Chromosomal Microarray Analysis in Pregnancies With Corpus Callosum or Posterior Fossa Anomalies.

OBJECTIVE: We investigated the detection rate of clinically significant chromosomal microarray analysis (CMA) results in pregnancies with sonographic diagnosis of fetal corpus callosum anomalies (CCA) or posterior fossa anomalies (PFA). METHODS: All CMA tests in pregnancies with CCA or PFA performed between January 2015 and June 2020 were retrospectively evaluated from the Israeli Ministry of Health database. The rate of CMA with clinically significant (pathogenic or likely pathogenic) findings was calculated and compared to a local Israeli cohort of 5,541 pregnancies with normal ultrasound. RESULTS: One hundred eighty-two pregnancies were enrolled: 102 cases with CCA and 89 with PFA (9 cases had both). Clinically significant CMA results were found in 7/102 of CCA (6.9%) and in 7/89 of PFA (7.9%) cases. The CMA detection rate in pregnancies with isolated CCA (2/57, 3.5%) or PFA (2/50, 4.0%) was lower than in nonisolated cases, including additional CNS and/or extra-CNS sonographic anomalies (CCA-5/45, 11.1%; PFA-5/39, 12.8%), but this was not statistically significant. However, the rate among pregnancies that had extra-CNS anomalies, with or without additional CNS involvement (CCA-5/24, 20.8%; PFA-5/29, 17.2%), was significantly higher compared to all other cases (p = 0.0075 for CCA; p = 0.035 for PFA). Risk of CMA with clinically significant results for all and nonisolated CCA or PFA pregnancies was higher compared to the background risk reported in the control cohort (p < 0.001), but was not significant for isolated cases. CONCLUSIONS: Our findings suggest that CMA testing is beneficial for the genetic workup of pregnancies with CCA or PFA, and is probably most informative when additional extra-CNS anomalies are observed.

Abnormal nuchal translucency followed by normal microarray analysis is associated with placental pathology-related complications.

OBJECTIVE: Identify placental pathology-related complications, labor and neonatal outcomes in pregnancies complicated by pathological nuchal translucency (NT) with normal microarray analysis. METHODS: A retrospective study in which all women with singleton pregnancy who demonstrated NT above 3 mm and a normal microarray analysis were matched to women with normal NT and a normal microarray analysis (2013-2019) in a single tertiary academic center. The following placental pathology-related parameters were measured: preeclampsia, oligohydramnios, suspected intrauterine growth restriction, abnormal Doppler studies or small for gestational age (SGA) neonates. The primary outcome was defined as a composite of complications related to placental pathology including preeclampsia and SGA neonate. Secondary outcomes were labor complications and neonatal morbidity. RESULTS: A total of 185 women were included in the study: of them, 47 presented an abnormal NT (study group) and 138 presented normal NT (controls). Groups did not significantly differ in baseline characteristics. Regarding primary outcome, all placental-related complications frequencies were higher in the study group, with a composite rate of 17.02% versus 6.52% in controls (p = 0.042%). Secondary outcomes did not differ between groups. CONCLUSIONS: Abnormal NT measurement presented in pregnancies with normal fetal microarray analysis is associated with higher rates of placental-related complications.

Expansion of the GRIA2 phenotypic representation: a novel de novo loss of function mutation in a case with childhood onset schizophrenia.

Childhood-onset schizophrenia (COS) is a rare form of schizophrenia with an onset before 13 years of age. There is rising evidence that genetic factors play a major role in COS etiology, yet, only a few single gene mutations have been discovered. Here we present a diagnostic whole-exome sequencing (WES) in an Israeli Jewish female with COS and additional neuropsychiatric conditions such as obsessive-compulsive disorder (OCD), anxiety, and aggressive behavior. Variant analysis revealed a de novo novel stop gained variant in GRIA2 gene (NM_000826.4: c.1522 G > T (p.Glu508Ter)). GRIA2 encodes for a subunit of the AMPA sensitive glutamate receptor (GluA2) that functions as ligand-gated ion channel in the central nervous system and plays an important role in excitatory synaptic transmission. GluA2 subunit mutations are known to cause variable neurodevelopmental phenotypes including intellectual disability, autism spectrum disorder, epilepsy, and OCD. Our findings support the potential diagnostic role of WES in COS, identify GRIA2 as possible cause to a broad psychiatric phenotype that includes COS as a major manifestation and expand the previously reported GRIA2 loss of function phenotypes.

Do human embryos have the ability of self-correction?

Human embryogenesis frequently coinciding with cell division mistakes contributing to pervasive embryonic aneuploidy/mosaicism. While embryo self-correction was elegantly demonstrated in mouse models, human studies are lacking. Here we are witness to human embryos ability to eliminate/expel abnormal blastomeres as cell debris/fragments. Each blastocyst and its corresponding debris were separated and underwent whole genome amplification. Seven of the 11 pairs of blastocysts and their corresponding cell debris/fragments revealed discordant results. Of the 9 euploid blastocysts, four showed euploid debris, while in the others, the debris were aneuploid. In the remaining pairs, the debris showed additional aneuploidy to those presented by their corresponding blastocyst. The observed ability of human embryos to self-correction doubts many invasive and non-invasive preimplantation testing for aneuploidy at the blastocyst stage, rendering high rate of false positive (discarding "good" embryos) by identifying the cell-free DNA originated from the expelled cell debris, as aneuploidy/mosaic blastocyst.

What have we learned from 691 prenatal chromosomal microarrays for ventricular septal defects?

INTRODUCTION: Ventricular septal defect (VSD) represents the most common type of congenital cardiac anomaly, affecting more than 1 in 300 live births. The objective of this study was to examine the incidence and nature of abnormal chromosomal microarray analysis (CMA) results in a large cohort of pregnancies with VSD. MATERIAL AND METHODS: Data acquisition was performed through the Ministry of Health computerized database. All CMA results performed due to VSD during 2013-2017 were included. The rates of clinically significant CMA results of cases with isolated and non-isolated VSD were compared with two control populations-a systematic review of 9272 pregnancies and a local cohort of 5541 fetuses with normal ultrasound. RESULTS: Overall, 691 CMA analyses performed due to a sonographic indication of VSD were detected. Of 568 pregnancies with isolated VSD, eight (1.4%) clinically significant copy number variants were detected, a nonsignificant difference compared with low risk pregnancies. Of the 123 pregnancies with non-isolated VSDs, 18 (14.6%) clinically significant CMA results were detected, a considerably increased risk compared with control pregnancies. Karyotype-detectable anomalies constituted 12 of the 18 abnormal CMA results in non-isolated VSD group (66.7%), a significantly higher proportion compared with 2 of 8 (25%) in isolated VSD cohort. CONCLUSIONS: The outcomes of our study, representing the largest number of CMA results in pregnancies with VSD, suggest that the rate of abnormal CMA findings in isolated VSD does not differ from pregnancies with normal ultrasound. This observation is true for populations undergoing routine common trisomy screening tests and early sonographic evaluation, as well as widely available non-invasive prenatal screening. Conversely, CMA analysis yields a high detection rate in pregnancies with non-isolated VSD. Our results question the recommendation to perform invasive prenatal testing for CMA in pregnancies with isolated VSD.

Chromosomal microarray findings in pregnancies with an isolated pelvic kidney.

Objective To examine the risk for abnormal chromosomal microarray analysis (CMA) results among fetuses with an apparently isolated pelvic kidney. Methods Data from all CMA analyses performed due to an isolated pelvic kidney reported to the Israeli Ministry of Health between January 2013 and September 2016 were retrospectively obtained. Risk estimation was performed comparing the rate of abnormal observed CMA findings to the general population risk, based on a systematic review encompassing 9272 cases and on local data of 5541 cases. Results Of 120 pregnancies with an isolated pelvic kidney, two gain-of-copy number variants suggesting microduplication syndromes were demonstrated (1.67%). In addition, three variants of unknown significance were detected (2.5%). Conclusion The risk for clinically significant CMA findings among pregnancies with an isolated single pelvic kidney was not significantly different compared to both control populations. The results of our study question the practice of routine CMA analysis in fetuses with an isolated pelvic kidney.

Post-childhood Presentation and Diagnosis of DiGeorge Syndrome.

BACKGROUND AND OBJECTIVES: The diversity of clinical presentations makes the diagnosis of DiGeorge syndrome (DGS) a diagnostic challenge. The objective of our study was to report the clinical presentation of DGS in the post-childhood period. METHODS: A retrospective study, investigating patients diagnosed clinically and genetically with DGS at Sheba Medical Center during the period of 2010-2013. Post-childhood period was defined as age >10 years. RESULTS: During the study period, 29 patients were diagnosed with DGS. Nine (31%) patients with DGS were diagnosed in their post-childhood period. The basis for clinical suspicion was diverse. However, once the suspicion was brought to attention, additional symptoms consistent with DGS were noted at up to 88% of patients who presented characteristic of facial features and developmental delay. CONCLUSION: Our research shows that diagnosing DGS patients in the post-childhood period is not uncommon. Characteristic facial features and developmental delay, although not leading presenting symptoms, are found very frequently in patients with DGS.

Chromosomal integrity of human preimplantation embryos at different days post fertilization.

INTRODUCTION: In order to investigate the dynamics of genomic alterations that occur at different developmental stages in vitro, we examined the chromosome content of human preimplantation embryos by molecular-cytogenetic techniques at the single-cell level, up to 13 days post fertilization. METHODS: The embryos were genetically analyzed several times during their development in culture; each embryo was first analyzed by FISH at 'Day 3' post fertilization, than during its growth in vitro and the third analysis was performed at development arrest, then the entire blastocyst was analyzed by comparative genomic hybridization (CGH/aCGH). RESULTS: We found that while on 'Day 3' only 31% of the embryos were detected as normal, on 'Day 5-6', 44% of the embryos were classified as normal and on 'Day 7', 57% were normal. On 'Days 8-13', 52% of the embryos were classified as chromosomally normal. One third of the embryos that were chromosomally abnormal on 'Day 3', were found to be normal at development arrest point. DISCUSSION: These dynamic changes that occur at early developmental stages suggest that testing a single blastomere at 'Day 3' post fertilization for PGD might inaccurately reflect the embryo ploidy and increase the risk of false aneuploidy diagnosis. Alternatively, blastocyst stage diagnosis may be more appropriate.

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia.

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the "candidate gene approach" we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

PURPOSE: Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. METHODS: Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. RESULTS: Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. CONCLUSIONS: Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Fish based preimplantation genetic diagnosis to prevent DiGeorge syndrome.

PURPOSE: To report the performance of fluorescence in-situ hybridization in the setting of preimplantation genetic diagnosis in order to diagnose embryos affected by DiGeorge syndrome. DESIGN: Case report. SETTING: Academic referral center. PATIENT: A 32 year-old female affected by DiGeorge syndrome. INTERVENTION(S): History and physical examination, karyotyping, amniocentesis, preimplantation genetic diagnosis, fluorescence in-situ hybridization. MAIN OUTCOME MEASURE(S): Avoidance of pregnancy with embryo affected by DiGeorge syndrome. RESULT(S): Termination of pregnancy with an affected embryo followed by fluorescence in-situ hybridization based preimplantation genetic diagnosis and delivery of healthy offspring. CONCLUSION(S): The combination of preimplantation genetic diagnosis with fluorescence in-situ hybridization is recommended to prevent pregnancies with DiGeorge syndrome affected embryos in properly selected patients.

Genetic alterations detected by comparative genomic hybridization and recurrence rate in epithelial ovarian carcinoma.

To assess the putative correlation between comparative genomic hybridization (CGH)-detectable genetic alterations in epithelial ovarian cancer and disease recurrence, conventional CGH was performed on 45 epithelial ovarian cancers: 26 tumors from sporadic, BRCA mutation noncarriers and 11 and 8 tumors from BRCA1 and BRCA2 mutation carriers, respectively. Relevant clinical data, including histology, grade, stage, size of residual tumor, recurrence, and survival, were obtained from outpatient and inpatient charts. Among the 45 cases, the most common regions involving gain of DNA copy number were 3q (n = 23; 51%), 8q (n = 21; 47%), and 1q (n = 14; 31%), and the most common regions with loss were 19 and 22 at 9 cases (20%) each, followed by 5q (n = 6; 13%). In multivariate analysis, the total number of genetic alterations was not associated with risk of recurrence, but gain in 5p was associated with a higher risk of recurrence (hazard ratio HR = 6.06, P = 0.0399), and gain in 1p as well as loss in 5q were associated with a significant decrease in recurrence (HR = 0.08, P = 0.0079, and HR = 0.10, P = 0.0143, respectively). Recurrence rate in patients with epithelial ovarian cancer is seemingly associated with specific genetic alterations detected by CGH, but the specific genes involved and the implications of these findings await further studies.

Proximal 19q trisomy: a new syndrome of morbid obesity and mental retardation.

AIMS: To report on the clinical and metabolic characteristic and the unique chromosomal defect of a mentally retarded and morbidly obese patient. METHODS: A 13-year follow-up, including insulin sensitivity, lipid profile and polysomnography studies and various therapeutic interventions are described. The presence of a supernumerary marker in karyotype preparation was further studied by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was used to identify the source of the chromosomal marker. RESULTS: Insulin resistance was found by the homeostatic model assessment (HOMA) and the quantitative insulin sensitivity check index (QUICKI). M-FISH identified euchromatin derived from chromosome 19, and CGH confirmed the FISH results and demonstrated that the supernumerary marker derived from 19q12 to 19q13.2. CONCLUSION: The clinical and metabolic characteristics in association with partial chromosomal trisomy differ our patient from the currently known syndromes of obesity and mental retardation. The metabolic impairments in this case can derive from unbalanced expression of several genes in the 19q12-19q13.2 region, genes that are related to adipose tissue homeostasis and insulin resistance. The clinical and genetic similarities to a previously reported case may suggest that partial 19q trisomy is a new syndrome of obesity and mental retardation.

Mapping of a gene causing brittle cornea syndrome in Tunisian jews to 16q24.

PURPOSE: To map the gene that causes brittle cornea syndrome (BCS). METHODS: Five patients from four families, all of Jewish Tunisian origin, were recruited into the study. Four of the five patients had red hair. DNA from the five patients and 104 control chromosomes was typed with seven 16q polymorphic markers surrounding the hair color gene, MC1R. RESULTS: A common haplotype in the homozygous state, comprising five markers spanning 4.7 Mb on chromosome 16q24, was found in all five patients but in none of the control subjects (P < 0.00001). CONCLUSIONS: The gene that causes BCS maps to a 4.7-Mb interval, between the markers D16S3423 and D16S3425 on 16q24.

Epidermal growth factor receptor gene amplification and expression in disseminated pediatric low-grade gliomas.

OBJECT: Pediatric low-grade gliomas (LGGs) are the largest group of central nervous system neoplasms in children. Although these tumors are generally benign, 5 to 10% of patients with pediatric LGGs present with leptomeningeal dissemination. The genetic and biological nature of these tumors is poorly understood. The authors looked for certain molecular abnormalities that may differentiate disseminated gliomas from the other pediatric LGGs. METHODS: Comparative genomic hybridization (CGH) was applied to 18 pediatric LGGs. Six cases featuring disseminated pediatric LGGs were compared with 12 control cases involving nondisseminated pediatric LGGs. Fluorescence in situ hybridization (FISH) analysis and immunohistochemical analysis were used to highlight further specific genetic targets. The CGH revealed multiple chromosomal abnormalities in five of six cases with disseminated gliomas and in six of 12 control cases. No correlation was found between the number of chromosomal abnormalities and dissemination status. Amplification of chromosome 7 was noted in four of six cases with disseminated gliomas as opposed to one of 12 control cases (p = 0.02). The FISH analysis revealed epidermal growth factor receptor (EGFR) amplification in one case negative to chromosome 7 amplification by CGH, raising the amplification cases to five of six (p = 0.0038). Immunohistochemical analysis for EGFR was positive in six of six cases and in two of 12 control cases (p = 0.0015). At the end of a mean follow-up period of 7.2 years, all patients with disseminated gliomas are alive with variable but slow disease progression. CONCLUSIONS: The high rate of EGFR gene amplification and protein expression in disseminated pediatric LGGs is intriguing and may have implications for our understanding of the role of EGFR in glioma genesis. Targeted therapies may be available for these children. Larger-scale studies are needed to establish further these findings.

In silico chromosomal clustering of genes displaying altered expression patterns in ovarian cancer.

Ovarian cancer, the leading cause of death due to gynecological malignancy, is diagnosed in most cases at an advanced stage. Combined with the paucity of symptoms of early-stage disease, the need to develop novel effective markers for the detection of potentially curable, early-stage disease is self-evident. Comprehensive analyses of somatic gene expression patterns in ovarian cancer were reported previously (n=17) and yielded substantial information on somatically altered genes, information that can potentially be useful in developing early detection markers. To further substantiate the role that these genes play in ovarian cancer tumorogenesis, we surveyed these reports and arranged the significantly altered genes from all reported studies by their chromosomal location (in silico chromosomal clustering). Subsequent comparison of this clustering to known genomic somatic alterations at the DNA level from data obtained using comparative genomic hybridization (CGH) was carried out. The major chromosomal regions that displayed overexpressed genes were correlated with the major CGH-detectable DNA amplification areas at 20q (harboring HE4, SLPI, MYBL2, UBE2C, and SDC4) and 1q (harboring MUC1). These genes may provide insights into ovarian cancer pathogenesis and may also prove to be useful as early detection tools.

Genomic analyses of primary and metastatic serous epithelial ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the western world. In 75% of patients, peritoneal metastases are found at the time of primary surgery. However, the genetic events leading to the development of ovarian tumors and to the genetic progression toward metastasis remain unclear. To gain insight into this issue, the types and patterns of DNA copy number changes were compared between primary ovarian tumors and their respective metastases by using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The genetic alterations (deletions and amplifications) detected by CGH were similar in the primary tumors and in their respective metastases. Moreover, the FISH results show a similar pattern of chromosomal abnormalities. Our results imply that the major gross genetic changes in ovarian cancer take place in the primary tumor, and the additional genetic changes that may occur in the metastases are not detectable by CGH.

Familial vs sporadic ovarian tumors: characteristic genomic alterations analyzed by CGH.

OBJECTIVE: Our purpose was to get an overview of the genetic events leading to the development of familial and sporadic ovarian tumors and to identify chromosomal regions that may contain genes important in tumor progression. METHODS: The comparative genomic hybridization (CGH) technique was employed in a total of 46 epithelial ovarian or peritoneal tumors: 27 sporadic tumors, 11 tumors disected from BRCA1 mutation (185delAG) carriers, and eight from BRCA2 mutation (6174delT) carriers (familial tumors). RESULTS: The average number of genetic alterations (deletions and amplifications) was significantly (alpha=0.0069) higher in familial tumors (9.17 +/- 4.25 alterations per tumor in the BRCA1 mutation carriers and 7.25 +/- 6.06 in the BRCA2 mutation carriers) compared to the sporadic group (4.26 +/- 3.61 alterations per tumor). The pattern of the chromosome amplifications resembled in the three groups and the most common amplifications detected were at chromosomes 8q, 3q, and 2q. The pattern of the chromosomal deletions varied between the groups. Among the BRCA1 group, the most common deletions were in chromosomes 9 and 19. The BRCA2 group showed a lower frequency of deletions. Deletion of chromosome 16 and 22 were the most frequent ones. No specific chromosomal deletion was significantly indicated in the sporadic group. CONCLUSIONS: Familial ovarian tumors exhibit a significantly higher number of chromosomal aberrations and genomic imbalances and nonrandom genetic changes were characterized in the BRCA1 and BRCA2 groups.

Comparative genomic hybridization analysis of craniopharyngiomas.

OBJECT: Craniopharyngioma is the most common childhood brain tumor and is thought to arise from embryonic remnants of the Rathke pouch. Some craniopharyngiomas are monoclonal in origin and hence presumably harbor somatic genetic alterations, although the precise molecular mechanisms involved in craniopharyngioma development are unknown. The goal of this study was to identify genetic alterations in craniopharyngiomas. METHODS: To gain insight into the molecular mechanisms involved in development of these tumors, the authors analyzed nine adamantinomatous craniopharyngiomas by using comparative genomic hybridization. Six tumors (67%) displayed at least one genomic alteration, and three had six or more alterations. Only two tumors displayed a decrease in DNA copy number, and in all others an increase in DNA copy number was noted. CONCLUSIONS: The authors conclude that a subset of craniopharyngiomas consists of monoclonal tumors arising from activation of oncogenes located at specific chromosomal loci.