Bernice E. Morrow

22q11.2 Deletion Syndrome

22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion disorder, estimated to result mainly from de novo non-homologous meiotic recombination events occurring in approximately 1 in every 1,000 fetuses. The first description in the English language of the constellation of findings now known to be due to this chromosomal difference was made in the 1960s in children with DiGeorge syndrome, who presented with the clinical triad of immunodeficiency, hypoparathyroidism and congenital heart disease. The syndrome is now known to have a heterogeneous presentation that includes multiple additional congenital anomalies and later-onset conditions, such as palatal, gastrointestinal and renal abnormalities, autoimmune disease, variable cognitive delays, behavioural phenotypes and psychiatric illness — all far extending the original description of DiGeorge syndrome. Management requires a multidisciplinary approach involving paediatrics, general medicine, surgery, psychiatry, psychology, interventional therapies (physical, occupational, speech, language and behavioural) and genetic counselling. Although common, lack of recognition of the condition and/or lack of familiarity with genetic testing methods, together with the wide variability of clinical presentation, delays diagnosis. Early diagnosis, preferably prenatally or neonatally, could improve outcomes, thus stressing the importance of universal screening. Equally important, 22q11.2DS has become a model for understanding rare and frequent congenital anomalies, medical conditions, psychiatric and developmental disorders, and may provide a platform to better understand these disorders while affording opportunities for translational strategies across the lifespan for both patients with 22q11.2DS and those with these associated features in the general population.

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.

MEIOTIC PACHYTENE ARREST IN MLH1-DEFICIENT MICE

Germ line mutations in DNA mismatch repair genes including MLH1 cause hereditary nonpolyposis colon cancer. To understand the role of MLH1 in normal growth and development, we generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals that the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis.

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.

VEGF : A modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.

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.

Association of codon 108/158 catechol‐O‐methyltransferase gene polymorphism with the psychiatric manifestations of velo‐cardio‐facial syndrome

Velo-cardio-facial-syndrome (VCFS) is a common congenital disorder associated with typical facial appearance, cleft palate, cardiac defects, and learning disabilities. The majority of patients have an interstitial deletion on chromosome 22q11. In addition to physical abnormalities, a variety of psychiatric illnesses have been reported in patients with VCFS, including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. The psychiatric manifestations of VCFS could be due to haploin-sufficiency of a gene(s) within 22q11. One candidate that has been mapped to this region is catechol-O-methyltransferase (COMT). We recently identified a polymorphism in the COMT gene that leads to a valine-->methionine substitution at amino acid 158 of the membrane-bound form of the enzyme. Homozygosity for COMT158met leads to a 3-4-fold reduction in enzymatic activity, compared with homozygotes for COMT158val. We now report that in a population of patients with VCFS, there is an apparent association between the low-activity allele, COMT158met, and the development of bipolar spectrum disorder, and in particular, a rapid-cycling form.

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.

Microduplication and triplication of 22q11.2: a highly variable syndrome.

22q11.2 microduplications of a 3-Mb region surrounded by low-copy repeats should be, theoretically, as frequent as the deletions of this region; however, few microduplications have been reported. We show that the phenotype of these patients with microduplications is extremely diverse, ranging from normal to behavioral abnormalities to multiple defects, only some of which are reminiscent of the 22q11.2 deletion syndrome. This diversity will make ascertainment difficult and will necessitate a rapid-screening method. We demonstrate the utility of four different screening methods. Although all the screening techniques give unique information, the efficiency of real-time polymerase chain reaction allowed the discovery of two 22q11.2 microduplications in a series of 275 females who tested negative for fragile X syndrome, thus widening the phenotypic diversity. Ascertainment of the fragile X-negative cohort was twice that of the cohort screened for the 22q11.2 deletion. We also report the first patient with a 22q11.2 triplication and show that this patients mother carries a 22q11.2 microduplication. We strongly recommend that other family members of patients with 22q11.2 microduplications also be tested, since we found several phenotypically normal parents who were carriers of the chromosomal abnormality.

Suppression of neural fate and control of inner ear morphogenesis by Tbx1

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. We show that in the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction or proper patterning of gene expression related to sensory organ morphogenesis (Otx1 and Bmp4, respectively). Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. We propose that Tbx1 acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst.