Ann S. G. Lee

Novel Mutations in ndh in Isoniazid-Resistant Mycobacterium tuberculosis Isolates

ABSTRACT Novel mutations in NADH dehydrogenase (ndh) were detected in 8 of 84 (9.5%) isoniazid (INH)-resistant isolates (T110A [n = 1], R268H [n = 7]), but not in 22 INH-susceptible isolates of Mycobacterium tuberculosis. Significantly, all eight isolates with mutations atndh did not have mutations at katG, kasA, or the promoter regions of inhA or ahpC, except for one isolate. Mutations in ndh appear to be an additional molecular mechanism for isoniazid resistance in M. tuberculosis.

Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity of Mycobacterium tuberculosis in Singapore

ABSTRACT Strain typing using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) is a powerful tool for studying the epidemiology and genetic relationships of Mycobacterium tuberculosis isolates. For this study, isolates from 291 patients in Singapore were genotyped by this method. One hundred sixty-six distinct MIRU-VNTR patterns were detected. One hundred sixty-two strains were grouped into 1 of 35 different MIRU-VNTR clusters and 131 isolates were unique. In this sample collection, 9 of the 12 MIRU-VNTR loci were moderately or highly discriminative according to their allelic diversities. The Hunter-Gaston discriminatory index was 0.975, indicating the high power of discrimination of MIRU-VNTR typing. By direct comparisons with previously typed MIRU-VNTR patterns and by genetic relationship analyses, we could identify and clearly define four epidemic groups of M. tuberculosis in our sample, corresponding to the W/Beijing, East-Africa-Indian, Haarlem, and Delhi genotype families. Furthermore, MIRU-VNTR typing was able to clearly distinguish ancestral and modern M. tuberculosis strains as defined by TbD1 genomic deletion analysis. These results indicate that MIRU-VNTR typing can be a useful first-line tool for studying the genetic diversity of M. tuberculosis isolates in a large urban setting such as Singapore.

Characterization of Ancestral Mycobacterium tuberculosis by Multiple Genetic Markers and Proposal of Genotyping Strategy

ABSTRACT Sixty-eight ancestral Mycobacterium tuberculosis isolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol. 42:1986-1993, 2004). These TbD1+ ancestral isolates were further characterized and typed in this study by IS6110 restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS6110 RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS6110 RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS6110 RFLP is proposed to achieve maximal discrimination for unrelated TbD1+ strains. This typing strategy may be useful in areas where TbD1+ ancestral strains are prevalent.

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis by High-Resolution Melting Analysis

ABSTRACT We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position −15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.

High Frequency of Mutations in the rpoB Gene in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Singapore

Rifampin (RIF) is a first-line antituberculosis drug. Resistance to RIF, in the majority of cases, has been associated with mutations within an 81-bp RIF resistance-determining region (RRDR) of the rpoB gene, which encodes the β subunit of the RNA polymerase ([8][1]). RIF acts by binding to the β

Detection of a novel Alu-mediated BRCA1 exon 13 duplication in Chinese breast cancer patients and implications for genetic testing

To the Editor: Large genomic rearrangements in the BRCA1 gene have been described in hereditary breast/ ovarian cancer (HBOC) families from Europe, America and Australia, accounting for up to 40% of all BRCA1 mutations (1–8). The frequency of large BRCA1 rearrangements in Asia is unknown. In Chinese, BRCA1 mutations are rare in breast cancer patients, ranging from 1.1 to 8.6% (9–13). To ascertain whether large genomic rearrangements of BRCA1may contribute to the mutational spectrum in Chinese breast cancer patients, we applied the multiplex ligation-dependent probe amplification (MLPA) technique (14) to screen 108 unrelated female Chinese breast cancer patients who tested negatively for mutations in the coding regions of BRCA1 by conventional methods. Five patients were from HBOC families, 22 had a family history of breast cancer only and the remaining 81 had early onset breast cancer (by age 40 years). Peripheral blood samples were obtained with prior informed consent according to the guidelines from the institutions’ ethics committees. MLPA profiles using the MLPA P002 test kit (MRC Holland, Amsterdam, Holland) on genomic DNA indicated that one patient (case MR0017, from a HBOC family) had a duplication of exon 13 and one patient (case BR74, with early onset breast cancer) had deletion of exon 13. When the samples were retested using the MLPA P087 (MRC Holland) confirmation kit, the duplication of exon 13 was confirmed for case MR0017. In contrast, case BR74 gave a normal result, indicating a possible false positive exon 13 deletion. On sequencing, case BR74 had a single nucleotide substitution in exon 13 at nucleotide 46278 (refSNP ID: rs1060915) situated two nucleotides proximal to the ligation site and within the region of probe ligation for the probe used in the P002 kit (14). This highlights the importance and necessity of confirmation testing done by additional methods (15). In total, large genomic rearrangements were detected in 20% (1/5) of Chinese HBOC families. Determination of the precise breakpoints involved in exon duplication in case MR0017 was done. Long-range polymerase chain reaction (PCR) specific for the 6-kb duplication in exon 13 of the BRCA1 gene, which is a founder mutation known to exist in families of Dutch or English origin (16, 17), was negative. Newly designed duplication-specific primers (Fig. 1a) produced a 1.12-kb fragment on PCR amplification only in the patient MR0017 (Fig. 1b). Sequencing of this fragment and alignment with the BRCA1 sequence showed that the duplication involved an 8463 bp region from nucleotides 41220–49682 (Fig. 1a,c). Both duplication breakpoints occurred within Alu repeats (Fig. 1a,c). Notably, both Alu repeats are oriented in the reverse sense compared with the BRCA1 transcript and share 87% homology with each other. Only two BRCA1 rearrangements involving Alu repeats oriented in the reverse sense have been reported in contrast to 22 rearrangements sharing the same orientation as the BRCA1 transcript (6, 18–20). Interestingly, sequence similarities of the 28-bp core sequence at the breakpoints were observed with two other core sequences (Fig. 1d), supporting the notion that recombination breaks occur at specific core sequences (21). On review of the clinical data, case MR0017, deceased at age 44 years, had bilateral breast cancer diagnosed at ages 32 and 41 years. She had initially been classified as having early onset breast cancer but has been reclassified as HBOC because one older sister was subsequently diagnosed with epithelial ovarian cancer at age 43 years. This sister had the same exon 13 duplication while three younger unaffected sisters had tested negative. Therefore, pedigree risk assessment for genetic testing is warranted as part of genetic counselling, Clin Genet 2006: 70: 80–82 # 2006 The Authors Printed in Singapore. All rights reserved Journal compilation # 2006 Blackwell Munksgaard

Discrimination of Single-Copy IS6110 DNA Fingerprints of Mycobacterium tuberculosis Isolates by High-Resolution Minisatellite-Based Typing

ABSTRACT Seven isoniazid-resistant isolates with mutations in the NADH dehydrogenase (ndh) gene were molecularly typed by IS6110-based restriction fragment length polymorphism analysis. All seven isolates with the R268H mutation had identical 1.4-kb IS6110 fingerprints. High-resolution minisatellite-based typing discriminated five of these isolates; two isolates were identical.

High-Resolution Melting Analysis for the Rapid Detection of Fluoroquinolone and Streptomycin Resistance in Mycobacterium tuberculosis

Background Molecular methods for the detection of drug-resistant tuberculosis are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with drug resistant tuberculosis. We aimed to develop and evaluate high-resolution melting (HRM) assays for the detection of mutations within gyrA, rpsL, and rrs, for the determination of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis (MTB). Methodology/Principal Findings A blinded series of DNA samples extracted from a total of 92 clinical isolates of MTB were analyzed by HRM analysis, and the results were verified using DNA sequencing. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing were 74.1% and 100.0% for the detection of fluoroquinolone resistance, and 87.5% and 100.0% for streptomycin resistance. Five isolates with low level resistance to ofloxacin had no mutations detected in gyrA, possibly due to the action of efflux pumps, or false negativity due to mixed infections. One fluoroquinolone-resistant isolate had a mutation in a region of gyrA not encompassed by our assay. Six streptomycin-resistant strains had undetectable mutations by HRM and DNA sequencing, which may be explained by the fact that not all streptomycin-resistant isolates have mutations within rpsL and rrs, and suggesting that other targets may be involved. Conclusion The HRM assays described here are potentially useful adjunct tests for the efficient determination of fluoroquinolone and streptomycin resistance in MTB, and could facilitate the timely administration of appropriate treatment for patients infected with drug-resistant TB.

LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast and colorectal cancer

Deletion of 11q23–q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2′-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Contribution of dfrA and inhA Mutations to the Detection of Isoniazid-Resistant Mycobacterium tuberculosis Isolates

ABSTRACT Screening of 127 isoniazid (INH)-resistant Mycobacterium tuberculosis isolates from Singapore for mutations within the dfrA and inhA genes revealed mutations in 0 and 5 (3.9%) isolates respectively, implying that mutations in dfrA do not contribute to the detection of INH-resistant M. tuberculosis and that mutations within inhA are rare. Thirty-seven (29%) of the 127 isolates had no mutations in any of the genes implicated in INH resistance (katG, kasA, and ndh; inhA and ahpC promoters), suggesting that there are new INH targets yet to be discovered.