Submicroscopic Imbalances Research Articles

Prenatally diagnosed 16p11.2 copy number variations by SNP Array: A retrospective case series

ObjectiveThe 16p11.2 copy number variations (CNVs) are increasingly recognized as one of the most frequent genomic disorders, with a broad spectrum of phenotypes. The fetal phenotype associated with 16p11.2 CNVs is poorly described. The current study presents prenatal series of 16p11.2 CNVs and provides a better understanding of this submicroscopic imbalance in prenatal diagnosis. MethodRetrospective case series were extracted from a single tertiary referral center performing prenatal single nucleotide polymorphism (SNP) array from April 2017 to December 2021. The maternal demographics, indication for amniocentesis, ultrasound findings, SNP array results, inheritance of the CNVs, and pregnancy outcomes were studied. ResultsWe indentified 30 fetuses carrying 16p11.2 CNVs, representing 0.35% (30/8578) of prenatal SNP array results. The series included 17 fetuses with a proximal deletion, 7 with a distal deletion, 4 with a proximal duplication, and 2 with a distal duplication. Prenatal ultrasound anomalies were reported in 80% of these cases. The most common presentation was vertebralanomalies (9/30). Other features noted in more than one fetus were increased nuchal translucency/nuchal fold (NT/NF) (5/30), absent/hypoplastic nasal bone (3/30), polyhydramnios (3/30), ventricular septal defect (VSD) (2/30), unilateral mild ventriculomegaly (2/30), fetal growth restriction (FGR) (2/30), right aortic arch (2/30). All the 9 vertebralanomalies were present in fetuses harboring proximal deletion (9/17). Familial transmission was confirmed in 44% of cases (11/25) and termination of pregnancy was requested in 62.1% (18/29) of cases. ConclusionThe 16p11.2 CNVs can have variable prenatal phenotypes and these CNVs are frequently inherited from parents with a milder or normal phenotype. Our results underline that vertebral deformities were frequent in cases of 16p11.2 proximal deletion, and further demonstrate the incomplete penetrance of the CNVs.

Abnormal chromosomes identification using chromosomal microarray

In this study, we presented a case series to highlight the chromosomal microarray (CMA) in identifying chromosomal abnormalities which is undetectable by conventional karyotyping or known abnormal chromosomes without clear diagnosis. Extensive studies showed that CMA was gradually accepted as a prenatal invasive testing during pregnancy. The aim of this study was to evaluate the diagnostic effect of CMA for foetuses with abnormal chromosomes unrecognised by conventional karyotyping. Pregnant women who need prenatal diagnosis with all indications were enrolled in this study. For aberrant cytogenetic findings that cannot be defined by routine karyotyping, single nucleotide polymorphism array (SNP-array) was used. Six cases with abnormal karyotype were included in the study. With higher resolution of translocation breakpoints, CMA could detect smaller chromosomal imbalances that were undetectable by karyotyping. This study highlights the value of CMA for the detection of submicroscopic abnormalities in foetuses that cannot be detected by conventional karyotyping. Impact Statement What is already known on this subject? Chromosomal microarray (CMA) offers additional diagnostic benefits by revealing submicroscopic imbalances or copy number variations (CNVs) that are too small to be identified on a standard G-banded chromosome preparation. What do the results of this study add? We added a case series to highlight the CMA in identifying chromosomal abnormalities not detectable by conventional karyotyping or known abnormal chromosomes without clear diagnosis. What are the implications of these findings for clinical practice and/or further research? This study highlights the value of CMA in the case of associated foetuses with submicroscopic abnormalities that cannot detect by conventional karyotyping.

Array-comparative Genomic Hybridization Results in Clinically Affected Cases with Apparently Balanced Chromosomal Rearrangements.

Carriers of apparently balanced chromosomal rearrangements (ABCRs) have a 2-3-fold higher risk of carrying an abnormal phenotype, when compared to the average population. Apparently balanced chromosomal rearrangements can be imbalanced at the submicroscopic level, and changes in the gene structure, formation of a new chimeric gene, gain or loss of function of the genes and altered imprinting pattern may also affect the phenotype. Chromosomal microarray (CMA) is an efficient tool to detect submicroscopic imbalances at the breakpoints as well as in the whole genome. We aimed to determine the effectiveness of array-comparative genomic hybridization (aCGH) application in phenotypically affected cases with ABCRs at a single center from Turkey. Thirty-four affected cases (13 prenatal, 21 postnatal) carrying ABCRs were investigated with CMA. In postnatal series, ABCRs were familial in 7 and de novo in 14 cases. Seven de novo cases were imbalanced (in postnatal series 33.3% and in de novo cases 50.0%). Out of 13 prenatal cases, five were familial and eight were de novo in origin and two de novo cases were imbalanced (in 15.4% prenatal series and in 25.0% de novo cases). No cryptic imbalance was observed in familial cases. The anomaly rates with array studies ranged between 14.3-25.0% in familial and between 20.0-57.5% in de novo cases of postnatal series in the literature. Studies focused on prenatal ABCR cases with abnormal ultrasound findings are limited and no submicroscopic imbalance was reported in the cohorts. When de novo postnatal or prenatal results were combined, the percentage of abnormalities detected by CMA was 40.9%. Taking this contribution into consideration, all ABCRs should be investigated by CMA even if the fetal ultrasound findings are normal.

Other genomic disorders and congenital heart disease

Congenital heart disease (CHD) is the common birth defect worldwide. Despite its recognized burden on public health, the etiology in the vast majority of individuals remains unknown. Chromosomal abnormality plays an important role, frequently observed as large cytogenetically visible rearrangement or small submicroscopic structural variation in the genome. Several genomic disorders are now recognized that are increasingly responsible for CHD with variable penetrance. Single gene disorders, epigenetic alterations, and environmental etiologies are also significant contributors. Our understanding of the genetic basis of CHD has increased exponentially with the escalating use of next generation sequencing to identify ever so small submicroscopic genomic imbalances at the level of coding exons in CHD. This review focuses on genomic disorders other than 22q11.2 deletion, that are major players in the etiology of human cardiac malformations.

Identification of novel genomic imbalances in Saudi patients with congenital heart disease

BackgroundQuick genetic diagnosis of a patient with congenital heart disease (CHD) is quite important for proper health care and management. Copy number variations (CNV), chromosomal imbalances and rearrangements have been frequently associated with CHD. Previously, due to limitations of microscope based standard karyotyping techniques copious CNVs and submicroscopic imbalances could not be detected in numerous CHD patients. The aim of our study is to identify cytogenetic abnormalities among the selected CHD cases (n = 17) of the cohort using high density oligo arrays.ResultsOur screening study indicated that six patients (~35%) have various cytogenetic abnormalities. Among the patients, only patient 2 had a duplication whereas the rest carried various deletions. The patients 1, 4 and 6 have only single large deletions throughout their genome; a 3.2 Mb deletion on chromosome 7, a 3.35 Mb deletion on chromosome 3, and a 2.78 Mb a deletion on chromosome 2, respectively. Patients 3 and 5 have two deletions on different chromosomes. Patient 3 has deletions on chromosome 2 (2q24.1; 249 kb) and 16 (16q22.2; 1.8 Mb). Patient 4 has a 3.35 Mb an interstitial deletion on chromosome 3 (3q13.2q13.31).Based on our search on the latest available literature, our study is the first inclusive array CGH evaluation on Saudi cohort of CHD patients.ConclusionsThis study emphasizes the importance of the arrays in genetic diagnosis of CHD. Based on our results the high resolution arrays should be utilized as first-tier diagnostic tool in clinical care as suggested before by others. Moreover, previously evaluated negative CHD cases (based on standard karyotyping methods) should be re-examined by microarray based cytogenetic methods.

Application of chromosomal microarray analysis for fetuses with ventricular septal defects

To explore the genetic etiology of fetuses with ventricular septal defects (VSD) using chromosomal microarray analysis (CMA). A total of 248 fetuses were divided into isolated VSD group, VSD with other cardiac and/or great vessels malformation group, VSD with extra-cardiac anomalies group (including malformation and sonographic soft markers), and VSD with both cardiac and extra-cardiac anomalies group. Standard karyotyping was carried out for all fetuses, and CMA was performed for 6 fetuses with an abnormal karyotype and a proportion of fetuses with a normal karyotype. All cases were followed up, and neonates were followed up until 1 year of age. Chromosomal abnormalities were identified in 60 (24.2%) of the 248 fetuses. For 6 of the fetuses subjected to further CMA analysis, the origin of abnormal chromosomes were clarified, among which 2 have overlapped with the critical region of Wolf-Hirschhorn syndrome. Candidate genes for VSD included WHSC1, LBX1, LDB3 and BBS10. For 143 fetuses with a normal karyotype, CMA has identified pathogenic copy number variations (CNVs) in 11 cases (7.7%). These included 9 well-known microdeletion or microduplication syndromes, including 22q11.2 microdeletion, 17p11.2 microdeletion (Smith-Magenis syndrome), 17p13.3 microdeletion (Miller-Dieker syndrome), 1p36 microdeletion, 1q21.1 microduplication and 4q deletion. Candidate genes for VSD included TBX1, LZTR1, FAT1, AKAP10, SKI, PRDM26, GJA5, ERCC4 and YWHAE. For 48.7% of the fetuses with benign CNVs, spontaneously closure has occurred within the first year of life. CMA may increase the detection rate of submicroscopic imbalances by 7.7%. No significant correlation between different groups of VSD and the pathogenic CNVs was observed. Whole-genome CMA should be recommended to the fetuses with VSD but a normal karyotype. Nearly half of VSDs with benign CNVs may close spontaneously within the first year of life.

Efficient and cost-effective genetic analysis of products of conception and fetal tissues using a QF-PCR/array CGH strategy; five years of data

BackgroundTraditional testing of miscarriage products involved culture of tissue followed by G-banded chromosome analysis; this approach has a high failure rate, is labour intensive and has a resolution of around 10 Mb. G-banded chromosome analysis has been replaced by molecular techniques in some laboratories; we previously introduced a QF-PCR/MLPA testing strategy in 2007. To improve diagnostic yield and efficiency we have now updated our testing strategy to a more comprehensive QF-PCR assay followed by array CGH. Here we describe the results from the last 5 years of service.MethodsFetal tissue samples and products of conception were tested using QF-PCR which will detect aneuploidy for chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. Samples that were normal were then tested by aCGH and all imbalance >1Mb and fully penetrant clinically significant imbalance <1Mb was reported.ResultsQF-PCR analysis identified aneuploidy/triploidy in 25.6% of samples. aCGH analysis detected imbalance in a further 9.6% of samples; this included 1.8% with submicroscopic imbalance and 0.5% of uncertain clinical significance. This approach has a failure rate of 1.4%, compared to 30% for G-banded chromosome analysis.ConclusionsThis efficient QF-PCR/aCGH strategy has a lower failure rate and higher diagnostic yield than karyotype or MLPA strategies; both findings are welcome developments for couples with recurrent miscarriage.

Application of chromosome microarray analysis for fetuses with multicystic dysplastic kidney

To explore the genetic etiology of fetuses with multicystic dysplastic kidney (MCDK) by chromosome microarray analysis (CMA). Seventy-two fetuses with MCDK were analyzed with conventional cytogenetic technique, among which 30 fetuses with a normal karyotype were subjected to CMA analysis with Affymetrix CytoScan HD arrays by following the manufacturer's protocol. The data was analyzed with ChAS software. Conventional cytogenetic technique has revealed three fetuses (4.2%) with identifiable chromosomal aberrations. CMA analysis has detected pathogenic CNVs in 5 fetuses (16.7%), which included two well-known microdeletion or microduplication syndromes, i.e., 17q12 microdeletion syndrome and Williams-Beuren syndrome (WBS) and three submicroscopic imbalances at 4q35.2, 22q13.33, and 1p33. PEX26, FKBP6, TUBGCP6, ALG12, and CYP4A11 are likely the causative genes. CMA can identify the submicroscopic imbalances unidentifiable by conventional cytogenetic technique, and therefore has a significant role in prenatal diagnosis and genetic counseling. The detection rate of pathogenic CNVs in fetuses with MCDK was 16.7% by CMA. 17q12 microdeletion syndrome and WBS are associated with MCDK. Mutations of PEX26, FKBP6, TUBGCP6, ALG12, and CYP4A11 genes may be the causes for MCDK.

Prenatal detection of 10q22q23 duplications: dilemmas in phenotype prediction

The phenotype for 10q22q23 duplication is diverse, ranging from intellectual disability and dysmorphism to normal development. Interpreting the clinical significance of the duplication identified in this region is difficult, especially in the prenatal setting. This study aimed to characterize the prenatal findings associated with this submicroscopic imbalance and discuss the dilemmas in predicting the phenotype of 10q22q23 duplications. This is a retrospective study of three cases of 10q22q23 duplications diagnosed prenatally by chromosomal microarray analysis. Detailed pregnancy outcome and pediatric follow-up were documented. The genotypic and phenotypic features of the reported cases were discussed. 10q22q23 duplications are associated with an unpredictable and variable phenotypic outcome. Despite there was no phenotype found to be shared by 50% of the duplication cases, congenital heart defects, hypotelorism, and developmental delays including speech and motor delay seem to be more common. The phenotype of 10q22q23 duplication is highly variable prenatally and postnatally. Identification of additional affected individuals with similar duplications is needed to provide further insights into the pathogenesis of this microduplication. © 2016 John Wiley & Sons, Ltd.

Clinical utility of array comparative genomic hybridisation in prenatal setting.

BackgroundThe objective of reported study was to evaluate the clinical utility of prenatal microarray testing for submicroscopic genomic imbalances in routine prenatal settings and to stratify the findings according to the type of fetal ultrasound anomaly.MethodsFrom July 2012 to October 2015 chromosomal microarray testing was performed in 218 fetuses with varying indications for invasive prenatal diagnosis: abnormal karyotype, ultrasound anomalies, pathogenic variant in previous pregnancy or carriership in a parent.ResultsThe detection rate in the group of fetuses with ultrasound anomalies was 10,0% for pathogenic copy number variants (CNVs), five of them being larger than 8 Mb and expected to be seen on prenatal karyotype. If only those pathogenic CNVs below the classical karyotype resolution are considered, chromosomal microarray testing provided an additional 7,7% diagnostic yield in here reported series. When stratified according to the ultrasound anomalies, the highest percentage of pathogenic CNVs were detected in the group of fetuses with multiple congenital anomalies (16,7%) and lowest in the group of isolated in utero growth restriction (6,3%). In the group of cases with isolated increased nuchal translucency we identified a small interstitial deletion of 16p24.1 involving FOXF1 gene. Prenatal aCGH also provided important insights into cases with seemingly balanced chromosomal rearrangements found on prenatal karyotype, where additional pathogenic CNV were discovered.ConclusionPrenatal chromosomal microarray testing significantly increases the diagnostic yield when compared with conventional karyotyping. The highest added value is shown in prenatal diagnostics in fetuses with abnormal ultrasound results. Variants of unknown significance and risk factor CNVs present important challenges and should be discussed with parents in advance, therefore pretest counseling prior to prenatal testing is very important.

Prenatal diagnosis of 1p34.3 interstitial microdeletion by aCGH in a fetus with jaw bone abnormalities

BackgroundInterstitial microdeletions in 1p are extremely rare, as very few cases have been reported postnatally and only one prenatally, yet. There is a variability of phenotypic findings such as hypotonia, facial dysmorphisms, mild microcephaly, with being most common developmental delay.Case presentationThe present case involved a female fetus with an interstitial deletion on 1p, presenting with micrognathia in the 2nd trimester routine ultrasound examination. Array-based comparative genomic hybridization (a-CGH) revealed a 2,7 Mb deletion located on 1p34.3 which could not be detected by standard karyotyping.ConclusionsThis is the first prenatal case of an interstitial deletion in 1p34.3 with facial dysmorphism detected by a-CGH. Due to the use of a-CGH techniques submicroscopic imbalances could be detected, and a refined genotype-phenotype correlation could be achieved.

Expanding non-invasive prenatal testing beyond chromosomes 21, 18, 13, X and Y.

Non-invasive prenatal testing (NIPT) based on cell-free DNA in maternal plasma is being expanded to include additional chromosome abnormalities beyond those involving chromosomes 21, 18, 13, X and Y. Review of population cytogenetic data provides insight into the likely number of additional abnormalities detectable. Additional clinically significant and cytogenetically recognizable abnormalities are present in less than 0.1% of newborns but clinically significant, or potentially significant, sub-microscopic imbalances are expected to be present in 1.7%. Cytogenetic studies on chorionic villus samples suggests that after excluding abnormalities involving chromosomes 21, 18, 13, X and Y, approximately 0.6% of NIPT results may be positive for an unbalanced abnormality attributable to mosaicism but most of these will not be confirmed at amniocentesis or in newborns. NIPT has also been developed for specific microdeletion syndromes and initial experience is now available. Laboratory procedures such as deeper sequencing and additional data analytics are rapidly evolving but even with existing protocols, it is already clear that NIPT does not necessarily need to be limited to trisomies 21, 18, 13 and the sex-chromosome abnormalities. Patient educational materials and genetic counseling services need to be available for women offered expanded NIPT.

Noninvasive Prenatal Testing and Detection of Maternal Cancer

Noninvasive prenatal testing (NIPT) to detect fetal aneuploidy (an abnormal number of chromosomes) usingmassive parallel sequencing of cell-free DNA (cfDNA) from maternal blood is gaining rapid acceptance in obstetrics, given its high sensitivity and specificity for the detection of trisomies 21, 18, and 13. Placental DNA, a surrogate of fetal DNA, enters the maternal bloodstream via apoptosis of trophoblasts in the intervillous space of the placenta,where itmixes with cfDNA of maternal origin. The fetal fraction in this mixture usually varies between 3% and 15% in the late first and second trimesters.NIPT represents a formofadvancedscreening, and positive results consistent with aneuploidy require confirmation by a diagnostic test usingmaterial obtained via amniocentesis or chorionic villus sampling (CVS).1,2 The sensitivity of NIPT for the detection of trisomies 21, 18, and 13 ranges between 91% and 99.2%; the false-positive rates range between 0.09% and 0.13%.3 False-positive results havebeen attributed to confinedplacentalmosaicism,maternal chromosomalmosaicism,maternalDNAcopynumbervariants, andmaternal organ transplant fromamale donor.4 Two case reports (in 2013 and 2015) suggested that the discordant results (between those of NIPT and amniocentesis) could be due to undiagnosed maternal cancer.5,6 The first case described a 37-year-old pregnant woman with an uncomplicated pregnancy who had NIPT performed at 13 weeks of gestation. The findings were suggestive of fetal aneuploidy for chromosomes 13 and 18 in 2 different maternal blood samples. Yet the fetal karyotype was 46,XY without evidence of submicroscopic genomic imbalances with wholegenome oligonucleotide microarray. After delivering a healthy infant, the patient reported pelvic pain found to be due to a pathologic fracture of the pubis; a biopsy showed metastatic neuroendocrine carcinoma of unknown origin. A separate lesion on the anterior vaginal wall proved to be a high-grade neuroendocrine small cell carcinoma. Tumor cells showed aneuploidy for chromosomes 13 and 18, consistent with the findings from the analysis of maternal blood assessed with NIPT.5 The second patient was a 27-year-old asymptomatic woman at 12 weeks of gestation whose NIPT results had abnormal genomic findings for chromosomes 8, 9, and 14 that were discordantwith a normal fetal karyotype from amniotic fluid cells. Moreover, maternal lymphocytes did not have evidence of aneuploidy ormosaicism. Given the genomic imbalances, amalignancywas suspected, and thepatientunderwent a whole-body diffusion magnetic resonance imaging (MRI) scan, which showed an anterior mediastinal mass and enlarged lymph nodes in the cervical and retroclavicular regions. The diagnosis of nodular sclerosis Hodgkin lymphoma wasmade,basedonabiopsyof themediastinalmass.Hodgkin/ Reed-Sternbergcells showedfindingsconsistentwith thoseobservedwith the analysis of cfDNA inmaternal blood.Basedon these observations, the authors investigated whether cfDNA couldbe informative innewlydiagnosedcasesofnodular sclerosis Hodgkin lymphoma in nonpregnant women (n = 4) and in nonpregnant patients who had a first relapse after therapy (n = 5). The results in 8 of the 9 cases showed genomic imbalances, and such findingswere validated by fluorescent in situ hybridization on Hodgkin/Reed-Sternberg cells in tissue specimens.6 Commonfeaturesof the2caseswere that thepatientswere youngasymptomaticpregnantwomen,hadabnormalNIPT results discrepant with the fetal karyotype (“false-positive NIPT”),were subsequently diagnosed tohave cancer, and the malignant cells showedevidenceofmultiple aneuploidies that mirrored the findings reported by maternal cfDNA analyzed for NIPT. A recent study reported that amongmore than4000pregnant women undergoing NIPT, 3 patients were found to have aberrant genome representation profiles that were reminiscent of cancer-related copy number variation. These women underwent whole-body diffusion-weighted MRI and were found to have cancer—the first had a high-grade serous ovarian carcinoma (stage IV with multiple metastases); the second, a follicular lymphoma; and the third, Hodgkin lymphoma. Analyses of the genome representation profile of the tumors showed that the copy-number variations detected in maternal plasma were present in the tumors.7 The report by Bianchi et al8 in this issue of JAMA is the first large-scale study that strengthens the premise that a false-positive NIPT result could be due to the presence of maternal malignancy based on the results of a case series of pregnant women who had NIPT and were subsequently diagnosed with cancer. This work leverages the information of a laboratory that had processed 125 426 samples from NIPT over 2.5 years with a test to detect the presence of aneuploidy for chromosomes 21, 18, and 13 (testing for sex chromosome aneuploidy was optional). Of these samples, 3% (3757) had NIPT results consistent with aneuploidy. The practice of the laboratory was to discuss all abnormal NIPT results with the referring clinician and recommend a diagnostic test, and clinicians were asked to inform the laboratory if the results Related article page 162 Opinion

Complex nature of apparently balanced chromosomal rearrangements in patients with autism spectrum disorder.

BackgroundApparently balanced chromosomal rearrangements can be associated with an abnormal phenotype, including intellectual disability and autism spectrum disorder (ASD). Genome-wide microarrays reveal cryptic genomic imbalances, related or not to the breakpoints, in 25% to 50% of patients with an abnormal phenotype carrying a microscopically balanced chromosomal rearrangement. Here we performed microarray analysis of 18 patients with ASD carrying balanced chromosomal abnormalities to identify submicroscopic imbalances implicated in abnormal neurodevelopment.MethodsEighteen patients with ASD carrying apparently balanced chromosomal abnormalities were screened using single nucleotide polymorphism (SNP) arrays. Nine rearrangements were de novo, seven inherited, and two of unknown inheritance. Genomic imbalances were confirmed by fluorescence in situ hybridization and quantitative PCR.ResultsWe detected clinically significant de novo copy number variants in four patients (22%), including three with de novo rearrangements and one with an inherited abnormality. The sizes ranged from 3.3 to 4.9 Mb; three were related to the breakpoint regions and one occurred elsewhere. We report a patient with a duplication of the Wolf-Hirschhorn syndrome critical region, contributing to the delineation of this rare genomic disorder. The patient has a chromosome 4p inverted duplication deletion, with a 0.5 Mb deletion of terminal 4p and a 4.2 Mb duplication of 4p16.2p16.3. The other cases included an apparently balanced de novo translocation t(5;18)(q12;p11.2) with a 4.2 Mb deletion at the 18p breakpoint, a subject with de novo pericentric inversion inv(11)(p14q23.2) in whom the array revealed a de novo 4.9 Mb deletion in 7q21.3q22.1, and a patient with a maternal inv(2)(q14.2q37.3) with a de novo 3.3 Mb terminal 2q deletion and a 4.2 Mb duplication at the proximal breakpoint. In addition, we identified a rare de novo deletion of unknown significance on a chromosome unrelated to the initial rearrangement, disrupting a single gene, RFX3.ConclusionsThese findings underscore the utility of SNP arrays for investigating apparently balanced chromosomal abnormalities in subjects with ASD or related neurodevelopmental disorders in both clinical and research settings.

Microduplication of chromosome Xq25 encompassing STAG2 gene in a boy with intellectual disability

Whole-genome microarray analysis is proven to be useful in the identification of submicroscopic copy number imbalances in families with intellectual disabilities. The first case of Xq25 duplication was identified using genome-wide array comparative genomic hybridization (array-CGH) in a 24-year-old patient with a syndromic intellectual disability. We report a 4-year-old boy with a de novo 591 kb duplication at Xq25. The duplication was first detected by a CytoScan HD array platform (Affymetrix, USA) and was confirmed by real-time quantitative PCR (qPCR) of the STAG2 gene, and by fluorescence in situ hybridization (FISH). The patient had clinical features partially consistent with published cases, including an intellectual disability and speech delay. The identification of this additional patient and a detailed analysis of duplications identified in other patient cohorts and absent in normal individuals support the existence of a rare pathological microduplication at Xq25 that encompasses STAG2.

Diagnostic utility of array-based Comparative Genomic Hybridization in a clinical setting

Background Submicroscopic genomic imbalances are a major cause of congenital and developmental abnormalities including unexplained Developmental Delay (DD), Intellectual Disability (ID), Autism Spectrum Disorders (ASD) and Multiple Congenital Anomalies (MCA). Submicroscopic imbalances are not visible at the resolution level offered by conventional cytogenetics techniques but could potentially be analysed with array based comparative genomic hybridization (aCGH) which offers high resolution scan of the genome. We present the motion backed by clinical evidence for the diagnostic utility of aCGH in a clinical settings for aforementioned cases.

Microdeletions/duplications involving TBX1 gene in fetuses with conotruncal heart defects which are negative for 22q11.2 deletion on fluorescence in‐situ hybridization

Conotruncal heart defects (CTD) are associated with del22q11.2 syndrome, which is often diagnosed by fluorescence in-situ hybridization (FISH). However, in those negative for del22q11.2 on FISH, the etiology is usually obscure. We aimed to use high-resolution array comparative genomic hybridization (array CGH) to clarify the underlying genetic causes in these cases. In this retrospective study, fetal samples of amniocytes or fibroblasts, taken either for prenatal diagnosis by amniocentesis or for postnatal survey after termination of pregnancy, were obtained from 45 fetuses with CTD and were investigated by cytogenetic analysis including karyotyping and FISH for del22q11.2 syndrome. Eight fetuses with no findings on karyotyping and FISH were investigated further by array CGH, real-time quantitative polymerase chain reaction (qPCR) and Sanger sequencing of TBX1. Array CGH revealed that three of the eight fetuses carried submicroscopic genomic imbalances. Of these, two cases showed similar small microdeletions/duplications in 22q11.2 (one 0.85 kb microdeletion and one 8.51 kb microduplication). The minimal shared region spanned exon 2 of TBX1, a candidate gene responsible for cardiovascular defects in del22q11.2 syndrome. In all eight cases, the array CGH results were confirmed by qPCR, and Sanger sequencing did not detect other molecular pathologies. Our findings indicate an association between TBX1 variations and fetal CTD. The results also demonstrate the power of array CGH to further scrutinize the critical gene(s) of del22q11.2 syndrome responsible for heart defects. Array CGH apparently has diagnostic sensitivity superior to that of FISH in fetuses with CTD associated with del22q11.2 (and dup22q11.2) syndrome.

High‐resolution analysis of copy number variants in adults with simple‐to‐moderate congenital heart disease

As patients with congenital heart disease (CHD) increasingly survive to childbearing age, it becomes important to understand the genetic origins of CHD. In children, CHD is frequently caused by chromosomal imbalances. We searched for submicroscopic imbalances in adults with CHD focusing on simple-to-moderate phenotypes, without associated dysmorphic features, a group not previously examined. A total of 100 Han Chinese adults with a diverse range of isolated CHD and 65 ethnically matched controls were screened using whole-genome array comparative genomic hybridization. Forty-five large (>100 kb) rare copy number variants (CNVs) were identified in 36/100 patients. These variants were not listed in the Database of Genomic Variants nor found in controls. In three of these genomic imbalances (22q11.2, 18q23, 3q21.3), genes that play an important role in cardiac development were implicated, including CRKL, NFATC1, PLXNA1, the latter has not been associated with human CHD before. This study detected a 0.7 Mb 22q11.2 deletion, which marginally overlapped the common 3 Mb 22q11.2 deletion, in one patient with a perimembranous ventricular septal defect without any extracardiac manifestation. Furthermore, we detected a novel inherited aberration dup (16q23.1). Although a causal relationship with CHD remains to be established, this CNVs profile provides a spectrum of genomic imbalances in this condition, and improves the CNV-phenotype correlations.

DOCK4 deletion at 7q31.1 in a de novo acute myeloid leukemia with a normal karyotype

Acute myeloid leukemia with a normal karyotype (NK-AML) has been assigned to an intermediate prognostic risk group. However, there is a marked variability in outcome within this group of AML, suggesting a significant biological and molecular heterogeneity. Chromosomal abnormalities may be cryptic by metaphase cytogenetics, but still have an impact on the process of leukemogenesis and/or the clinical outcome of NK-AML. Therefore, we analyzed the genomic complement of NK-AML cases to search for the presence of submicroscopic genomic imbalances. We applied high-resolution oligo-based aCGH analysis to a cohort of AML patients with a normal karyotype, and FISH to validate the aCGH findings. Relative gene expression levels were examined by qPCR. High-resolution aCGH analysis of 21 NK-AML patients revealed one patient with a rare submicroscopic deletion at 7q31.1. This female patient exhibited a rapid disease progression and a dismal outcome. The deletion was mono-allelic, approximately 189,1 kb in size, and encompassed the major 3' part of the DOCK4 gene. The expression level of the DOCK4 gene in her leukemic cells was decreased when compared to CD34+ cells from healthy controls. When compared to AML patients with -7/del(7q) as the sole chromosomal anomaly, the DOCK4 expression level was found to be similarly low. This is the first report of an acquired partial deletion of the DOCK4 gene in a patient with de novo NK-AML. DOCK4 is a strong tumor suppressor candidate, required for GTPase activation in signal transduction pathways controlling cellular proliferation, adhesion, migration and phagocytosis. Our findings may be relevant for understanding of the process of leukemogenesis and the response to therapy in a subset of NK-AML patients.

GW24-e1706 Genetic analysis of copy number variants in chinese adults with isolated congenital heart disease

ObjectivesWith more patients with congenital heart disease (CHD) surviving to childbearing age, it becomes even more critical to understand the genetic origins of CHD. Chromosomal imbalances have been identified in...