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Cytogenetic studies of the parents of a girl with the DiGeorge (or velocardiofacial) syndrome, who carried a deletion at 22q11.2, revealed an unexpected rearrangement of both 22q11.2 regions in the unaffected father. He carried a 22q11.2 deletion on one copy of chromosome 22 and a reciprocal 22q11.2 duplication on the other copy of chromosome 22. Genetic compensation, which is consistent with the normal phenotype of the father, was shown through quantitative-expression analyses of genes located within the genetic region associated with the DiGeorge syndrome. This finding has implications for genetic counseling and represents a case of genetic compensation in a human genomic disorder.
Methods
Case Report
The diagnosis of 22q11DS was suspected at birth in a female child of healthy parents because of seizures due to severe and persistent hypocalcemia, lack of the thymus, and facial dysmorphism. No cardiac malformation was apparent. We obtained written informed consent from all family members studied.
Molecular Cytogenetic Analysis
Fluorescence in situ hybridization (FISH) assays were performed on cultured peripheral-blood lymphocytes obtained from the proband, her parents, and her grandfather, with the use of the following probes: N25 from Cytocell covering DGCR14 (the DiGeorge syndrome critical region gene 14) and partially covering DGCR2 (the DiGeorge syndrome critical region gene 2) and CLCTL1 (Homo sapiens clathrin, heavy chain–like 1); TUPLE1 from Vysis, a probe partially covering HIRA (the HIR histone cell-cycle regulation defective homolog A [Saccharomyces cerevisiae]) from Vysis, and T-box 1 gene (TBX1) from Kreatech. The probes covered the 22q11.2 region and were used according to the manufacturer's instructions. FISH analyses of 900 interphase nuclei from buccal mucosa of the proband's father only were performed with the use of the TBX1 probe, as previously described.14
Genomic DNA Analysis
Genomic DNA was extracted from peripheral-blood specimens with the use of the FlexiGene DNA kit (Qiagen). A quantitative multiplex polymerase-chain-reaction (PCR) assay of short fluorescent fragments (QMPSF), adapted from Jacquet et al.,15 was used to assess the numbers of copies of seven genes in total. Five are located within the 3-Mb genetic region associated with the DiGeorge syndrome, called the DiGeorge syndrome critical region — GSCL (the goosecoid homeobox 2 gene), TBX1, ARVCF (the armadillo repeat gene deletes in velocardiofacial syndrome), ZNF74 (the zinc-finger protein 74 gene), and SNAP29 (the synaptosomal-associated protein, 29-kD gene) — and two genes are located on either side of the 3-Mb region — USP18 (the ubiquitin-specific peptidase 18 gene) and UBE2L3 (the gene for ubiquitin-conjugating enzyme E2L 3). The microsatellite markers D22S420, D22S941, D22S264, D22S303, D22S257, and D22S533 were studied with the use of dye-labeled primers, capillary electrophoresis on a genetic analyzer (ABI3100, Applied Biosystems), and GeneScan software, version 3.7 (PE Applied Biosystems).
Gene-Expression Analysis
RNA was extracted from peripheral-blood lymphocytes with the use of Trizol reagent (Invitrogen) and was subjected to reverse transcription, with the use of avian myeloblastosis virus reverse transcriptase (AMV-RT, Finzyme). Expression of DGCR8 (the DiGeorge syndrome critical region gene 8) and DGCR6L (the DiGeorge syndrome critical region gene 6–like gene), as well as expression of USP18, located at the proximal boundary outside the DiGeorge syndrome critical region, was assessed by means of a real-time quantitative PCR assay. The procedure used the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the TATA box binding protein (TBP) as reference genes, QuantiTect Primer Assays (Qiagen), and a LightCycler 480 System (Roche).16
Results
FISH with the use of probes covering the 22q11.2 region confirmed the 22q11.2 microdeletion in the proband (Figure 1A and 1D). We carried out the same analyses in the parents, to determine whether the deletion was sporadic or inherited. Normal staining of TBX1 was observed on metaphasic and interphasic chromosomal spreads of peripheral-blood cells from the mother (Figure 1B and 1E). Unexpectedly, TBX1 staining in cells from the father revealed the absence of signal on one chromosome 22 and an enlarged signal on its homologue in all observed metaphases (Figure 1C). In 86% of interphasic nuclei from the father's cells, two distinct TBX1 probes were clearly observed, always close together (Figure 1F), in contrast with their random location in the mother's interphasic nuclei (Figure 1E). Similar results were obtained with the N25 and TUPLE1 probes (data not shown).
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We used a QMPSF assay to determine the numbers of copies of various genes in the region of interest (Figure 2A). The assay confirmed, in the index case, the classic 3-Mb microdeletion between low-copy repeat A and low-copy repeat D on chromosome 22 and, in the father, the presence of two copies of each of the genes tested within the 22q11.2 region (Figure 2B).
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To determine the origin of the rearrangements observed in the father, we analyzed the segregation of six microsatellite markers on chromosome 22 within the family (Figure 3). The detection of alleles of different parental origin for the pericentromeric marker D22S420 in the proband's father (Figure 3) allowed us to rule out paternal uniparental disomy of chromosome 22. Maternal uniparental disomy of chromosome 22 in the proband's father can also be ruled out, on the basis of the microsatellite alleles observed for markers D22S941 and D22S264 in the proband's father, paternal aunt, and paternal grandfather.
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The data for the father of a child with 22q11DS are consistent with a 22q11.2 deletion on one chromosome 22 and a 22q11.2 duplication on the other. As expected, the expression of genes located within the genetic region associated with the DiGeorge syndrome was reduced by half in the affected proband. Conversely, expression levels of these genes in the father's lymphocytes were similar to those observed in the mother's lymphocytes, suggesting that the phenotypic effects of the 22q11.2 deletion in the father were balanced by the 22q11.2 duplication. This compensatory effect has already been shown in a mouse model of the human 22q11.2-deletion region: congenital heart defects found in mice that are heterozygous for a deletion encompassing the murine region homologous to the human 22q11.2 DiGeorge syndrome critical region are corrected by cross-breeding with mice that are heterozygous for a Tbx1 duplication.18 Beckmann et al.19 recently hypothesized that copy-number variation might explain reduced penetrance of some disease-causing mutations. These authors suggested that the phenotype of a dominant loss-of-function mutation could be rescued by the gain of a copy-number variation, resulting in increased gene expression. Our study validates this prediction and suggests that, considering the density of copy-number variations in the human genome, the presence of compensatory copy-number variations may explain why some loss-of-function mutations have low penetrance.
The simultaneous duplication and deletion in the proband's father can be explained by nonallelic homologous recombination occurring either at a prezygotic or postzygotic stage. One hypothesized mechanism is nonallelic homologous recombination between low-copy repeat A and low-copy repeat D on chromosome 22, occurring during the first meiosis in one grandparental germ line, followed by meiotic nondisjunction and uniparental heterodisomy after trisomy rescue in chromosome 22 (Fig. 1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). However, this hypothesis is not supported by the results of microsatellite analyses within the family. Fertilization between a grandparental gamete carrying a de novo deletion and another carrying a de novo duplication is possible but very unlikely, given that the estimated rate of de novo, locus-specific mutation is approximately 1.25x10–4 for the 22q11.2 deletion.20 The father's genotype could also have resulted from a postzygotic, nonallelic homologous recombination (Fig. 2 in the Supplementary Appendix) occurring soon after conception and potentially resulting in a perfectly balanced 22q11.2 deletion–duplication genotype in the embryo and in chromosomal placental mosaicism (Fig. 3 in the Supplementary Appendix). This hypothesis is compelling, since it requires only one event.
Our study emphasizes the importance of FISH analyses, especially of interphasic nuclei, which quickly provided the correct cytogenetic diagnosis of the father's chromosomal rearrangement. Comparative genomic hybridization by means of microarray analysis would have failed to detect this balanced anomaly.
Genetic compensation in the context of genomic disorders has tremendous clinical consequences for genetic counseling, given the 100% risk of unbalanced outcomes. Our case report highlights the importance of performing genetic investigations in both parents of any child presenting with a genomic disorder, even if the parents have normal phenotypes.
No potential conflict of interest relevant to this article was reported.
We thank the proband and her family for their participation; Prof. J.R. Lupski, Prof. J.S. Beckmann, and Dr. W. Gu for scientific exchanges and expert advice; and J. Auguste for technical assistance and J. Flori for computer assistance.
Source Information
From the Departments of Cytogenetics (N.C.-C., F.G.-L., M.A., C.G., E.S., E.F.), Neurology (G.R.), Medical Genetics (B.D.), Biochemistry and Molecular Biology (E.G.), and Pediatrics (P.K.), Strasbourg University Hospital, Strasbourg; the Department of Genetics, Rouen University Hospital and University of Rouen, Rouen (P.S.-V., S.F., T.F.); and the Department of Pediatrics, Saverne Hospital, Saverne (A.S.) — all in France.
Address reprint requests to Dr. Flori at Service de Cytogénétique, Hôpital de Hautepierre, Ave. Molière, 67098 Strasbourg, France, or at elisabeth.flori{at}chru-strasbourg.fr.
References
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