(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features.

Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene. The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities. As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model. The mouse displayed reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype.

A submicroscopic deletion in Xq26 associated with familial situs ambiguus.

Abnormal left-right-axis formation results in heterotaxy, a multiple-malformation syndrome often characterized by severe heart defects, splenic abnormalities, and gastrointestinal malrotation. Previously we had studied a large family in which a gene for heterotaxy, HTX1, was mapped to a 19-cM region in Xq24-q27.1. Further analysis of this family has revealed two recombinations that place HTX1 between DXS300 and DXS1062, an interval spanning approximately 1.3 Mb in Xq26.2. In order to provide independent confirmation of HTX1 localization, a PCR-based search for submicroscopic deletions in this region was performed in unrelated males with sporadic or familial heterotaxy. A cluster of sequence-tagged sites failed to amplify in an individual who also had a deceased, affected brother. FISH identified the mother as a carrier of the deletion, which arose as a new mutation from the maternal grandfather. The deletion interval spans 600-1,100 kb and lies wholly within the 1.3-Mb region identified by recombination. Discovery of this deletion supports localization of HTX1 to Xq26.2 and reveals the first molecular-genetic abnormality associated with human left-right-asymmetry defects.

DMD-specific FISH probes are diagnostically useful in the detection of female carriers of DMD gene deletions.

The challenge of Duchenne muscular dystrophy (DMD) carrier identification resides in the ability to identify the presence of a mutant gene over the background contributed by the normal allele. Current diagnosis of carrier status when a deletion has been identified in a proband is based on an analysis of a gene dosage. We present a diagnostic strategy that uses fluorescence in situ hybridization (FISH) to detect female carriers with major deletions in the dystrophin gene. We screened a human X-chromosome-derived genomic library with a full-length dystrophin cDNA and isolated 15 dystrophin-specific cosmids that contain DMD gene exons. Six cosmids were further tested as FISH probes in control individuals and subsequently applied on chromosomes from eight males with DMD and known deletions and on samples from three female carriers. As expected, X chromosomes in normal females displayed four signals, two for the DMD-specific probe and two for the X-chromosome centromeric probe. Hybridization on chromosomal spreads from carriers of deletions revealed only one signal from the DMD-specific probe and two from the control centromeric probe. Males carrying deletions showed no DMD-specific signal for the deleted exons tested. Our data indicate that FISH could represent an alternative method for the detection of female carriers with DMD gene deletions.

FISH in the evaluation of pleural and ascitic fluids.

Pleural and ascitic fluids (PAF) are complications of both nonmalignant and malignant conditions, such as congestive heart failure and chronic infections, as well as neoplasias, such as mesothelioma, lymphoma, and adenocarcinomas of the lung, ovary, endometrium, breast, colon, stomach, and pancreas. Differentiation between malignant and nonmalignant PAF is not always easy to assess on the basis of clinical, cytologic, and other criteria. A review of the chromosomal anomalies in neoplasms which can cause PAF revealed aneusomies of chromosomes 1, 3, 7, 8, 10, and 11 in about 40% to 80% of these malignancies. We performed FISH using centromere-specific probes for chromosomes 1, 3, 7, 8, 10, and 11 and chromosomal analysis on PAF cells from 21 patients, including 3 with ovarian cancer, 2 with lymphoma, 5 with adenocarcinoma of unknown origin, 1 with breast cancer, and 10 with atypical lymphocytosis of unknown cause. The results indicate a) a high correspondence between FISH and the clinical diagnosis (9 of the 11 cases of malignant fluid showing FISH abnormalities); b) that FISH is more sensitive than cytogenetics in detecting abnormal clones (10 vs. 6); and c) that FISH is a valuable adjunct to cytology in the interpretation of atypical lymphocytosis (3 of the 10 cases were shown to be abnormal by FISH). Thus, the FISH technique can be a very useful adjunct to conventional cytogenetics in yielding crucial information on the origin of PAF.

Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome.

Nearly all cases of fragile X syndrome result from expansion of a CGG trinucleotide repeat found in the 5' untranslated portion of the FMR1 gene. Methylation of the expanded repeats correlates with down-regulation of transcription of FMR1; thus fragile X syndrome is postulated to be due to a loss of function of the FMR1 gene product, and this has been demonstrated at the protein level. However, the nature of the mutation offers the possibility of methylation spreading to adjacent genes with consequent loss of expression and contribution to the phenotype. Deletions of FMR1 and flanking sequence (some of substantial size) have been reported in patients with phenotypes consistent with a diagnosis of fragile X-syndrome, however, none is strictly intragenic. We report here the identification of two different intragenic loss of function mutations in FMR1: a single de novo nucleotide deletion in a young male patient (IJ) and an inherited two basepair change in an Adult male (SD), each with classical features of fragile X syndrome.

Deletion 6q in three cases of mixed-type liposarcoma in addition to t(12;16)(q13;p11)

We report the cytogenetic findings in three mixed liposarcoma following short-term cultures. During the course of cytogenetic investigation of various types of liposarcomas, we observed an interstitial deletion of the long arm of chromosome 6 together with the translocation (12;16)(q13;p11) in three tumors. Translocation (12;16) is associated with myxoid and mixed (myxoid/round cell) liposarcomas, although deletion of chromosome 6 has been observed in only a few of these tumors. Our findings suggest that del(6), as an additional change in myxoid liposarcoma, is probably related to tumor progression.

Involvement of chromosome 7 in Wilms tumor.

Cytogenetic and molecular analysis of Wilms tumors have led to the identification of two regions on the short arm of chromosome 11 (11p13 and 11p15) involved in tumor development. Recent studies have provided evidence that an additional locus on 16q is also involved. Further molecular testing may reveal additional loci associated with the development or progression of this tumor. Reports of single chromosome abnormalities in tumors generally pinpoint regions of interest that may be involved in the etiology of the tumor. We present an additional case of Wilms tumor with an isochromosome 7q as the sole cytogenetic change, resulting in loss of 7p and gain of 7q material.

FISH studies of urinary cells of patients with bladder cancer.

Generally, bladder cancers are characterized by complex and numerous chromosome changes that vary from tumor to tumor. Nevertheless, certain chromosome changes recur with a consistency, (eg, +7, +8, -9, and -Y). In applying fluorescence in situ hybridization (FISH) studies to urinary cells in bladder cancer we chose probes for these chromosomes as well as those for chromosomes 10 and 11. A probe for the X chromosome was used for female patients in place of the Y. In the present study, we show that FISH of urine samples can detect the presence of cancer cells in transitional cell carcinoma (TCC) of the bladder of any grade and stage, including carcinomas in situ (CIS). We analyzed 27 samples from 25 patients (three were from the same patient); 24 samples were recurrent or newly diagnosed TCC and 3 were CIS. Our results show that FISH of urine samples is a reliable test for the detection of bladder cancer cells, regardless of the grade and stage of the tumor, and that a correlation appeared to exist between invasiveness of the tumor and the number of abnormalities in such tumor.

Trisomy 5 in long-term cultures from bone marrow of patients with solid tumors.

Long-term cultures of bone marrow from 15 cases diagnosed previously with primary solid tumors were analyzed cytogenetically. Of these cases, 10 had normal karyotypes and five had chromosomal abnormalities. Trisomy 5 was found in four cases, three with trisomy 5 as the only change and one with trisomy 5 and trisomy 12. These results suggest that trisomy 5 may be a nonrandom change associated with an in vitro or in vivo phenomenon.

Chromosome abnormalities in breast fibroadenomas.

Cytogenetic analysis of 25 breast fibroadenomas (FA) showed clonal chromosome alterations in three cases. Insertion (12;?) (q15;?) and deletion (2) (q14q31 or q32) were detected as a sole change in cases 1 and 3, respectively. Case 2 displayed the karyotype 45,XX,t(1;8;16)(q25;q23;q22-23), add (7)(p14), rea(15), -17. The present findings are discussed together with the reports on FA in the literature.

Cytogenetic findings in 19 malignant bone tumors.

BACKGROUND: The majority of karyotypes observed in osteosarcomas (OS) and chondrosarcomas (CS) are complex. Specific chromosomal abnormalities have not yet been characterized in either tumor except for a ring chromosome in parosteal OS. The purpose of this study was to determine recurrent chromosomal abnormalities and establish a possible correlation between the cytogenetic changes and the pathologic findings. METHODS: Ten OS and nine CS were cytogenetically analyzed. Tumor samples were obtained from patients having a resection or incisional biopsy. Cytogenetic study of short term cell cultures included harvesting and G-banding, which were performed by routine methodologies. RESULTS: Clonal abnormalities were observed in six OS and six CS. Modal chromosome numbers ranged from near diploid to near tetraploid in both types of tumors. The structural rearrangements observed in OS involved mostly chromosomes 1, 2, 6, 12, and 17. Nonreciprocal translocations were the most frequent event. Two OS had a single clonal abnormality involving 11p15 and 14q32, respectively. Double minute chromosomes were observed in three cases. In CS, the most frequent structural abnormalities were nonreciprocal translocations and deletions involving numerous chromosomes. Rearrangements of 1p together with other abnormalities were observed in four CS. CONCLUSIONS: The karyotypes were usually complex consisting of numerical and structural changes, particularly in high grade tumors. Rearrangements of 11p15 and 14q32 in OS and possibly 1p in CS were found as primary cytogenetic aberrations. Cytogenetic analysis in more cases of OS and CS together with molecular studies are necessary to characterize further the consistent genetic changes in these tumors.

Cytogenetic findings in 21 malignant melanomas.

Cytogenetic analysis was performed on 21 tumor samples of malignant melanoma to identify the presence of consistent chromosome abnormalities. Four cases had a normal karyotype, and 17 were cytogenetically abnormal. Numerical chromosome alterations were observed in 15 tumors: 12 were hyperdiploid and three were hypodiploid. The most frequent losses consisted of chromosomes 5, 9, 17 and Y. The structural abnormalities were usually complex, consisting mainly of nonreciprocal translocations and deletions affecting 1p, 1q, 3p, and 9p. This study adds further data to previously reported melanoma cases, confirming that chromosomes 1, 3, 6, and 9 are nonrandomly affected.

A new approach in the diagnosis and follow-up of bladder cancer. FISH analysis of urine, bladder washings, and tumors.

The aim of the present study was to ascertain whether fluorescence in situ hybridization (FISH) of urine could be a useful approach in bladder cancer. Herein, we present the cytogenetic and FISH findings in patients with and without bladder cancer. The samples examined with FISH consisted of urine, bladder washings, and tumor tissue, when available. The results obtained show that the FISH technique, particularly when used on urine, is a very useful tool in the diagnosis, early detection, and management of bladder cancer.

Relation between regular intake of phenacetin-containing analgesics and laboratory evidence for urorenal disorders in a working female population of Switzerland.

A study group of 623 employed Swiss women aged 30-49 years showing objective evidence of intake of phenacetin-containing analgesics, and a control group of 621 comparable women showing no such intake, were observed for 4 years (1969-72) for laboratory evidence of urorenal disorders. In both study and control groups morbidity was low. There was no difference between the study and control groups with respect to subsequent proteinuria, bacteriuria, and haematuria. The 4-year incidence of low urine specific gravity after overhight thirsting was significantly higher in the study group than in the control group (3-8% v. 0-8%) and the incidence of raised serum-creatinine was also significantly higher in the study group (2-9% v. 0-4%). However, when the study group was further subdivided into a sub-group showing evidence of high intake of phenacetincontaining analgesics and one showing low intake, only the high-intake subgroup had an incidence of raised serum-creatinine (5-4%) significantly higher than the control group (0-4%), whereas the low-intake subgroup had an incidence (0-4%) similar to the control group.