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.     

Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.     

The psychopathological phenotype of velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is mostly associated with deletions of chromosome 22q11, and is thought to be characterized by an increased frequency of major psychiatric disorders. Sixteen patients adults with VCFS and psychiatric symptoms were evaluated using a semi-structured investigation of history, symptoms, signs and behaviour. All available data were used in consensus meetings to obtain a classifiable diagnostic category. In contrast to other reports, no categorical diagnosis could be established. Instead, a quite specific psychological, behavioural and psychopathological constellation emerged that should most adequately be denominated as a VCFS-psychiatric syndrome. It is concluded that VCFS is associated with a specific psychopathological syndrome.     

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.     

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.     

Molecular Definition of the 22q11 Deletions in Velo-Cardio-Facial Syndrome

Velo-cardio-facial syndrome (VCFS) is a common genetic disorder among individuals with cleft palate and is associated with hemizygous deletions in human chromosome 22q11. Toward the molecular definition of the deletions, we constructed a physical map of 22q11 in the form of overlapping YACs. The physical map covers >9 cM of genetic distance, estimated to span 5 Mb of DNA, and contains a total of 64 markers. Eleven highly polymorphic short tandem-repeat polymorphic (STRP) markers were placed on the physical map, and 10 of these were unambiguously ordered. The 11 polymorphic markers were used to type the DNA from a total of 61 VCFS patients and 49 unaffected relatives. Comparison of levels of heterozygosity of these markers in VCFS patients and their unaffected relatives revealed that four of these markers are commonly hemizygous among VCFS patients. To confirm these results and to define further the breakpoints in VCFS patients, 15 VCFS individuals and their unaffected parents were genotyped for the 11 STRP markers. Haplotypes generated from this study revealed that 82% of the patients have deletions that can be defined by the STRP markers. The results revealed that all patients who have a deletion share a common proximal breakpoint, while there are two distinct distal breakpoints. Markers D22S941 and D22S944 appear to be consistently hemizygous in patients with deletions. Both of these markers are located on a single nonchimeric YAC that is 400 kb long. The results also show that the parental origin of the deleted chromosome does not have any effect on the phenotypic manifestation     

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.     

Analysis of TBX1 variation in patients with psychotic and affective disorders

A significant portion of patients with 22q11 deletion syndrome (22q11DS) develop psychiatric disorders, including schizophrenia and other psychotic and affective symptoms, and the responsible gene/s are assumed to also play a significant role in the etiology of nonsyndromic psychiatric disease. The most common psychiatric diagnosis among patients with 22q11DS is schizophrenia, thought to result from neurotransmitter imbalances and also from disturbed brain development. Several genes in the 22q11 region with known or suspected roles in neurotransmitter metabolism have been analyzed in patients with isolated schizophrenia; however, their contribution to the disease remains controversial. Haploinsufficiency of the TBX1 gene has been shown to be sufficient to cause the core physical malformations associated with 22q11DS in mice and humans and via abnormal brain development could contribute to 22q11DS-related and isolated psychiatric disease. 22q11DS populations also have increased rates of psychiatric conditions other than schizophrenia, including mood disorders. We therefore analyzed variations at the TBX1 locus in a cohort of 446 white patients with psychiatric disorders relevant to 22q11DS and 436 ethnically matched controls. The main diagnoses included schizophrenia (n = 226), schizoaffective disorder (n = 67), bipolar disorder (n = 82), and major depressive disorder (n = 29). We genotyped nine tag SNPs in this sample but did not observe significant differences in allele or haplotype frequencies in any of the analyzed groups (all affected, schizophrenia and schizoaffective disorder, schizophrenia alone, and bipolar disorder and major depressive disorder) compared with the control group. Based on these results we conclude that TBX1 variation does not make a strong contribution to the genetic etiology of nonsyndromic forms of psychiatric disorders commonly seen in patients with 22q11DS.     

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.     

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.     

Genomic disorders on 22q11

The 22q11 region is involved in chromosomal rearrangements that lead to altered gene dosage, resulting in genomic disorders that are characterized by mental retardation and/or congenital malformations. Three such disorders-cat-eye syndrome (CES), der(22) syndrome, and velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS)-are associated with four, three, and one dose, respectively, of parts of 22q11. The critical region for CES lies centromeric to the deletion region of VCFS/DGS, although, in some cases, the extra material in CES extends across the VCFS/DGS region. The der(22) syndrome region overlaps both the CES region and the VCFS/DGS region. Molecular approaches have revealed a set of common chromosome breakpoints that are shared between the three disorders, implicating specific mechanisms that cause these rearrangements. Most VCFS/DGS and CES rearrangements are likely to occur by homologous recombination events between blocks of low-copy repeats (e.g., LCR22), whereas nonhomologous recombination mechanisms lead to the constitutional t(11;22) translocation. Meiotic nondisjunction events in carriers of the t(11;22) translocation can then lead to offspring with der(22) syndrome. The molecular basis of the clinical phenotype of these genomic disorders has also begun to be addressed. Analysis of both the genomic sequence for the 22q11 interval and the orthologous regions in the mouse has identified >24 genes that are shared between VCFS/DGS and der(22) syndrome and has identified 14 putative genes that are shared between CES and der(22) syndrome. The ability to manipulate the mouse genome aids in the identification of candidate genes in these three syndromes. Research on genomic disorders on 22q11 will continue to expand our knowledge of the mechanisms of chromosomal rearrangements and the molecular basis of their phenotypic consequences.     

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.     

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.     

The behavioural phenotype in velo-cardio-facial syndrome (VCFS): from infancy to adolescence

In this contribution the current status and recent findings of the behavioural phenotype in VCFS (22q11 deletion) are discussed with regard to motor development, cognition and neurodevelopment, and behaviour and temperament. Motor: hypotonia in infancy, gross-motor milestones are delayed, problems with coordination and balance from preschool age on, problems with tempo/speed during adolescence. Cognition and neurodevelopment: learning disabilities (82-100%), intellectual disability (45%), better verbal abilities than performal abilities, poor attention and concentration, visuo-perceptual-spatia problems, good (auditory) memory. An important subgroup of children (55%) has a non-verbal learning disability (NLD). Behaviour and social-emotional development AD(H)D, withdrawn and shy, person-dependent social problems in relationships with peers, anxious, risk for child psychiatric problems as well as for the development of psychiatric problems during adolescence and early adulthood. Information on the behavioural phenotype in VCFS (22q11 deletion) is of great importance to clinicians as an aid to syndrome diagnosis, but even more to parents because it offers immense direct practical value to the management of the behaviour of their child. Appropriate counseling and information on the long-term expectations, and better insight in the behaviour will lead to the development of realistic ways of coping with their child.     

Chromosome 22q11 deletions in patients with selected outflow tract malformations

The incidence of 22q11 deletions and its effect on the phenotype were established in 170 patients with selected outflow tract malformations and transposition of the great arteries (conotruncal defects). Cases were seen both prospectively and retrospectively. All patients had a dysmorphological evaluation by the clinical geneticist and a cytogenetic analysis including FISH analysis for 22q11 deletions. A chromosomal abnormality was present in 29 patients, including a 22q11 deletion in 22/170 patients (13%). The 22q11 deletion was found in 11% of tetralogy of Fallot, in 11% of pulmonary atresia and VSD, in 44% of pulmonary atresia. VSD and collateral arteries, in 20% of truncus arteriosus, in 60% of interrupted aortic arch and in 25% patients with aberrant subclavian artery. They were absent in double outlet right ventricle or in transposition of the great arteries. No parental deletion was found. All patients had clinical characteristics of the velocardiofacial syndrome. This study confirms a high incidence of chromosome 22q11 deletions in patients with selected outflow tract malformations, with great clinical impact for further management and genetic counseling.     

Heterozygosity of murine Crkl does not recapitulate behavioral dimensions of human 22q11.2 hemizygosity

Deletions in 22q11.2 human chromosome are known to be associated with psychiatric disorders, such as intellectual disability, schizophrenia, autism spectrum disorder, and anxiety disorders. This copy number variation includes a 3.0 Mb deletion and a nested proximal 1.5 Mb hemizygous deletion in the same region. Evidence indicates that the distal 22q11.2 region outside the nested 1.5 Mb deletion also might be contributory in humans. However, the precise genetic architecture within the distal region responsible for psychiatric disorders remains unclear, and this issue cannot be experimentally evaluated beyond the correlation in humans. As CRKL (CRK-like Proto-Oncogene, Adaptor Protein) is one of the genes encoded in the distal 22q11.2 segment and its homozygous deletion causes physical phenotypes of 22q11.2 hemizygous deletion, we tested the hypothesis that its murine homolog Crkl contributes to behavioral phenotypes relevant to psychiatric disorders in mice. Congenic Crkl heterozygosity reduced thigmotaxis, an anxiety-related behavior, in an inescapable open field, but had no apparent effect on social interaction, spontaneous alternation in a T-maze, anxiety-like behavior in an elevated plus maze, or motor activity in an open field. Our data indicate that the heterozygosity of murine Crkl does not recapitulate social deficits, working memory deficits, repetitive behavior traits or hyperactivity of human 22q11.2 hemizygous deletion. Moreover, while 22q11.2 hemizygous deletion is associated with high levels of phobia and anxiety in humans, our data suggest that Crkl heterozygosity rather acts as a protective factor for phobia-like behavior in an open field.     

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.