H. D. Hager

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

SummaryThe localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes

SummaryTwo cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter→Xp22.2::Yq11→Y qter) contained in the Chinese hamster x human hybrid cell line 445 x 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.

Isoform of fibronectin mediates bone loss in patients with primary biliary cirrhosis by suppressing bone formation.

Osteoporosis is a major cause of morbidity and decreased quality of life in patients with chronic cholestatic liver disease. It is established that this osteoporosis results from decreased bone formation, but the mechanisms for the interaction between liver and bone remain elusive. The aim of this study was to test the hypothesis that an increase in the production of cellular fibronectins during liver disease may result in decreased osteoblast‐mediated mineralization and thus explain the decrease in bone formation. We performed a prospective cross‐sectional study in patients with primary biliary cirrhosis and matched controls, followed by experiments on human and mouse osteoblasts in culture and injections in mice in vivo. In patients with primary biliary cirrhosis, the oncofetal domain of fibronectin correlated significantly with the decrease in osteocalcin, a marker of bone formation (r = −0.57, p < 0.05). In vitro, amniotic fluid fibronectin (aFN) containing mainly the oncofetal domain and EIIIA domain resulted in decreased osteoblast‐mediated mineralization in human osteoblasts (69% decrease at 100 μg/ml; p < 0.01) and mouse osteoblasts (71% decrease; p < 0.05). Removing the EIIIA domain from aFN similarly suppressed mineralization by osteoblasts (78% decrease; p < 0.05). Injection of labeled aFN in mice showed that it infiltrates the bone, and its administration over 10 days resulted in decreased trabecular BMD (17% drop; p < 0.05), mineralizing surface (30% drop; p < 0.005), and number of osteoblasts (45% drop; p < 0.05). Increased production of a fibronectin isoform containing the oncofetal domain and its release in the circulation in patients with primary biliary cirrhosis is at least partially responsible for the decrease in bone formation seen in these patients. This establishes that a molecule that has thus far been viewed as an extracellular matrix protein exerts hormone‐like actions.

Study of 30 patients with unexplained developmental delay and dysmorphic features or congenital abnormalities using conventional cytogenetics and multiplex FISH telomere (M-TEL) integrity assay

Abstract. Cryptic subtelomeric chromosome rearrangements are a major cause of mild to severe mental retardation pointing out the necessity of sensitive screening techniques to detect such aberrations among affected patients. In this prospective study a group of 30 patients with unexplained developmental retardation and dysmorphic features or congenital abnormalities were analysed using the recently published multiplex FISH telomere (M-TEL) integrity assay in combination with conventional G-banding analysis. The patients were selected by one or more of the following criteria defined by de Vries et al.: (a) family history with two or more affected individuals, (b) prenatal onset growth retardation, (c) postnatal growth abnormalities, (d) facial dysmorphic features, (e) non-facial dysmorphism and congenital abnormalities. In addition, we included two patients who met these criteria and revealed questionable chromosome regions requiring further clarification. In four patients (13.3%) cryptic chromosome aberrations were successfully determined by the M-TEL integrity assay and in two patients with abnormal chromosome regions intrachromosomal aberrations were characterized by targetted FISH experiments. Our results accentuate the requirement of strict selection criteria prior to patient testing with the M-TEL integrity assay. Another essential precondition is high-quality banding analysis to identify structural abnormal chromosomes. The detection of familial balanced translocation carriers in 50% of the cases emphasizes the significance of such an integrated approach for genetic counselling and prenatal diagnosis.

Multiplex FISH telomere integrity assay identifies an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three mentally retarded individuals

Abstract. Cryptic rearrangements involving the terminal regions of chromosomes are suspected to be the cause of idiopathic mental retardation in a significant number of cases. This finding highlights the necessity of a primary screening test for such chromosome aberrations. Here we present a multiplex fluorescence in situ hybridization telomere integrity assay which allows the detection of submicroscopic aberrations in the telomeric regions of all chromosomes. This novel approach identified an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three cases of unexplained mental retardation and dysmorphic features. The symptoms of the patients represent neither the classical dup(3q)- nor cri du chat syndrome, although all affected individuals demonstrate several features of both syndromes. The identification of two balanced translocation carriers emphasizes the significance of the telomere integrity assay for genetic counseling and prenatal diagnosis

Position of chromosomes in the human interphase nucleus

SummaryThe problem of localization of chromosomes in relation to each other in the interphase nucleus of human lymphocytes was investigated by analysis of chromatid and chromosome aberrations observed in lymphocyte cultures of three patients with Fanconis anemia, one patient with Blooms syndrome, and in Trenimon-treated (Trenimon, Bayer) normal cells. Distribution of open gaps and breaks is highly correlated with chromosome length and distribution of breaks involved in chromatid translocations in Fanconis anemia and in Trenimontreated cells. Both correlations are much lower in Blooms syndrome. In Fanconis anemia and in normal cells after Trenimon-treatment, the majority of chromatid translocations are between nonhomologous chromosomes, whereas in Blooms syndrome mainly homologous chromosomes are involved. Statistical localization of chromosomes in relation to each other in the three-dimensional space by multidimensional scaling gives results consistent with the limited amount of independent evidence.

Three new 46,XX male patients: A clinical, cytogenetic and molecular analysis

BACKGROUND XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. AIM To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. PATIENTS AND METHODS We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. RESULTS Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. CONCLUSIONS In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

The problem of partial endoreduplication

SummaryPartial endoreduplication (PE) as defined by Lejeune et al. (1966) has only been found in a few instances. Similar configurations, also called PEs, seem to originate from a different process. A series of 12 PEs is presented in this paper, discovered in metaphases from healthy individuals, and in patients with or without chromosome-breakage syndrome and after treatment with chromosome-breaking agents. Interpretations of the microscopic appearance of each configuration led to the conclusion that there are three different modes of origin for such rare events, one being true partial endoreduplication, the second a partial pseudoendoreduplication, and the third a homologous triradial chromatid translocation.

Clinical, cytogenetic and molecular investigations in three patients with Wolf‐Hirschhorn syndrome

Clinical, cytogenetic and molecular studies were performed in three patients with Wolf‐Hirschhorn syndrome (WHS). In all cases the altered chromosome 4 appeared to be the result of a de novo deletion. Cytogenetic investigations located the breakpoint at 4p15.3 and 4p13. With cytogenetic methods it was not possible to decide whether these deletions were terminal or interstitial. DNA methods also failed to define a distal breakpoint within the 4p16.3 region which might have indicated an interstitial deletion. According to the literature, the paternal chromosome 4 is preferentially deleted in most patients with WHS. DNA analysis with polymorphic markers out of the 4p16.3 region revealed that in two of the cases reported here the deleted segment was of paternal and in one case of maternal origin.

Survival up to age 10 years in a patient with partial duplication 6q: case report and review of the literature.

Partial duplication of chromosome 6q has been recognized as a distinct dysmorphic syndrome with severe psychomotor and growth retardation, typical craniofacial features including microcephaly and microstomia, neck webbing, congenital contractures, and variable internal malformations. Most patients have died in the first year of life. We describe the clinical features and disease course in a boy with a duplication of 6q23.3-qter who lived up to the age of 10 years and discuss similarities with other patients.