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Mutations of UFD1L Are Not Responsible for the Majority of Cases of DiGeorge Syndrome/Velocardiofacial Syndrome without Deletions within Chromosome 22q11

1999; Elsevier BV; Volume: 65; Issue: 1 Linguagem: Inglês

10.1086/302468

1537-6605

Roy Wadey, Judith M. McKie, Charalambos Papapetrou, Helen Sutherland, Frans P. Lohman, Jan Osinga, Ingrid Frohn, Robert M.W. Hofstra, Carel Meijers, Francesca Amati, Emanuela Conti, Antonio Pizzuti, Bruno Dallapiccola, Giuseppe Novelli, Peter Scambler,

Tracheal and airway disorders

To the Editor: Deletions of chromosome 22q11 are associated with a wide spectrum of congenital malformation, encompassed by the acronym “CATCH22” (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, and hypocalcemia on chromosome 22), including velocardiofacial syndrome (VCFS; MIM 192430), DiGeorge syndrome (DGS; MIM 188400), and conotruncal-anomaly face (Emanuel et al. Emanuel et al., 1998Emanuel BS Budarf BS Scambler PJ The genetic basis of conotruncal heart defects: the chromosome 22q11.2 deletion.in: Rosenthal N Harvey R Heart development. Academic Press, San Diego1998: 463-478Google Scholar). The major anomalies include outflow-tract congenital heart defects, hypoplasia of the parathyroids and thymus, craniofacial dysmorphism, and learning/behavioral problems (Ryan et al. Ryan et al., 1997Ryan AK Goodship JA Wilson DI Philip N Levy A Siedel 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 (931) Google Scholar). Many of these are thought to be due to a defective neural-crest contribution during development. The DiGeorge chromosomal region (DGCR) is entirely cloned (Carlson et al. Carlson et al., 1997Carlson C Sirotkin H Pandita R Goldberg R McKie J Wadey R Patanjali S et al.Molecular definition of 22q11 deletions in 151 VCFS patients.Am J Hum Genet. 1997; 61: 620-629Abstract Full Text PDF PubMed Scopus (286) Google Scholar) and sequenced, and several genes have been reported mapping to the region. Mutation screens of genes mapping to the proximal end of this region, termed the “minimal DiGeorge chromosomal region” (MDGCR; Gong et al. Gong et al., 1996Gong W Emanuel BS Collins J Kim DH Wang Z Chen F Zhang G et al.A transcription map of the DiGeorge and velo-cardio-facial syndrome critical region on 22q11.Hum Mol Genet. 1996; 5: 789-800Crossref PubMed Scopus (115) Google Scholar), have been negative (Wadey et al. Wadey et al., 1995Wadey R Daw S Taylor C Atif U Kamath S Halford S O'Donnell H et al.Isolation of a gene encoding an integral membrane protein from the vicinity of a balanced translocation breakpoint associated with the DiGeorge syndrome.Hum Mol Genet. 1995; 4: 1027-1034Crossref PubMed Scopus (45) Google Scholar; Gong et al. Gong et al., 1997Gong W Emanuel BS Galili N Kim DH Roe B Driscoll DA Budarf ML Structural and mutational analysis of a conserved gene (DGSI) from the minimal DiGeorge syndrome critical region.Hum Mol Genet. 1997; 6: 267-276Crossref PubMed Scopus (26) Google Scholar; Gottlieb et al. Gottlieb et al., 1997Gottlieb S Emanuel BS Driscoll DA Sellinger B Wang Z Roe B Budarf ML The DiGeorge syndrome minimal critical region contains a Goosecoid-like (GSCL) homeobox gene, which is expressed early in human development.Am J Hum Genet. 1997; 60: 1194-1201PubMed Google Scholar; Lindsay et al. Lindsay et al., 1998Lindsay EA Harvey EL Scambler PJ Baldini A ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene.Hum Mol Genet. 1998; 7: 629-635Crossref PubMed Scopus (22) Google Scholar). Attention therefore has turned to the regions adjacent and distal to the MDGCR. Recently, the gene UFD1L was proposed as the major gene haploinsufficient in this group of syndromes (Yamagishi et al. Yamagishi et al., 1999Yamagishi H Garg V Matsuoka R Thomas T Srivastava D A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects.Science. 1999; 283: 1158-1161Crossref PubMed Scopus (229) Google Scholar). UFD1L is downstream of dHAND, a gene known to be involved in control of the development of structures affected in DGS, and Ufd1l is expressed in the branchial arches, frontonasal mass, and outflow tract. In addition, a single patient has been reported with a de novo deletion affecting UFD1L and the neighboring gene, CDC45L2 (Yamagishi et al. Yamagishi et al., 1999Yamagishi H Garg V Matsuoka R Thomas T Srivastava D A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects.Science. 1999; 283: 1158-1161Crossref PubMed Scopus (229) Google Scholar). CDC45 is required for initiation of DNA replication in yeast, and CDC45 mutants are nonviable. However, CDC45L2 expression is not altered in dHAND −/− embryos. On the basis of these findings, Yamagishi and colleagues concluded that UFD1L haploinsufficiency (perhaps with some contribution from CDC45L2) causes DGS. We conducted mutation screens, in both UFD1L and CDC45L2, as a three-center collaboration. UFD1L was screened by direct sequencing of 12 patients in London, by direct sequencing of all exons and 900 bp of the 5′ UTR in 20 patients in Rome, and by DGGE of 7 patients' DNA in Rotterdam. Local ethical review and consenting procedures were followed. The majority of patients were chosen on the basis of the presence of two or more features of the 22q11 deletion syndromes, but with no detectable deletion of 22q11 or of the DGSII region of 10p13 (Daw et al. Daw et al., 1996Daw SCM Taylor C Kraman M Call K Mao J Meitinger T Lipson A et al.A common region of 10p deleted in DiGeorge and velo-cardio-facial syndrome.Nat Genet. 1996; 13: 458-460Crossref PubMed Scopus (149) Google Scholar). The Rome series contained six patients with an isolated (i.e., nonsyndromic) interrupted aortic arch, a congenital heart defect commonly associated with the deletion. These patients were included because point mutations may be associated with a narrower spectrum of malformation than deletion and—since UFD1L was specifically identified as a dHAND target—because congenital heart defects might be especially significant. The previously described patient with a balanced 2;22 translocation in association with DGS (patient ADU; Augusseau et al. Augusseau et al., 1986Augusseau S Jouk S Jalbert P Priur M DiGeorge syndrome and 22q11 rearrangements.Hum Genet. 1986; 74: 206Crossref PubMed Scopus (90) Google Scholar) was also screened. UFD1L primers and conditions are available from the collaborating centers, and the genomic organization of UFD1L and the resources for exon PCR amplification have been described elsewhere by Novelli et al. (Novelli et al., 1998Novelli G Mari A Amati F Colosimo A Sangiuolo F Bengala M Conti E et al.Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L).Biochem Biophys Acta. 1998; 1396: 158-162PubMed Google Scholar). In London, 24 patients were similarly screened for CDC45L2 mutations; primers and PCR conditions are available on request, and genomic organization has been published previously (McKie et al. McKie et al., 1998McKie JM Wadey R Sutherland H Taylor K Scambler PJ Direct selection of conserved cDNAs from the DiGeorge critical region: isolation of a novel CDC45-like gene.Genome Res. 1998; 8: 834-841PubMed Google Scholar). No mutations of either gene were detected. We did, however, detect a number of sequence variants. Within the 5′UTR of UFD1L we found a single polymorphic sequence, initially detected by SSCP and subsequently shown to involve an A→G transition, located at the −277 position (with respect to the first base of the initiation codon). Screening of 25 unrelated controls generated a heterozygosity value of .40. Within CDC45L2 we detected an A→G transition 22 bp upstream of exon 17 (at intron 16, with heterozygosity of .3) and a G→T transversion 24 bp into intron 18 (heterozygosity of .5). In addition, Southern analysis of 42 patients was conducted, with four different restriction-enzyme digests (HindIII, EcoRI, KpnI, and BamHI), in an attempt to ascertain rearrangements similar to the UFD1L/CDC45L2 deletion reported elsewhere. This analysis included all of the London patients screened for point mutations, as well as an additional 18 patients. No rearrangements or deletions were detected, although four RFLPs were observed. Finally, mice with hemizygous targeted mutations of Ufd1l were normal (A. Baldini, personal communication). Where does this leave the molecular genetics of the 22q11 deletion syndromes? Interpretation of current data must consider that, although ≥10% of deletions are inherited (Ryan et al. Ryan et al., 1997Ryan AK Goodship JA Wilson DI Philip N Levy A Siedel 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 (931) Google Scholar), there is no good evidence for inheritance of DGS/VCFS in nondeletion cases. Furthermore, there are a large number of potential phenocopies of the condition (Emanuel et al. Emanuel et al., 1998Emanuel BS Budarf BS Scambler PJ The genetic basis of conotruncal heart defects: the chromosome 22q11.2 deletion.in: Rosenthal N Harvey R Heart development. Academic Press, San Diego1998: 463-478Google Scholar). It is therefore possible that only a fraction of nondeleted cases have an etiology related to chromosome 22q11. Therefore, UFD1L must still be regarded as a good candidate for contributing to this complex multiple-malformation syndrome. However, it should be kept in mind that a number of genes might be acting to produce a combined haploinsufficiency, especially since other genes within the DGCR are also expressed in affected tissues. In the case of HIRA, for instance, the protein is known to interact with PAX3, a gene required for conotruncal septation in the mouse (Magnaghi et al. Magnaghi et al., 1998Magnaghi P Roberts C Lorain S Lipinski M Scambler PJ HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with pax3.Nat Genet. 1998; 20: 74-77Crossref PubMed Scopus (107) Google Scholar), and antisense attenuation of HIRA expression in chicks yields an increased incidence of persistent truncus arteriosus (Farrell et al. Farrell et al., 1999Farrell M Stadt H Wallis K Scambler PJ Hixon RL Wolfe R Leatherbury L et al.Persistent truncus arteriosus is associated with decreased expression of HIRA by cardiac neural crest cells in chick embryos.Circ Res. 1999; 84: 127-135Crossref PubMed Scopus (69) Google Scholar). However, as with UFD1L, mutations within HIRA have not been detected. Another consideration is the presence of distinct (i.e., nonoverlapping) rearrangements of 22q11, associated with very similar DGS-like phenotypes (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; 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: 1377-1881PubMed Google Scholar; Sutherland et al. Sutherland et al., 1996Sutherland HF Wadey R McKie JM Taylor C Atif U Johnstone KA Halford S et al.Identification of a novel transcript disrupted by a balanced translocation associated with DiGeorge syndrome.Am J Hum Genet. 1996; 59: 23-31PubMed Google Scholar; Rauch et al. Rauch et al., 1999Rauch A Pfieffer RA Leipold G Singer H Tigges M Hofbeck M A novel 22q11.2 microdeletion in DiGeorge syndrome.Am J Hum Genet. 1999; 64: 658-666Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Perhaps haploinsufficiency of more than one gene can cause the syndrome, or long-range effects induced by the rearrangements can down-regulate the expression of the relevant gene(s). The role of combinations of genes during development is being tested by chromosome engineering in the mouse (Lindsay and Baldini Lindsay and Baldini, 1998Lindsay EA Baldini A Congenital heart defects and 22q11 deletions: which genes count?.Mol Med Today. 1998; 4: 350-357Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), although it is conceivable that long-range effects will confuse analysis in the murine system. In agreement with other commentators (Baldini Baldini, 1999Baldini A DiGeorge syndrome: is the solution in HAND?.Nat Genet. 1999; 21: 246-247Crossref PubMed Scopus (9) Google Scholar; Hagmann Hagmann, 1999Hagmann M A gene that scrambles your heart.Science. 1999; 283: 1091-1093Crossref PubMed Google Scholar), we think it is too early to call “Closing Time” (Heller Heller, 1996Heller JL Closing time. Simon and Schuster, New York1996Google Scholar) on “CATCH22” (Heller Heller, 1955Heller JL Catch 22. Simon and Schuster, New York1955Google Scholar). We would like to thank the families and clinicians who made the study possible. Support was from the Birth Defects Foundation and the British Heart Foundation (to P.J.S.), Telethon Foundation grant E. 723 (to B.D. and G.N.), and the Dutch Heart Foundation and the Sophia Foundation for Medical Research (to C.M.). We would like to thank Drs. Antonio Baldini and Elizabeth Lindsay for patient referrals, helpful discussion, and providing critical data prior to publication. Access to PCR conditions can be obtained at the e-mail addresses that follow: [email protected] (for C.M.), [email protected] (for R.W.), and [email protected] (for G.N.).