GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype-phenotype analysis of five families with deletions in the Williams syndrome region

Most individuals with Williams syndrome (WS) have a 1.6 Mb deletion in chromosome 7q11.23 that encompasses the elastin (ELN) gene, while most families with autosomal dominant supravalvar aortic stenosis (SVAS) have point mutations in ELN. The overlap of the clinical phenotypes of the two conditions (cardiovascular disease and connective tissue abnormalities such as hernias) is due to the effect of haploinsufficiency of ELN. SVAS families often have affected individuals with some WS facial features, most commonly in infancy, suggesting that ELN plays a role in WS facial gestalt as well. To find other genes contributing to the WS phenotype, we studied five families with SVAS who have small deletions in the WS region. None of the families had mental retardation, but affected family members had the Williams Syndrome Cognitive Profile (WSCP). All families shared a deletion of LIMK1, which encodes a protein strongly expressed in the brain, supporting the hypothesis that LIMK1 hemizygosity contributes to impairment in visuospatial constructive cognition. While the deletions from the families nearly spanned the WS region, none had a deletion of FKBP6 or GTF2I, suggesting that the mental retardation seen in WS is associated with deletion of either the centromeric and/or telomeric portions of the region. Comparison of these five families with reports of other individuals with partial deletions of the WS region most strongly implicates GTF2I in the mental retardation of WS.     

A novel human homologue of the Drosophila frizzled wnt receptor gene binds wingless protein and is in the Williams syndrome deletion at 7q11.23

Williams syndrome (WS) is a developmental disorder with a characteristic personality and cognitive profile that is associated, in most cases, with a 2 Mb deletion of part of chromosome band 7q11.23. By applying CpG island cloning methods to cosmids from the deletion region, we have identified a new gene, called FZD3. Dosage blotting of DNA from 11 WS probands confirmed that it is located within the commonly deleted region. Sequence comparisons revealed that FZD3, encoding a 591 amino acid protein, is a novel member of a seven transmembrane domain receptor family that are mammalian homologs of the Drosophila tissue polarity gene frizzled. FZD3 is expressed predominantly in brain, testis, eye, skeletal muscle and kidney. Recently, frizzled has been identified as the receptor for the wingless (wg) protein in Drosophila. We show that Drosophila as well as human cells, when transfected with FZD3 expression constructs, bind Wg protein. In mouse, the wg homologous Wnt1 gene is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon. The potential function of FZD3 in transmitting a Wnt protein signal in the human brain and other tissues suggests that heterozygous deletion of the FZD3 gene could contribute to the WS phenotype.      

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.     

Myelomeningocele and Waardenburg syndrome (type 3) in patients with interstitial deletions of 2q35 and the PAX3 gene: Possible digenic inheritance of a neural tube defect

From a spina bifida clinic we have identified two patients with a syndrome of myelomeningocele and Waardenburg syndrome type 3 (WS3). The patients each possess a single, de novo, interstitial deletion of chromosome 2 (2q35–36.2), including the PAX3 gene. Deletion of PAX3 was confirmed by fluorescence in situ hybridization (FISH). Analysis with PAX3 and flanking microsatellites shows that the deleted interval of chromosome 2 is of paternal origin and is at least 2 and 6 cM in the two patients. Interstitial deletions in this region result in the Waardenburg syndrome (WS1), but have not been associated with neural tube defects (NTDs). Although other etiologies have not been formally excluded, these patients raise the possibility of a digenic etiology of their NTDs via a genetic interaction of the deleted PAX3 gene with a second unidentified locus. Am. J. Med. Genet. 75:401-408, 1998. © 1998 Wiley-Liss, Inc.     

Expression and mutation analysis of BRUNOL3, a candidate gene for heart and thymus developmental defects associated with partial monosomy 10p

Partial monosomy 10p is a rare chromosomal aberration. Patients often show symptoms of the DiGeorge/velocardiofacial syndrome spectrum. The phenotype is the result of haploinsufficiency of at least two regions on 10p, the HDR1 region associated with hypoparathyroidism, sensorineural deafness, and renal defects (HDR syndrome) and the more proximal region DGCR2 responsible for heart defects and thymus hypoplasia/aplasia. While GATA3 was identified as the disease causing gene for HDR syndrome, no genes have been identified thus far for the symptoms associated with DGCR2 haploinsufficiency. We constructed a deletion map of partial monosomy 10p patients and narrowed the critical region DGCR2 to about 300 kb. The genomic draft sequence of this region contains only one known gene, BRUNOL3 ( NAPOR, CUGBP2, ETR3). In situ hybridization of human embryos and fetuses revealed as well as in other tissues a strong expression of BRUNOL3 in thymus during different developmental stages. BRUNOL3 appears to be an important factor for thymus development and is therefore a candidate gene for the thymus hypoplasia/aplasia seen in partial monosomy 10p patients. We did not find BRUNOL3 mutations in 92 DiGeorge syndrome-like patients without chromosomal deletions and in 8 parents with congenital heart defect children.     

Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.      

Delineation of the common critical region in Williams syndrome and clinical correlation of growth,heart defects,ethnicity, and parental origin

Williams syndrome (WS) is a neurodevelopmental disorder with a variable phenotype. Molecular genetic studies have indicated that hemizygosity at the elastin locus (ELN) may account for the cardiac abnormalities seen in WS, but that mental retardation and hypercalcemia are likely caused by other genes flanking ELN. In this study, we defined the minimal critical deletion region in 63 patients using 10 microsatellite markers and 5 fluorescence in situ hybridization (FISH) probes on chromosome 7q, flanking ELN. The haplotype analyses showed the deleted cases to have deletions of consistent size, as did the FISH analyses using genomic probes for the known ends of the commonly deleted region defined by the satellite markers. In all informative cases deleted at ELN, the deletion extends from D7S489U to D7S1870. The genetic distance between these two markers is about 2 cM. Of the 51 informative patients with deletions, 29 were maternal and 22 were paternal in origin. There was no evidence for effects on stature by examining gender, ethnicity, cardiac status, or parental origin of the deletion. Heteroduplex analysis for LIMK1, a candidate gene previously implicated in the WS phenotype, did not show any mutations in our WS patients not deleted for ELN. LIMK1 deletions were found in all elastin-deletion cases who had WS. One case, who has isolated, supravalvular aortic stenosis and an elastin deletion, was not deleted for LIMK1. It remains to be determined if haploinsufficiency of LIMK1 is responsible in part for the WS phenotype or is simply deleted due to its close proximity to the elastin locus. Am. J. Med. Genet. 78:82–89, 1998. © 1998 Wiley-Liss, Inc.     

An extended region of biallelic gene expression and rodent-human synteny downstream of the imprinted H19 gene on chromosome 11p15.5

There is increasing evidence for chromosomal domains containing multiple imprinted genes and for domain-wide disruption of imprinting in certain diseases. In a majority of Wilms' tumors (WTs) there is an abnormal bipaternal pattern of expression at three imprinted loci, H19, IGF2 and KIP2, clustered on chromosome 11p15.5. We previously described biallelic expression of L23MRP, 40 kb downstream of H19. Here we map two additional genes, the first encoding a ubiquitously expressed RNA, 2G7, and the second encoding the fast isoform of skeletal muscle troponin-T (TNNT3), in the 55 kb of DNA downstream of L23MRP. 2G7 RNA is spliced and polyadenylated but lacks long open reading frames. 2G7 and TNNT3 are biallelically expressed in mid-fetal and adult human tissues and 2G7 shows persistent expression in WTs. The rat homologue of L23MRP is highly conserved and lies within 85 kb of H19 in a region of rat chromosome 1 which also contains IGF2 and TNNT3. Parallel expression of H19 and TNNT3 in different adult skeletal muscle types suggests that these genes may share an enhancer. These data outline multiple contiguous loci downstream of H19 which escape functional imprinting in humans. The rodent-human synteny of this region may facilitate a search for an imprinting domain boundary.