Spectrum of germ-line RB1 gene mutations in Malaysian patients with retinoblastoma.

PURPOSE: The availability of molecular genetic testing for retinoblastoma (RB) in Malaysia has enabled patients with a heritable predisposition to the disease to be identified, which thus improves the clinical management of these patients and their families. In this paper, we presented our strategy for performing molecular genetic testing of the RB1 gene and the findings from our first 2 years of starting this service. METHODS: The peripheral blood of 19 RB probands, including seven bilateral and 12 unilateral cases, was obtained, and genomic DNA was extracted. Analysis of the RB1 exons and the promoter region was conducted first using PCR and direct sequencing. Next, multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients whom the first results were negative. For patients whom either the first or second method results were positive, parental samples were analyzed to determine the origin of the mutation. RESULTS: Ten RB1 mutations were identified in ten (52.6%) of the 19 probands (seven bilateral and three unilateral cases), of which 30.0% (3/10) was identified with MLPA. The detection rates in the bilateral and unilateral cases were 100.0% (7/7) and 25.0% (3/12), respectively. Three new RB1 mutations were discovered, two in patients with bilateral RB and one in patient with unilateral RB. Interestingly, all mutations detected with the PCR-sequencing method were predicted to create a premature stop codon. Eight mutations were proven to be de novo while one mutation was inherited from the mother in a family with a positive history of RB. CONCLUSIONS: Our results confirmed the heterogeneous nature of RB1 mutations and the predominantly de novo origin. The high prevalence of pathogenic truncating mutations was evident among local patients with RB. The combination of PCR sequencing and MLPA is recommended for sensitive identification of heritable RB cases.

Genotypic characterization of Streptococcus pneumoniae serotype 19F in Malaysia.

Streptococcus pneumoniae is an epidemiologically important bacterial pathogen. Recently, we reported the antibiotic susceptibility patterns of a limited collection of pneumococcal isolates in Malaysia with a high prevalence of erythromycin resistant strains. In the present study, 55 of the pneumococcal isolates of serotype 19F were further analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The generated genotypic patterns were then correlated with the antibiograms previously reported. Forty-seven different PFGE profiles (PTs) were obtained, showing that the isolates were genetically diverse. MLST identified 16 sequence types (STs) with ST-236 being predominant (58.2%), followed by ST-81 (10.3%). Among the ST-236 isolates, 22 were erythromycin resistant S. pneumoniae (ERSP) and 15 were trimethoprim/sulfamethoxazole (TMP/SMX) resistant, while among ST-81, four isolates were ERSP and two were TMP/SMX resistant. The high prevalence of erythromycin resistant serotype 19F isolates of ST-236 in this study has also been reported in other North and South East Asian countries.

Characterization of multidrug-resistant and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains from Malaysian hospitals.

The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla(SHV), 19 harboured bla(CTX-M), 5 harboured bla(OXA-1) and 4 harboured bla(TEM-1). Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5'CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla(SHV), bla(CTX-M) and bla(TEM-1)) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.