Implementation of genetic screening test to reduce the incidence of dapsone hypersensitivity syndrome among patients with leprosy in Papua, Indonesia: a study protocol.

INTRODUCTION: The mainstay of leprosy treatment is multidrug treatment (MDT), which contains rifampicin, dapsone and clofazimine. The occurrence of dapsone hypersensitivity syndrome (DHS), a sudden, potentially fatal and traumatic adverse reaction due to dapsone, may affect treatment adherence and may result in fatality if untreated. Before MDT administration, screening for HLA-B*13:01 in patients with leprosy can potentially reduce DHS risk. The study aims to assess the effectiveness of using a screening test for HLA-B*13:01 in reducing the incidence of DHS and to evaluate the feasibility of using the quantitative PCR-based screening tool as DHS predictors before dapsone administration using individual patient testing in a referral centralised-lab model. METHODS AND ANALYSIS: A total of 310 newly diagnosed patients with leprosy will be recruited from health centres in two highly endemic districts in Indonesia. Dried blood will be taken on filter paper as the specimen receptacle to collect DNA from the patients and transported at room temperature to the leprosy referral laboratory before MDT administration. Checking for HLA-B*13:01 from human DNA is performed using the Nala PGx 1301 V.1 kit. The results will be shared with the leprosy health workers on the site via phone call and courier. Patients with a positive test result will be treated with MDT without dapsone, and patients with a negative result will be treated with complete MDT. Physical examination (weight, height, skin, muscle and nerve function examination), complete blood tests (including renal function test) will be carried out at baseline. Follow-up will be performed at the fourth and eighth weeks to observe any development of adverse drug reactions. ETHICS AND DISSEMINATION: The ethical approval for the study was issued by the Ethical Committee of the National Institute of Health Research and Development, Ministry of Health, Indonesia. Written informed consent will be sought from all participants.

Approach to inherited hypertrichosis: A brief review.

Hypertrichosis refers to the growth of hair, of an excessive amount and thickness, on any part of the body. It must be distinguished from hirsutism which is characterized by excess growth of hair in androgen-dependent areas on the upper lip, chin, chest, linea alba, thigh and axilla. Hypertrichosis may be localized or generalized, and congenital or acquired. Excess hair growth has a psychological impact on the child as well as the parents due to the cosmetic disfigurement it produces. Current treatment options are limited and not wholly satisfactory. Treatment should be customized according to the area, nature and amount of hair growth, age of the patient and personal preferences. In addition, when hypertrichosis occurs as a component of a syndrome, multidisciplinary management is required to address the associated systemic features. A detailed review of inherited generalized hypertrichosis is presented here with emphasis on clinical clues to identifying complex syndromes with multisystem involvement.

Prediction of leprosy in the Chinese population based on a weighted genetic risk score.

Genome wide association studies (GWASs) have revealed multiple genetic variants associated with leprosy in the Chinese population. The aim of our study was to utilize the genetic variants to construct a risk prediction model through a weighted genetic risk score (GRS) in a Chinese set and to further assess the performance of the model in identifying higher-risk contact individuals in an independent set. The highest prediction accuracy, with an area under the curve (AUC) of 0.743 (95% confidence interval (CI): 0.729-0.757), was achieved with a GRS encompassing 25 GWAS variants in a discovery set that included 2,144 people affected by leprosy and 2,671 controls. Individuals in the high-risk group, based on genetic factors (GRS > 28.06), have a 24.65 higher odds ratio (OR) for developing leprosy relative to those in the low-risk group (GRS≤18.17). The model was then applied to a validation set consisting of 1,385 people affected by leprosy and 7,541 individuals in contact with leprosy, which yielded a discriminatory ability with an AUC of 0.707 (95% CI: 0.691-0.723). When a GRS cut-off value of 22.38 was selected with the optimal sensitivity and specificity, it was found that 39.31% of high risk contact individuals should be screened in order to detect leprosy in 64.9% of those people affected by leprosy. In summary, we developed and validated a risk model for the prediction of leprosy that showed good discrimination capabilities, which may help physicians in the identification of patients coming into contact with leprosy and are at a higher-risk of developing this condition.

A Review on Dapsone Hypersensitivity Syndrome Among Chinese Patients with an Emphasis on Preventing Adverse Drug Reactions with Genetic Testing.

AbstractDapsone is a bactericidal and bacteriostatic against Mycobacterium leprae, a causative agent of leprosy. Dapsone is also applied in a range of medical fields because of its anti-inflammatory and immunomodulatory effects. Dapsone hypersensitivity syndrome (DHS) is a rare yet serious adverse drug reaction (ADR) caused by dapsone involving multiple organs. We performed a systematic review of published articles describing dapsone-induced hypersensitivity syndrome, including all Chinese articles and the latest literature available in online databases published between October 2009 and October 2015. We determined the prevalence, clinical characteristics, and mortality rate of DHS. Importantly, we also summarized the recent advances in genetic testing allowing prediction of ADRs. In an initial systematic electronic search, we retrieved 191 articles. Subsequently, these articles were further filtered and ultimately 84 articles (60 Chinese case reports, 21 non-Chinese articles, and three epidemiological studies) were selected, which included 877 patients. The prevalence of DHS among Chinese patients was 1.5% with a fatality rate of 9.6%. Early withdrawal of dapsone and appropriate treatment reduced the fatality rate. Most importantly, genetic screening for the HLA-B*13:01 allele among high-risk populations showed a significant utility as a useful genetic marker to DHS. In conclusion, this review discusses the epidemiological and clinical characteristics of DHS among Chinese patients, which may help physicians to understand this syndrome.

Occurrence of FFZ genes in yeasts and correlation with fructophilic behaviour.

Fructophily has been described in yeasts as the ability to utilize fructose preferentially when fructose and glucose are available in the environment. In Zygosaccharomyces bailii and Zygosaccharomyces rouxii, fructophilic behaviour has been associated with the presence of a particular type of high-capacity and low-affinity fructose transporters designated Ffz. In this study, a PCR screening was performed in several yeasts using degenerate primers suitable to detect FFZ-like genes. In parallel, fructophilic character was evaluated in the same strains by comparing the relative consumption rate of fructose and glucose. For all the strains in which FFZ-like genes were detected, fructophilic behaviour was observed (25 strains). Results show that FFZ genes are ubiquitous in the Zygosaccharomyces and Starmerella clades. Strains of Lachancea fermentati, Torulaspora microellipsoides and Zygotorulaspora florentina were not fructophilic and did not harbour FFZ genes. It is of note that these new species were recently removed by taxonomists from the Zygosaccharomyces clade, supporting the view that the presence of FFZ-like genes is a main characteristic of Zygosaccharomyces. Among the strains tested, only Hanseniaspora guilliermondii NCYC2380 was an exception, having a preference for fructose in medium with high sugar concentrations, despite no FFZ-like genes being detected in the screening. Furthermore, this study supports the previous idea of the emergence of a new family of hexose transporters (Ffz facilitators) distinct from the Sugar Porter family.

Parallel detection of ancient pathogens via array-based DNA capture.

DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone.

Lack of effects of the TNF-alpha and IL-10 gene polymorphisms in Mexican patients with lepromatous leprosy.

Several human genetic variants have been associated with susceptibility or resistance to leprosy. The aim of this study was to assess whether gene polymorphisms of -308 G/A TNF-alpha and -819 T/C IL-10 are associated with lepromatous leprosy in Mexican mestizos patients from northwest Mexico. We genotyped these polymorphisms by means of polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) in 68 patients with lepromatous leprosy and 144 healthy Mexican Mestizos controls. We found that the -308G TNF-alpha allele was predominant in both cases (94.3%) and controls (92.3%) without statistical significance and the frequencies of -819C IL-10 allele were also similar for the cases (56.0%) and controls (59.0%). These negative findings suggest that other genes or polymorphisms may be important in the susceptibility to leprosy infection in the Mexican mestizos.

Absence of nucleotide-binding oligomerization domain-containing protein 2 variants in patients with leprosy and tuberculosis.

Crohn's disease-associated NOD 2 variants (Arg702Trp and 3020insC) were found to be monomorphic (wild), and 7 subjects were heterozygous for Gly908Arg SNP in 263 patients with tuberculosis, 260 patients with leprosy and 270 healthy controls residing in northern Indian states. This is the first report to suggest the minimal role of these variants in susceptibility/resistance to TB and leprosy in this population.

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.

Molecular diagnostics in genodermatoses - simplified.

The field of genetics in dermatology has progressed at an astonishing rate. Most of the known single gene disorders have at least been mapped to a particular chromosomal region and the causative genes have been identified and studied in many of them. However, most research work in genetics relating to genodermatoses has been confined to the western population. Very few reports, if any, have been published from Indian studies. A first step may be to develop a registry to link most of these cases providing a full description of the clinical phenotype. We would next need to attempt genetic analysis of these conditions thereby detecting any novel mutations in known and unknown genes different from the western population. This would help in designing indigenous assays appropriate to the Indian population. The review describes various techniques used in a molecular biology/ genetics laboratory with special focus on polymerase chain reaction (PCR), gene sequencing, genotyping and DNA micro arrays. Gene identification strategies have also been described with appropriate examples in dermatology.

Ninjurin 1 asp110ala single nucleotide polymorphism is associated with protection in leprosy nerve damage.

Leprosy is the major cause of non-traumatic neuropathy. Herein, we investigated the role of ninjurin 1, an adhesion molecule involved in nerve regeneration in leprosy. Our results demonstrated that M. leprae stimulates in vitro up-regulation of ninjurin mRNA in cultured Schwann and blood cells as well as in vivo mRNA and protein expression in leprosy nerve biopsies. A polymorphism (asp110ala) was investigated in a case-control study (1123 individuals) and no association was found with leprosy per se or with disseminated forms. Nevertheless, ala110 was associated with functional nerve impairment (OR=2.42; p=0.02 for ala/ala) and with lower mRNA levels. Our data suggests that asp110ala could be a valuable genetic marker of nerve damage in leprosy.

Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians.

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 x 10(-5); D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

Purification of Mycobacterium leprae RNA for gene expression analysis from leprosy biopsy specimens.

Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.

Purification of Mycobacterium leprae RNA for gene expression analysis from leprosy biopsy specimens

Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.

Pathogenesis of nerve damage in leprosy: genetic polymorphism regulates the production of TNF alpha.

Studies carried out over the last decade have strongly suggested that TNF alpha both overtly participates in the cell-mediated immune response against Mycobacterium leprae, and is overproduced during reaction. In addition, reactions are intimately related to the onset of nerve damage. Finally, TNF alpha has been implicated in the pathogenesis of many human and experimental autoimmune peripheral neuropathies that, as in leprosy, result in demyelination and axonal lesions. Because of recent findings associating human TNF alpha mutant alleles at the -308 position with increased production of TNF alpha in many immunological and infectious diseases, an investigation of the role of TNF2 in predisposing leprosy patients to reaction has been undertaken. Analysis of 300 patients with leprosy--210 multibacillary and 90 paucibacillary--has shown that the percentage of reactional patients was similar among both carriers and non-carriers of the TNF2 allele. However, a separate analysis of 57 carriers of TNF2 found that reactions occurred much more frequently among heterozygous than among homozygous patients. Moreover, the frequency of neuritis was somewhat greater among the heterozygous patients than among the non-carriers. Enhanced serum levels of TNF alpha have been noted in both TNF-1 and TNF-2 mutant patients in the course of leprosy reaction. Our observations to date suggest that other factors not related to the presence of the mutant gene may lead to the TNF alpha hyper-responsiveness observed during reaction.