Genetic and epigenetic defects at the 6q24 imprinted locus in a cohort of 13 patients with transient neonatal diabetes: new hypothesis raised by the finding of a unique case with hemizygotic deletion in the critical region.

BACKGROUND: Transient neonatal diabetes (TND) is a rare form of diabetes usually present in the first few days after birth that resolves within 1 year but that has a tendency to recur later in life. It can be associated with chromosome 6 paternal uniparental disomy (UPD), paternal duplications or loss of maternal methylation at the 6q24 imprinted locus. OBJECTIVE: To report on a cohort of 13 sporadic TND cases, including five with birth defects (congenital abnormalities of heart, brain and bone) and eight without. RESULTS: The hallmarks of diabetes were similar in patients with or without 6q24 defects. The chromosome 6 abnormalities in our patients (n = 13) included 2 of 13 (approximately 15.4%) cases of paternal UPD6, 2 of 11 (approximately 18%) cases of complete and 3 of 11 (approximately 27%) cases of partial loss of the maternal methylation signature upstream of ZAC1-HYMAI imprinted genes in non-UPD cases, and 1 of 13 (approximately 7.7%) cases of hemizygotic deletion. CONCLUSION: The deletion was found in a patient with severe congenital abnormalities. This genetic lesion was not reported previously. The hypothesis of an effect on regulatory elements critical for imprinting and tissue-specific gene expression in early development by the deletion is raised. The data presented here may contribute to the diagnosis and the understanding of imprinting in the region.

HLA-DRB1 and DQB1 genotypes in patients with insulin-dependent neonatal diabetes mellitus. A study of 13 cases.

Insulin-dependent neonatal diabetes mellitus (NDM) is a rare form of diabetes with a heterogeneous genetic background. The HLA-DRB1 and DQB1 genotypes were determined for 13 patients with NDM, from 9 unrelated families. Four patients had permanent NDM (PNDM) and 9 patients had transient NDM (TNDM). No excess of HLA susceptibility markers for type 1 diabetes (IDDM) was observed in this series of patients, whatever the forms of diabetes PNDM or TNDM. Paternal isodisomy of chromosome 6 was observed in two TNDM cases. These observations are consistent with the current hypothesis that there is a recessive susceptibility gene, at least in the transient form of the disease, unlinked to the MHC locus on chromosome 6. Although established in a short series, our results do not support an additive role of IDDM1 in the progression of the disease.

Wolcott-Rallison syndrome: a case with endocrine and exocrine pancreatic deficiency and pancreatic hypotrophy.

Clinical analysis and genetic investigations of new cases of Wolcott-Rallison syndrome are needed to evaluate the role of the gene(s) directly or indirectly implicated in pancreas development and in the aetiology of the syndrome.

Variable features of transient neonatal diabetes mellitus with paternal isodisomy of chromosome 6.

We describe two patients who suffered transient neonatal diabetes mellitus (TDNM), due to paternal isodisomy of chromosome 6. One patient, now 5 years old, had severe intra-uterine growth retardation, but recovered normal growth parameters. The other patient, currently 12 years old, had a normal birth weight but showed impaired post-natal growth; in addition to TNDM the patient presented with cardiac and thyroid abnormalities. These cases may suggest that the clinical phenotype of TNDM is more variable than previously believed. The contribution of genetic and epigenetic factors needs to be determined to elucidate the phenotype-genotype relationships of this disease.

Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.

Dynamin (Dyn) is a member of a novel group of GTPases which was initially identified as a microtubule-binding protein with a role in vectorial movement. Three distinct Dyn-encoding genes (DYN I, II and III), with a neuronal-, ubiquitous or testis-specific expression, respectively, have been identified in rat. In man, only DYN I has so far been characterized. We have previously isolated a genomic DNA fragment implicated in the correction of mitomycin C hypersensitivity of cells from a Fanconi anemia patient belonging to genetic complementation group D (FA(D)). Using this probe, we have cloned a human complementary DNA designated hDYN II encoding a ubiquitous Dyn isoform. The predicted protein consists of 866 amino acids (97.5 kDa). Dyn proteins exhibit a high degree of evolutionary conservation: hDyn II is 98% identical to rat Dyn II and 73% identical to hDyn I. A unique 3.6-kb transcript is found in all human tissues examined and it is more abundant in skeletal muscle and heart. This transcript is also expressed in tissue-culture cells. The hDYN II message is present and not mutated in the FA(D) patient studied. In addition to the GTP-binding domain and motifs associated with regulatory function, the hDyn II protein contains a noticeable number of concensus motifs for p34Cdc2 kinase phosphorylation which may indicate a potential role at the G2/mitosis transition. The sequence reported here should allow a more complete analysis of Dyn function(s) in man.

Identification and chromosomal localization of a DNA fragment implicated in the partial correction of the Fanconi anemia group D cellular defect.

Fanconi anemia (FA) cells, complementation group D, which had been transfected with mouse genomic DNA were partially corrected for their mitomycin C (MMC) hypersensitivity. A genomic DNA fragment which complements the resistance of FA(D) cells to MMC close to normal level has been cloned; it has no correcting activity in FA group A cells. It contains two highly conserved regions between the mouse and human genome, which flank mouse repeated DNA. This DNA fragment detects a 3.6-4-kb mRNA transcript in human cells. Moreover this fragment maps to chromosome 11q23, a region of particular interest since several genes involved in the control of major cellular functions are located in this area. This DNA fragment may belong to a gene directly or indirectly involved in FA(D) function.

Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation.

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA. Phenotypic dissociation of chromosomal and cellular hypersensitivity to DNA cross-linking agents.

Transfectants obtained by mouse DNA-mediated gene transfer in Fanconi anemia (FA) primary fibroblasts from the genetic complementation groups A and B were examined for the frequencies of chromosomal aberrations and cytotoxicity following treatments by cross-linking agents. Cells from group A (FA 150), which is the most sensitive to such agents, are partially corrected for both the chromosomal and cellular hypersensitivity to 8-methoxypsoralen photoaddition. In contrast, after treatment with mitomycin C (MMC), only the chromosomal sensitivity is re-established to a near normal level. The opposite is true for FA group B cells (FA 145), i.e. cell survival to MMC is partially corrected, whereas the frequency of MMC-induced chromosomal aberration remains close to that of the untransfected cells. The partial phenotypic correction of the two end points examined is interpreted as indicating either a gene dosage effect or the necessity of introducing more than one gene type in order to achieve complete recovery of a normal phenotype. The phenotypic dissociation between the clastogenic and cellular hypersensitivity to cross-linking agents may offer the opportunity of isolating separately the responsible gene(s) by conventional rescue techniques.

Fanconi's anemia: genetic and molecular aspects of the defect.

Several features of Fanconi's anemia (FA) are reported: relative inefficiency in the processing of DNA cross-links and monoadducts, hypomutability at the two loci analysed, complementation of the cytogenetic defect by cocultivation with mouse cells and homology of the FA group A with the mouse cellular mutant MCS attempts to clone and characterise a DNA fragment which complements the defect. The relation between observed features and predisposition to leukemia are discussed.

An abnormal expression of a tumor-activated multigenic set in cells from cancer prone patients with inherited Fanconi's anemia (FA) and retinoblastoma (Rb).

Normal skin fibroblasts from patients genetically predisposed for cancer express certain phenotypic properties usually associated with transformed cells, suggesting that these cells are engaged in a cancer progression process. Previously, we have shown that a subgenomic fraction, which we called Ta DNA ('tumor-activated DNA') was preferentially expressed in all human tumor cells (Hanania et al., Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6504-6508; Hanania et al, EMBO J., 2 (1983) 1621-1624). Here, we show that Ta DNA transcripts are present in Fanconi's anemia (FA) and retinoblastoma (Rb) fibroblasts, at a level hardly lower than in tumor cells. In contrast, a few Ta DNA transcripts can be detected in normal cells.

Two complementation groups of Fanconi's anemia differ in their phenotypic response to a DNA-crosslinking treatment.

The two genetic complementation groups reported for Fanconi's anemia (FA) correspond to two phenotypic classes as characterized by measurements of the rate of DNA semiconservative synthesis after 8-methoxypsoralen photoaddition. This test allows a rapid genetic classification of FA patients which appears to be a prerequisite for investigations of the biochemical defect(s) in FA.

Abnormal response to DNA crosslinking agents of Fanconi anemia fibroblasts can be corrected by transfection with normal human DNA.

Primary skin fibroblast cell lines from patients with Fanconi anemia were cotransfected with UV-irradiated pSV2neo plasmids and high molecular weight DNA from normal human cells. Restoration of a normal cellular resistance to mitomycin C (MMC) was observed provided that a Fanconi anemia cell line is selected for DNA-mediated transformation (neo gene) and that at least two successive rounds of transfection are performed. Cells were selected by taking advantage of the higher proliferation rate and plating efficiency of the MMC resistant transformants. As estimated from reconstruction experiments, the frequency of transfer of MMC resistance lies between 1 and 30 X 10(-7). The MMC resistance phenotype was maintained for at least 10 generations following transfection. Evidence for DNA-mediated transformation also includes the recovery of a normal pattern of DNA semiconservative synthesis after treatment with 8-methoxypsoralen and 365-nm UV irradiation, and the presence of exogenous pSV2neo DNA sequences was shown by Southern blot analysis. The acquired MMC resistance is probably due to the presence of DNA from normal cells. Indeed, sensitivity to MMC was maintained when Fanconi anemia cells were cotransfected with the UV-irradiated pSV2neo plasmid mixed with their own DNA or with yeast or salmon sperm DNA. These negative results also render unlikely the selection of spontaneous MMC resistant revertants in transfection of Fanconi anemia cells with normal DNA. These experiments establish the prerequisites for the isolation of the gene(s) involved in the response to DNA crosslinking lesions in human cells.

DNA semi-conservative synthesis in normal and Fanconi anemia fibroblasts following treatment with 8-methoxypsoralen and near ultraviolet light or with X-rays.

The effect of treatment with 8-methoxypsoralen (8-MOP) plus near-UV radiation (UVA) or with X-rays on the rate of DNA semi-conservative synthesis of fibroblasts from 10 Fanconi anemia (FA), two heterozygous, and three normal cell lines was studied. Following treatments with either X-rays or low doses of 8-MOP plus UVA leading to a majority of monoadducts over cross-links per genome, the FA and heterozygous cell lines were indistinguishable from normals: the transient inhibition of semi-conservative DNA synthesis was followed by the recovery of a normal rate of synthesis. In contrast treatment with higher (but not saturating) doses of 8-MOP plus UVA allowed us to distinguish two classes among the FA cell lines. One class demonstrated a pattern of recovery similar to that of heterozygous and normal cell lines. This indicates that in such cell lines, the predominant lesion in this condition, the cross-links, do not arrest DNA synthesis and are likely to be normally repaired. Another class of FA cell lines did not show a recovery of a normal rate of DNA synthesis even after prolonged post-treatment incubation and although the proportion of cells in S phase was similar to that of the strains of the first category. This indicates that in such cell lines the repair of cross-links is inhibited at some step which is not necessarily the incision one.

Low dose rate ionizing radiation induces increased growth capacities of d-deletion retinoblastoma skin fibroblasts.

Skin fibroblasts from normal children and three children with a 13q deletion retinoblastoma (Rb) were exposed to cumulative low doses of gamma rays. The typical response of normal donors was a reduction in the lifespan of irradiated fibroblasts, the precocity of the decline being inversely related to the dose received. In contrast, the lifespan of one Rb cell line (Rb1) was prolonged; irradiated cells with an increased growth potential showed a higher number of cells at confluency and more cells were entering DNA synthesis phase than in non-irradiated cells. Another Rb cell line (Rb2) demonstrated a normal lifespan following irradiation but foci were observed in irradiated cultures. Cytogenetic analysis revealed no selection of abnormal clones in these cell populations. The third Rb line examined (Rb3) responded like a normal cell line. We suggest that irradiated skin fibroblasts derived from some patients with Rb are in certain cases able to express abnormal growth capacities which may be one of the manifestations of the high susceptibility of the individual's stromal cells to carcinogenic agents.

[Stimulation by ionizing radiation of the proliferative potential of fibroblasts from children with del(13q14) retinoblastoma]. / Stimulation par des radiations ionisantes du potentiel prolifératif de fibroblastes d'enfants atteints de del (13q14) rétinoblastome.

Skin fibroblasts from normal children and two children with a 13q14 deletion retinoblastoma (Rb) were submitted to fractionated doses of gamma radiations. Irradiation reduced the population doublings in normal fibroblasts and the decline was inversely related to the dose. An increase in population doublings was obtained with one of the Rb cell lines. Foci appeared in the irradiated culture of the other Rb donor. It is suggested that fibroblasts from patients with Rb are able to express some phenotypical properties of transformed cells, perhaps related to factors rendering them more susceptible to carcinogens.

Effect of growth arrest on the doubling potential of human fibroblasts in vitro: a possible influence of the donor.

Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi's anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to instability of the cells.

Effect of low dose rate ionizing radiation on the division potential of cells in vitro. VIII. Cytogenetic analysis of human fibroblasts.

Human embryonic and adult cells were irradiated with fractionated doses of low dose rate ionizing radiation starting early during their lifespan. Adult cells were found to be more sensitive than fetal cells to ionizing radiation in terms of the number of cells produced during the lifespan of the control and the irradiated cultures. Phase-III adult control cells had fewer chromosomal aberrations than phase-III embryonic control cells. After irradiation there was an increase in chromosomal aberrations in adult cells but no increase in embryonic cells beyond those found in the control cultures. It is suggested that cells that have a higher potential for chromosomal rearrangements survive better after low dose rate ionizing radiation.

Comparison between the radiosensitivity of human, mouse and chicken fibroblast-like cells using short-term endpoints.

We have performed a comparative study of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to our survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells.