heart digeorge ventricular 22ql 1 origin

Br Heart J 1991;66:308-12

DiGeorge syndrome with isolated aorticcoarctation and isolated ventricular septal defectin three sibs with a 22ql 1 deletion of maternalorigin

D I Wilson, I E Cross, J A Goodship, S Coulthard, A H Carey, P J Scambler, H H Bain,A S Hunter, P E Carter, J Burn

AbstractDiGeorge syndrome was diagnosed in aninfant who had an interrupted aorticarch, hypoparathyroidism, and low Tlymphocyte numbers. Two siblings hadheart defects that are not commonlydescribed in DiGeorge syndrome (amembranous ventricular septal defectand coarctation of the aorta respec-tively). These siblings did not haveevidence of thymic dysfunction orhypoparathyroidism. Chromosomeanalysis showed that the mother, whosecardiovascular examination was normal,and her three offspring with heart defectshad a 22qll interstitial deletion, whichwas confirmed by molecular analysis.This family suggests that 22qll dele-

tions can cause apparently isolated heartdefects and that the range of thesedefects may be wider than previouslyrecognised. Once the genes that aredeleted in this family are characterisedthey will be useful candidate genes in theinvestigation of isolated cardiac malfor-mations.

Department ofHuman Genetics,University ofNewcastle upon TyneD I WilsonI E CrossJ A GoodshipS CoulthardJ BurnDepartment ofBiochemistry andMolecular Genetics,St Mary's HospitalMedical School,LondonA H CareyP J ScamblerDepartment ofPaediatric Cardiology,Freeman Hospital,Newcastle upon TyneH H BainA S HunterDepartment ofPaediatrics, WestCumberland Hospital,CumbriaP E CarterCorrespondence toDr D I Wilson, DepartmentofHuman Genetics,19 Claremont Place,Newcastle upon TyneNE2 4AA.

Accepted for publication4 July 1991

Though genetic defects are known to be a

major cause of heart malformation the preciserelation is poorly understood. One approachto the search for the genetic basis of heartdefects is via syndromes with knownchromosomal localisations.

In 1965 DiGeorge described congenitalabsence of the thymus and parathyroid glandsfound at necropsy in three children, and in a

fourth child with congenital hypopara-thyroidism and defective cellular immunity.'In subsequent years it was recognised thatcongenital heart defects and a characteristicfacial appearance were often associated withthese findings.2" The combination of aplasia,or hypoplasia, of the thymus and parathyroidglands together with cardiac defects and dys-morphic facial features became known as

DiGeorge syndrome.The cardiac defects most commonly found

in DiGeorge syndrome are interrupted aorticarch type B and truncus arteriosus.4 Type Binterrupted aortic arch occurs in over 30% ofcases of DiGeorge syndrome, truncus arter-iosus in 25% of cases, and tetralogy of Fallotin 20%.7 An aberrant right subclavian arteryarising from the descending aorta, a rightsided aortic arch, and transposition of the

great arteries are also well recognised cardiacabnormalities in DiGeorge syndrome.The dysmorphic features seen in the syn-

drome are hypertelorism with short palpebralfissures, a small mouth and short philtrum,retrognathia, and low set, posteriorly rotatedears.2

In 1981 de la Chapelle described a familywith members carrying a balanced chromo-some rearrangement, t(20;22)(ql l;q 1). Fourchildren in this pedigree had DiGeorge syn-drome; all had the same unbalanced trans-location products resulting in monosomy for22pter-22qll and trisomy for 20pter-20qll.8The following year a further three unrelatedpatients with DiGeorge syndrome were repor-ted who were monosomic for 22pter-22ql .9Two of these children had inherited un-balanced forms of familial translocations andthe third child had a de novo unbalancedtranslocation. There have been additionalreports of affected children with unbalancedtranslocations with chromosome 22 involve-ment.>"" The minimal region of deletioncommon to all these cases is 22ql 1. Morerecently a child with DiGeorge syndromeand an interstitial deletion of 22ql 1 has beendescribed.14We report a family in which an interstitial

deletion of 22ql 1 was transmitted from amother to three offspring. One child has thecomplete DiGeorge syndrome phenotype.The remaining three individuals with thecytogenetic deletion have a range of clinicalfeatures but none fulfils the diagnostic criteriaof DiGeorge syndrome.

Patients and methodsCASE 1The proposita, the youngest member of thesibship, was born at 40 weeks' gestation andweighed 2-46 kg. She had bilateral talipesequinovarus and a dysmorphic appearancewith small mouth, short philtrum, and low setposteriorly rotated ears (fig 1). She becameunwell at three days of age with poor peri-pheral perfusion and cardiac failure. Echocar-diography showed an interrupted aortic archtype B and a perimembranous ventricularseptal defect. The total blood T lymphocytecount was low (580 x 106/1, reference values> 1500 x 106/1). Surgical repair and patchclosure of the ventricular septal defect were

performed. The postoperative period wascomplicated by necrotising enterocolitis and

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DiGeorge syndrome with isolated aortic coarctation and isolated ventricular septal defect in three sibs with a 22ql 1 deletion of maternal origin 309

Figure 1 Small mouthand short philtrum incase 1.

Figure 2 Short philtrumand narrow upslantingpalpebralfissures in themother.

declined further cardiological investigation.The mother was mildly dysmorphic having ashort philtrum and narrow upslanting palpe-bral fissures (fig 2). Her four pregnancies wereuncomplicated. Alcohol and drug ingestionduring these pregnancies was denied and therewas no history of pregnancy loss. The firstchild, born at 40 weeks' gestation weighing3-5 kg, was well and not dysmorphic. Theirother two children had undergone surgicalcorrection of heart malformations.

CASE 2The second child was born at 41 weeks' gesta-tion and weighed 2-53 kg. He was dysmor-phic, with narrow palpebral fissures and a higharched palate (fig 3), and had a right inguinalhernia. He presented with failure to thrive attwo months of age and was found to have amembranous ventricular septal defect. Thiswas confirmed by echocardiography and car-diac catheterisation. The ventricular septaldefect required surgical repair when he was 4years old. At 11 years of age he had had nosymptoms of immunodeficiency or hypocal-caemia and his serum calcium concentrationand total blood T lymphocyte count werewithin normal limits.

CASE 3The third child was born at 39 weeks' gesta-tion and weighed 2-9 kg (fig 4). He had a smallmouth, poorly modelled philtrum, overfoldedpinnae, and bilateral talipes equinovarus.Bilateral inguinal hernias were repaired at 7days of age. He did not thrive postoperativelyand coarctation of the aorta and a persistentductus arteriosus were diagnosed when he was4 weeks old. At operation a hypoplasticisthmus was identified between the left carotid

hypocalcaemic seizures (total serum calcium1-15 mmol/l, normal range 2 28-2-65 mmol/l).She was given calcium supplementation untilshe was six weeks old. Renal ultrasoundshowed that she had no left kidney.The parents were unrelated white

Europeans. Neither had a history of frequentinfections, seizures, or heart disease in child-hood. Clinical examination of the cardiovas-cular system in both parents was normal; they

Figure 3 Narrow;palpebralfissuresin.case..:

Figure 3 Narrow palpebralfissures in ca.se 2.



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Figure 4 Small mouthand poorly modelledphiltrum in case 3.

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artery and the left subclavian artery. This was

repaired and the ductus arteriosus was ligated.Twenty four hours after the operation routinebiochemical investigations showed that hisserum calcium was 2-06 mmol/l (referencerange 2-28-2-65 mmol/l). He was symptom

free and the serum calcium returned to thenormal range within 48 hours without specifictreatment. He had two seizures two weeksafter the thoracotomy. Blood glucose, cal-cium, and magnesium were normal at the timeof these seizures and no cause was identified.At the age of 4 he was well and his Tlymphocyte and calcium values were withinnormal limits.

CYTOGENETIC STUDIESChromosome preparations were made from 72hour phytohaemagglutinin stimulated cell cul-tures from peripheral blood from members ofthe family. Cultures were synchronised by a

pulse of thymidine administered 21 hoursbefore harvesting. Colcemid was added foronly 8-15 minutes before harvesting to obtainchromosomes of sufficiently high resolution to

visualise band 22ql1.22-that is at least 850bands per haploid set.'5 Preparations were Gbanded with trypsin and Leishman stain.Chromosome analysis showed apparently

normal karyotypes in the father and eldestdaughter of the family. The mother and theremaining three children (cases 1,2, and 3) hada small interstitial deletion of the proximal longarm of chromosome 22, del(22)(ql 1.21ql 1.23)(fig 5).

MOLECULAR STUDIES

Quantitative Southern blot analysis was carriedout with the probe KI-182 3 (D22S134) whichwas generated from the flow-sort-enriched

Figure 5 Chromosome 22 photomicrographs from case 2with adjacent ideograms. The normal chromosome is onthe left and the deleted chromosome on the right. Bandql 1.22 is nbsentfrom the chromosome on the right.

chromosome 22 library LL22NSO1 and local-ised to the region deleted in DiGeorge syn-drome patients."DNA was extracted from venous blood by

salting out'7 and digested with PvuII. Electro-phoresis was carried out through a 0-8%agarose gel at 2 volts/cm for 18 hours. TheDNA was transferred to Hybond N+ mem-brane (Amersham International) by alkali blot-ting following the manufacturer's protocol.Probes were labelled by random priming.'8 Thefilter was hybridised with KI-182-3 and arhodopsin control probe. Hybridisation andwashing conditions were as describedpreviously.16The signal intensity with KI-182-3 in the

father and eldest child in the sibship wascomparable to that in equally loaded controllanes. The KI-182-3 signal intensity in themother and in cases 1, 2, and 3 was half theintensity in normal control tracks with equalDNA loading (fig 6). This was confirmed bylaser densitometry.

DiscussionFour members ofthis family have an interstitialdeletion at chromosome 22q1 1. The youngestchild fulfills the clinical criteria for the diagnosisofDiGeorge syndrome. She had a typical heartdefect, hypocalcaemia, low T lymphocytenumbers, and the dysmorphic features asso-ciated with the condition. Her mother and twoof her siblings have the same cytogenetic dele-tion but do not have DiGeorge syndrome.The molecular analysis of DNA from this

family confirms the cytogenetic findings. TheKI-182-3 signal intensity in the father andnormal sister were equivalent to the normalcontrol sample showing the presence of twocopies of the DNA sequence. The signal inten-sity in the mother, proposita, and her siblingswith congenital heart disease was half that inthe equivalent normal control track showingthat they have only one copy of KI-182-3(D22S134). Thus a deletion of chromosome22qll was demonstrated by two independentmethods in the proposita, her mother, and thetwo siblings with congenital heart disease.A common feature in the range of cardio-

vascular malformations in DiGeorge syndromeis a disturbance of the outflow tract of theembryonic heart or impaired development of

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DiGeorge syndrome with isolated aortic coarctation and isolated ventricular septal defect in three sibs with a 22ql I deletion of maternal origin

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ggp0b;: ;0^ -:t;: bbV.d4Is~; 7.2 kb..%.:

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*W 3.5 kb

1 2 3 4 .5 6 7 8 9 10 11

Figure 6 Autoradiograph showing the quantitative Southern blot analysis. TheKI-182-3 band is at 3-5 kb and the control band at 7 2 kb. Tracks 3 to 7 contain 3 jg,

5 jg, 7 jg, 9 jg, and 11 jg ofDNA from a normal control. Track 1 is DNA from thefather, track 2from the oldest (normal) child in the sibship, track 8from case 2, track 9from case 3, track 10from case 1 and track 11 from the mother. The intensity of theKI-182 3 band in the father and eldest child is comparable with that in the normalcontrol. The intensity of the KI-182 3 band is clearly reduced in the mother, case 1,case 2, and case 3.

the branchial arch arteries or both. Clinicalexamination of the cardiovascular system in themother was normal, though this does not

exclude congenital heart defects. The heartdefects in the brothers were an isolated mem-branous ventricular septal defect and a coarc-

tation of the aorta. While ventricular septaldefects are commonly found in DiGeorgesyndrome in association with other cardiacabnormalities they are unusual as an isolatedfinding.47 We found only one previous reportof coarctation of the aorta in DiGeorgesyndrome."'The older boy (case 2) did not have any signs

of hypocalcaemia at any stage. His serum

calcium was not measured in the neonatalperiod, but after surgical repair of his ven-

tricular septal defect his serum calciumremained within the normal range. In case 3

serum calcium was found to be slightly low

after cardiac surgery but returned to normalquickly without specific treatment. Transienthypocalcaemia is often seen after cardiac sur-

gery in children and can be non-specific. Therewas no history of symptoms of hypocalcaemiain the mother.The index case had the characteristic dys-

morphic appearance seen in DiGeorge syn-drome. However, the facial features in patientswith DiGeorge syndrome are subtle and vari-able. The mildly dysmorphic appearance of themother and other affected siblings was compat-ible with DiGeorge syndrome.DiGeorge originally described children with

an immunodeficiency and the index case had alow T lymphocyte count. T lymphocyte num-bers were not measured at the time of presenta-tion in her siblings but were within normallimits later in childhood and they had not hadrecurrent infections. This suggests that they

did not have important thymic involvement.De la Chapelle described four affected

individuals in one family in which a 20;22translocation was segregating.8 Greenberg et alreported a case of DiGeorge syndrome asso-ciated with partial monosomy of 22q resultingfrom an unbalanced translocation, der(22),t(4;22)(q35.2;ql 1.2).2o The first child in thesibship described by Greenberg had died attwo months and was thought to have had acongenital heart defect; the second child wasfound at necropsy to have truncus arteriosus,thymic aplasia and parathyroid hypoplasia; andthe third child was the proband. The motherwas found to carry the same unbalancedchromosome rearrangement. Cardiovascularexamination was normal but she was noted tohave hypertelorism and on investigation shehad low T lymphocyte numbers and a reducedresponse to T cell mitogens compared with anormal control. Augusseau et al reported afamily in which a phenotypically normal femalewith a balanced translocation (46,XX,t(2;22)(ql4;ql 1)) had a child with the same karyotypewith hypertelorism, micrognathia, severe co-arctation of the aorta, and hypocalcaemia.'9The woman's sister carried the same trans-location and had one pregnancy terminatedbecause of a fetal cardiac malformation. It ispossible that a further chromosome re-arrangement may have occurred and led to thedifferent phenotype in the children reported byAugusseau and Greenberg. However, it is morelikely that the same cytogenetic abnormalityresulted in differing phenotypes withinfamilies-both in these families and in thefamily we have presented here.There have been previous reports of

DiGeorge syndrome occurring in more thanone individual in families with no detectablecytogenetic abnormality. Rohn et al described afamily in which a father and two male offspringhad features of DiGeorge syndrome.2' Bothinfants had truncus arteriosus, hypocalcaemia,T lymphocyte dysfunction, and facial featurestypical of DiGeorge syndrome. Their fatherwas mildly dysmorphic and had an abnormalparathyroid response to a phosphate loadingtest but a normal cardiovascular system.Chromosome analysis in the father and his sonswas normal but a molecular deletion of 22q1 1has recently been detected in the second childin this sibship.22 Keppen et al have describedanother family with dominant transmissionwhere cytogenetic analysis did not show anyabnormality.23 The infant in this family had aninterrupted aortic arch type B and hypocal-caemia. His father had a history of neonatalseizures secondary to hypocalcaemia, recurrentinfections throughout his life, and cardiaccatheterisation at the age of 14 had shown aright sided aortic arch. There is now evidenceof a molecular deletion of 22ql 1 in this man.22A family of three children with truncusarteriosus, hypocalcaemia, and hypoplasia ofthe thymus and clinically normal parents hasbeen reported.24 The authors suggestedautosomal recessive inheritance in this familybut an alternative explanation would be non-

penetrance, as occurred in the present report,

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or a group of cells with a new dominantmutation in the gonads of one of the parents-that is, germline mosaicism.DiGeorge syndrome is a clinical diagnosis

based on finding evidence of thymic and para-thyroid hypoplasia, outflow tract defects of theheart, and a dysmorphic appearance. In thefamily we present, the cytogenetic deletionassociated with DiGeorge syndrome caused awide variation in phenotype. The youngest girlhas all four features of DiGeorge syndrome.The two boys have heart defects which are notoften found in DiGeorge syndrome and themother has no apparent cardiac abnormality.The phenotypic variation in this family and thefamilies discussed above is evidence againstDiGeorge syndrome being a contiguous genedefect. Both in the familial cases in which no

cytogenetic abnormality was identified andin the families reported by Greenberg,Augusseau, and ourselves where individualswith the same apparent cytogenetic abnor-mality have different phenotypes the parent hasthe mildest phenotype and does not fulfil thediagnostic criteria of DiGeorge syndrome. It isclearly important to exclude clinical andcytogenetic abnormalities in parents of chil-dren with complete DiGeorge syndrome. Inthe past individuals with the milder end of theclinical spectrum were more likely to havereproduced than those with completeDiGeorge syndrome. The incidence ofDiGeorge syndrome in offspring of affectedindividuals may, in part, be answered by theBritish Heart Foundation study of offspring.25The findings in this family suggest that in

some individuals with isolated heart defects,including ventricular septal defects or coarc-tations, the cause of the heart defect is adeletion of chromosome 22ql 1. This hasobvious implications for the risk of recurrencein their offspring.

This work was supported by grants from the British HeartFoundation and the Borwick Trust. We also thank Mrs J Brownwho carried out chromosome analysis in two of the familymembers. Dr J Nathans kindly provided the rhodopsin probeused as the control probe in the dosage experiments. Weobtained written permission from the family for publication ofthe photographs.

1 Cooper MD, Peterson RDA, Good RA. A new concept of thecellular basis of immunity. J Pediatr 1965;67:907-8.

2 Freedom RM, Rosen FS, Nadas AS. Congenital cardiovas-cular disease and anomalies of the third and fourthpharyngeal pouch. Circulation 1972;46:165-72.

3 Finley JP, Collins GF, De Chadarevian JP, Williams RL.DiGeorge syndrome presenting as severe congenital heartdisease in the newborn. Can Med Assoc J 1977;116:635-40.

4 Conley ME, Beckwith JB, Mancer JFK, Tenckhoff L. Thespectrum of the DiGeorge syndrome. J Pediatr 1979;94:883-90.

5 Moerman P, Goddeeris P, Lauwerijns J, Van Der HauwaertLG. Cardiovascular malformations in DiGeorge syn-drome (congenital absence or hypoplasia of the thymus).Br Heart J 1980;44:452-9.

6 Moerman P, Dumoulin M, Lauweryns J, Van Der HauwaertLG. Interrupted right aortic arch in DiGeorge syndrome.Br Heart J 1987;58:274-8.

7 Van Mierop LHS, Kutsche LM. Cardiovascular anomaliesin DiGeorge syndrome and importance ofneural crest as apossible pathogenetic factor. AmJ Cardiol 1986;58:133-7.

8 De la Chapelle A, Herva R, Koivisto M, Aula P. A deletion inchromosome 22 can cause DiGeorge syndrome. HumGenet 1981;57:253-6.

9 Kelley RI, Zackai EH, Emanuel BS, Kistenmacher M,Greenberg F, Punnett HH. The association of theDiGeorge anomalad with partial monosomy ofchromosome 22. J Pediatr 1982;101:197-200.

10 Schwanitz G, Zerres K. Partial monosomy 22 as result of anX/22 translocation in a newborn with DiGeorge syn-drome. Ann Genet 1987;30:80-4.

11 Faed MJW, Robertson J, Swanson Beck J, Cater JI, Bose B,Madlom MM. Features of diGeorge syndrome in a childwith 45,XX,-3,-22, + der(3),t(3;22)(p25;ql 1). J MedGenet 1987;24:225-34.

12 Dallapiccola B, Marino B, Giannotti A, Valorani G.DiGeorge anomaly associated with partial deletion ofchromosome 22: report of a case with X/22 translocationand review of the literature. Ann Genet 1989;32:92-6.

13 Anneren G, Gustafsson J, Sunnegardh J. DiGeorge syn-drome in a child with partial monosomy of chromosome22. Ups J Med Sci 1989;94:47-53.

14 Mascarello JT, Bastian JF, Jones MC. Interstitial deletion ofchromosome 22 in a patient with the diGeorge malforma-tion sequence. Am J Med Genet 1989;32:112-4.

15 Standing Committee on Human Cytogenetic Nomenclature.An international systemfor human cytogenetic nomenclature( 1985). Basle: S Karger, 1985.

16 Carey AH, Roach S, Williamson R, et al. Localization of 27DNA markers to the region ofhuman chromosome 22ql 1-pter deleted in patients with the DiGeorge syndrome andduplicated in the der22 syndrome. Genomics 1990;7:299-306.

17 Miller SA, Dykes DD, Polesky HF. A simple salting outprocedure for extracting DNA from human nucleatedcells. Nucleic Acids Res 1988;16:1215.

18 Feinberg AP, Vogelstein B. A technique for radiolabellingDNA restriction endonuclease fragments to high specificactivity. Anal Biochem 1984;137:266-7.

19 Augusseau S, Jouk S, Jalbert P, Prieur M. DiGeorgesyndrome and 22q II rearrangements. Hum Genet 1986;74:206.

20 Greenberg F, Crowder WE, Paschall V, Colon-Linares J,Lubianski B, Ledbetter DH. Familial DiGeorge syn-drome and associated partial monosomy of chromosome22. Hum Genet 1984;65:317-9.

21 Rohn RD, Leffell MS, Leadem P, Johnson D, Rubio T,Emanuel BS. Familial third-fourth pharyngeal pouchsyndrome with apparent autosomal dominant transmis-sion. J Pediatr 1984;105:47-51.

22 Scambler PJ, Carey AH, Wyse RKH, et al. Microdeletionswithin 22ql 1 associated with sporadic and familialDiGeorge syndrome. Genomics 1991;10:201-6.

23 Keppen LD, Fasules JW, Burks AW, Gollin SM, SawyerJR, Miller CH. Confirmation of autosomal dominanttransmission of the DiGeorge malformation complex. JPediatr 1988;113:506-8.

24 Raatikka M, Rapola J, Tuuteri L, Louhimo I, Savilahti E.Familial third and fourth pharyngeal pouch syndromewith truncus arteriosus:DiGeorge syndrome. Pediatrics1981;67:173-5.

25 Burn J, Coffey R, Little J, et al. Recurrence risks in theoffspring of adults born with major heart defects; firstresults of a British collaborative study [abstract]. Am JHum Genet 1990;47:121.

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