Ayala Aviram-Goldring

Obsessive-compulsive disorder in patients with velocardiofacial (22q11 deletion) syndrome

The study of neurogenetic microdeletion syndromes provides an insight into the developmental psychopathology of psychiatric disorders. The aim of the study was to evaluate the prevalence of psychiatric disorders, especially obsessive‐compulsive disorder (OCD), in patients with velocardiofacial syndrome (VCFS), a 22q11 microdeletion syndrome. Forty‐three subjects with VCFS of mean age 18.3 ± 10.6 years were comprehensively assessed using semi‐structured psychiatric interview and the Yale–Brown obsessive compulsive scale (Y‐BOCS). Best estimate diagnoses were made on the basis of information gathered from subjects, parents, teachers, and social workers. Fourteen VCFS subjects (32.6%) met the DSM‐IV criteria for OCD. OCD had an early age of onset and generally responded to fluoxetine treatment. It was not related to mental retardation. The most common obsessive‐compulsive symptoms were contamination, aggression, somatic worries, hoarding, repetitive questions, and cleaning. Sixteen of the 43 patients (37.2%) had attention‐deficit/hyperactivity disorder (ADHD), and 7 (16.2%) had psychotic disorder. The results of our study suggest that there is a strong association between VCFS and early‐onset OCD. This finding may be significant in the understanding of the underlying genetic basis of OCD.

Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter’s syndrome

OBJECTIVE The aim of the study was to determine the potential risk for fetal chromosomal anomalies in non-mosaic Klinefelters syndrome patients undergoing IVF and intracytoplasmic sperm injection. DESIGN Individually collected spermatozoa were isolated from wet testicular tissue preparations and fixed on glass slides using micromanipulation. Their nuclei were analyzed for chromosomes X, Y, and 18 by fluorescent in situ hybridization. SETTING Assisted reproductive technology program. PATIENT(S) Consenting patients with non-mosaic Klinefelters syndrome undergoing testicular biopsy and IVF (fresh specimens) or following such treatment (cryopreserved specimens). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The rates of numerical chromosome abnormalities for chromosomes X, Y, and 18 among spare testicular sperm and the pregnancy outcome following treatment. RESULT(S) Testicular sperm were found in 8 of 20 patients. Four couples became pregnant following embryo replacement. Sperm chromosomes were analyzed in five patients. One hundred and five sperm of 112 analyzed (93.7%) were normal with X to Y ratio of 50:55 (NS) respectively. Among the 112 sperm tested, seven (6.3%) demonstrated chromosomal abnormalities, of which five were related to the sex chromosomes and two to chromosome 18. One set of triplets, one set of twins, and two singletons (four males and three females) with normal karyotypes were born. CONCLUSION(S) Most of the testicular sperm retrieved from Klinefelters syndrome patients demonstrates a normal pattern of sex chromosome segregation. Therefore, the risk of transmitting numerical sex chromosome abnormalities is relatively low and probably comparable with the rates found in other severe male factor infertility patient groups.

Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in vitro fertilization with intracytoplasmic sperm injection

OBJECTIVE To investigate the potential paternal contribution to the risk of fetal chromosomal anomalies after intracytoplasmic sperm injection (ICSI). DESIGN Spermatozoa isolated from testicular tissue and ejaculated specimens of consenting patients undergoing testicular biopsy and ICSI were analyzed for chromosomes X, Y, and 18 by FISH. SETTING Assisted reproductive technology program. PATIENT(S) Consenting patients undergoing testicular biopsy and ICSI, severe oligozoospermic patients, and normal fertile donors. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The rate of chromosome abnormalities in testicular sperm with regard to the type of azoospermia and ejaculated sperm compared to healthy men. RESULT(S) The mean serum levels of FSH in the groups with nonobstructive azoospermia (n = 9), obstructive azoospermia (n = 10), severe oligozoospermia (n = 9), and the normal donors (n = 6) were 17.5 +/- 8.2 (P<.05), 3.5 +/- 2.6, 14.6 +/- 3.5 (P<.05), and 3.1 +/- 0.4 IU/mL, respectively. The corresponding rates of sperm chromosome abnormalities among these groups were 19.6% (P<.001), 8.2% (P<.001), 13.0% (P<.001), and 1.6%, respectively. The corresponding rates of disomy among these groups were 7.8% (12 of 153 spermatozoa), 4.9% (18 of 367), 6.2% (109 of 1,751), and 1% (5 of 500 spermatozoa), respectively. Errors in chromosomes X and Y were significantly more common than in chromosome 18. CONCLUSION(S) The present findings demonstrate a linkage between gonadal failure (high serum FSH levels) and the occurrence of sperm chromosome aneuploidies. Our findings may explain the increased incidence of sex chromosome abnormalities found after IVF in the severe male factor patient population. Genetic screening during pregnancy or before embryo replacement should be considered carefully.

Association of the low-activity COMT 158Met allele with ADHD and OCD in subjects with velocardiofacial syndrome.

Velocardiofacial syndrome (VCFS) is caused by a microdeletion in chromosome 22 and is a risk factor for the development of schizophrenia and other psychiatric disorders. The catechol-O-methyltransferase (COMT), residing in the 22q11.2 microdeletion region, is a major candidate gene for genetic susceptibility to neuropsychiatric disorders in VCFS. Individuals with VCFS carrying the low-activity allele (COMTL) are expected to have the lowest possible COMT activity since they have only a single copy of the gene. We explored the possibility that COMTL is associated with psychiatric disorders commonly found in VCFS. Fifty-five unrelated individuals with VCFS underwent psychiatric evaluation and were genotyped for the COMT 158Val/Met polymorphism coding for COMT high/low-activity alleles. The COMTL allele was significantly more prevalent in VCFS subjects with attention deficit hyperactivity disorder (ADHD) (73.9% vs. 33.3%, OR 5.67, chi2=7.76, p=0.005) and obsessive-compulsive disorder (OCD) (78.6% vs. 33.3%, OR 7.33, chi2=7.24, p=0.007) than in the control group (VCFS subjects without OCD, ADHD and schizophrenia/schizoaffective (SZ/SZaff) disorder). The results of this study suggest that greatly reduced COMT activity, as expected in VCFS COMTL individuals may be a risk factor for psychiatric sequelae in this population. Future longitudinal studies focusing on additional COMT polymorphic sites and other candidate genes from the deleted region will elucidate the molecular pathways leading to schizophrenia and other psychiatric disorders in VCFS.

Analysis of fetal blood cells in the maternal circulation: challenges, ongoing efforts, and potential solutions.

The invasive procedures amniocentesis and chorionic villus sampling (CVS) are routinely applied in pregnancies at risk for fetal abnormalities and the results obtained are the gold standard for prenatal diagnosis. Because these methods of fetal cell procurement involve a 0.5-2% risk for fetal loss, they are recommended mainly in cases at high risk for fetal genetic or cytogenetic abnormalities. The development of a reproducible, reliable, noninvasive method based on retrieval of rare fetal cells from the maternal circulation will render testing feasible for the general population. Despite intensive investigation, a satisfactory, clinically acceptable method has not yet emerged. Several cell types have been targeted to this end, mostly nucleated red blood cells (NRBC), CD34+ hematopoietic progenitors, and trophoblasts. Although these cell types have been unequivocally proven to be present in the maternal circulation, each bears a significant disadvantage, rendering their application in clinical testing currently impossible: NRBC cannot be expanded in culture, thereby ruling out metaphase chromosome analysis, an essential component of prenatal diagnosis. CD34+ cells do posses the potential for in vitro proliferation, however, they have been found to persist in the maternal circulation after delivery, thereby complicating diagnosis in consecutive pregnancies. Trophoblasts are not consistently detected in the maternal circulation. Moreover, due to the lack of a definitive fetal cell marker and a reliable sorting method, foolproof fetal cell identification of any of these cell types is not possible. This report outlines the obstacles that impede development of a method for noninvasive fetal cell sampling for prenatal genetic diagnosis, along with a description of our efforts to analyze simultaneously two fetal blood cell types, NRBC and CD34+ cells in maternal blood during pregnancy, and the problems encountered. This work and that of others lead us to suggest potential future directions to help develop this important technique.

Molecular cytogenetic studies in three patients with partial trisomy 2p, including CGH from paraffin-embedded tissue.

We report on three cases of partial trisomy 2p in which the identification and exact localization of the duplicated chromosome segment was possible only by application of molecular cytogenetic techniques. These included fluorescence in situ hybridization by use of wcp2, N-myc, and subtelomeric 2p probes and comparative genomic hybridization with DNA isolated from blood samples, frozen fetal tendon, and formalin fixed, paraffin-embedded fetal lung tissue. Two of the cases concerned fetuses of gestational week 20 and 24 with duplication of nonoverlapping terminal (2pter-->p24) and more proximal (2p25-->p23) segments and with distinctly different phenotypes. The third case was due to a de novo inverted duplication of 2p25-->p23, with loss of the subtelomeric region of 2p. This 53-month-old girl was a Bloom syndrome carrier. The patient had prenatal growth failure, borderline microcephaly, dilated lateral horns of the cerebral ventricles, transient cortical blindness, myopia, muscle hypotonia, and dilatation of the left renal collecting system. Dermal cysts were found on the glabella, the soles of both feet, and the vocal cord, causing respiratory embarrassment. Previously reported cases of pure trisomy 2p are reviewed, in an attempt to correlate clinical findings to overlapping regions in 2p. These cases illustrate the effectiveness of molecular cytogenetic methods in resolving subtle chromosomal aberrations in order to coordinate more accurately a chromosome regionspecific phenotype.

Detection of numerical chromosome aberrations by comparative genomic hybridization.

At least 50 per cent of all first‐trimester spontaneous abortions are cytogenetically abnormal, including trisomy, monosomy X, triploidy, tetraploidy and structural chromosome anomalies. Traditionally, the detection of aneuploidy in fetal tissues is performed by tissue sampling, cell culturing, metaphase spread preparation, and conventional banding analyses. This is a tedious, laborious and time‐consuming process, prone to errors due to external contamination, culture failure and selective growth of maternal cells. In the present study, we applied the CGH technique in the detection of numerical chromosome abnormalities in 50 placentae of spontaneously aborted fetuses. CGH detected six different types of trisomy (trisomy 8, 15, 16, 18, 22 and 21), one double trisomy (involving chromosomes 14 and 21), and one monosomy X. Overall, nine samples (18 per cent) harboured numerical chromosome aberrations. Aneuploidy was detected in eight samples by CGH and in six samples by conventional cytogenetic analysis. In only one case, CGH failed to detect a mosaic for trisomy revealed by conventional cytogenetic analysis.

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.

Congenital diaphragmatic hernia in a family segregating a reciprocal translocation t(5;15)(p15.3;q24)

Congenital diaphragmatic hernia (CDH) is a relatively common malformation of unknown cause with high mortality due to hypoplasia of the lungs and pulmonary hypertension. We studied a family in which two fetuses had CDH, and two pregnancies resulted in first trimester missed abortions. Both fetuses with CDH had an apparently normal karyotype. In a subsequent pregnancy, fluorescent in situ hybridization analysis of amniocytes showed a balanced translocation 46,XY, t(5;15) (p15.3;q24) also present in the mother and in a normal child, suggesting that the diaphragmatic hernia in the first two fetuses was caused by a cryptic unbalanced translocation. This hypothesis is supported by a previous observation of CDH in a distal deletion of 15q as part of a multiple congenital anomalies syndrome. It is suggested that a gene distal to 15q21 is important for the normal development of the diaphragm.

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.