Holger Hoehn

The Fanconi anaemia group G gene FANCG is identical with XRCC9

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; Refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Cytogenetics of Werner’s syndrome cultured skin fibroblasts: variegated translocation mosaicism

Skin fibroblast-like (FL) cells from patients with Werners syndrome (adult progeria) regularly demonstrate frequent pseudodiploidy involving variable structural rearrangements that are clonal: variegated translocation mosaicism (VTM). Ninety-two percent of 1,538 metaphases from 29 independent strains derived from five patients with Werners syndrome demonstrated this cytogenetic abnormality. In contrast, only eight (8.4%) of 95 non-Werners syndrome FL cell cultures demonstrated VTM: seven with low-grade VTM (approximately 5% of 300 metaphases), and one with VTM affecting 90-100% of metaphases. Unlike the cytogenetic abnormalities observed in the terminal stages of normal FL cell cultures, VTM occurs throughout the entire lifespan of Werners syndrome cultures. Ten of the identifiable break points in 1,005 banded metaphases accounted for 27% of all definable rearrangements. Baseline sister chromatid exchanges were not increased. Cocultivation of Werners syndrome and normal strains did not induce VTM in the normal strain. The relationship between VTM and the reduced growth potential of Werners syndrome FL cells is not yet understood, nor is the relationship between these in vitro abnormalities and the presumptive single gene defect that causes the progeroid clinical manifestations of Werners syndrome.

BrdU—Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis☆

Unlike other techniques, flow cytometric analysis of BrdU-quenched 33258 Hoechst fluorescence may be used to measure cell activation and the G1, S, and G2/M compartment distributions in each of three successive cell cycles after growth stimulation of human peripheral blood lymphocytes. Cell cycle kinetic curves can be constructed from the BrdU-Hoechst flow data which allow the simultaneous assessment of growth fraction, lag-time, compartment exit rate, compartment duration, and compartment arrest. Applications of this new versatile technique include the evaluation of drug and growth factor effects, cell aging, and diagnosis in medicine and immunology.

Chapter 2. Morphological and Biochemical Heterogeneity of Amniotic Fluid Cells in Culture

Publisher Summary This chapter describes the morphological and biochemical heterogeneity of amniotic fluid cells in culture. Midgestation amniotic fluid cells constitute a precious source of human cells for the purpose of cell biological investigation. Several properties contribute to the diagnostic and scientific value of these cells, the most important of which are the presence in a single amniotic fluid specimen of multiple, morphologically and biochemically distinct cell types that are all derived from the fetus or from fetal membranes and thus are isogenic. It is found that in addition to variable amounts of cellular debris and anucleate particles, second-trimester amniotic fluid contains between 103 and 105 nucleated cells per milliliter of fluid. All epithelial surfaces in direct or indirect contact with the amniotic cavity are possible sites of origin of these cells. According to their behavior in the culture environment, nucleated amniotic fluid cells can be operationally divided into three separate categories. The first category includes cells that remain afloat even after prolonged periods of incubation. It is found that amniotic fluid F-type cells have the greatest in vitro growth potential of all colony-forming cells.

Cultivated cells from diagnostic amniocentesis in second trimester pregnancies. I. Clonal morphology and growth potential.

Extract: Amniotic fluid obtained transabdominally for prenatal diagnosis (mean length of amenorrhea 16.1 weeks) contained between 10 and 10 cells/ml, the great majority of which were squamous; their labeling index with ( H)thymidine was < 2 × 10-3. An average of 3.5 clones ( > 10 cells)/ml fluid grew; of these, an average of 1.5 grew for >20 population doublings (CPD). Of 288 clones analyzed from 20 cases, 271 could be classified into one of three groups: group I, 24 were typical fibroblast-like cells (F cells) comparable with control clones from neonatal dermis with macroscopic “ribbing” growth pattern of megaclones, preponderance of spindle-shaped cells growing in parallel arrays, and high growth potential (mean of selected clones = 53 CPD); group II, 67 were typical epithelioid types (E cells) consisting of cells with intimate cell-to-cell contact which were resistant to trypsin detachment, with poor subcloning efficiencies and poor growth potential (mean of selected clones 14.5 CPD); group III, the predominant class with 180 clones, was of a type not described previously (AF cells); their megaclones had “bulls-eye” growth patterns, individual cells were pleomorphic, and growth potentials were intermediate to those of E and F cells. This tripartite classification holds for the majority of samples, but individual variation is suggested by the observation of exceptional clonal types sharing characteristics of both the E and AF class.Speculation: The AF cell is the predominant type in most diagnostic amniotic fluid cultures; heretofore, such cultures were widely regarded as consisting primarily of fibroblast and epithelial-like cells. Skin fibroblast cultures are therefore inappropriate controls for the interpretation of constitutive and induced levels of enzymes in such cultures. Established amniotic fluid cell cultures are derived from a single or a few clonable cells with high growth potential and therefore may not always be representative of the fetus.

Hypomorphic Mutations in the Gene Encoding a Key Fanconi Anemia Protein, FANCD2, Sustain a Significant Group of FA-D2 Patients with Severe Phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

SummaryThe growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 55% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence or cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Genotype-phenotype correlations in Fanconi anemia

Although still incomplete, we now have a remarkably detailed and nuanced picture of both phenotypic and genotypic components of the FA spectrum. Initially described as a combination of pancytopenia with a limited number of physical anomalies, it was later recognized that additional features were compatible with the FA phenotype, including a form without detectable malformations (Estren-Dameshek variant). The discovery of somatic mosaicism extended the boundaries of the FA phenotype to cases even without any overt hematological manifestations. This clinical heterogeneity was augmented by new conceptualizations. There was the realization of a constant risk for the development of myelodysplasia and certain malignancies, including acute myelogenous leukemia and squamous cell carcinoma, and there was the emergence of a distinctive cellular phenotype. A striking degree of genetic heterogeneity became apparent with the delineation of at least 12 complementation groups and the identification of their underlying genes. Although functional genetic insights have fostered the interpretation of many phenotypic features, surprisingly few stringent genotype-phenotype connections have emerged. In addition to myriad genetic alterations, less predictable influences are likely to modulate the FA phenotype, including modifier genes, environmental factors and chance effects. In reviewing the current status of genotype-phenotype correlations, we arrive at a unifying hypothesis to explain the remarkably wide range of FA phenotypes. Given the large body of evidence that genomic instability is a major underlying mechanism of accelerated ageing phenotypes, we propose that the numerous FA variants can be viewed as differential modulations and compression in time of intrinsic biological ageing.

WRN mutations in Werner syndrome patients: genomic rearrangements, unusual intronic mutations and ethnic-specific alterations

Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere.

Growth Characteristics of Werner Syndrome Cells in Vitro

Cultured skin fibroblast-like (FL) cells from patients with Werner syndrome grow poorly: reduced growth potential has been reported by more than 11 laboratories for cultures from more than 26 patients, using many different tissue culture media. This characteristic is of importance to research in Werner syndrome for several reasons. It remains to be determined whether poor growth is a primary or a secondary manifestation of the basic molecular defect, and the relationship is not yet clear between reduced growth of cultured skin fibroblasts and the other manifestations of Werner syndrome in vitro and in vivo. This characteristic also makes it difficult to obtain sufficient growth for clonal studies or for biochemical studies that require a large number of cells.