Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

Next-generation sequencing-assisted diagnosis of a case of leprosy misdiagnosed as erythema multiforme.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that is mainly transmitted through droplets from the nose and mouth of untreated patients. Owing to the lack of specific serological markers and clinical manifestations, leprosy can be easily confused with other skin lesion-related diseases and is difficult to distinguish. CASE PRESENTATION: This study introduces and summarises the diagnosis and treatment process of a case of leprosy misdiagnosed as erythema multiforme for a long time. A 43-year-old female was admitted to our hospital because of "repeated fever with superficial lymphadenopathy and systemic rash in May". The diagnosis of the patient was based on the two main clinical characteristics of superficial lymphadenopathy and systemic pleomorphic erythema by using a combination of multiple samples of lymph nodes and skin, routine pathological examination, immunohistochemistry, acid-fast, silver hexamine, periodic acid-Schiff (PAS) staining, and second-generation gene sequencing of fresh biopsy tissue. The patient was treated with dapsone, rifampicin, and clofazimine at the Institute of Dermatology and Venereal Diseases. After treatment for 1 year, her temperature returned to normal, the area of facial erythema decreased, and the volume of axillary lymph nodes had gradually reduced. CONCLUSIONS: In conclusion, special pathological staining and second-generation gene sequencing show promising advantages in distinguishing leprosy from other skin lesion-related diseases.

Deep resequencing identifies candidate functional genes in leprosy GWAS loci.

Leprosy is the second most prevalent mycobacterial disease globally. Despite the existence of an effective therapy, leprosy incidence has consistently remained above 200,000 cases per year since 2010. Numerous host genetic factors have been identified for leprosy that contribute to the persistently high case numbers. In the past decade, genetic epidemiology approaches, including genome-wide association studies (GWAS), identified more than 30 loci contributing to leprosy susceptibility. However, GWAS loci commonly encompass multiple genes, which poses a challenge to define causal candidates for each locus. To address this problem, we hypothesized that genes contributing to leprosy susceptibility differ in their frequencies of rare protein-altering variants between cases and controls. Using deep resequencing we assessed protein-coding variants for 34 genes located in GWAS or linkage loci in 555 Vietnamese leprosy cases and 500 healthy controls. We observed 234 nonsynonymous mutations in the targeted genes. A significant depletion of protein-altering variants was detected for the IL18R1 and BCL10 genes in leprosy cases. The IL18R1 gene is clustered with IL18RAP and IL1RL1 in the leprosy GWAS locus on chromosome 2q12.1. Moreover, in a recent GWAS we identified an HLA-independent signal of association with leprosy on chromosome 6p21. Here, we report amino acid changes in the CDSN and PSORS1C2 genes depleted in leprosy cases, indicating them as candidate genes in the chromosome 6p21 locus. Our results show that deep resequencing can identify leprosy candidate susceptibility genes that had been missed by classic linkage and association approaches.

Use of High-Throughput Sequencing to Identify Fungal Communities on the Surface of Citri Reticulatae Pericarpium During the 3-Year Aging Process.

Citri Reticulatae Pericarpium (CRP) is a natural product that is used widely in food and is an ingredient in traditional Chinese medicine. CRP improves gradually with aging; this process typically takes 3 years or more. During the aging process, CRP can be colonized with fungi and mildew. Molds and mildew may result in an increased flavonoid content; however, this has been observed only in response to fungi of the genera Penicillium and Aspergillus. As fungal colonization may alter the quality and properties of CRP, it is critical to have an understanding of the fungal communities detected on the surface of CRP during the aging process. We used a high-throughput sequencing (HiSeq) platform to sequence internal transcribed spacers (ITS) region to identify the contaminants associated with CRP during the 3-year aging process. We also evaluated the distribution of the dominant fungi of the genera Aspergillus and Penicillium over time. At the phylum level, we identified Ascomycota (36.26%) and Basidiomycota (18.98%), along with smaller populations of Mucoromycota, Glomeromycota, and Mortierellomycota. At the genus level, the fungi detected include Wallemia (12.40%), Cystofilobasidium (4.62%), Zasmidium (4.52%), Cladosporium (3.72%), Hanseniaspora (3.55%), Fusarium (3.49%), Kurtzmaniella (2.03%), Candida (1.74%), Passalora (1.47%), Ceramothyrium (1.33%), Mucor (1.07%), and Aspergillus (1.03%). Fungi of the genus Penicillium were detected primarily during the first year of storage. By contrast, fungi of the genus Aspergillus were not detected during the early stages (fresh peel-8 months), but appeared gradually at later stages of the aging process. Taken together, our results indicate that HiSeq is an effective method to study the changes in fungal communities that develop on the CRP surface over time. These findings provide a basis for further research into the correlation between dominant fungi and the mechanisms underlying the successful aging of CRP.

Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Biochemical changes and microbial community dynamics during spontaneous fermentation of Zhacai, a traditional pickled mustard tuber from China.

Zhacai is a traditional fermented vegetable that has been consumed in China for centuries. It is currently manufactured by spontaneous fermentation and therefore mostly relies on the activities of autochthonous microorganisms. Here, we characterized microbial community dynamics and associated biochemical changes in 12% salted Zhacai during a 90-day spontaneous fermentation process using high-throughput sequencing and chromatography-based approaches to identify associations between microorganisms and fermentation characteristics. Amplicon sequencing targeting bacterial 16S rRNA genes revealed that bacterial communities were dominated by halophilic bacteria (HAB, i.e., Halomonas and Idiomarina) and lactic acid bacteria (LAB, i.e., Lactobacillus-related genera and Weissella) after 30 days of fermentation. In addition, the relative abundances of the fungal genera Debaryomyces, Sterigmatomyces, and Sporidiobolus increased as fermentation progressed. Concomitantly, pH decreased while titratable acidity increased during fermentation, along with associated variation in biochemical profiles. Overall, the levels of organic acids (i.e., lactic and acetic acid), free amino acids (i.e., alanine, lysine, and glutamic acid), and volatiles (i.e., alcohols, esters, aldehydes, and ketones) increased in mature Zhacai. In addition, the abundances of Lactobacillus-related species, Halomonas spp., Idiomarina loihiensis, as well as that of the yeast Debaryomyces hansenii, were strongly correlated with increased concentrations of organic acids, amino acids, biogenic amines, and volatiles. This study provides new detailed insights into the succession of microbial communities and their potential roles in Zhacai fermentation.

Multi-omic detection of Mycobacterium leprae in archaeological human dental calculus.

Mineralized dental plaque (calculus) has proven to be an excellent source of ancient biomolecules. Here we present a Mycobacterium leprae genome (6.6-fold), the causative agent of leprosy, recovered via shotgun sequencing of sixteenth-century human dental calculus from an individual from Trondheim, Norway. When phylogenetically placed, this genome falls in branch 3I among the diversity of other contemporary ancient strains from Northern Europe. Moreover, ancient mycobacterial peptides were retrieved via mass spectrometry-based proteomics, further validating the presence of the pathogen. Mycobacterium leprae can readily be detected in the oral cavity and associated mucosal membranes, which likely contributed to it being incorporated into this individual's dental calculus. This individual showed some possible, but not definitive, evidence of skeletal lesions associated with early-stage leprosy. This study is the first known example of successful multi-omics retrieval of M. leprae from archaeological dental calculus. Furthermore, we offer new insights into dental calculus as an alternative sample source to bones or teeth for detecting and molecularly characterizing M. leprae in individuals from the archaeological record. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Leprosy in a low-incidence setting : Case report relevant to metagenomic next generation sequencing applications.

Leprosy is a disease caused by Mycobacterium leprae that results in disability. In 2000 the World Health Organization announced that leprosy had been eradicated. In nonendemic areas diagnosing leprosy is becoming a challenge for inexperienced clinicians. This case involves a male patient suffering from chronic numbness, hand deformity and recurrent erythema. Skin biopsy revealed granuloma and acid-fast staining of short-rod bacteria. Peripheral venous blood was subjected to metagenomic next generation sequencing and bioinformatics analysis, which revealed 3 unique sequence reads of M. leprae. Paraffin-embedded tissue and fresh samples scraped from skin lesions were subjected to in-house PCR targeting 16S rRNA, hsp65, rpoB, rpoT, ribF-rpsO, and mmaA. Sanger sequencing of amplicons from fresh samples and paraffin-embedded tissue verified the presence of M. leprae. For inexperienced clinicians in nonendemic areas nucleic acid amplification tests, such as in-house PCR, are helpful for diagnosing leprosy but sequence reads from metagenomic next generation sequencing may also provide evidence when interpreted cautiously.

Is there any still undisclosed biodiversity in Ciauscolo salami? A new glance into the microbiota of an artisan production as revealed by high-throughput sequencing.

Ciauscolo is a fermented sausage with the Protected Geographical Indication (PGI) status. To disclose the microbial ecology of a model Ciauscolo salami manufacture during its natural fermentation, viable counting, amplicon-based sequencing and real-time PCR were applied. The volatilome during fermentation was also characterized. The results allowed previously undetected species to be discovered. The core microbiota was composed by Lactobacillus algidus, Leuconostoc carnosum, Lactobacillus sakei, Debaryomyces hansenii, Glomus hyderabadensis, Tilletiopsis washingtonensis, and Kurtzmaniella zeylanoides. Salmonella spp. and Listeria monocytogenes were absent in all the samples; moreover, multiplex real-time PCR revealed the absence of the target genes bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP), encoding botulinic toxins. Volatilome, deeply depending on microbiological metabolism, was characterized by spices-derived components. Limonene, sabinene, α- and ß-pinene, 3-carene, and α-thujene were the most represented monoterpene hydrocarbons, whereas ß- and α-copaene were the most represented sesquiterpene hydrocarbons. Allyl methyl sulphide and diallyl disulphide were the major aliphatic sulphur compounds, together with diallyl sulphide and allyl methyl disulphide.

Into the wild: new yeast genomes from natural environments and new tools for their analysis.

Genomic studies of yeasts from the wild have increased considerably in the past few years. This revolution has been fueled by advances in high-throughput sequencing technologies and a better understanding of yeast ecology and phylogeography, especially for biotechnologically important species. The present review aims to first introduce new bioinformatic tools available for the generation and analysis of yeast genomes. We also assess the accumulated genomic data of wild isolates of industrially relevant species, such as Saccharomyces spp., which provide unique opportunities to further investigate the domestication processes associated with the fermentation industry and opportunistic pathogenesis. The availability of genome sequences of other less conventional yeasts obtained from the wild has also increased substantially, including representatives of the phyla Ascomycota (e.g. Hanseniaspora) and Basidiomycota (e.g. Phaffia). Here, we review salient examples of both fundamental and applied research that demonstrate the importance of continuing to sequence and analyze genomes of wild yeasts.

Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens.

The last century has witnessed progress in the study of ancient infectious disease from purely medical descriptions of past ailments to dynamic interpretations of past population health that draw upon multiple perspectives. The recent adoption of high-throughput DNA sequencing has led to an expanded understanding of pathogen presence, evolution, and ecology across the globe. This genomic revolution has led to the identification of disease-causing microbes in both expected and unexpected contexts, while also providing for the genomic characterization of ancient pathogens previously believed to be unattainable by available methods. In this review we explore the development of DNA-based ancient pathogen research, the specialized methods and tools that have emerged to authenticate and explore infectious disease of the past, and the unique challenges that persist in molecular paleopathology. We offer guidelines to mitigate the impact of these challenges, which will allow for more reliable interpretations of data in this rapidly evolving field of investigation.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts.

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85-23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.

Missense Variants in HIF1A and LACC1 Contribute to Leprosy Risk in Han Chinese.

Genome-wide association studies (GWASs) and genome-wide linkage studies (GWLSs) have identified numerous risk genes affecting the susceptibility to leprosy. However, most of the reported GWAS hits are noncoding variants and account for only part of the estimated heritability for this disease. In order to identify additional risk genes and map the potentially functional variants within the GWAS loci, we performed a three-stage study combining whole-exome sequencing (WES; discovery stage), targeted next-generation sequencing (NGS; screening stage), and refined validation of risk missense variants in 1,433 individuals with leprosy and 1,625 healthy control individuals from Yunnan Province, Southwest China. We identified and validated a rare damaging variant, rs142179458 (c.1045G>A [p.Asp349Asn]) in HIF1A, as contributing to leprosy risk (p = 4.95 × 10-9, odds ratio [OR] = 2.266). We were able to show that affected individuals harboring the risk allele presented with multibacillary leprosy at an earlier age (p = 0.025). We also confirmed the association between missense variant rs3764147 (c.760A>G [p.Ile254Val]) in the GWAS hit LACC1 (formerly C13orf31) and leprosy (p = 6.11 × 10-18, OR = 1.605). By using the population attributable fraction, we have shown that HIF1A and LACC1 are the major genes with missense variants contributing to leprosy risk in our study groups. Consistently, mRNA expression levels of both HIF1A and LACC1 were upregulated in the skin lesions of individuals with leprosy and in Mycobacterium leprae-stimulated cells, indicating an active role of HIF1A and LACC1 in leprosy pathogenesis.

Amino Acid Variants of HLA-DRB1 Confer Susceptibility to Dapsone Hypersensitivity Syndrome in Addition to HLA-B*13:01.

Dapsone hypersensitivity syndrome is a rare yet severe adverse drug reaction caused by dapsone, a principal drug in multidrug therapy for leprosy. HLA-B*13:01 has been identified as a strong risk factor of dapsone hypersensitivity syndrome; however, its low positive predictive value indicated that additional genetic variants may be involved in the disease development. To discover contributing genetic variants within HLA loci in addition to HLA-B*13:01, we performed a high-coverage next-generation sequencing (NGS)-based HLA typing analysis in 103 dapsone-hypersensitive and 857 dapsone-tolerant HLA-B*13:01-positive leprosy patients in a Chinese population. Five amino acid variants in high linkage disequilibrium of HLA-DRB1 were significantly associated with dapsone hypersensitivity syndrome (positions 133, 142, -17, 11, and 13). DRB1*16:02 and DRB1*15:01 tagged by these risk-conferring amino acid residues were associated at a nominal significance level. This study identifies five amino acid variants within HLA-DRB1 that are in high linkage disequilibrium and significantly associated with dapsone hypersensitivity syndrome in a Chinese population.

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.

Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases.

BACKGROUND: Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. METHODOLOGY: DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. PRINCIPAL FINDINGS: In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. CONCLUSIONS/SIGNIFICANCE: This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

Common variants in the PARL and PINK1 genes increase the risk to leprosy in Han Chinese from South China.

Leprosy is a chronic infectious and neurological disease caused by Mycobacterium leprae, an unculturable pathogen with massive genomic decay and dependence on host metabolism. We hypothesized that mitochondrial genes PARL and PINK1 would confer risk to leprosy. Thirteen tag SNPs of PARL and PINK1 were analyzed in 3620 individuals with or without leprosy from China. We also sequenced the entire exons of PARL, PINK1 and PARK2 in 80 patients with a family history of leprosy by using the next generation sequencing technology (NGS). We found that PARL SNP rs12631031 conferred a risk to leprosy (Padjusted = 0.019) and multibacillary leprosy (MB, Padjusted = 0.020) at the allelic level. rs12631031 and rs7653061 in PARL were associated with leprosy and MB (dominant model, Padjusted < 0.05) at the genotypic level. PINK1 SNP rs4704 was associated with leprosy at the genotypic level (Padjusted = 0.004). We confirmed that common variants in PARL and PINK1 were associated with leprosy in patients underwent NGS. Furthermore, PARL and PINK1 could physically interact with each other and were involved in the highly connected network formed by reported leprosy susceptibility genes. Together, our results showed that PARL and PINK1 genetic variants are associated with leprosy.