Ninjurin 1 gene asp110ala genetic variants as a susceptibility factor in nerve damage leprosy patients of India

Accepted Manuscript Ninjurin 1 gene asp110ala genetic variants as a susceptibility factor in nerve damage leprosy patients of south India

Arun Sundaramoorthy, Kavitha Hemachandran, Chandirasekar Ramachandran, Sasikala Keshavarao, Calistus Jude AL, Vimala Karuppaiya, Abilash Valsala Gopalakrishnan PII: DOI: Reference:

S2214-5400(16)30099-8 doi: 10.1016/j.mgene.2016.12.012 MGENE 289

To appear in:

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Received date: Revised date: Accepted date:

23 August 2016 12 November 2016 29 December 2016

Please cite this article as: Arun Sundaramoorthy, Kavitha Hemachandran, Chandirasekar Ramachandran, Sasikala Keshavarao, Calistus Jude AL, Vimala Karuppaiya, Abilash Valsala Gopalakrishnan , Ninjurin 1 gene asp110ala genetic variants as a susceptibility factor in nerve damage leprosy patients of south India. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mgene(2016), doi: 10.1016/j.mgene.2016.12.012

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ACCEPTED MANUSCRIPT Ninjurin 1 gene asp110ala genetic variants as a susceptibility factor in nerve damage leprosy patients of south India Arun Sundaramoorthya,*, Kavitha Hemachandranb, Chandirasekar Ramachandranc, Sasikala Keshavaraoa, Calistus Jude ALd Vimala Karuppaiyae, Abilash Valsala

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Human Molecular Genetics Laboratory, Department of Zoology, Bharathiar University,

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a

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Gopalakrishnanf

Coimbatore-641 046, Tamil Nadu, India

Department of Zoology, Kongunadu Arts and Science College, Coimbatore-641 029,

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b

Department of Zoology,Vasavi Arts and Science College, Erode, Tamil Nadu, India

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c

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India

Department of Biotechnology, School of Science, Kristu Jayanthi College, K.

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Narayanapura, Kothanur (Post), Bangalore - 560077, Karnataka, India e

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Tamil Nadu, India

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Department of Zoology, School of Life Sciences, Periyar University, Salem-636 011,

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Division of Bio Molecules and Genetics, School of Bio Science and Technology, VIT-

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University, Vellore-632 014, Tamil Nadu, India Corresponding author: S. Arun; e-mail address: [email protected] ABSTRACT Background: Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae, the obligate intracellular bacillus that attacks cutaneous tissue and can damage the pheripheral nervous system. 1

ACCEPTED MANUSCRIPT Aim: To determine the association between the Ninjurin 1 gene asp110ala (rs2275848) polymorphism in nerve damage leprosy patients. Meterials and Methods: We carried the 234 leprosy patients along with equal number of controls with age and gender-matched were recruited. Genotyping was done by

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polymerase chain reaction/restriction fragment length polymorphism and confirmation of

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sequence.

Results: The CC genotype (ala/ala) had a higher risk of developing nerve disability when

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compared those carrying the AA genotype (asp/asp) and the variation observed were

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statistically significant at P<0.05.

Conclusion: The Ninjurin 1 gene asp110ala genetic variation may be a risk of nerve

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damage among leprosy patients in south India.

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Abbreviations

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Ninj1: ninjurin 1; DG: disability grade; PCR: polymerase chain reaction; RFLP:

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restriction fragment length polymorphism; PB: paucibacillary; MB: multibacillary; UN:

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ulnar; WHO: World Health Organization Keywords: leprosy, Ninjurin 1 gene, asp110ala polymorphism, disability evaluation, disability grade

1. INTRODUCTION Leprosy is a chronic infectious disease an obligate intracellular pathogen of Mycobacterium leprae that principally damage macrophages, Schwann cells and peripheral nerves cells. The World Health Organization (WHO) classification of Borderline 2

ACCEPTED MANUSCRIPT Lepromatous (BL) and Lepromatous Leprosy (LL) individuals represented as multibacillary (MB) patients, whereas the Borderline Tuberculoid (BT) and Tuberculoid leprosy (TT) individuals represented the paucibacillary (PB) patients. Reactions in leprosy are as a result of spontaneous fluctuations in the clinical state due to the dynamic nature of the immune

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response to M. leprae (Britton et al., 2004). Leprosy diagnosis was determined and

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followed by the Ridley and Jopling classification (Ridley and Jopling, 1966). The

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diagnosis and treatment of leprosy is easy and most endemic countries are striving to fully integrate leprosy services into existing general health proplem. In the official reports

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received from 103 countries and territories, the global registered prevalence of leprosy at

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the end of the first quarter of 2014 stood at 180,464 cases, while the number of new cases detected during 2013 was 215,557 small numbers of cases were excesting in Europe

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(WHO, 2013). In India totally 127,295 cases were reported across the country in 2011-

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12. According to the Ministry of Health and Family Welfare in Uttar Pradesh recorded the highest number of cases at 24,627 in 2011-12, followed by 17,892 in Maharashtra and

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17,801 in Bihar (The Hindu business line, 2012).

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Genes play on important role in this disease in particularly immune genes is a vital

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responsibility in a number of biological activities including proliferation, development, homeostasis, regeneration, repair and inflammation (Okada and Murakam, 1998). Genetic influence is a main role on the disease-associated mortality in particular infectious diseases has long been observed since the 1980s (Sorensen et al., 1988). On exposure to M. leprae, a strong innate immune response may help prevent the disease to establish itself in the host. In addition, the insufficient innate immune response may allow for an infection and thus be a determining genetic factor (Scollard et al., 2006). Family based studies, segregation

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ACCEPTED MANUSCRIPT analyses and twin studies are provide evidence that, in addition to environmental and exposure factors and host genetic factors influence to leprosy susceptibility (da Silva et al., 2009). The candidate genes, single nucleotide polymorphisms (SNPs) may be important factor in leprosy susceptibility and outcome of the disease severity (Moraes et

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al., 2004). The genetic evidence studies in Indian leprosy patients TAP1, LRRK2 and

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RIPK2 immune gene variant as a susceptibility factor (Patrick Marcinek et al., 2013;

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Vidyagouri Shinde et al., 2013)

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The Ninjurin 1 gene, which encodes the protein, is located in chromosome long arm of 9q22. In the earlier results are controversial, and the association between this

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SNPs and leprosy still needs further investigation of the Ninjurin 1 gene (Moraes et al.,

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2006). The earlier Indian leprosy patients study comprised of 303 new multibacillary (MB) leprosy patients who were recruited to risk factors for the reactions and nerve

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damage in leprosy (van Brakel et al., 2005). Leprosy patient when present the grade

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disability is one of the key for clinical parameters that indicate nerve impairment (Pimentel et al., 2004). It has been suggested that the Ninjurin 1 gene, could play a role in

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nerve regeneration since it is upregulated during nerve damage, promoting neurite

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outgrowth (Araki and Milbrandt, 1996; Kubo et al., 2002). The aim of this study was to evaluate the importance of Ninjurin 1 gene single nucleotide polymorphism (SNPs) on leprosy susceptibility and association of nerve damage, either PB or MB, case and controls of south Indian leprosy patients. 2. MATERIALS AND METHODS 2.1. Subject recruitment

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ACCEPTED MANUSCRIPT The recruited 234 patients, 164 Males and 70 Females with the average age of 43.7±24.45 were included in this study. All the blood samples were collected from Sacred heart leprosy hospital, Kumbakkonam, Tamil Nadu and equal number of controls were collected. The WHO classified the leprosy LL, BL and BB individuals represent the

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multibacillary group [n=154, Male/Female ratio=118/36, Mean age=46.4±26.1 years],

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whereas, BT, TT and I individuals represented the paucibacillary group [n=80,

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Male/Female ratio=46/34, Mean age=41±22.8 years]. The controls Male/Female ratio=153/81, age: 40.1±20.1 years were collected at same. Informed written consent was

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obtained from all subjects throuth questioner were collected [family history, leprosy types

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and other related diseases]. The 3 ml of blood samples were collected from leprosy patients and healthy controls using the K3 EDTA as anticoagulant tube stored at -20 oC

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until DNA isolation. This study was approved by the institutional human ethical

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committee at bharathiar university, coimbatore-641 046, Tamil nadu, India.

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2.2. Genomic DNA Extraction

The genomic DNA was extracted from human blood utilizing the Salt out method

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(Miller et al., 1998). All blood samples were processed within a week time. In the

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extracted DNA were diluted to working concentrations 50 ng/ml. The isolated DNA was stored at -20 °C.

2.3. Amplification of Ninjurin1 gene The

PCR

was

performed

with

the

following

primer:

forward

5′-

GCGACTGAGAGCCCTGTAATG -3′ and reverse 5′-AGCGAGCAACCCCAGGACT3′. The 10X PCR Master Mix: 18 µl (PCR Emerald Amp GT), Forward Primer: 0.15 µl/2pm, Reverse Primer: 0.15 µl/2pm, Nuclease free water: 4.7 µl and Template DNA: 5

ACCEPTED MANUSCRIPT 2µl/50 ng. All the necessary components were added to the PCR tube. It was gently vortexed and centrifuged to collect all the drops to the bottom then followed the thermal cycle program. Initial Denaturation: 95 °C-5 Minutes for 1 cylcle, Denaturation: 95 °C-30 seconds, Annealing: 55 °C-45 seconds, Extension: 72 °C-1.5 Minutes for 30 cylcle, Final

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2.4. Restriction fragment length polymorphism (RFLP)

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extension: 72 °C-10 Minutes and Holding at 4 °C-10 Minutes.

Restriction digestion was performed using Hae-III restriction enzyme and

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followed the components of 10X HiBuffer H4: 5 µl, Hae III Enzyme: 1 U, DNA Concentration: 1 µg (1µl), Nuclease free water: 18 µl and total volume 25 µl. The 339bp

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amplified product was digested. Reactions were incubated overnight at 37 °C and

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digested fragments were separated in 2% ethidium bromide stained agarose gels. A HaeIII cleavage site is generated when the C allele (asp110) is present, in way that the

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genotypes were easily ascertained by the size of the fractionated fragments (339bp for the A

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allele; 150+189bp for the C allele).

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2.5. Polymorphic sites were confirmed using sequencing analysis The 1 µl of the purified products were sequenced using the Big Dye terminator v.

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1.1 cycle sequencing kit (Applied Biosystems, USA) and ABI 3130XL and ABI 3730 DNA sequencer, according to the manufacturer’s instructions (Sanger et al., 1977). 2.9. STATISTICAL ANALYSIS The allele frequencies and genotype distributions of SNPs into patients versus controls; MB versus PB and DG=0 versus DG>0 were carried out and Chi-square tested by the software Minitab (Version 15). Odds ratios and 95% CI adjusted. The statistically

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ACCEPTED MANUSCRIPT significant was followed by p<0.05. In addition, different genetic models were obtained form the genotype vs. heterozygous+homozygous. 3. RESULTS The asp110ala SNPs versus leprosy neural damage, measured herein by the disability

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grade (DG), were evaluated in leprosy patients in Table 1. The immune gene asp110ala

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region were amplified the PCR product and hae III restriction digestion products were run at 2% agarose gel electrophoresis then observed the genotypic allele variants. The

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genotypic frequencies of asp110ala SNPs were initially compared between leprosy patients and the control groups, using the OR (95% CI) Z-P-Value in C-Allele frequency

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0.03 and C Allele carrier χ2 (P) in 0.04 statistically significant differences was observed

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the nerve damage leprosy patients. Also, no differences were observed in the frequency of asp110ala SNPs between MB and PB groups displayed in Table 2. Exhibited

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chromatograms of leprosy patients Ninjurin 1 gene, exon 4 asp110ala variations

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compared with the controls groups.

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The frequency of allele AA (asp110) was 0.54 % in patients with disability grade (DG>0) and 0.52 % in patients absence of disability grade (DG=0). The allele CC

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(ala110) was 0.17 % in patients DG>0 and 0.08 % in DG=0 in controls. The allele frequencies of CC (ala110) was found at increased frequency in leprosy patients with sensory or motor nerve impairment (DG>0) compared with patients showing no disability due to (DG=0) CC carrier of leprosy patients. In addition, leprosy patients with the CC polymorphic genotype (ala/ala) had a higher risk (OR=2.79), OR (95% CI) Z, P<0.02 of developing nerve disability when compared to grade disability. In spite of the loss of allele dose effect, results confirmed the association of asp110ala to DG development 7

ACCEPTED MANUSCRIPT (P<0.05) and ala110 CC carriers as the risk group OR 0.85 (95% CI) in Table 3. Indicating that genetic association of Ninjurin 1 acts as an independent in nerve damaged in leprosy. These results strongly suggest an association between the ala110 allele and development of nerve damage in leprosy patients.

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4. DISCUSSION

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Leprosy is a chronic infectious disease of the skin and nerves. Although decreased the prevalence in last two decades, the number of new case detection rates remains high

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as far as in 130 countries, with India contributing to half of the new cases detected worldwide (Wkly Epidemiol Rec, 2011). Host immune responses (Monot et al., 2009)

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and genetic factors had been shown to influence the clinical spectrum of leprosy (Zhang

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et al., 2009; Vannberg et al., 2011). The earlirer study, susceptibility locus was having been mapped using linkage analysis of case-control studies on south Indian leprosy patient

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chromosomes in 10p13 and 20p12 (Ruby Siddiqui et al., 2001; Kerrie Tosh et al., 2002). The

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association study for HLA-DRB1 alleles with susceptibility to leprosy, the MICA and MICB genes variation were identified and stronger effect in south Indian population, the gene is

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9q22 chromosomal region, which had been previously linked to hereditary

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sensoryneuropathies type I and type II (Tosh et al., 2006), although no clear association with NINJ1 as been confirmed as of yet (Mandich et al., 1999). Hence, set out to assess the function of the adhesion molecule Ninjurin 1 in the interaction of M. leprae with the human host. The genetics factors leading to different immune responses is clearly observed for leprosy susceptibility, as well as for its clinical course and type of disease; moreover, specific molecular variants could be important to define increased individual risk for 8

ACCEPTED MANUSCRIPT disease after exposition to M. leprae (Cardoso et al., 2007). A non synonymous A/C transversion in its third exon results on amino acid exchanged in asp110ala the protein position (Graca et al., 2012; Lee et al., 2010). The ninjurin protein is transported down the length of the axon to the site of injury, where it accumulates (Lee et al., 2010). In the

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present study was to evalute the risk of Ninjurin 1 gene with nerve damage leprosy

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patients. The studies find out statistically significant association for the DG leprosy

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patients. Leprosy with C allele (ala110) was more susceptible to develop nerve damage, assessed by DG. Therefore, polymorphism in this gene could be related to nerve damage

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in leprosy patients.

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Globally the study is third to associate polymorphism in the Ninjurin 1 gene among the

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leprosy nerve damage. The first and second study reported this polymorphism and its relationship with protection in leprosy nerve damage (Cardoso et al., 2007; Graca et al.,

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2012). They also found a significant association between the C allele and nerve damage in

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leprosy patients. To our knowledge, this study is the first report to associate polymorphisms in the Ninjurin 1 gene with leprosy nerve damage in Tamil Nadu, South

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India. The south Indian leprosy patients Ninurin 1 gene asp110ala sequence variant was

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accepted for the genbank (id-kp256370). As genetic variants of the Ninjurin induced by the nerve injury in leprosy and have a strong role for neuronal recovery by increasing cell adhesion and Schwann cells regeneration (Graca et al., 2012). The ninjurin gene plays a role in nerve regeneration and in the formation and function of other tissues, ans the gene novel adhesion molecule, is induced by the nerve injury (Araki et al., 1996). These studies indicate that the Ninjurin 1 is an important adhesion molecule and could also be participating in many neurodegenerative diseases. The results of this present investigation

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ACCEPTED MANUSCRIPT strengthen previous findings (Graca et al., 2012) on this polymorphism association with nerve damage in leprosy patients. In addition, in present findings Ninj1 gene as carriers of the CC genotype (ala/ala) had a higher risk (ORs) of developing disability. Together, these data encourage future efforts towards the establishment of a Ninj1 asp110ala

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genotyping as a prognostic marker of nerve damage.

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5. CONCLUSION

The investigation was findouted in association between the C allele (ala110) and

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nerve damage in leprosy patients. Moreover, the study population, carriers of the CC genotype (ala/ala) had a higher risk (ORs) of developing disability. In the polymorphism,

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which is easily determined by minimally invasive methods, as a genetic marker, opens new

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perspectives on the approaches to be used in the clinic and the possibility of identifying patients at high risk for the development of more aggressive disease compared with controls.

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6. ACKNOWLEDGEMENT

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This study was partially supported by the University Grant Commission (UGC), Government of India, file no.F.4-1/2006 (BSR)/7-24/2007 (BSR). The Department of

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Science and Technology (DST), Government of India, supported for presented the

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research findings in ESPID Conference-2014, official memorandum no: SB/ITSY/0495/2014-15.

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ACCEPTED MANUSCRIPT WHO, Weekly epidemiological record, Releve epidemiologique hebdomadaire., 2013. No. 35 (88), 365–380. The Hindu, Business Line, http://www.thehindubusinessline.com/news/india-topsinnumber-of-leprosy-cases, 2012.

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Okada, H., Murakam, S., 1998. Cytokine expression in periodontal health and disease.

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Scollard, D.M., Adams, L.B., Gillis, T.P., Krahenbuhl, J.L., Truman, R.W., Williams, D.L., 2006. The Continuing Challenges of Leprosy. Clinical Microbiology Reviews.

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Moraes, M.O., Pacheco, A.G., Schonkeren, J.J., Vanderborght, P.R., Nery, J.A., 2004. Santos, A.R., Interleukin-10 promoter single-nucleotide polymorphisms as markers

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ACCEPTED MANUSCRIPT Vidyagouri Shinde., Patrick Marcinek., Deepa Selvi Rani., Sharada Ramaseri Sunder., Sundaramoorthy Arun et al., 2013. Genetic evidence of TAP1 gene variant as a susceptibility factor inIndian leprosy patients. Human Immu. 74, 803–807. Moraes, M.O., Cardoso, C.C., Vanderborght, P.R., Pacheco, A.G., 2006. Genetics of host

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van Brakel, W.H., Nicholls, P.G., Das, L., Barkataki, P., Suneetha, S.K., 2005. The

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INFIR Cohort Study: investigating prediction, detection and pathogenesis of neuropathy and reactions in leprosy, methods and baseline results of a cohort of

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multibacillary leprosy patients in north India. Lepr. Rev. 76, 14–34.

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Pimentel, M.I., Nery, J.A., Borges, E., Goncalves, R.R., Sarno, E.N., 2004. Impairments in multibacillary leprosy; a study from Brazil. Lepr. Rev. 75, 143–152.

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Araki, T., Milbrandt, J., 1996. Ninjurin, a novel adhesion molecule, is induced by nerve

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ACCEPTED MANUSCRIPT Monot, M., Honore, N., Garnier, T., Zidane, N., Sherafi, D., Paniz-Mondolfi, A., Matsuoka, M., Taylor, G.M., Donoghue, H.D., et al., 2009. Comparative genomic and phylogeographic analysis of Mycobacterium leprae. Nat. Genet. 41, 1282–1289. Zhang, F.R., Huang, W., Chen, S.M., Sun, L.D., Liu, H. Li, Y., 2009. Genome wide

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Vannberg, F.O., Chapman, S.J., Hill, A.V., 2011. Human genetic susceptibility to

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leprosy in India maps to chromosome 10p13. Nature genetics. 27.

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Kerrie Tosh., Sarah Meisner., Ruby Siddiqui., et al., 2002. A Region of Chromosome 20 Is Linked to Leprosy Susceptibility in a South Indian Population. The Journal of Infectious

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Mandich, P., Bellone, E., Di Maria, E., Pigullo, S., Pizzuti, A., Schenone, A., Soriani, S., Varese, A., Windebank, A.J., Ajmar, F., 1999. Exclusion of the ninjurin gene as a

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candidate for hereditary sensory neuropathies type I and type II. Am. J. Med. Genet. 83, 409–410.

Cardoso, C.C., Martinez, A.N., Guimarães, P.E., Mendes, C.T., Pacheco, A.G., de liveira, R.B., Teles, R.M., Illarramendi, X., Sampaio, E.P., Sarno, E.N., Dias-Neto, E., Moraes, M.O., 2007. Ninjurin 1 asp110ala single nucleotide polymorphism is

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ACCEPTED MANUSCRIPT associated with protection in leprosy nerve damage. J. Neuroimmunol. 190, 131– 138. Graca, G.R., Paschoal, V.D., Cordeiro-Soubhia, R.M., Tonelli-Nardi, S.M., Machado, R.L., Kouyoumdjian, J.A., Baptista Rossit, A.R., 2012. NINJURIN1 single

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nucleotide polymorphism and nerve damage in lepros. Infect. Genet Evol. 12(3),

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Lee, H.J., Ahn, B.J., Shin, M.W., Choi, J.H., Kim, K.W., 2010. Ninjurin1 a potential adhesion molecule and its role in inflammation and tissue remodeling. Mol. Cells.

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ACCEPTED MANUSCRIPT Table 1: Preoperative characteristics of clinical classification and disability grade (DG) of leprosy patients

DG=0

DG>0

Total

56 (23.9)

24 (10.3)

80 (34.2)

Multibacillary

77 (32.9)

77 (32.9) 51 (21.8)

BL

31 (13.24)

154 (65.8)

BB

16 (06.8)

19 (8.1)

BT

30 (12.8)

08 (3.4)

TT

16 (06.8)

11 (4.7)

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26 (11.1)

05 (2.1)

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14 (5.9)

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07 (2.99)

82 (35.04)

23 (10.3)

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LL

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Paucibacillary

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(Ridley and Jopling)

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Clinical forms of classification

49 (20.9) 24 (10.3) 37 (15.8) 19 (8.1)

DG, disability grade; LL, lepromatous leprosy; BL, borderline lepromatous; BB,

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borderline; TT, tuberculoid leprosy; I, indeterminate

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ACCEPTED MANUSCRIPT Table 2: Genotype and allelic distribution among paucibacillary (PB), multibacillary (MB) leprosy patients and healthy controls OR (95% CI) Z-P-Value

χ2(P)

136 112 Reference (0.58) (0.48) 1.4(0.9457 68 (0.30) 2.23)1.68(0.09) (0.24) 54 (0.23) 1.10(0.6441 (0.2) 1.88)0.36(0.71)

45 (0.56) 23 (0.3) 12 (0.15)

91 (0.59) 35 (0.23) 28 (0.2)

OR (95% CI) Z-P-Value

Reference 0.75(0.39-1.42) 0.87(0.38) 1.53(0.65-3.61) 0.9(0.32)

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Allele frequency A C C Carriers

5.05 (0.07)

PB MB (n=80) (n=154)

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Controls (n=234)

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Genotype frequency AA AC CC

Patients (n=234)

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Subjects

χ2(P)

1.15 (0.5)

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313 294 Reference 113 217 Reference (0.69) (0.62) 1.34(1.02(0.71) (0.7) 1.03(0.67139 176 1.77)2.14(0.03)** 6.22 46 91 (0.3) 1.56)0.13(0.89) 0.05 (0.31) (0.38) 0.98(0.69(0.04)* (0.29) 63 0.93(0.54(0.97) 98 122 1.39)0.09(0.92) 34 (0.41) 1.61)0.24(0.81) (0.42) (0.52) (0.43) MB, multibacillary; PB, paucibacillary, OR, Odds ratio. CI: 95% Confidence interval.

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*Significantly different (Chi-square (χ2) value) with compared to the controls.

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** Significant different at p<0.05 level (Z statistic p value).

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ACCEPTED MANUSCRIPT Table 3: Frequency of asp110ala polymorphism in leprosy patients according to

OR(95% CI)Z(P)

72(0.54) 51 (0.38) 10 (0.08)

53 (0.52) 31 (0.31) 17 (0.17)

Reference 0.82(0.46-1.46)0.62(0.51) 2.79(1.13-6.87)2.24(0.02)** 5.30(0.07)

195 (0.73) 71 (0.27) 61 (0.46)

137 (0.68) 65 (0.32) 48 (0.48)

Reference 1.30(0.87-1.94)1.29(0.19) 0.85(0.51-1.42)0.58(0.55)

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DG>0 (n=101)

1.7(0.42)

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Genotype frequency AA AC CC Allele frequency A C C Carriers

DG=0 (n=133)

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Subjects

ChiSquare (PValue)

DG, Grade Disability, OR, Odds ratio. CI: 95% Confidence interval.

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*Significantly different compared with the controls (Chi-square (χ2) value).

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** Significant different at p < 0.05 level (Z statistic p value).

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disab ility

ACCEPTED MANUSCRIPT Highlights 

First report to associate the asp110ala polymorphisms in the Ninj1 gene with nerve damage in Tamil Nadu, South India.

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Ninjurin 1 gene CC Homozygous as a risk of leprosy.

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Genetic variants were observed in leprosy patient’s disability grade, DG>2

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followed by DG>1.

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