A deletion and a duplication in distal 22q11.2 deletion syndrome region. Clinical implications and review

DiGeorge syndrome (DGS) and Velocardiofacial syndrome (VCFS) are genetic disorders affecting pharyngeal and neurobehavioural development [1] that result in conotruncal congenital heart defects (CHD), velopharyngeal insufficiency, hypoparathyroidism, thymic aplasia or hypoplasia, craniofacial dysmorphism, learning difficulties and psychiatric disorders [2, 3]. Interstitial microdeletions in 22q11.2 have been identified as the underlying cause in most cases of DGS [4], VCFS [5] and apparently isolated conotruncal CHD [6].

Deletions in 22q11.2 cluster into a standard 3 Mb deletion in 87% of the cases, a smaller, proximally nested 1.5 Mb deletion in 7% and other atypical deletions, nested, overlapping or adjacent to the typically deleted region (TDR) [7, 8]. By non-allelic homologous recombination (NAHR) after asynchronous replication [9], large low-copy repeats in 22q11.2 (LCR22s A to D) mediate recurrent deletions [7], whereas recently described uncommon deletions are flanked by smaller LCRs (E to H) [10] or alternative breakpoints [5, 11‐24].

Different point mutations [25, 26], balanced translocation breakpoints [27‐29] and shortest regions of deletion overlap (SRO) [11‐16, 30, 31] in 22q11.2 have been compared in order to identify candidate genes for the 22q11.2 deletion syndrome phenotype. However, no clear genotype-phenotype correlation has been found [5, 32] and identical alterations, even within members of the same family, show high phenotypic diversity and variable expressivity or incomplete penetrance [33‐35]. Systematic clinical sorting of patients with non-overlapping deletions has recently shown that an ascertainment bias could be eclipsing different phenotypes or even what would be different syndromes [19, 23, 36].

22q11.2 duplication syndrome has also been recently characterized as a different clinical entity [37] with features overlapping 22q11.2 deletion syndrome [38]. Since fewer duplications have been reported, it is suspected that the diagnosis of this condition is also biased [39‐43], a fact supported by its clinical diversity, generally ranging from a milder, cognitive/behavioral to an apparently normal phenotype in these patients [41, 43‐48]. This lower severity suggests that duplications with sizes that range from 3 to 6 Mb are less deleterious than deletions [37] and therefore are more likely to be inherited at reduced penetrance [43]. Again, LCRs work as recombination substrates for these rearrangements [37], and different sizes have also been described [43, 49].

In this paper we present two patients referred to us for genetic diagnosis of 22q11.2 deletion syndrome. The first patient was found to harbor an atypical deletion and the second one an inherited atypical duplication in the distal segment of the TDR covering LCRs C and D [7]. We discuss screening diagnostic strategies for patients referred for 22q11.2 deletion testing as well as the clinical implications of these findings for a potential genotype-phenotype correlation.

Samples and pictures from patients and their families were obtained after informed consent. Ethical approval was obtained for this study from the IRB at Hospital Universitario La Paz in Madrid (HULP-CEIC-PI347). Research was performed in compliance with the Declaration of Helsinki.

Karyotype and FISH analysis with probe N25 in both patients and their parents showed normal results.

However, MLPA testing of both patients and their parents with kits P023B, P250 and P324-A1 showed a deletion of 5 probes (SNAP29, LZTR1, LCRD probe 06317-L10754, HIC2 and LCRD probe 06321-L05796) on Patient 1 (Figure 2a, b), and a duplication of 3 probes (SNAP29, LZTR1 and LCRD probe 06317-L10754) on Patient 2 (Figure 2a, b) and his mother, uncle, two aunts and maternal grandfather (not shown). These MLPA loci fall within a shortest segment of 675.1 kb for the deletion and 207.4 kb for the duplication, both including the region between LCR22-C and LCR22-D [7]. On examination, the mother, uncle, two aunts and maternal grandfather of Patient 2 were all phenotypically normal.

On the other hand, microsatellite segregation studies were informative for STRs D22S311, D22S1709 and CATCH 38 in Patient 1, and confirmed a de novo paternal deletion. STR CATCH 42 showed a triallelic result for both Patient 2 and his mother, whereas CATCH 11 showed such a result only in the mother. Fluorescence intensity of the alleles of markers CATCH 41, D22S311 and CATCH 13 suggested a trisomic result (Table 1).

SNP genotyping by array-CGH narrowed down the deletion size to 1092.027 kb (rs165626-rs12170039) in Patient 1 (Figure 3a) and the duplication size to 377.634 kb (rs17819969-rs11703181) for Patient 2 (Figure 3b). These results are consistent with MLPA and STRs studies. Other genomic copy number variations (CNV) observed in these patients had been previously described in the normal population [53, 54] according to the Database of Genomic Variants [55] (data not shown). The relative location of each marker and the deleted and duplicated segments are shown in Figure 4.

The whole phenotypic spectrum of 22q11.2 deletion syndrome is contributed by multiple dosage-sensitive genes across the 22q11.2 region, that are required for normal development [reviewed in [1]]. Moreover, non-overlapping, atypical deletions do have significantly overlapping phenotypes, suggesting either, several candidate genes for the syndrome [13, 14, 56], a common developmental pathway [17] or a positional gene effect in 22q11.2 [17, 57‐59]. On the other hand, phenotypes shown by identical deletions in 22q11.2 have been suggested to be modified by parental imprinting [60], unbalanced regulatory effects [15, 59], recessive mutations or polymorphisms unmasked by hemizygosity [59, 60], environmental factors [60] or stochastic events during morphogenesis [15, 61]. Susceptibility to other syndromes in patients with 22q11.2 deletions has also been proposed [58, 62, 63]. Phenotypic discordance has been observed in monozygotic twins with 22q11.2 deletion syndrome [33, 64], a finding that is consistent with both a genetic and an environmental influence.

In the recent years, several candidate genes reproducing part of the phenotype in animal models [13, 14, 26, 56, 65] or suspected to change disease predisposition [66‐69] have been identified in 22q11.2. In this context, correlations made within a contiguous gene syndrome will always be subjected to phenotype exceptions.

Rauch et al [19] described a likely genotype-phenotype correlation, by comparing the phenotypes of non-overlapping deletions in 22q11.2 (Table 2). They concluded that deletions covering the whole TDR or the proximally nested 1.5 Mb segment result in a phenotype characterized by conotruncal CHDs and typical VCFS, mainly by effects of haploinsufficiency of TBX1, a gene previously involved in the expression of these features when mutated [26], whereas distally nested deletions including CRKL would present with a milder, atypical phenotype with uncommon CHDs, developmental delay and mental impairment. Likewise, distal deletions which are adjacent to, and do not include the TDR, would generally have associated non-specific CHDs and very uncommon features of 22q11.2 deletion syndrome such as choanal atresia. Within these deletions, the shortest proximal segment has been associated with mild learning difficulties [19]. Recently, additional characteristic features of this new condition such as prematurity, prenatal and postnatal growth retardation, developmental delay, mild skeletal abnormalities and characteristic facial dysmorphic features have been described [23] (Table 2).

The absence of a genotype-phenotype correlation highlights the incomplete penetrance associated with alterations in the 22q11.2 region. As an example, an apparently identical atypical deletion in two reported familial cases was found to present both inter- and intrafamilial variability [18, 19]. This atypical deletion which spanned the B-D segment, and which should have been mainly involved in learning disability and mental retardation, was found to be associated with a conotruncal CHD in a child of a deleted father with normal phenotype [18] (Table 2). Whereas, in the second case, the child developed a more neurobehavioural phenotype, with an undiagnosed father with schizophrenia [19]. Both patients had mild facial dysmorphism with overlapping features of DGS/VCFS.

In line with this, the only two reported distally nested C-D deletions similar to that found in Patient 1 are also phenotypically discordant [21, 70]. Both were located within this segment that should be associated with the neurobehavioural expression. The patient from D'Angelo et al [21] had mild facial features and a complex neurodevelopmental, behavioural and psychiatric spectrum, with a major depressive disorder in her transmitting mother, but the patient described by Kurahashi et al [70] had a typical conotruncal anomaly face syndrome with pulmonary atresia, tetralogy of Fallot, and facial dysmorphic features. Our patient had a non-specific CHD and mild facial features, barely suggestive of the 22q11.2 deletion syndrome (Table 2). All these findings, within such a small sample size, do not reveal strong evidences but suggest that genetic changes in 22q11.2 are incompletely penetrant [19] or that maybe genotype-phenotype correlations are invalid for this specific region [21].

The reason for all these exceptions in the distal TDR might be the presence of CRKL, a gene that has been involved in multisystemic affection of cardiac neural crest derivatives when null-mutated in mice [65]. Otherwise, other genes in this region seem to be responsible for a more particular phenotype: certain SNP haplotypes within genes PIK4CA [66, 67] and SNAP29 [68, 69] have been recently suggested to confer a higher risk of developing schizophrenia (Figure 4).

Recently, high resolution molecular diagnostic techniques have contributed to map the rearrangements in 22q11.2 more precisely. In fact, high resolution comparative genomic hybridization (HR-CGH), real-time quantitative PCR (qPCR) and high density MLPA (HD-MLPA) have shown that around 7% of the typical 3 Mb A-D deletions have atypical distal breakpoints covering HIC2, suggesting that this gene located within LCR22-D might be involved in the phenotypic variability among these patients [20, 71, 72]. Hic2 may be required for heart development in mouse [Lammerts van Beuren K and Scambler PJ, personal communication]. HIC2 might be also involved in the phenotypic variability of Patient 1 and other patients reported to carry the C-D deletion [21, 70], as well as in patients with other atypical deletions [15, 17, 19, 20, 58].

Screening for duplications at 22q11.2 has no standardized procedure, as these patients show clinical manifestations, only some of which are compatible with 22q11.2 deletion syndrome within a generally milder phenotype [37, 41, 43, 48]. Different duplication sizes have been reported [43, 48, 49], all of them showing a higher tendency to be inherited than deletions [43]. Phenotype correlations to the sizes and locations of these duplications are even harder to find than in deletions, as the whole clinical spectrum is still unknown and many of these individuals overlap normality [41, 43].

Patient 2 and his maternal relatives are carriers of an atypical duplication that maps distal within the TDR. Unlike common duplications [43, 63, 73], this novel rearrangement, the smallest one reported to date, has therefore unknown clinical implications. Polymorphisms and copy number variants can be defined as alterations of a small region known to contain no critical genes and that can be inherited from healthy individuals [41, 43]. However, as proposed for TBX1, other genes in 22q11.2 may have clinical significance when present in a trisomic dosage [43, 74, 75]. On the other hand, four other carriers of the family of Patient 2 show normal phenotype, and the patient shows cleft palate, a potentially multifactorial malformation which may be less associated with 22q11.2 duplications than thought [40]. The case that is most similar to this duplication was described by Fan et al [76] in a patient with mental retardation or developmental delay associated to other malformations, inherited from a phenotypically normal father. This 479 kb duplication also included PCQAP, another gene with a potential involvement in schizophrenia susceptibility [77, 78].

Novel and rare rearrangements in such a complex chromosomal region with highly uncertain phenotypic consequences, have uncertain implications for genetic counseling. High intrafamilial variability and reduced penetrance are phenotypic effects that hamper the distinction between pathologic change and polymorphism [79]. In this context, the possibility that the phenotypes of our patients might not be caused by the observed rearrangements should be reminded. Also, small deletions and duplications occurring from generation to generation in this rearranged segment cannot be excluded [80].

Despite having occurred in the same region, our two rearrangements, as far as they have been molecularly characterized, are significantly different (Figure 4). The deletion is more than twice the size of the duplication and includes HIC2 and the whole LCRs C and D, which are nearly intact in the duplication. This variation in the breakpoints location highlights potential differences in the molecular mechanisms mediating these rearrangements. In fact, the deletion is too large to be a C-D rearrangement and is possibly not mediated by non-allelic homologous recombination between LCRs.

Even for identical rearrangements, genetic counseling and clinical prognosis is still complex. Genotype-phenotype correlations made so far in 22q11.2 are compatible with a clinical outcome modified by environmental/stochastic events [15, 60, 61] and eclipsed by regulatory factors [15, 59]. At this moment, the molecular diagnostic techniques we have used are the best screening procedure for patients with this whole spectrum of overlapping clinical features, suspected of having any kind of rearrangement in 22q11.2 resulting in a copy number imbalance [71, 81, 82].

In summary, we report on the third atypical deletion and the first atypical duplication in the vicinity of low-copy repeats C and D in distal 22q11.2 deletion region. The breakpoints location and sizes make it possible that both rearrangements may be mediated by different mechanisms or sequences. A complex network of modifying factors may influence the phenotypic outcome of these chromosomal aberrations. A systematic literature review and genotyping of phenotype-associated polymorphisms can help anticipating the clinical evolution of patients and understanding the prognosis and predisposition to this disease.