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Pathogen Evolution Group Research Areas :
I have been involved in Tuberculosis research since 1997 beginning with contribution to the improved sensitivity of culture methods for M. tuberculosis complex and the rapid diagnosis of mycobacterial diseases using molecular methods. A rapid diagnostic test kit has been developed by our group that uses specific primers targeted against universal motifs in the IS1081. This test kit is being evaluated on patients with tuberculosis using different kinds of clinical samples ranging from blood to semen and the results are very promising.
[Ahmed N, Mohanty A K, Batish V K, Mukhopadhyay U. and Grover S (1998). PCR-based Rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis. J. Clinical Microbiol. 36 : 3094-3095] Global networking for molecular epidemiology of M. tuberculosis We understand that in the context of genome sequence utilization, there is a need to develop new approaches to link epidemiology and biology of tubercle bacilli with reference to evolutionary history, metabolic details, virulence and drug resistance profiles. We have been using geographically distinct strains from South America, Europe and Australia to test any possible cross talks between pathogen genotype diversity and host diversity in the world. AmpliBASE MT: An electronic resource for genomic heterogeneity in Mycobacterium tuberculosis The AmpliBASE MT, corresponds to an
object oriented database of genotypic signatures (amplified fragment length
polymorphism (AFLP) patterns) of M. tuberculosis strains originating
from India and the other parts of the world. It aims at examining DNA segments
distributed over the entire genome of M. tuberculosis and was constructed
on the top of an Oracle database with a JAVA client server. The genotypic
signatures for all the pathogenic strains have been generated using universal
AFLP approach of Keygene and Applied Biosystems. Each of the records represents
a digitized electropherogram along with a detailed case sheet of the strain.
The database when loaded in a web browser enables the user to systematically
compare the available genotypes with the standard H37Rv genotype (which
is provided as a clickable image map with full sequence annotation for
all markers). Currently the database contains more than 1500 records that
represent electronic genotypes of representative outbreak strains from
different parts of India, The Netherlands, Peru, Australia, Italy, Canada,
Iran, Vietnam, Tanzania and UK. It also contains relevant information on
outbreak patterns and strain characters such as the drug sensitivity profiles,
IS6110 copy numbers, Spoligotypes, RFLP cluster type, patient info, date
of isolation etc. This database allows Inter-laboratory exchange of data
towards faster comparisons and to rule out possible laboratory contaminations
with other slow growing mycobacteria and the possibilities of duplicate
strain accessions. It may also provide information on geographic partitioning
of genotypes and important diagnostic markers for a particular epidemiological
setting. With a web enabled Oracle/JAVA backbone, the first release of
the database has been made available via CDFD www server (http://www.cdfd.org.in)
and is expected to be very useful for epidemiologists, clinicians and the
research fraternity in the area of molecular diagnosis and surveillance.
 &nb sp; Copy right 2002 CDFD, Hyderabad. All rights reserved AmpliBASE Portal at www.cdfd.org.in/amplibase
Geographic partitioning of M. tuberculosis gene pool: DNA fragments from 136 different gene loci, were analyzed robustly at the genome sequence interface. Detailed analysis revealed 5 regional clusters, indicating specific, bio-geographic partitioning of isolates worldwide. Genomic diversity in our strains was mostly due to base substitutions in important genes such as aconitase, phosphate transport protein, amidase, permease, several members of the PE and PPE family, putative regulators of transcription, hypotheticals and conserved hypothetical proteins, etc. Also, more traditional approaches such as IS6110 RFLPs and newer methods like MIRU-typing have also been used. All the results have been compiled as a browsable databank of DNA fingerprint profiles. (Published on internet www.cdfd.org.in/amplibase). We understand from our large scale genotyping program that FAFLP data are very helpful in identification of strain types in the context of whole genome profiles. Precisely fractionated fragments are amenable to robust analysis at the genome sequence interface. This is essentially because MTB has a clonal genomic structure and the profiles therefore can be easily compared with the genome sequence data without much ambiguity. It has been therefore, proposed that a single fragment difference in FAFLP may sometimes define a new strain, if, a given context is supported. [Ahmed N, Siddiqi N, Banerjee S, Prachee, Alam M, Katoch V M and Hasnain S E. (2001) The Post-genomic face of TB Research: New insights on microevolution, molecular epidemiology and drug resistance. Proceedings of Round Table Conference on Tuberculosis, Ranbaxy Research Foundation, New Delhi, India, pp 123-128.] Is there any AIDS associated strain of M. tuberculosis? In another finding, isolates from AIDS patients revealed a distinct genotype based on a differential detection of atleast 12 genomic loci including 3 important ORFs (Rv3343c, Rv3902c and Rv0929). This suggests an increased risk of infection with a defined strain group for HIV/AIDS patients (Please see figure on the next page). This indicates higher risk for recent transmission amongst patients with AIDS as the age group of patients in HIV group was between 21-45 years. The high degree of clustering found in the HIV patient's isolates suggests that Frequent travel and social links among young, mobile, transgender Peruvians of economically productive age group in Lima raises concern that the 'possibly HIV-associated' type of M. tuberculosis identified in this study may be circulating in other cities of Peru Ahmed N, Caviedes L, Sheen P, Alam M, Gilman R H and Hasnain S E. 2002, unpublished Mycobacterium pinnipedii : A new member of Mtb complex from sea lions! A missing link? Fluorescent amplified-fragment length polymorphism (FAFLP) was applied to isolates from four Australian and six Argentinean seals and compared with FAFLP pattern for standard strains belonging to the M. tuberculosis complex. The FAFLP profiles derived from EcoRI/MseI restricted fragments of blind coded DNA samples differentiated the seal bacillus from other members of the M. tuberculosis complex. Based on the differential amplification of a total of 131 genomic loci representing important genes, seal bacilli appear to have diverged significantly from other members of the M. tuberculosis complex. We describe the suitability of as many as 18 highly polymorphic FAFLP markers for the rapid identification of the seal bacillus strain. Our data additionally supports the previous hypothesis that the seal bacillus occupies a unique taxonomic position within the M. tuberculosis complex. [Ahmed N, Alam M, Cataldi A, Cousins D V and Hasnain S E. 2002, unpublished]
Fig 4: FAFLP profiles of M. tuberculosis, M. bovis and the seal bacillus (M. pinnipedii)
B) The Helicobacter pylori genome program: Genomic analysis reveals distinct geographic partitioning of Helicobacter pylori populations Earlier studies on different insertion deletion motifs in H. pylori genome indicated highly heterogeneous structure of this gastric pathogen worldwide. This interpretation was tested and further extended by chromosome - wide scan of base substitutions in and around the EcoRI and MseI restriction sites in more than 400 independent isolates of H. pylori from five continents. The fluorescent amplified fragment length polymorphism (FAFLP) profiles comprised of hundreds of well resolved fragments generated from EcoRI/MseI restricted DNA. A total of 198 FAFLP fragments were found to be highly informative. The strains were found to be segregated in regional clusters indicating geographic partitioning of H. pylori genotypes worldwide. Of the five most common amplified fragment sets (we call them as amplitypes), type A predominated in the strains from south India. Type B was exclusively observed among Japanese strains; while type C was found mainly in the strains from India and Europe, type D predominated in the African strains. South American strains from Peru shared most of the genomic landmarks with their European counterparts from Spain and England and were considered as amplitype E strains. The genotypes of South Indian strains were distinct, without major affinity with strains from Africa, East Asia and South America. The strains from native Peruvians were most similar to those from Spaniards than Asians. The 'amplitype' diversity revealed in the study was found to be congruent with the results obtained by gene specific PCR and sequencing of loci associated with pathogenesis such as vac-A and cag-PAI. This data may be significant to speculate about the age of H. pylori genotype in South Asia/East Asia and the West and to predict the time and circumstances under which the H. pylori might have got associated with humans. Genomic differences found to date between various strains in this study should encourage further analyses of strains from relatively understudied geographic regions. Such "geographic genomics" may unravel new possibilities towards our understanding of microbial pathogenesis and the host-component. Genetic profiling of Pseudomonas aeruginosa Genome sequence based fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for fingerprinting and subtyping multiple drug resistant isolates of Pseudomonas aeruginosa from post-surgical endophthalmitis. The FAFLP profiles derived from EcoRI/MseI restricted fragments differentiated clinical isolates and were found to be extremely reproducible. Seventeen different amplification patterns (amplitypes) were observed for all the 42 isolates from 11 different patients. Also, we were able to genotype the isolates based on the differential amplification of a total of 31 FAFLP markers representing genomic fragments from the P. aeruginosa chromosome. These data appear to provide clues to the genetic diversity of endopthalmitis associated strains and could be converted into digitized fingerprints suitable for electronic manipulations and archiving.
[Ahmed N, Bal A, Khan A A, Alam M, Kagal A, Arjunwadkar V, Rajput A, Majeed A A, Rahman S A, Banerjee S, Joshi S, Bharadwaj R (2002) Whole genome fingerprinting and genotyping of multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from endophthalmitis patients in India. Infection Genet. Evol. 2: 00-00 (In press)] D) Single nucleotide polymorphisms in Ocular Disorders Primary congenital glaucoma example (In Collaboration with L V Prasad Eye Institute, Hyderabad) Primary congenital glaucoma (PCG) is a severe form of glaucoma, which causes near total/complete blindness in childhood. Though several cases of PCG with varying severity and manifestations have been identified in India, the genetic etiology remains unknown. Twenty two members of 5 clinically well-characterized consanguineous families were studied. The primary candidate gene CYP1B1 was amplified, sequenced, and analyzed in controls and patients to identify the disease-causing mutations. Five distinct mutations were identified in the coding region of CYP1B1 in 8 patients of 5 PCG families, of these three were novel and include a novel homozygous frameshift, compound heterozygous missense and other known mutations. One family showed pseudo-dominance whereas others were autosomal recessive with full penetrance. In contrast to all known CYP1B1 mutations, the frameshift identified by us is of special significance because all functional motifs are missing. This, therefore, represents a rare example of a natural functional CYP1B1 "knockout" resulting in a null allele (both patients are blind).
Figure 6: Genomic analysis, validation
and computational modelling of SNPs in CYP1B1 gene in South Indian Pedigrees
The molecular mechanism leading to the development of PCG is unknown. Since CYP1B1 knock-out mice did not show glaucoma phenotype, the functional "knock-out" identified here has important implications in elucidating the pathogenesis of PCG. Further understanding of how this molecular defect leads to PCG could influence the development of specific therapies. This is the first study describing the molecular basis of PCG from the Indian subcontinent and has profound clinical implications in multiple ways - in diagnosis, genetic counseling, genotype-phenotype correlations and prognosis. Hence it is a step forward in preventing this devastating childhood blindness. [Panicker S G, Reddy ABM, Mandal A K, Ahmed N, Nagarajaram H A, Hasnain SE and Balasubramanian D (2002). Identification of novel mutations causing familial primary congenital glaucoma in Indian pedigrees. Investigative Ophthalmol. Visual Sci. 00:0000 (In press) Ataur Rasheed M, Vemuganti G K, Honawar S G, Ahmed N, Hasnain S E and Kannabiran C. (2002) Mutational analysis of the RB1 gene in Indian patients with Retinoblastoma. Ophthalmic Genet. 00:0000 (In press)] CONTACT INFORMATION
Last updated on : Monday, 13th June, 2005. |
