Interstitial deletions in DiGeorge syndrome detected with microclones from 22q11

DiGeorge syndrome in humans is charaterized by immunodeficiency, heart defects, mental retardation and facial dysmorphism; cytogenetic analysis has shown that deletions at 22q11 occur in approximately 25% of cases. To generate DNA markers from this region, we have microdissected and microcloned band q11 of human Chromosome (Chr) 22. Nineteen thousand clones were obtained from material dissected from 20 chromosome fragments. Seventeen of 61 clones analyzed (28%) were repetitive, 27 (44%) gave no signal, and 17 (28%) detected single copy sequences of which ten mapped to Chr 22. Two of these were found to be deleted in patients with DiGeorge syndrome and either monosomy for 22q11-pter or visible interstitial deletions of 22q11. These two markers are also hemizygous in patients with no visible chromosomal abnormality, demonstrating that submicroscopic deletions are common in DiGeorge syndrome patients.     

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.     

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.     

Familial DiGeorge syndrome and associated partial monosomy of chromosome 22

Summary Partial monosomy of 22q due to an unbalanced 4;22 translocation was seen in a 2-month-old male with Type I truncus arterious, dysmorphic features, and T-cell abnormalities. The family history revealed a previous sib with Type I truncus arteriosus, thymic aplasia, and parathyroid hypoplasia noted on postmortem examination, consistent with DiGeorge syndrome. Evaluation of the asymptomatic mother of these two patients revealed partial T-cell deficiency and the same unbalanced translocation with deletion of proximal 22qll. These findings provide further evidence that some cases of complete or partial DiGeorge syndrome are associated with monosomy of the proximal long arm of chromosome 22, and they may explain many, if not all, familial cases of the syndrome.Supported in part by National Foundation-March of Dimes Grant No. 2-161/C-331. Funds from the Texas Department of Health through PL94-278 National Genetic Diseases Act, from the Robert J. Kleberg, Jr. Center for Human Genetics, and USPHS Grant No. RR-05425.     

Digeorge anomaly associated with partial deletion of chromosome 22. Report of a case with X/22 translocation and review of the literature

Partial monosomy of 22q, resulting from a de novo unbalanced translocation t(X;22)(q28;q11) was detected in a newborn female with manifestations of the DiGeorge anomaly including multiple anomalies, type I truncus arteriosus, T-cell abnormalities, thymic aplasia and parathyroid hypoplasia noted on postmortem examination. Although DiGeorge anomaly is causally heterogeneous, our patient, together with 18 previously known cases, confirm that partial monosomy of the proximal long arm of chromosome 22 is the single most common cause of this polytopic developmental field defect.     

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.     

Cytogenetic findings in a prospective series of patients with DiGeorge anomaly.

High-resolution cytogenetics analysis of peripheral blood lymphocytes was done prospectively on 27 of 28 patients with features of DiGeorge anomaly. Twenty-two patients (81%) had normal chromosome studies with no detectable deletion in chromosome 22. Five patients (18%) had demonstrable chromosome abnormalities. Three patients had monosomy 22q11, one due to a 4q;22q translocation, one due to a 20q;22q translocation, and one due to an interstitial deletion of 22q11. One patient had monosomy 10p13, and one patient had monosomy 18q21.33, although the latter had subsequent resolution of T-cell defects. These findings are consistent with the heterogeneity of DiGeorge anomaly but confirm the association with monosomy 22q11 in some cases. However, monosomy 10p13 may also lead to this phenotype. Because of these associated chromosome findings, cytogenetic analyses should be done on patients with suspected DiGeorge anomaly. This is particularly important since many of the abnormalities involving chromosome 22 are translocations that can be familial with a higher recurrence risk. Since only one subtle, interstitial deletion of chromosome 22 was observed, it is not clear whether high-resolution cytogenetic analysis is cost beneficial for all such patients.     

The deletions of 22q11--the Portuguese experience

The patients with a chromosome 22q11 deletion have a variable phenotype which includes DiGeorge (DG) and Velocardiofacial (VCF) syndromes. The aim of the present study is to characterize the phenotype of DG and VCF using facial biometry in 12 portuguese patients. We found 4/12 patients with the DG phenotype: 3/4 had telecanthus, small mouth and retrognathia; 1/4 had telecanthus, short nose with bulbous tip and a normal mouth. These patients had major cardiac defects associated with hypoplastic or absent thymus and monosomy 22q11. We did not find velopharyngeal insufficiency in patients with the so called DG phenotype 8/12 patients had the VCF phenotype: typical facies with variable features. Four of these had velopharyngeal insufficiency and learning disabilities. Four patients had cardiac defects and 5/8 had monosomy 22q11. Probably this clinical variability is due to mutations in critical genes involved in embryonic development.     

Congenital heart defects in patients with DiGeorge/velocardiofacial syndrome and del22q11

Congenital heart defects (CHDs) are found in 75% of patients with DiGeorge/velocardiofacial (DG/VCF) syndromes with deletion 22q11.2 (del22q11). The purpose of this study was to analyse clinical features and, particularly, types and subtypes of CHDs associated with del22q11 in our series of patients and in those reported in other studies. All patients with CHD and del22q11 present major or minor clinical features of DG/VCF syndrome. Many children, particularly in the neonatal age, have only a "subtle" phenotype, so that accurate phenotypical evaluation is mandatory for selecting patients with CHD at risk for del22q11. Conotruncal cardiac defects are the most common CHDs in patients with DG/VCF syndrome, but other defects can also occur. Peculiar anatomical subtypes are found in patients with del22q11. They are frequently complex, consisting in malalignment with deficiency of the infundibular septum and anomalies of the aortic arch and pulmonary arteries.     

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.     

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.     

GNB1L, a gene deleted in the critical region for DiGeorge syndrome on 22q11, encodes a G-protein beta-subunit-like polypeptide

CATCH 22 syndromes, which include DiGeorge syndrome and Velocardiofacial syndrome, are the most common cause of congenital heart disease which involve microdeletion of 22q11. Using a strategy including EST searching, PCR amplification and 5'-RACE, we have cloned a 1487 bp cDNA fragment from human heart cDNA library. The cloned GNB1L cDNA encodes a G-protein beta-subunit-like polypeptide, and the GNB1L gene is located in the critical region for DiGeorge syndrome. A comparison of GNB1L cDNA sequence with corresponding genomic DNA sequence revealed that this gene consists of seven exons and spans an approximately 60 kb genomic region. Northern blot analysis revealed GNB1L is highly expressed in the heart.     

Molecular genetic analysis of the DiGeorge syndrome among Korean patients with congenital heart disease

The DiGeorge syndrome (DGS) is a developmental defect of the third and fourth pharyngeal pouches, which is associated with congenital heart defects, hypoparathyroidism, cell-mediated immunodeficiency, velo-pharyngeal insufficiency and craniofacial dysmorphism. The aetiological factor in a great majority of DGS cases is monosomy for the chromosomal region 22q11. To analyze DGS at the molecular level, a new molecular probe (DGCR680) encompassing the ADU balanced translocation breakpoint was prepared. When 13 Korean patients with DGS-type congenital heart disease were analyzed with this probe, 9 turned out to have a deletion at this locus, and all of them except one exhibited a typical facial dysmorphism associated DGS. Though only 9 independent patients were detected to have a deletion at the locus using the commercial probe N25 (D22S75), which maps at about 160 kb from the ADU breakpoint to the telomeric end, results from fluorescence in situ hybridization revealed a deletion in all cases tested at this locus. Two patients who had a deletion at the locus D22S75 but not at DGCR680 did not exhibit any DGS-type facial abnormalities. This result implies that the 680 bp probe covering the ADU translocation breakpoint might be a candidate for a molecular marker that can distinguish a specific phenotype, such as facial features associated with the DiGeorge syndrome. This study also suggested that systematic approaches with several small DNA probes along the DGCR could help to dissect the complex phenotypes associated with the DiGeorge syndrome, such as cardiac defects, abnormal faces, thymic hypoplasia, cleft palate, and hypocalcemia, etc.     

A novel, single nucleotide polymorphism-based assay to detect 22q11 deletions

Velocardiofacial syndrome, DiGeorge syndrome, and conotruncal anomaly face syndrome, now collectively referred to as 22q11deletion syndrome (22q11DS) are caused by microdeletions on chromosome 22q11. The great majority ( approximately 90%) of these deletions are 3 Mb in size. The remaining deleted patients have nested break-points resulting in overlapping regions of hemizygosity. Diagnostic testing for the disorder is traditionally done by fluorescent in situ hybridization (FISH) using probes located in the proximal half of the region common to all deletions. We developed a novel, high-resolution single-nucleotide polymorphism (SNP) genotyping assay to detect 22q11 deletions. We validated this assay using DNA from 110 nondeleted controls and 77 patients with 22q11DS that had previously been tested by FISH. The assay was 100% sensitive (all deletions were correctly identified). Our assay was also able to detect a case of segmental uniparental disomy at 22q11 that was not detected by the FISH assay. We used Bayesian networks to identify a set of 17 SNPs that are sufficient to ascertain unambiguously the deletion status of 22q11DS patients. Our SNP based assay is a highly accurate, sensitive, and specific method for the diagnosis of 22q11 deletion syndrome.     

A human gene similar to Drosophila melanogaster peanut maps to the DiGeorge syndrome region of 22q11

A Drosophila-related expressed sequence tag (DRES) with sequence similarity to the peanut gene has previously been localized to human chromosome 22q11. We have isolated the cDNA corresponding to this DRES and show that it is a novel member of the family of septin genes, which encode proteins with GTPase activity thought to interact during cytokinesis. The predicted protein has P-loop nucleotide binding and GTPase motifs. The gene, which we call PNUTL1, maps to the region of 22q11.2 frequently deleted in DiGeorge and velo-cardio-facial syndromes and is particularly highly expressed in the brain. The mouse homologue, Pnutl1, maps to MMU16 adding to the growing number of genes from the DiGeorge syndrome region that map to this chromosome.     

Combined trisomy 9P and Shprintzen syndrome resulting from a paternal t(9;22)

The authors report on a female infant with partial trisomy 9 (pter-->q12) together with partial monosomy 22 (pter-->q11.23) that included DiGeorge critical region (DGCR), as a result of adjacent-2 disjunction. In addition to the clinical features characteristic of trisomy 9p syndrome, the patient had Truncus arteriosus type A2, bilateral hydronephrosis, palatal anomaly, retrognathia, and laryngeal hypotonia, which are likely to be attributed to 22q11.2 deletion. This patient appears to be the first reported case with such unbalanced translocation resulting from a paternal reciprocal translocation. For live birth, the risk for male carrier is 8.7-17.4%. It is important to consider this higher risk when counseling. Precise study concerning the presence of the DGCR can facilitate in the better understanding of the condition.     

The phenotypic spectrum of the 10p deletion syndrome versus the classical DiGeorge syndrome

We reviewed 36 patients with a deletion of the short arm of chromosome 10 and a partial DiGeorge syndrome. We compared the phenotypes observed in these del(10p) patients with the classical DiGeorge phenotype associated with del(22q11), pointing out both similarities and differences. Some features, such as sensorineural hearing loss, seem to be highly associated with a deletion of 10p but are absent in the classical DiGeorge spectrum caused by del(22q11).     

Isolation of anonymous DNA markers for human chromosome 22q11 from a flow-sorted library,and mapping using hybrids from patients with DiGeorge syndrome

Summary DiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.     

Developing models of DiGeorge syndrome

DiGeorge syndrome is a common congenital disorder characterized by neural-crest-related developmental defects. Mouse models of DiGeorge syndrome have been created that recapitulate defects seen in human patients. Here, the genetic pathways regulating cardiac neural crest development are reviewed and the evidence implicating TBX1 and other genes on chromosome 22q11 in the pathogenesis of DiGeorge syndrome is summarized.