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A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot

A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot
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  ARTICLE A variant in the carboxyl-terminus of connexin40 alters GAP junctions and increases risk fortetralogy of Fallot Valentina Guida 1,11 , Rosangela Ferese 1,2,11 , Marcella Rocchetti 3 , Monica Bonetti 4 , Anna Sarkozy  1,12 ,Serena Cecchetti 5 , Vania Gelmetti 1 , Francesca Lepri 6 , Massimiliano Copetti 7 , Giuseppe Lamorte 1 ,Maria Cristina Digilio 6 , Bruno Marino 8,9 , Antonio Zaza 3 , Jeroen den Hertog  4,10 , Bruno Dallapiccola 6 and Alessandro De Luca* ,1 GJA5   gene (MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40. In humans,copy number variants of chromosome 1q21.1 have been associated with variable phenotypes comprising congenital heartdisease (CHD), including isolated TOF. In mice, the deletion of  Gja5   can cause a variety of complex CHDs, in particular of thecardiac outflow tract, corresponding to TOF in many cases. In the present study, we screened for mutations in the  GJA5   gene178 unrelated probands with isolated TOF. A heterozygous nucleotide change (c.793C 4 T) in exon 2 of the gene leading to thep.Pro265Ser variant at the carboxyl-terminus of the protein was found in two unrelated sporadic patients, one with classicanatomy and one with pulmonary atresia. This  GJA5   missense substitution was not observed in 1568 ethnically-matchedcontrol chromosomes. Immunofluorescent staining and confocal microscopy revealed that cells expressing the mutant proteinform sparse or no visible gap-junction plaques in the region of cell–cell contact. Moreover, analysis of the transfer of the gapjunction permanent tracer lucifer yellow showed that cells expressing the mutant protein have a reduced rate of dye transfercompared with wild-type cells. Finally, use of a zebrafish model revealed that microinjection of the  GJA5  -p.Pro265Ser mutantdisrupts overall morphology of the heart tube in the 37% (22/60) of embryos, compared with the 6% (4/66) of the  GJA5  wild-type-injected embryos. These findings implicate  GJA5   gene as a novel susceptibility gene for TOF. European Journal of Human Genetics   (2013)  21,  69–75; doi:10.1038/ejhg.2012.109; published online 20 June 2012 Keywords:  congenital heart disease; tetralogy of Fallot; 1q21.1;  GJA5  ; connexin 40 INTRODUCTION Tetralogy of Fallot (TOF, MIM no. 187500) is the most commoncyanotic heart defect (MIM no. 217095); it is estimated to occur in2.5–3.5 per 10000 live births, and accounts for 6.8% of all congenitalheart disease (CHD). 1 TOF can arise as the consequence of prenatalinfections, maternal illnesses, and maternal therapeutic and non-therapeutic drug exposures. A proportion of TOF cases is associatedwith chromosome imbalances; B 15% are heterozygous for chromosome22q11.2 deletion, and nearly 7% have trisomy 21 (Down syndrome). 2,3 Non-syndromic TOF can be caused by dominant mutations intranscriptional regulators (  NKX2.5 , 4,5 GATA4 , 6,7  ZFMP2/FOG2 , 8 FOXH1 9 ) and in genes involved in signal transduction (  NOTCH1 , 10  JAG1, 11,12 )  CFC1 , 9 TDGF1 , 9 GDF1 , 13 and  NODAL 14 ). However,mutations in these genes have been related also to other CHDs. 8,9,15–17 In addition, large  de novo  copy number variants have been implicated asa cause of non-syndromic TOF. 18 Among these, a copy number variantat chromosome 1q21.1 was found in 1% of non-syndromic sporadicTOF cases. 18 Recently, duplications at chromosome 1q21.1 have beenassociated with a  B 30-fold increase in the risk of TOF. 19 GJA5  gene(MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40 (Cx40), and is highly expressed in thehuman outflow tract. 18 A systematic study of the anatomy and histology of Cx40-deleted mice has revealed a variety of complex CHDs in theseanimals, with the most common malformations being of the conotruncaltype, corresponding to TOF in many cases. 20 In humans, abnormalitiesin the  GJA5  gene have been associated with atrial fibrillation. 21–23 In the present study, we evaluated whether  GJA5  mutations mightbe pathogenic in isolated TOF, by gene screening in a series of non-syndromic TOF patients. MATERIALS AND METHODS Study subjects The study cohort included 178 patients with non-syndromic TOF. Onehundred and forty-seven cases had TOF with classic anatomy and 31 had 1 Mendel Laboratory, Casa Sollievo della Sofferenza Hospital, IRCCS, San Giovanni Rotondo, Italy;  2 Department of Experimental Medicine, ‘Sapienza’ University, Rome, Italy; 3 Biotechnology and Biosciences Department, University of Milan-Bicocca, Milan, Italy;  4 Hubrecht Institute, KNAW and University Medical Centre Utrecht, Utrecht, TheNetherlands;  5 Section of Molecular and Cellular Imaging, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita `, Rome, Italy;  6 Medical Genetics, Cytogeneticsand Pediatric Cardiology, Bambino Gesu ` Children Hospital, IRCCS, Rome, Italy;  7 Unit of Biostatistics, Casa Sollievo della Sofferenza Hospital, IRCCS, San Giovanni Rotondo, Italy; 8 Pediatric Cardiology, Department of Pediatrics, ‘Sapienza’ University, Rome, Italy;  9 Eleonora Lorillard Spencer-Cenci Foundation, Rome, Italy;  10 Institute Biology Leiden, LeidenUniversity, Leiden, The Netherlands*Correspondence: Dr A De Luca, Istituto CSS-Mendel, Viale Regina Margherita 261, Rome 00198, Italy. Tel: +39 06 44160510; Fax: +39 06 44160548;E-mail: 11 These authors contributed equally to this work. 12 Current address: Northern Genetic Service, Institute of Human Genetics, Newcastle University, International Center for Life, Newcastle upon Tyne, UKReceived 30 January 2012; revised 26 April 2012; accepted 27 April 2012; published online 20 June 2012 European Journal of Human Genetics (2013) 21,  69–75 &  2013 Macmillan Publishers Limited All rights reserved 1018-4813/13  TOF with pulmonary atresia. All patients were sporadic with the exception of four familial cases with TOF with classic anatomy. All patients underwentclinical assessment, electrocardiogram, echocardiogram, and chest-X-ray studies. Diagnosis of TOF was obtained by echocardiogram in all children.All patients showed viscero-atrial situs solitus, d-ventricular loop, andnormally related great arteries. Patients showed neither major nor minorextracardiac features of DiGeorge syndrome/velo-cardio-facial-syndrome,neither deletion of 22q11 and 10p13 DiGeorge syndrome regions, which wereexcluded using standard molecular protocols. Moreover, these patients hadbeen previously analyzed for mutations in the  GATA4 ,  NKX2.5 ,  ZFPM2/FOG2 , GDF1 ,  ISLET1,  and  JAG1  genes by denaturing high performance liquidchromatography (DHPLC). 8,12 The study cohort also included 784 unrelatednormal control individuals obtained as ethnically matched anonymoussamples from the Mendel Institute (Italians of Caucasian srcin). Ethicalapproval for this study was obtained from the Ethics Committees of theparticipating institutions, and informed consent for the genetic analyses wasobtained from all patients or their legal guardians. Molecular studies To search for mutations in the  GJA5  gene, the entire coding region of the gene,together with exon–intron boundaries, was PCR amplified from genomicDNA. Primers were designed based on the cDNA in GenBank (NM_005266.5)and the corresponding genomic regions. PCR products were screened by DHPLC by use of the Wave 3500 HT System (Transgenomic, Omaha,NE, USA) at column temperatures recommended by Navigator software,version (Transgenomic). Amplimers with abnormal elution profileswere purified (Microcon PCR (Millipore, Billerica, MA, USA)) and thensequenced bidirectionally using the ABI BigDye Terminator Sequencing Kitv.3.1 (Applied Biosystems, Foster City, CA, USA) and ABI Prism 3130xlGenetic Analyzers (Applied Biosystems). Primer pair sequences, as well as PCR and DHPLC analysis settings, are available upon request. The HomoloGenetool of NCBI ( was used to analyze the level of conservation of sequence variants in orthologous genes. Numbering for the mutation started atthe adenine nucleotide (A) in the ATG initiation codon. Genotyping of   GJA5 c.793C 4 T variant in healthy controls was performed on genomic DNA using acustom TaqMan genotyping assay (Forward primer 5 0 -CCCCTCTGTGGGCATAGTC-3 0 ; reverse primer 5 0 -GCCATTCTCCAGGCACTGATTA-3 0 ; AppliedBiosystems), according to manufacturer’s protocol on a 7900HT real-timePCR platform (Applied Biosystems). The reactions were cycled with standardTaqMan conditions (2min at 50 1 C, 10min at 95 1 C, 40 cycles with 15s at 95 1 C,and annealing/extension at 60 1 C for 1min). The genotypes were called withthe SDS 2.2.2 software package (Applied Biosystems). Statistical analyses The occurrence of a mutation in a subject was assumed to follow a binomialdistribution, for which the power to detect at least one mutation was equal toone minus the probability to observe zero events. Study power for differentscenarios according to a set of hypothesized true mutation prevalence wasprovided (ie, from 2 to 10 per 1000 subjects). According to these scenarios asample size of 784 subjects has a power 4 99% to detect at least one mutationwith a type I error ( a ) equal to 0.05, assuming a mutation prevalence of 1% (Supplementary Figure S1).The prevalence difference of mutation between patients and controls wastested using the Fisher’s exact test. A  P  -value  o 0.05 was considered asstatistically significant.The Fisher’s exact test was used to compare the frequency distributions forthe morphology of the heart in zebrafish experiments. The significantthreshold was set at  P  ¼ 0.05. Plasmids constructs The single-base change resulting in the p.Pro265Ser amino-acid substitutionwas introduced into a full-length cDNA of the wild-type human  GJA5  cDNAcloned into a p-yellow fluorescence protein (YEFP)-N1 tagged plasmid, kindly provided by MH Gollob (Departement of Medicine, University of OttawaHeart Institute, Ottawa, Canada), by site-directed mutagenesis with the use of the QuickChange Site II-direct mutagenesis kit (Stratagene, La Jolla, CA, USA).Both wild-type and mutated human  GJA5  cDNAs were subsequently subcloned at the C-terminus of pCDNA6/V5HIS plasmid by using primersCx40 forward (5 0 -CACACAGGATTCATGGGCGATTGGAGCTTCCTG-3 0 )and Cx40 reverse (5 0 -CACACACTCGAGCACTGATAGGTCATCTGACCT-3 0 ),including restriction enzyme site for cloning. Localization studies For indirect immunofluorescent staining, murine neuroblastoma (N2A) cells,a gap-junction-deficient cell line, were transfected with  GJA5 -pYEFP-N1wild-type or mutated plasmid using lipofectamine 2000 (Invitrogen LifeTechnologies, Carlsbad, CA, USA), as indicated by the manufacturer protocol.Cells were blocked and fixed in 4% paraformaldeide at 24–48h aftertransfection. Nucleus was highlighted with Hoechst and the cells wereexamined using a fluorescence microscope (PCM Ecplise TE300, NikonInstruments, Tokyo, Japan).For confocal laser scanning microscopy, 3  10 3 N2A cells were seeded onglass coverslips, and transiently transfected with either wild-type or mutated GJA5 - pCDNA6/V5HIS plasmid, as described below. Cells were fixed after24–48h with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100,and stained with mouse monoclonal anti-V5 antibody (Life Technologies,Paisley, UK), followed by green-fluorescent Alexa Fluor-488 goat anti-mouseantibody (Molecular Probes, Eugene, OR, USA); actin cytoskeleton wasdetected by red-fluorescent Alexa Fluor-594 phalloidin; (Molecular Probes,Eugene, OR). The extensively rinsed cover glass was then mounted on themicroscope slide with Vectashield Mounting Medium with DAPI (VectorLaboratories, Burlingame, CA, USA). Imaging was performed on a Leica TCSSP2 AOBS confocal microscope (Leica Microsystems, Mannheim, Germany),using excitation spectral laser lines at 405, 488, and 594nm, tuned with anacousto-optical tunable filter. Signals from different fluorescent probes weretaken in sequential scanning mode. Dye transfer studies Dye transfer through gap junction channels was investigated using transiently transfected N2A cells; experiments were performed 24h after transfection withwild-type or mutated constructs. We patch-clamped only adjacent cells withYEFP-positive gap junctions localized at the cell membrane level in bothexperimental groups. Cells plated on glass coverslips were superfused at roomtemperature with Tyrode solution containing (m M ): 154 NaCl, 4 KCl, 2 CaCl 2 ,1 MgCl 2 , 5 HEPES-NaOH, 5.5 D-glucose, adjusted to pH 7.35. Lucifer Yellow (LY) CH (2m M ) (Molecular Probes, Eugene, OR, USA) was dissolved in thepipette solution containing (m M ): 110 K þ -aspartate, 23 KCl, 0.4 CaCl 2 (calculated free Ca 2 þ ¼ 10–7 M ), 3 MgCl 2 , 5 HEPES KOH, 1 EDTA KOH, 5ATP-Na salt, pH 7.3. Large patch electrodes (about 1.5 M O ) were used tofacilitate the diffusion of LY in the cytosol of the patched cell after achievementof the whole-cell configuration, detected by monitoring membrane capacitance(Multiclamp 700B, Axon Instruments, Inverurie, Scotland). Measurementswere performed with a Nikon TE200 inverted microscope at   63 magnifica-tion (Nikon Instruments). Cells were excited with a xenon lamp (100W)through suitable bandpass filters and a gray filter to limit dye bleaching. Cellimages were acquired once in bright field mode and every minute influorescence mode by a MOTIC 2300 camera. Image acquisition and analysiswere respectively controlled and performed by ImageJ software (NationalInstitutes of Health, Bethesda, MD, USA); exposure time and gain factor weremaintained constant throughout all measurements. Dye permeation throughgap-junction channels was investigated using N2A cell pairs. Fluorescence inthe injected and recipient cell was monitored at 1min intervals for 15min afterpatch rupture; cell autofluorescence was eliminated by digital subtraction of images acquired before dye dialysis (cell attached configuration). The timecourse of dye transfer to injected cell and, secondarily, to the recipient one wasobtained by measuring fluorescence changes over time. The fluorescence ateach time point was expressed as percent of the fluorescence measured at15min from the injected cell. Injected cell fluorescence at 15min, taken as areference, was assumed to result from equilibration with pipette solution andtherefore to represent a LY concentration of 2m M . LY concentration at eachtime-point was calculated from the fluorescence normalized to the reference GJA5   gene in tetralogy of Fallot V Guida  et al  70 European Journal of Human Genetics  one. The number of LY molecules (MN LY ) corresponding to each LYconcentration is: MN LY ¼ Vol  C  N, where Vol indicates cell volume(assumed 0.75pL),  C   is LY concentration and  N   is Avogadro’s number.LY flow rate is expressed as the change over time of MN LY  present in therecipient cell. Means were compared by two-way ANOVA. Statistical signifi-cance was defined as  P  o 0.05. Data are expressed as mean ± SE of independentdeterminations. Zebrafish and  in situ  hybridization experiments Zebrafish were kept and the embryos were staged as described before. 24 Allprocedures involving experimental animals were performed in compliancewith local animal welfare laws, guidelines, and policies.  In situ  hybridizationswere done essentially as described 25 using probe specific for myocardial markercardiac myosin light chain 2. 26 Embryos were cleared in methanol andmounted in 100% glycerol before pictures were taken. Phenylthiourea wasadded to suppress pigmentation in developing embryos at 20hpf. RNA and injections pCS2 þ  Gja5  constructs were linearized with  Not  I enzyme and cappedmRNA prepared with the Message Machine kit (Ambion, Austin, TX, USA).mRNA was diluted in nuclease-free water, and 1nl per embryo was injected atthe 1- to 2-cell stage. RESULTS Mutation analysis One hundred and seventy-eight unrelated TOF probands (eithersporadic ( n ¼ 174) or familial ( n ¼ 4)) with isolated TOF with( n ¼ 31) or without ( n ¼ 147) pulmonary atresia were screened formutations in  GJA5  gene by DHPLC. Amplicons bearing heterodu-plexes were then analyzed by DNA sequencing. A heterozygousnucleotide change (c.793C 4 T) in exon 2 of the gene leading to thep.Pro265Ser variant was found in two unrelated sporadic patientswith TOF, one with classic anatomy (TF131) and one with pulmonary atresia (AP18) (Figures 1a and b). None of them disclosed any conduction defect at electrocardiogram. The p.Pro265Ser substitutionwas found to alter an amino-acid residue that was highly conservedacross multiple species, including chimpanzee, dog, cows, mouse,rat, chicken, and zebrafish (GenBank accession numbers:XP_001156907.1, NP_001017442.1, NP_001071490.1, NP_032147.1,NP_062153.1, NP_990835.1, NP_001007214.1, (Figure 1c). Family members were unable to becontacted to verify if p.Pro265Ser had  de novo  occurrence or wastransmitted by parents. The p.Pro265Ser missense change was notobserved in 784 unaffected, unrelated, ethnically matched controlsubjects (Italians of Caucasian srcin) (1568 control chromosomes),a sample with a power  4 99% to detect a variant with 1% prevalencesuch as p.Pro265Ser substitution. Significantly, the prevalence of thissubstitution in patients was statistically different from that foundin controls (2/178 (1.12)  versus  0/784 (0.00),  P  ¼ 0.03). In additionto p.Pro265Ser variant, mutation analysis disclosed four previously reported  GJA5  polymorphisms (c.-188C 4 T (rs1775479); c.-185A 4 G(rs1692140); c.531C 4 T, p.Tyr123Tyr (rs2232191); c.*53A 4 G(rs1692141)). Figure 1  DHPLC elution profiles ( a ) and sequence electropherograms ( b ) of the novel  GJA5   variant p.Pro265Ser. The position of the mutated nucleotide isindicated by an arrow. ( c ) Phylogenetic conservation analysis of  GJA5   p.Pro265Ser variant. The mutant amino-acid residue 265 is highlighted in yellow. GJA5   gene in tetralogy of Fallot V Guida  et al  71 European Journal of Human Genetics  Protein localization studies To determine if the p.Pro265Ser variant had an effect on proteinlocalization, N2A cells deficient in gap junctional communicationwere transiently transfected with either the wild-type or thep.Pro265Ser mutant constructs directly ‘tagged’ with YEFP at thecarboxyl-terminus (CT) end of the protein and visualized using bothindirect immunofluorescent staining and confocal microscopy.Following 24h incubation, cells expressing wild-type Cx40 showedcytoplasmic punctate staining and aggregation at the plasmamembrane of large gap-junction plaques in the region of cell–cellcontact (Figure 2a). A different cellular distribution was observed forthe p.Pro265Ser variant. Cells expressing this variant formed sparse orno visible gap-junction plaques and had intracellular retentionof Cx40 within a wide array of punctuate structures (Figure 2b).Similar staining patterns were obtained using confocal microscopy (Figures 2c and d). Dye transfer studies In order to explore whether functional intercellular channels areformed, in particular by the mutant proteins that traffic to the plasmamembrane, the transfer of the gap junction permeant tracer LYbetween cells transfected either with the wild-type or mutantconstruct was investigated. Dye transfer efficiency was quantified by measuring the rate of dye transfer between adjoining cells. As shownin Figure 3a, LY dialyzed into a cell transfected with wild-type  GJA5 cDNAs diffused easily to the adjacent cell reaching equilibration inabout 13–15min. As LY does not diffuse through the N2A cellmembrane, the intercellular diffusion of the dye suggests the presenceof permeable gap junctions, in this case formed by Cx40. The rate of fluorescence increment in injected cells was similar between cellstransfected with mutant and wild-type ( n ¼ 14 for both) constructsthus their results were pooled. On the other hand, the rate of fluorescence increment in the recipient cell was lower steep in thepresence of mutated gap junctions ( P  o 0.05). For instance, at 13minthe relative fluorescence dye intensity was 18.9 ± 3.4% (correspondingto 2.9  10 5 dye molecules per second) and 9.2 ± 2.7% (correspondingto 1.3  10 5 dye molecules per second) with wild-type and mutatedconstructs, respectively, ( P  o 0.05, Figure 3b). Zebrafish experiments Previous data have shown that Cx40 has an important role incardiomyocytes of the atria, cardiac conduction system, and endothe-lial cells of large arteries. It has already been demonstrated that Cx40knockout mice exhibit cardiac electrophysiological and morphologicalphenotypes. 27 We used the zebrafish embryo model system toelucidate the role of mutant  GJA5  in heart patterning  in vivo .To this end, we injected synthetic mRNA encoding mutant GJA5 -p.Pro265Ser or as a control wild-type  GJA5  into zebrafishembryos at the 1- to 2-cell stage and assessed the morphology of theheart. Aberrant heart morphology was apparent in embryos-expressing mutant  GJA5  at 24h post fertilization.  In situ hybridization analysis for cardiac myosin light chain 2, a cardiacmyosin marker, showed that the overall morphology of the heart tubewas disrupted in the 37% (22/60) of   GJA5 - p.Pro265Ser-injectedembryos, compared with the 6% (4/66) of   GJA5 -wild-type-injectedembryos (Fisher’s exact test,  P  o 0.0001) (Figure 4). Noteworthy, themutant  GJA5 -injected embryos did not display gross developmentaldefects and had normal body size compared with wild-type  GJA5 -injected embryos. These data demonstrate that mutant  GJA5 -p.Pro265Ser has a dominant effect over endogenous wild-type  GJA5 and disrupts normal heart morphology. DISCUSSION Deficient or improper gap junction channel function caused by mutations in the genes encoding connexin proteins has beenassociated with a variety of diseases, such as peripheral neuropathy,oculo-dento-digital dysplasia, sensorineural deafness, skin disorders,and cataracts. 28 This is the first report of   GJA5  variant in TOFpatients and in general in CHDs. Various  GJA5  defects other thanp.Pro265Ser were previously associated with familial and sporadicatrial fibrillation. 21–23 Similarly, different mutations of cardiactranscription factor  NKX2.5  have been identified in kindreds withconduction abnormalities (atrioventricular block) and concurrentcongenital heart malformations, primarily secundum atrial septaldefect, as well as in patients with sporadic TOF. 4,15,29 In the presentstudy, variant p.Pro265Ser was found in B 1% of TOF patients. Thelow incidence of p.Pro265Ser variant in the cohort we studied may be consistent with the high degree of heterogeneity of this CHD. Figure 2  Differential localization of the wild-type and  GJA5   p.Pro265Sermutant proteins in N2A cells. N2A cells were transfected with plasmidsencoding either wild-type  GJA5   or the  GJA5   mutant p.Pro265Ser, taggedwith YEFP at the CT. In cells expressing YEFP-tagged  GJA5   wild-typegap-junction-like structures were readily seen at cell–cell interfaces (whitearrows) ( a ). However, cells expressing YEFP-tagged p.Pro265Ser mutantfailed to assemble gap junctions or formed sparse gap junctions ( b ).Confocal microscopic analysis of the wild-type and  GJA5-   p.Pro265Sermutant proteins in N2A cells. N2A cells expressing wild-type  GJA5   formedlarge gap-junction plaques between adjacent cells ( c ). In contrast, N2Acells transfected with the p.Pro265Ser mutant showed formation ofpunctuate gap-junction-like structures at cell–cell interfaces ( d ). GJA5   gene in tetralogy of Fallot V Guida  et al  72 European Journal of Human Genetics  On analysis of 230 patients with both syndromic and non syndromicTOF, genetic abnormalities were identified in the 18% of patients. 30 The most common genetic alteration was 22q11.2 deletion associatedwith DiGeorge/VCFS syndrome. Single-gene mutations in  JAG1 ,  NKX2.5 , and  TBX1  were found in 1.3%, 0.9%, and 0.4% of thecohort, respectively. In a parallel study, we found  JAG1 ,  NKX2.5 , and  ZPFM2/FOG2  mutations in 2.7, 1.1, and 0.6% of sporadic patientswith isolated TOF. 8,12 Clearly, TOF is a heterogeneous CHD, withsmall contributions from different loci. Present results further expandthe genetic heterogeneity of this defect. GJA5  gene mutated in TOF maps to the 1q21.1 chromosomalregion. Microdeletion 1q21.1 (del 1q21.1) and the reciprocal micro-duplication 1q21.1 (dup 1q21.1) are newly recognized genomicdisorders, characterized by developmental delay, neuropsychiatricabnormalities, dysmorphic features, and a variety of congenitalmalformations, including CHDs. 31–33 In addition, both del 1q21.1and dup 1q21.1 have been found in patients with non-syndromicCHD. 18,34,35 A recent study of 512 patients with apparently isolatedsporadic TOF using an array designed to detect deletions orduplications revealed that 5/512 individuals had a deletion orduplication of chromosome 1q21.1, encompassing  GJA5  gene. 18 Amore recent study further defined the relationship between 1q21.1rearrangements and isolated TOF showing that 1q21.1 duplicationsconfer an odds ratio for TOF of   B 30. 19 Furthermore, within the1q21.1 region, the same authors identified three smaller overlappingduplications in three patients with TOF in which  GJA5  gene was theonly gene in common. Moreover, they found a  GJA5  triplication inone patient with pulmonary atresia. 19 Similarly, we identified  GJA5 p.Pro265Ser variant in one patient with TOF and one with TOF andpulmonary atresia, which is considered the extreme end of theanatomic spectrum of TOF and is a cardiac defect with a very highgenetic involvement. 36,37 Taken together, present and previousfindings strongly suggest that  GJA5  is the key gene in the etiology of cardiovascular diseases found in patients with 1q21.1 chromosomeimbalances.In agreement with a pathogenic role of Cx40 mutations in CHDs,previous studies on animal models have demonstrated that this genehas a major role in the cardiac conduction system and in heartmorphogenesis.  Cx40   gene is strongly expressed in both atria andventricles in early stages of development. 38 Kirchhoff   et al  39 have Figure 3  Dye transfer (LY) through gap-junction channels in N2A cell pairs transfected with either wild-type (WT), or mutated (MUT) constructs.( a ) Examples of the intercellular diffusion of the dye at different time points after patch rupture (time 0); the bright field image and the fluorescence one at15min after patch rupture are superimposed. ( b ) Average time course of absolute (left panel) and relative (right panel) fluorescence; in each panel theupper curve refers to the injected cell (data from WT and MUT were pooled) and the lower ones to WT and MUT recipient cells, respectively. Figure 4  Expression of mutant  GJA5  -p.Pro265Ser disrupts zebrafish heartmorphology in zebrafish  in vivo  .  In situ   hybridization staining for cardiacmyosin (cardiac myosin light chain 2) in 48hpf zebrafish embryos that wereinjected at the 1- to 2-cell stage with wild-type  GJA5   (left) or mutant  GJA5  -p.Pro265Ser (right). The overall morphology of the heart tube is disruptedin  GJA5  -p.Pro265Ser-injected embryos compared with  GJA5  -wild-type-injected embryos. Dorsal views are shown with anterior to the top.A, atrium; v, ventricle. GJA5   gene in tetralogy of Fallot V Guida  et al  73 European Journal of Human Genetics
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