American Journal of Medical Genetics 113:346–350 (2002)

Apparently Unrelated Cytogenetic AbnormalitiesAmong 462 Probands Referred for the Detectionof del(22q) by FISH

Arabella Smith,* Luke St. Heaps, and Lisa RobsonDepartment of Cytogenetics, Children’s Hospital at Westmead, Westmead, Australia

Laboratory-based reports of the cytogeneticabnormalities detected during the course oftesting for deletion del(22q) are scant. Wereport our findings from the testing withFISH of 462 patients suspected to havedel(22q) between 1994 and 2000. Of these,447 had a normal karyotype. An apparentlyunrelated cytogenetic abnormality was de-tected in 15 (3.2%). Two of these abnormali-ties involved reciprocal translocation withchromosome 22q and one of these showeddel(22q) with FISH. The other abnormali-ties included sex chromosome aneuploidiesand unbalanced rearrangements of variouschromosomal segments. There was no com-monality among these abnormalities and nocorrelation with other reported cases. Amongthose with a normal karyotype, an unexpect-ed deletion of the control arylsulphatase A(ARSA) probe was found, providing a definitefrequency of 1/262 for ARSA deletions amongpatients suspected to have del(22q). Of the462 referrals, 48 (10%) had one or more addi-tional diagnoses, and in this group, 4 (8%)had del(22q) and 2 (4%) had an apparentlyunrelated cytogenetic abnormality. The datahighlight the importance of initial cytogene-tic analysis in patients suspected of del(22q)and negates the use of interphase FISHscreening by itself for del(22q). The findingof 3.2% unrelated cytogenetic abnormalitiesis noteworthy. FISH should be used in anystructural rearrangement to ascertain if therelevant locus is deleted or not. The contin-ued reporting of patients diagnosed withdel(22q) found to have an unrelated cytoge-netic abnormality will expand the phenoty-

pic spectrum and possible gene mappingmay refine phenotypic specificity.� 2002 Wiley-Liss, Inc.

KEY WORDS: velocardiofacial syndrome;Di George syndrome; ARSAdeletion

INTRODUCTION

Deletion of chromosome 22(q11.2) produces a broadspectrum of clinical manifestations, including men-tal retardation, cleft lip/palate, velopharyngeal dysfunc-tion, cardiovascular abnormalities, characteristic facialappearance, growth retardation, immunodeficiency,hypocalcemia, speech and language delay, and a com-mon neuropsychological profile [Goldberg et al., 1993;Scambler, 1994; Ryan et al., 1997; Swillen et al., 1997],features overlapping several specialties. The name DiGeorge syndrome has been given for the spectrum ofabnormalities encompassing mainly congenital heartdisease (CHD), thymic problems, and hypoparathyroid-ism [Scambler, 1994], while Shprintzen syndrome in-cludes the dysmorphic facial features, cleft palate, shortstature, and mental disability [Shprintzen, 1994]. Velo-cardiofacial syndrome (VCFS) [Goldberg et al., 1993],CHARGE syndrome1 [Devriendt et al., 1998], and cono-truncal face anomaly [Goodship, 1993] also share featu-res. There is no feature specific for del(22q), and theseverity can vary among affected individuals fromsevere to apparently normal [Lipson et al., 1991;Goldberg et al., 1993; Ryan et al., 1997]. Intrafamilydifferences have been described with apparently iden-tical deletions [Devriendt et al., 1997; Kasprzak et al.,1998]. The phenotype may alter with age and the fullphenotype may not yet be delineated [Levy et al., 1997;Weinzimer et al., 1998]. Thus, making the diagnosis ofdel(22q) clinically with confidence may be difficult.

Thedisorder results froman interstitialmicrodeletionwithinthechromosome22q11.2region,spanningaregionof � 2Mb [Scambler, 1994]. The known deleted loci in-clude 30 different genes, none of which at present are

1Coloboma, heart disease, atresia (choanal), retardation/renal,growth delay, ear (deafness).

*Correspondence to: Dr. Arabella Smith, Children’s Hospital atWestmead, Locked Bag 4001, Westmead NSW 2145, Australia.E-mail: ellies@chw.edu.au

Received 12 March 2002; Accepted 6 June 2002

DOI 10.1002/ajmg.10800

� 2002 Wiley-Liss, Inc.

considered to be solely responsible for the completephenotype [Baldini, 1999]. However, there is a consis-tent deletion interval [Driscoll et al., 1992], for whichFISH probes are commercially available, and FISH hasbeen shown to be a reliable way of detecting del(22q)[Ravnan et al., 1996].

We reviewed the referrals for FISH detection ofdel(22q) from 1994 to the end of 2000. We present herethe cytogenetic abnormalities detected among the 462referrals to our laboratory and compare them withprevious studies.

MATERIALS AND METHODS

Over the 6.5-year period from July 1994 to the end ofDecember 2000, we tested 462 probands with FISH forthedetection of del(22q). Specimens (all peripheral blood)were accompanied by a referral form and additional cli-nical information was sometimes requested, verbally orbyquestionnaire.Consistent detailswerenot alwayspro-vided, so the referring information has some limitations.

Cytogeneticswas performed onall patients as thefirststep. Standard cytogenetic analysis (15 cells) was ap-plied to most cases, but more cells were analyzed whenmosaicism was evident and less than 15 cells whengrowthwassuboptimal.FragileXtestingwasperformedwhen requested.

FISH was performed on the suspension remainingafter the cytogenetic harvest [Smith and Robson, 1999].FISH was undertaken over this time with three dif-ferent probes, determined by commercial availability.Advanced techniques, e.g., CGHand SKY, were not per-formed. The probes used for FISHwereD22S75/D22S39(Oncor, Gaithersburg, MD) in the initial 163 patients,TUPLE1/ARSA (Vysis, Downers Grove, IL) in 262 pati-ents,andTUPLE1(Cytocell,Banbury,Oxfordshire,U.K.)in 37 probands. With probe D22S75, the control probewasD22S39, at a terminally placed locus; with TUPLE1(Vysis), the control probe was the Aryl sulphatase Alocus (ARSA), localized subtelomerically on 22q; andwith TUPLE1 (Cytocell), the control probe was the 22qsubtelomere.Although the threedifferent control probeswere terminally localized within 22q13.3, we could notestablish their precise order and presumed that the 22qsubtelomere was the most distal [Smith et al., 2000].

RESULTS

Of the 462 referrals requestingFISH for patientswithfeatures suggestive of del(22q), 261 came from clinicalgeneticists (57%), 42 from cardiologists (9%), 60 fromgeneral pediatricians (13%), 47 from neonatologists(10%), and 19 from doctors in developmental disability(4%); there was a miscellaneous group of 33 patients(7%), mainly with single indications. The sex of thepatients was equal, 232 males and 230 females. The agerange was from 2 weeks to 57 years, but 98% of theprobands were in the pediatric age group.

Cytogenetics was normal in 447 patients and abnor-mal in 15 (3%). The abnormal karyotypes are shown inTable I. In seven patients (cases 1–7), the cytogeneticabnormality detected was considered by the referringdoctor as insufficient to account for the phenotype and

FISH for del(22q) was performed. One of these (patient6) was deleted. In eight patients (cases 8–15; Table I),the cytogenetic abnormality was able to describe thephenotype and FISH for del(22q) was not performed.

There was no commonality among the cytogeneticabnormalities in these 15 patients. Two patients hadX chromosome aneuploidy, one had an apparently bal-anced reciprocal translocation (patient 3), while theremainder had unbalanced karyotypes due to structuralrearrangements, resulting in deletion or duplication ofdifferent chromosome regions (Table I). In two (patients2 and 6), translocation involved chromosome 22, atbreakpointsq11.2-12. Inpatient2,probeD22S75wasnotdeleted, but present in three copies as a result of mal-segregation at meiosis from the paternal balanced car-rier parent. Subsequent testing with GABRB3 (Oncor)localized to 15q11-13 showed deletion at this locus(paternal in origin) and the patient was diagnosed withPrader-Willi syndrome. In patient 6, FISHwith D22S75showed deletion on the der(17) within the translocatedsegment; further study with the Miller-Dieker D17S379(Oncor) and p53 (Vysis) probes, localized within 17p13,showed both intact on the der(17) chromosome. Thus,this unbalanced t(17;22) had resulted in del(22q).

Of those with a normal karyotype, FISH showeddel(22q) in 97 probands (21%). However, in one patientwith a normal karyotype, FISH did not show a TUPLE1deletion, but anunexpected deletion in the control probeARSA, and, on a subsequent hybridization, also deletionof the 22q subtelomere (Cytocell). This patient was an8-year-old female, with high palate, speech problems,tapering fingers, and hypermetropia. The mother wasnormal, the father not tested. While ARSA deficiency ismostly due to a gene mutational defect associated withmetachromatic leukodystrophy, deletion has been de-fined in a few patients with features that overlap withdel(22q) [Nahara et al., 1992; Nesslinger et al., 1994].The frequency of ARSA deletions detected by FISHis unknown, but in our cohort we found that 1/262(but likely 1/462) patients suspected of del(22q) had anARSA deletion instead.

Due to overlapping phenotypes, an additional diag-nosiswas sought at the same time as del(22q) in 48 of the462 probands (10%; Table II). All had normal karyo-types. Four (8.5%) were deleted for del(22q), including 2(of 4) with possible Turner syndrome and 1 (of 31) withpossible fragile X. The true fragile X status of thesepatients is unknown. Patients 31 and 32 showed anormal methylation pattern.

DISCUSSION

In examining the issue of cytogenetic abnormalityother than interstitial deletion among patients referredwith features suggestive of del(22q), two types of abnor-mality are evident. The first is translocation of an appar-ently unrelated chromosome with chromosome 22q, forexample, t(20;22) [de la Chapelle et al., 1981], t(X;22)[Schwanitz and Zerres, 1987], and t(4;22) [Reddy et al.,1996].Notall of thesecaseshavebeenexaminedbyFISH,leavingsomedoubtas towhether thephenotypehadcon-tributions from the concomitant reciprocal duplication.

Cytogenetic Abnormalities 347

In ourpatient 6with t(17;22), FISHshoweddel(22q), butnot del(17p). In a patient with the t(15;22) [Jaquez et al.,1997], FISH was performed with probes from 22q andchromosome 15 and the patient had del(22q) but notPrader-Willi syndrome. This is in contrast to our patient2 with a similar karyotype, who did not have del(22q)but did have Prader-Willi syndrome (Table I). Withcurrent probe availability, it would seem mandatory toperform FISH in any translocation involving 22q toestablish if the critical locus is deleted or not.

The second type of abnormality is that in which chro-mosome 22 is not involved at all. Those cases in whichmonosomy 10p15 has been confirmed have led to thenotion of another locus critical for the del(22q) pheno-type. Here, monosomy of a 2 Mb region has been asso-ciated with hypoparathyroidism, mental retardation,dysmorphic face, short stature, sensorineural deafness,and renal disease, but not usually congenital heartdisease [Schuffenhauer et al., 1998].We had one patientwith deletion within the short arm of chromosome 10,althoughnot at theprecise breakpoints attributed to10pmonosomy (patient 9). A possible third locus has beensuggested at chromosome 4q34, as patients with thismicrodeletionhave learning difficulties, palatal and car-diac abnormalities [Tsai et al., 1999]. We did not haveany patient with abnormality of chromosome 4.

The frequency of apparently unrelated cytogeneticabnormalities in patients with features suggestive ofdel(22q) is not known. Apart from some individual casereports, as mentioned above, there was only one otherlaboratory-based study we could find that provided data

similar to ours. In that study, unrelated cytogeneticabnormalitieswere found in4of100cases [Ravnanetal.,1996], a frequency very similar to ours (3%). The fourpatients comprised a 2-month-old boy with the karyo-type 46,XY,dup(4)(q31.3q33?) de novo; a 3-month-oldgirl with 46,XX,del(8)(p23.1) de novo; a 3-year-old boywith46,XY,-1,þder(1)t(1;DorG)(p36;p12); anda6-year-old girl with 45,X/46,X,i(Xq)/47,X,i(Xq),i(Xq). Theseabnormalities are different from those we detected.Maybe these patients represent a group of dyschromo-somal patientswithwhole genome imbalance [Kirchhoffet al., 2000] and nonspecificmental and growth retarda-tion and dysmorphic facial features.

While the frequency of apparently unrelated cytoge-netic abnormalities, in patients fromaspecialist referralbase, is relatively low (3–4%), it is not far from the yieldof subtelomerics in mentally retarded and dysmorphicpatients [Knight et al., 1999] andhigher than theyield offragile X in the same type of population [de Vries et al.,1998]. It is important to identify this group, as somecasesmaybe familial, as in two fromour cohort (patients2 and 12; Table I). Furthermore, breakpoint mappingstudies may lead to the identification of genes causingspecific phenotypic features [Bugge et al., 2000], thusproviding more precise diagnostic criteria.

ACKNOWLEDGMENTS

We thank Dr. Alison Colley for information on thepatient with ARSA deletion, the many collaboratingdoctors for clinical information, and cytogeneticists at

TABLE I. Cytogenetic Abnormalities Detected*

Number Age/sex Karyotype Clinical notes

FISH for del(22q) performed1 1 month/f 46,XX,add(17)(q23.3)[15] ?del(22q) ?Pierre Robin sequence; 22q not deleted2 3 years/f 46,XX,-15,þder(22)t(15;22)(q15;q12)[25]pat Dysmorphic features, VSD, hypotonic baby, severe DD; 22q

not deleted, but present in three copies; PWS locusdeleted; the 27-year-old normal father was a balancedcarrier 46,XY,t(15;22)(q15;q12)[15]

3 2 years/f 46,XX,t(3;11)(p22.2;q24.2)[15] Dysmorphic features, VSD, pulmonary stenosis; 22q notdeleted

4 11 years/f 47,XX,þmar[6]/46,XX[44] ?del(22q) ?Opitz-Frias; 22q not deleted; origin of marunresolved

5 7 years/m 47,XY,þmar[45]/46,XY[5] DD, hypotonia face, speech delay and nasal speech; 22q notdeleted; origin of mar unresolved

6 2 years/f 45,XX,der(17)t(17;22)(p13;q11.2),-22[15]de novo DD, tetralogy of fallot, microcephaly; 22q deleted;Miller-Diecker locus not deleted; mother 38 and father39 years, both normal phenotype and cytogenetics

7 1 month/m 47,XXY [15] Congenital heart disease; 22q not deletedFISH for del(22q) not performed

8 4 days/f 46,XX,add(2)(q37)[20] ?VCFS9 2 days/f 46,XX,del(10)(p12.2p13)[15] Low-set posteriorly rotated ears, small philtrum, thin lips,

almond-shaped eyes, no CHD10 8 years/f 46,XX,del(3)(q21.3q22.2)[15] Cleft palate, unfolded ear helices, hypoplastic lobes,

transient pulmonary stenosis, facial gestalt11 4 years/m 46,XY,del(16)(q11.2q13)[10] DD, velopharyngeal insufficiency, mild dysmorphism12 18 years/f 46,XX,add(11)(p15.5)[10] ?VCFS13 3 years/f 46,XX,dup(5)(p13.3p14.3)[12]mat Heart VSD, speech problems, large head; the 26-year-old

mother had the same unbalanced karyotype, facialhypotonia, and developmental delay

14 3 years/f 46,X,?del(X)(p21.3p21.3)[10] Unusual ears, almond-shaped eyes15 19 years/f 45,X[7]/46,XX[43] CHD, hyponasal speech, learning problems

*CHD, congenital heart disease; DD, developmental delay; VSD, ventricular septal defect.

348 Smith et al.

the Department of Cytogenetics, Children’s Hospital atWestmead, for cytogenetic testing.

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TABLE II. Other Referral Indications*

Age/sex Additional referral FISH 22q

7 days/f Trisomy 13 Not deleted2 weeks/f Trisomy 21 Deleted3 days/f Turner Deleted18 days/f Turner Deleted21 days/f Turner Not deleted6 weeks/f Turner Not deleted4 weeks/f Williams Not deleted10 months/f Cat eye Not deleted4 years/m Cri-du-chat Not deleted1 month/f a Pierre-Robin Not deleted9 years/f Rett Not deleted5 years/f Opitz G Not deleted3 years/m Opitz G Not deleted11 years/f b Opitz-Frias Not deleted9 years/m Opitz-Frias/FraX Not deleted5 years/m FraX Not deleted2 years/m FraX Not deleted9 years/f FraX Not deleted4 years/m FraX Not deleted8 years/m FraX Not deleted2 years/m FraX Not deleted9 years/f FraX Not deleted6 years/m FraX Not deleted5 years/m FraX Not deleted4 years/f FraX Not deleted2 years/m FraX Not deleted6 years/m FraX Not deleted4 years/m FraX Not deleted7 years/m FraX Not deleted5 years/m FraX Not deleted8 years/m FraX Not deleted17 years/m FraX Deleted3 years/f FraX Not deleted5 years/f FraX Not deleted9 years/m FraX Not deleted4 years/m FraX Not deleted6 years/m FraX Not deleted3 years/m FraX Not deleted5 years/m FraX Not deleted11 years/f FraX Not deleted3 years/m FraX Not deleted4 years/m FraX Not deleted5 years/m FraX Not deleted3 years/m FraX Not deleted8 years/m FraX Not deleted4 years/m FraX Not deleted4 years/m FraX/AS Not deleted4 years/f FraX/PWS Not deleted

*The word ‘‘syndrome’’ is not included under ‘‘additional referral’’ but isimplied.aRefers to patient 1.bRefers to patient 4, with chromosomal abnormality (Table I).

Cytogenetic Abnormalities 349

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