A joint cross-border investigation of a cluster of multidrug-resistant tuberculosis in Austria, Romania and Germany in 2014 using classic, genotyping and whole genome sequencing methods: lessons learnt.

Molecular surveillance of multidrug-resistant tuberculosis (MDR-TB) using 24-loci MIRU-VNTR in the European Union suggests the occurrence of international transmission. In early 2014, Austria detected a molecular MDR-TB cluster of five isolates. Links to Romania and Germany prompted the three countries to investigate possible cross-border MDR-TB transmission jointly. We searched genotyping databases, genotyped additional isolates from Romania, used whole genome sequencing (WGS) to infer putative transmission links, and investigated pairwise epidemiological links and patient mobility. Ten isolates from 10 patients shared the same 24-loci MIRU-VNTR pattern. Within this cluster, WGS defined two subgroups of four patients each. The first comprised an MDR-TB patient from Romania who had sought medical care in Austria and two patients from Austria. The second comprised patients, two of them epidemiologically linked, who lived in three different countries but had the same city of provenance in Romania. Our findings strongly suggested that the two cases in Austrian citizens resulted from a newly introduced MDR-TB strain, followed by domestic transmission. For the other cases, transmission probably occurred in the same city of provenance. To prevent further MDR-TB transmission, we need to ensure universal access to early and adequate therapy and collaborate closely in tuberculosis care beyond administrative borders.

Pharmacoepigenetics of depression: no major influence of MAO-A DNA methylation on treatment response.

The monoamine oxidase A (MAO-A) gene has been suggested to be involved in the pathogenesis as well as the pharmacological treatment of major depressive disorder. In the present analysis, for the first time a pharmacoepigenetic approach was applied investigating the influence of DNA methylation patterns in the MAO-A regulatory and exon1/intron1 region on antidepressant treatment response. 94 patients of Caucasian descent with major depressive disorder (f = 61; DSM-IV) were analyzed for DNA methylation status at 43 MAO-A CpG sites via direct sequencing of sodium bisulfite treated DNA extracted from blood cells. Patients were also genotyped for the functional MAO-A VNTR. Clinical response to antidepressant treatment with escitalopram was assessed by intra-individual changes of HAM-D-21 scores after 6 weeks of treatment. Apart from two CpG sites, male subjects showed no or only very minor methylation. In female patients, lower methylation at two individual CpG sites in the MAO-A promoter region was nominally associated with impaired response to antidepressant treatment after 6 weeks (GRCh37/hg19: CpG 43.514.063, p = 0.04; CpG 43.514.684, p = 0.009), not, however, withstanding correction for multiple testing. MAO-A VNTR genotypes did not influence MAO-A methylation status. The present pilot data do not suggest a major influence of MAO-A DNA methylation on antidepressant treatment response. However, the presently observed trend towards CpG-specific MAO-A gene hypomethylation-possibly via increased gene expression and consecutively decreased serotonin and/or norepinephrine availability-to potentially drive impaired antidepressant treatment response in female patients might be worthwhile to be followed up in larger pharmacoepigenetic studies.

Molecular surveillance of multi- and extensively drug-resistant tuberculosis transmission in the European Union from 2003 to 2011.

The European Centre for Disease Prevention and Control (ECDC) initiated a project on the molecular surveillance of multi- and extensively drug-resistant tuberculosis (MDR-/XDR-TB) transmission in the European Union (EU) in the period from 2009 to 2011. In total, 2,092 variable number of tandem repeat (VNTR) patterns of MDR-/XDR-TB Mycobacterium tuberculosis isolates were collected, originating from 24 different countries in the period 2003 to 2011. Of the collected VNTR patterns, 45% (n=941) could be assigned to one of the 79 European multiple-country molecular fingerprint clusters and 50% of those (n=470) belonged to one extremely large cluster caused by Beijing strains of one genotype. We conclude that international transmission of MDR-/XDR-TB plays an important role in the EU, especially in the eastern part, and is significantly related to the spread of one strain or clone of the Beijing genotype. Implementation of international cluster investigation in EU countries should reveal underlying factors of transmission, and show how TB control can be improved regarding case finding, contact tracing, infection control and treatment in order to prevent further spread of MDR-/XDR-TB in the EU.

Male-mediated developmental toxicity.

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

Human dopamine transporter gene: differential regulation of 18-kb haplotypes.

AIM: Since previous functional studies of short haplotypes and polymorphic sites of SLC6A3 have shown variant-dependent and drug-sensitive promoter activity, this study aimed to understand whether a large SLC6A3 regulatory region, containing these small haplotypes and polymorphic sites, can display haplotype-dependent promoter activity in a drug-sensitive and pathway-related manner. MATERIALS & METHODS: By creating and using a single copy number luciferase-reporter vector, we examined regulation of two different SLC6A3 haplotypes (A and B) of the 5´ 18-kb promoter and two known downstream regulatory variable number tandem repeats by 17 drugs in four different cellular models. RESULTS: The two regulatory haplotypes displayed up to 3.2-fold difference in promoter activity. The regulations were drug selective (37.5% of the drugs showed effects), and both haplotype and cell type dependent. Pathway analysis revealed at least 13 main signaling hubs targeting SLC6A3, including histone deacetylation, AKT, PKC and CK2 α-chains. CONCLUSION: SLC6A3 may be regulated via either its promoter or the variable number tandem repeats independently by specific signaling pathways and in a haplotype-dependent manner. Furthermore, we have developed the first pathway map for SLC6A3 regulation. These findings provide a framework for understanding complex and variant-dependent regulations of SLC6A3.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner.

AIM: TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION: These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae.

G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.

An engineered zinc finger protein reveals a role for the insulin VNTR in the regulation of the insulin and adjacent IGF2 genes.

An engineered zinc finger protein (eZFP) was isolated from a library based on its ability to activate expression of the endogenous insulin gene in HEK-293 cells. Using a panel of insulin promoter constructs, the eZFP was shown to act through the variable number of tandem repeat (VNTR) region located 365 base pairs upstream of the transcription start site. The eZFP also activated expression of the IGF2 gene that lies close to INS on chromosome 11p15. These results demonstrate that the INSVNTR controls expression of the insulin and IGF2 genes and provide a mechanistic explanation for previous studies that demonstrated an association between INSVNTR genotypes and placental levels of IGF2.

Serotonin transporter gene (5-Htt): association analysis with temporal lobe epilepsy.

Two functional polymorphisms, a 44bp insertion/deletion polymorphism in the 5' regulatory region and a variable number of tandem repeat polymorphisms in the second intron of the serotonin transporter gene (5-HTT), were previously identified and suggested to modulate transcription. The current study was designed to determine the contribution of these polymorphisms in the 5-HTT gene to susceptibility to temporal lobe epilepsy (TLE). Two hundred and seventy six patients with TLE, and 309 age- and sex-matched healthy controls from Calabria (Southern Italy) were studied. Patients and controls were genotyped using the WAVE TM DNA Fragment Analysis System for the insertion/deletion polymorphism in the promoter region (5-HTTLPR), and the GENESCAN TM System for the variable number tandem repeat (VNTR) in the second intron of the 5-HTT gene (5-HTTVNTR). The program UNPHASED was used to compare genotype, allele and haplotype frequencies between cases and controls, including age and gender as covariates in the model. No significant differences between cases and controls were observed for 5-HTTLPR, but a significant association was obtained for the 5-HTTVNTR polymorphism, both modeling genotypes (P-value=0.0145) or alleles (P-value=0.0086). Patients with TLE showed lower frequencies of the 10 repeat at 5-HTTVNTR than the controls (26.2% in patients versus 40.8% in controls). The frequency of homozygous individuals for the 10 allele was observed to be lower among patients than the controls (5.2% of patients were 10/10 versus 18.8% of controls). Haplotype analysis did not increase the evidence for association. These results suggest that the serotonin transporter gene may play a role in the etiology of TLE.

Differential regulation of the serotonin transporter gene by lithium is mediated by transcription factors, CCCTC binding protein and Y-box binding protein 1, through the polymorphic intron 2 variable number tandem repeat.

The serotoninergic pathways are possible targets for the action of lithium, a therapeutic agent for treatment of bipolar affective disorders. This study aimed to investigate the molecular mechanisms regulating human serotonin transporter gene (SLC6A4) expression by lithium and, specifically, the role of the variable number tandem repeat (VNTR) polymorphic region in intron 2, which is potentially a predisposing genetic factor for bipolar affective disorders. We demonstrated that addition of lithium to human JAr cells led to changes in the levels of SLC6A4 mRNA and protein. Additional investigations revealed that the intron 2 VNTR domain was a potential target for mediation of a transcriptional response to lithium. Properties of two transcription factors, CCCTC binding protein (CTCF) and Y-box binding protein 1 (YB-1), previously shown to be involved in the regulation of SLC6A4 VNTR, were found to be modulated by LiCl. Thus, levels of CTCF and YB-1 mRNA and protein were altered in vivo in response to LiCl. Furthermore, CTCF and YB-1 showed differential binding to the polymorphic alleles of the VNTR on exposure to LiCl. Our data suggest a model in which differential binding of CTCF and YB-1 to the allelic variants of the intron 2 VNTR can be regulated by lithium and in part result in differential and even aberrant expression of SLC6A4. Our study of the regulation of the SLC6A4 VNTR by lithium may improve the understanding of psychiatric disorders and enable the development of novel therapies for conditions such as bipolar affective disorder to target only the at-risk allele.

Transgenerational transmission of radiation- and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes.

This article provides a broad overview of our earlier studies on the induction of tumors and congenital anomalies in the progeny of X-irradiated or chemically treated mice and our subsequent (published, hitherto unpublished and on-going) investigations aimed at identifying potential relationships between genetic changes induced in germ cells and the adverse effects manifest as tumors and congenital anomalies using cytogenetic and molecular approaches. The earlier studies document the fact that tumors and congenital anomalies can be induced by irradiation or treatment with certain chemicals such as urethane and that these phenotypes are heritable i.e., transmitted to generations beyond the first generation. These findings support the view that transmissible induced genetic changes are involved. The induced rates of congenital abnormalities and tumors are about two orders of magnitude higher than those recorded in the literature from classical mutation studies with specific locus mutations. The cytogenetic studies addressed the question of whether there were any relationships between induced translocations and induced tumors. The available data permit the inference that gross chromosomal changes may not be involved but do not exclude smaller induced genetic changes that are beyond the resolution of the techniques used in these studies. Other work on possible relationship between visible chromosomal anomalies (in bone marrow preparations) and tumors were likewise negative. However, there were indications that some induced cytogenetic changes might underlie induced congenital anomalies, i.e., trisomies, deletions and inversions were observed in induced and transmissible congenital anomalies (such as dwarfs, tail anomalies). Studies that explored possible relationships between induction of minisatellite mutations at the Pc-3 locus and tumors were negative. However, gene expression analysis of tumor (hepatoma)-susceptible offspring of progeny descended from irradiated male mice showed abnormal expression of many genes. Of these, only very few were oncogenes. This lends some support to our hypothesis that cumulative changes in gene expression of many genes, which perform normal cellular functions, may contribute to the occurrence of tumors in the offspring of irradiated or chemically treated mice.

Eating disorders with binge-eating behaviour are associated with the s allele of the 3'-UTR VNTR polymorphism of the dopamine transporter gene.

OBJECTIVE: The dopaminergic system is associated with feelings of pleasure and reward and with positive hedonic processes related to food, sexual activity and certain substances. Because it is recognized that patients who have eating disorders with binge-eating behaviour have a high comorbidity of substance dependence, we examined the association between the variable number of tandem repeats (VNTR) polymorphism in the 3; untranslated region of the dopamine transporter gene (DAT1) and eating disorders with binge-eating behaviour. METHODS: The subjects were 90 female Japanese patients with eating disorders diagnosed using DSM-IV; they were compared with 115 healthy female controls. Genomic DNA was extracted from whole blood, and standard polymerase chain reaction testing was performed. We compared the frequencies of a short allele (7 or 9 repeats) and a long allele (10 or 11 repeats) in both groups. RESULTS: In the group who had an eating disorder with binge-eating behaviour, the frequency of a short allele was significantly higher compared with the control group. CONCLUSION: It seems plausible that the association between the DAT1 VNTR and binge-eating behaviour indicates that dysregulation of dopamine reuptake may act as a common pathophysiologic mechanism in eating disorders with binge-eating behaviour and in disorders related to substance use.

Direct analysis by small-pool PCR of MS205 minisatellite mutation rates in sperm after mutagenic therapies.

We have used small-pool PCR to analyse mutation in samples of sperm taken from men after mutagenic therapy. Small-pool PCR uses direct analysis of germline DNA at a highly unstable tandem-repeated "minisatellite" locus to measure rates of length-change mutation in individual sperm samples. The advantages of this approach are that the normal mutation rate is extremely high (about 0.4% per gamete at the locus analysed here), so that relatively small increases in mutation rate can be detectable in individual samples. It is known from work on sperm from untreated individuals that different alleles at this locus have different mutation rates. For this reason, we have analysed the germline mutation rates in sperm samples from two men, in each case comparing a post-treatment sample with a pre-treatment sample from the same individual. We find no evidence for altered mutation in the post-treatment sample, suggesting that the repopulation of the germ-cell compartment after treatment may be subject to stringent mechanisms for the detection and elimination of germ-cell damage.

[Minisatellite instability induced by okadaic acid].

Okadaic acid (OA) is an inhibitor of serine/threonine protein phosphatase (PP) and a tumor promoter in mouse skin carcinogenesis. According to Carcinogenesis Division, National Cancer Center Research Institute, OA induces various genetic alterations, such as loss of exogenous genes, sister chromatid exchanges and diphtheria toxin resistant mutants, although there is no evidence showing that it interacts with DNA directly or produces active oxygen under the conditions used. In this study, minisatellite, which is a hotspot of recombination, was investigated regarding the induction of alteration and instability by OA. It was also attempted to elucidate the roles of minisatellite instability in carcinogenesis. NIH3T3 cells were cultured either with or without OA, subcloned and DNA from each clone was subjected to fingerprint analysis using the Pc-1 minisatellite probe. The frequency of minisatellite recombination was 29% in OA-treated cells, as opposed to 3% in nontreated cells. Furthermore, OA-treated cells exhibited tumorigenicity in nude mice. Minisatellite fingerprint analysis of clones obtained from the tumors revealed that those tumors had acquired minisatellite instability. These mechanisms may be involved in tumor promotion by OA.

Molecular typing of methicillin-resistant Staphylococcus aureus by polymerase chain reaction: distribution of the typed strains in hospitals.

Three methods for molecular typing of methicillin-resistant Staphylococcus aureus using polymerase chain reaction (PCR) were compared. The method which amplified the variable number of tandem repeat of dru sequences grouped the isolates into six types. Whereas, the method examining restriction fragment length polymorphism of coagulase gene and the method using arbitrarily primed-PCR showed poor diversity in typing. We investigated the distribution of dru types in two hospitals. Obvious concentration of a type in one ward was not recognized in our hospital. In the other hospital, a rare type was detected from the inpatients in the pediatrics ward. It suggested that the infection was an epidemic. We also found that some patients were infected with more than two strains. Even if two isolates show the same type, it does not necessarily mean that they originated from one clone. However, this method brings meaningful information on nosocomial infection, more easily than other genotyping.