ß2007Wiley-Liss,Inc.American Journal of Medical Genetics Part A143A:2738–2743(2007)
Clinical Report
Complex Balanced Translocation
t(1;5;7)(p32.1;q14.3;p21.3)and Two Microdeletions del(1)(p31.1p31.1)and del(7)(p14.1p14.1)in a Patient With Features of Greig Cephalopolysyndactyly
and Mental Retardation
Katarzyna Borg,1Beata Nowakowska,1,2Ewa Obersztyn,1Sau Wai Cheung,2
Joanna Brycz-Witkowska,3Lech Korniszewski,4Tadeusz Mazurczak,1
PawełStankiewicz,1,2*and Ewa Bocian1
1Department of Medical Genetics,Institute of Mother and Child,Warsaw,Poland
2Department of Molecular and Human Genetics,Baylor College of Medicine,Houston,Texas
3Novum Fertility Clinic,Cytogenetic Laboratory,Warsaw,Poland
4Institute of Physiology and Pathology of Hearing,Outpatient Genetic Clinic,Warsaw,Poland
Received26January2007;Accepted11July2007
Complex chromosome rearrangements(CCRs)are rare struc-tural abnormalities that involve at least two chromosomes and more than two breakpoints and are often associated with developmental delay,mental retardation,and congenital anomalies.We report on a de novo,apparently balanced translocation t(1;5;7)(p32.1;q14.3;p21.3)involving three chro-mosomes in a7-year-old boy with severe psychomotor retardation,neonatal muscular hypertonia,congenital heart defect,polysyndactyly of hands and feet,and dysmorphic features resembling Greig cephalopolysyndactyly syndrome. Analysis of the chromosome breakpoints usingfluorescence in situ hybridization(FISH)with locus-specific BAC clones and long-range PCR products did not identify chromosome imbalance at any of the interrogated regions.High-resolution comparative genomic hybridization(HR-CGH)and array CGH (aCGH)revealed two additional cryptic de novo deletions, del(1)(p31.1p31.1)and del(7)(p14.1p14.1),respectively,that are not associated with the translocation breakpoints.FISH and polymorphic marker analyses showed that the deletion on derivative chromosome1is between4.2and6.1Mb,and the deletion on derivative chromosome7is approximately5.1Mb, and that both are paternal in origin.The deletion on chromosome7p encompass
es the GLI3gene that is causative for the Greig cephalopolysyndactyly,Pallister–Hall and some cases of Acrocallosal syndromes.We discuss the potential mechanisms of formation of the described CCR.
ß2007Wiley-Liss,Inc.
Key words:complex chromosomal rearrangement;Greig cephalopolysyndactyly;GLI3gene;array and high-resolu-tion comparative genomic hybridization;interchromosomal effect;microdeletion
How to cite this article:Borg K,Nowakowska B,Obersztyn E,Cheung SW,Brycz-Witkowska J,Korniszewski L, Mazurczak T,Stankiewicz P,Bocian E.2007.Complex balanced translocation t(1;5;7)(p32.1;q14.3;p21.3)and two microdeletions del(1)(p31.1p31.1)and del(7)(p14.1p14.1)in a patient with features of Greig cephalopolysyndactyly and mental retardation.Am J Med Genet Part A143A:2738–2743.
单增李斯特菌Katarzyna Borg and Beata Nowakowska contributed equally to this work.
Grant sponsor:Polish Ministry of Scientific Research and Information Technology;Grant number:PBZ-KBN-122/P05/2004-01-1;Grant spon-sor:National Institute of Child Health and Develop
ment;Grant number: PO1HD39420;Grant sponsor:Baylor College of Medicine Mental Retardation Research Center;Grant number:HD24064.
*Correspondence to:PawełStankiewicz,M.D.,Ph.D.,Department of Molecular and Human Genetics,Baylor College of Medicine,One Baylor Plaza,Rm.S921,Houston,TX77030.E-mail:pawels@bcm.edu
DOI
10.1002/ajmg.a.32017
INTRODUCTION
卡波940
Complex chromosome rearrangements(CCRs)are rare structural abnormalities that involve more than two breakpoints and exchange of genetic material between two or more chromosomes[Kleczkowska et al.,1982].Typically,CCRs are three-way trans-locations with one breakpoint in each chromosome; however,CCRs with up tofifteen breakpoints have been reported[H
ouge et al.,2003].The frequency of anomalies in carriers of apparently balanced trans-locations is approximately6%[Warburton,1991]. In such cases,the phenotypic abnormalities are thought to result from disruption of a gene(s)at chromosome breakpoint(s),additional cryptic rear-rangements,or position effect[Borg et al.,2002; McMullan et al.,2002;Astbury et al.,2004;Kleinjan and van Heyningen,2005;Johnson et al.,2006;Yue et al.,2006].Several apparently balanced rearrange-ments have been studied usingfluorescence in situ hybridization(FISH)and array comparative genomic hybridization(aCGH)methods.Additional complex-ities or genome imbalances such as loss,gain or inversion at or near the breakpoint have been identified in10–60%of cases[Kirchhoff et al.,2001; Borg et al.,2002,2005;Vissers et al.,2003;Astbury et al.,2004;Tyson et al.,2004;Shaw-Smith et al., 2004;Gribble et al.,2005;Cheung et al.,2005]. Microdeletions not directly involved in the trans-location breakpoints but situated on derivative chromosomes have been reported in only two patients[Tyson et al.,2004;Gribble et al.,2005]. We report on a7-year-old boy with severe psycho-motor retardation,neonatal hypertonia,congenital heart defect,polysyndactyly of hands and feet and dysmorphic features in whom we found a de novo, complex translocation t(1;5;7)(p32.1;q14.3;p21.3) and two cryptic deletions del(1)(p31.1p31.1)and del(7)(p14.1p14.1).
MATERIALS AND METHODS
Clinical Report
The proband is a7-year-old boy who presented at birth with dysmorphic features and multiple congenital anomalies.He was the3,500g,appropriate for gestational age,product of a38week gestation delivered vaginally and born to a35-year-old mother whose pregnancy was not complicated by maternal illness,toxic exposure,medication or substance use. The father of the pregnancy was36years old and the family history was negative for congenital anomalies or mental retardation.Chorioamnionitis was suspected at delivery.Apgar scores were6and9at1and5min, respectively.Clinical examination revealed an ap-parently normal head circumference with facial dysmorphisms including micrognathia,apparent hypertelorism,wide nasal bridge,bulbous nasal tip,thin upper lip,relatively large anterior fontanel, preaxial polydactyly of hands with bilateral duplication and partial cutaneous syndactyly of the distalfirst phalanx and preaxial polydactyly with partial cuta-neous syndactyly of the second and third toes.Neuro-logical assessment revealed horizontal nystagumus, generalized hypertonia,and brisk deep tendon reflexes.There was a high-pitched cry.Postnatal cranial ultrasound revealed mild cerebral ventricular dilatationand acyst ofseptumpellucidum.Echocardio-graphy showed a ventricular septal defect and a secundum atrial septum defect.Abdominal ultrasound revealed duplication of both ureters.Radiographs of the extremi
ties confirmed bilateral duplication of halluces andfirst metatarsals.A diagnosis of Saethre–Chotzen syndrome was suspected(Fig.1).
The patient was reassessed at age7years and was noted to have severe psychomotor retardation with spastic tetraparesis and absent speech.Clinical examination revealed microcephaly(OFC51cm [À1.2SD])with low set,posteriorly rotated ears,high forehead,apparent hypertelorism,broad nasal root, downslanting palpebralfissures,and synophrys.The neck was short and there was hirsutism of the back and limbs.Growth parameters were significant for a length of114cm(À1.91SD)and of weight28.2kg (þ1.1SD)and BMI(þ3.89SD).Although the patient had several features of Greig cephalopolysyndactyly syndrome(GCPS,OMIM175700),the clinical picture was complex and more severe than observed in GCPS.Some symptoms resembling Acrocallosal syndrome(ACLS;OMIM200990)were also observed. Imaging of the central nervous system was
not
F IG.1.Patient at age2months.Note(a)hypertelorism,wide nasal bridge, bulbous nasal tip,thin upper lip,(b)micrognathia,(c)preaxial polydactyly of left hand with bilateral duplication and partial cutaneous syndactyly of the distal first phalynx,and(d)preaxial polydactyly of toes.[Colorfigure can be viewed in the online issue,which is available at www.interscience.wiley.]
结晶紫
COMPLEX BALANCED REARRANGEMENT IN A PATIENT WITH GCPS AND MR2739
performed,thus the presence of agenesis of corpus callosum or other anomalies could not be excluded.
Cytogenetic,FISH,and Whole
Genome Analysis
The karyotype analysis of the proband and his parents were performed on peripheral blood lympho-cytes using the standard GTG banding technique. FISH studies with whole chromosome painting (wcp)probes for chromosomes1,5,and7(Oncor) and subtelomeric probes(Chromoprobe Multip-robe-T System,Cytocell Technologies,Cambridge, UK)were performed on metaphase spreads accord-ing to the manufacturers’specifications.Chromo-some breakpoints were mapped using FISH with39 region-specific BAC clones selected from the phys-ical maps of the regions of interest using the UCSC Browser(genome.ucsc.edu/)and obtained from the Sanger Institute(www.sanger. ac.uk).
We also performed FISH with6,000-8,000bp in size PCR products as probes to narrow the trans-loc
ation breakpoint at7p21.3.The primers were designed using Primer3program(frodo.wi. mit.edu/).PCR reaction was performed using the LA PCR Kit Ver.2.1(Takara Bio,Inc.,Otsu,Shiga,Japan) on Gradient Thermal Cycler(BIO-RAD,Hercules, CA)with reaction profiles:948C(1min),45cycles at of988C(20sec),688C(11min),and728C(10min). Products were labeled with digoxigenin in nick-translation reaction(DIG-Nick Translation Mix, Roche,Mannheim,Germany)and visualized with rhodamine-labeled antibodies(Sigma,Munnich, Germany).
HR-CGH was performed as described by Kirchhoff et al.[1998].The result of HR-CGH analysis was verified by FISH with11BAC clones.Array CGH designed to cover genomic regions of75known genomic disorders including Greig syndrome region, all41subtelomeric regions,and43pericentromeric regions(Bayor College of Medicine,Chromosome Microarray Analysis,V.5,www.bcm.edu/ cma/assets/abnormalities.pdf)was used as previ-ously described[Cheung et al.,2005]and the results were verified by FISH analysis using15BAC clones. Parental origin was assigned by using nine dinucleo-tide repeat microsatellite markers from the UCSC browser.PCR primers were designed used Primer3 program.The products were amplified using Taq PCR Core Kit(Qiagen,Hilden,Germany)and were separated on8%polyacrylamide gels. Int
erspersed repeat sequences within the down-loaded DNA sequence of the clones were eliminated by RepeatMasker(genome.ucsc.edu)and the repeat masked genomic sequences were analyzed for the presence of low-copy repeats(LCRs)using NCBI BLAST2(bi.v/blast/ bl2seq/bl2.html).
RESULTS
The chromosome banding analysis in the pro-band at the550band resolution level revealed an apparently balanced complex translocation t(1;5;7)(p32;q15;p22)that was verified by FISH with the wcp and subtelomeric probes(Fig.2a,b).The parental chromosome analyses were normal.The 1p32.1breakpoint was spanned by BAC clone RP11-13N22.The breakpoints at5q14.3and7p21.3were narrowed to$400kb(between BAC clones RP11-1029H20and RP11-10N5and RP11-125A23and RP11-482M1,respectively).Four genes were identi-fied at the breakpoints.Two of them,CYP2J2 (1p32.1)and NXPH1(7p21.3)appeared to be good candidate genes responsible for the heart defect in our patient.FISH analysis with two CYP2J2-specific long-range PCR probes narrowed the breakpoint to approximately10kb fragment that includes part of CYP2J2.
Array CGH identified a deletion of three BAC clones (RP11-260M3,RP11-706L12,and RP11-2J17)at7p14.1 (Fig.2c).HR-CGH verified the presence of the7p14.1 submicroscopic deletion(Fig.2d)and unexpectedly revealed a second microdeletion at1p31.1(Fig.2e). The latter deletion was not detected by the aCGH because no clones from this region were present on the microarray.We mapped the breakpoints of both deletions using26BAC clones and defined their sizes to between4.2and6.1Mb for del(1)(p31.1p31.1)and $5.1Mb for del(7)(p14.1p14.1).The proximal break-point of the1p31.1deletion was mapped between BAC clones RP11-653A5and RP11-243K11and the distal breakpoint between RP11-70K7and RP11-42O15.The proximal breakpoint of the7p14.1deletion was localized between the BAC clones RP11-2J17 and RP11-429N13clones and the distal breakpoint mapped between clones RP11-786M13and RP11-164E6.Interestingly,both deletions were localized on the translocation derivative chromosomes1and7, approximately15Mb from the1p32.1translocation breakpoint and28Mb from the7p21.3translocation breakpoint.
The two microsatellite markers analyzed for del(1) (28AT and26GT)and three for del(7)(24GT,23AC and22CA)were informative and showed only one maternal allele,indicating that both deletions are of the paternal origin.
DNA sequence analysis of the genomic regions surrounding the chromosome translocation break-po
ints using NCBI BLAST2revealed no significant similarity or evidence for LCRs.
DISCUSSION
Abnormal phenotypes observed in persons who harbor apparently balanced chromosomal rear-rangements are thought to result from disruption of a gene(s)at chromosome breakpoint(s),additional
2740BORG ET AL.
genomic imbalance undetected by the routine karyotyping,or position effect.Recently,genome analyses using techniques of higher resolution such as HR-CGH and aCGH have enabled identification of previously undetected submicroscopic imbalances at the translocation breakpoints or in other genomic regions in patients with CCR [Kirchhoff et al.,2001;Borg et al.,2002,2005;Astbury et al.,2004;Gribble et al.,2005;Cheung et al.,2005].CCRs with sub-microscopic deletions on the derivative translocation chromosomes,not localized at the breakpoints,are very rare,and to date,only two cases with such deletion have been published [Tyson et al.,2004;Gribble et al.,2005].Two microdeletions identified in our patient were also localized outside the trans-location breakpoint regions on the derivative chro-mosomes 1and 7.
The deletion on chromosome 7harbors the GLI3gene encoding a zinc-finger transcription factor that plays an important role during embryogenesis via the sonic hedgehog signaling pathway.Loss of function or haploinsufficiency of GLI3due to point mutation,deletions or disruption by chromosome breakpoints have been shown to cause GCPS [Elson et al.,2002;Biesecker,2006;Schwarzbraun et al.,2006].Muta-tions in GLI3have been found also in allelic PHS,preaxial polydactyly type IV,and postaxial poly-dactyly types A1and B.Clinical symptoms of these disorders overlap also with ACLS.
The proband presented with many features of the GCPS clinical spectrum,including scaphocephaly,high forehead,frontal bossing,apparent hypertelor-ism,broad nasal root,preaxial polydactyly of hands with duplication of distal phalanges of the thumbs,partial cutaneous syndactyly of fingers 3and 4,preaxial polydactyly of feet with broad halluces and partial cutaneous syndactyly,cardiac defect and hirsutism.In GCPS,preaxial (as opposed to post-axial)polydactyly of hands is rare whereas preaxial polydactyly of feet is common.In contrast,preaxial polysyndactyly of hands is found more often in ACLS.Other features common to ACLS in the proband include congenital heart disease,severe mental retardation,prominent forehead,and hypertelorism.However,he did not manifest seizures or macro-cephaly,which are typical for patients with ACLS,yet are seen in only 52%of GCPS patients [Gorlin et al.,2001].Severe MR and muscle hypertonia with
spastic
F I
东北大学校长活捉东南大学校长G .2.Three-way translocation and two cryptic deletions identified by array and HR-CG H and FISH methods.a :FISH with whole chromosome 1(green)and 7(red)painting probes.b :G-banded rearranged chromosomes with the breakpoints (black arrows)defined by FISH with region-specific BAC probes.Red arrows indicate position of two identified cryptic interstitial deletions at 7p14.1and 1p31.1.c :Array CGH profile showing the deletion of three BAC clones at 7p14.1(arrow).Results of HR-CGH analysis demonstrating two microdeletions at chromosomes (d )7p and (e )1p (red vertical bars).[Color figure can be viewed in the online issue,which is available at www.interscience.wiley.]
COMPLEX BALANCED REARRANGEMENT IN A PATIENT WITH GCPS AND MR
2741
tetraparesis present in the proband are relatively rare in the classic form of GCPS.Severe MR has been described more frequently in GCPS patients with chromosomal deletions involving GLI3.These cases have been designated as GCPS contiguous gene syndrome.To date,more than20patients with
features characteristic of GCPS and GLI3deletions ranging in size from151kb to15Mb have been reported[Johnston et al.,2003,2005;Schwarzbraun et al.,2006].Johnston et al.[2003]concluded that such patients are likely to have cognitive deficits and more often have agenesis of corpus callosum, craniosynostosis,cardiac defects,likely as a result of deletion of additional genes.
The7p14.1deletion harbors a C7orf10gene. Interestingly,this gene has been proposed to be responsible for the Russell-Silver syndrome[Naka-bayashi et al.,2002].However,our patient does not present any features characteristic for RSS,suggest-ing a different function of the gene.
We hypothesize that the intellectual disability and cardiac defects in our patient may be due to deletion or disruption of the other genes localized at trans-location breakpoints regions or within the deletions. Molecular and cytogenetic analyses indicate that the described CCR arose during spermatogenesis. Thisfinding corroborates previous observations that CCRs usually arise in spermatogenesis whereas transmission of these anomalies is more common through oogenesis in subsequent generations as opposed to male constitutional carriers[Batista et al., 1994].We propose two models of formation of the described CCR(Fig.3).As the microdeletions in our patient and in other reports[Tyson et al.,2004; Gribble et al.,2005]were present on the derivative chromosomes and not on the normal homologues a one step rather than two step mechanism is more plausible.Microd
eletions might have arisen simulta-neously before or during DNA replication phase in an early stage of meiosis I.The alternative multi-step models are less parsimonious.
In conclusion,we present a patient with a complex clinical phenotype whose genomic compliment was only elucidated following application of aCGH and HR-CGH despite him having an abnormal G-banded
停电宝
chromosome analysis.Furthermore,our analysis of this patient revealed that haploinsufficiency of GLI3 is responsible for much of his craniofacial,skeletal, and neurological phenotype.Thisfinding would not be at all apparent by conventional cytogenetic analysis.Utilization of aCGH and HR-CGH in the clinical laboratory is essential for diagnosis,prog-nosis,management and risk assessment in patients with multiple congenital anomalies.We predict that wider application of array CGH technique will identify more cases of imbalances in apparently balanced aberrations,thus helping to better correlate genotypes with phenotypes and unravel the mech-anisms of their formation.
ACKNOWLEDGMENTS
We gratefully acknowledge the Wellcome Trust Sanger Institute for providing BAC clones.We thank Dr. A.L.Beaudet and Dr.L.Potocki for helpful discussions and suggestions and Joanna Pietrzak for te
chnical assistance.
REFERENCES
Astbury C,Christ LA,Aughton DJ,Cassidy SB,Kumar A,Eichler EE,Schwartz S.2004.Detection of deletions in de novo ‘‘balanced’’chromosome rearrangements:Further evidence for their role in phenotypic abnormalities.Genet Med6:
F IG.3.Schematic representation of two proposed models of the origin of the described CCR.a:In a two-step model,a three-way balanced complex translocation arosefirst either in a primordial cell or in an early phase of meiosis I.The hexagonal pachytene structure of the translocation chromosomes1,5, and7exposed the chromosomes to additional tensions near the translocation breakpoint that resulted in the two submicroscopic deletions.b:In a one-step mechanism,both microdeletions occurred on the derivative chromosomes1 and7concurrently with the three-way translocation.Arrows indicate the deleted fragments.
2742BORG ET AL.
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