Single nucleotide polymorphism analysis of European archaeological M. leprae DNA

Abstract

Background: Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA) from medieval bones and single nucleotide polymorphism (SNP) typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.

Methods and findings: Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs) in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3).

Conclusions: These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.

Figure 1. 3% agarose gel showing clear bands for the aDNA extracts taken from sample G483, 2A and 3A, matching the size of the M. leprae positive control DNA following amplification with RLEP primers 2 & 4 (111 bp), Croatian samples 1A and 4A did not show a matching band and were not analysed further.

Key: 1. M. lep pos control DNA. 2. 2A (Croatia). 3. 3A (Croatia). 4. 4A (Croatia). 5. 1A (Croatia). 6. Extraction blank 1. 7. Extraction blank 2. 8. Water control 1. 9. Water control 2 (run with positive control). 10. G483 (Denmark). 11. 100 bp ladder. N.B. The M. leprae positive control DNA (lane 1) was amplified separately from the extractions to avoid contamination.

Figure 2. Sequence of SNP 14676 showing a “C” (highlighted in yellow) for aDNA extracted from St John's Timber Hill skeletal sample 11784 (rhino-max).

Figure 3. 3% agarose gel stained with ethidium bromide visualising the reproduction of amplification of aDNA extraction from skeletal UK samples 11784, 11287 and 11503, following amplification with SNP-2935685 (151 bp).

1. Sample 11784. 2. Blank well. 3. Sample 11287. 4. Blank well. 5. Sample 11503. 6. Extraction blank. 7. M. leprae control DNA. 8. Negative water control. 9. 100 bp ladder.

Figure 4. Sequence of SNP2935685 showing a “C” (highlighted in yellow) for aDNA extracted from Croatian skeletal sample 2A (rhino-max).