Isolation and characterization of a novel gene containing WD40 repeats from the region deleted in velo-cardio-facial/DiGeorge syndrome on chromosome 22q11

Three congenital disorders, cat-eye syndrome (CES), der(22) syndrome, and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), result from tetrasomy, trisomy, and monosomy, respectively, of part of 22q11. They share a 1.5-Mb region of overlap, which contains 24 known genes. Although the region has been sequenced and extensively analyzed, it is expected to contain additional genes, which have thus far escaped identification. To understand completely the molecular etiology of VCFS/DGS, der(22) syndrome, and CES, it is essential to isolate all genes in the interval. We have identified and characterized a novel human gene, located within the 1.5-Mb region deleted in VCFS/DGS, trisomic in der(22) syndrome and tetrasomic in CES. The deduced amino acid sequence of the human gene and its mouse homologue contain several WD40 repeats, but lack homology to known proteins. We termed this gene WDR14 (WD40 repeat-containing gene deleted in VCFS). It is expressed in a variety of human and mouse adult and fetal tissues with substantial expression levels in the adult thymus, an organ hypoplastic in VCFS/DGS.     

A complex rearrangement on chromosome 22 affecting both homologues; haplo-insufficiency of the Cat eye syndrome region may have no clinical relevance

The presence of highly homologous sequences, known as low copy repeats, predisposes for unequal recombination within the 22q11 region. This can lead to genomic imbalances associated with several known genetic disorders. We report here a developmentally delayed patient carrying different rearrangements on both chromosome 22 homologues, including a previously unreported rearrangement within the 22q11 region. One homologue carries a deletion of the proximal part of chromosome band 22q11. To our knowledge, a ‘pure’ deletion of this region has not been described previously. Four copies of this 22q11 region, however, are associated with Cat eye syndrome (CES). While the phenotypic impact of this deletion is unclear, familial investigation revealed five normal relatives carrying this deletion, suggesting that haplo-insufficiency of the CES region has little clinical relevance. The other chromosome 22 homologue carries a duplication of the Velocardiofacial/DiGeorge syndrome (VCFS/DGS) region. In addition, a previously undescribed deletion of 22q12.1, located in a relatively gene-poor region, was identified. As the clinical features of patients suffering from a duplication of the VCFS/DGS region have proven to be extremely variable, it is impossible to postulate as to the contribution of the 22q12.1 deletion to the phenotype of the patient. Additional patients with a deletion within this region are needed to establish the consequences of this copy number alteration. This study highlights the value of using different genomic approaches to unravel chromosomal alterations in order to study their phenotypic impact.     

Der(22) syndrome and velo-cardio-facial syndrome/DiGeorge syndrome share a 1.5-Mb region of overlap on chromosome 22q11

Derivative 22 (der[22]) syndrome is a rare disorder associated with multiple congenital anomalies, including profound mental retardation, preauricular skin tags or pits, and conotruncal heart defects. It can occur in offspring of carriers of the constitutional t(11;22)(q23;q11) translocation, owing to a 3:1 meiotic malsegregation event resulting in partial trisomy of chromosomes 11 and 22. The trisomic region on chromosome 22 overlaps the region hemizygously deleted in another congenital anomaly disorder, velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Most patients with VCFS/DGS have a similar 3-Mb deletion, whereas some have a nested distal deletion endpoint resulting in a 1.5-Mb deletion, and a few rare patients have unique deletions. To define the interval on 22q11 containing the t(11;22) breakpoint, haplotype analysis and FISH mapping were performed for five patients with der(22) syndrome. Analysis of all the patients was consistent with 3:1 meiotic malsegregation in the t(11;22) carrier parent. FISH-mapping studies showed that the t(11;22) breakpoint occurred in the same interval as the 1.5-Mb distal deletion breakpoint for VCFS. The deletion breakpoint of one VCFS patient with an unbalanced t(18;22) translocation also occurred in the same region. Hamster-human somatic hybrid cell lines from a patient with der(22) syndrome and a patient with VCFS showed that the breakpoints occurred in an interval containing low-copy repeats, distal to RANBP1 and proximal to ZNF74. The presence of low-copy repetitive sequences may confer susceptibility to chromosome rearrangements. A 1.5-Mb region of overlap on 22q11 in both syndromes suggests the presence of dosage-dependent genes in this interval.     

Computational analysis and refinement of sequence structure on chromosome 22q11.2 region: application to the development of quantitative real-time PCR assay for clinical diagnosis

The low-copy repeat (LCR) is a new class of repetitive DNA element and has been implicated in many human disorders, including DiGeorge/velocardiofacial syndrome (DGS/VCFS). It is now recognized that nonallelic homologous recombination (NAHR) through LCRs flanking the chromosome 22q11.2 region leads to genome rearrangements and results in the DGS/VCFS. To refine the structure and content of chromosome 22q11.2 LCRs, we applied computational analysis to dissect region-specific LCRs using publicly available sequences. Nine distinct duplicons between 1.6 and 65 kb long and sharing >95% sequence identity were identified. The presence of these sequence motifs supports the NAHR mechanism. Further sequence analysis suggested that the previously defined 3-Mb deletion may actually comprise two deletion intervals of similar size close to each other and thus indistinguishable when using fluorescence in situ hybridization (FISH) analysis. The differentially deleted regions contain several hypothetical proteins and UniGene clusters and may partially explain the clinical heterogeneity observed in DGS/VCFS patients with the 3-Mb common deletion. To implement further sequence information in molecular medicine, we designed a real-time quantitative PCR assay and validated the method in 122 patients with suspected DGS/VCFS. The assay detected 28 patients with chromosome 22q11.2 deletion later confirmed using FISH. Our results indicated that the developed assay is reliable as well as time and cost effective for clinical diagnosis of chromosome 22q11.2 deletion. They also suggest that this methodology can be applied to develop a molecular approach for clinical detection and diagnosis of other genomic disorders.     

22q11.2 distal deletion: a recurrent genomic disorder distinct from DiGeorge syndrome and velocardiofacial syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent ~3 Mb deletion or a smaller, less common, ~1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the ~3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a ~1.4 Mb or ~2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.     

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.     

The role of neural crest during cardiac development in a mouse model of DiGeorge syndrome

The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation.     

Molecular genetic study of the frequency of monosomy 22q11 in DiGeorge syndrome

It is well established that DiGeorge syndrome (DGS) may be associated with monosomy of 22q11-pter. More recently, DNA probes have been used to detect hemizygosity for this region in patients with no visible karyotypic abnormality. However, DGS has also been described in cases where the cytogenetic abnormality does not involve 22q11; for instance, four cases of 10p- have been reported. In this study we have prospectively analyzed patients, by using DNA markers from 22q11, to assess the frequency of 22q11 rearrangements in DGS. Twenty-one of 22 cases had demonstrable hemizygosity for 22q11. Cytogenetic analysis had identified interstitial deletion in 6 of 16 cases tested; in 6 other cases no karyotype was available. When these results are combined with those from our previous studies, 33 of 35 DGS patients had chromosome 22q11 deletions detectable by DNA probes.     

22q11 deletions in isolated and syndromic patients with tetralogy of Fallot

Tetralogy of Fallot (TF) is a congenital conotruncal heart defect commonly found in DiGeorge (DGS) and velo-cardio-facial (VCFS) syndromes. The deletion of chromosome 22q11 (de122q11) is a well established cause of DGS and VCFS, and it has been demonstrated also in sporadic or familial cases of TF. In order to investigate the prevalence of de122q11 in patients with TF, we analyzed the DNA of 137 consecutive patients with syndromic and isolated TF, using the HD7k probe, which detects hemizygosity for the D22S134 locus. De122q11 has been detected in 11/26 (42%) syndromic patients. Evidence for hemizygosity was obtained in all patients with DGS and in 8/15 patients with VCFS. None of the 107 patients with isolated TF had de122q11. Our experience suggests that children with TF and de122q11 always present major or minor extracardiac anomalies. These features, including subtle facial dysmorphisms, should be checked routinely in patients with TF and other conotruncal heart defects.     

High-density single nucleotide polymorphism array analysis in patients with germline deletions of 22q11.2 and malignant rhabdoid tumor

Malignant rhabdoid tumors are highly aggressive neoplasms found primarily in infants and young children. The majority of rhabdoid tumors arise as a result of homozygous inactivating deletions or mutations of the INI1 gene located in chromosome band 22q11.2. Germline mutations of INI1 predispose to the development of rhabdoid tumors of the brain, kidney and extra-renal tissues, consistent with its function as a tumor suppressor gene. We now describe five patients with germline deletions in chromosome band 22q11.2 that included the INI1 gene locus, leading to the development of rhabdoid tumors. Two patients had phenotypic findings that were suggestive but not diagnostic for DiGeorge/Velocardiofacial syndrome (DGS/VCFS). The other three infants had highly aggressive disease with multiple tumors at the time of presentation. The extent of the deletions was determined by fluorescence in situ hybridization and high-density oligonucleotide based single nucleotide polymorphism arrays. The deletions in the two patients with features of DGS/VCFS were distal to the region typically deleted in patients with this genetic disorder. The three infants with multiple primary tumors had smaller but overlapping deletions, primarily involving INI1. The data suggest that the mechanisms underlying the deletions in these patients may be similar to those that lead to DGS/VCFS, as they also appear to be mediated by related, low copy repeats (LCRs) in 22q11.2. These are the first reported cases in which an association has been established between recurrent, interstitial deletions mediated by LCRs in 22q11.2 and a predisposition to cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Clinical and molecular description of the prenatal diagnosis of a fetus with a maternally inherited microduplication 22q11.2 of 2.5 Mb

Microduplications of 22q11.2 have been recently characterized as a new genomic duplication syndrome showing an extremely variable phenotype ranging from normal or mild learning disability to multiple congenital defects and sharing some overlapping features with DiGeorge/Velocardiofacial syndrome (DGS/VCFS). We report on the prenatal diagnosis of a 22q11.2 microduplication in a fetus with normal development that was referred for chromosomal analysis at 17 weeks of gestation because of advanced maternal age. Pregnancy was the result of an IVF-ICSI attempt after 4 years of infertility, mainly due to severe oligoasthenoteratospermia of the father. Amniocentesis was undertaken and cytogenetic analysis revealed an apparently normal male karyotype. Multiple Ligation-dependent Probe Amplification (MLPA) revealed a microduplication in the 22q11.2 chromosome region. Parental analysis showed that the 22q11.2 microduplication has been inherited from the otherwise healthy mother. Analysis with high resolution array-CGH showed that the size of the microduplication is 2.5 Mb and revealed the genes that are duplicated, including the TBX1 gene. The parents elected to continue with the pregnancy and the infant is now five months old and shows normal development.     

Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is the most common microdeletion syndrome in humans. It occurs with an estimated frequency of 1 in 4, 000 live births. Most cases occur sporadically, indicating that the deletion is recurrent in the population. More than 90% of patients with VCFS and a 22q11 deletion have a similar 3-Mb hemizygous deletion, suggesting that sequences at the breakpoints confer susceptibility to rearrangements. To define the region containing the chromosome breakpoints, we constructed an 8-kb-resolution physical map. We identified a low-copy repeat in the vicinity of both breakpoints. A set of genetic markers were integrated into the physical map to determine whether the deletions occur within the repeat. Haplotype analysis with genetic markers that flank the repeats showed that most patients with VCFS had deletion breakpoints in the repeat. Within the repeat is a 200-kb duplication of sequences, including a tandem repeat of genes/pseudogenes, surrounding the breakpoints. The genes in the repeat are GGT, BCRL, V7-rel, POM121-like, and GGT-rel. Physical mapping and genomic fingerprint analysis showed that the repeats are virtually identical in the 200-kb region, suggesting that the deletion is mediated by homologous recombination. Examination of two three-generation families showed that meiotic intrachromosomal recombination mediated the deletion.     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies. Received: 8 July 1997 / Accepted 11 August 1997     

In situ hybridization and translocation breakpoint mapping. III. DiGeorge syndrome with partial monosomy of chromosome 22

We have performed in situ hybridization of a probe for the lambda IGLC constant region to metaphase spreads from two DiGeorge syndrome (DGS)-related chromosomal rearrangements with breakpoints in 22q11. In this study we have demonstrated that the breakpoints are proximal to the lambda IGLC constant region cluster. Thus, at the molecular level, DGS-related breakpoints can be distinguished from the 22q11 breakpoint of CML, but not from the 8;22 translocation of Burkitt lymphoma or from the 21;22 translocations that we have previously studied.     

UFD1L and CDC45L: a role in DiGeorge syndrome and related phenotypes?

Molecular genetics is contributing to the understanding of normal and abnormal cardiovascular development and morphogenesis. Deletions of chromosome 22q11.2 have been associated with distinct phenotypes that result from a failure to form derivatives of third and fourth branchial arches, including DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). The biochemical mechanisms underlying these phenotypes remain undetermined. A recent study provides new insight into the mechanism by which gene deletions produce the DGS and VCFS phenotypes.