HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3.

HIRA maps to the DiGeorge/velocardiofacial syndrome critical region (DGCR) at 22q11 (refs 1,2) and encodes a WD40 repeat protein similar to yeast Hir1p and Hir2p. These transcriptional co-repressors regulate cell cycle-dependent histone gene transcription, possibly by remodelling local chromatin structure. We report an interaction between HIRA and the transcription factor Pax3. Pax3 haploinsufficiency results in the mouse splotch and human Waardenburg syndrome (WSI and WSIII) phenotypes. Mice homozygous for Pax3 mutations die in utero with a phenocopy of DGS, or neonatally with neural tube defects. HIRA was also found to interact with core histones. Thus, altered stoichiometry of complexes containing HIRA may be important for the development of structures affected in WS and DGS.

A common region of 10p deleted in DiGeorge and velocardiofacial syndromes.

DiGeorge (DGS, MIM 188400) and velocardiofacial (VCFS, MIM 192430) syndromes may present many clinical problems including cardiac defects, hypoparathyroidism, T-cell immunodeficiency and facial dysmorphism. They are frequently associated with deletions within 22q11.2, but a number of cases have no detectable molecular defect of this region. A number of single case reports with deletions of 10p suggest genetic heterogeneity of DGS. Here we compare the regions of hemizygosity in four patients with terminal deletions of 10p (one patient diagnosed as having hypoparathyroidism and three as DGS) and one patient with a large interstitial deletion (diagnosed as VCFS). Fluorescence in situ hybridization (FISH) analysis demonstrates that these patients have overlapping deletions at the 10p13/10p14 boundary. A YAC contig spanning the shortest region of deletion overlap (SRO) has been assembled, and allows the size of SRO to be approximated to 2 Mb. As with deletions of 22q11, phenotypes vary considerably between affected patients. These results strongly support the hypothesis that haploinsufficiency of a gene or genes within 10p (the DGSII locus) can cause the DGS/VCFS spectrum of malformation.

Mutation and deletion of the pseudoautosomal gene SHOX cause Leri-Weill dyschondrosteosis.

Leri-Weill Dyschondrosteosis (LWD; OMIM 127300) is a dominantly inherited skeletal dysplasia characterized by disproportionate short stature with predominantly mesomelic limb shortening. Expression is variable and consistently more severe in females, who frequently display the Madelung deformity of the forearm (shortening and bowing of the radius with dorsal subluxation of the distal ulna). The rare Langer Mesomelic Dysplasia (LD; OMIM 249700), characterized by severe short stature with hypoplasia/aplasia of the ulna and fibula, has been postulated to be the homozygous form of LWD (refs 4-6). In a six-generation pedigree with LWD, we established linkage to the marker DXYS6814 in the pseudoautosomal region (PAR1) of the X and Y chromosomes (Z max=6.28; theta=0). Linkage analysis of three smaller pedigrees increased the lod score to 8.68 (theta=0). We identified submicroscopic PAR1 deletions encompassing the recently described short stature homeobox-containing gene SHOX (refs 7,8) segregating with the LWD phenotype in 5 families. A point mutation leading to a premature stop in exon 4 of SHOX was identified in one LWD family.

A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis.

Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.

The gene for familial polyposis coli maps to the long arm of chromosome 5.

The inherited genetic defect in adenomatous polyposis has been localized to a small region on the long arm of chromosome 5. Sixteen DNA marker loci were used to construct a linkage map of the chromosome. When five kindreds segregating a gene for adenomatous polyposis coli were characterized with a number of the markers, significant linkage was found between one marker and the disease gene. Linkage analysis determined the location of the defective gene within a primary genetic map of chromosome 5.

Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by multiple clinical features that include pigmentary retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. BBS is considered an autosomal recessive disorder, and recent positional cloning efforts have identified two BBS genes (BBS2 and BBS6). We screened our cohort of 163 BBS families for mutations in both BBS2 and BBS6 and report the presence of three mutant alleles in affected individuals in four pedigrees. In addition, we detected unaffected individuals in two pedigrees who carry two BBS2 mutations but not a BBS6 mutation. We therefore propose that BBS may not be a single-gene recessive disease but a complex trait requiring three mutant alleles to manifest the phenotype. This triallelic model of disease transmission may be important in the study of both Mendelian and multifactorial disorders.

Deletions of human chromosome 22 and associated birth defects.

Investigations into the genetic basis of DiGeorge syndrome have shown that in the majority of cases there are DNA deletions from the long arm of chromosome 22, at 22q11. Similar deletions are now known to be present in a wide range of conditions with overlapping clinical features, and are an important cause of familial congenital heart defect. Deletions within 22q11 have also been identified in individuals with no clinical complications.

Molecular study of 33 families with Fraser syndrome new data and mutation review.

Fraser syndrome (FS) is an autosomal recessive malformation disorder characterized by cryptophthalmos, syndactyly, and abnormalities of the respiratory and urogenital tract. FS is considered to be the human equivalent of the murine blebbing mutants: in the mouse mutations at five loci cause a phenotype that is comparable to FS in humans, and thus far mutations in two syntenic human genes, FRAS1 and FREM2, have been identified to cause FS. Here we present the molecular analysis of 48 FS patients from 18 consanguineous and 15 nonconsanguineous families. Linkage analysis in consanguineous families indicated possible linkage to FRAS1 and FREM2 in 60% of the cases. Mutation analysis identified 11 new mutations in FRAS1 and one FREM2 mutation. Manifestations of these patients and previously reported cases with an FRAS1 mutation were compared to cases without detectable FRAS1 mutations to study genotype-phenotype correlations. Although our data suggest that patients with an FRAS1 mutation have more frequently skull ossification defects and low insertion of the umbilical cord, these differences are not statistically significant. Mutations were identified in only 43% of the cases suggesting that other genes syntenic to murine genes causing blebbing may be responsible for FS as well.

RhoE regulates actin cytoskeleton organization and cell migration.

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.

Loss of heterozygosity for the short arm of chromosome 7 in sporadic Wilms tumour.

Cytogenetic analysis of Wilms tumours (WT) have shown that abnormalities involving chromosome 7 occur in approximately 25% of tumours. In some cases, these abnormalities involve deletions of the short arm, and are seen as the sole cytogenetic change, strongly suggesting the presence of a tumour suppressor gene in this location. Since loss of heterozygosity (LOH) studies have been crucial in defining chromosomal regions involved in Wilms tumorigenesis, we have analysed 40 sporadic Wilms tumours using a panel of 10 microsatellite polymorphic markers distributed along the length of the chromosome arm. In our series, four tumours (10%) showed allelic loss for 7p markers which is twice the background rate of LOH in WT. The shortest common region of overlap of LOH was located between markers D7S517-D7S503 in band 7p21-15. In one tumour there was evidence for a homozygous, interstitial deletion at a locus within this region. These findings provide strong evidence for the existence of a tumour suppressor gene involved in Wilms tumorigenesis and defines the critical region of the chromosome involved.

Cloning of HOXD1 from unfertilised human oocytes and expression analyses during murine oogenesis and embryogenesis.

We describe the cloning of HOXD1 in human unfertilised oocytes and detailed expression analyses during mouse oogenesis and embryogenesis. The cDNA of 1991bp has an open reading frame of 987bp encoding a protein of 329 amino acids. A comparison of the amino acid sequence with the mouse homologue revealed an overall homology of 85.5% with 99% identity within the homeodomain. Expression was detected in unfertilised human oocytes and 2-, 4-, 8-cell and blastocyst stage embryos. Expression analyses in mature mouse ovaries, early embryos and isolated gut revealed expression in the oocytes of the primary and secondary ovarian follicles, and in embryonal mesodermal derivatives such as dermatomes, urogenital tubercle, tail bud, kidney, ovaries, testes and enteric mesoderm adjacent to the caecum where expression was up-regulated in vitro in response to increasing doses of retinoic acid. Our observations indicate a possible role for HOXD1/Hoxd1 in the ovarian oocytes and the establishment of mesodermal derivatives during embryogenesis.

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.

A locus for asphyxiating thoracic dystrophy, ATD, maps to chromosome 15q13.

Asphyxiating thoracic dystrophy (ATD), or Jeune syndrome, is a multisystem autosomal recessive disorder associated with a characteristic skeletal dysplasia and variable renal, hepatic, pancreatic, and retinal abnormalities. We have performed a genome wide linkage search using autozygosity mapping in a cohort of four consanguineous families with ATD, three of which originate from Pakistan, and one from southern Italy. In these families, as well as in a fifth consanguineous family from France, we localised a novel ATD locus (ATD) to chromosome 15q13, with a maximum cumulative two point lod score at D15S1031 (Zmax=3.77 at theta=0.00). Five consanguineous families shared a 1.2 cM region of homozygosity between D15S165 and D15S1010. Investigation of a further four European kindreds, with no known parental consanguinity, showed evidence of marker homozygosity across a similar interval. Families with both mild and severe forms of ATD mapped to 15q13, but mutation analysis of two candidate genes, GREMLIN and FORMIN, did not show pathogenic mutations.

Replication and extension studies of inflammatory bowel disease susceptibility regions confirm linkage to chromosome 6p (IBD3).

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestine, commonly diagnosed as either ulcerative colitis (UC) or Crohn's disease (CD). Epidemiological studies have consistently shown that both genetic and environmental factors influence the pathogenesis of IBD. A number of genome scans have been conducted in cohorts of IBD families with affected sibling pairs (ASPs) to identify chromosomal regions that harbour IBD susceptibility genes. Several putative linked loci have been identified, including two loci on chromosomes 16 and 12, IBD1 and IBD2, which have subsequently been replicated by independent region-specific studies. We have conducted both a replication study on another linkage region, chromosome 6p (IBD3), and extension studies on two other regions, chromosomes 3p and 7q. Microsatellite markers across each region were genotyped in 284 IBD ASPs from 234 families. A nonparametric peak multipoint LOD score of 3.0 was observed near D6S291, replicating the previous linkage to chromosome 6p (IBD3). Nominal evidence of linkage was observed at both the 3p and 7q regions.

Isolation and genomic characterization of the TUPLE1/HIRA gene of the pufferfish Fugu rubripes.

In an effort to obtain a small genomic construct for the generation of a HIRA transgenic mouse, we have isolated and sequenced the Fugu TUPLE1/HIRA gene. We have compared the gene organization and the proteins encoded in pufferfish and human and also searched for conserved DNA sequences that might be important in gene regulation. The pufferfish gene spans approx. 9 kb, which is approx. 11 times smaller than the human gene, owing to the reduced size of the introns. Like its human counterpart, it is organized into 25 exons. The majority of the splice sites are in identical positions to those found in the human gene, however, for three internal exons the positions of the splice sites are not directly comparable. The coding regions are almost identical in size and show a high degree of similarity, especially at the amino and carboxy termini. Comparisons of 5' and 3' sequences failed to detect similarities or sequences involved in regulation.

No evidence for allelic association between schizophrenia and a polymorphism determining high or low catechol O-methyltransferase activity.

OBJECTIVE: Catechol O-methyltransferase (COMT) inactivates catecholamines by methylating their m-hydroxy group. Some previous studies using biochemical methods have found higher levels of COMT activity in schizophrenic patients. Recently, the genetic polymorphism that underlies variation in COMT activity, which results in the creation of a NlaIII restriction site in the low-activity allele, has been elucidated. METHOD: This study investigated this polymorphism in 78 unrelated schizophrenic patients and 78 comparison subjects matched for age and ethnicity. High-molecular-weight DNA was isolated from lymphocytes with routine procedures, and each individual was typed for high and low COMT activity. RESULTS: The frequency of the NlaIII polymorphism was 0.51 in the schizophrenic patients and 0.53 in the comparison subjects, and no significant allelic or genotypic associations were observed. CONCLUSIONS: There was no evidence for variation in COMT activity between a group of schizophrenic patients and matched comparison subjects.

Submicroscopic deletions at 22q11.2: variability of the clinical picture and delineation of a commonly deleted region.

DiGeorge anomaly (DGA) and velo-cardiofacial syndrome (VCFS) are frequently associated with monosomy of chromosome region 22q11. Most patients have a submicroscopic deletion, recently estimated to be at least 1-2 Mb. It is not clear whether individuals who present with only some of the features of these conditions have the deletion, and if so, whether the size of the deletion varies from those with more classic phenotypes. We have used fluorescence in situ hybridization (FISH) to assess the deletion status of 85 individuals referred to us for molecular analysis, with a wide range of DGA-like or VCFS-like clinical features. The test probe used was the cosmid sc11.1, which detects two loci about 2 Mb apart in 22q11.2. Twenty-four patients carried the deletion. Of the deleted patients, most had classic DGA or VCFS phenotypes, but 6 deleted patients had mild phenotypes, including 2 with minor facial anomalies and velopharyngeal incompetence as the only presenting signs. Despite the great phenotypic variability among the deleted patients, none had a deletion smaller than the 2-Mb region defined by sc11.1. Smaller deletions were not detected in patients with particularly suggestive phenotypes who were not deleted for sc11.1, even when tested with two other probes from the DGA/VCFS region.

Velo-cardio-facial syndrome: a review of 120 patients.

A series of earlier reports has described the velo-cardio-facial syndrome (VCFS), a syndrome of multiple anomalies including cleft palate, heart malformations, facial characteristics, and learning disabilities. The patients reported previously were primarily ascertained from a craniofacial program at a large tertiary medical center. Recent reports, including a companion paper in this issue, suggest that this common syndrome of clefting is also a common syndrome of congenital heart defect (CHD) which is expressed as familial examples of DiGeorge sequence. Appreciation of more severely affected cases of VCFS and the detection of mild expressions have led to a broadening of the phenotypic spectrum of the syndrome. The purpose of this report is to describe the full spectrum of VCFS, including several new manifestations and to compare the VCFS phenotype with published cases of "familial DiGeorge sequence" which are now thought to represent examples of VCFS.

Dual-probe fluorescence in situ hybridization assay for detecting deletions associated with VCFS/DiGeorge syndrome I and DiGeorge syndrome II loci.

Over 90% of patients with DiGeorge syndrome (DGS) or velocardiofacial syndrome (VCFS) have a microdeletion at 22q11.2. Given that these deletions are difficult to visualize at the light microscopic level, fluorescence in situ hybridization (FISH) has been instrumental in the diagnosis of this disorder. Deletions on the short arm of chromosome 10 are also associated with a DGS-like phenotype. Since deletions at 22q11.2 and at 10p13p14 result in similar findings, we have developed a dual-probe FISH assay for screening samples referred for DGS or VCFS in the clinical laboratory. This assay includes two test probes for the loci, DGSI at 22q11.2 and DGSII at 10p13p14, and centromeric probes for chromosomes 10 and 22. Of 412 patients tested, 54 were found to be deleted for the DGSI locus on chromosome 22 (13%), and a single patient was found deleted for the DGSII locus on chromosome 10 (0. 24%). The patient with the 10p deletion had facial features consistent with VCFS, plus sensorineural hearing loss, and renal anomalies. Cytogenetic analysis showed a large deletion of 10p [46, XX,del(10)(p12.2p14)] and FISH using a 10p telomere region-specific probe confirmed the interstitial nature of the deletion. Analysis for the DGSI and the DGSII loci suggests that the deletion of the DGSII locus on chromosome 10 may be 50 times less frequent than the deletion of DGSI on chromosome 22. The incidence of deletions at 22q11.2 has been estimated to be 1 in 4000 newborns; therefore, the deletion at 10p13p14 may be estimated to occur in 1 in 200,000 live births.