Characterization of an expressible nonclassical class I HLA gene.

Screening of a human cosmid library representing genomic DNA from an individual homozygous for the HLA-DR2 B7 A2 haplotype yielded 109 class I HLA-specific clones. One cosmid clone, Ice 6.23, had a full-length nonclassical class I gene within a 5.4-kb HindIII fragment. The Ice 6.23-5.4H gene was cloned into the unique NotI site of an expression vector pSV2.Not, a derivative of pSV2neo, which was constructed to contain a second SV40 early region promoter adjacent to an introduced NotI site. The resulting construct was transfected into the P815-B2M cell line, a derivative of the mouse mastocytoma P815 (HTR) line which expressed human beta2-microglobulin following stable transfection with a cloned human beta2-microglobulin gene. Following transfection the Ice 6.23-5.4 H gene was found to be expressed at both the mRNA and cell surface product levels. DNA sequencing of this gene suggests that it is allelic to the HLA-6.0 gene clone (HLA-G) of Geraghty et al. (Proceedings of the National Academy of Sciences USA, 84:9145, 1987); thereby revealing a HindIII restriction fragment length polymorphism at the HLA-G locus. An extraordinarily high degree of sequence similarity (99.92%) between these two genes, which derive from unrelated HLA haplotypes, suggests strong conservative selection pressure at the HLA-G locus. A flanking single copy sequence probe 4 kb distant from the Ice 6.23-5.4H gene was used to generate long-range restriction mapping at the HLA-G locus.     

In-depth analysis of spatial cognition in Williams syndrome: A critical assessment of the role of the LIMK1 gene

The LIM kinase1 protein (LIMK1) is thought to be involved in neuronal development and brain function. However, its role in spatial cognition in individuals with Williams syndrome (WS) is currently ambiguous, with conflicting reports on the cognitive phenotypes of individuals who do not have classic WS but harbour partial deletions including LIMK1. Two families with partial WS deletions have been described with deficits in visuospatial cognition (Frangiskakis, J. M., Ewart, A. K., Morris, C. A., Mervis, C. B., Bertrand, & J., Robinson, et al. (1996). LIM-kinase 1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell, 86, 59-69), in contrast to others with similar partial deletions who did not display spatial impairments (Tassabehji, M., Metcalfe, K., Karmiloff-Smith, A., Carette, M. J., Grant, J., & Dennis, N., et al. (1999). Williams syndrome: Use of chromosomal microdeletions as a tool to dissect cognitive and physical phenotypes. American Journal of Human Genetics, 64, 118-125). To determine the role of LIMK1 in the highly penetrant visuospatial deficits associated with classic WS, it is essential to investigate the discrepancies between the two studies. Previous research used a standardised task to measure spatial cognition, which may not pick up subtle impairments. We therefore undertook more extensive testing of the spatial cognition of two adults with partial genetic deletions in the WS critical region (LIMK1 and ELN only), who had not displayed spatial impairments in the previous study, and compared them to two high-functioning adults with WS matched on verbal ability. All participants completed a broad battery of 16 perceptual and constructive spatial tests, and the clear-cut spatial difficulties observed in the WS group were not found in the partial deletion group. These findings rule out the claim that the deletion of one copy of LIMK1 is alone sufficient to result in spatial impairment, but leave open the possibility that LIMK1 contributes to the WS cognitive deficits if deleted in combination with other genes within the WS deletion. We conclude that a deeper assessment of WS at the genetic level is required before the contribution of specific genes to phenotypic outcomes can be fully understood.     

Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal / carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for ΔF508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, ΔF508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G>T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes. © 1996 Wiley-Liss, Inc.     

Isolation and characterisation of GTF2IRD2, a novel fusion gene and member of the TFII-I family of transcription factors, deleted in Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a developmental disorder with characteristic physical, cognitive and behavioural traits caused by a microdeletion of approximately 1.5 Mb on chromosome 7q11.23. In total, 24 genes have been described within the deleted region to date. We have isolated and characterised a novel human gene, GTF2IRD2, mapping to the WBS critical region thought to harbour genes important for the cognitive aspects of the disorder. GTF2IRD2 is the third member of the novel TFII-I family of genes clustered on 7q11.23. The GTF2IRD2 protein contains two putative helix-loop-helix regions (I-repeats) and an unusual C-terminal CHARLIE8 transposon-like domain, thought to have arisen as a consequence of the random insertion of a transposable element generating a functional fusion gene. The retention of a number of conserved transposase-associated motifs within the protein suggests that the CHARLIE8-like region may still have some degree of transposase functionality that could influence the stability of the region in a mechanism similar to that proposed for Charcot-Marie-Tooth neuropathy type 1A. GTF2IRD2 is highly conserved in mammals and the mouse ortholgue (Gtf2ird2) has also been isolated and maps to the syntenic WBS region on mouse chromosome 5G. Deletion mapping studies using somatic cell hybrids show that some WBS patients are hemizygous for this gene, suggesting that it could play a role in the pathogenesis of the disorder.     

Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome

Klippel-Feil syndrome (KFS) is a congenital disorder of spinal segmentation distinguished by the bony fusion of anterior/cervical vertebrae. Scoliosis, mirror movements, otolaryngological, kidney, ocular, cranial, limb, and/or digit anomalies are often associated. Here we report mutations at the GDF6 gene locus in familial and sporadic cases of KFS including the recurrent missense mutation of an extremely conserved residue c.866T>C (p.Leu289Pro) in association with mirror movements and an inversion breakpoint downstream of the gene in association with carpal, tarsal, and vertebral fusions. GDF6 is expressed at the boundaries of the developing carpals, tarsals, and vertebrae and within the adult vertebral disc. GDF6 knockout mice are best distinguished by fusion of carpals and tarsals and GDF6 knockdown in Xenopus results in a high incidence of anterior axial defects consistent with a role for GDF6 in the etiology, diversity, and variability of KFS.     

Elastin: genomic structure and point mutations in patients with supravalvular aortic stenosis

We describe the complete exon-intron structure of the human elastin (ELN) gene located at chromosome 7q11.23. There are 34 exons occupying approximately 47 kb of genomic DNA. All exons are in-frame, allowing exon skipping without disrupting the reading frame. Microsatellites are located in introns 17 and 18. Deletions of all or large parts of the ELN gene have been previously reported in two patients with supravalvular aortic stenosis (SVAS), and SVAS is also a frequent feature of Williams syndrome, where patients are hemizygous for ELN. We list primer pairs for amplifying each exon, with flanking intron, from genomic DNA to allow detection of point mutations in the ELN gene. We show that some patients with isolated SVAS have point mutations that are predicted to lead to premature chain termination. Knowledge of the genomic structure will allow more extensive mutation screening in genomic DNA of patients with SVAS and other conditions.