Genetic abnormalities detected by comparative genomic hybridization in a human endometriosis-derived cell line.

Comparative genomic hybridization (CGH) was used in parallel with fluorescence in-situ hybridization (FISH) and conventional karyotyping to perform a genome-wide survey of DNA gains and losses in the endometriosis-derived permanent cell line, FbEM-1. The cytogenetic analysis showed a complex karyotype with numerical changes and multiple chromosome aberrations, including the der(1) complement marker exhibiting a large homogenous staining region (HSR). The chromosomal rearrangement interpreted as der(5) t(5;6)(q34;p11) was found in the majority of the metaphases indicating a clonal abnormality. Repeated CGH experiments demonstrated over-representation of chromosomes 1, 2, 3, 5, 6p, 7, 16, 17q, 20, 21q and 22q, while chromosomes 6q, 9, 11p, 12, 13q, 18 and X were under-represented. Using DNA from the original endometriotic tissues, including a peritoneal implant and ovarian endometrioma, CGH analysis revealed loss of DNA copy number on 1p, 22q and chromosome X, while gain was found on chromosomal arms 6p and 17q. FISH analysis confirmed that the gain at 17q includes amplification of the proto-oncogene HER-2/neu in 16% of the FbEM-1 nuclei and in 12% of cells from the primary ovarian endometrioma tissue. These findings demonstrate that FbEM-1 cells share certain molecular cytogenetic features with the original tissue and suggest that chromosomal instability is important in the development of endometriosis.

Narrowing the Duane syndrome critical region at chromosome 8q13 down to 40 kb.

Duane syndrome (MIM 126800) is an autosomal dominant disorder characterised by primary strabismus and other ocular anomalies, associated with variable deficiency of binocular sight. We have recently identified a < 3 cM smallest region of deletion overlap (SRO) by comparing interstitial deletions at band 8q13 in two patients (one described by Vincent et al, 1994, and the other by Calabrese et al, 1998). Here we report on another patient with Duane syndrome carrying a reciprocal translation t(6;8)(q26;q13). FISH and PCR analyses using a YAC contig spanning the SRO narrowed the Duane region to a < 1 cM interval between markers SHGC37325 and W14901. In addition, the identification and mapping of two PAC clones flanking the translocation breakpoint, allowed us to further narrow the critical region to about 40 kb. As part of these mapping studies, we have also refined the map position of AMYB, a putative candidate gene, to 8q13, centromeric to Duane locus. AMYB is expressed in brain cortex and genital crests and has been previously mapped to 8q22.

Cellular and genetic constitution of human endometriosis tissues.

For many years, endometriosis has been an enigmatic and confusing disorder, but there have been recent contributions to the subject, provided by modern techniques in cellular and molecular biology, regarding the cell lineage involved, the stage of differentiation, and genomic features. This review deals mainly with the cellular, cytochemical, cytogenetic, and molecular cytogenetic features of primary endometriotic lesions and cultured endometriotic cells. The FbEM-1 cell line, taken as an in vitro model, showed cell proliferation and differentiation features suggesting an immature endometriosis-related cell lineage. Chromosomal analysis of these cells demonstrate a complex karyotype including a rearrangement interpreted as der(5) t(5q34;6p11) indicating a clonal cell proliferation. Data of recurrent DNA sequence copy number alterations detected by the comparative genomic hybridization in a series of primary endometriotic lesions also are described. Predominant recurrent anomalies were found on chromosome 1p and 22q in 50% of the studied samples. Additional losses were seen on chromosomes 5p(33%), 6q(27%), 7p(22%), 9q(22%), and 1q(22%), as well as on 17q segments in one case. Gain of DNA sequences was seen on chromosomes 6q, 7q, and 17q. The potential role of the genetic changes identified are discussed in relation to the putative oncogenes and/or tumor suppressor genes possibly involved in development of endometriosis.

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization.

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.

45,X/46,XY mosaicism: report of 27 cases.

OBJECTIVES: There exist substantial differences between prenatally and postnatally diagnosed cases of 45,X/46,XY mosaicism. Ninety percent of prenatally diagnosed cases show a normal male phenotype, whereas the postnatally diagnosed cases show a wide spectrum of phenotypes. This 10% risk of an abnormal outcome in prenatally diagnosed cases requires further attention. The purpose of the present study is to provide more information on the postnatally diagnosed 45,X/46,XY mosaicism cases. To date, only a few series have been reported. An accurate diagnosis in these patients is essential not only to their follow-up, but also to providing appropriate genetic counselling and subsequent prenatal diagnosis to their parents. METHODS: The clinical, cytogenetic, endocrinologic, histologic and molecular biological findings of 27 patients with 45, X/46,XY mosaicism are analyzed. RESULTS: The reported cases showed a wide spectrum of phenotypes as Turner syndrome, mixed gonadal dysgenesis (MGD), male pseudohermaphroditism (MPH) and apparently normal male. However, Ulrich-Turner stigmata were the most common features found in this series. Patients with MGD or MPH presented with various degrees of sex reversal such as hypospadias and/or abnormal internal genitalia. No correlation between the proportion of the 45,X/46,XY cell lines in the blood or the fibroblasts and the phenotype was found. Mild mental retardation was present in 4 of the patients and 2 patients showed signs of autism. CONCLUSIONS: Two major points are emphasized in this series: 1) the presence in 7 histologically analyzed streak gonads of a homogeneous 45,X chromosomal complement suggests that the invasion of the primitive genital ridge by a such a cell line may induce abnormal gonadal development; 2) 3 males, apparently normal at birth, developed late onset abnormalities such as dysgenetic testes leading to infertility, Ulrich-Turner stigmata, dysmorphic features, and mild mental retardation. These data indicate the importance of an accurate clinical and histologic evaluation of any patient presenting with 45, X/46,XY mosaicism.

Failure of testicular development associated with a rearrangement of 9p24.1 proximal to the SNF2 gene.

In 46,XY individuals, testes are determined by the activity of the SRY gene (sex-determining region Y), located on the short arm of the Y chromosome. The other genetic components of the cascade that leads to testis formation are unknown and may be located on the X chromosome or on the autosomes. Evidence for the existence of several loci associated with failure of male sexual development is indicated by reports of 46,XY gonadal dysgenesis associated with structural abnormalities of the X chromosome or of autosomes (chromosomes 9, 10, 11 and 17). In this report, we describe the investigation of a child presenting with multiple congenital abnormalities, mental retardation and partial testicular failure. The patient had a homogeneous de novo 46,XY,inv dup(9)(pter-->p24.1::p21.1-->p23.3::p24.1-->qter) chromosome complement. No deletion was found by either cytogenetic or molecular analysis. The SRY gene and DSS region showed no abnormalities. Southern blotting dosage analysis with 9p probes and fluorescent in situ hybridisation data indicated that the distal breakpoint of the duplicated fragment was located at 9p24.1, proximal to the SNF2 gene. We therefore suggest that a gene involved in normal testicular development and/or maintenance is present at this position on chromosome 9.

A duplication of distal Xp associated with hypogonadotrophic hypogonadism, hypoplastic external genitalia, mental retardation, and multiple congenital abnormalities.

An unusual familial case of three sibs with a partial duplication of distal Xp sequences is described. The proband, an 18 year old boy, showed mental retardation, severe dysmorphic features, hypogonadotrophic hypogonadism (HHG), and hypoplastic external genitalia. His karyotype was 46,Y,inv dup(X) (p22.11-->p 22.32). The proband has two sisters each with the same inv dup(Xp) chromosome. Both sisters presented with short stature but were otherwise phenotypically normal. The abnormal X chromosome was inactive in the majority of cells examined. Southern blot dosage analysis indicated a duplication of distal Xp sequences. The proximal breakpoint is located between DXS28 and DXS41, and is therefore at least 2 Mb distal to the DSS locus. The relationship between the phenotype and the Xp duplication is discussed.

A novel mutation in the putative DNA helicase XH2 is responsible for male-to-female sex reversal associated with an atypical form of the ATR-X syndrome.

We describe a pedigree presenting X-linked severe mental retardation associated with multiple congenital abnormalities and 46,XY gonadal dysgenesis, leading in one family member to female gender assignment. Female carriers are unaffected. The dysmorphic features are similar to those described in the alpha-thalassemia and mental retardation (ATR-X) syndrome, although there is no clinical evidence of alpha-thalassemia in this family. In addition, the family had other clinical features not previously observed in the ATR-X syndrome, including partial optic-nerve atrophy and partial ocular albinism. Mutations in a putative DNA helicase, termed XH2, have been reported to give rise to the ATR-X syndrome. We screened the XH2 gene for mutations in affected members of the family and identified a 4-bp deletion at an intron/exon boundary that removes an invariant 3' splice-acceptor site. The mutation cosegregates with the syndrome. The genomic deletion causes missplicing of the pre-mRNA, which results in the loss of 8 bp of coding sequence, thereby generating a frameshift and a downstream premature stop codon. Our finding increases the range of clinical features associated with mutations in the XH2 gene.

Testicular development in an SRY-negative 46,XX individual harboring a distal Xp deletion.

A case of a true hermaphrodite presenting with a karyotype of 46,X,del(X)(p21.1-->pter) is described. The testis-determining gene, SRY, was not detected in DNA prepared from either peripheral blood lymphocytes or from a gonad biopsy. The patient also presented with a series of discrete somatic abnormalities, including abnormal skin and retinal pigmentation, and mental retardation. The extent of the Xp deletion was mapped by Southern blotting. X chromosome replication studies of lymphoblast cells prepared from the patient indicated that the deleted X chromosome was inactivated in all cells examined. It is suggested that the phenotype of the patient is caused by the unmasking of a recessive allele(s) on the grossly intact X chromosome. The relationship between the Xp deletion, the intersex phenotype, and the possible role of an Xp locus involved in human sex determination is discussed.

Gonadal dysgenesis in del(18p) syndrome.

We report on a girl with syndromal gonadal dysgenesis and a de novo del(18p). Genetic factors controlling gonadal development are located not only on the X chromosome, but also on autosomes. The present case suggests that one of these genes is situated on 18p. We conclude that patients with del(18p) syndrome should be evaluated for gonadal dysgenesis.

Is Rett syndrome a chromosome breakage syndrome?

Lymphocytes from venous blood from 15 girls with Rett syndrome (RTS), 7 girls with RTS "forme fruste," and 46 unrelated control females were examined. All subjects had a normal karyotype using RHG and RTBG technique. The frequency of gaps and breaks was determined for each group. A significantly higher (P < 0.01) frequency of chromosome breakage was observed in RTS subjects compared to controls. This work suggests that an increased tendency to chromosome breakage may be part of a genetically determined disorder in RTS patients.

De novo t(X;21)(q28;q11) in a girl with phenotypic features of Williams-Beuren syndrome.

We describe a female infant with mental retardation and some of the phenotypic features of Williams-Beuren syndrome. Chromosome analysis showed t(X;21)(q28;q11). Diagnosis, inactivation of the X chromosome, and possible involvement of the translocation breakpoints in the pathogenesis of this syndrome are discussed.

An antisense interferon-beta RNA abolishes repression of c-fos gene expression.

To determine the cellular functions which are modified when interferon-beta (IFN-beta) gene expression is inhibited, a plasmid allowing the constitutive expression of RNA complementary to IFN-beta mRNA was constructed and stably introduced into L929 cells. Some of the selected clones expressing this antisense IFN-beta mRNA, named L-ASI, were unable to produce IFN-beta and lost the ability to arrest in the G0 phase of the cell cycle. Indeed, the usual transrepression of the c-fos gene observed in quiescent cells was blocked in IFN-beta antisense L-ASI clones and the c-fos gene was permanently stimulated. This overexpression of c-fos was not modified in response to protein kinase C agonists such as phorbol esters, but increased in response to the adenylate cyclase activator forskolin. In addition, the ability to induce major histocompatibility class I genes following recombinant IFN-beta treatment was impaired in antisense IFN-beta L-ASI clones, suggesting an important alteration of this cell with regard to the interferon system. Unexpectedly, the tumorigenicity of the clones was significantly diminished. We postulate that IFN-beta antisense RNA blocks the repression of the c-fos gene and thus prevents the arrest of cells in the G0 phase of the cycle.

Segregation of three reciprocal translocations in the same family: t(3;4), t(5;10), and t(15;21).

A male infant with static antenatal encephalopathy and epilepsy was found to have a duplication of 5p12----5pter and deficiency of 10p13----10pter. Each of his parents was a carrier of a balanced reciprocal translocation. A third translocation was found in the maternal grandfather. The pedigree of each translocation and the segregation of parental reciprocal translocations are discussed.

Immunosuppressive effects of isoprinosine in man: a comparison to chlorambucil effects in multiple sclerosis.

Immunological and clinical functions were studied over a 2 year period in conjunction with a placebo controlled trial of isoprinosine and chlorambucil in 21 patients with exacerbating remitting multiple sclerosis. Laboratory and clinical evaluations were performed at 3 month intervals and during relapses. In placebo-treated patients, the decrease in circulating T8+ cells was maximum during relapses, T lymphocyte function was impaired, and five of the six patients experienced clinical worsening. Chlorambucil treatment was responsible for a decrease in circulating T4+ and T8+ cells; nevertheless, T lymphocyte function was slightly improved during relapses. The alterations of delayed hypersensitivity responses were not accompanied by improvement in relapse rate or in intensity and major side effects: mainly infections with leukopenia and thrombocytopenia. During isoprinosine therapy, a regulation of circulating T lymphocytes and cell proliferation occurred. The higher level of circulating T cells was related to the increase in T4+ and T8+ cells, which did not decrease during relapses. The absence of Leu 7+ cell modifications suggest that NK were numerically unaffected by isoprinosine therapy and that in vivo regulation of circulating T suppressor cells was performed by this treatment. Four out of seven patients did not experience any relapse during the duration of the trial. In relapsing patients, the frequency and duration of the relapses were significantly different from that of other patients. A reduction of the disease progression was observed without any side effects. While no conclusion can be drawn on the long-term effectiveness, the results of this pilot study are consistent indicators of the immunological and clinical beneficial effects of isoprinosine therapy in patients with exacerbating remitting multiple sclerosis.

Immunological effects of isoprinosine as a pulse immunotherapy in melanoma and ARC patients.

Immunomodulatory effect of Isoprinosine are presented in melanoma and HTLV-III/LAV infected patients. Isoprinosine (50 mg/kg) was used as a pulse immunotherapy according to two different schedules: A) 5 days every 15 days and B) 5 days every 15 days for 2 months, then 5 days every 2 months. The patients' immunological profiles were tested before and during the treatment in terms of T-cell subsets, cell number requirement for PHA-induced proliferation, and delayed hypersensitivity reaction to recall antigens. Primary malignant melanoma patients are randomized between surgery alone or associated to isotherapy (schedule A or B). Schedule A, after an initial improvement of surgery-induced immune deficiency, is responsible for an immunodepression, whereas schedule B determines a prolonged restoration in immune responses in melanoma and AIDS related complex or Kaposi sarcoma patients as well. In vitro effects of Isoprinosine on HTLV-III/LAV infection are presented. These data exhibit 1) the need of an immunological follow-up during isotherapy and 2) the immunological benefit of a pulse immunotherapy during acquired immunodeficiencies related to cancer surgery or to HTLV-III/LAV infection in man.