Chromosomal aberrations and gene expression profiles in non-small cell lung cancer.

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

Studies on sperm chromosomes in patients with severe male factor infertility undergoing assisted reproductive technology treatment.

The aim of the study was to determine the rate of chromosome abnormalities in testicular sperm after intracytoplasmic sperm injection due to severe male factor infertility. The study groups included patient with non-obstructive azoospermia (n=9), obstructive azoospermia (n=10), Klinefelter's syndrome (n=5) and normal controls (n=6, groups I-VI, respectively). The mean serum levels of FSH 17.5+/-8.2 (P<0.05), 3.5+/-2.6, 29.8+/-13.0 (P<0.05) and 3.1+/-0.4 mIU/ml, respectively. The rates of chromosome abnormalities were 19.6% (P<0.001), 8.2% (P<0.001), 6.3 and 1.6%, respectively. Chromosomes X and Y were significantly more involved in the aneuploidy than chromosome 18 in groups I and II. The present findings demonstrate a linkage between gonadal failure (high serum FSH levels) and sperm chromosome abnormalities. Our findings may explain the increased incidence of perinatal sex chromosome abnormalities found in severe male factor patients. Patients with non-mosaic Klinefelter's syndrome have comparable risk for sex chromosomes aneuploidy as the rest of the patients with azoospermia. Therefore, genetic screening during pregnancy or before embryo replacement should be carefully considered in severe male factor patient following in vitro fertilization (IVF).

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.

Use of interphase fluorescence in situ hybridization in third trimester fetuses with anomalies and growth retardation.

In the last few years, attention has been focused on the use of interphase fluorescence in situ hybridization (FISH) for prenatal diagnosis with chromosome-specific DNA probes in the second trimester. This technique is accurate, rapid, and detects the most common aneuploidies. We present a preliminary study using FISH technique on uncultured amniotic cells derived from 30 fetuses with ultrasonographic evidence of intrauterine growth retardation (IUGR) in the third trimester. Fifteen fetuses were males and 15 were females. Seven fetuses (23.3%) had abnormal chromosomal constitution: five (18.6%) had trisomy 21, one (2.35%) had trisomy 18, and one (2.35%) showed a mosaic trisomy 18. No abnormalities were detected in the other 23 fetuses. Amniocentesis combined with FISH appears to be a safe, rapid, and accurate alternative to blood sampling in the third trimester, reducing the clinical and emotional stress of the time required to complete chromosome analysis by routine cytogenetics.

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.

Caudal dysplasia sequence with penile enlargement: case report and a potential pathogenic hypothesis.

The clinical spectrum of caudal dysplasia sequence (CDS) is noted for its diversity. The origin of CDS remains unknown, though poorly controlled gestational diabetes has been implicated in some cases. Here we describe the case of a newborn with CDS associated with penile enlargement (PE). The main anomalies included anal atresia, agenesis of the kidneys and of the sacrococcygeal vertebrae, dysgenesis of lumbar vertebrae, and bilateral cryptorchidism. Penile enlargement (7 cm), a rather unusual finding, has so far not been reported in association with CDS. Chromosomal analysis failed, and the neonate died 30 min after birth. Comparative genomic hybridization analysis using stored DNA showed a balanced normal male DNA content, which negates chromosomal losses or gains as a cause of CDS and/or PE. PE due to virilizing-type adrenal hyperplasia, caused by common mutations in the genes encoding for the adrenal enzymes 21-hydroxylase and 11-hydroxylase, was ruled out. We report on a previously unpublished case of the coexistence of PE and severe CDS and propose a possible pathogenetic hypothesis of this association.

Comparative genomic hybridization analysis of radiation-associated and sporadic meningiomas.

Ionizing irradiation to the skull is a known risk factor for meningioma development. To gain insight into the molecular mechanisms that underlie radiation-associated meningioma (RAM), we characterized the somatic genetic alterations in 16 RAMs by using comparative genomic hybridization and compared the pattern of alterations with 17 nonradiation-associated meningiomas (non-RAM). Most tumors (29/33;87.9%) displayed at least one DNA copy number alteration, and 11 out of 33 (33%) exhibited four or more changes. The mean number of DNA copy number changes was similar in RAMs (2.4+/-1.9) and in non-RAMs (2.5+/-1.9). The most common DNA losses were noted in chromosome 22 (56.2% in RAM, and 47% in non-RAM) and chromosome 1 (37.5% in RAM and 35.3% in non-RAM), with no significant differences between the two groups. Noteworthy, gain in DNA copy number of chromosomes 8 and 12 was detected in two RAM tumors only. In conclusion, no significant differences were noted between RAMs and non-RAMs regarding the number of genetic changes and the extent and frequency of chromosomes 1 and 22 losses. These preliminary data suggest that the tumorogenic pathways of meningioma formation are similar, regardless of previous skull irradiation.

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 Klinefelter's 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 Klinefelter's 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 Klinefelter's 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.

Advanced FISH with directly labeled X, Y and 18 DNA probes as a tool for rapid prenatal diagnosis.

OBJECTIVE: To examine a rapid technique for identification and determination of fetal sex chromosomes and one autosome (chromosome 18) in uncultured amniotic fluids using fluorescence in situ hybridization (FISH) with directly labeled DNA probes and ignoring the use of proteinase K and Rnase. STUDY DESIGN: Twenty-five amniotic samples taken from pregnant women who were in their 18th gestational week and had advanced maternal age were studied for analysis of sex chromosomes and chromosome 18 with the FISH technique as well as standard cytogenetic analysis. RESULTS: Four hours after amniocentesis was performed, we identified the sex of the fetuses and disomy of chromosome 18 in a minimal sample of uncultured amniotic fluid by using directly labeled DNA probes for chromosomes X, Y and 18 (VYSIS) and ignoring the use of proteinase K and Rnase. CONCLUSION: The possibility of shortening the time required for identification of aneuploidies in a second-trimester fetus is useful in cases where fetal anomalies are ultrasonically diagnosed at a relatively advanced gestational age.

Cognition, psychosocial adjustment and coping in familial cases of velocardiofacial syndrome.

Velocardiofacial syndrome (VCFS) is characterized by both physical manifestations and neuropsychiatric disabilities. About 6-28% of cases are familial. The aim of the present study was to compare the clinical characteristics of subjects with familial and nonfamilial VCFS, with a special focus on cognitive and psychiatric disabilities. In addition, the complexities of coping with the disease in families in which both a parent and children are affected were highlighted in case vignettes. Sixteen patients from six families with VCFS were compared to 63 subjects with nonfamilial VCFS for physical parameters, IQ, and rate of major psychiatric disorders. After controlling for the effect of age, IQ was significantly lower in the familial compared to the nonfamilial group of VCFS patients. Rate of psychiatric disorders was similarly high in both groups. The familial group had fewer cardiac and palate anomalies. A significant negative correlation was found between IQ and age. Most of the adults with familial VCFS were neuropsychiatrically disabled. Thus, although familial VCFS seems to be associated with a milder physical phenotype than nonfamilial VCFS, the neuropsychiatric deficits are significant in both types, at all ages.

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.

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. The successful application of the CGH technique to the detection of aneuploidy in spontaneous abortions, demonstrates the utility of using this technique to screen prenatally for numerical chromosome abnormalities. Our preliminary data support the application of CGH to the clinical genetics setting, at least as a complementary tool to the traditional cytogenetic techniques.

Detection of chromosomal aberration in fetuses arising from recurrent spontaneous abortion by comparative genomic hybridization.

Chromosomal abnormalities (mostly aneuploidy) account for approximately 50% of fetal losses in the first 8-15 weeks of gestation. Cytogenetic analysis of aborted fetal material depends on conventional tissue culturing and karyotyping. This technique is laborious and is subject to problems including external contamination, culture failure and selective growth of maternal cells. Comparative genomic hybridization (CGH) was used to determine the chromosomal constitution of 27 fetuses arising from recurrent spontaneous abortion. In 12 samples, the CGH results were compared to the results obtained by conventional cytogenetic techniques. Correlation was found in 75% of samples. Overall, CGH detected chromosomal abnormalities in 48% of the samples, including trisomies, monosomies, and partial chromosome gains and losses. The preliminary data in this study show that CGH can be added, at least as a complementary method, to the traditional cytogenetic techniques used in the investigation of recurrent spontaneous abortions.

Deletion patterns of the STS gene and flanking sequences in Israeli X-linked ichthyosis patients and carriers: analysis by polymerase chain reaction and fluorescence in situ hybridization techniques.

BACKGROUND: Deletion of the entire steroid sulfatase (STS) gene is the most common molecular defect in X-linked ichthyosis (XLI) patients. Usually, additional flanking sequences are also missing. The aim of this study was to estimate the extent of deletions in an ethnically heterogeneous population of Israeli XLI patients. METHODS: Multiplex polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques were applied in the analysis of blood samples of 24 patients and amniotic cells of seven affected fetuses from 22 unrelated families. RESULTS: In 19 families, a large deletion of the 2-3 megabase was found. It included the whole STS gene and spanned adjacent areas up- and downstream between the loci DXS 1139 and DXS 1132. Two unrelated families of Iraqi ancestry had a partial deletion of the gene and its centromeric adjacent sequence. In another family, the telomeric end of the extragenic segment was only partially missing. Application of FISH on metaphase blood cells and interphase amniotic cells confirmed the diagnosis of XLI in all patients, except the three with partial intragenic deletion. In those cases, the remaining fraction of the gene was sufficient to provide a false negative result. Diagnosis of carriers and prenatal diagnosis in uncultured cells was applicable only by FISH. CONCLUSIONS: Our study revealed a remarkable heterogeneity in the deletion pattern among Israeli patients with XLI. This heterogeneity could not be attributed to specific ethnic groups because of the small size of the study group. More studies involving patients of various ancestries should be carried out. In addition, this study demonstrated the usefulness of the FISH technique in the prenatal diagnosis of fetuses with suspected XLI.