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A Novel 22q11.2 Microdeletion in DiGeorge Syndrome

1999; Elsevier BV; Volume: 64; Issue: 2 Linguagem: Inglês

10.1086/302235

1537-6605

Anita Rauch, Rudolf A. Pfeiffer, Georg Leipold, H. Singer, Monika Tigges, Michael Hofbeck,

Tracheal and airway disorders

To the Editor: DiGeorge syndrome (DGS; MIM 188400) is a multiple-malformation syndrome characterized by aplasia or hypoplasia of the thymus; immunodeficiency, aplasia, or hypoplasia of the parathyroid glands; conotruncal cardiac defects; and typical facial anomalies(DiGeorge DiGeorge, 1965DiGeorge AM Discussion on Cooper MD, Peterson RDA, Good RA (1965): a new concept of the cellular basis of immunology.J Pediatr. 1965; 67: 907-908Abstract Full Text PDF Google Scholar; Conley et al. Conley et al., 1979Conley ME Beckwith JB Mancer JFK Tenckhoff I The spectrum of DiGeorge syndrome and importance of neural crest as a possible pathogenetic factor.J Pediatr. 1979; 94: 883-890Abstract Full Text PDF PubMed Scopus (323) Google Scholar). Despite causal heterogeneity (Lammer and Opitz Lammer and Opitz, 1986Lammer EJ Opitz JM The DiGeorge anomaly as a developmental field defect.Am J Med Genet Suppl. 1986; 2: 113-127Crossref PubMed Google Scholar), ∼90% of patients with DGS have hemizygosity of an ∼1.5–3-Mb region within 22q11.2 (Driscoll et al. Driscoll et al., 1990Driscoll DA Budarf ML McDermid H Emanuel BS Molecular analysis of DiGeorge syndrome: 22q11 interstitial deletions.Am J Hum Genet Suppl. 1990; 47: A215Google Scholar; Scambler et al. Scambler et al., 1991Scambler PJ Carey AH Wyse RKH Roach S Dumanski JP Nordenskjold M Williamson R Microdeletions within 22q11 associated with sporadic and familial DiGeorge syndromes.Genomics. 1991; 10: 201-206Crossref PubMed Scopus (194) Google Scholar; Driscoll et al. Driscoll et al., 1992aDriscoll DA Budarf ML Emanuel BS A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11.Am J Hum Genet. 1992a; 50: 924-933PubMed Google Scholar). Because of phenotypic overlap, the same deletion was demonstrated in the majority of patients with velo-cardio-facial syndrome (VCFS; MIM 192430) (Driscoll et al. Driscoll et al., 1992bDriscoll DA Spinner NB Budarf ML McDonald-McGinn DM Zackai EH Goldberg RB Shprintzen RJ et al.Deletions and microdeletions of 22q11.2 in velo-cardio-facial syndrome.Am J Med Genet. 1992b; 44: 261-268Crossref PubMed Scopus (326) Google Scholar; Carlson et al. Carlson et al., 1997bCarlson C Sirotkin H Pandita R Goldberg R McKie J Wadey R Patanjali SR et al.Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.Am J Hum Genet. 1997b; 61: 620-629Abstract Full Text PDF PubMed Scopus (290) Google Scholar), which was initially characterized by hypernasal speech caused by cleft palate, cardiac anomalies, learning disabilities, and typical facial appearance (Shprintzen et al. Shprintzen et al., 1978Shprintzen RJ Goldberg RB Lewin ML Sidoti EJ Berkman MD Argamaso RV Young D A new syndrome involving cleft palate, cardiac anomalies, typical facies, and learning disabilities: velo-cardio-facial syndrome.Cleft Palate J. 1978; 15: 56-62PubMed Google Scholar, Shprintzen et al., 1981Shprintzen RJ Goldberg RB Young D Wolford L The velo-cardio-facial syndrome: a clinical and genetic analysis.Pediatrics. 1981; 67: 167-172PubMed Google Scholar). In addition, 22q11.2 deletions were observed in cases fitting within the spectrum of Cayler syndrome (MIM 125520) (Giannotti et al. Giannotti et al., 1994Giannotti A Digilio MC Marino B Mingarelli R Dallapiccola B Cayler cardiofacial syndrome and del 22q11: part of the CATCH22 phenotype.Am J Med Genet. 1994; 53: 303-304Crossref PubMed Scopus (93) Google Scholar), Takao conotruncal anomaly face syndrome (contained in MIM 188400) (Burn et al. Burn et al., 1993Burn J Takao A Wilson DI Cross I Momma K Wadey R Scambler PJ et al.Conotruncal anomaly face syndrome is associated with the deletion within chromosome 22q11.J Med Genet. 1993; 30: 822-824Crossref PubMed Scopus (234) Google Scholar), Noonan syndrome (MIM 163915) (Wilson et al. Wilson et al., 1993Wilson DI Britton SB McKeown C Kelly D Cross IE Strobel S Scambler PJ Noonan's and DiGeorge syndrome with monosomy 22q11.Arch Dis Child. 1993; 68: 187-189Crossref PubMed Scopus (23) Google Scholar), Kousseff syndrome (MIM 245210) (Nickel et al. Nickel et al., 1994Nickel RE Pillers D-AM Merkens M Magenis RE Driscoll DA Emanuel BS Zonana J Velo-cardio-facial syndrome and DiGeorge sequence with meningomyelocele and deletions of the 22q11 region.Am J Med Genet. 1994; 52: 445-449Crossref PubMed Scopus (50) Google Scholar), and Opitz GBBB syndrome (MIM 145410) (McDonald-McGinn et al. McDonald-McGinn et al., 1995McDonald-McGinn DM Driscoll DA Bason L Christensen K Lynch D Sullivan K Canning D et al.Autosomal dominant "Opitz" GBBB syndrome due to a 22q11.2 deletion.Am J Med Genet. 1995; 59: 103-113Crossref PubMed Scopus (123) Google Scholar). Meanwhile, it became evident that deletion 22q11.2 is associated with a phenotypic spectrum that may present as one of the aforementioned syndromes or any condition in between them that has considerable inter- and intrafamilial variability (De Silva et al. De Silva et al., 1995De Silva D Duffty P Booth P Auchterlonie I Morrison N Dean JCS Family studies in chromosome 22q11 deletion: further demonstration of phenotypic heterogeneity.Clin Dysmorphol. 1995; 4: 294-303Crossref PubMed Scopus (22) Google Scholar; Leana-Cox et al. Leana-Cox et al., 1996Leana-Cox J Pangkanon S Eanet KR Curtin MS Wulfsberg EA Familial DiGeorge/velocardiofacial syndrome with deletions of chromosome area 22q11.2: report of five families with a review of the literature.Am J Med Genet. 1996; 65: 309-316Crossref PubMed Scopus (73) Google Scholar; Devriendt et al. Devriendt et al., 1997Devriendt K Van Hoestenberghe R Van Hole C Devlieger H Gewillig M Moerman P Van den Berghe H et al.Submicroscopic deletion in chromosome 22q11 in trizygous triplet siblings and their father.Clin Genet. 1997; 51: 246-249Crossref PubMed Scopus (11) Google Scholar; Ryan et al. Ryan et al., 1997Ryan AK Goodship JA Wilson DI Philip N Levy A Seidel H Schuffenhauer S et al.Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study.J Med Genet. 1997; 34: 798-804Crossref PubMed Scopus (947) Google Scholar). Therefore, 22q11.2 microdeletion is one of the most common genetic defects, with an estimated incidence of >1 in 5,000 (Wilson et al. Wilson et al., 1994Wilson DI Cross IE Wren C Scambler PJ Burn J Goodship J Minimum prevalence of chromosome 22q11 deletions.Am J Hum Genet Suppl. 1994; 55: A169Google Scholar; Tezenas Du Montcel et al. Tezenas Du Montcel et al., 1996Tezenas Du Montcel S Mendizabal H Ayme S Levy A Philip N Prevalence of 22q11 microdeletion.J Med Genet. 1996; 33: 719Crossref PubMed Google Scholar). According to Carlson et al. (Carlson et al., 1997bCarlson C Sirotkin H Pandita R Goldberg R McKie J Wadey R Patanjali SR et al.Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.Am J Hum Genet. 1997b; 61: 620-629Abstract Full Text PDF PubMed Scopus (290) Google Scholar), 90% of patients with deletions have a common 3-Mb deletion, 8.5% have a proximal 1.5-Mb deletion, and 3% have unique nested proximal deletions, which may define a proximal shortest region of overlap within the commonly deleted 3-Mb region. Because of two patients with small deletions in the distal part of the 3-Mb deletion, a distal shortest region of overlap within the deleted region may also be defined (Kurahashi et al. Kurahashi et al., 1997Kurahashi H Tsuda E Kohamma R Nakayama T Masuno M Imaizumi K Kamiya T et al.Another critical region for deletion of 22q11: a study of 100 patients.Am J Med Genet. 1997; 72: 180-185Crossref PubMed Scopus (60) Google Scholar; O'Donnell et al. O'Donnell et al., 1997O'Donnell H McKeown C Gould C Morrow B Scambler P Detection of an atypical 22q11 deletion that has no overlap with the DiGeorge syndrome critical region.Am J Hum Genet. 1997; 60: 1544-1548Abstract Full Text PDF PubMed Google Scholar). However, there is no obvious correlation between the site or size of the deletion and the severity of the clinical manifestations, and position effects have been taken into consideration (Carlson et al. Carlson et al., 1997bCarlson C Sirotkin H Pandita R Goldberg R McKie J Wadey R Patanjali SR et al.Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.Am J Hum Genet. 1997b; 61: 620-629Abstract Full Text PDF PubMed Scopus (290) Google Scholar; Kurahashi et al. Kurahashi et al., 1997Kurahashi H Tsuda E Kohamma R Nakayama T Masuno M Imaizumi K Kamiya T et al.Another critical region for deletion of 22q11: a study of 100 patients.Am J Med Genet. 1997; 72: 180-185Crossref PubMed Scopus (60) Google Scholar; O'Donnell et al. O'Donnell et al., 1997O'Donnell H McKeown C Gould C Morrow B Scambler P Detection of an atypical 22q11 deletion that has no overlap with the DiGeorge syndrome critical region.Am J Hum Genet. 1997; 60: 1544-1548Abstract Full Text PDF PubMed Google Scholar). We describe here a novel 22q11.2 microdeletion in a family with mild to severe phenotype. This deletion is adjacent to but does not overlap with the known deletions. Nevertheless, it shows similar clinical characteristics and may therefore give a clue to the mechanisms and genes involved in phenotype determination in 22q11.2 deletions. Patient III:3 was primarily investigated in the context of a study of incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch (Rauch et al. Rauch et al., 1998bRauch A Hofbeck M Leipold G Klinge J Trautmann U Kirsch M Singer H et al.Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.Am J Med Genet. 1998b; 78: 322-331Crossref PubMed Scopus (63) Google Scholar). Within that study, she was the only patient with symptoms of the DGS/VCFS spectrum who did not have the 22q11.2 deletion and, therefore, prompted further analysis. Phenotype assessment of the patient, her parents, and her sibs was performed before molecular studies and included dysmorphologic analysis of lymphocyte subpopulations by flow cytometry on an Orthoscan, by means of fluorochrome-labeled antibodies against CD3, CD4, CD8, and CD19; and surface immunoglobulin (according to Becton-Dickinson). Diphtheria toxoid and tetanus toxoid were measured after vaccination, by means of a commercially available enzyme-linked immunosorbent assay (ELISA) (ABICAP; Abion). Parathyroid hormone levels were determined by chemoluminescence-ELISA (Nichols) of the patient's sera. Cardiac status was established by echocardiography and angiography in the patient and by echocardiography only in the patient's parents and sibs. Flexible transnasal pharyngoscopy was performed in the patient's sister and mother, to exclude velopharyngeal insufficiency. In the patient, conventional karyotyping of GTG-banded chromosomes from peripheral T lymphocytes and fibroblasts was performed at an ∼550-band level, according to Mitelman Mitelman, 1995Mitelman F ISCN: an international system for human cytogenetic nomenclature. S Karger, Basel1995Google Scholar, pp 14–21). The patient, both of her sibs, and her mother were investigated by FISH with the DNA probes D22S75 (ONCOR), cHKAD26 (Kurahashi et al. Kurahashi et al., 1994Kurahashi H Akagi K Karakawa K Nakamura T Dumanski JP Sano T Okada S et al.Isolation and mapping of cosmid markers on human chromosome 22, including one within the submicroscopically deleted region of DiGeorge syndrome.Hum Genet. 1994; 93: 248-254Crossref PubMed Scopus (25) Google Scholar, Kurahashi et al., 1997Kurahashi H Tsuda E Kohamma R Nakayama T Masuno M Imaizumi K Kamiya T et al.Another critical region for deletion of 22q11: a study of 100 patients.Am J Med Genet. 1997; 72: 180-185Crossref PubMed Scopus (60) Google Scholar) (kindly provided by the Japanese Cancer Research Resources Bank) and bacterial artificial chromosome (BAC) 438P22 (see below) on metaphase chromosomes from peripheral T lymphocytes. In the patient, FISH was performed with the additional probes Tuple1 (VYSIS), M-bcr/abl, m-bcr/abl (ONCOR), and BAC 458J22. The DNA probes D22S75 and cHKAD26 were also analyzed in metaphases from fibroblasts of the patient. The commercially produced probes were used according to the manufacturer's instructions, or two-color FISH was performed with the critical probe biotin-labeled and with a digoxigenin-labeled centromeric 14/22 probe (ONCOR), as described elsewhere (Rauch et al. Rauch et al., 1996Rauch A Trautmann U Pfeiffer RA Clinical and molecular cytogenetic observations in three cases of "trisomy 12p syndrome".Am J Med Genet. 1996; 63: 243-249Crossref PubMed Scopus (55) Google Scholar). The Research Genetics human BAC DNA pools, release IV, were screened with the polymorphic marker D22S425 (see below), according to manufacturer's instructions. Positive BACs were tested and amplified according to Research Genetics guidelines. Two–color fiber-FISH was performed with BAC 438P22 and 458J22, on fixed cultured T lymphocytes from a healthy control, as described elsewhere (Fidlerova et al. Fidlerova et al., 1994Fidlerova H Senger G Kost M Sanseau P Sheer D Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization.Cytogenet Cell Genet. 1994; 65: 203-205Crossref PubMed Google Scholar). The patient and her parents had been tested before, for the following 10 short–tandem-repeat polymorphism (STRP) markers from the 22q11.2 region: D22S264 (Marineau et al. Marineau et al., 1992Marineau C Aubry M Julien J-P Rouleau GA Dinucleotide repeat polymorphism at the D22S264 locus.Nucleic Acids Res. 1992; 20: 1430Google Scholar); D22S311 and D22S306 (Porter et al. Porter et al., 1993Porter JC Ram KT Puck JM Twelve new polymorphic microsatellites on human chromosome 22.Genomics. 1993; 15: 57-61Crossref PubMed Scopus (13) Google Scholar); D22S427 (Gyapay et al. Gyapay et al., 1994Gyapay G Morissette J Vignal A Dib C Fizames C Millasseau P Marc S et al.The 1993-94 Généthon human genetic linkage map.Nat Genet. 1994; 7: 246-339Crossref PubMed Scopus (1972) Google Scholar); D22S941 and D22S944 (Morrow et al. Morrow et al., 1995Morrow B Goldberg R Carlson C Das Gupta R Sirotkin H Collins J Dunham I et al.Molecular definition of the 22q11 deletions in velo-cardio-facial syndrome.Am J Hum Genet. 1995; 56: 1391-1403PubMed Google Scholar); and D22S1638, D22S1648, D22S1623, and D22S308 (Carlson et al. Carlson et al., 1997aCarlson C Papolos D Pandita RK Faedda GL Veit S Goldberg R Shprintzen R et al.Molecular analysis of velo-cardio-facial syndrome patients with psychiatric disorders.Am J Hum Genet. 1997a; 60: 851-859PubMed Google Scholar,Genome Database), as described elsewhere (Rauch et al. Rauch et al., 1998bRauch A Hofbeck M Leipold G Klinge J Trautmann U Kirsch M Singer H et al.Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.Am J Med Genet. 1998b; 78: 322-331Crossref PubMed Scopus (63) Google Scholar). Subsequently, the patient, her sibs, her parents, and her maternal grandparents were tested for STRPs at the loci D22S311 (Genome Database 190609), D22S1709 (Genome Database 5865052), D22S306 (Genome Database 190620), D22S308 (Genome Database 190623), D22S425 (Genome Database 199610), D22S303 (Genome Database 190616), D22S257 (Genome Database 180549), D22S301 (Genome Database 190613), D22S156 (Genome Database 177327), TOP1P2 (Genome Database 159908), D22S1144 (Genome Database 606049; SangerCentre bK929C8), and D22S1167 (Genome Database 610902; Sanger Centre bK373H7), by PCR amplification of DNA extracted from fresh peripheral blood and separation on 6% denaturing polyacrylamide gels (41 cm) in a Li-cor (MWG-Biotech) sequencer, as described elsewhere (Rauch et al. Rauch et al., 1998bRauch A Hofbeck M Leipold G Klinge J Trautmann U Kirsch M Singer H et al.Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.Am J Med Genet. 1998b; 78: 322-331Crossref PubMed Scopus (63) Google Scholar). Additional mapping information about the STRP markers was obtained by both a search of the BLAST database by means of the PCR primer sequences and data produced by the Chromosome 22 Mapping Group at the Sanger Centre, which were obtained from the World Wide Web. In addition to interrupted aortic arch type B, patient III:3 had truncus arteriosus communis type A4, T-cell deficiency, Pseudomonas aeruginosa sepsis, hypoplasia of halluces and toenails, choanal stenosis, retrognathia, and ear anomalies (fig. 1a–d). After repair of her congenital heart defect, the patient died neonatally, from heart failure and sepsis. Dysmorphologic analysis revealed subtle anomalies in her sister and mother, whereas her brother and father appeared normal. Minor anomalies in the mother included external strabismus, retrognathia, posteriorly angulated ears, broad neck with low posterior hairline, short 5th fingers (Dubois sign), and a high-arched palate with a minimal nick in the uvula (fig. 1e and f). Her occipitofrontal circumference (OFC) was 52 cm (<3d centile), and her height was 159 cm (10th centile). She had recurrent bronchitis and otitis media, but immunologic investigations revealed normal results. Despite some learning problems, she attended regular school. Her voice was normal. The 12-year-old sister (III:1) also showed mild retrognathia; thin vermillion border of the upper lip; low-set, posteriorly angulated ears with overfolded helices; high-arched palate with a minimal nick in the uvula; and mild muscular hypotonia (fig. 1g and h). Her OFC was 52.3 cm (25th centile), and her height was 143.8 cm (10th centile). She had a history of recurrent bronchitis, but immunologic investigations revealed normal results. She attends a special school because of minor learning difficulties. Her voice is normal. Echocardiographic, immunologic, endocrine, and pharyngoscopic studies in the parents and sibs of the patient did not show any abnormalities. There were neither attention-deficit/hyperactivity disorders nor behavioral or psychiatric problems in any family members. Karyotyping and FISH with the probes D22S75, Tuple1, and cHKAD26, of chromosomes from T lymphocytes and fibroblasts of the patient, did not reveal any chromosomal aberration or microdeletion in the commonly deleted 22q11.2 region. FISH with the probes D22S75 and cHKAD26, in the patient's mother and sibs, also showed normal signals on both chromosomes 22. STRP analyses of seven loci within (D22S1638, D22S941, D22S1648, D22S944, D22S1623, D22S264, and D22S311) and two loci flanking (D22S427 and D22S306) the 22q11.2 deletion region in the patient and her parents demonstrated heterozygosity of five markers in the patient. Four markers showed only one allele, but the parental allele constellation was uninformative. At two of the uninformative markers, the patient's mother was heterozygous. At one further distal marker, D22S308, the patient had not inherited the maternal allele. Subsequent STRP analyses with additional distal markers, in the patient and her family, demonstrated a deletion of D22S308, D22S425, D22S303, and D22S257 in the patient, her mother (II:1), and her older sister (III:1), whereas markers D22S301, D22S156, TOP1P2, D22S1144, and D22S1167 were informative for heterozygosity (fig. 2). Two BAC addresses—438P22 and 458J22—were identified by library screen with D22S425 and were confirmed by PCR from single clones. FISH with BAC 438P22 showed only a signal on one chromosome 22 in 50 metaphases from the patient (fig. 3), her sister, and her mother, whereas in her brother, father, and maternal grandparents signals on both chromosomes 22 were seen. In addition, FISH with the probes M-bcr/abl and m-bcr/abl showed a deletion of the BCR (D22S257) signal on one of the chromosomes 22 in the patient. FISH with BAC 458J22 (D22S425) in the patient showed signals on both chromosomes 22, but one signal appeared weaker than the other, in most of the 30 analyzed metaphases. Fiber-FISH with both BACs in a control revealed a relatively short signal by BAC 438P22, which was located at one end of the very long but at least two-times-interrupted signal by BAC 458J22 (fig. 4). Both BACs were negative for markers D22S1709, D22S308, D22S303, D22S257, D22S301, D22S156, TOP1P2, D22S1144, and D22S1167. Therefore, BAC 458J22 does not span either of the deletion breakpoints. On interphase nuclei from a control, BAC 458J22 appeared only rarely as two signals; most of the time it gave split signals. These findings could be explained if BAC 458J22 contained repetitive elements that led to a signal on both chromosomes 22 despite deletion of D22S425.Figure 4Fiber-FISH with the D22S425-positive BAC clones 438P22 (red) and 458J22 (green), suggesting the existence of repetitive elements within BAC 458J22.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have demonstrated a novel microdeletion at 22q11.2 in a patient with DGS who had neither a deletion in the known 3-Mb 22q11.2 deletion region nor any other detectable chromosomal aberration such as deletion 10p. The deletion most probably comprises the loci D22S306, D22S308, D22S425, D22S303, and D22S257 in all affected family members. According to the physical map provided by Morrow et al. (Morrow et al., 1995Morrow B Goldberg R Carlson C Das Gupta R Sirotkin H Collins J Dunham I et al.Molecular definition of the 22q11 deletions in velo-cardio-facial syndrome.Am J Hum Genet. 1995; 56: 1391-1403PubMed Google Scholar), the size of the deletion should be ∼2 Mb; however, the exact physical distance to the distal breakpoint is not known. The presented deletion is distal not only to the commonly deleted 22q11.2 region but also to the small distal deletions described by Kurahashi et al. (Kurahashi et al., 1996Kurahashi H Nakayama T Osugi Y Tsuda E Masuno M Imaizumi K Kamiya T et al.Deletion mapping of 22q11 in CATCH22 syndrome: identification of a second critical region.Am J Hum Genet. 1996; 58: 1377-1381PubMed Google Scholar, Kurahashi et al., 1997Kurahashi H Tsuda E Kohamma R Nakayama T Masuno M Imaizumi K Kamiya T et al.Another critical region for deletion of 22q11: a study of 100 patients.Am J Med Genet. 1997; 72: 180-185Crossref PubMed Scopus (60) Google Scholar) and O'Donnell et al. (O'Donnell et al., 1997O'Donnell H McKeown C Gould C Morrow B Scambler P Detection of an atypical 22q11 deletion that has no overlap with the DiGeorge syndrome critical region.Am J Hum Genet. 1997; 60: 1544-1548Abstract Full Text PDF PubMed Google Scholar) (fig. 5). Since this novel deletion is adjacent to the commonly deleted region, a position effect on genes located in the commonly deleted region, or vice versa, may explain the DGS/VCFS phenotype in both the patient and those with the common or published small distal 22q11.2 deletions. Since four of the markers (i.e., D22S306, D22S308, D22S425, and D22S303) from our novel deletion region are within the immunoglobulin light-chain (IGLC) region, immunodeficiency in the affected family members may partly be explained by a reduced number of possible combinations during differentiation of antibody-forming cells. However, the cause of T-cell deficiency, which is the primary immunologic finding in DGS, remains unclear. One should also be aware that deletions of the IGLC region in differentiated B-lymphocytes is not a pathological finding and could lead to misdiagnosis of a germ-line deletion. Since it has been shown that 22q11 contains several low-copy repeats (Collins et al. Collins et al., 1997aCollins JE Mungall AJ Badcock KL Fay JM Dunham I The organization of the gamma-glutamyl transferase genes and other low copy repeats in human chromosome 22q11.Genome Res. 1997a; 7: 522-531PubMed Google Scholar), one might also consider that there are either similar genes or several copies of critical genes within the common and the presented deletion regions. Therefore, the search for such similar genes might give a clue to the answer to the question of whether any of the many genes already known in the commonly deleted region might have a major impact on the pathogenesis in 22q11.2 microdeletion syndromes—and, if so, which ones. Recently, Chen et al. (Chen et al., 1997Chen K-S Manian P Koeuth T Potocki L Zhao Q Chinault AC Lee CC et al.Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome.Nat Genet. 1997; 17: 154-163Crossref PubMed Scopus (324) Google Scholar) identified flanking repeat sequences within the Smith-Magenis syndrome critical region, which may lead to this common microdeletion via chromosomal recombinations. Accordingly, Morrow et al. (Morrow et al., 1997Morrow BE Edelmann L Ferreira J Pandita RK Carlson CG Procter JE Jackson M et al.A duplication on chromosome 22q11 is the basis for the common deletion that occurs in velo-cardio-facial syndrome patients.Am J Hum Genet Suppl. 1997; 61: A7Google Scholar) mentioned a duplicated element within the breakpoints of the common 3-Mb deletion in VCFS/DGS patients. The proximal breakpoint of the 22q11.2 deletion in the family that we studied is in the same region as the distal breakpoint of the known deletion (Carlson et al. Carlson et al., 1997bCarlson C Sirotkin H Pandita R Goldberg R McKie J Wadey R Patanjali SR et al.Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients.Am J Hum Genet. 1997b; 61: 620-629Abstract Full Text PDF PubMed Scopus (290) Google Scholar) and, therefore, may lie within this repeat sequence. According to the corrected low–copy-repeat map provided by Collins et al. (Collins et al., 1997bCollins JE Mungall AJ Badcock KL Fay JM Dunham I The organization of the gamma-glutamyl transferase genes and other low copy repeats in human chromosome 22q11 [erratum].Genome Res. 1997b; 7: 942Google Scholar), this repeat sequence also occurs between the genomic markers D22S257 and D22S301, which flank the distal deletion breakpoint in the atypical deletion presented here. Therefore, it is conceivable that an intra- or interchromosomal rearrangement because of repeated elements at the deletion breakpoints has led to the presented deletion as it is postulated in the common deletion. The repetitive nature of the 22q11 region may also give rise to misinterpretation of results, as could have easily happened with the FISH results of BAC 458J22, which appeared to be not deleted but which, on the basis of Fiber-FISH, seems to contain a multiple repeated element in addition to the specific deleted sequence. Affected family members with the presented microdeletion show several symptoms that are typical of the common 22q11.2 microdeletion: interrupted aortic arch type B; immunodeficiency; hypotonia; mild short stature; microcephaly; short neck; learning difficulties; small, squared-off ears with overfolded helices; retrognathia; high-arched palate; choanal stenosis; and limb anomalies (Ryan et al. Ryan et al., 1997Ryan AK Goodship JA Wilson DI Philip N Levy A Seidel H Schuffenhauer S et al.Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study.J Med Genet. 1997; 34: 798-804Crossref PubMed Scopus (947) Google Scholar; Rauch et al. Rauch et al., 1998bRauch A Hofbeck M Leipold G Klinge J Trautmann U Kirsch M Singer H et al.Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.Am J Med Genet. 1998b; 78: 322-331Crossref PubMed Scopus (63) Google Scholar). However, the typical facial gestalt, nearly always seen in the common 22q11.2 deletion, was not evident in this family. Therefore, the characteristic facial gestalt of the common deletion should occur because of unique and probably contiguous genes from that region. The search for this novel 22q11.2 microdeletion in patients with the 22q11.2 deletion phenotype but without the common deletion will further delineate differences and similarities in both phenotype and genotype and could lead to a better understanding of mechanisms in the pathogenesis of DGS/VCFS. Because the patient's mother and sister have only some minor anomalies, the novel microdeletion shows a clinical variability similar to that of the known 22q11.2 deletion. To explain the inter- and even intrafamilial variability (De Silva et al. De Silva et al., 1995De Silva D Duffty P Booth P Auchterlonie I Morrison N Dean JCS Family studies in chromosome 22q11 deletion: further demonstration of phenotypic heterogeneity.Clin Dysmorphol. 1995; 4: 294-303Crossref PubMed Scopus (22) Google Scholar; Leana-Cox et al. Leana-Cox et al., 1996Leana-Cox J Pangkanon S Eanet KR Curtin MS Wulfsberg EA Familial DiGeorge/velocardiofacial syndrome with deletions of chromosome area 22q11.2: report of five families with a review of the literature.Am J Med Genet. 1996; 65: 309-316Crossref PubMed Scopus (73) Google Scholar; Devriendt et al. Devriendt et al., 1997Devriendt K Van Hoestenberghe R Van Hole C Devlieger H Gewillig M Moerman P Van den Berghe H et al.Submicroscopic deletion in chromosome 22q11 in trizygous triplet siblings and their father.Clin Genet. 1997; 51: 246-249Crossref PubMed Scopus (11) Google Scholar), additional factors have been taken into consideration, such as imprinting, recessive mutations or polymorphisms unmasked by hemizygosity, unbalanced regulatory effects, a second-hit theory, and environmental factors (Hall Hall, 1993Hall JG CATCH 22.J Med Genet. 1993; 30: 801-802Crossref PubMed Scopus (53) Google Scholar; Dallapiccola et al. Dallapiccola et al., 1996Dallapiccola B Pizzuti A Novelli G How many breaks do we need to CATCH on 22q11?.Am J Hum Genet. 1996; 59: 7-11PubMed Google Scholar; Hatchwell Hatchwell, 1996Hatchwell E Monozygotic twins with chromosome 22q11 deletion and discordant phenotype.J Med Genet. 1996; 33: 261Crossref PubMed Google Scholar). However, the observation of MZ twins with a concordant phenotype and 22q11.2 deletion strongly argues in favor of a predominant genetic determination of the 22q11.2 deletion phenotype (Rauch et al. Rauch et al., 1998aRauch A Hofbeck M Leipold G Bähring S Pfeiffer RA Monozygotic twins concordant for Cayler syndrome.Am J Med Genet. 1998a; 75: 113-117Crossref PubMed Scopus (14) Google Scholar). Since both affected sisters have inherited the deletion from their mother, the considerable clinical variation cannot be explained by imprinting. Moreover, both sisters share the same paternal haplotype at the remaining wild-type chromosome 22, which makes the unmasking of different recessive mutations or polymorphisms by hemizygosity unlikely. Therefore, several types of 22q11.2 hemizygosity might result in a susceptibility to certain syndromes, the expression of which might be dependent on other factors, unlinked to this region. We thank the family for their cooperation and patience, and we thank Silke Appel (Berlin) for help with the BAC screening.