Repeat expansion and methylation-sensitive triplet-primed polymerase chain reaction for fragile X mental retardation 1 gene screening in institutionalised intellectually disabled individuals

INTRODUCTION@#Fragile X syndrome (FXS) is the most prevalent X-linked intellectual disability (ID) and a leading genetic cause of autism, characterised by cognitive and behavioural impairments. The hyperexpansion of a CGG repeat in the fragile X mental retardation 1 (FMR1) gene leads to abnormal hypermethylation, resulting in the lack or absence of its protein. Tools for establishing the diagnosis of FXS have been extensively developed, including assays based on triplet-primed polymerase chain reaction (TP-PCR) for detection and quantification of the CGG trinucleotide repeat expansion, as well as determination of the methylation status of the alleles. This study aimed to utilise a simple, quick and affordable method for high sensitivity and specificity screening and diagnosis of FXS in institutionalised individuals with ID.@*METHODS@#A total of 109 institutionalised individuals at the Center for Social Rehabilitation of Intellectual Disability Kartini, Temanggung, Central Java, Indonesia, were screened in a three-step process using FastFrax™ Identification, Sizing and Methylation Status Kits.@*RESULTS@#Two samples that were classified as indeterminate with respect to the 41-repeat control at the identification step were subsequently determined to be non-expanded by both sizing and methylation status analyses. Two samples classified as expanded at the identification step were determined to carry full mutation expansions > 200 repeats that were fully methylated using sizing and methylation status analyses, respectively, yielding a disease prevalence of 1.83%.@*CONCLUSION@#Repeat expansion and methylation-specific TP-PCR is practical, effective and inexpensive for the diagnosis of FXS, especially in high-risk populations of individuals with ID of undetermined aetiology.

A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria.

Review on Molecular Diagnostic Techniques in Friedreich’s Ataxia.

Friedreich’s ataxia is a commonly inherited neurodegenerative disease with an autosomal recessive pattern of inheritance, and was described by Nikolaus Friedreich first in 1863. Friedreich’s ataxia is caused due to hyperexpansion of the intronic GAA trinucleotide repeats or mutations in the FXN gene on chromosome 9q13. This gene codes for a mitochondrial protein, frataxin, which is highly conserved in many species and has functions in iron-sulfur cluster biosynthesis. Friedreich’s ataxia mainly results from a deficiency of the frataxin protein, due to mutations in the FXN gene. Formation of sticky DNA, formation of DNA-RNA hybrid and epigenetic changes, including methylation of DNA and histone modifications, are the proposed mechanisms for disruption of FXN gene expression. Most cases of Friedreich’s ataxia are homozygous and caused due to expansion of the GAA trinucleotide repeat in the first intron of the FXN gene, however, some cases can be heterozygous, with GAA expansion in one allele and point mutation or deletion in the FXN gene on the other allele. Therefore, diagnosis of the disease based on only the clinical symptoms becomes difficult. Molecular diagnosis is, therefore, important, in order to detect GAA repeat expansions as well as mutations in the FXN gene. This review represents an overview of the molecular diagnostic studies in Friedreich’s ataxia, including an overview of the disease, as well as the gene and protein involved in the disease and techniques that can be useful in diagnosis of the Friedreich’s ataxia. The described methods include tools that are based on analysis of DNA as well as analysis of mRNA and protein levels. A brief description of mutations found in compound heterozygous Friedreich’s ataxia patients, is also provided.

AP-PCR and Antimicrobial Susceptibility Patterns of Fusobacterium nucleatum Associated with Chronic Periodontitis among Patients at Lagos University Teaching Hospital.

Aims: To isolate, identify and evaluate the genetic diversity and antimicrobial susceptibility of F. nucleatum recovered from Nigerian patients with chronic periodontitis. Study Design: Cross-sectional design. Place and Duration of Study: Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, between January 2007 and July 2008. Methodology: We analyzed F. nucleatum species recovered from Nigerian patients with chronic periodontitis. Bacterial identification was done using colonial morphology; Grams stain reaction, conventional biochemical tests, API 20-A and Polymerase chain reaction (PCR). The minimum inhibitory concentration (MIC) of 6 antibiotics was determined by agar dilution method on Brucella blood agar while the bacterial genetic diversity was studied using the Arbitrarily Primed-PCR (AP-PCR) method with the arbitrary primer OPA-05. The interrelationship and genetic similarity matrix among the isolates was determined and by Numerical taxonomy and multivariate analysis system (NTSYS-pc) statistical package. Results: We obtained 48 isolates of F. nucleatum from 50 Nigerian patients (28 males and 22 females) with chronic periodontitis. They were susceptible to metronidazole, clindamycin, cefoxitin, tetracycline, amoxicillin and clavulanate. One was resistant to amoxicillin (MIC >32 μg/ml) and produced β-lactamase. The isolates were further placed into five groups (A, B, C, D and E) based on their AP-PCR profile. Conclusion: The AP-PCR analysis showed heterogeneity among strains. By using APPCR, we observed a single β-lactamase producing clone resistant to amoxicillin which eventually formed a distinct group showing that such genetic difference may have contributed to the formation of a separate clone.

Molecular epidemiology profile of methicillin-resistant Staphylococcus aureus in ICU ward / 中国实用内科杂志

Objective To investigate molecular epidemiology profile of methicillin-resistant Staphylococcus aureus (MRSA) in ICU ward.Methods Twenty-five MRSA strains were typed by arbitrarily primed PCR (AP-PCR).Results Ten different AP-PCR patterns (A-G) were found among 25 MRSA strains.Most of MRSA in ICU ward were A and B pulsotype.Conclusion Hospital acquired MRSA is multi-resistant to antibiotics.A and B pulsotype MRSA outbreak occures in ICU ward.

Delineation of chromosome translocations by fluorescence in situ hybridization / 第二军医大学学报

Objective:To delineate the G-banding-sug gested chromosome translocations by fluorescence in situ hybridization (FISH ) technique. Methods: Locus-specific probes, generated by degen erate oligonucleotide-primed PCR (DOP-PCR) technique from yeast artificial chr omosomes (YACs) mapping the regions in question, were used for FISH tests. Results: Among the 2 cases unresolved by G-banding, FISH confirm ed that one had a balanced translocation between chromosome 11 and chromosome 13 , the other had an unbalanced translocation between chromosome 6 and chromosome X.Conclusion: Because of its high sensitivity and specificity, FISH technique is a powerful adjunct to chromosome banding techniques, particula rly for the delineation of subtle chromosome rearrangement(s) and the origin of segment(s).

Delineation of chromosome translocations by fluorescence in situ hybridization / 第二军医大学学报

Objective:To delineate the G-banding-sug gested chromosome translocations by fluorescence in situ hybridization (FISH ) technique. Methods: Locus-specific probes, generated by degen erate oligonucleotide-primed PCR (DOP-PCR) technique from yeast artificial chr omosomes (YACs) mapping the regions in question, were used for FISH tests. Results: Among the 2 cases unresolved by G-banding, FISH confirm ed that one had a balanced translocation between chromosome 11 and chromosome 13 , the other had an unbalanced translocation between chromosome 6 and chromosome X.Conclusion: Because of its high sensitivity and specificity, FISH technique is a powerful adjunct to chromosome banding techniques, particula rly for the delineation of subtle chromosome rearrangement(s) and the origin of segment(s).

Delineation of chromosome translocations by fluorescence in situ hybridization / 第二军医大学学报

Objective:To delineate the G-banding-sug gested chromosome translocations by fluorescence in situ hybridization (FISH ) technique. Methods: Locus-specific probes, generated by degen erate oligonucleotide-primed PCR (DOP-PCR) technique from yeast artificial chr omosomes (YACs) mapping the regions in question, were used for FISH tests. Results: Among the 2 cases unresolved by G-banding, FISH confirm ed that one had a balanced translocation between chromosome 11 and chromosome 13 , the other had an unbalanced translocation between chromosome 6 and chromosome X.Conclusion: Because of its high sensitivity and specificity, FISH technique is a powerful adjunct to chromosome banding techniques, particula rly for the delineation of subtle chromosome rearrangement(s) and the origin of segment(s).