Microarray Comparative Genomic Hybridization Research Articles

Lissencephaly in an epilepsy cohort: Molecular, radiological and clinical aspects.

Lissencephaly is a rare malformation of cortical development due to abnormal transmantle migration resulting in absent or reduced gyration. The lissencephaly spectrum consists of agyria, pachygyria and subcortical band heterotopia. In this study we compared genetic aetiology, neuroradiology, clinical phenotype and response to antiepileptic drugs in patients with epilepsy and lissencephaly spectrum malformations. The study group consisted of 20 patients - 13 males and 7 females, aged 18 months to 21yearsat the time of data collection. Genetic testing was performed by oligonucleotide array comparative genomic hybridization (microarray), multiplex ligation-dependent probe amplification (MLPA), targeted gene panels and whole exome/genome sequencing. All neuroradiological investigations were re-evaluated and the malformations were classified by the same neuroradiologist. Clinical features and response to anti-epileptic drugs (AEDs) were evaluated by retrospective review of medical records. In eleven patients (55%) mutations in PAFAH1B1 (LIS1) or variable microdeletions of 17p13.3 including the PAFAH1B1 gene were detected. Four patients (20%) had tubulin encoding gene mutations (TUBA1A, TUBG1 and TUBGCP6). Mutations in DCX, DYNC1H1, ADGRG1 and WDR62 were identified in single patients. In one patient, a possibly pathogenic intragenic deletion in TRIO was detected. A clear radiologic distinction could be made between tubulinopathies and PAFAH1B1 related lissencephaly. The majority of the patients had therapy resistant epilepsy and epileptic spasms was the most prominent seizure type. The best therapeutic response to seizure control in our cohort was obtained by the ketogenic diet, vigabatrin, clobazam, phenobarbital and valproate. The most common genetic aetiologies in our cohort of 20 individuals with epilepsy and lissencephaly spectrum were intragenic deletions or single nucleotide mutations in PAFAH1B1 or larger deletions in 17p13.3, encompassing PAFAH1B1, followed by mutations in tubulin encoding genes. Radiological findings could reliably predict molecular results only in agyria with a posterior to anterior gradient. Radiological and molecular findings did not correlate consistently with severity of clinical outcome or therapeutic response.

Intellectual disability and dysmorphisms in a child with 14q32.31-q32.33 deletion: a case report

INTRODUCTION: Intellectual disability and global developmental delay are frequent causes of genetic consultation requested by pediatricians. Around 30% of severe intellectual disability and 70% of mild intellectual disability do not present a defined diagnosis. OBJECTIVES: Chromosome microarray comparative genomic hybridization test is now the standard diagnosis for patients presenting unexplained global developmental delay or intellectual disability, autism spectrum disorders, and multiple congenital anomalies due to the tests sensibility, increasing the chance for a diagnosis and elucidating size and region affected. CASE REPORT: This paper reports a girl with intellectual disability, prominent hypotonia, postnatal growth retardation and several dysmorphisms with a small subtelomeric 14q terminal deletion whose diagnosis was sought during six years and only became possible through the chromosome microarray test.

The Association of Scoliosis and NSD1 Gene Deletion in Sotos Syndrome Patients.

A retrospective comparative study. The aim of this study was to examine the NSD1 abnormalities in patients diagnosed with Sotos syndrome and its correlation with the presence, severity, and progression of associated scoliosis. Scoliosis has been reported in approximately 30% of patients diagnosed with Sotos syndrome, a genetic disorder characterized by a distinctive facial appearance, learning disability, and overgrowth. Sotos syndrome is mainly attributed to NSD1 haploinsufficiency, but with ethnical differences in genetic profile: NSD1 microdeletions are frequently identified in Japanese Sotos patients whereas intragenic mutations are more frequently found in non-Japanese patients. Although possible genotype-phenotype correlations have been proposed, the genotype of Sotos syndrome patients suffering from scoliosis has not been examined. The medical records and spinal radiographs of 63 consecutive Sotos syndrome patients at a single center were reviewed. Fluorescent in situ hybridization or microarray comparative genomic hybridization and DNA sequencing or multiplex ligation-dependent probe amplification were performed to detect 5q35 microdeletion involving the NSD1 gene and intragenic mutations of the NSD1 gene, respectively. The phenotypes of all cases and radiological assessments for the presence and progression of scoliosis were studied. NSD1 abnormalities were identified in 55 patients (87%): microdeletion in 34 patients (54%) and intragenic mutation in 22 patients (33%). Scoliosis was observed in 26 patients (41%), with a significantly higher ratio of microdeletions than mutations. The 10 patients with progressive scoliosis all had NSD1 microdeletions. Scoliosis was a common phenotypical trait in children with Sotos syndrome and its presence as well as progression were higher in cases with NSD1 microdeletions. Although all Sotos syndrome patients should be monitored for scoliosis, clinicians should be made aware that patients with NSD1 microdeletions have a higher probability of scoliosis development and progression that may require early intervention.Level of Evidence: 3.

Xp;Yq Unbalanced Translocation with Pseudoautosomal Region Aberrations in a Natural Two-Generation Transmission.

Translocations involving X and Y chromosomes rarely occur in humans and may affect reproductive function. We investigated an Xp:Yq unbalanced translocation with pseudoautosomal region (PAR) aberrations in a natural two-generation transmission. We report the case of an azoospermic male and his fertile mother without any other abnormal clinical phenotypes, except for short stature. Cytogenetic methods, including karyotyping and fluorescence in situ hybridization (FISH), revealed the translocation. Chromosomal microarray comparative genomic hybridization (array-CGH) was used to investigate the regions of Xp partial deletion and Yq partial duplication. Final chromosome karyotypes in the peripheral blood of the infertile male and his mother were 46,Y,der(X)t(X;Y)(p22.33;q11.22) and 46,X,der(X)t(X;Y)(p22.33;q11.22), respectively. Short-stature-homeobox gene deletion was responsible for the short stature in both subjects. PAR aberrations and AZFc duplication may be a direct genetic risk factor for spermatogenesis. This report further supports the use of routine karyotype analysis, FISH-based technology, and array-CGH analysis to identify derivative chromosomes in a complex rearrangement.

In vitro fertilization outcomes after preimplantation genetic testing for chromosomal structural rearrangements comparing fluorescence in-situ hybridization, microarray comparative genomic hybridization, and next-generation sequencing

ObjectiveTo compare in vitro fertilization (IVF) outcomes for preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) using various testing platforms.DesignRetrospective cohort.SettingLarge academic IVF center.Patient(s)Fifty-one balanced translocation carriers undergoing IVF with PGT-SR who completed a total of 91 cycles, including 31 fluorescence in-situ hybridization (FISH), 24 microarray comparative genomic hybridization (aCGH), and 36 next-generation sequencing (NGS) testing cycles.Intervention(s)PGT-SR.Main Outcome Measure(s)Primary outcome of live-birth rate and secondary outcomes including implantation rate, clinical loss rate, and percentages of normal or balanced, unbalanced, and aneuploid embryos detected.Result(s)There was no statistically significant difference in LBR, though there was a tendency toward a higher LBR for NGS testing (14 of 19, 73.7%) compared with FISH (8 of 18, 44.4%) and aCGH (10 of 20, 50.0%). The implantation rate was statistically significantly higher for NGS (16 of 20, 80.0%) compared with FISH (11 of 25, 44.0%) and aCGH (16 of 30, 53.3%). There was no statistically significant difference in clinical pregnancy losses. There was a lower percentage of normal or balanced embryos with FISH (12.5%) compared with aCGH (23.7%) and with NGS (20.7%).Conclusion(s)This is the first report of PGT-SR outcomes for translocation carriers directly comparing PGT-SR using FISH, aCGH, and NGS. Our findings suggest an improvement in pregnancy outcomes parallel to the advancement in technology and are reassuring for continued use of NGS for this population.

Abstract 5461: Tacc1 is amplified in a wide range of pediatric malignancy

Abstract While fluorescence in situ hybridization (FISH) is traditionally used to detect copy number variations (CNV) at known pathogenic chromosomal loci, the comparative genomic hybridization (CGH) microarray is a platform capable of simultaneous detection of CNVs at thousands of loci. CGH microarrays are typically used to assess loci of known prognostic significance; however loci variants of uncertain significance (VUS) are frequently present. To identify potentially actionable targets, we retrospectively analyzed VUS loci from 192 cases of cancer or genetic disorders using CGH data generated for treatment guidance at the Texas Tech University Health Sciences Center Cytogenetics Laboratory, a CLIA-certified Children's Oncology Group reference lab serving the West Texas population. The majority of cases were of pediatric malignancies, though some adult cancers and benign hematological diseases were included. Patient DNA was hybridized onto CytoSure Constitution V3 arrays (60k loci), scanned with the Agilent microarray D scanner, and analyzed by CytoSure Interpret Software. TACC1, TM2D2, KAT6A and ADAM32 were knocked down in H358 lung cancer (p53-null), MCF7 (ER+) and MDA-MB-231 (triple-negative) breast cancer, and B16-F0 murine melanoma cell lines using siRNA, followed by evaluation of growth inhibition by MTT assay. Colcemid block was used to evaluate metaphase reduction and chromosomal abnormalities following TACC1 knockdown. We found 794 distinct CNVs, the most frequent being 14q32.22 (112 cases), 14q11.2 (100 cases), 8p11.22 (98 cases), 15q11.1-15q11.2 (83 cases), and 8p23.1 (77 cases). In particular, the 8p11.22 locus was amplified in many pediatric cancers, including acute lymphoblastic leukemia (18 gains/2 losses), lymphoma (6/1), neuroblastoma (5/0), pediatric breast tumors (5/0), and Wilm's tumor (4/0). Linkage of TACC1, TM2D2, KAT6A and ADAM32, all genes residing at the 8p11.22 locus, was indicated by a similar average 5-year overall survival rate (75.3 ± 1.7%, n=253) among patients harboring alterations in these genes in The Cancer Genome Atlas (TCGA) database. Knockdown of TACC1, TM2D2, and KAT6A reduced growth of H358, MCF7, MDA-MB-231, and B16-F0 cells (P < 0.05). ADAM32 depletion inhibited H358, MCF7, and MDA-MB-231 cells (P < 0.05), but not B16-F0 cells. Because TACC1 function is linked to mitosis through the aurora kinases, we subsequently studied metaphase of B16-F0 cells after TACC1 depletion, found an 80% reduction metaphase cells, and increased morphological abnormalities. In summary, we report a high frequency of 8p11.2 gains in our pediatric oncology patients and demonstrate that evaluation of growth inhibition of cancer cells following knockdown of genes at VUS loci may help to identify new cancer therapy targets. These results further suggest that TACC1 could be a target for treating triple negative breast cancer, melanoma, and p53-null lung cancer. Citation Format: Aditya Rajan, Sharda P. Singh, Santhosh Chavali, Chhanda Bose, Vijay Tonk, Sanjay Awasthi. Tacc1 is amplified in a wide range of pediatric malignancy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5461.

Detection of chromosomal abnormalities in spontaneous miscarriage by low‑coverage next‑generation sequencing.

Chromosomal abnormalities (CAs) can cause spontaneous miscarriage and increase the incidence of subsequent pregnancy loss and other complications. Presently, CAs are detected mainly by array comparative genomic hybridization (CGH) and single nucleotide polymorphism microarrays. The present study developed a low-coverage next-generation sequencing method to detect CAs in spontaneous miscarriage and assess its clinical performance. In total, 1,401 patients who had experienced an abortion were enrolled in the present study and divided into two groups. In group I, 437 samples that had been previously validated by array CGH were used to establish a method to detect CAs using a semiconductor sequencing platform. In group II, 964 samples, which were not verified, were assessed using established methods with respect to clinical significance. Copy number variant (CNV)-positive and euploidy samples were verified by array CGH and short tandem repeat profiling, respectively, based on quantitative fluorescent PCR. The low-coverage sequencing method detected CNVs >1 Mb in length and a total of 3.5 million unique reads. Similar results to array CGH were obtained in group I, except for six CNVs <1 Mb long. In group II, there were 341 aneuploidies, 195 CNVs, 25 mosaicisms and 403 euploidies. Overall, among the 1,401 abortion samples, there were 536 aneuploidies, 263 CNVs, 34 mosaicisms, and 568 euploidies. Trisomies were present in all autosomal chromosomes. The most common aneuploidies were T16, monosomy X, T22, T15, T21 and T13. Furthermore, one tetrasomy 21, one CNV associated with Wolf-Hirschhorn syndrome, one associated with DiGeorge syndrome and one associated with both Prader-Willi and Angelman syndromes were identified. These four cases were confirmed by short tandem repeat profiling and array CGH. Quantitative fluorescent PCR revealed nine polyploidy samples. The present method demonstrated equivalent efficacy to that of array CGH in detecting CNVs >1 Mb, with advantages of requiring less input DNA and lower cost.

An efficient genetic test flow for multiple congenital anomalies and intellectual disability

Genetic test has enabled the diagnosis of multiple congenital anomalies and/or intellectual disabilities. However, because of the phenotypic variability in these disorders, selection of an appropriate genetic test can be difficult and complex. For clinical examination, particularly in clinical facilities, a simple and standardized system is needed. We compared microarray comparative genomic hybridization and clinical exome sequencing in terms of diagnostic yield, cost, and time required to reach a definitive diagnosis. After first performing G-banding for 200 patients with multiple congenital anomalies and/or intellectual disability, as a subsequent genetic test, microarray and Clinical Exome Sequencing were compared in diagnostic yield, cost, and time required. There was no obvious difference in the diagnostic rate between the two methods; however, clinical exome sequencing was superior in terms of cost and time. In addition, clinical exome sequencing could sufficiently identify Copy Number Variants, and even smaller Copy Number Variants could be identified. Clinical exome sequencing should be implemented earlier as a genetic test for undiagnosed patients with multiple congenital anomalies and/or intellectual disabilities. Our results can be used to establish inspection methods in clinical facilities.

Y/15 and Y/22 Derivative Chromosomes in Couples with Reproductive Failures: Algorithm of Preimplantation Genetic Testing and Specificity of Inheritance

The present study reports on the results of preimplantation genetic testing and IVF outcomes in four families, where one of the spouses is a carrier of Y/15 or Y/22 derivative chromosome: three cases of male carriers and one—of female carrier. In the cases of male carriers, we detected the predominant inheritance of the derivative chromosomes by the embryos and no abnormalities in sex chromosome segregation. In the case of female carrier, we did not observe either predominant inheritance of derivative chromosome by the embryos or effect of derivative chromosome on sex chromosome segregation. In all families, we revealed a high frequency of embryonic aneuploidies of chromosomes other than those involved in translocation. To exclude inheritance of the derivative chromosome by the offspring, preimplantation genetic testing should be recommended: fluorescent in situ hybridization (FISH), which allows identification of the Y-chromosome satellite DNA, followed by next generation sequencing (NGS) or microarray comparative genomic hybridization (aCGH) to detect other genomic and chromosomal abnormalities.

Microarray analysis of cumulus cells in women with ovarian endometriosis undergoing intracytoplasmic sperm injection

Objective: The aim of this study was to find the significantly altered genes in cumulus cells of women with ovarian endometriosis by using microarray and quantitative polymerase chain reaction analysis. Methods: Thirty women with ovarian endometriosis and 30 age–body mass index matched controls (women with infertility as a result of pure male factor) were enrolled in this study. Cumulus cells from study participants who underwent controlled ovarian hyperstimulation were isolated mechanically. Microarray comparative genomic hybridization was used to compare the transcriptome of cumulus cells from women with ovarian endometriosis and controls. According to the different expression levels in the microarrays and their putative functions, KRAS, ZNF322, and SDHA were selected and analyzed by real-time quantitative polymerase chain reaction. Results: There was no significant difference in the basal conditions between patients with endometriosis and controls, such as age, body mass index, basal follicle stimulating hormone and estradiol levels, and total gonadotrophin dosage. The gene expression profile of cumulus cells from patients with endometriosis was significantly different from that of controls. A total of 295 genes were significantly up- or down-regulated (p-value &lt; 0.05 and absolute fold change &gt; 1.5). For all of the genes adjusted p-value was found to be 0.999. Polymerase chain reaction analysis showed that KRAS and ZNF322 mRNA levels in the cumulus cells of patients with ovarian endometriosis were significantly up-regulated compared to controls (fold changes: 3.05 and 3.22, respectively). Conclusion: KRAS and ZNF322 mRNA levels in the cumulus cells of patients with ovarian endometriosis were significantly up-regulated.

Chromosomal alterations of pediatric malignancy in a West Texas population

Background: Comparative genomic hybridization (CGH) microarrays are used forgenome-wide evaluation of copy number variations (CNV) of known prognostic significance;however, unannotated variants of uncertain significance (VUS) are frequently present. Toidentify potentially actionable targets, we retrospectively analyzed VUS loci using CGH datafrom 192 cases of cancer or genetic disorders treated at TTUHSC.Methods: DNA was hybridized onto CytoSure Constitution V3 arrays, scanned with theAgilent microarray D scanner, and analyzed by CytoSure Interpret Software.Results: We found 794 distinct CNVs, the most frequent being 14q32.22 (112rearrangements), 14q11.2 (100), 8p11.2 (98), 15q11.1-q11.2 (83), and 8p23.1 (77). In particular,8p11.22 alterations were found in many pediatric tumors, with gain/loss ratio of 4.7. Linkage ofTACC1, TM2D2, KAT6A and ADAM32 was indicated by a similar 5-year survival rate of 75.3%(n = 253), which was greater than in unaltered cases (62.2%, n = 15,809 cases) in The CancerGenome Atlas database.Conclusion: Knockdown of genes occurring at variants of uncertain significance (VUS)loci may help identify new therapy targets.

Development and Validation of a 34-Gene Inherited Cancer Predisposition Panel Using Next-Generation Sequencing.

The use of genetic testing to identify individuals with hereditary cancer syndromes has been widely adopted by clinicians for management of inherited cancer risk. The objective of this study was to develop and validate a 34-gene inherited cancer predisposition panel using targeted capture-based next-generation sequencing (NGS). The panel incorporates genes underlying well-characterized cancer syndromes, such as BRCA1 and BRCA2 (BRCA1/2), along with more recently discovered genes associated with increased cancer risk. We performed a validation study on 133 unique specimens, including 33 with known variant status; known variants included single nucleotide variants (SNVs) and small insertions and deletions (Indels), as well as copy-number variants (CNVs). The analytical validation study achieved 100% sensitivity and specificity for SNVs and small Indels, with 100% sensitivity and 98.0% specificity for CNVs using in-house developed CNV flagging algorithm. We employed a microarray comparative genomic hybridization (aCGH) method for all specimens that the algorithm flags as CNV-positive for confirmation. In combination with aCGH confirmation, CNV detection specificity improved to 100%. We additionally report results of the first 500 consecutive specimens submitted for clinical testing with the 34-gene panel, identifying 53 deleterious variants in 13 genes in 49 individuals. Half of the detected pathogenic/likely pathogenic variants were found in BRCA1 (23%), BRCA2 (23%), or the Lynch syndrome-associated genes PMS2 (4%) and MLH1 (2%). The other half were detected in 9 other genes: MUTYH (17%), CHEK2 (15%), ATM (4%), PALB2 (4%), BARD1 (2%), CDH1 (2%), CDKN2A (2%), RAD51C (2%), and RET (2%). Our validation studies and initial clinical data demonstrate that a 34-gene inherited cancer predisposition panel can provide clinically significant information for cancer risk assessment.

Embryos derived from donor or patient oocytes are not different for in vitro fertilization outcomes when PGT allows euploid embryo selection: a retrospective study

BackgroundAt our facilities, patients that received embryos using donor oocyte during in vitro fertilization (IVF), usually have had at least one failed attempt to produce at least one euploid embryo with their own oocytes; however, the current debate between using donor over patient oocytes remains inconclusive. We examined the aneuploidy rate and IVF clinical outcomes from embryos derived from either donor or patient oocytes.MethodsRetrospectively, 973 cycles were examined of patients who underwent a standard IVF protocol. Chromosomal content was determined using Pre-implantation Genetic Testing (PGT) by either microarray-comparative genomic hybridization or Next-generation sequencing from either Day 3 (blastocysts) or Day 5 (trophectoderm) embryo biopsies, respectively. Embryo implantation was confirmed by serum β-hCG (> 10 m IU/mL/Day 14), whereas clinical pregnancy by a fetal heartbeat (Week 6.5–8).ResultsEmbryos derived from donor oocytes presented with more monosomies than embryos derived from patient oocytes (41.2% vs. 25.4%, p < 0.05, respectively); however, only Trisomy 7 (0.4% vs. 2.3%, p < 0.05) and Trisomy in X (0.7% vs. 2.3%, p < 0.05) were significantly less present when compared to patient oocyte derived embryos. Interestingly, rates for embryo implantation (46.7% vs. 50.8%, p = 0.35), clinical pregnancy (38.5% vs. 43.1%, p = 0.30), and live birth (30.5% vs. 30.5%, p = 0.99) were similar for embryos derived from donor and patient oocytes. These results did not change when adjusted for the number of embryos implanted.ConclusionHere, we show no significant differences in achieving pregnancy when using donor oocytes. Taking into consideration that aneuploidy rates are > 30% in embryos, independent of the oocyte origin, PGT should be recommended with donor oocytes as well.

Yield of comparative genomic hybridization microarray in pediatric neurology practice.

ObjectiveThe present study investigated the diagnostic yield of array comparative genomic hybridization (aCGH) in a large cohort of children with diverse neurologic disorders as seen in child neurology practice to test whether pathogenic copy number variants (CNVs) were more likely to be detected in specific neurologic phenotypes.MethodsA retrospective cross-sectional analysis was performed on 555 children in whom a genetic etiology was suspected and who underwent whole-genome aCGH testing between 2006 and 2012. Neurologic phenotyping was performed using hospital medical records. An assessment of pathogenicity was made for each CNV, based on recent developments in the literature.ResultsForty-seven patients were found to carry a pathogenic CNV, giving an overall diagnostic yield of 8.59%. Certain phenotypes predicted for the presence of a pathogenic CNV, including developmental delay (odds ratio [OR] 3.69 [1.30–10.51]), cortical visual impairment (OR 2.73 [1.18–6.28]), dysmorphism (OR 2.75 [1.38–5.50]), and microcephaly (OR 2.16 [1.01–4.61]). The combination of developmental delay/intellectual disability with dysmorphism and abnormal head circumference was also predictive for a pathogenic CNV (OR 2.86 [1.02–8.00]). For every additional clinical feature, there was an increased likelihood of detecting a pathogenic CNV (OR 1.18 [1.01–1.38]).ConclusionsThe use of aCGH led to a pathogenic finding in 8.59% of patients. The results support the use of aCGH as a first tier investigation in children with diverse neurologic disorders, although whole-genome sequencing may replace aCGH as the detection method in the future. In particular, the yield was increased in children with developmental delay, dysmorphism, cortical visual impairment, and microcephaly.

Detection of large rearrangements in a hereditary pan-cancer panel using next-generation sequencing

BackgroundHealthcare providers increasingly use information about pathogenic variants in cancer predisposition genes, including sequence variants and large rearrangements (LRs), in medical management decisions. While sequence variant detection is typically robust, LRs can be difficult to detect and characterize and may be underreported as a cause for hereditary cancer risk. This report describes the outcomes of hereditary cancer genetic testing using a comprehensive strategy that employs next-generation sequencing (NGS) for LR detection, coupled with LR confirmation using repeat hybrid capture NGS, microarray comparative genomic hybridization (microarray-CGH), and/or multiplex ligation-dependent probe amplification (MLPA).MethodsSequencing and LR analysis were conducted in a consecutive series of 376,159 individuals who received clinical testing with a hereditary pan-cancer gene panel from September 2013 through May 2017. NGS dosage analysis was used to evaluate potential deletions or duplications, with controls in place to exclude pseudogene reads. Samples positive for a putative LR based on NGS were confirmed using a comprehensive approach that included targeted microarray-CGH and/or MLPA analysis, with further examination as needed to ascertain the nature of the LR.ResultsA total of 3461 LRs were identified and classified as a deleterious mutation (DM), suspected deleterious mutation (SDM) or variant of uncertain significance. Pathogenic LRs (DM/SDM) accounted for the majority of LRs (67.7%), the largest proportion of which were deletions (86.1%), followed by duplications (11.3%), insertions (1.8%), triplications (0.5%), and inversions (0.3%). Several cases presented illustrate that the laboratory approach employed here can ensure consistent identification and accurate characterization of LRs. In the absence of this comprehensive testing strategy, 9% of LRs identified in this testing population might have been missed, potentially leading to inappropriate medical management in as many as 210 individuals referred for hereditary cancer testing.ConclusionsThese data show that copy number analysis using NGS coupled with confirmatory testing reliably detects and characterizes LRs. Further, LRs comprise a substantial proportion (7.2%) of pathogenic variants identified by the test. A robust and accurate LR identification strategy is an essential component of a high-quality genetic testing program, enabling clinicians to optimize patient medical management decisions.

Embryos Arising from Apronuclear (0PN) and Unipronuclear (1PN) Have Similar Euploidy Rates with Those from 2PN and Should be Considered for Transfer

Background: Fertilisation assessment is routinely made at 16–18 hours post-ICSI and 18–20 hours post-insemination. However, the absence of pronuclei (PN) during standard fertilisation assessment does not necessarily indicate fertilisation failure. The aim of this study is to assess the chromosomal status of blastocysts derived from 0PN and 1PN zygotes as well as to assess the clinical outcome after transfer of such embryos. Methods: In this study, we use microarray comparative genomic hybridisation (MaCGH) or next generation sequencing (NGS) to analyse the chromosomal status of 271 blastocysts (204 from 2PN, 41 from 0PN, 26 from 1PN) obtained from 42 patients who underwent conventional IVF (cIVF) and ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Results: Euploidy was confirmed in 126 (126/204; 61.8%), 31 (31/41; 75.6%) and 18 (18/26; 69.2%) 2PN-, 0PN- and 1PN-derived blastocysts respectively while the remaining 96 blastocysts displayed various chromosomal abnormalities. A Y-chromosome was observed in 0PN-derived blastocysts (19/41; 46.3%) and 1PN-derived blastocysts (13/26; 50%), indicating that sperm had penetrated the oocyte and not due to parthenogenetic activation. Four euploid 0PN-derived blastocysts were transferred to 4 patients and 3 healthy live births were achieved. Four euploid 1PN-derived blastocysts were transferred to 4 patients and 1 on-going pregnancy was achieved. Conclusion(s): 0PN- and 1PN-derived zygotes can be chromosomally normal and result in healthy live births. Such zygotes should not be discarded but instead be subjected to extended culture with PGT-A to ascertain the chromosomal and ploidy status and be considered for transfer.

MicroRNAs profiling in fibroblasts derived from patients with Gorlin syndrome.

Gorlin syndrome (GS) is a hereditary disorder with tumorigenicity, caused by constitutive hyperactivity of hedgehog signaling. Smoothened (SMO) antagonists have been effectively used in the clinical treatment of hedgehog signaling-related cancer. However, these treatments have led to problematic side effects, including severe adverse reactions and drug resistance from additional somatic mutations. We profiled microRNAs in GS fibroblasts to explore a novel therapeutic target for controlling hyper-activated hedgehog signaling. To identify GS-related microRNAs, we analyzed dermal fibroblasts from five patients with GS and three normal controls. We used microarray comparative genomic hybridization to screen 632 human microRNAs in GS fibroblasts. We identified 16 down- and 19 upregulated microRNAs with over twofold change in expression. We validated the increased expression of four microRNAs, confirming hsa-miR-196a-5p downregulation and hsa-miR-4485 upregulation using real-time PCR. Moreover, hsa-miR-196a-5p is complementary to sites in the 3' UTR of MAP3K1, which exhibits upregulated expression at mRNA and protein levels in GS fibroblasts. In addition, hedgehog signal induction with exogenous components decreased miR-196a-5p expression and increased map3k1 expression in a mouse mesenchymal cell line. Given that MAP3K1 has been reported to activate hedgehog signaling, hsa-miR-196a-5p may contribute to the positive feedback loop in this pathway.

Williams–Beuren syndrome in Mexican patients confirmed by FISH and assessed by aCGH

Williams-Beuren syndrome (WBS) has a prevalence of 1/7500-20000 live births and results principally from a de novo deletion in 7q11.23 with a length of 1.5 Mb or 1.8 Mb. This study aimed to determine the frequency of 7q11.23 deletion, size of the segment lost, and involved genes in 47 patients with a clinical diagnosis of WBS and analysed by fluorescence in situ hybridization (FISH); among them, 31 had the expected deletion. Micro-array comparative genomic hybridization (aCGH) confirmed the loss in all 18 positive-patients tested: 14 patients had a 1.5 Mb deletion with the same breakpoints at 7q11.23 (hg19: 72726578-74139390) and comprising 24 coding genes from TRIM50 to GTF2I. Four patients showed an atypical deletion: two had a 1.6 Mb loss encompassing 27 coding genes, from NSUN5 to GTF2IRD2; another had a 1.7 Mb deletion involving 27 coding genes, from POM121 to GTF2I; the remaining patient presented a deletion of 1.2 Mb that included 21 coding genes from POM121 to LIMK1. aCGH confirmed the lack of deletion in 5/16 negative-patients by FISH. All 47 patients had the characteristic facial phenotype of WBS and 45 of 47 had the typical behavioural and developmental abnormalities. Our observations further confirm that patients with a classical deletion present a typical WBS phenotype, whereas those with a high (criteria of the American Association of Pediatrics, APP) clinical score but lacking the expected deletion may harbour an ELN point mutation. Overall, the concomitant CNVs appeared to be incidental findings.

Results of preimplantation genetic testing for aneuploidies as a basis for parental karyotypes correction

Introduction Chromosomal abnormalities are the most common genetic cause of infertility. An information about karyotypes of patients undergoing in vitro fertilization (IVF) is not always known. Therefore, IVF patients can be potential carriers of structural chromosomal rearrangements. We report three cases, in which results of preimplantation genetic testing for aneuploidies (PGT-A) allowed us to suspect the presence of parental reciprocal translocations. In each case, the parental reciprocal translocation was confirmed. Material & Methods PGT-A was performed by microarray comparative genomic hybridization (aCGH) (24sure, Illumina) or by next generation sequencing (NGS) (VeriSeq, Illumina). Indications for preimplantation testing were advanced maternal age (case 1 and 2) and paternal pericentric inversion of chromosome 10 (case 3). Parental karyotypes were not known before PGT-A in cases 1 and 2. In case 3 patients’ karyotypes was known before PGT-A and were 46,XX and 46,XY,inv(10)(p11.2;q22). The trophectoderm biopsy was performed at 5-6 day of embryo development. Analysis of parental karyotypes after obtaining PGT results was performed by standard cytogenetic study or by fluorescence in situ hybridization (FISH) on peripheral blood lymphocyte culture. The choice of method of parental karyotypes analysis was determined by size of the embryo chromosomal imbalance. Results PGT-A results showed a nonrandom pattern of segmental loses and gains in each case (table 1). These facts were the basis for parental karyotypes analysis. It was confirmed, that in each case one of the couple was the carrier of reciprocal translocation. In case 1 a paternal reciprocal translocation between chromosomes 11 and 22 was found – 46,XY,t(11;22)(q23.3;q13). In case 2 a maternal reciprocal translocation between chromosomes 10 and 12 was found – ish t(10;12)(D10S2290-, D12S158+; D10S2290+, D12S158-). In case 3 a maternal reciprocal translocation between chromosomes 2 and 12 was found - ish t(2;12)(D2S2147-,D12S399+; D2S2147+, D12S399-). Table 1 Case № Number of embryos Number of embryos with nonrandom pattern of segmental abnormalities Chromosomes involved in nonrandom segmental abnormalities Case 1 5 3 Chromosomes 11 and 22 Case 2 4 4 Chromosomes 10 and 12 Case 3 4 3 Chromosomes 2 and 12 Conclusions It is very importantto draw attention to pattern of segmental chromosomal abnormalities of embryos. 24-chromosome aneuploidy testing is not only an actual method for detection of embryo chromosomal imbalance, but in some cases also can help to correct parental karyotypes.

Prenatal diagnosis and genetic analysis of increased fetal nuchal translucency due to 2p15-16.1 microdeletion: a case report

We reported the prenatal molecular diagnosis and pregnant outcome of a fetus with increased nuchal translucency. The ultrasound findings of the gravida at 12+5 gestational weeks indicated that the fetal nuchal translucency thickness was 4.5 mm, and non-invasive prenatal testing suggested as low risk. Amniocentesis was performed at 18 gestational weeks. Fetal chromosomal karyotype was normal but chromosome microarray comparative genomic hybridization analysis identified a 1.878 Mb deletion on chromosome 2p15-16.1. No copy number variation was found in the parents. The microdeletion was also verified by multiplex ligation-dependent probe amplification. Literature reported that chromosome 2p15-16.1 microdeletion syndrome was characterized by mental retardation, language developmental disorder, microcephaly and so on. This case we reported here was a de novo 2p15-16.1 microdeletion which contained the critical region and genes of 2p15-16.1 microdeletion syndrome and was inferred to be a pathogenetic mutation. The gravida chose to terminate the pregnancy after genetic consultation. Key words: Chromosomes, human, pair 2; Chromosome deletion; Nuchal translucency measurement; Prenatal diagnosis; Pregnancy outcome