Multiple strain infection of Mycobacterium leprae in a family having 4 patients: A study employing short tandem repeats.

BACKGROUND: Leprosy is a slow, chronic disorder caused by Mycobacterium leprae. India has achieved elimination of leprosy in December 2005 but new cases are being detected and continue to occur in some endemic pockets. The possible ways of transmission of leprosy is not fully understood and is believed that leprosy is transmitted from person to person in long term contact. Studying the transmission dynamics is further complicated by inability to grow M. leprae in culture medium and lack of animal models. More than one family members were found to be affected by leprosy in some highly endemic pockets. This study reported the transmission pattern of leprosy in a family having 4 patients. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the transmission of leprosy in a single family having 4 patients using microsatellite typing. DNA was isolated from slit skin smear samples taken from the patients and the isolated DNA were amplified using microsatellite loci TA11CA3. The amplified products were sequenced using Sanger's sequencing methods and the copy number variation in the microsatellite loci between strains were elucidated by multiple sequence alignment. The result showed that all the 4 members of the family acquired infection from 3 different strains of M. leprae from 3 different sources. The elder and middle daughters were infected by same types of strains having the repeat unit TA13CA3 and could have acquired the infection from social contacts of leprosy cases while the father and younger daughter were infected by strains with the repeat unit TA12CA3 and TA11CA3 and could have acquired infection from social contacts. CONCLUSIONS/SIGNIFICANCE: The study suggested that three family members viz, elder daughter, father and younger daughter could be infected by M. leprae from 3 different sources and the history of the disease and genetic analysis showed that the middle daughter acquired infection from her elder sister in due course of contact. This study implies that the transmission of leprosy not only occurred amongst the house hold members but also has been transmitted from social and neighborhood contacts in long term association with the them.

The mtDNA replication-related genes TFAM and POLG are associated with leprosy in Han Chinese from Southwest China.

BACKGROUND: The pathogen Mycobacterium leprae of leprosy is heavily dependent on the host energy metabolites and nutritional products for survival. Previously we and others have identified associations of several mitochondrion-related genes and mitochondrial DNA (mtDNA) copy number alterations with leprosy and/or its subtype. We hypothesized that genetic variants of mtDNA replication-related genes would affect leprosy. OBJECTIVE: We aimed to identify genetic associations between the mtDNA replication-related genes TFAM, POLG and leprosy. METHODS: Genetic association study was performed in 2898 individuals from two independent sample sets in Yunnan Province, China. We first screened 7 tag SNPs of TFAM and POLG in 527 leprosy cases and 583 controls (Sample I). Expression quantitative trait loci (eQTL) analysis and differential mRNA expression were analyzed to discern potential effect of risk variants. The entire exon region of TFAM and POLG were further analyzed in 798 leprosy cases and 990 controls (Sample II; 4327 East Asians from the ExAC dataset was included as a reference control) by using targeted gene sequencing for fine mapping potentially causal variants. RESULTS: Two tag SNPs of TFAM (rs1049432, P=0.007) and POLG (rs3176238, P=0.006) were associated with multibacillary leprosy (MB) in Sample I and the significance survived correction for multiple comparisons. SNPs rs1937 of TFAM (which was linked with rs1049432) and rs61756401 of POLG were associated with leprosy, whereas no potentially causative coding variants were identified in Sample II. The eQTL analysis showed that rs1049432 was a significant cis eQTL for TFAM in nerve tissue (P=1.20×10-12), and rs3176238 was a significant cis eQTL for POLG in nerve (P=3.90×10-13) and skin tissues (P=2.50×10-11). Consistently, mRNA level of POLG was differentially expressed in leprotic skin lesions. CONCLUSIONS: Genetic variants of TFAM and POLG were associated with leprosy in Han Chinese, presumably by affecting gene expression.

IL12B SNPs and copy number variation in IL23R gene associated with susceptibility to leprosy.

BACKGROUND: Genome-wide studies have identified both human leucocyte antigen (HLA) and non-HLA regions in association with leprosy. Involvement of novel functional loci within these regions has been proposed by us earlier. METHODS: We investigated the role of 23 single nucleotide polymorphisms (SNPs) in IL12B and IL12RB2 in a total of 2345 individuals from India, using MassArray platform, along with the copy number variations in IL23R, IL12RB2 and IL10 genes in a representative set of 257 individuals, using real-time PCR. RESULTS: SNP rs2853694 in IL12B gene (AA vs AC+CC, p=2.6E-04, OR=1.42 (1.17-1.70)) showed an association with leprosy. Pairwise interaction analysis followed by combined analysis of multiple SNPs identified that IL12B, TNF and BTNL2-DRA inter-genic SNPs provided a major risk towards leprosy (p=2.6E-08, OR=3.94 (2.43-6.38)), showing a further increase (p=3.6E-14) for combined risk genotype interactions. On the other hand, IL12B, BAT1, NFKBIL1 and LTA SNPs together showed a disposition towards protection (p=0.000011, OR=0.32 (0.19-0.53)) with a further increase (p=6.38E-10) for combined protective genotype-interactions. Copy number variation analysis showed an increased copy number of the IL23R gene (PB=36.4%, controls=20.2%; p=0.026) associated with the pauci-bacillary form of leprosy, which correlated with a trend towards enhanced expression in memory T cells in a preliminary observation. CONCLUSIONS: The observations made here highlight the importance of interaction between specific genetic backgrounds of immune response related genes in the outcome of Mycobacterium leprae infection.

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2.

OBJECTIVES: To perform a comprehensive population genetic study of PARK2. PARK2 mutations are associated with juvenile parkinsonism, Alzheimer disease, cancer, leprosy, and diabetes mellitus, yet ironically, there has been no comprehensive study of PARK2 in control subjects; and to resolve controversial association of PARK2 heterozygous mutations with Parkinson disease (PD) in a well-powered study. METHODS: We studied 1,686 control subjects (mean age 66.1 ± 13.1 years) and 2,091 patients with PD (mean onset age 58.3 ± 12.1 years). We tested for PARK2 deletions/multiplications/copy number variations (CNV) using semiquantitative PCR and multiplex ligation-dependent probe amplification, and validated the mutations by real-time quantitative PCR. Subjects were tested for point mutations previously. Association with PD was tested as PARK2 main effect, and in combination with known PD risk factors: SNCA, MAPT, APOE, smoking, and coffee intake. RESULTS: A total of 0.95% of control subjects and 0.86% of patients carried a heterozygous CNV mutation. CNV mutations found in 16 control subjects were all in exons 1-4, sparing exons that encode functionally critical protein domains. Thirteen patients had 2 CNV mutations, 5 had 1 CNV and 1 point mutation, and 18 had 1 CNV mutation. Mutations found in patients spanned exons 2-9. In whites, having 1 CNV was not associated with increased risk (odds ratio 1.05, p = 0.89) or earlier onset of PD (64.7 ± 8.6 heterozygous vs 58.5 ± 11.8 normal). CONCLUSIONS: This comprehensive population genetic study in control subjects fills the void for a PARK2 reference dataset. There is no compelling evidence for association of heterozygous PARK2 mutations, by themselves or in combination with known risk factors, with PD.