Another Critical Region for Deletion of 22q11: AStudy of 100 Patients

Hiroki Kurahashi,1,2* Etsuko Tsuda,3 Rikako Kohama,1 Takahiro Nakayama,1 Mitsuo Masuno,4Kiyoshi Imaizumi,4 Tetsuro Kamiya,3 Tetsuya Sano,2 Shintaro Okada,2 and Isamu Nishisho1

1Division of Clinical Genetics, Department of Medical Genetics, Biomedical Research Center, Osaka UniversityMedical School, Osaka, Japan

2Department of Pediatrics, Osaka University Medical School, Osaka, Japan3Department of Pediatrics, National Cardiovascular Center, Osaka, Japan4Department of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan

Deletions at 22q11.1-q11.2 present with vari-able manifestations usually referred to asDiGeorge or velo-cardio-facial syndrome.We previously reported that deletions ob-served in patients with the syndrome can besubgrouped into three types (common largedeletion, proximal deletion, and distal dele-tion) and demonstrated the presence of asecond critical region for the syndrome. Inorder to characterize further the secondcritical region, a 22q11 deletion map wasconstructed from the data of 100 patients,using 12 DNA markers scattered in the com-mon large deletion, and then a phenotype-genotype correlation was analyzed. The sec-ond critical region was found to correspondto the distal deletion encompassing theHCF2, cHKAD26, and D22S935 loci, and theproximal and distal deletions do not overlapeach other. Although it seems that this con-dition is a contiguous gene syndrome, thephenotype of patients with these two typesof deletion was indistinguishable from thatof patients with the common large deletion.Thus, it is plausible that several genes lo-cated in the two segments corresponding tothe two deleted regions are involved in thesame developmental pathway or in an ex-tremely long-range position effect. Am. J.Med. Genet. 72:180–185, 1997.© 1997 Wiley-Liss, Inc.

KEY WORDS: del 22q11 syndrome; contigu-ous gene syndrome; positioneffect

INTRODUCTION

DiGeorge syndrome (DGS; MIM 188400) is a devel-opmental field defect that involves the third and fourthpharyngeal pouches, and is characterized by absence ofthymus and parathyroid glands, and congenital heartand facial abnormalities. Cytogenetic and molecularanalyses have demonstrated a consistent relationshipbetween 22q11.2 deletion and the syndrome [Green-berg et al., 1988; Fibson et al., 1990]. Patients withvelo-cardio-facial syndrome (VCFS; MIM192430), cono-truncal anomaly face syndrome (CTAF), familial con-genital cardiac abnormality (FCCA), and nonsyn-dromic conotruncal abnormality (CCA) have been re-ported with a 22q11 deletion similar to that of DGS[Scambler et al., 1992; Wilson et al., 1992; Burn et al.,1993; Goldmuntz et al., 1993].

DGS was first thought to be a contiguous gene syn-drome, multiple genes being responsible for the pheno-typic differences among the patients [Emanuel, 1988].However, most patients share a common large deletionof 2<4,33>4Mb, irrespective of their phenotypes, andthe severity of symptoms does not seem to correlatewith the extent of the deletions [Lindsay et al., 1995].In addition, patients suspected of carrying the same22q11 deletion in a given family show variable pheno-types from complete DGS to no clinical symptoms [Wil-son et al., 1991]. Thus, it is generally accepted that del22q11 syndrome is caused by identical gene(s) in thecommon deletion, while some other factors may influ-ence the phenotypic variability. Based on the deletionsin rare cases of unbalanced translocations, the shortestregion of overlap (SRO) has been defined [Halford etal., 1993a]. A breakpoint in one DGS patient (patientADU) carrying a balanced translocation was locatedwithin SRO, suggesting that DGS is a single gene dis-order [Augusseau et al., 1986]. Recently, a transcriptdisrupted by the balanced translocation in the patient

Contract grant sponsor: Grants-in-Aid for Scientific Researchfrom the Ministry of Education, Science and Culture of Japan;Contract grant sponsor: Osaka Community Foundation.

*Correspondence to: Hiroki Kurahashi, M.D., Ph.D., Division ofClinical Genetics, Department of Medical Genetics, BiomedicalResearch Center, Osaka University Medical School, 2-2 Yamad-aoka, Suita, Osaka 565, Japan. E-mail: kura@clgene.med.osaka-u.ac.jp

Received 18 July 1996; Accepted 28 April 1997

American Journal of Medical Genetics 72:180–185 (1997)

© 1997 Wiley-Liss, Inc.

ADU was identified, suggesting a major gene for thesyndrome [Budarf et al., 1995]. However, previously,we identified a CTAF patient with a small deletion lo-cated within the common large deletion, but not in SROor at the ADU-breakpoint [Kurahashi et al., 1996]. An-other patient with a deletion not involving the ADU-breakpoint was also reported [Levy et al., 1995].

In this study, we reexamined patients with del 22q11syndrome by constructing a refined 22q11 deletion mapto delineate further the second critical region for thedel 22q11 syndrome. The relationship between the phe-notype and the extent of deletion was also analyzed asto whether the del22q11 syndrome is a contiguous genesyndrome.

MATERIALS AND METHODSPatients

This study comprised of 100 patients with some ofthe following clinical manifestations: conotruncalanomaly, thymic abnormality, hypocalcemia, and/orthe facial appearence characteristic of syndromes suchas DGS, VCFS, CTAF, FCCA, and CCA. Of the 100patients, 80 were those reported in our preliminarystudy [Kurahashi et al., 1996]. All patients but one hada normal karyotype. The exceptional patient had ashort-arm terminal deletion due to an unbalancedtranslocation involving the paracentromeric region ofchromosome 22. The patients were divided into sevenclinical groups, according to their clinical manifesta-tions reflecting the components of the del 22q11 syn-drome. The definition and the number of patients ineach group are shown in Table I. Roughly, patientsclinically diagnosed as DGS are included in groups 1, 2,and 3, and those diagnosed as VCFS and CTAF are ingroups 4 and 5.

DNA Probes and Cell Lines

Fourteen probes were used in this study, 12 of whichwere capable of detecting hemizygosity in patients withdel 22q11 syndrome. Six markers added to those usedin our previous work [Kurahashi et al., 1996] includedcos40 (corresponding to the ZNF74 locus, generouslydonated by Dr. M. Aubry) [Aubry et al., 1993], DVL22,E0472 (D22S933), N122B5 (D22S934), HCF2, and849E9R (D22S935). Probes were generated by PCRamplification using the reported sequence [Morrow etal., 1995; Pizzuti et al., 1996]. Location of two cosmid

clones, cosG1 and cosD1 (D22S938), corresponded re-spectively to the proximal end of YAC 966A8 and thedistal end of YAC 872F9 (CEPH Mega YAC library),the former being located just proximal and the latterdistal to the endpoint of the common large deletion. AnADU-breakpoint spanning clone, ADUBP, and threeother markers, N25, DVL22, and TUPLE1, are all lo-cated within SRO. The order of the 14 markers wascen-cosG1-ADUBP-N25(D22S75)-DVL22-TUPLE1-cos71-D0832-E0472(D22S933 ) -cos40(ZNF74 ) -N 1 2 2 B 5 ( D 2 2 S 9 3 4 ) - H C F 2 - c H K A D 2 6 -849E9R(D22S935)-cosD1(D22S938)-tel. A marker,pEKZ19.3 (D9S17), was used as an internal diploidstandard for dosage analysis, and the other, cHKAD-7,located at 22q13 was used for the identification of chro-mosome 22 for fluorescence in situ hybridization(FISH) [Wolff et al., 1987; Kurahashi et al., 1994].

Two cell lines were obtained from the Human Ge-netic Mutant Cell Repository (Coriell Institute forMedical Research, Camden, NJ). GM10888 is a Chi-nese hamster/human somatic cell hybrid that containshuman chromosome 22 as its only human component.GM00980 is a fibroblast cell line from a VCFS patientwith a deletion that demarcates the distal boundary ofSRO.

Quantitative Hybridization

Quantitative hybridization was performed for theanalysis of deletion at all the loci but N25. Southernhybridization was carried out with the standardmethod. When a test probe was a cosmid, its subfrag-ments were used as probes. The intensity of autoradio-graphic signals was measured densitometrically with amicrocomputer imaging device (Imaging Research Inc.,St. Catharines, Ontario, Canada). The mean intensitywas calculated from the autoradiographic signals oftriplicated membranes. Corrected density for varia-tions in DNA loading was calculated by a density ratioof each test probe to the control probe. Finally, locuscopy numbers were deduced by comparing the valuesfor patients with those for normal healthy individuals.When the locus copy number is lower than 1.5, thesubject was judged to represent a deletion.

Fluorescence In Situ Hybridization

Two-color FISH was performed for the analysis ofdeletion at the 14 loci. Lymphocyte cultures from thepatients were treated with the thymidine synchroniza-

TABLE I. Relationship Between Groups of Patients and Types of Deletion*

Group

Clinical characteristics Type of deletion Percent ofpatients with

deletionCa A T Cl H 1 2 3 No deletion

1 + + + + + 4 0 0 1 802 + + + − + 6 1 0 3 703 − + + − + 2 0 0 1 1004 + + − + − 6 0 0 3 675 + + − − − 23 3 1 18 606 + − − − − 2 0 0 25 77 − + − − − 0 1 0 0 100Total 43 5 1 51 49

*Ca, cardiac defect; A, abnormal face; T, thymic hypoplasia; Cl, cleft palate; H, hypocalcemia.

Deletion Mapping in del22q11 Syndrome 181

tion/BrdU release technique. The cells were arrestedwith colcemid and fixed with the standard method.Metaphase chromosomes were denatured at 758°C for4 min in 70% formamide/2X SSC and dehydrated withethanol. Two-color FISH was carried out as previouslydescribed [Inazawa et al., 1994]. Test probes and con-trol probes (0.5 mg each) were labeled by means of nick-translation with biotin-16-dUTP and digoxigenin-16-dUTP, respectively, and denatured at 758°C in 100%formamide containing 2.5–10 mg of CotI DNA (BRL,Gaithersburg, MD). The denatured probes were thenhybridized to the metaphase chromosomes at 378°C for16 hr in a hybridization mixture containing 50% form-amide, 10% dextran sulfate, and 2X SSC. Hybridizedslides were washed at 378°C for 15 min in 50% form-amide/2X SSC. Hybridization signals were cytochemi-cally detected with FITC-avidin (Boehringer Man-nheim, Penzberg, Germany) and anti-digoxigenin-r h o d a m i n e , w h i l e t h e c h r o m o s o m e s w e r ecounterstained with DAPI (Sigma, St. Louis, MO) in ananti-fade solution.

RESULTS

FISH and/or quantitative hybridization showed that49 of the 100 patients gave evidence of hemizygositywith at least one of the 14 loci. The extent of the dele-tion was then determined in the 49 patients (Figs. 1, 2).In 48 of the 49 patients, the deletions did not involvecosG1 and cosD1, which are both located just outsidethe common large deletion. The exceptional patientwith the unbalanced translocation appeared to behemizygous for a segment corresponding to cosG1. The

interstitial deletions in 43 (88%) of 49 patient encom-passed all the 12 loci that correspond to the extent ofthe common large deletion (referred to as type 1 dele-tion). Six patients were found to have unusual dele-tions. Five of them, including the translocation patient,were hemizygous for a segment encompassing theADUBP and E0472 loci, while they were dizygous forloci distal to cos40 (type 2 deletion). The remaining onepatient who was not found to be hemizygous at any of

Fig. 1. FISH analysis for 22q11 deletion on metaphase chromosomes of a patient with conotruncal anomaly face syndrome. Biotin-labelled test probesgive FITC green-signals at the common large deletion (arrows), and digoxigenin-labelled control probes rhodamine red-signals at 22q13 (arrowheads).With ADUBP probe, both green and red signals were detected on both chromosomes 22 of the patient, indicating that the locus had not been deleted (A),whereas only a red signal appears with cHKAD26 probe, indicating that it is deleted (B).

Fig. 2. Quantitative hybridization for deletion, using ADUBP (A), cos77(B), N122B5 (C), cHKAD26 (D), and pEKZ19.3 (E) probes. Lane 1:GM10888; lane 2: healthy individual with a normal karyotype; lanes 3, 4,and 5: patients with type 1, 2, and 3 deletion, respectively; lane 6:GM00980.

182 Kurahashi et al.

the seven centromeric loci, including the loci locatedwithin SRO, was hemizygous for three telomeric loci,HCF2, cHKAD-26, and 849E9R (type 3 deletion). Sincethe patients with type 2 and type 3 deletion were dizy-gous for the cos40 and N122B5 loci, the two types ofdeletion did not overlap each other. From these results,a deletion map was made (Fig. 3). The map indicatesthat the second critical region is located at around thetelomeric loci, including HCF2, cHKAD26, and849E9R.

In order to analyze a relationship between the dele-tion extent and the clinical manifestations, the pa-tients were divided into seven groups (Table I). Mostpatients with a deletion had minor facial anomalies(96%) or cardiac anomalies (94%), and were distributedover all seven groups. The frequency of deletion washigher among patients in groups 1, 2, and 3, who wereclinically diagnosed as DGS. Among patients with iso-lated CCA (group 6), the deletion frequency was rela-tively low. The phenotypes of patients with the com-mon large deletion (type 1 deletion) varied from group1 to group 6. Five patients with type 2 deletion be-longed to group 2 (one), group 5 (three), and group 7(one). Therefore, the distribution was not significantlydifferent from that of type 1 deletion. A patient (J24)with CTAF (group 5) had a type 3 deletion, althoughthe facial anomalies of this patient were milder thanthose of other CTAF patients. The detailed phenotypesof the patients with two types of unusual deletion areshown in Table II. No specific phenotype other thanthat of patients with the common large deletion wasfound.

DISCUSSIONSince 22q11 deletion was observed in patients with

DGS, VCFS, CTAF, FCCA, or nonsyndromic CCA, a

positional cloning strategy was undertaken by manyinvestigators to identify the putative disease-causinggene. Although several cDNAs were isolated from theSRO [Halford et al., 1993a; Demuczuk et al., 1995;Budarf et al., 1995; Pizzuti et al., 1996; Goldmuntz etal., 1996; Lindsay et al., 1996], it remained unclearwhether haploinsufficiency of the gene causes the del22q11 syndrome. The fact that the affected tissues in-volved are multiple may not contradict a hypothesisthat the disorder is caused by a single gene, if it en-codes a protein related to neural crest migration, whichis shared in common by the several defective parts ofthe body [Mierop et al., 1986]. This assumption is sup-ported by the observation of patient ADU, whose trans-location breakpoint is within the SRO. Recently, tran-scripts disrupted by the translocation of patient ADUhave been identified, and it was expected that the char-acterization of the gene would lead to the understand-ing the biological nature of the syndrome. However,one of our patients had a small deletion located within

Fig. 3. Deletion map for deletions in patients with del 22q11 syndrome. The loci are listed at the top, and deletions subgrouped into three types areon the left. White areas indicate dizygosity, and black areas hemizygosity. GM00980 is a cell line demarcating the distal boundary of SRO. Note that thetype 3 deletion does not contain ADUBP or SRO. Arrows indicate two del 22q11 critical regions deduced from this deletion map.

TABLE II. Clinical Manifestations of Six Patients With aUnique Deletion

Patients J2 J37 J42 J59 U1 J24

Telecanthus + + + + − −Low-set ears − − − − − −Flat nasal bridge + + + + − +Cleft palate − − − − − −Nasal voice + + + + − −Micrognathia + − − − − −Thymic hypoplasia − − − − + −Hypocalcemia − − − − + −Conotruncal anomaly + + + − + +Anomaly of aortic arch − + + − − −Clinical group 5 5 5 7 2 5Type of deletion 2 2 2 2 2 3

Deletion Mapping in del22q11 Syndrome 183

the common large deletion but outside SRO andADUBP. Thus, the deleted region in our patient mayalso be critical for the del 22q11 phenotype, and thissuggests that two or more genes are involved in thedevelopment of the del 22q11 syndrome.

In this study, we examined more patients and moreloci to construct the deletion map than in our prelimi-nary study [Kurahashi et al., 1996]. Of the 49 patientswith deletion, 43 (88%) were identified as having thetype 1 deletion. Although the extent of deletion was thesame among these 43 patients, as far as the 12 lociexamined are concerned the phenotype of Type 1 dele-tion patients varied (Table I). The facial appearance ofthese patients diagnosed originally as VCFS was verysimilar to that of DGS patients [Shprintzen. et al.,1981]. Patients with CCA are often compromised byinfections. The reduced number of T cells and abnormalT-cell subset proportions in peripheral blood were re-ported in CCA patients, suggesting that they may havethymic dysfunctions [Radford et al., 1986]. Some CTAFpatients have a nasal voice, suggesting velo-pharyngeal insufficiency [Burn et al., 1993]. It is likelythat the phenotypic difference among these disorders isnot a result of the number of affected tissues, but aresult of the severity of the illness. Therefore, the dis-orders may be caused by haploinsufficiency of identicalgene(s) in the common deletion, and some epigeneticfactors may induce variable phenotypes in these disor-ders.

Five patients with the proximal deletion (type 2 de-letion) were hemizygous only for the seven centromericloci, including SRO and ADUBP. The patient with thechromosomal translocation was judged to have a type 2deletion. Since this patient only had the del 22q11 phe-notype, it is unlikely that the paracentromeric (22pter-q11) deletion proximal to the DGS critical region re-sults in a specific phenotype. The clinical manifesta-tions of patients with type 2 deletion were alsononspecifically distributed among the seven clinicalgroups. Since these patients were hemizygous for SROand ADUBP, their phenotype is likely caused bygene(s) located at these regions.

Only one patient (J24), as shown in our previousstudy [Kurahashi et al., 1996], had a type 3 deletion,confined to the second critical region, although thistelomeric region was found to be deleted in all but fivepatients with type 2 deletion. Since patients with type2 or type 3 deletion were dizygous for the cos40 andN122B5 loci, the two deletions do not overlap. The re-gion deleted in the patient J24 may also be critical forthe del 22q11 phenotype, independent from both SROand the translocation breakpoint of the ADU-patient,because the deletion is far from SRO with a distance ofat least 1Mb as shown in a pulsed field gel electropho-resis analysis (data not shown). Thus, the possibilitythat the del 22q11 syndrome is a contiguous gene syn-drome has not been ruled out. Patient J24 was diag-nosed as CTAF, although the facial changes weremilder than those of other CTAF patients with type 1or type 2 deletion. It was known that the phenotype ofthe patient with the balanced translocation or thosewhose deletions demarcate SRO were not severe [Fu etal., 1976; Augusseau et al., 1986]. It is hypothesized

that either the putative disease-causing genes in eachregion may play similar roles in the embryogenesis ofaffected organs or the two genes may function througha regulatory cascade during an early development ofthe same organ. The deletion at either or both of theregions may cause a similar phenotype; however, thedeletion of only one of the regions may be compensatedfor by genes in the remaining region or by those in anunknown region in the common deletion, resulting in amilder phenotype. Alternatively, a cis-acting elementmay be located in either or both of the regions and mayhave a very long-range position effect on the gene(s) atanother region. Because dysfunction of the elementmay alter the function of the gene in another region,the deletion at either locus would result in a similarphenotype. Functional analysis of genes expressed inboth regions may become a clue to elucidate the natureof the syndrome.

ACKNOWLEDGMENTS

We thank Drs. Y. Makita and T. Takeuchi for pro-viding peripheral blood samples of patients, Dr. S. Hal-ford for providing D0832, and SRL Inc. for technicalassistance.

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