Analyzing a relatively large 22q11.2DS cohort allowed us to compare the FISH and MLPA methods, perform molecular analysis of the 22q11.2 deletion in our patients and examine the phenotype-genotype relationship.
The MLPA technique confirmed all the deletions that were previously detected by FISH in our patients while the FISH assay failed to detect one atypical deletion because it was outside the location of the FISH probes. Thus the MLPA is clearly superior to FISH as a diagnostic tool and a useful method for deletion characterization, essential for genotype-phenotype analysis. CGH and SNP array techniques can also detect atypical deletions but their costs are substantially higher. Other studies have recommended using MLPA as a diagnostic tool
[8, 21]. However, MLPA is limited in identifying the deletion boundary that is determined by the availability of probes in the deletion breakpoints. The MLPA kit that we used (P250-A1) lacked probes in the candidate PRODH gene located in the proximal breakpoint which was found to be associated with psychotic disorders in 22q11.2DS
[22, 23]. To solve this problem we used qPCR technique and found that the PRODH gene was deleted in most (94.4%) of the 22q11.2DS individuals, as was found previously
[6, 24]. The MLPA kit released after we performed our study (P324-A2) already contained several PRODH gene probes.
Molecular characteristics of the 22q11.2 deletion
We identified 5 types of 22q11.2 deletions. According to the results of the specific MLPA kit used, the proximal starting point for all deletions, except one, was located in the CLTCL1 gene probe, near LCR A (Figure
2). The distal breakpoints of the deletions were located in LCRs B, C, D and D+ thus determining the size and location of the deletions. Ninety seven out of 110 individuals (88.2%) carried the typical ~3Mb deletion (LCR A-D) consistent with frequencies found in other studies (77–88.7%)
[20, 21, 24–27].
The largest deletion (LCR A-D+), a variant of the 3Mb deletions, was present in 3.63% of the subjects compared to 4.5-6.5% in previous reports
[8, 24]. The prevalence of the proximal deletions of 1.5Mb (LCR A-B) and 2Mb (LCR A-C), each 3.63% in our subjects, varied largely in other studies
[20, 21, 24–27], probably due to different sample sizes and resolution of the mapping methods used.
Studying the genotype-phenotype relationship in 22q11.2DS is a formidable task. The difficulties are obvious: there are up to 200 possible clinical symptoms
, the clinical variability is large even in individuals with the same type of deletion and the statistical power is low because most individuals harbor the common 3Mb TDR and only a few with other entities. Yet, it can potentially shed light on the role of the genes that contribute to the physical and psychiatric symptoms expressed in this disorder. The quantitative comparison between 97 individuals with TDR and those with four different types of nonTDR deletions along several physical and neuropsychiatric-cognitive parameters did not reveal statistically significant differences. This could be due to methodological limitations such as sample size, and the complexity of the genetic and epigenetic mechanisms governing the phenotype in 22q11.2DS. Our results are supported by most previous studies that could not demonstrate a correlation between the size and the location of the deletion and the clinical features of the subjects
[24, 25, 28–30]. However, Rauch et al. (2005) demonstrated a correlation between deletion characteristics and phenotypic expression by assessment of individuals with typical and atypical 22q11.2 deletions, showing that atypical CHD and mild dysmorphism are recognizable feature of atypical distal deletions
. Recently, a case-report of monozygotic twins differing in their deletion size and clinical expression was published, indicating a possible role of deletion characteristics for the phenotype
In accordance with previous studies
 hypocalcemia was found in 27.7% of patients with large deletions (3Mb, LCR A-D and LCR A-D+) but was absent in those carrying the proximal nested deletions (1.5 and 2Mb). Interestingly, neither was it observed in patients with distal nested deletions
. Although the number of cases is small, this observation may suggest that haploinsufficiency of genes both at the proximal and the distal part of 22q11.2 is required in order to cause hypocalcemia.
The presented clinical data of each individual carrying a non-TDR deletion are not in themselves informative but may be of help in the effort to build a common data base. More informed and educated conclusions could be made on this intriguing question by applying unified molecular methods for mapping the 22q11.2 deletion (high resolution methods such as CGH and SNP arrays and next generation sequencing) and using agreed clinical parameters and scoring system for characterizing the phenotype.
Individuals without 22q11.2 deletion
Before the era of molecular diagnosis patients were diagnosed as VCFS (22q11.2DS) solely based on their clinical symptoms. When molecular genetics methods were introduced it turned out that in about 20% of the patients classified as VCFS no deletion could be detected
[20, 21] and the question of the etiology of their phenotype is still unknown. Interestingly, when we examined a subgroup of these individuals it appeared that the variety and severity of clinical symptoms in these subjects are very similar to those found in confirmed 22q11.2DS patients. Subjects may have been misdiagnosed because they had a combination of relatively common symptoms, such as cardiac anomalies and neuropsychiatric disorders, that when considered together phenocopy the 22q11.2DS. It is also possible that these patients do carry very small deletions or even point mutations which are below the resolution of the methods used or they have other microdeletion or microduplication syndromes.
The case with atypical nested distal deletion
Patient VC901 carries a rare, small deletion that does not include the nested proximal 1.5Mb region which harbors several important candidate genes, thought to be sufficient for producing the syndrome
. This case portrays the complexity of the molecular mechanisms controlling the 22q11.2DS phenotype, indicating the possibility of redundancy in causative genes. Since the "classical" candidate genes are not deleted in this person other candidate genes residing in the nested distal region may be responsible for the physical features and neuropsychiatric manifestations. For example, of the candidate genes that have been shown to be involved in cardiac malformations (HIRA, UFD1L, TBX1 and CRKL)
[35–39]CRKL is one that is located in the distal deletion region and has been found to affect cardiac neural crest derivatives in mice
. Its loss may be the causative event for the CHD in this case. In a similar manner, the neuropsychiatric phenotype may be affected by the deletion of PIK4CA and SNAP29 from the distal region which have been demonstrated to be associated with schizophrenia
[40–43], and not by the putative schizophrenia genes PRODH, COMT, DGCR8 and ZDHHC8. Another genetic mechanism that may affect the phenotype involves control elements in the deleted region that act by cis mechanisms on the expression of the non-deleted candidate genes as in the dosage-sensitive interaction between Tbx1and Crkl in mice
The longitudinal neuropsychiatric follow-up of this patient has raised the intriguing question of whether multiple genes in the 22q11.2 region are responsible for the emergence of schizophrenia in 22q11.2DS patients. Her psychiatric developmental trajectory is typical of 22q11.2DS namely, anxiety and subthreshold positive psychotic symptoms during childhood evolving to a full psychotic disorder in adulthood. Yet her cognitive developmental trajectory is not typical, as her IQ scores did not decline and even improved a little during young adulthood, whereas in typical 22q11.2DS patients there is a cognitive decline especially of verbal IQ [45, 46]. This might suggest that the neuropsychiatric manifestations in 22q11.2DS are governed by several sets of genetic elements. It is tempting to speculate that genes such as PIK4CA and SNAP29 that are deleted in our patient may be involved in the emergence of psychotic "positive symptoms" while others, such as PRODH and COMT that are not deleted, play a role in the cognitive decline process.
Two other 22q11.2DS cases with similar distally nested deletion have been reported but they are not readily comparable to the present case because of the probands' young age. One was a patient with TOF and facial dysmorphology, last assessed at 9 months of age
, and the other a 6 year-old with typical facial features, significant developmental delay and increased anxiety suggestive of possible early signs of psychiatric illness