Testing the possible negative association of type 1 diabetes and atopic disease by analysis of the interleukin 4 receptor gene.

Variations in the interleukin 4 receptor A (IL4RA) gene have been reported to be associated with atopy, asthma, and allergy, which may occur less frequently in subjects with type 1 diabetes (T1D). Since atopy shows a humoral immune reactivity pattern, and T1D results from a cellular (T lymphocyte) response, we hypothesised that alleles predisposing to atopy could be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D. We genotyped seven exonic single nucleotide polymorphisms (SNPs) and the -3223 C>T SNP in the putative promoter region of IL4RA in up to 3475 T1D families, including 1244 Finnish T1D families. Only the -3223 C>T SNP showed evidence of negative association (P=0.014). There was some evidence for an interaction between -3233 C>T and the T1D locus IDDM2 in the insulin gene region (P=0.001 in the combined and P=0.02 in the Finnish data set). We, therefore, cannot rule out a genetic effect of IL4RA in T1D, but it is not a major one.

A SNP resource for human chromosome 22: extracting dense clusters of SNPs from the genomic sequence.

The recent publication of the complete sequence of human chromosome 22 provides a platform from which to investigate genomic sequence variation. We report the identification and characterization of 12,267 potential variants (SNPs and other small insertions/deletions) of human chromosome 22, discovered in the overlaps of 460 clones used for the chromosome sequencing. We found, on average, 1 potential variant every 1.07 kb and approximately 18% of the potential variants involve insertions/deletions. The SNPs have been positioned both relative to each other, and to genes, predicted genes, repeat sequences, other genetic markers, and the 2730 SNPs previously identified on the chromosome. A subset of the SNPs were verified experimentally using either PCR-RFLP or genomic Invader assays. These experiments confirmed 92% of the potential variants in a panel of 92 individuals. [Details of the SNPs and RFLP assays can be found at http://www.sanger.ac.uk and in dbSNP.]

Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder that involves tissues derived from the embryonic neural crest. Besides the functional gene on chromosome arm 17q, NF1-related sequences (pseudogenes) are present on a number of chromosomes including 2, 12, 14, 15, 18, 21, and 22. We elucidated the complete nucleotide sequence of the NF1 pseudogene on chromosome 22. Only the middle part of the functional gene but not exons 21-27a, encoding the functionally important GAP-related domain of the NF1 protein, is presented in this pseudogene. In addition to the two known NF1 pseudogenes on chromosome 14 we identified two novel variants. A phylogenetic tree was constructed, from which we concluded that the NF1 pseudogenes on chromosomes 2, 14, and 22 are closely related to each other. Clones containing one of these pseudogenes cross-hybridised with the other pseudogenes in this subset, but did not reveal any in situ hybridisation with the functional NF1 gene or with NF1 pseudogenes on other chromosomes. This suggests that their hybridisation specificity is mainly determined by homologous sequences flanking the pseudogenes. Strong support for this concept was obtained by sequence analysis of the flanking regions, which revealed more than 95% homology. We hypothesise that during evolution this subset of NF1 pseudogenes initially arose by duplication and transposition of the middle part of the functional NF1 gene to chromosome 2. Subsequently, a much larger fragment, including flanking sequences, was duplicated and gave rise to the current NF1 pseudogene copies on chromosomes 14 and 22.

The DNA sequence of human chromosome 22.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation). NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion. To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome. In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22. We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene. However, the GAP-related domain itself is not represented in this locus. In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21. These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus.

A bacterial artificial chromosome-based framework contig map of human chromosome 22q.

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.

Assignment of the beta B1 crystallin gene (CRYBB1) to human chromosome 22 and mouse chromosome 5.

By using primers complementary to the rat beta B1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences. CRYBB1 was assigned to the group 5 region in 22q11.2-q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products of CRYBB1 were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other beta crystallin genes (beta B2(-1), beta B3, and beta A4) have previously been mapped to the same regions in human and mouse. We demonstrate that the beta B1 and beta A4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major beta crystallins genes that are expressed in the bovine lens.

A high-density YAC contig map of human chromosome 22.

We have constructed a high-resolution clone map of human chromosome 22 which integrates the available physical and genetic information, establishing a single consensus. The map consists of all classes of DNA landmarks ordered on 705 yeast artificial chromosomes (YACs) at an average landmark density of more than one per 70 kilobases. This map represents the practical limits of currently available YAC resources and provides the basis for determination of the entire gene content and genomic DNA sequence of human chromosome 22.