysis in an affected individual. We present haplotype and mutational analyses of our collection of 20 unrelated EPM1 patients and families from different ethnic groups. We identify four different mutations, the most common of which consists of an unstable approximately 600-900 bp insertion which is resistant to PCR amplification. This insertion maps to a 12-bp polymorphic tandem repeat located in the 5' flanking region of the STFB gene, in the region of the promoter. The size of the insertion varies between different EPM1 chromosomes sharing a common haplotype and a common origin, suggesting some level of meiotic instability over the course of many generations. This dynamic mutation, which appears distinct from conventional trinucleotide repeat expansions, may arise via a novel mechanism related to the instability of tandemly repeated sequences.

Genomic structure of the human GT334 (EHOC-1) gene mapping to 21q22.3.

Several inherited diseases have been mapped to the distal tip of human chromosome 21. In our recent efforts to clone candidate genes for some of these disorders, we have assembled a cosmid and BAC contig spanning 770 kb. We have identified expressed sequences from this contig by means of a cDNA hybrid selection scheme. We present here the isolation, cDNA sequence, genomic organization, and polymorphisms analysis of one such expressed sequence, GT334, which had been identified independently and designated EHOC-1. GT334 is split into 23 exons, and spans an estimated 95 kb of genomic DNA. A pseudogene of the histone H2AZ gene has been identified, and maps within the third intron. We have identified an ORF potentially encoding a protein 1259 amino acids in length, longer than that described in the EHOC-1 gene. The GT334 gene was screened for single base pair changes using single-strand conformation polymorphism (SSCP) analysis and we have identified seven sequence variations within this gene. These polymorphisms can be used as markers in the genetic mapping of other diseases localized to this region.

Isolation and genomic structure of a human homolog of the yeast periodic tryptophan protein 2 (PWP2) gene mapping to 21q22.3.

As part of efforts to identify candidate genes for disease mapping to the 21q22.3 region, we have assembled a 770-kb cosmid and BAC contig containing eight tightly linked markers. These cosmids and BACs were restriction mapped using eight rare cutting enzymes, with the goal of identifying CpG-rich islands. One such island was identified by the clustering of NotI, EagI, SstII, and BssHII sites, and corresponded to the NotI linking clone LJ104 described previously. A 7.6-kb HindIII fragment containing this CpG-rich island was subcloned and partially sequenced. A homology search using the sequence obtained from either side of the NotI site identified an expressed sequence tag with homology to the yeast periodic tryptophan protein 2 (PWP2). Several cDNAs corresponding to the human PWP2 gene were identified and partially sequenced. Northern blot analysis revealed a 3.3-kb transcript that was well expressed in all tissues tested. A cDNA consensus of 3157 bp was obtained, and an open reading frame potentially encoding 919 amino acid residues was identified. The predicted protein shows 42% identity and 57% similarity at the amino acid level to the yeast PWP2 protein, which is a member of the WD-repeat containing superfamily, and potentially encodes a G-protein beta subunit. The PWP2 gene is split into 21 exons, ranging in size from 53 to 516 bp, and spans an estimated 25 kb. The gene is transcribed in a 21cen-->21qter direction, with its 5' end mapping approximately 195 kb proximal to the 5' end of the phosphofructokinase-liver isoform gene. Four single base-pair polymorphisms were identified using single-stranded conformation polymorphism analysis. Possible functions of the protein based on homology to other members of the WD-repeat-containing family are discussed.

Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3.

As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region. A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection. We present here the cDNA sequence of one such gene (GT335) mapping to this region. The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length. GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons. Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions. We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions. This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism.