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Open AcceResearch articleA deletion and a duplication in distal 22q112 deletion syndrome region Clinical implications and reviewLuis Fernaacutendez12 Juliaacuten Nevado12 Fernando Santos12 Damiagrave Heine-Suntildeer3 Victor Martinez-Glez12 Sixto Garciacutea-Mintildeaur12 Rebeca Palomo12 Alicia Delicado12 Isidora Loacutepez Pajares12 Mariacutea Palomares12 Luis Garciacutea-Guereta4 Eva Valverde5 Federico Hawkins5 and Pablo Lapunzina12

Address 1Instituto de Geneacutetica Meacutedica y Molecular (INGEMM) Hospital Universitario La Paz Paseo de la Castellana 261 28046 Madrid Spain 2CIBER de Enfermedades Raras (CIBERER) Madrid Spain 3Seccioacute de Genegravetica Hospital Universitari Son Dureta Palma de Mallorca Spain 4Servicio de Cardiologiacutea Infantil Hospital Universitario La Paz Madrid Spain and 5Servicio de Neonatologiacutea Hospital Universitario La Paz Madrid Spain

Email Luis Fernaacutendez - lfernandezhulpsaludmadridorg Juliaacuten Nevado - jnevadohulpsaludmadridorg Fernando Santos - fsantoshulpsaludmadridorg Damiagrave Heine-Suntildeer - dheinehsdes Victor Martinez-Glez - vmartinezghulpsaludmadridorg Sixto Garciacutea-Mintildeaur - sixtogarciasaludmadridorg Rebeca Palomo - rebecapfhotmailcom Alicia Delicado - adelicadohulpsaludmadridorg Isidora Loacutepez Pajares - ilopezhulpsaludmadridorg Mariacutea Palomares - mpalomareshulpsaludmadridorg Luis Garciacutea-Guereta - lgueretaarrakises Eva Valverde - evalverdehulpsaludmadridorg Federico Hawkins - fhawkinshulpsaludmadridorg Pablo Lapunzina - plapunzinahulpsaludmadridorg

Corresponding author

AbstractBackground Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity andvariable expressivity The most frequent clinical features include conotruncal congenital heart defects velopharyngealinsufficiency hypocalcemia and a characteristic craniofacial dysmorphism The etiology in most patients is a 3 Mb recurrentdeletion in region 22q112 However cases of infrequent deletions and duplications with different sizes and locations have alsobeen reported generally with a milder slightly different phenotype for duplications but with no clear genotype-phenotypecorrelation to date

Methods We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs and dysmorphic facialfeatures not clearly suggestive of 22q112 deletion syndrome and a newborn male infant with cleft lip and palate and upslantingpalpebral fissures Karyotype FISH MLPA microsatellite markers segregation studies and SNP genotyping by array-CGH wereperformed in both patients and parents

Results Karyotype and FISH with probe N25 were normal for both patients MLPA analysis detected a partial de novo 11 Mbdeletion in one patient and a novel partial familial 04 Mb duplication in the other Both of these alterations were located at adistal position within the commonly deleted region in 22q112 These rearrangements were confirmed and accuratelycharacterized by microsatellite marker segregation studies and SNP array genotyping

Conclusion The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlationin the vicinity of the LCRC-LCRD interval of the 22q112 chromosomal region whereas the high presence of duplications innormal individuals supports their role as polymorphisms We suggest that any hypothetical correlation between the clinicalphenotype and the size and location of these alterations may be masked by other genetic andor epigenetic modifying factors

Published 2 June 2009

BMC Medical Genetics 2009 1048 doi1011861471-2350-10-48

Received 19 December 2008Accepted 2 June 2009

This article is available from httpwwwbiomedcentralcom1471-23501048

copy 2009 Fernaacutendez et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

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BMC Medical Genetics 2009 1048 httpwwwbiomedcentralcom1471-23501048

BackgroundDiGeorge syndrome (DGS) and Velocardiofacial syn-drome (VCFS) are genetic disorders affecting pharyngealand neurobehavioural development [1] that result inconotruncal congenital heart defects (CHD) velopharyn-geal insufficiency hypoparathyroidism thymic aplasia orhypoplasia craniofacial dysmorphism learning difficul-ties and psychiatric disorders [23] Interstitial microdele-tions in 22q112 have been identified as the underlyingcause in most cases of DGS [4] VCFS [5] and apparentlyisolated conotruncal CHD [6]

Deletions in 22q112 cluster into a standard 3 Mb dele-tion in 87 of the cases a smaller proximally nested 15Mb deletion in 7 and other atypical deletions nestedoverlapping or adjacent to the typically deleted region(TDR) [78] By non-allelic homologous recombination(NAHR) after asynchronous replication [9] large low-copy repeats in 22q112 (LCR22s A to D) mediate recur-rent deletions [7] whereas recently described uncommondeletions are flanked by smaller LCRs (E to H) [10] oralternative breakpoints [511-24]

Different point mutations [2526] balanced translocationbreakpoints [27-29] and shortest regions of deletion over-lap (SRO) [11-163031] in 22q112 have been comparedin order to identify candidate genes for the 22q112 dele-tion syndrome phenotype However no clear genotype-phenotype correlation has been found [532] and identi-cal alterations even within members of the same familyshow high phenotypic diversity and variable expressivityor incomplete penetrance [33-35] Systematic clinicalsorting of patients with non-overlapping deletions hasrecently shown that an ascertainment bias could be eclips-ing different phenotypes or even what would be differentsyndromes [192336]

22q112 duplication syndrome has also been recentlycharacterized as a different clinical entity [37] with fea-tures overlapping 22q112 deletion syndrome [38] Sincefewer duplications have been reported it is suspected thatthe diagnosis of this condition is also biased [39-43] afact supported by its clinical diversity generally rangingfrom a milder cognitivebehavioral to an apparently nor-mal phenotype in these patients [4143-48] This lowerseverity suggests that duplications with sizes that rangefrom 3 to 6 Mb are less deleterious than deletions [37] andtherefore are more likely to be inherited at reduced pene-trance [43] Again LCRs work as recombination substratesfor these rearrangements [37] and different sizes havealso been described [4349]

In this paper we present two patients referred to us forgenetic diagnosis of 22q112 deletion syndrome The first

patient was found to harbor an atypical deletion and thesecond one an inherited atypical duplication in the distalsegment of the TDR covering LCRs C and D [7] We dis-cuss screening diagnostic strategies for patients referredfor 22q112 deletion testing as well as the clinical implica-tions of these findings for a potential genotype-phenotypecorrelation

MethodsSamples and pictures from patients and their familieswere obtained after informed consent Ethical approvalwas obtained for this study from the IRB at Hospital Uni-versitario La Paz in Madrid (HULP-CEIC-PI347) Researchwas performed in compliance with the Declaration ofHelsinki

Patient 1A male infant was the first child of healthy non-consan-guineous parents of age 39 and 36 years Due to menstrualdysfunction in the mother artificial insemination wasperformed with an oocyte donation from a woman ofunknown age The child was born at 37 weeks of gestationand weighed 2450 g (10thndash25th centile) At age 3 monthshe was referred to Pediatric Cardiology because of respira-tory distress and an ASD (ostium secundum type)together with multiple muscular and perimembranousVSDs He was examined by a clinical geneticist and notedto have some dysmorphic features such as a triangularfacies with a slighty small chin and a broad nose (Figure1a b) He had a poor head control which he finallyachieved at the age of 4 months Cardiovascular surgerywas performed at that time At age 7 months he was re-evaluated his growth parameters were height 625 cm (lt5th centile) weight 5540 g (lt 3rd centile) and OFC 41 cm(5ndash10th centile) He was not sitting unsupported yet

Patient 2A male newborn was born to healthy 37 year-old parentsafter Cesarean section due to lack of progress at 33 weeksof gestation Birthweight was 2290 g (75th centile) Hewas referred to the Neonatology intensive care unitbecause of respiratory distress He was noted to have aright cleft lip with a complete cleft palate and upslantingpalpebral fissures with no other significant features onexamination He made good progress and was finally dis-charged two weeks later He was reviewed in clinic at agefive months His development was within the normalrange His growth parameters were height 62 cm weight6500 g and OFC 425 cm (all measurements between the10th and 25th centiles) (Figure 1c) His cleft lip had beenrepaired and the cleft palate operation was scheduled forthe age of nine months A later review at age 13 monthsand a half showed a good progress and a normal develop-ment

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Karyotype and FISHCytogenetic analysis was performed after standard har-vesting of peripheral blood lymphocytes Metaphasechromosomes were G-banded Fluorescence in situhybridization was performed on peripheral blood lym-phocytes with probe N25 (D22S1660ndashD22S1646)(Kreatech Diagnostics Amsterdam The Netherlands) Aminimum of 15 metaphase cells were assessed under afluorescence microscope (Leica Microsystems WetzlarGermany)

MLPAMultiplex ligation-dependent probe amplification wasperformed on DNA from peripheral blood lymphocytesextracted with Puregene DNA Isolation Kit (Gentra Sys-tems Minneapolis MN) Kits P023B P250 and P324-A1for DGSVCFSCES (MRC-Holland Amsterdam TheNetherlands) were used The three kits test 65 loci on22q11 (8 on the CES region 37 within the TDR and 20adjacent distal to the TDR) 2 on 22q13 7 on 4q 1 on 7p5 on 8p 2 on 9q 9 on 10p 1 on 10q 6 on 17p and 2 on18q most of them involved in the phenotypes of DGSand VCFS Data analysis was made against up to 5 controlsamples using the MRC Coffalyser v8 and v9 software [50]or an in-house Excel spreadsheet

Microsatellite segregation studiesEighteen microsatellite markers (CATCH 10 CATCH 20CATCH 42 CATCH 41 CATCH 11 D22S311 CATCH 12CATCH 13 CATCH 14 CATCH 39 D22S1709 CATCH38 CATCH 37 CATCH 36 CATCH 35 D22S446bisD22S306 D22S303) spanning the region in the vicinityof LCR22-C and LCR22-D were studied Markers namedCATCH were designed by Torres-Juan et al [51]

Array Hybridization profiling and Data AnalysisDNAs were quantified using PicoGreen (Invitrogen Cor-poration Carlsbad CA) and a genome-wide scan of620901 tag SNPs was conducted on the probands usingthe Illumina Human610-Quad BeadChip according tothe manufacturers specifications (Illumina San DiegoCA) GenCall scores lt 015 at any locus were consideredno calls Image data was analyzed using the Chromo-some Viewer tool contained in Beadstudio 32 (IlluminaSan Diego CA) The metric used was the log R ratio whichis the log (base 2) ratio of the observed normalized Rvalue for a SNP divided by the expected normalized Rvalue [52] In addition an allele frequency analysis wasapplied for all SNPs All genomic positions were basedupon NCBI Build 36 (dbSNP version 126)

ResultsKaryotype and FISH analysis with probe N25 in bothpatients and their parents showed normal results

However MLPA testing of both patients and their parentswith kits P023B P250 and P324-A1 showed a deletion of5 probes (SNAP29 LZTR1 LCRD probe 06317-L10754HIC2 and LCRD probe 06321-L05796) on Patient 1 (Fig-ure 2a b) and a duplication of 3 probes (SNAP29 LZTR1and LCRD probe 06317-L10754) on Patient 2 (Figure 2ab) and his mother uncle two aunts and maternal grand-father (not shown) These MLPA loci fall within a shortestsegment of 6751 kb for the deletion and 2074 kb for theduplication both including the region between LCR22-Cand LCR22-D [7] On examination the mother uncletwo aunts and maternal grandfather of Patient 2 were allphenotypically normal

On the other hand microsatellite segregation studies wereinformative for STRs D22S311 D22S1709 and CATCH 38in Patient 1 and confirmed a de novo paternal deletionSTR CATCH 42 showed a triallelic result for both Patient2 and his mother whereas CATCH 11 showed such aresult only in the mother Fluorescence intensity of thealleles of markers CATCH 41 D22S311 and CATCH 13suggested a trisomic result (Table 1)

SNP genotyping by array-CGH narrowed down the dele-tion size to 1092027 kb (rs165626-rs12170039) inPatient 1 (Figure 3a) and the duplication size to 377634

Facial appearance of Patients 1 and 2Figure 1Facial appearance of Patients 1 and 2 Patient 1 (a b) Patient 2 (c)



kb (rs17819969-rs11703181) for Patient 2 (Figure 3b)These results are consistent with MLPA and STRs studiesOther genomic copy number variations (CNV) observedin these patients had been previously described in the nor-mal population [5354] according to the Database ofGenomic Variants [55] (data not shown) The relativelocation of each marker and the deleted and duplicatedsegments are shown in Figure 4

DiscussionThe whole phenotypic spectrum of 22q112 deletion syn-drome is contributed by multiple dosage-sensitive genesacross the 22q112 region that are required for normaldevelopment [reviewed in [1]] Moreover non-overlap-ping atypical deletions do have significantly overlappingphenotypes suggesting either several candidate genes forthe syndrome [131456] a common developmental

pathway [17] or a positional gene effect in 22q112[1757-59] On the other hand phenotypes shown byidentical deletions in 22q112 have been suggested to bemodified by parental imprinting [60] unbalanced regula-tory effects [1559] recessive mutations or polymor-phisms unmasked by hemizygosity [5960]environmental factors [60] or stochastic events duringmorphogenesis [1561] Susceptibility to other syndromesin patients with 22q112 deletions has also been pro-posed [586263] Phenotypic discordance has beenobserved in monozygotic twins with 22q112 deletionsyndrome [3364] a finding that is consistent with both agenetic and an environmental influence

In the recent years several candidate genes reproducingpart of the phenotype in animal models [1314265665]or suspected to change disease predisposition [66-69]

MLPA results of Patients 1 and 2Figure 2MLPA results of Patients 1 and 2 Gene dosage of Patient 1 and Patient 2 observed by MLPA Kit P250 (a) showed deletion of probes SNAP29 LZTR1 and HIC2 in Patient 1 and duplication of probes SNAP29 and LZTR1 in Patient 2 whereas kit P324-A1 (b) showed deletion of both LCRD probes in Patient 1 and duplication of only the proximal one in Patient 2 Data from both kits were analysed with Coffalyser v8 and v9 respectively


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nal aunt 2b Maternal uncle 2 Maternalgrandfather 2

122 122 122

160 160 160

N N N

93 99 101 93 99 101 93 101

169 173 169 173 169 173

1 195 199 191 195 199 195 199

249 253 249 253 249 253

116 116 116

138 143 138 143 138 143

dup dup dup

8 216 227 208 216 227 216 220 227

4 102 106 94 102 106 94 102

dup dup dup

6 128 132 126 128 132 126 132

159 159 159 163

230 245 230 245 230 245

dup dup dup

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048 Table 1 Alleles of STR markers studied in Patient 1 and father and Patient 2 and relatives

Position Patient 1 Father 1 Patient 2 Mother 2 Father 2 Maternal aunt 2a Mater

CATCH 10 19116496 120 122 NA 120 122 122 120 122 122

CATCH 20 19245111 160 160 NA 160 160 162 160

PCQAP 19266745 N N N N N N

LCR-C 19354000ndash19417000

CATCH 42 19449222 99 93 99 93 97 101 93 99 101 97 99 93 99 101

CATCH 41 19450208 NA 169 173 169 173 169 173 173 177 169 173

CATCH 11 19469542 197 195 197 195 199 191 195 199 195 199 191 195 199 19

D22S311 19503575 251 249 255 251 253 249 253 251 249 253

CATCH 12 19566292 116 116 116 116 116 116

CATCH 13 19567154 143 138 143 138 143 138 143 143 138 143

SNAP29 19572014 del N dup dup N dup

CATCH 14 19595454 212 212 215 200 216 227 208 216 227 200 210 208 216 227 20

CATCH 39 19640114 106 106 110 94 102 110 94 102 106 106 110 94 102 106 9

LZTR1 19679191 del N dup dup N dup

D22S1709 19735440 120 128 126 128 132 126 128 132 128 132 126 128 132 12

CATCH 38 19745025 157 159 159 159 155 159 159

CATCH 37 19759833 NA 226 234 230 245 230 245 230 245 230 245

LCR-D 19777000

LCRD 06317- 19779435 del N dup dup N dup

BM

C M L10754

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109 109 109

N N N

N N N

136 143 136 143 136 143

157 161 157 161 157 161

N N N

106 106 106

212 222 212 222 212

CRs () The shortest informative deletion and icates haploinsufficiency or trisomy two alleles ates non informative results del deletion dup

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CATCH 36 20126412 109 109 109 109 109 109

HIC2 20130631 del N N N N N

LCR-D 20242000

LCRD 06321-L05796

20247101 del N N N N N

CATCH 35 20341833 136 136 150 136 150 136 143 143 150 136 143

D22S446bis 20343665 157 157 185 157 180 157 161 165 180 157 161

PPIL2 20379687 N N N N N N

D22S306 20887523 106 106 NA NA NA 106

D22S303 21599366 212 222 212 222 NA NA NA 212 222

Alleles are expressed in fragment bp STR markers are located according to the NCBI Build 361 together with the MLPA probes () and the Lduplication segments by MLPA and STRs are D22S311-LCRD 06321-L05796 and CATCH 42-LCRD 06317-L10754 respectively Bold text indlisted in bold mean that homozygosity is suspected in one of them so there are really three alleles Regular text indicates biallelism Italic indicduplication N normal NA not available

Table 1 Alleles of STR markers studied in Patient 1 and father and Patient 2 and relatives (Continued)

BM

C M



Array-CGH SNP genotyping of Patients 1 and 2Figure 3Array-CGH SNP genotyping of Patients 1 and 2 Chromosome 22 plot of Patients 1 (a) and 2 (b) The upper panels show the genic dosage (Log R ratio) and the homozygousheterozygous distribution (B allele frequency) for all the SNPs geno-typed Both parameters are used together for interpretation of deletions and duplications The region showing copy number alterations is zoomed in below indicating the flanking SNPs and the genes in the region A copy number decrease and loss of heterozygosity was found in Patient 1 (pink shading a) whereas a copy number increase with four different allele populations was found in Patient 2 (blue shading b)


have been identified in 22q112 In this context correla-tions made within a contiguous gene syndrome willalways be subjected to phenotype exceptions

Rauch et al [19] described a likely genotype-phenotypecorrelation by comparing the phenotypes of non-over-lapping deletions in 22q112 (Table 2) They concludedthat deletions covering the whole TDR or the proximallynested 15 Mb segment result in a phenotype character-ized by conotruncal CHDs and typical VCFS mainly byeffects of haploinsufficiency of TBX1 a gene previouslyinvolved in the expression of these features when mutated[26] whereas distally nested deletions including CRKLwould present with a milder atypical phenotype withuncommon CHDs developmental delay and mentalimpairment Likewise distal deletions which are adjacentto and do not include the TDR would generally haveassociated non-specific CHDs and very uncommon fea-tures of 22q112 deletion syndrome such as choanalatresia Within these deletions the shortest proximal seg-

ment has been associated with mild learning difficulties[19] Recently additional characteristic features of thisnew condition such as prematurity prenatal and postna-tal growth retardation developmental delay mild skeletalabnormalities and characteristic facial dysmorphic fea-tures have been described [23] (Table 2)

The absence of a genotype-phenotype correlation high-lights the incomplete penetrance associated with altera-tions in the 22q112 region As an example an apparentlyidentical atypical deletion in two reported familial caseswas found to present both inter- and intrafamilial varia-bility [1819] This atypical deletion which spanned the B-D segment and which should have been mainly involvedin learning disability and mental retardation was foundto be associated with a conotruncal CHD in a child of adeleted father with normal phenotype [18] (Table 2)Whereas in the second case the child developed a moreneurobehavioural phenotype with an undiagnosed father

Detailed map of the distal TDR in 22q112Figure 4Detailed map of the distal TDR in 22q112 The relative location of the informative MLPA probes (blue font) STRs (black font) and SNPs (gray small-sized font) sizing the shortest deletion and duplication size are shown above the line Green boxes indicate LCRs purple boxes indicate the discussed genes and the pink bar and the blue bar below the line indicate the deleted and the duplicated segment respectively Cen centromere tel telomere



with schizophrenia [19] Both patients had mild facialdysmorphism with overlapping features of DGSVCFS

In line with this the only two reported distally nested C-D deletions similar to that found in Patient 1 are also phe-notypically discordant [2170] Both were located withinthis segment that should be associated with the neurobe-havioural expression The patient from DAngelo et al [21]had mild facial features and a complex neurodevelop-mental behavioural and psychiatric spectrum with amajor depressive disorder in her transmitting mother butthe patient described by Kurahashi et al [70] had a typicalconotruncal anomaly face syndrome with pulmonaryatresia tetralogy of Fallot and facial dysmorphic featuresOur patient had a non-specific CHD and mild facial fea-tures barely suggestive of the 22q112 deletion syndrome(Table 2) All these findings within such a small sample

size do not reveal strong evidences but suggest thatgenetic changes in 22q112 are incompletely penetrant[19] or that maybe genotype-phenotype correlations areinvalid for this specific region [21]

The reason for all these exceptions in the distal TDR mightbe the presence of CRKL a gene that has been involved inmultisystemic affection of cardiac neural crest derivativeswhen null-mutated in mice [65] Otherwise other genesin this region seem to be responsible for a more particularphenotype certain SNP haplotypes within genes PIK4CA[6667] and SNAP29 [6869] have been recently suggestedto confer a higher risk of developing schizophrenia (Fig-ure 4)

Recently high resolution molecular diagnostic techniqueshave contributed to map the rearrangements in 22q112

Table 2 Observed clinical features in atypical deletions of the LCR intervals in 22q112

LCR interval Rauch 2005 [19] Ben-Shachar 2008 [23]

Garciacutea-Mintildeaur 2002 [18]

Kurahashi 1996 [70]

DAngelo 2007 [21] Patient 1

- ctCHD - ctCHDA-B - typical VCFS - typical VCFS

- Atypical CHD - ctCHDB-C - Mild

dysmorphism- Mild facial dysmorphism

- DD - Father normal

- Learning disabilities

- CTAFS TOF PA

- Mild facial features - Atypical CHD

C-D - Facial dysmorphism

- Neurodevelopmental behavioural and psychiatric spectrum disorder

- Atypical facial features

- Mother with depressive disorder

- Prematurity- Mild DD - Mild DD

- Prenatal and postnatal GR

- Choanal atresia

- Learning difficulties andor DD

D-E - Atypical CHD

- Specific skeletal abnormalities- Characteristic facial dysmorphism- Higher risk for TA

E-F

CTAFS conotruncal anomaly face syndrome ctCHD conotruncal congenital heart defect DD developmental delay GR growth retardation PA pulmonary atresia TA truncus arteriosus TOF tetralogy of Fallot VCFS Velocardiofacial syndrome



more precisely In fact high resolution comparativegenomic hybridization (HR-CGH) real-time quantitativePCR (qPCR) and high density MLPA (HD-MLPA) haveshown that around 7 of the typical 3 Mb A-D deletionshave atypical distal breakpoints covering HIC2 suggestingthat this gene located within LCR22-D might be involvedin the phenotypic variability among these patients[207172] Hic2 may be required for heart developmentin mouse [Lammerts van Beuren K and Scambler PJ per-sonal communication] HIC2 might be also involved inthe phenotypic variability of Patient 1 and other patientsreported to carry the C-D deletion [2170] as well as inpatients with other atypical deletions [1517192058]

Screening for duplications at 22q112 has no standardizedprocedure as these patients show clinical manifestationsonly some of which are compatible with 22q112 deletionsyndrome within a generally milder phenotype[37414348] Different duplication sizes have beenreported [434849] all of them showing a higher ten-dency to be inherited than deletions [43] Phenotype cor-relations to the sizes and locations of these duplicationsare even harder to find than in deletions as the wholeclinical spectrum is still unknown and many of these indi-viduals overlap normality [4143]

Patient 2 and his maternal relatives are carriers of an atyp-ical duplication that maps distal within the TDR Unlikecommon duplications [436373] this novel rearrange-ment the smallest one reported to date has thereforeunknown clinical implications Polymorphisms and copynumber variants can be defined as alterations of a smallregion known to contain no critical genes and that can beinherited from healthy individuals [4143] However asproposed for TBX1 other genes in 22q112 may have clin-ical significance when present in a trisomic dosage[437475] On the other hand four other carriers of thefamily of Patient 2 show normal phenotype and thepatient shows cleft palate a potentially multifactorialmalformation which may be less associated with 22q112duplications than thought [40] The case that is most sim-ilar to this duplication was described by Fan et al [76] in apatient with mental retardation or developmental delayassociated to other malformations inherited from a phe-notypically normal father This 479 kb duplication alsoincluded PCQAP another gene with a potential involve-ment in schizophrenia susceptibility [7778]

Novel and rare rearrangements in such a complex chro-mosomal region with highly uncertain phenotypic conse-quences have uncertain implications for geneticcounseling High intrafamilial variability and reducedpenetrance are phenotypic effects that hamper the distinc-tion between pathologic change and polymorphism [79]In this context the possibility that the phenotypes of our

patients might not be caused by the observed rearrange-ments should be reminded Also small deletions andduplications occurring from generation to generation inthis rearranged segment cannot be excluded [80]

Despite having occurred in the same region our two rear-rangements as far as they have been molecularly charac-terized are significantly different (Figure 4) The deletionis more than twice the size of the duplication and includesHIC2 and the whole LCRs C and D which are nearlyintact in the duplication This variation in the breakpointslocation highlights potential differences in the molecularmechanisms mediating these rearrangements In fact thedeletion is too large to be a C-D rearrangement and is pos-sibly not mediated by non-allelic homologous recombi-nation between LCRs

Even for identical rearrangements genetic counseling andclinical prognosis is still complex Genotype-phenotypecorrelations made so far in 22q112 are compatible with aclinical outcome modified by environmentalstochasticevents [156061] and eclipsed by regulatory factors[1559] At this moment the molecular diagnostic tech-niques we have used are the best screening procedure forpatients with this whole spectrum of overlapping clinicalfeatures suspected of having any kind of rearrangement in22q112 resulting in a copy number imbalance[718182]

ConclusionIn summary we report on the third atypical deletion andthe first atypical duplication in the vicinity of low-copyrepeats C and D in distal 22q112 deletion region Thebreakpoints location and sizes make it possible that bothrearrangements may be mediated by different mecha-nisms or sequences A complex network of modifying fac-tors may influence the phenotypic outcome of thesechromosomal aberrations A systematic literature reviewand genotyping of phenotype-associated polymorphismscan help anticipating the clinical evolution of patients andunderstanding the prognosis and predisposition to thisdisease

List of abbreviations usedArray-CGH Array-based comparative genomic hybridiza-tion ASD Atrial septal defect CES Cat Eye syndromeCHD Congenital heart defect CNV Copy number varia-tion DGS DiGeorge syndrome FISH Fluorescence in situhybridization HD-MLPA High density multiplex liga-tion-dependent probe amplification HR-CGH High res-olution comparative genomic hybridization IRBInstitutional Review Board LCR Low-copy repeat MLPAMultiplex ligation-dependent probe amplificationNAHR Non-allelic homologous recombination OFCOccipitofrontal circumference qPCR Quantitative



polymerase chain reaction SNP Single nucleotide poly-morphism SRO Shortest region of overlap TDR Typi-cally deleted region VCFS Velocardiofacial syndromeVSD Ventricular septal defect

Competing interestsThe authors declare that they have no competing interests

Authors contributionsLF carried out the molecular genetic studies and wrote themanuscript JN carried out the molecular genetic studiesand participated in the draft of the manuscript FS carriedout the clinical genetic characterization of Patient 2 DHScarried out the microsatellites design and participated inthe draft of the manuscript VMG carried out the SNParray and CNV data analysis and interpretation SGM andRP carried out the clinical genetic characterization of thefamily of Patient 2 AD ILP and MP carried out the cytoge-netic and FISH studies LGG carried out the cardiologicalevaluation and follow-up of Patient 1 EV and FH carriedout the clinical evaluation and follow-up of Patient 2 PLconceived the study carried out the clinical genetic char-acterization of Patients 1 and 2 and participated in thedraft of the manuscript All authors read and approved thefinal manuscript

AcknowledgementsWe thank patients and relatives for their kind cooperation Written con-sent for publication was obtained from the patients parents We thank Anna Gonzaacutelez-Neira from the Centro Nacional de Genotipado (CEGEN) for the Illumina Bead Scanner hybridization of samples and data analysis sup-port This work was enclosed in the project Estudio cliacutenico citogeneacutetico y molecular del siacutendrome de microdelecioacuten 22q112 Caracterizacioacuten molecular de la region 22q112 supported by the Fundacioacuten de Investi-gacioacuten Meacutedica Mutua Madrilentildea Madrid Spain to PL The CIBER de Enfermedades Raras is an initiative of the ISCIII

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6 Thomas JA Graham JM Chromosome 22q11 deletion syn-drome an update and review for the primary pediatricianClin Pediatr 1997 36253-266

7 Shaikh TH Kurahashi H Saitta SC OHare AM Hu P Roe BA Dris-coll DA McDonald-McGinn DM Zackai EH Budarf ML Emanuel BSChromosome 22-specific low copy repeats and the 22q112deletion syndrome genomic organization and deletion end-point analysis Hum Mol Genet 2000 9489-501

8 Saitta SC Harris SE Gaeth AP Driscoll DA McDonald-McGinn DMMaisenbacher MK Yersak JM Chakraborty PK Hacker AM ZackaiEH Ashley T Emanuel BS Aberrant interchromosomalexchanges are the predominant cause of the 22q112 dele-tion Hum Mol Genet 2004 13417-428

9 Baumer A Riegel M Schinzel A Non-random asynchronous rep-lication in 22q112 favours unequal meiotic crossovers lead-ing to the human 22q112 deletion J Med Genet 200441413-420

10 Shaikh TH OConnor RJ Pierpont ME McGrath J Hacker AM Nim-makayalu M Geiger E Emanuel BS Saitta SC Low copy repeatsmediate distal chromosome 22q112 deletions sequenceanalysis predicts breakpoint mechanisms Genome Res 200717482-491

11 Kurahashi H Tsuda E Kohama R Nakayama T Masuno M ImaizumiK Kamiya T Sano T Okada S Nishisho I Another critical regionfor deletion of 22q11 a study of 100 patients Am J Med Genet1997 72180-185

12 ODonnell H McKeown C Gould C Morrow B Scambler P Detec-tion of an atypical 22q11 deletion that has no overlap withthe DiGeorge syndrome critical region Am J Hum Genet 1997601544-1548

13 Yamagishi H Garg V Matsuoka R Thomas T Srivastava D A molec-ular pathway revealing a genetic basis for human cardiac andcraniofacial defects Science 1999 2831158-1161

14 McQuade L Christodoulou J Budarf M Sachdev R Wilson MEmanuel B Colley A Patient with a 22q112 deletion with nooverlap of the minimal DiGeorge syndrome critical region(MDGCR) Am J Med Genet 1999 8627-33

15 Amati F Conti E Novelli A Bengala M Digilio MC Marino B Gian-notti A Gabrielli O Novelli G Dallapiccola B Atypical deletionssuggest five 22q112 critical regions related to the DiGeorgevelo-cardio-facial syndrome Eur J Hum Genet 1999 7903-909

16 Lu JH Chung MY Betau H Chien HP Lu JK Molecular character-ization of tetralogy of fallot within DiGeorge critical regionof the chromosome 22 Pediatr Cardiol 2001 22279-284

17 Saitta SC McGrath JM Mensch H Shaikh H Zackai EH Emanuel BSA 22q112 deletion that excludes UFD1L and CDC45L in apatient with conotruncal and craniofacial defects Am J HumGenet 1999 65562-566

18 Garcia-Mintildeaur S Fantes J Murray RS Porteous MEM Strain L BurnsJE Stephen J Warner JP A novel atypical 22q112 distal deletionin father and son J Med Genet 2002 39E62

19 Rauch A Zink S Zweier C Thiel T Koch A Rauch R Lascorz J Huumlff-meier U Weyand M Singer H Hofbeck M Systematic assessmentof atypical deletions reveals genotype-phenotype correlationin 22q112 J Med Genet 2005 42871-876

20 Weksberg R Stachon AC Squire JA Moldovan L Bayani J Meyn SChow E Bassett AS Molecular characterization of deletionbreakpoints in adults with 22q11 deletion syndrome HumGenet 2007 120837-845

21 DAngelo CS Jehee FS Koiffmann CP An inherited atypical 1 Mb22q112 deletion within the DGSVCFS 3 Mb region in a childwith obesity and aggressive behavior Am J Med Genet A 2007143A1928-1932

22 Nogueira SI Hacker AM Bellucco FT Christofolini DM KulikowskiLD Cernach MC Emanuel BS Melarango MI Atypical 22q112deletion in a patient with DGSVCFS spectrum Eur J MedGenet 2008 51226-230

23 Ben-Shachar S Ou Z Shaw CA Belmont JW Patel MS Hummel MAmato S Tartaglia N Berg J Sutton VR Lalani SR Chinault AC Che-ung SW Lupski JR Patel A 22q112 distal deletion a recurrentgenomic disorder distinct from DiGeorge syndrome andvelocardiofacial syndrome Am J Hum Genet 2008 82214-221

24 Uddin RK Zhang Y Siu VM Fan YS OReilly RL Rao J Singh SMBreakpoint associated with a novel 23 Mb deletion in theVCFS region of 22q11 and the role of Alu (SINE) in recurringmicrodeletions BMC Med Genet 2006 718

25 Gong W Gottlieb S Collins J Blescia A Dietz H Goldmuntz EMcDonald-McGinn DM Zackai EH Emanuel BS Driscoll DA BudarfML Mutation analysis of TBX1 in non-deleted patients with



features of DGSVCFS or isolated cardiovascular defects JMed Genet 2001 38E45

26 Yagi H Furutani Y Hamada H Sasaki T Asakawa S Minoshima S Ich-ida F Joo K Kimura M Imamura S Kamatani N Momma K Takao ANakazawa M Shimizu N Matsuoka R Role of TBX1 in humandel22q112 syndrome Lancet 2003 3621366-1373

27 Augusseau S Jouk S Jalbert P Prieur M DiGeorge syndrome and22q11 rearrangements Hum Genet 1986 74206

28 Lindsay EA Halford S Wadey R Scambler PJ Baldini A Molecularcytogenetic characterization of the DiGeorge syndromeregion using fluorescence in situ hybridization Genomics 199317403-407

29 Holmes SE Riazi MA Gong W McDermid HE Sellinger BT Hua AChen F Wang Z Zhang G Roe B Gonzalez I McDonald-McGinnDM Zackai E Emanuel BS Budarf ML Disruption of the clathrinheavy chain-like gene (CLTCL) associated with features ofDGSVCFS a balanced (2122)(p12q11) translocation HumMol Genet 1997 6357-367

30 Halford S Wadey R Roberts C Daw SC Whiting JA ODonnell HDunham I Bentley D Lindsay E Baldini A Francis F Lehrach H Wil-liamson R Wilson DI Goodship J Cross I Burn J Scambler PJ Isola-tion of a putative transcriptional regulator from the regionof 22q11 deleted in DiGeorge syndrome Shprintzen syn-drome and familial congenital heard disease Hum Mol Genet1993 22099-2107

31 Novelli G Amati F Dallapiccola B UFD1L and CDC45L a role inDiGeorge syndrome and related phenotypes Trends Genet1999 15251-254

32 Lindsay EA Goldberg R Jurecic V Morrow B Carlson C Kucherlap-ati RS Shprintzen RJ Baldini A Velo-cardio-facial syndrome fre-quency and extent of 22q11 deletions Am J Med Genet 199557514-522

33 Yamagishi H Ishii C Maeda J Kojima Y Matsuoka R Kimura M TakaoA Momma K Matsuo N Phenotypic discordance in monozy-gotic twins with 22q112 deletion Am J Med Genet 199878319-321

34 McDonald-McGinn DM Tonnesen MK Laufer-Cahana A Finucane BDriscoll DA Emanuel BS Zackai EH Phenotype of the 22q112deletion in individuals identified through an affected relativecast a wide FISHing net Genet Med 2001 323-29

35 Fernaacutendez L Lapunzina P Pajares IL Criado GR Garciacutea-Guereta LPeacuterez J Quero J Delicado A Higher frequency of uncommon15ndash2 Mb deletions found in familial cases of 22q112 deletionsyndrome Am J Med Genet 2005 13671-75

36 Mikhail FM Descartes M Piotrowski A Andersson R de Staringhl TDKomorowski J Bruder CE Dumanski JP Carroll AJ A previouslyunrecognized microdeletion syndrome on chromosome 22band q112 encompassing the BCR gene Am J Med Genet A2007 143A2178-2184

37 Ensenauer RE Adeyinka A Flynn HC Michels VV Lindor NM Daw-son DB Thorland EC Lorentz CP Goldstein JL McDonald MT SmithWE Simon-Fayard E Alexander AA Kulharya AS Ketterling RPClark RD Jalal SM Microduplication 22q112 an emerging siacuten-drome clinical cytogenetic and molecular analysis of thir-teen patients Am J Hum Genet 2003 731027-1040

38 Portnoiuml MF Lebas F Gruchy N Ardalan A Biran-Mucignat V MalanV Finkel L Roger G Ducrocq S Gold F Taillemite JL Marlin S22q112 duplication syndrome two new familial cases withsome overlapping features with DiGeorgevelocardiofacialsyndromes Am J Med Genet A 2005 13747-51

39 Brunet A Gabau E Perich RM Valdesoiro L Brun C Caballiacuten MRGuitart M Microdeletion and microduplication 22q112screening in 295 patients with clinical features of DiGeorgevelocardiofacial syndrome Am J Med Genet A 2006140A2426-2432

40 Sivertsen Lie RT Wilcox AJ byholm F Vindenes H Haukanes BIHouge G Prevalence of duplications and deletions of the22q11 DiGeorge syndrome region in a population-basedsample of infants with cleft palate Am J Med Genet A 2007143A129-134

41 Courtens W Schramme I Laridon A Microduplication 22q112a benign polymorphism or a syndrome with a very large clin-ical variability and reduced penetrance ndash Report of two fam-ilies Am J Med Genet A 2008 146758-763

42 Dempsey MA Schwartz S Waggoner DJ Mosaicismdel(22)(q112q112)dup(22)(q112q112) in a patient with

features of 22q112 deletion syndrome Am J Med Genet A 2007143A1082-1086

43 Ou Z Berg JS Yonath H Enciso VB Miller DT Picker J Lenzi TKeegan CE Sutton VR Belmont J Chinault AC Lupski JR CheungSW Roeder E Patel A Microduplications of 22q112 are fre-quently inherited and are associated with variable pheno-types Genet Med 2008 10267-277

44 Edelmann L Pandita RK Spiteri E Funke B Goldberg R PalanisamyN Chaganti RS Magenis E Shprintzen RJ Morrow BE A commonmolecular basis for rearrangement disorders on chromo-some 22q11 Hum Mol Genet 1999 81157-1167

45 Yobb TM Somerville MJ Willatt L Firth HV Harrison K MacKenzieJ Gallo N Morrow BE Shaffer LG Babcock M Chernos J Bernier FSprysak K Christiansen J Haase S Elyas B Lilley M Bamforth SMcDermid HE Microduplication and triplication of 22q112 ahighly variable syndrome Am J Hum Genet 2005 76865-876

46 Yu S Cox K Friend K Smith S Buchheim R Bain S Liebelt J Thomp-son E Bratkovic D Familial 22q112 duplication a three-gener-ation family with a 3-Mb duplication and a familial 15-Mbduplication Clin Genet 2008 73160-164

47 De la Rochebrochard C Joly-Heacutelas G Goldenberg A Durand ILaquerriegravere A Ickowicz V Saugier-Veber P Eurin D Moirot HDiguet A de Kergal F Tiercin C Mace B Marpeau L Frebourg TThe intrafamilial variability of the 22q112 microduplicationencompasses a spectrum from minor cognitive deficits tosevere congenital anomalies Am J Med Genet A 2006140A1608-1613

48 Wentzel C Fernstroumlm M Oumlhrner Y Annereacuten G Thuresson ACClinical variability of the 22q112 duplication syndrome EurJ Med Genet 2008 51(6)501-10

49 Alberti A Romano C Falco M Caligrave F Schinocca P Galesi O SpallettaA Di Benedetto D Fichera M 15 Mb de novo 22q1121 micro-duplication in a patient with cognitive deficits and dysmor-phic facial features Clin Genet 2007 71177-182

50 MRC-Holland Multiplex Ligation-dependent Probe Amplifi-cation [httpwwwmlpacomWebFormsWebFormMainaspxTag=fNPBLedDVp38pCxU2h0mQ||]

51 Torres-Juan L Rosell J Saacutenchez-de-la-Torre M Fibla J Heine-SuntildeerD Analysis of meiotic recombination of 22q112 a regionthat frequently undergoes deletions and duplications BMCMed Genet 2007 814

52 Simon-Sanchez J Scholz S Fung HC Matarin M Hernandez D GibbsJR Britton A de Vrieze FW Peckham E Gwinn-Hardy K Crawley AKeen JC Nash J Borgaonkar D Hardy J Singleton A Genome-wideSNP assay reveals structural genomic variation extendedhomozygosity and cell-line induced alterations in normalindividuals Hum Mol Genet 2007 161-14

53 Pinto D Marshall C Feuk L Scherer SW Copy-number variationin control population cohorts Hum Mol Genet 2008 17166-167

54 Perry GH Ben-Dor A Tsalenko A Sampas N Rodriguez-Revenga LTran CW Scheffer A Steinfeld I Tsang P Yamada NA Park HS KimJI Seo JS Yakhini Z Laderman S Bruhn L Lee C The fine-scale andcomplex architecture of human copy-number variation AmJ Hum Genet 2008 82685-695

55 Database of Genomic Variants A curated catalogue of struc-tural variation in the human genome [httpprojectstcagcavariationsource=hg18]

56 Guris DL Fantes J Tara D Druker BJ Imamoto A Mice lacking thehomologue of the human 22q112 gene CRKL phenocopyneurocristopathies of DiGeorge syndrome Nat Genet 200127293-298

57 Sutherland HF Wadey R McKie JM Taylor C Atif U Johnstone KAHalford S Kim UJ Goodship J Baldini A Scambler PJ Identificationof a novel transcript disrupted by a balanced translocationassociated with DiGeorge syndrome Am J Hum Genet 19965923-31

58 Rauch A Pfeiffer RA Leipold G Singer H Tigges M Hofbeck M Anovel 22q112 microdeletion in DiGeorge syndrome Am JHum Genet 1999 64659-666

59 Dallapiccola B Pizzuti A Novelli G How many breaks do weneed to CATCH on 22q11 Am J Hum Genet 1996 597-11

60 Hall GJ CATCH 22 J Med Genet 1993 30801-80261 Kurnit DM Layton WM Matthysse S Genetics chance and mor-

phogenesis Am J Hum Genet 1987 41979-99562 Digilio MC Marino B Capolino R Angioni A Sarkozy A Roberti MC

Conti E de Zorzi A Dallapiccola B Familial recurrence of non-



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syndromic congenital heart defects in first degree relativesof patients with deletion 22q112 Am J Med Genet 2005134A158-164

63 Heine-Suntildeer D Armengol L Torres-Juan L de la Fuente M Garciacutea-Algas F Fernaacutendez L Reyero M Juan M Tubau A Peacuterez-Granero ABernueacutes M Govea N Lapunzina P Estivill X Rosell J Clinical andmolecular characterization of deletions duplications andmutations in the 22q112 region [abstract] Eur J Hum Genet2008 16(Suppl 2)

64 Goodship J Cross I Scambler P Burn J Monozygotic twins withchromosome 22q11 deletion and discordant phenotype JMed Genet 1995 32746-748


66 Jungerius BJ Hoogendoorn ML Bakker SC Vant Slot R Bardoel AFOphoff RA Wijmenga C Kahn RS Sinke RJ An association screenof myelin-related genes implicates the chromosome 22q11PIK4CA gene in schizophrenia Mol Psychiatry 2008131060-1068

67 Vorstman JAS Chow EW Ophoff RA van Engeland H Beemer FAKahn RS Sinke RJ Bassett AS Association of the PIK4CA schiz-ophrenia-susceptibility gene in adults with the 22q112 dele-tion syndrome Am J Med Genet B Neuropsychiatr Genet 2009150B430-433

68 Saito T Guan F Papolos DF Rajouria N Fann CS Lachman HM Pol-ymorphism in SNAP29 gene promoter region associatedwith schizophrenia Mol Psychiatry 2001 6605

69 Wonodi I Hong LE Avila MT Buchanan RW Carpenter WT Jr StineOC Mitchell BD Thaker GK Association between polymor-phism of the SNAP29 gene promoter region and schizophre-nia Schizophr Res 2005 78339-341

70 Kurahashi H Nakayama T Osugi Y Tsuda E Masuno M Imaizumi KKamiya T Sano T Okada S Nishisho I Deletion mapping of22q11 in CATCH22 syndrome identification of a secondcritical region Am J Hum Genet 1996 581377-1381

71 Jalali GR Vorstman JA Errami A Vijzelaar R Biegel J Shaikh TEmanuel BS Detailed analysis of 22q112 with a high densityMLPA probe set Hum Mutat 2008 29433-440

72 Urban AE Korbel JO Selzer R Richmond T Hacker A Popescu GVCubells JF Green R Emanuel BS Gernstein MB Weissman SM Sny-der M High-resolution mapping of DNA copy alterations inhuman chromosome 22 using high-density tiling oligonucle-otide arrays Proc Natl Acad Sci USA 2006 1034534-4539

73 Blennow E Lagerstedt K Malmgren H Sahleacuten S Schoumans J Ander-lid B Concurrent microdeletion and duplication of 22q112Clin Genet 2008 7461-67

74 Zweier C Sticht H Aydin-Yaylaguumll I Campbell CE Rauch A HumanTBX1 missense mutations cause gain of function resulting inthe same phenotype as 22q112 deletions Am J Hum Genet2007 80510-517

75 Torres-Juan L Rosell J Morla M Vidal-Pou C Garciacutea-Algas F de laFuente MA Juan M Tubau A Bachiller D Bernues M Perez-GraneroA Govea N Busquets X Heine-Suntildeer D Mutations in TBX1 gen-ocopy the 22q112 deletion and duplication syndromes anew susceptibility factor for mental retardation Eur J HumGenet 2007 15658-663

76 Fan YS Jayakar P Zhu H Barbouth D Sacharow S Morales A CarverV Benke P Mundy P Elsas LJ Detection of pathogenic gene copynumber variations in patients with mental retardation bygenomewide oligonucleotide array comparative genomichybridization Hum Mutat 2007 281124-1132

77 De Luca A Conti E Grifone N Amati F Spalletta G Caltagirone CBonaviri G Pasini A Gennarelli M Stefano B Berti L Mittler G Asso-ciation study between CAG trinucleotide repeats in thePCQAP gene (PC2 glutamineQ-rich-associated protein)and schizophrenia Am J Med Genet B Neuropsychiatr Genet 2003116B32-35

78 Sandhu HK Hollenbeck N Wassink TH Philibert RA An associa-tion study of PCQAP polymorphisms and schizophrenia Psy-chiatr Genet 2004 14169-172

79 Mefford H Sharp A Baker C Itsara A Jiang Z Buysse K Huang SMaloney V Crolla J Baralle D Collins A Mercer C Norga K de RavelT Devriendt K Bongers E de Leeuw N Reardon W Gimelli S BenaF Hennekam R Male A Gaunt L Clayton-Smith J Simonic I Park S

Mehta S Nik-Zainal S Woods C Firth H Parkin G Fichera M Reit-ano S Lo Giudice M Li K Casuga I Broomer A Conrad B Schwerz-mann M Raumlber L Gallati S Striano P Coppola A Tolmie J Tobias ELilley C Armengol L Spysschaert Y Verloo P De Coene A GoosensL Mortier G Speleman F van Binsbergen E Nelen M HochstenbachR Poot M Gallagher L Gill M McClellan J King MC Regan R SkinnerC Stevenson R Antonarakis S Chen C Estivill X Menten B GimelliG Gribble S Schwartz S Sutcliffe J Walsh T Knight S Sebat JRomano C Schwartz C Veltman J de Vries B Vermeesch J Barber JWillatt L Tassabehji M Eichler E Recurrent rearrangements ofchromosome 1q211 and variable pediatric phenotypes NEngl J Med 2008 3591685-1699

80 Wieser R Fritz B Ullmann R Muumlller I Galhuber M Storlazzi CTRamaswamy A Christiansen H Shimizu N Rehder H Novel rear-rangement of chromosome band 22q112 causing 22q112microdeletion syndrome-like phenotype and rhabdoidtumor of the kidney Hum Mutat 2005 2678-83

81 Fernaacutendez L Lapunzina P Arjona D Loacutepez Pajares I Garciacutea-GueretaL Elorza D Burgueros M De Torres ML Mori MA Palomares MGarciacutea-Alix A Delicado A Comparative study of three diagnos-tic approaches (FISH STRs and MLPA) in 30 patients with22q112 deletion syndrome Clin Genet 2005 68373-378

82 Vorstman JA Jalali GR Rappaport EF Hacker AM Scott C EmanuelBS MLPA a rapid reliable and sensitive method for detec-tion and analysis of abnormalities of 22q Hum Mutat 200627814-821

Pre-publication historyThe pre-publication history for this paper can be accessedhere

httpwwwbiomedcentralcom1471-23501048prepub


Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA

Microsatellite segregation studies

Array Hybridization profiling and Data Analysis

Results

Discussion

Conclusion

List of abbreviations used

Competing interests

Authors contributions

Acknowledgements

References

Pre-publication history


BackgroundDiGeorge syndrome (DGS) and Velocardiofacial syn-drome (VCFS) are genetic disorders affecting pharyngealand neurobehavioural development [1] that result inconotruncal congenital heart defects (CHD) velopharyn-geal insufficiency hypoparathyroidism thymic aplasia orhypoplasia craniofacial dysmorphism learning difficul-ties and psychiatric disorders [23] Interstitial microdele-tions in 22q112 have been identified as the underlyingcause in most cases of DGS [4] VCFS [5] and apparentlyisolated conotruncal CHD [6]

Deletions in 22q112 cluster into a standard 3 Mb dele-tion in 87 of the cases a smaller proximally nested 15Mb deletion in 7 and other atypical deletions nestedoverlapping or adjacent to the typically deleted region(TDR) [78] By non-allelic homologous recombination(NAHR) after asynchronous replication [9] large low-copy repeats in 22q112 (LCR22s A to D) mediate recur-rent deletions [7] whereas recently described uncommondeletions are flanked by smaller LCRs (E to H) [10] oralternative breakpoints [511-24]

Different point mutations [2526] balanced translocationbreakpoints [27-29] and shortest regions of deletion over-lap (SRO) [11-163031] in 22q112 have been comparedin order to identify candidate genes for the 22q112 dele-tion syndrome phenotype However no clear genotype-phenotype correlation has been found [532] and identi-cal alterations even within members of the same familyshow high phenotypic diversity and variable expressivityor incomplete penetrance [33-35] Systematic clinicalsorting of patients with non-overlapping deletions hasrecently shown that an ascertainment bias could be eclips-ing different phenotypes or even what would be differentsyndromes [192336]

22q112 duplication syndrome has also been recentlycharacterized as a different clinical entity [37] with fea-tures overlapping 22q112 deletion syndrome [38] Sincefewer duplications have been reported it is suspected thatthe diagnosis of this condition is also biased [39-43] afact supported by its clinical diversity generally rangingfrom a milder cognitivebehavioral to an apparently nor-mal phenotype in these patients [4143-48] This lowerseverity suggests that duplications with sizes that rangefrom 3 to 6 Mb are less deleterious than deletions [37] andtherefore are more likely to be inherited at reduced pene-trance [43] Again LCRs work as recombination substratesfor these rearrangements [37] and different sizes havealso been described [4349]

In this paper we present two patients referred to us forgenetic diagnosis of 22q112 deletion syndrome The first

patient was found to harbor an atypical deletion and thesecond one an inherited atypical duplication in the distalsegment of the TDR covering LCRs C and D [7] We dis-cuss screening diagnostic strategies for patients referredfor 22q112 deletion testing as well as the clinical implica-tions of these findings for a potential genotype-phenotypecorrelation

MethodsSamples and pictures from patients and their familieswere obtained after informed consent Ethical approvalwas obtained for this study from the IRB at Hospital Uni-versitario La Paz in Madrid (HULP-CEIC-PI347) Researchwas performed in compliance with the Declaration ofHelsinki

Patient 1A male infant was the first child of healthy non-consan-guineous parents of age 39 and 36 years Due to menstrualdysfunction in the mother artificial insemination wasperformed with an oocyte donation from a woman ofunknown age The child was born at 37 weeks of gestationand weighed 2450 g (10thndash25th centile) At age 3 monthshe was referred to Pediatric Cardiology because of respira-tory distress and an ASD (ostium secundum type)together with multiple muscular and perimembranousVSDs He was examined by a clinical geneticist and notedto have some dysmorphic features such as a triangularfacies with a slighty small chin and a broad nose (Figure1a b) He had a poor head control which he finallyachieved at the age of 4 months Cardiovascular surgerywas performed at that time At age 7 months he was re-evaluated his growth parameters were height 625 cm (lt5th centile) weight 5540 g (lt 3rd centile) and OFC 41 cm(5ndash10th centile) He was not sitting unsupported yet

Patient 2A male newborn was born to healthy 37 year-old parentsafter Cesarean section due to lack of progress at 33 weeksof gestation Birthweight was 2290 g (75th centile) Hewas referred to the Neonatology intensive care unitbecause of respiratory distress He was noted to have aright cleft lip with a complete cleft palate and upslantingpalpebral fissures with no other significant features onexamination He made good progress and was finally dis-charged two weeks later He was reviewed in clinic at agefive months His development was within the normalrange His growth parameters were height 62 cm weight6500 g and OFC 425 cm (all measurements between the10th and 25th centiles) (Figure 1c) His cleft lip had beenrepaired and the cleft palate operation was scheduled forthe age of nine months A later review at age 13 monthsand a half showed a good progress and a normal develop-ment




















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122 122 122

160 160 160

N N N

93 99 101 93 99 101 93 101

169 173 169 173 169 173

1 195 199 191 195 199 195 199

249 253 249 253 249 253

116 116 116

138 143 138 143 138 143

dup dup dup

8 216 227 208 216 227 216 220 227

4 102 106 94 102 106 94 102

dup dup dup

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CATCH 10 19116496 120 122 NA 120 122 122 120 122 122

CATCH 20 19245111 160 160 NA 160 160 162 160


LCR-C 19354000ndash19417000

CATCH 42 19449222 99 93 99 93 97 101 93 99 101 97 99 93 99 101

CATCH 41 19450208 NA 169 173 169 173 169 173 173 177 169 173

CATCH 11 19469542 197 195 197 195 199 191 195 199 195 199 191 195 199 19

D22S311 19503575 251 249 255 251 253 249 253 251 249 253

CATCH 12 19566292 116 116 116 116 116 116

CATCH 13 19567154 143 138 143 138 143 138 143 143 138 143


CATCH 14 19595454 212 212 215 200 216 227 208 216 227 200 210 208 216 227 20

CATCH 39 19640114 106 106 110 94 102 110 94 102 106 106 110 94 102 106 9


D22S1709 19735440 120 128 126 128 132 126 128 132 128 132 126 128 132 12

CATCH 38 19745025 157 159 159 159 155 159 159

CATCH 37 19759833 NA 226 234 230 245 230 245 230 245 230 245

LCR-D 19777000


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157 161 157 161 157 161

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106 106 106

212 222 212 222 212


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CATCH 36 20126412 109 109 109 109 109 109


LCR-D 20242000

LCRD 06321-L05796

20247101 del N N N N N

CATCH 35 20341833 136 136 150 136 150 136 143 143 150 136 143

D22S446bis 20343665 157 157 185 157 180 157 161 165 180 157 161

PPIL2 20379687 N N N N N N

D22S306 20887523 106 106 NA NA NA 106

D22S303 21599366 212 222 212 222 NA NA NA 212 222



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D-E - Atypical CHD


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BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References




















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122 122 122

160 160 160

N N N

93 99 101 93 99 101 93 101

169 173 169 173 169 173

1 195 199 191 195 199 195 199

249 253 249 253 249 253

116 116 116

138 143 138 143 138 143

dup dup dup

8 216 227 208 216 227 216 220 227

4 102 106 94 102 106 94 102

dup dup dup

6 128 132 126 128 132 126 132

159 159 159 163

230 245 230 245 230 245

dup dup dup

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CATCH 10 19116496 120 122 NA 120 122 122 120 122 122

CATCH 20 19245111 160 160 NA 160 160 162 160


LCR-C 19354000ndash19417000

CATCH 42 19449222 99 93 99 93 97 101 93 99 101 97 99 93 99 101

CATCH 41 19450208 NA 169 173 169 173 169 173 173 177 169 173

CATCH 11 19469542 197 195 197 195 199 191 195 199 195 199 191 195 199 19

D22S311 19503575 251 249 255 251 253 249 253 251 249 253

CATCH 12 19566292 116 116 116 116 116 116

CATCH 13 19567154 143 138 143 138 143 138 143 143 138 143


CATCH 14 19595454 212 212 215 200 216 227 208 216 227 200 210 208 216 227 20

CATCH 39 19640114 106 106 110 94 102 110 94 102 106 106 110 94 102 106 9


D22S1709 19735440 120 128 126 128 132 126 128 132 128 132 126 128 132 12

CATCH 38 19745025 157 159 159 159 155 159 159

CATCH 37 19759833 NA 226 234 230 245 230 245 230 245 230 245

LCR-D 19777000


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136 143 136 143 136 143

157 161 157 161 157 161

N N N

106 106 106

212 222 212 222 212


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ics

2009

10

48ht

tp

ww

wb

iom

edce

ntra

lcom

147

1-23

501

048

CATCH 36 20126412 109 109 109 109 109 109


LCR-D 20242000

LCRD 06321-L05796

20247101 del N N N N N

CATCH 35 20341833 136 136 150 136 150 136 143 143 150 136 143

D22S446bis 20343665 157 157 185 157 180 157 161 165 180 157 161

PPIL2 20379687 N N N N N N

D22S306 20887523 106 106 NA NA NA 106

D22S303 21599366 212 222 212 222 NA NA NA 212 222



BM

C M




















Kurahashi 1996 [70]







- CTAFS TOF PA








- Choanal atresia


D-E - Atypical CHD


E-F

































































































BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References


o

f 13

(pag

e nu

mbe

r not

for c

itatio

n pu

rpos

es)


122 122 122

160 160 160

N N N

93 99 101 93 99 101 93 101

169 173 169 173 169 173

1 195 199 191 195 199 195 199

249 253 249 253 249 253

116 116 116

138 143 138 143 138 143

dup dup dup

8 216 227 208 216 227 216 220 227

4 102 106 94 102 106 94 102

dup dup dup

6 128 132 126 128 132 126 132

159 159 159 163

230 245 230 245 230 245

dup dup dup

edic

al G

enet

ics

2009

10

48ht

tp

ww

wb

iom

edce

ntra

lcom

147

1-23

501



CATCH 10 19116496 120 122 NA 120 122 122 120 122 122

CATCH 20 19245111 160 160 NA 160 160 162 160


LCR-C 19354000ndash19417000

CATCH 42 19449222 99 93 99 93 97 101 93 99 101 97 99 93 99 101

CATCH 41 19450208 NA 169 173 169 173 169 173 173 177 169 173

CATCH 11 19469542 197 195 197 195 199 191 195 199 195 199 191 195 199 19

D22S311 19503575 251 249 255 251 253 249 253 251 249 253

CATCH 12 19566292 116 116 116 116 116 116

CATCH 13 19567154 143 138 143 138 143 138 143 143 138 143


CATCH 14 19595454 212 212 215 200 216 227 208 216 227 200 210 208 216 227 20

CATCH 39 19640114 106 106 110 94 102 110 94 102 106 106 110 94 102 106 9


D22S1709 19735440 120 128 126 128 132 126 128 132 128 132 126 128 132 12

CATCH 38 19745025 157 159 159 159 155 159 159

CATCH 37 19759833 NA 226 234 230 245 230 245 230 245 230 245

LCR-D 19777000


BM

C M L10754

Page

6 o

f 13

(pag

e nu

mbe

r not

for c

itatio

n pu

rpos

es)

109 109 109

N N N

N N N

136 143 136 143 136 143

157 161 157 161 157 161

N N N

106 106 106

212 222 212 222 212


edic

al G

enet

ics

2009

10

48ht

tp

ww

wb

iom

edce

ntra

lcom

147

1-23

501

048

CATCH 36 20126412 109 109 109 109 109 109


LCR-D 20242000

LCRD 06321-L05796

20247101 del N N N N N

CATCH 35 20341833 136 136 150 136 150 136 143 143 150 136 143

D22S446bis 20343665 157 157 185 157 180 157 161 165 180 157 161

PPIL2 20379687 N N N N N N

D22S306 20887523 106 106 NA NA NA 106

D22S303 21599366 212 222 212 222 NA NA NA 212 222



BM

C M




















Kurahashi 1996 [70]







- CTAFS TOF PA








- Choanal atresia


D-E - Atypical CHD


E-F

































































































BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References


o

f 13

(pag

e nu

mbe

r not

for c

itatio

n pu

rpos

es)

109 109 109

N N N

N N N

136 143 136 143 136 143

157 161 157 161 157 161

N N N

106 106 106

212 222 212 222 212


edic

al G

enet

ics

2009

10

48ht

tp

ww

wb

iom

edce

ntra

lcom

147

1-23

501

048

CATCH 36 20126412 109 109 109 109 109 109


LCR-D 20242000

LCRD 06321-L05796

20247101 del N N N N N

CATCH 35 20341833 136 136 150 136 150 136 143 143 150 136 143

D22S446bis 20343665 157 157 185 157 180 157 161 165 180 157 161

PPIL2 20379687 N N N N N N

D22S306 20887523 106 106 NA NA NA 106

D22S303 21599366 212 222 212 222 NA NA NA 212 222



BM

C M




















Kurahashi 1996 [70]







- CTAFS TOF PA








- Choanal atresia


D-E - Atypical CHD


E-F

































































































BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References





















Kurahashi 1996 [70]







- CTAFS TOF PA








- Choanal atresia


D-E - Atypical CHD


E-F

































































































BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References
































































































BioMedcentral


























Abstract

Background

Methods

Results

Conclusion

Background

Methods

Patient 1

Patient 2

Karyotype and FISH

MLPA



Results

Discussion

Conclusion


Competing interests


Acknowledgements

References


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