I Med Genet 1993 30: 813-817

Prevalence of 22q1 1 microdeletions in DiGeorgeand velocardiofacial syndromes: implications forgenetic counselling and prenatal diagnosis

Deborah A Driscoll, Joshua Salvin, Beatrice Sellinger, Marcia L Budarf,Donna M McDonald-McGinn, Elaine H Zackai, Beverly S Emanuel

AbstractDeletions of chromosome 22ql 1 havebeen seen in association with DiGeorgesyndrome (DGS) and velocardiofacialsyndrome (VCFS). In the present study,we analysed samples from 76 patientsreferred with a diagnosis of either DGSor VCFS to determine the prevalence of22qll deletions in these disorders. Usingprobes and cosmids from the DiGeorgecritical region (DGCR), deletions of22qll were detected in 83% of DGS and68% of VCFS patients by DNA dosageanalysis, fluorescence in situ hybridisa-tion, or by both methods. Combined withour previously reported patients, dele-tions have been detected in 88% of DGSand 76% ofVCFS patients. The results ofprenatal testing for 22qll deletions byFISH in two pregnancies are presented.We conclude that FISH is an efficient anddirect method for the detection of 22qlldeletions in subjects with features ofDGS and VCFS as well as in pregnanciesat high risk for a deletion.(J Med Genet 1993;30:813-17)

Division ofReproductiveGenetics, Departmentof Obstetrics andGynecology, Hospitalof the University ofPennsylvania, 3400Spruce Street,Philadelphia, PA19104, USA.D A Driscoll

Division of HumanGenetics andMolecular Biology,Department ofPediatrics, TheChildren's Hospitalof Philadelphia,Philadelphia, PA19104, USA.J SalvinB SellingerM L BudarfD M McDonald-McGinnE H ZackaiB S Emanuel

Correspondence toDr Driscoll.

Received 16 June 1993.Revised version accepted10 July 1993.

DiGeorge syndrome (DGS), a developmentalfield defect of the third and fourth pharyngealpouches, is characterised by thymic aplasia orhypoplasia, absent or hypoplastic parathyroidglands, and conotruncal cardiac malformations.The aetiology is presumed to be heterogeneous.There have been reported cases of autosomaldominant, autosomal recessive, and X linkedinheritance.' Chromosomal abnormalitiesare not uncommon in patients with DGS. Themajority of cytogenetically abnormal DGSpatients initially reported had unbalancedtranslocations with monosomy 22pter-.ql 1 .2-11

More recently, using high resolution bandingtechniques, visible interstitial deletions of22q1 1.2 (del(22)(ql 1.21ql 1.23)) have beenidentified in approximately 25% of DGSpatients.'2 On the basis of the cytogeneticabnormalities, it was hypothesised that theDiGeorge critical region (DGCR) lies within22q1 1."3 Molecular studies have borne outthis suggestion as we and others have shownthat cytogenetically normal DGS patients havemicrodeletions of 22ql 1.*158 Our initial studybegan to delineate the DGCR. This region,bounded by locus D22S75 (N25) proximallyand D22S259 (pR32) distally, was consistentlydeleted in the first 14 DGS patients westudied."More recently, we and others have shown

that patients with velocardiofacial syndrome(VCFS) also have deletions of this same regionof 22ql 1 .1920 VCFS is an autosomal dominantdisorder characterised by palatal abnormalit-ies, cardiac defects, learning disabilities, and atypical facial appearance.21-2' The presence offeatures common to DGS, such as neonatalhypocalcaemia and decreased lymphoid tissue,in some patients with VCFS suggested thatthese two disorders might share a commonaetiology and pathogenesis.24 Cytogeneticstudies using high resolution banding tech-niques detected interstitial deletions of22ql 1.2 in approximately 20% of VCFSpatients.'9 Molecular studies using either res-triction fragment length polymorphisms(RFLP) or dosage analysis detected 22ql 1microdeletions in the majority of patientsreported by our group and others.'820

Since our initial reports we have evaluatedan additional 36 patients presumed to haveDGS and 40 patients with the suspected dia-gnosis ofVCFS to determine the prevalence of22q1 1 deletions in association with these dis-orders. In addition, we have applied a fluores-cence in situ hybridisation (FISH) assay usingcosmids from the DiGeorge critical region(DGCR) to detect 22q1 1 deletions directly inpatients as well as in pregnancies at risk for adeletion. In this report we describe the resultsofDNA dosage and FISH studies, discuss theimplications for genetic counselling, and de-scribe the application of FISH for the prenataldiagnosis of 22q1 1 microdeletion syndromes.

Materials and methodsPATIENTSThirty-six patients with the presumed dia-gnosis of DGS and four parents of DGSpatients with a 22ql 1 deletion were referred toour laboratory for molecular analysis. Fortypatients with suspected VCFS were also ana-lysed for evidence of a 22qll deletion. Twocases were studied for prenatal diagnosis. Case1. One of our previously reported, deletionpositive VCFS patients (VCF-10)'9 elected tohave an amniocentesis during her second preg-nancy. Amniocentesis was performed at 16weeks' gestation. Cultured amniocytes wereobtained for DNA analysis as well as FISH.Case 2. The normal parents of a deletionpositive VCFS patient requested that FISHstudies be performed to exclude a 22q1 1 dele-tion in a subsequent pregnancy. This studywas performed as an adjunct to routine cytoge-netic studies performed for advanced maternal

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age. Cultured chorionic villi were sent to ourlaboratory for analysis.

DNA ANALYSISDNA was extracted from lymphoblastoid orfibroblast cell lines and cultured amniocytes byroutine methods. Southern blot analysis fordosage studies was performed as previouslydescribed.'5 Probes N25 (D22S75) and R32(D22S259), the most proximal and distalmarkers delineating the DGCR, respectively,were used in this study. Quantitative hybridis-ation of genomic DNA from the patients and anormal control was performed in triplicate todetermine copy number, as previously de-scribed. 5

FLUORESCENCE IN SITU HYBRIDISATION (FISH)Cosmids specific to loci D22S75 and D22S259were isolated from a flow sorted chromosome22 cosmid library (kindly provided by Pdejong). Cosmid cos82 which maps to the longarm of chromosome 22 was used as a controlprobe (kindly provided by H Vissing). Meta-phase spreads prepared from lymphoblastoidcell lines, peripheral blood lymphocytes, cul-tured amniocytes, or chorionic villi were cohy-bridised with biotinylated- 11 -d-UTP labelledtest probes and the control probe, cos82, andvisualised with fluoresceinated avidin.Twenty-five metaphase spreads were scoredfor copy number at each test locus.

ResultsDIGEORGE SYNDROMEIn the present study, 30 of 36 patients referredwith the diagnosis ofDGS were hemizygous atlocus D22S75 (N25) or D22S259 (R32) orboth by either dosage analysis, FISH, or by acombination of both methods (table). Dosageanalysis identified 22ql 1 deletions in 19patients. FISH detected a 22qll deletion innine patients. Two patients appeared deletedby both dosage and FISH. Six patients,studied by dosage analysis, did not appear tobe deleted at either locus D22S75 or D22S259.

VELOCARDIOFACIAL SYNDROMEMicrodeletions of 22ql 1 were detected in 27 ofthe 40 patients referred with the diagnosis ofVCFS (table). Hemizygosity at one or bothloci, D22S75 and D22S259, was shown in 12patients by dosage analysis, seven by FISH,and eight by a combination of dosage andFISH. Thirteen patients suspected of havingVCFS did not appear to have a deletion, seven

Detection of 22ql l microdeletions by dosage analysisand FISH of DGS and VCFS patients.

Deletions detected/number studied (%)

by dosage, three by FISH, and three by bothdosage and FISH.

FAMILY STUDIESMolecular analysis of the parents of five pro-

bands, four with DGS and one with VCFS,showed a 22ql 1 deletion in one parent in eachcase (fig 1A-E). One of these parents was

hemizygous at D22S75 and D22S259 bydosage analysis (fig 1D). FISH indicatedhemizygosity at these loci in the remainingparents. Three parents had mild learning dis-abilities but no history of congenital heartdisease, neonatal hypocalcaemia, immune defi-ciency, or palatal abnormalities (fig lA-C). Inaddition to a learning disability, the mother ofthe fourth proband with DGS had a cleftpalate (fig 1D). The parent of the child withVCFS also carries the diagnosis of VCFS; shehas a cleft palate, learning disorder, and facialdysmorphism consistent with VCFS (fig 1E).

PRENATAL STUDIES

Case 1

DNA was obtained from the cultured amnio-cytes from the pregnancy at 50% risk forVCFS and the associated 22ql 1 deletion.Dosage analysis showed the presence of two

copies of D22S75 (N25) in contrast to a singlecopy in both the affected mother and heraffected daughter. FISH with an N25 specificcosmid confirmed the presence of two alleles inthe fetus. Hence, the fetus did not appear tohave inherited the maternal chromosome 22bearing the deletion.

Fig 2A shows an example of metaphasechromosomes from the mother with VCFSafter hybridisation with N25 and cos82 cos-

mids. The chromosomes 22 are identified withcos82 marking the distal long arm of bothhomologues. A hybridisation signal for N25 isvisualised on only one of the chromosomes 22,consistent with a deletion. In contrast, in fig2B, signals for both cosmids are seen on bothchromosomes 22 of the at risk fetus. In addi-tion to molecular studies, we recommendedthat a level II ultrasound examination and fetalechocardiogram be performed during thepregnancy to rule out a cleft palate and a

congenital heart defect in the fetus. Therewere no apparent congenital anomalies seen on

ultrasonography. The mother declined to havean echocardiogram. She was delivered of a

normal appearing liveborn infant by spontan-eous vaginal delivery at 36 weeks' gestation.There was no evidence of a palatal abnormalityor facial dysmorphism. Dosage studies on

DNA obtained from a sample of cord blood as

well as FISH of peripheral blood lymphocytesconfirmed the absence of a 22ql deletion inthe infant.

Case 2FISH studies of metaphase chromosomesfrom the chorionic villus sample showed two

copies of both D22S75 and D22S259. Thisfinding was in contrast to results obtained in a

Method applied DGS VCFS

Dosage 19/25 12/19FISH 9/9 7/10Dosage+FISH 2/2 8/11Total 30/36 (83%) 27,40 (68%)

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Prevalence of 22ql l microdeletions in DiGeorge and velocardiofacial syndromes

A

DGS

D

DGS

B

E

DGS DGS

GVCFS

C1CEl E

C

DGS

DGS, VCFS affected

3 Learning disability ormild mental retardation

; Cleft palate

Characterisi) facies

D Congenital

Figure I Pedigrees of the five familial cases (A-E).

Figure 2 Fluorescence in situ hybridisation with chromosome 22 cosmids. The casmidswere labelled with biotinylated-11-dUTP and visualised with fluoresceinated avidin.The chromosomes 22 are identified with cos82 marking the distal long arm and the testprobe is a cosmid for N25, from the DGCR. (A) Metaphase chromosomes frommother affected with VCFS hybridised with cosmids cos82 and N25. A hybridisationsignal for N25 is seen on only one of the chromosomes 22 which is consistent with adeletion. (B) Hybridisation of cos82 and N25 to metaphase chromosomes from her atrisk fetus. A hybridisation signal for N25 is present on each chromosome 22.

tic VCFS

heart defect

study of the affected sib in whom we hadpreviously shown a deletion of both loci. Thepregnancy is continuing.

DiscussionInitial studies by our laboratory and othersreported an association between DGS, VCFS,and 22q1 1 deletions.5 1619 In these studies over90% of the patients studied had either cyto-genetically visible interstitial deletions or sub-microscopic deletions of 22ql 1. We have ex-amined an additional 76 patients with eitherDGS or VCFS to determine the prevalence of22ql 1 deletions in these disorders. In thepresent study, 83% (30 of 36) of presumedDGS patients and 68% (27 of 40) of putativeVCFS patients have 22qll deletions. If weinclude the 14 DGS and 14 VCFS patients wepreviously reported, then deletions weredetected in 88% of patients referred with adiagnosis of DGS and 76% with VCFS.The percentage of affected patients with a

22ql 1 deletion is less than previouslyreported. If patients are included in the pre-sent study who do not have either DGS orVCFS, we may have underestimated the trueprevalence of 22qll deletions in these twogroups of patients. It is of interest that thedeletion appears to be more frequentlydetected in patients referred with the diagnosisofDGS. This may reflect the clinician's abilityto diagnose DGS accurately in contrast to theirability to recognise VCFS.The diagnosis of DGS is based on the pres-

ence of three findings: a congenital heart de-fect, hypocalcaemia, and a small or absentthymus. The clinical criteria for establishing adiagnosis of VCFS are not as restricted and itis likely that this study included patients inwhom the diagnosis of VCFS was suspectedbut might not be corroborated. However, werelied on the clinical impressions of the refer-ring geneticists and so the reported frequencyof deletions in the population we present inthis report may reflect the actual likelihood ofdetecting a deletion in clinical practice. Thehigh frequency with which deletions wereinitially detected may reflect the small numberof patients studied or the patient sample. Theinitial sample was derived from fewer clini-cians and based on stricter inclusion criteria.Therefore, this study, which examines a largenumber of patients referred from numerouspractising clinicians, provides us with a betterestimate of the prevalence of 22ql 1 deletionsin patients suspected to have DGS and VCFS.The majority of patients appear to be

deleted for the entire DiGeorge critical region(DGCR). We have not excluded the possibilitythat the non-deletion patients may havesmaller deletions within the DGCR. Studiesare in progress to determine whether thesepatients have deletions internal to the twomarkers (N25 and R32) used in this study.These disorders may also result from pointmutations within critical genes in this region.As genes are identified, the non-deletedpatients will be studied for the presence ofpoint mutations.

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Alternatively, another locus may be impli-cated, such as a locus on lOp. There have beenseveral reports of lOp monosomy in associationwith DGS.625 Recently, molecular analysis oftwo DGS patients with cytogenetic deletionsof lOp did not show a deletion of 22qll,suggesting that another locus is involved in thepathogenesis of DGS (unpublished results).Teratogens, such as retinoic acid and alcohol,and maternal diabetes have been associatedwith the DiGeorge anomaly.' Molecular stud-ies by our laboratory failed to detect a 22ql1microdeletion in two infants with DGS born toinsulin dependent mothers, supporting thehypothesis that DGS is causally heterogeneousand that this represents a phenocopy of the22q1 1 microdeletion syndrome.26Approximately 8% of the patients studied

showed familial transmission of the 22qll de-letion. Deletions were detected in seven par-ents of patients with 22ql 1 deletions, five inthis study and two in our previous report.'9Three parents were also diagnosed as havingVCFS. However, three parents had only alearning disability or mild mental retardationand were only ascertained after the birth of anaffected child. Such a mild phenotype mayreflect which genes in 22ql 1 are deleted. As yetwe have not detected a difference in the size ofthe deletion between numerous affected parentsand their affected offspring. However, now thatadditional probes are available within andflanking the DGCR in 22ql 1, studies are inprogress to identify the deletion boundaries inthese families to determine if changes in the sizeof the deletion during meiosis accounts for theobserved phenotypic differences. Phenotypicvariability may also be explained by parent oforigin of the deletion chromosome, geneticbackground, or in utero environment. We haverecently identified highly polymorphic shorttandem repeat polymorphisms in the DGCRwhich will enable us easily to examine theeffect of parent of origin on the phenotype.

Recent studies suggest that several cyto-genetically based disorders, such as Miller-Dieker and Wolf-Hirschhorn syndromes arebetter assessed for the presence of a microdele-tion or cryptic translocation by FISH ratherthan routine cytogenetic analysis.27-29 We haveestablished a FISH assay using cosmids forloci in the DGCR and have successfully usedthis assay to detect deletions in DGS andVCFS patients.30 Microdeletions of 22ql 1have been detected in a total of 16 patientsusing FISH with cosmids specific for N25 andR32. FISH confirmed the results of dosageanalysis in 10 patients, two with DGS andeight with VCFS. This highly sensitive assayeliminates the need for high resolution cytoge-netic analysis to detect interstitial deletions of22ql 1 in patients at high risk for a deletion.

In addition, we have shown the usefulness ofFISH for prenatal testing for the detection of22ql 1 deletions. FISH can be performed bothon cultured amniocytes and chorionic villi. Itdoes not require DNA extraction and timeconsuming Southern blotting necessary fordosage analysis. Hence, results are available

faster. Traditionally, prenatal diagnosis ofDGS and VCFS relied on ultrasonographyand fetal echocardiography during the secondtrimester to detect the presence of a cleft palateor congenital heart defect. Detection of anaffected fetus is limited by the ability to detectone of these features sonographically and,since the clinical features are variable, a nor-mal sonogram would not definitively excludethe diagnosis of DGS or VCFS.

Patients with a 22ql 1 microdeletion whohave a 50% risk of transmitting the deletion totheir offspring can now be offered FISH forthe prenatal detection of a 22ql1 deletion asearly as 10 to 12 weeks' gestation by chorionicvillus sampling (CVS). Although normal par-ents of offspring with a de novo deletion arepresumably at a low risk for a recurrence insubsequent pregnancies, we have not excludedthe small possibility of germline mosaicism fora 22ql 1 deletion in these persons. In theabsence of studies to dispute this possibility,these families might consider prenatal testingfor reassurance, as did the second family pre-sented in this report.We had previously suggested that molecular

and cytogenetic evaluation of the fetus for a22qll microdeletion should be offered to theparents when a conotruncal heart malforma-tion is detected prenatally.3' Recent studieshave shown that 22ql 1 microdeletions occur in20 to 30% of newborns with non-familial,isolated conotruncal cardiac malformations,including truncus arteriosus, interruptedaortic arch, and tetralogy of Fallot.3233 Thesefindings support our previous recommendation.These findings also suggest that, in additionto subjects with clinical features diagnosticof DGS and VCFS, persons with conotruncalheart defects need to be screened for 22q1 1microdeletions to determine the aetiology of thedisorder, identify other family members at riskfor a deletion, and to assess the risk to theiroffspring.

Subjects with a 22ql 1 deletion have a 50%risk of transmitting the deletion to their off-spring and should be offered genetic counsell-ing. Furthermore, since the phenotype of the22ql 1 microdeletion syndromes is variable,the fetus with a deletion is at risk for thespectrum of malformations seen in both DGSand VCFS. At present we are unable to predictthe clinical outcome based on a deletion of thetwo markers used in the present studies. Thecorrelation between the genotype and pheno-type will require a detailed molecular analysisof the deleted region to determine which re-gion or genes specify individual features of thephenotype.

The authors wish to thank Dr J Biegel forinvaluable assistance with laboratory handlingof patient specimens. We also thank G Lechand M Dunn for their technical assistance.This work was supported in part by NIHgrants HD26979 and CA39926 and March ofDimes Birth Defects Foundation Basic Re-search grant 1-FY91-106.

1 Lammer EJ, Opitz JM. The DiGeorge anomaly as a de-

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