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Investigation of meiotic rearrangements in DGS/VCFS patients with a microdeletion 22q11.2
  1. * Institut für Humangenetik der Universität Bonn, Wilhelmstrasse 31, 53111 Bonn, Germany
  2. Department of Paediatric Cardiology, Johanniter-Kinderklinik, St Augustin, Germany
  3. Medizinische Klinik IV des Universitätsklinikums der RWTH, Aachen, Germany
  1. Dr Trost, DTrost{at}

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Editor—Microdeletions in 22q11.2 are associated in 80-90% of cases with DiGeorge syndrome (DGS, MIM 188400) or velocardiofacial syndrome (VCFS, MIM 192430) and occur with an estimated frequency of 1/4000 live births.1 Most deletions are the result of a de novo event, although probably 6-28% of them are familial.2 The phenotype of the patients is mainly characterised by conotruncal heart defect, cleft palate, immune deficiency, neonatal hypocalcaemia, and facial dysmorphism. The number of clinical symptoms varies substantially and their reduced expression can lead to a mild phenotype.3 There does not seem to be a correlation between the presence or the size of a microdeletion and the clinical manifestation of the syndrome. Molecular analyses have shown that most patients have a deletion of about 1.5 or 3 Mb.4 5 The length of the delineated minimal critical region for a DGS/VCFS phenotype, however, is only 480 kb.6 Reports of patients with a DGS/VCFS-like phenotype having a deletion in 10p7 led to the definition of a second critical region, DGSII. However, the incidence of 10p deletions is low in comparison to the rate of microdeletions in 22q.8 9 The high rate of sporadic microdeletions in 22q11.2 provides evidence for frequent meiotic rearrangements as a molecular basis for the development of this structural aberration.

In order to ascertain such rearrangements in patients with a 22q11.2 deletion, we performed haplotype analyses on five patients and their unaffected relatives using 11 polymorphic STRP markers from the DGS/VCFS critical region in 22q11.2 (fig 1). Furthermore, the haplotype analyses enabled us to determine the extent of the deletions in deletion carriers and the parental origin of the abnormal chromosome.

Figure 1

Pedigrees of the investigated families. The index patients are indicated by arrows. Bars and numbers symbolise the individual chromosomes and the haplotypes of the tested STRP markers. Marker names are given on the left. In the case of a meiotic crossover, the abnormal chromosome 22 shows STRP markers from both chromosomes of the parent of origin. Unclear recombination breakpoints are indicated by white bars. Black symbol = microdeletion 22q11.2, black spot indicates parent of origin.

Microdeletion analysis was performed by fluorescence in situ hybridisation (FISH) on metaphase chromosomes prepared from fresh peripheral blood samples using DNA probes D22S75 (Oncor, Illkirch) or TUPLE1 (Vysis, Downers Grove, IL) from the DGS/VCFS critical region on 22q11.2.

In order to haplotype patients and their family members the parents and, if available, the grandparents of origin were analysed with 11 STRP markers using standard methods. Primer information was obtained from the Genome Data Base (GDB). If the grandparents of origin were not available, haplotyping was performed with the results obtained from healthy sibs of the patient and the parent of origin. A total of 30 family members were included in the study. The family pedigrees are shown in fig 1.

The STRP analyses allowed us to determine the deletion sizes in 22q11.2 of the investigated probands (fig 1). Patients F1-7, F2-8, F4-3, F4-8, and F5-3 had deletions of the 3 Mb type and patient F3-4 has a 1.5 Mb deletion. The parental origin of the aberrant chromosome was determined to be four times paternal and once maternal.

Haplotype analyses were performed to ascertain the developmental mechanism of the microdeletion. In four families (F1, F2, F3, F5), a parental unequal crossover was proven by the exchange of parental marker alleles flanking the deleted region (fig 1 and 2). In family F4, the underlying mechanism could be either an unequal crossover or an intrachromosomal rearrangement, as the microsatellite markers proximal to the monosomic area did not allow us to distinguish between these mechanisms.

Figure 2

STRP analyses of markers from 22q11.2 in family F2 show that the deletion in patient F2-8 (black symbol) has developed from an unequal crossover event. The grandparental marker alleles (a grandpaternal, b grandmaternal) are given below. D22S427 defines the proximal breakpoint, D22S308 the distal breakpoint of the microdeletion, and D22S941 is located within the deleted area. In patient F2-8 the flanking alleles are derived grandmaternally proximal (b) and grandpaternally distal (a) to the deletion which confirms a meiotic crossover in the parent of origin (*F2-3).

Additional crossover events are present in family F5 where proband F5-4 shows a rearrangement between D22S264 and D22S311 (fig 1) and in family F2 in probands F2-6 and F2-7 between D22S303 and D22S257 (fig 1).

In this study we were able to analyse in detail the 22q11.2 deletions present in five patients and one father from five unrelated families. Although there are differences in deletion size, it was not possible to delineate any significant correlation with the phenotypic manifestations. This is especially conspicuous in family F4 in which the father and son share an identical deletion. In this case the father displays only slight dysmorphic facial features and a cleft palate, while his son is more severely affected with ventricular septal defect (VSD), cerebral malformations, T cell defect, and marked developmental delay. This variation may be the result of the different maternal haplotype in each of them (fig 1, patients F4-3 and F4-6).

All proximal deletion breakpoints are flanked by the STRP marker D22S427. The distal breakpoint, however, is variable, being flanked in four cases by D22S306 and in one case each by D22S311 or D22S308. These findings are in agreement with previously defined deletion breakpoints where most patients showed a deleted interval of approximately 3 Mb flanked by D22S247 and D22S306.6 The genetic distance over this region is approximately 6 cM according to the Généthon and GDB linkage maps.

In our study the deletions were of maternal (n=1) as well as of paternal (n=4) origin which does not confirm the bias towards maternally derived deletions found in other studies.10 The haplotype analyses of the investigated families show that four of five deletions are the result of an unequal meiotic crossover event. In these cases the markers flanking the deletion breakpoints are derived from different parental chromosomes (fig 1 and 2). In family F4, it is not clear from the present data if the underlying event involved homologous pairing of chromosomes or exchanges between sister chromatids (fig 1). In our sample of five families, no statistical significance can be calculated, but the data confirm the findings from a previous investigation that the DiGeorge critical region (DGCR), though located near the centromere of chromosome 22, is subject to numerous meiotic recombinations, many of which lead to the formation of a microdeletion.11 Patient F5-4 displays a crossover near D22S264 and D22S311 (fig 1). This is an interesting finding because these markers are located at common distal deletion breakpoints6 and underlines the presence of crossover mediating elements at the breakpoints of 22q11.2 deletions.5 12-14

The mechanisms of microdeletion formation have been investigated in other syndromes as well. The critical region for Prader-Willi/Angelman syndrome (PWS/AS) (15q11-q13) is subject to above average rates of recombination and sex specific hotspots have been described.15 The deletions were caused by both intra- and interchromosomal recombination in the PWS/AS families investigated.16 17 The results obtained for deletions in 7q11.23 associated with Williams-Beuren syndrome (WBS) suggest that the majority of microdeletions in this region are caused by unequal crossover events.11 18 19 In comparison, in most informative DGS/VCFS families, the microdeletion 22q11.2 was associated with a crossover but an intrachromosomal rearrangement cannot be excluded in the remaining cases.5 11


We thank all families participating in this investigation. The study was supported by “Richard-Winter-Stiftung”, Stuttgart, Germany.