Familial Kleefstra syndrome due to maternal somatic mosaicism for interstitial 9q34.3 microdeletions.

The Kleefstra syndrome (Online Mendelian Inheritance in Man 607001) is caused by a submicroscopic 9q34.3 deletion or by intragenic euchromatin histone methyl transferase 1 (EHMT1) mutations. So far only de novo occurrence of mutations has been reported, whereas 9q34.3 deletions can be either de novo or caused by complex chromosomal rearrangements or translocations. Here we give the first descriptions of affected parent-to-child transmission of Kleefstra syndrome caused by small interstitial deletions, approximately 200 kb, involving part of the EHMT1 gene. Additional genome-wide array studies in the parents showed the presence of similar deletions in both mothers who only had mild learning difficulties and minor facial characteristics suggesting either variable clinical expression or somatic mosaicism for these deletions. Further studies showed only one of the maternal deletions resulted in significantly quantitative differences in signal intensity on the array between the mother and her child. But by investigating different tissues with additional fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) analyses, we confirmed somatic mosaicism in both mothers. Careful clinical and cytogenetic assessments of parents of an affected proband with an (interstitial) 9q34.3 microdeletion are merited for accurate estimation of recurrence risk.

Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes.

Infantile juvenile polyposis is a rare disease with severe gastrointestinal symptoms and a grave clinical course. Recently, 10q23 microdeletions involving the PTEN and BMPR1A genes were found in four patients with infantile juvenile polyposis. It was hypothesized that a combined and synergistic effect of the deletion of both genes would explain the condition. Subsequently, however, a patient with a larger 10q23 deletion including the same genes but with a mild clinical phenotype was identified. Here, we present four additional patients with 10q23 microdeletions involving the PTEN and BMPR1A genes. The sizes of the deletions were analyzed using single nucleotide polymorphism array analysis. All patients had macrocephaly, dysmorphic features, retardation and congenital abnormalities. One patient developed colorectal cancer. However, only one case had disease onset before 2 years of age and severe symptoms requiring colectomy. No clear correlation was found between ages at onset or severity of gastrointestinal symptoms and the sizes of the deletions. We conclude that patients with 10q23 microdeletions involving the PTEN and BMPR1A genes have variable clinical phenotypes, which cannot be explained merely by the deletion sizes. The phenotypes are not restricted to severe infantile juvenile polyposis but include childhood-onset cases with macrocephaly, retardation, mild gastrointestinal symptoms and possibly early-onset colorectal cancer.

Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission.

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.

Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.

We present a boy with blepharophimosis, ptosis, epicanthus inversus, microcephaly, mild mental retardation, and growth delay. Chromosomal analysis revealed a male karyotype with an interstitial deletion in the long arm of chromosome 3. DNA-analysis showed that the deletion is of maternal origin and encompasses the region between markers D3S1535 and D3S1593. The deletion contains not only the FOXL2 gene, but also the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR). Mutations in FOXL2 have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). ATR has been identified as a candidate gene for Seckel syndrome, an autosomal recessive syndrome that comprises growth retardation, microcephaly, and mental retardation. We hypothesize that our patient has a contiguous gene syndrome and that the non-BPES-associated abnormalities (microcephaly, mild mental retardation, and growth delay) are the result of the deletion of the maternal ATR gene. However, it has not yet been excluded that haploinsufficiency of some other gene in this region plays a role.

Atypical progeroid syndrome: an unknown helicase gene defect?

We describe a boy with chromosomal breakage syndrome, who died of hepatocellular carcinoma at the age of 17 years. Other findings included growth retardation, bilateral cataracts, premature graying of hair and elevated levels of urinary hyaluronic acid. Intellectual functions were normal. Although some manifestations were suggestive of Werner syndrome, the diagnosis could not be confirmed by molecular investigations. Therefore, this patient probably represents a provisionally unique syndrome, perhaps due to a mutation in a related (helicase) gene.

[Williams syndrome: new insights into genetic etiology, pathogenesis and clinical aspects]. / Williams-syndroom: nieuwe inzichten in genetische etiologie, pathogenese en kliniek.

Williams syndrome (WS) is a developmental disorder characterized by distinct facial features, congenital heart disease, mental retardation and a gregarious personality. The majority of people with this disorder have a submicroscopic deletion of genes in chromosome band 7q11.23. This deletion can be detected using fluorescence in situ hybridization (FISH). Although the condition is usually sporadic a few familial cases with autosomal dominant inheritance have been described. A clinical scoring system has been developed by Selicorni with which a diagnosis of 'Williams syndrome' can be made; in all patients in whom the diagnosis was made in this way FISH results were positive.

Mobilisation of haemopoietic progenitors in CML: a second course of intensive chemotherapy does not improve Ph-negativity in stem cell harvests.

We collected peripheral blood stem cells (PBSC) in 19 early chronic phase CML patients following each of two consecutive cycles of intensive chemotherapy (CT) to evaluate whether an additional cycle of CT would increase Philadelphia (Ph)-negativity of the PBSC harvest. Autologous SCT (autoSCT) was performed if a major cytogenetic response (MCR) of the PBSC harvest was obtained. CT consisted of cytarabine 200 mg/ m2/day (days 1-7)/idarubicin 12 mg/m2/day (days 1-2) (cycle one) and cytarabine 2000 mg/m2/day (days 1-6)/amsacrine 120 mg/m2/day (days 1-3) (cycle two). One patient died of fungal pneumonia after the first cycle. Stem cells were harvested in 18 patients after cycle one and in 16 patients after cycle two. After the first cycle, all patients showed a cytogenetic response of their graft (MCR in eight patients: three complete, five partial), after cycle two, seven patients obtained an MCR (one complete, six partial). Seven patients became eligible for autoSCT. All patients proceeded with IFNalpha maintenance. Currently, 16 patients are alive. At the latest cytogenetic examination of bone marrow, four patients showed an MCR and four a minor response. In conclusion, although a second cycle of CT may contribute to elimination of leukemia residing in the patient, it appeared to be ineffective in improving the Ph-negativity of the PBSC graft.

Aberrant Fanconi anaemia protein profiles in acute myeloid leukaemia cells.

Fanconi anaemia (FA) is an autosomal recessive disease strongly predisposing to bone marrow failure and acute myeloid leukaemia (AML). Four FA genes, corresponding to complementation groups A, C, F and G, have been cloned, but the molecular functions of the corresponding proteins are unknown. The high risk of AML in FA patients suggests that the 'FA pathway' helps to prevent AML in non-FA individuals. We examined 10 AML cell lines, as well as primary cells from 15 AML patients representing the French-American-British subclasses M1-M5a, for possible deficiencies in the 'FA pathway'. Cellular lysates were analysed for the presence of the FA proteins FANCA, FANCC, FANCF and FANCG, as well as the complexes reported to be formed between these proteins, using immunoprecipitation and Western blot analysis. Aberrant protein profiles were observed in five of the 10 cell lines and in 11 of the 15 primary AML samples. Aberrations, that included absence or reduced presence of FA proteins and/or their complexes, were noted in the subclasses M1-M4, but not in M5a (n = 3). Our results suggest that a significant proportion of general AML is characterized by a disturbance of the 'FA pathway' that may represent an early event in the development of this type of leukaemia.

Recombination in a balanced complex translocation of a mother leading to a balanced reciprocal translocation in the child. Review of 60 cases of balanced complex translocations.

We report an unusual case of a balanced reciprocal translocation with a recombinant chromosome which has arisen from a familial balanced complex translocation. Fluorescence in situ hybridization studies were essential for the identification of the breakpoints. A review of 60 cases of balanced complex translocations (BCT) has revealed three cases similar to ours. Carriers of BCT have a high risk of having spontaneous abortions or a child with an unbalanced karyotype. Certain types of balanced rearrangements involving an insertion can give rise to a simpler balanced translocation as a result of crossover. Our observations support the assumption that the chance that a de novo balanced complex translocation is associated with an abnormal phenotype increases with the number of breakpoints.

Kabuki syndrome in son and low grade mosaic 45,X/46,XX in mother.

We report on a two-year-old boy with Kabuk syndrome and a normal male karyotype whose mother is a low grade mosaic 45,X/46,XX. We hypothesized that the son's Kabuki syndrome might have been caused by gonosomal uniparental (paternal) disomy DNA analysis proved this hypothesis to be incorrect. A review of twelve patients with Kabuki syndrome or Kabuki-syndrome-like features and chromosome abnormalities is presented.

Multiparameter in situ analysis of trophoblast cells in mixed cell populations by combined DNA and RNA in situ hybridization.

We developed a non-radioactive assay for simultaneous detection of cytoplasmic mRNA and nuclear genomic DNA in fetal trophoblast cells by sequential in situ hybridization. Trophoblast-specific mRNA is detected with a digoxigenin-labeled RNA probe complementary to HLA-G, followed by visualization through the generation of stable contrast-rich DAB/Ni complexes. Genomic target DNA is subsequently visualized in labeled cells by fluorescent in situ hybridization using biotin-labeled chromosome-specific DNA probes. Simultaneous visualization of both targets is made possible using a fluorescence microscope with FITC filter and conventional brightfield light. This method allows detection of trophoblast cells within a mixed cell population and, at the same time, analysis of chromosome anomalies in the trophoblast cells identified. For prenatal diagnosis of fetal cells enriched from maternal peripheral blood during pregnancy, this multiparameter in situ analysis of immobilized fetal trophoblast cells will be very useful.

Cytogenetic alterations associated with P-glycoprotein- and non-P-glycoprotein-mediated multidrug resistance in SW-1573 human lung tumor cell lines.

Multidrug resistance can be induced in mammalian cells by selection with a single cytotoxic agent. Overproduction of the energy-dependent drug efflux pump P-glycoprotein, encoded by the mdr1 gene, has been identified as the cause of one form of multidrug resistance. The molecular basis of other forms of multidrug resistance is unknown. Doxorubicin selection of the human squamous lung cancer cell line SW-1573 resulted in multidrug-resistant sublines in which a non-P-glycoprotein-mediated form of multidrug resistance precedes mdr1 expression. Here we present a cytogenetic analysis of both non-P-glycoprotein-mediated multidrug-resistant and P-glycoprotein-mediated multidrug-resistant sublines derived from SW-1573. Three independently derived non-P-glycoprotein-mediated multidrug-resistant sublines showed a heterozygous deletion of the short arm of chromosome 2 (p23-pter), whereas alterations of chromosome 7 were present in the P-glycoprotein-mediated multidrug-resistant cell lines. In one series of clonally derived P-glycoprotein-mediated multidrug-resistant sublines, mdr1 overexpression was accompanied by various markers of chromosome 7 with breakpoints at 7q22, the mdr1 gene being known to be located at 7q21.1. Our data suggest that in SW-1573 cells acquisition of non-P-glycoprotein-mediated multidrug resistance is accompanied by a specific deletion or a translocation involving the short arm of chromosome 2, whereas in the emergence of P-glycoprotein-mediated multidrug resistance a rearrangement of the long arm of chromosome 7 is a critical event.

Genetic transfer of non-P-glycoprotein-mediated multidrug resistance (MDR) in somatic cell fusion: dissection of a compound MDR phenotype.

A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) has been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell line SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D, and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). They accumulate less drug than the parental cells and their resistance is not due to the MDR1-encoded P-glycoprotein, as the resistant cell lines have lost the low amount of MDR1 mRNA detectable in parental cells. Here we show that the resistant cell lines also contain less topoisomerase II mRNA and enzyme activity than the parental cells. This might contribute to the resistance of these lines to drugs interacting with topoisomerase II, such as doxorubicin, daunorubicin, and VP16-213, but cannot account for the resistance to the other drugs. We have tested whether all properties of the non-Pgp MDR cell lines cosegregate in somatic cell fusions between lethally gamma-irradiated, resistant donor cells and drug-sensitive acceptor cells. Whereas a MDR phenotype with reduced drug accumulation and the loss of MDR1 P-glycoprotein mRNA were cotransferred to the acceptor cells, the decrease in topoisomerase II gene expression was not. We conclude that the MDR phenotype, the reduced drug accumulation, and the loss of MDR1 P-glycoprotein mRNA are genetically linked. They might be due to a single dominant mutation, which does not cause the alteration in topoisomerase II.

Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line.

Two different mechanisms that contribute to multidrug resistance (MDR) were found in derivatives of the human squamous lung cancer cell line SW-1573. The parental cell line has a low amount of mdr1 P-glycoprotein mRNA. In three independent selections for doxorubicin resistance, MDR variants arose in which mdr1 P-glycoprotein mRNA and protein was not detectable. Selection on higher doxorubicin concentrations gave rise to variants containing high levels of mdr1 mRNA, due to transcriptional activation of the mdr1 gene. Upon continued selection for higher levels of doxorubicin resistance, the mdr1 gene became amplified, resulting in an additional increase in the level of mdr1 mRNA. The cross-resistance pattern of the sublines that lack mdr1 P-glycoprotein expression is different from that seen in the mdr1 overexpressing cells. Both types of MDR cell lines are resistant to doxorubicin, daunorubicin, etoposide, colchicine, gramicidin D, and vincristine. However, in the non-P-glycoprotein-mediated MDR cell lines, resistance levels are lower and a preferential resistance for etoposide is seen.

Cytogenetic toxicity of paraquat and streptonigrin in Fanconi's anemia.

We reported earlier that the frequency of chromosomal aberrations observed in Fanconi's anemia lymphocyte cultures depends on the oxygen tension during growth of the cultures, suggesting that "activated oxygen species" (superoxide, O2-; hydrogen peroxide, H2O2; hydoxyl radical, OH; and singlet oxygen, 1O2) or other reactive products generated during oxygen metabolism may be involved in the production of chromosomal damage in this syndrome. Paraquat and streptonigrin, agents that have been proposed as model compounds exerting cellular toxicity through overproduction of superoxide, were tested for their clastogenic potency in lymphocyte cultures from healthy controls and patients with Fanconi's anemia. Paraquat, at concentrations that severely affected mitotic activity (100-200 micrograms/ml), appeared to be a weak clastogen in human lymphocytes, whereas a clastogenic effect of streptonigrin was demonstrable already at a concentration as low as 0.005 microgram/ml. The results indicate that Fanconi's anemia lymphocytes fail to exhibit an increased sensitivity to the antimitotic and clastogenic effects of paraquat and streptonigrin. This suggests that intracellular superoxide is not critically involved in the generation of spontaneous chromosomal aberrations in Fanconi's anemia. We infer from these and previous data that singlet oxygen (1O2) may be a critical contributor to the chromosomal breakage in this disorder.

Oxygen toxicity and chromosomal breakage in ataxia telangiectasia.

Toxic effects of ionizing radiation and elevated O2 levels (hyperoxia) are both thought to be mediated by oxidizing free radicals. In view of the reported hypersensitivity of ataxia telangiectasia (A-T) cells to the clastogenic effect of ionizing radiation, the chromosomal sensitivity of A-T cells to hyperoxic culture conditions was investigated in unirradiated and G0-irradiated A-T lymphocyte cultures. Unlike Fanconi's anaemia lymphocytes, which tend to respond to oxygen, especially after treatment with mitomycin C, both non-irradiated and G0-irradiated A-T lymphocytes failed to show an effect. We conclude that the excessive spontaneous and radiation-induced chromosomal breakage in A-T does not result from a deficiency in the cellular defences against the clastogenic effect of hyperoxia.

DNA damage by chemically generated singlet oxygen.

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (1O2) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10(8) daltons) was exposed to 120 mol m-3 1O2 (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for 1O2-induced interstrand crosslinking was obtained. The capacity of 1O2 to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage phi X174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of 1O2-exposure were assessed by examining the plaque-forming capacity of ss and ds phi X174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,) we infer that 1O2-induced inactivation of phi X174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.