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Robertsonian translocations (RTs) result from fusion of 2 acrocentric chromosomes (e.g., 13, 14, 15, 21, 22) and consequential losses of segments of the p arms containing 47S rDNA clusters and transcription factor binding sites. Depending on the position of the breakpoints, the size of these losses vary considerably between types of RTs. The prevalence of RTs in the general population is estimated to be around 1 per 800 individuals, making RTs the most common chromosomal rearrangement in healthy individuals. Based on their prevalence, RTs are classified as “common,” rob(13;14) and rob(14;21), or “rare” (the 8 remaining nonhomologous combinations). Carriers of RTs are at an increased risk for offspring with chromosomal imbalances or with uniparental disomy. RTs are generally regarded as phenotypically neutral, although, due to RTs formation, 2 of the 10 ribosomal rDNA gene clusters, several long noncoding RNAs, and in the case of RTs involving chromosome 21, several mRNA encoding genes are lost. Nevertheless, recent evidence indicates that RTs may have a significant phenotypic impact. In particular, rob(13;14) carriers have a significantly elevated risk for breast cancer. While RTs are easily spotted by routine karyotyping, they may go unnoticed if only array-CGH and NextGen sequencing methods are applied. This review first discusses possible molecular mechanisms underlying the particularly high rates of RT formation and their incidence in the general population, and second, likely causes for the elevated cancer risk of some RTs will be examined.

Craniosynostosis refers to a condition during early development in which one or more of the fibrous sutures of the skull prematurely fuse by turning into bone, which produces recognizable patterns of cranial shape malformations depending on which suture(s) are affected. In addition to cases with isolated cranial dysmorphologies, craniosynostosis appears in syndromes that include skeletal features of the eyes, nose, palate, hands, and feet as well as impairment of vision, hearing, and intellectual development. Approximately 85% of the cases are nonsyndromic sporadic and emerge after de novo structural genome rearrangements or single nucleotide variation, while the remainders consist of syndromic cases following mendelian inheritance. By karyotyping, genome wide linkage, and CNV analyses as well as by whole exome and whole genome sequencing, numerous candidate genes for craniosynostosis belonging to the FGF, Wnt, BMP, Ras/ERK, ephrin, hedgehog, STAT, and retinoic acid signaling pathways have been identified. Many of the craniosynostosis-related candidate genes form a functional network based upon protein-protein or protein-DNA interactions. Depending on which node of this craniosynostosis-related network is affected by a gene mutation or a change in gene expression pattern, a distinct craniosynostosis syndrome or set of phenotypes ensues. Structural variations may alter the dosage of one or several genes or disrupt the genomic architecture of genes and their regulatory elements within topologically associated chromatin domains. These may exert dominant effects by either haploinsufficiency, dominant negative partial loss of function, gain of function, epistatic interaction, or alteration of levels and patterns of gene expression during development. Molecular mechanisms of dominant modes of action of these mutations may include loss of one or several binding sites for cognate protein partners or transcription factor binding sequences. Such losses affect interactions within functional networks governing development and consequently result in phenotypes such as craniosynostosis. Many of the novel variants identified by genome wide CNV analyses, whole exome and whole genome sequencing are incorporated in recently developed diagnostic algorithms for craniosynostosis.

Intragenic deletions of the contactin-associated protein-like 2 gene (CNTNAP2) have been found in patients with Gilles de la Tourette syndrome, intellectual disability (ID), obsessive compulsive disorder, cortical dysplasia-focal epilepsy syndrome, autism, schizophrenia, Pitt-Hopkins syndrome, stuttering, and attention deficit hyperactivity disorder. A variety of molecular mechanisms, such as loss of transcription factor binding sites and perturbation of penetrance and expressivity, have been proposed to account for the phenotypic variability resulting from CNTNAP2mutations. Deletions of both CNTNAP2alleles produced truncated proteins lacking the transmembrane or some of the extracellular domains, or no protein at all. This observation can be extended to heterozygous intragenic deletions by assuming that such deletion-containing alleles lead to expression of a Caspr2 protein lacking one or several extracellular domains. Such altered forms of Capr2 proteins will lack the ability to bridge the intercellular space between neurons by binding to partners, such as CNTN1, CNTN2, DLG1, and DLG4. This presumed effect of intragenic deletions of CNTNAP2, and possibly other genes involved in connecting neuronal cells, represents a molecular basis for the postulated neuronal hypoconnectivity in autism and probably other neurodevelopmental disorders, including epilepsy, ID, language impairments and schizophrenia. Thus, CNTNAP2may represent a paradigmatic case of a gene functioning as a node in a genetic and cellular network governing brain development and acquisition of higher cognitive functions.

Complex chromosome rearrangements (CCRs) are currently defined as structural genome variations that involve more than 2 chromosome breaks and result in exchanges of chromosomal segments. They are thought to be extremely rare, but their detection rate is rising because of improvements in molecular cytogenetic technology. Their population frequency is also underestimated, since many CCRs may not elicit a phenotypic effect. CCRs may be the result of fork stalling and template switching, microhomology-mediated break-induced repair, breakage-fusion-bridge cycles, or chromothripsis. Patients with chromosomal instability syndromes show elevated rates of CCRs due to impaired DNA double-strand break responses during meiosis. Therefore, the putative functions of the proteins encoded by ATM,BLM, WRN,ATR, MRE11, NBS1, and RAD51in preventing CCRs are discussed. CCRs may exert a pathogenic effect by either (1) gene dosage-dependent mechanisms, e.g. haploinsufficiency, (2) mechanisms based on disruption of the genomic architecture, such that genes, parts of genes or regulatory elements are truncated, fused or relocated and thus their interactions disturbed - these mechanisms will predominantly affect gene expression - or (3) mixed mutation mechanisms in which a CCR on one chromosome is combined with a different type of mutation on the other chromosome. Such inferred mechanisms of pathogenicity need corroboration by mRNA sequencing. Also, future studies with in vitro models, such as inducible pluripotent stem cells from patients with CCRs, and transgenic model organisms should substantiate current inferences regarding putative pathogenic effects of CCRs. The ramifications of the growing body of information on CCRs for clinical and experimental genetics and future treatment modalities are briefly illustrated with 2 cases, one of which suggests KDM4C(JMJD2C)as a novel candidate gene for mental retardation.

AbstractBased on genomic rearrangements and copy number variations, the contactin-associated protein-like 2 gene (CNTNAP2)has been implicated in neurodevelopmental disorders such as Gilles de la Tourette syndrome, intellectual disability, obsessive compulsive disorder, cortical dysplasia-focal epilepsy syndrome, autism, schizophrenia, Pitt-Hopkins syndrome, and attention deficit hyperactivity disorder. To explain the phenotypic pleiotropy of CNTNAP2alterations, several hypotheses have been put forward. Those include gene disruption, loss of a gene copy by a heterozygous deletion, altered regulation of gene expression due to loss of transcription factor binding and DNA methylation sites, and mutations in the amino acid sequence of the encoded protein which may provoke altered interactions of the CNTNAP2-encoded protein, Caspr2, with other proteins. Also exome sequencing, which covers <0.2 of the CNTNAP2genomic DNA, has revealed numerous single nucleotide variants in healthy individuals and in patients with neurodevelopmental disorders. In some of these disorders, disruption of CNTNAP2may be interpreted as a susceptibility factor rather than a directly causative mutation. In addition to being associated with impaired development of language, CNTNAP2may turn out to be a central node in the molecular networks controlling neurodevelopment. This review discusses the impact of CNTNAP2mutations on its functioning at multiple levels of the combinatorial genetic networks that govern brain development. In addition, recommendations for genomic testing in the context of clinical genetic management of patients with neurodevelopmental disorders and their families are put forward.© 2015 S. Karger AG, Basel

AbstractWe present 2 cases with multiple de novo supernumerary marker chromosomes (sSMCs), each derived from a different chromosome. In a prenatal case, we found mosaicism for an sSMC(4), sSMC(6), sSMC(9), sSMC(14) and sSMC(22), while a postnatal case had an sSMC(4), sSMC(8) and an sSMC(11). SNP-marker segregation indicated that the sSMC(4) resulted from a maternal meiosis II error in the prenatal case. Segregation of short tandem repeat markers on the sSMC(8) was consistent with a maternal meiosis I error in the postnatal case. In the latter, a boy with developmental/psychomotor delay, autism, hyperactivity, speech delay, and hypotonia, the sSMC(8) was present at the highest frequency in blood. By comparison to other patients with a corresponding duplication, a minimal region of overlap for the phenotype was identified, with CHRNB3and CHRNA6as dosage-sensitive candidate genes. These genes encode subunits of nicotinic acetylcholine receptors (nAChRs). We propose that overproduction of these subunits leads to perturbed component stoichiometries with dominant negative effects on the function of nAChRs, as was shown by others in vitro. With the limitation that in each case only one sSMC could be studied, our findings demonstrate that different meiotic errors lead to multiple sSMCs. We relate our findings to age-related aneuploidy in female meiosis and propose that predivision sister-chromatid separation during meiosis I or II, or both, may generate multiple sSMCs.© 2015 S. Karger AG, Basel

AbstractAlthough autism spectrum disorder (ASD) shows a high degree of heritability, only a few mutated genes and mostly de novo copy number variations (CNVs) with a high phenotypic impact have as yet been identified. In families with multiple ASD patients, transmitted CNVs often do not appear to cosegregate with disease. Therefore, also transmitted single nucleotide variants which escape detection if genetic analyses were limited to CNVs may contribute to disease risk. In several studies of ASD patients, CNVs covering at least one gene of the contactin gene family were found. To determine whether there is evidence for a contribution of transmitted variants in contactin genes, a cohort of 67 ASD patients and a population-based reference of 117 healthy individuals, who were not related to the ASD families, were compared. In total, 1,648 SNPs, spanning 12.1 Mb of genomic DNA, were examined. After Bonferroni correction for multiple testing, the strongest signal was found for a SNP located within the CNTN5gene (rs6590473 [G], p = 4.09 × 10-7; OR = 3.117; 95 CI = 1.603-6.151). In the ASD cohort, a combination of risk alleles of SNPs in CNTN6 (rs9878022 [A]; OR = 3.749) and in CNTNAP2 (rs7804520 [G]; OR = 2.437) was found more frequently than would be expected under random segregation, albeit this association was not statistically significant. The latter finding is consistent with a polygenic disease model in which multiple mutagenic mechanisms, operatingconcomitantly, elicit the ASD phenotype. Altogether, this study corroborates the possible involvement of contactins in ASD, which has been indicated by earlier studies of CNVs.© 2014 S. Karger AG, Basel

Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements.

One of the aims of clinical genetics is to identify gene mutations or genomic rearrangements that may underlie complex presentations of phenotypic features, such as multiple congenital malformations and mental retardation. During the decade after publication of the first article on array-based comparative genome hybridization, this technique has supplemented karyotyping as the prime genome-wide screening method in patients with idiopathic multiple congenital malformations and mental retardation. The use of this novel, discovery-based, approach has dramatically increased the detection rate of genomic imbalances. Array-based comparative genome hybridization detects copy number changes in the genome of patients and healthy subjects, some of which may represent phenotypically neutral copy number variations. This prompts the need for properly distinguishing between those copy number changes that may contribute to the clinical phenotype amid a pool of neutral copy number variations. We briefly review the characteristics of copy number changes in relation to their clinical relevance. Second, we discuss several published workflow schemes to identify copy number changes putatively contributing to the phenotype, and third, we propose a three-step procedure aiming to rapidly evaluate copy number changes on a case-by-case basis as to their potential contribution to the phenotype of patients with idiopathic multiple congenital malformations and mental retardation. This workflow is gene-centered and should aid in identification of disease-related candidate genes and in estimating the recurrence risk for the disorder in the family.

One of the aims of clinical genetics is to identify gene mutations or genomic rearrangements that may underlie complex presentations of phenotypic features, such as multiple congenital malformations and mental retardation. During the decade after publication of the first article on array-based comparative genome hybridization, this technique has supplemented karyotyping as the prime genome-wide screening method in patients with idiopathic multiple congenital malformations and mental retardation. The use of this novel, discovery-based, approach has dramatically increased the detection rate of genomic imbalances. Array-based comparative genome hybridization detects copy number changes in the genome of patients and healthy subjects, some of which may represent phenotypically neutral copy number variations. This prompts the need for properly distinguishing between those copy number changes that may contribute to the clinical phenotype amid a pool of neutral copy number variations. We briefly review the characteristics of copy number changes in relation to their clinical relevance. Second, we discuss several published workflow schemes to identify copy number changes putatively contributing to the phenotype, and third, we propose a three-step procedure aiming to rapidly evaluate copy number changes on a case-by-case basis as to their potential contribution to the phenotype of patients with idiopathic multiple congenital malformations and mental retardation. This workflow is gene-centered and should aid in identification of disease-related candidate genes and in estimating the recurrence risk for the disorder in the family.

High resolution genomic copynumber analysis has shown that inherited and de novo copynumber variations contribute significantly to autism pathology, and that identification of small chromosomal aberrations related to autism will expedite the discovery of risk genes involved. Here, we report a microduplication of chromosome 15q11.2, spanning only four genes, cosegregating with autism in a Dutch pedigree, identified by SNP microarray analysis, and independently confirmed by FISH and MLPA analysis. Quantitative RTPCR analysis revealed over 70 increase in peripheral blood mRNA levels for the four genes present in the duplicated region in patients, and RNA in situ hybridization on mouse embryonic and adult brain sections revealed that two of the four genes, CYFIP1and NIPA1, were highly expressed in the developing mouse brain. These findings point towards a contribution of microduplications at chromosome 15q11.2 to autism, and highlight CYFIP1and NIPA1as autism risk genes functioning in axonogenesis and synaptogenesis. Thereby, these findings further implicate defects in dosagesensitive molecular control of neuronal connectivity in autism. However, the prevalence of this microduplication in patient samples was statistically not significantly different from control samples 0.94 in patients vs. 0.42 controls, P  0.247, which suggests that our findings should be interpreted with caution and indicates the need for studies that include large numbers of control subjects to ascertain the impact of these changes on a population scale. © 2009 WileyLiss, Inc.

A 10‐year‐old boy with vermis hypoplasia, dilatation of the fourth ventricle, enlarged cisterna magna and aplasia of the corpus callosum, consistent with the Dandy‐Walker complex (DWC), and slight facial dysmorphisms, severe motor and mental retardation is presented. By combining data obtained by karyotyping, array‐CGH, FISH, and multiplex ligation‐mediated probe amplification (MLPA) we identified a 5 Mb deletion of the 1q44 → qter region resulting from a paternal t(1;20)(q44;q13.33). This smallest 1q44 deletion reported so far, enabled us to significantly narrow down the number of candidate genes for the DWC in this region. Since the ZNF124 transcription factor is strongly expressed in the fetal brain it may represent a candidate gene for the DWC at 1q44. © 2007 Wiley‐Liss, Inc.

Hypoparathyroidism‐retardation‐dysmorphism (HRD) or Sanjad–Sakati syndrome (SSS) (OMIM 241410) is a rare autosomal recessive (AR) inherited condition, characterized by congenital hypoparathyroidism (hypoPTH), retardation, seizures, and a typical facial dysmorphism, consisting of prominent forehead, deep‐set eyes, and abnormal external ears. This disorder has been mapped to the long arm of chromosome 1 (1q42‐q43) and mutations in the gene coding for tubulin‐specific chaperone E (TBCE) have been identified as the cause of the disease. Mutations in the same gene were also reported in patients with AR Kenny–Caffey syndrome (KCS). We report on a 4½‐year‐old girl with congenital hypoPTH, seizures, developmental delay, and a facial dysmorphism, compatible with HRD syndrome. Mutation analyses revealed no mutations in the TBCE gene. In addition, normal TBCE protein and alpha‐tubulin immunostaining were observed in a lymphoblastoid line derived from the patient, excluding the TBCE gene as the causative gene of the syndrome in this patient. A de novo microduplication of probe RP11‐262I1 on 4q35 in the proposita was detected by microarray analyses, but this could not be confirmed by additional studies. We review and discuss the clinical findings of our case and those of the other reported cases with SSS and AR KCS. We conclude that a second gene locus for this disorder seems probable and that 4q35 needs further evaluation as a candidate region. © 2006 Wiley‐Liss, Inc.

Array‐based comparative genomic hybridization allows high‐resolution screening of copy number abnormalities in the genome, and becomes an increasingly important tool to detect deletions and duplications in tumor and post‐natal cytogenetics. Here we illustrate that genomic arrays can also provide novel clues regarding the structural basis of chromosome rearrangement, including instability and mechanisms of formation of ring chromosomes. We also showed that array results might impact the recurrence risks for relatives of affected individuals. Our data indicate that chromosome rearrangements frequently involve more breaks than current cytogenetic models assume. © 2004 Wiley‐Liss, Inc.

Drug sensitivity is commonly determined by assays that utilize colony formation to discriminate between surviving and lethally treated cells. These assays require cells with high plating efficiency that form discernible colonies, are time-consuming and laborious, and require manual counting of large numbers of colonies. To overcome these drawbacks, we developed a flow cytometric technique that assays survival of proliferative capacity in cultured cells. Labeling with bromodeoxyuridine for 72 h followed by bivariate Hoechst 33258/ethidium bromide flow cytometry allows discrimination of nonproliferating cells from those that have undergone one to three divisions. Addition of an internal standard, chicken erythrocyte nuclei, permits determination of total cell number. To validate our assay, we used flow and colony-forming assays to determine the sensitivity of cell lines derived from Werner syndrome patients and unaffected individuals to 4-nitroquinoline-1-oxide (4NQO) and camptothecin. The flow and colony-forming assays yielded comparable sensitivity for each drug and essentially identical increases in drug sensitivity exhibited by Werner syndrome cells. Our results indicate that the flow assay is a less laborious surrogate for colony-forming assays. The flow technique will also facilitate the analysis of drug sensitivity in cells that are not amenable to colony-forming assays. Cytometry 48:1–5, 2002. © 2002 Wiley-Liss, Inc.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytesin vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ∼50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFκB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant α-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

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AbstractNo abstract available

Geriatricians want to know the extent to which inborn genetic variations influence health span and life span in their patients. Current research on this subject is largely confined to the very basic issues of identifying, mapping, and cloning relevant genes and of investigating their modes of action. Here we summarize the status of such basic research on a gene (WRN), null mutations at which cause a striking seg‐mental propertied syndrome, the Werner syndrome (WS). The views of clinicians and basic scientists concerning the significance of WRN gene action for normal aging are polarized. Some regard it as the most important aging gene and refer to the syndrome explicitly as one of premature aging. Others regard WS as an entirely inappropriate model for aging because WS exhibits many clinical and cell biological discordances with normal aging. Moreover, WS has a major impact on reproductive fitness and, hence, does not escape the forces of natural selection. By contrast, senescent phenotypes of normal (usual) aging can be defined as those that do escape the forces of natural selection. We here give a more balanced view. Work has only just begun on the potential significance of a range of variants at the WS locus, including “leaky” mutations, heterozygosity for null mutations, and polymorphisms, some of which may indeed escape the force of natural selection and thus contribute to the burden of illness in old age. We are also at a very early stage of knowledge concerning genes coding for WRN interacting proteins, variations at which may also be of significance for gerontology and geriatric medicine. J Am Geriatr Soc 47:1136–1144,1999.

The possible relationships between changes in mitochondrial membrane potential and other mitochondrial functions during apoptosis remain controversial. To detect concomitant changes in mitochondrial function during apoptosis, we performed correlated multiparameter flow cytometry after simultaneous cell staining with several dyes. After camptothecin treatment, nonapoptotic cells exhibited a concomitant rise in mitochondrial membrane potential [8-(4'-chloromethyl) phenyl-2, 3, 5, 6, 11, 12, 14, 15-octahydro-1H, 4H, 10H, 13H-diquinolizino-8H-xanthylium chloride, or CMXRos; CMXRos fluorescence divided by MitoTracker Green fluorescence], NADH level (ultraviolet-excited blue autofluorescence), and oxidative turnover (H2-CMXRos oxidation). Frankly apoptotic cells showed a decreased mitochondrial membrane potential, NADH level, and oxidative turnover. Oxidative turnover was not sensitive to antimycin A treatment, which suggests that H2-CMXRos oxidation in these cells may be due to lipid peroxidation. In addition, frankly apoptotic cells showed lower cardiolipin levels (by nonyl-acridine orange staining). The efficiency of energy transfer between nonyl-acridine orange and CMXRos was slightly lower in camptothecin-treated nonapoptotic cells and reduced to zero in frankly apoptotic cells. We conclude that, in an initial phase of camptothecin-induced apoptosis, mitochondrial activity is increased and a subtle loss of structural integrity of the mitochondrial membranes takes place. In frankly apoptotic cells, all measured parameters of mitochondrial collapse and lipid peroxidation occurs. Cytometry 35:311–317, 1999. © 1999 Wiley-Liss, Inc.

Fluorescein diacetate analogs 3a–3care shown to react selectively with protein sulfhydryl groups at neutral pH in aqueous solution, by means of substitution at the perfluorinated ring. The acetate moieties are cleaved by treatment with aqueous carbonate or by endogenous esterases, giving a fluorescent protein conjugate.

BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. Thein vitrogrowth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein–Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity towardcis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity towardtrans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations ofo-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations ofo-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.

The β-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected β-galactosidase activity vs. lysosomal enzyme activity. Among these substrates, 5-chloromethylfluoresecein di-β-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity. An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death. In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold. Incubation with probenecid, on the other hand, showed little effect. Cytometry 28:36–41, 1997. © 1997 Wiley-Liss, Inc.