C9orf72 hexanucleotide repeat expansion in Indian patients with ALS: a common founder and its geographical predilection

Journal Pre-proof C9orf72 Hexanucleotide repeat expansion in Indian ALS patients: A common founder and its geographical predilection. Uzma Shamim, Sakshi Ambawat, Jyotsna Singh, Aneesa Thomas, Chevula PradeepChandra-Reddy, Varun Suroliya, Bharathram Uppilli, Shaista Parveen, Pooja Sharma, Shankar Chanchal, Saraswati Nashi, Veeramani Preethish-Kumar, Seena Vengalil, Kiran Polavarapu, Muddasu Keerthipriya, Niranjan Prakash Mahajan, Neeraja Reddy, Priya Treesa Thomas, Arun Sadasivan, Manjusha Warrier, Malika Seth, Sana Zahra, Aradhana Mathur, Atchayaram Nalini, Achal K. Srivastava, Deepti Vibha, Mohammed Faruq PII:

S0197-4580(19)30453-1

DOI:

https://doi.org/10.1016/j.neurobiolaging.2019.12.024

Reference:

NBA 10750

To appear in:

Neurobiology of Aging

Received Date: 15 November 2019 Accepted Date: 27 December 2019

Please cite this article as: Shamim, U., Ambawat, S., Singh, J., Thomas, A., Pradeep-Chandra-Reddy, C., Suroliya, V., Uppilli, B., Parveen, S., Sharma, P., Chanchal, S., Nashi, S., Preethish-Kumar, V., Vengalil, S., Polavarapu, K., Keerthipriya, M., Mahajan, N.P., Reddy, N., Thomas, P.T., Sadasivan, A., Warrier, M., Seth, M., Zahra, S., Mathur, A., Nalini, A., Srivastava, A.K, Vibha, D., Faruq, M., C9orf72 Hexanucleotide repeat expansion in Indian ALS patients: A common founder and its geographical predilection., Neurobiology of Aging (2020), doi: https://doi.org/10.1016/j.neurobiolaging.2019.12.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

C9orf72 Hexanucleotide repeat expansion in Indian ALS patients: A common founder and its geographical predilection. Authors: Uzma Shamim1$, Sakshi Ambawat1$, Jyotsna Singh1, Aneesa Thomas2, Chevula Pradeep-ChandraReddy3, Varun Suroliya2, Bharathram Uppilli1, Shaista Parveen1, Pooja Sharma1, Shankar Chanchal1, Saraswati Nashi3, Veeramani Preethish-Kumar3, Seena Vengalil3, Kiran Polavarapu3, Muddasu Keerthipriya3, Niranjan Prakash Mahajan3, Neeraja Reddy3, Priya Treesa Thomas4, Arun Sadasivan4, Manjusha Warrier4, Malika Seth, Sana Zahra, Aradhana Mathur1, Atchayaram Nalini3, Achal K Srivastava2, Deepti Vibha2, Mohammed Faruq1* 1

Genomics and Molecular Medicine, CSIR - Institute of Genomics and Integrative Biology, Delhi – 110007

2

Department of Neurology, Neuroscience Centre, All India Institute of Medical Sciences, New Delhi

3

Neurology Department, National Institute of Mental Health and Neurosciences, Bengaluru, India

4

Psychiatric social work, National Institute of Mental Health and Neurosciences, Bengaluru, India

$ equal contribution *Corresponding Author

Dr Mohammed Faruq, MBBS, PhD Senior Scientist Genomics and Molecular Medicine, CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), Mall Road, New Delhi, India. Tel: 91-11-27666156; Fax: +91-11-27667471 Email: [email protected], [email protected] ABSTRACT Hexanucleotide repeat expansion in C9orf72 is defined as a major causative factor for familial Amyotrophic Lateral Sclerosis (ALS). The mutation frequency varies dramatically among populations of different ethnicity, however in majority of cases, C9orf72 mutant has been described on a common founder haplotype. We assessed its frequency in a study cohort involving 593 clinically and electro-physiologically defined ALS

cases. We also investigated the presence of reported Finnish haplotype among the mutation carriers. The identified common haplotype region was further screened in 192 (carrying 2-6 G4C2 repeats) and 96 (≥7 repeats) control chromosomes. The G4C2 expansion was observed in 3.2% (19/593) of total cases where 9/19 (47.4%) positive cases belonged to Eastern region of India. Haplotype analysis revealed 11 G4C2-Ex carriers shared the common haplotype (haplo-A) background spanning a region of ~90kbp (SNP895021rs11789520) including rs3849942 (a well-known global at-risk allele for G4C2 expansion). The other three G4C2-Ex cases had a different haplotype (haplo-B) with core difference from haplo-A at G4C2-Ex flanking 31 kbp region between rs3849942 and rs11789520 SNPs (allele 'C' of rs3849942 which is a non-risk allele). This study establishes the prevalence of C9orf72 expansion in Indian ALS cases providing further evidence for geographical predilection. The global core risk haplotype predominated C9orf72 expansion positive ALS cases yet the existence of a different haplotype suggests a second lineage (haplo B) which may have arisen from the TCT haplotype background or may imply a unique haplotype among Asians. The association of risk haplotype with normal intermediate C9orf72 alleles reinforced its role in conferring instability to C9orf72G4C2 region. We thus present an effective support to interpret future burden of ALS cases in India.

Key words: C9ORF72, ALS, Indian Population, Haplotype analysis

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurogenerative disorder characterized by a spectrum of clinical symptoms including relentlessly progressive weakness in limbs and bulbar muscles, hyperreflexia, spasticity of arms or legs and respiratory failure (Gibson et al., 2017;Nguyen et al., 2018). It is one of the most common motor neuron disorders resulting from progressive upper and lower motor neuron dysfunction in the brain stem and spinal cord (Rowland and Shneider, 2001). The genetic etiology leading to ALS is complex due to significant sharing of major causative genes (C9orf72, SOD1, TARDBP and FUS) with FTD

(Frontotemporal Dementia) and other neurogenerative disorders, despite the pathological distribution being different. Similarly, the phenotypic spectrum of C9orf72 mutation has been found to be variable including Parkinsonian, Huntington's disease-like and dementia features (Cooper-Knock et al., 2014;Hsiao et al., 2014). Since the discovery of hexanucleotide repeat expansion in C9orf72 gene, various studies have shown that GGCCCC; [G4C2]n) expansion in the first intron of C9orf72 gene is the most common genetic mutation leading to ALS (Jesus-Hernandez et al., 2011;Renton et al., 2011). The presence of C9orf72 expansion is found in approximately 18-50% of familial ALS cases and 5-7% of sporadic ALS cases worldwide (Gendron and Petrucelli, 2018;Smith et al., 2013;Stewart et al., 2012). Interestingly, various studies have deliberated that the frequency of hexanucleotide repeat expansion in C9orf72 gene causing ALS greatly depends on ethnicity and geographical background (Gijselinck et al., 2018). However, a common risk haplotype (Finnish-haplotype for c9orf72 expansion) has been found to be associated with G4C2-expansion repeats across the globe. Ancestrally, the origin of this shared haplotype has been dated back to ~6000 years ago, and a strong linkage to one of the haplotype allele rs3849942 has been observed consistent across different ethnic and geographical groups. In addition, a recent study by Zou et al., authors have noted a significant difference in the mutation spectrum of ALS between European and Asian populations (Zou et al., 2017). The reports of C9orf72-G4C2 expansion mutation screening among Indian ALS patients are limited. Although few earlier studies have shown its absence (Majounie et al., 2012; Vats et al., 2017) among ALS patients (small sample size), while recently, Narain et al has shown four C9orf72 expansion mutation carrier among a cohort of 131 ALS patients (Narain et al., 2017). Together, these initial findings highlight the necessity for large scale study for the assessment of C9orf72-G4C2 expansion mutation among ALS patients across India and its geographical distribution. In this study, we have investigated a large clinical cohort of ALS patients from two major tertiary care hospitals from Northern and Southern part of India to show the occurrence of C9orf72 expansion and determined its geographical distribution and any differences. The identified G4C2 expansion carriers were also evaluated by haplotype analysis for the presence of common founder in Indian C9orf72–G4C2 expansion repeats. To date, this is the most extensive survey from India interrogating the predictive deleterious occurrence of C9orf72 in the Indian ALS patient population.

Materials and Methods:

Patient Recruitment: A total of 593 patients enrolled in the CSIR-GOMED project (between June 2016Dec2018) for genetic screening for ALS were included in the study which was approved by Ethics Committee of the various Institutions that contributed to the samples. We also included 362 neurologically healthy subjects as controls in this study. Written informed consent was obtained from all patients and controls for molecular genetic testing and research. All patients were clinically examined and evaluated by neuromuscular experts (AN, AS) and broadly, the patients were identified as having clinical symptoms of definitive ALS (n=514), probable ALS (n=62), and ALS-FTD (n=17). The diagnosis of ALS was established according to the revised El Escorial criteria (Brooks et al., 2000). The clinical and demographic details of patients were submitted along with blood samples. They were further categorized according to demographic (age, race, gender, family history including first degree relatives/sporadic) and detailed clinical data (age of onset, first clinical symptom, site of onset and total duration of illness). Familial ALS (f-ALS) cases were also distinguished from sporadic ALS (s-ALS) cases. Detailed clinical report was not available for n=71 patient samples. However, complete genetic analysis was done for these samples and included in the study cohort. The demography and clinical characteristics of patients are outlined in Table 1. The other subjects included for the study were 480 Late Onset Cerebellar Ataxia (LOCA) and Huntington Disease (HD) phenocopies which were genetically negative for the respective mutations. Genetic Analyses: RP-PCR for estimation of hexanucleotide expansion in C9orf72 gene: Genomic DNA was extracted from peripheral blood lymphocytes using modified salting out method ((Miller et al., 1988). A modified RPPCR protocol was followed for the detection of the expanded allele of C9orf72 (Biasiotto et al., 2017). PCR was carried out in a 12 µl reaction, containing 100 ng DNA, 5 µl FailSafe G reagent (Lucigen), 2U G2 Hot start Polymerase (Promega), 0.25 mM 7-deaza-GTP, 0.4 µM FAM-labelled forward primer, 0.125 µM reverse primer and 0.4 µM anchor tail reverse primer. Heat pulse extension PCR cycle was used for the amplification of C9orf72 repeat region (Orpana et al., 2012). The PCR product was mixed with HiDi and size marker, denatured and analysed using an automated capillary electrophoresis based 3500 Genetic Analyzer and GeneMapper software (ABI, Foster City). An extended saw-tooth pattern signifying greater than 30 repeats was considered positive for C9orf72 expansion. Furthermore, Coriell Institute for Medical Research provided us with C9orf72 positive DNA sample which was included in all sample runs as positive control. All 593 ALS samples, 48 controls and 480 LOCA and HD samples were screened for the presence of C9orf72 expansion.

Estimation of normal repeat length distribution in C9orf72 and ATXN2 alleles: Fluorescence-based PCR amplification with Fam-labelled primers was performed to assess the normal range of G4C2 repeats in C9orf72 as well as ATXN2-CAG repeats for all ALS patients and controls included in the study. The amplified and labeled PCR products were analyzed by capillary electrophoresis on ABI sequencer as described above. The sequences of all primers used in this study are summarized in Table S1. Repeat lengths for ATXN2 ranging between 29 and 33 were considered to be of intermediate length and contributory to increased risk for ALS (Neuenschwander et al., 2014). Targeted sequencing of SOD1: The entire coding region and intron-exon junctions of the SOD1 gene were amplified by polymerase chain reaction with appropriate primer pairs (Table S1). The amplified PCR products were subjected to Sanger sequencing using ABI 3730- genetic analyzer (ABI, Foster City). Haplotype and population structure assessment using Genome wide genotyping data: Genotyping was performed in n=14 expansion positive ALS cases and n=10 control samples using Affymetrix Axiom Genotyping Array. After performing Quality Control on samples for exclusion of SNPs with low call rates, Genotype analysis was done using Axiom Analysis suite software. From the dataset obtained, genotype calls were selected for region flanking the G4C2 repeats (~200kbp region and 205 SNPs) followed by selection of markers with MAF (minor allele frequency) of 0.05 shortlisting 118 SNPs. Case control analysis was performed for frequency difference assessment of each of the SNP marker with 10% significance (due to lower sample size, significance threshold was lowered to less stringency to include the markers reported in Finnish Haplotype block). Finally, 48 SNPs were selected for haplotype construction with Phase analysis tools, Phase version2.1 (Stephens et al., 2001). The final selected set of markers (48 SNPs) included 10 Finnish haploblock markers flanking G4C2 repeats (Laaksovirta et al., 2010). The initial haplotype analysis was focused only on the 10 Finnish haploblock markers (Laaksovirta et al., 2010) to infer the presence of Finnish haplotype in the G4C2 positive cases. Furthermore, Haplotypes were also constructed taking into consideration the SNPs (rs868856-rs774359) forming the core haploblock in close proximity of G4C2 expansion. Input files were created with excel tool. Haplotype phase construction was performed with following parameters; 100, 10, 100 (number of iterations, thinning interval and burn-in). From the output files, frequencies of the predicted haplotypes were calculated for expansion and nonexpansion G4C2 alleles in the cases and controls. The genotype dataset for G4C2 flanking region (~200kbp) of case and control was also used for visualization of Linkage disequilibrium patterns in Haploview (Barrett et al., 2005).

Principal Component Analysis (PCA): The genotyped subjects (G4C2 positive cases and controls, 14:10) were analyzed by PCA to observe any clustering within the groups (interrelatedness) and also with other global populations. For PCA using Affymetrix genome-wide dataset, the initial filtering of variants was done using PLINK ((Purcell et al., 2007) for missing genotype rate and excess heterozygosity. The final quality checked 560854 variants were considered for further downstream analysis. The genotyped dataset from 1000 genome population was obtained (www.internationalgenome.org) for referencing purpose and PCA was done using smartpca module of EIGENSOFT software (Patterson et al., 2006) while R ggplot was used for the visualization.

Targeted Genotyping of 3 risk haplotype associated SNPs using Single Base Extension (SBE)/ SNaPshot assay: SNaPshot assay is a genotyping technique for detection of SNPs at specific positions in the genome. The core haplotype block and its association with G4C2 repeats was interrogated by genotyping 3 marker SNPs (rs3849942, rs774359 and rs2453554) in C9orf72 negative ALS cases and controls. For each marker, the region containing the SNP was amplified using PCR and specific primers (Table S1). Subsequently, the ‘internal primer’ (table S1) designed to anneal at the sequence adjacent to the SNP position led to a complementary dye-conjugated dideoxy nucleotide base to anneal at the 3′ end of the internal primer thus terminating the reaction at the position of variant allele. Finally, capillary electrophoresis was performed to determine the nucleotide present at that position. The G4C2 Expansion negative ALS cases as well as controls carrying normal short repeats (2-6) and normal intermediate repeats (≥7) were taken as follows: ALS CASES Normal Short Repeats (2-6) Normal Intermediate repeats (≥7)

n=95 n=48

CONTROLS n= 95 n=48

The genotype obtained for all G4C2 Expansion negative ALS cases and controls were tabulated along with their G4C2 repeat numbers. This data was used for the haplotype analysis using Phase analysis tool, Phase version2.1 (Stephens et al., 2001). In addition, five remaining G4C2 positive cases (left out from genome wide genotyping) were as well taken for 3 marker-based haplotype analysis. Statistical analysis: p values were calculated for the distribution of G4C2 allele frequencies by paired student’s t test.

RESULTS C9orf72 Pathological Expansion: Genetic screening for C9orf72 expansion conducted in the ALS cohort provided the following findings. G4C2 expansion was observed in 3.2% (19/593) of the total cases including eighteen cases with pure ALS manifestations, whereas only one patient belonged to the ALS-FTD phenotype. The clinical details of positive cases are shown in Table 1. The frequency of occurrence of the expansion in f-ALS cases is 10.7% (3/28), whereas 2.8% (16/565) were tested positive from s-ALS group. The age of onset among C9orf72 positive patients was highly variable ranging from 34-65years and not found to be segregating with f-ALS cases having early onset. The distribution of the G4C2 expansion across different geographical regions of India was highly significant with 47.4% (9/19) of cases belonging to the eastern belt of India. The remaining positive cases were divided among northern, western and southern regions comprising of 4, 2 and 4 cases each. Moreover, only one case of LOCA out of 480 patients screened with LOCA and HD phenocopies, detected positive for the presence of C9orf72 expansion. Screening for SOD1 mutations: A preliminary investigation of 138 s-ALS cases for non-synonymous mutations in SOD1 gene was essentially negative implying the absence of SOD1 mutations as major causative gene for s-ALS from Indian subcontinent. Subsequently, screening of 20 f-ALS cases (negative for C9orf72 expansion) identified only one patient positive for homozygous I114T allele of SOD1 gene. The chromatogram depicting the mutation has been shown in Figure S1. Distribution of Normal C9orf72 G4C2 and intermediate ATNX2 Repeat Length: Sizing the normal C9orf72 allele in both C9orf72 expansion negative ALS cases as well as controls revealed no significant difference (p=0.97) in the repeat lengths in both cohorts as depicted in Figure 1. It was found to fall between 2-24 for both cases and controls. The frequency distribution of G4C2 repeats in G4C2-ex negative ALS cases (n=558) and controls (n=357) has been in shown in Table S2. The most common repeat length was 2 followed by near equal presence of 4, 5, 6,7 and 8 repeats (Figure 1). When comparing the normal intermediate alleles between cases and controls, we failed to detect any difference in allele frequencies. However, a substantial number of subjects in both the cases and control groups carried normal intermediate alleles. The ATXN2 intermediate repeat length of 29-32 known for contributing to the risk for ALS was harbored by 1.5% of the total ALS cases, which falls well below the observed frequency in other population-based studies (Corrado et al., 2011;Elden et al., 2010). Also, there was no difference observed

when comparing the presence of intermediate ATXN2 allele in cases and control group. However, association of ALS with ATXN2 repeats differed between cases and controls when restricted to alleles between 30 and 33 repeats with higher frequency seen in ALS cases. Also, none of C9orf72 positive ALS cases contained the intermediate ATXN2 allele. Association of G4C2 expansion carriers with Finnish at-risk haplotype The initial haplotype analysis using 10 Finnish haploblock markers (spanning in a region of ~200 kbp) have shown sharing of a haplotype region between rs868856-rs774359-G4C2 (~84 kb region) in 11 G4C2 (represented by AATTC) expanded chromosomes while three G4C2-Ex alleles had a different allelic haplotype in the core region of 31kbp between rs2814707 and rs2453554. Thus, in this core region the majority of G4C2-Ex alleles had TTC haplotype (haplotype-A) and three had CCT haplotype (Haplotype-B), Figure-2A, Table-S3. The frequency comparison of the predicted haplotypes in G4C2-Ex/G4C2-NonEx alleles in 84 kbp region is shown in Figure-2B, where it is observed that the Finnish risk haplotype (AA-TTC) segregates with the G4C2 expansion alleles (p≤0.0001), Table-S4. This risk haplotype was observed with two non-expansion alleles. The other three G4C2-Ex alleles had AA-CCT haplotype. Furthermore, the non-expanded alleles (controls) depicted majorly GG-CCT haplotype (74%). Therefore it is suggestive that a recombination event in the past has allowed the occurrence of CCT (core haplotype) with G4C2 expanded alleles as well, since the 5’ flanking region represented by rs868856 and rs7046653 (AA) is of finnish risk haplo-block category was present in all the G4C2 alleles. For extended haplotype analysis 48 SNPs (available from Affymetrix genotype data) flanking the G4C2 alleles were chosen and that analysis also have shown similar results as described above (Table-S5). A haploblock-5 (38 kbp region carrying the core haplotype and also the 3’ region of G4C2 repeats) (Table-S5), with risk alleles was shown to be segregated with the same 11 G4 C2-Ex alleles while three G4C2-Ex allele had haplotypic alleles of non-risk variants (Table- S5). The Linkage Disequilibrium (LD) patterns observed in cases and controls (Figure-S2) also depicted strong LD blocks that coincide with the haplo blocks of haplotype analysis. LD pattern in case and controls separately have shown subtle differences at few SNPs, while, striking similarities in LD blocks pattern proximal to rs3849942 was observed. The later observation shows allelic flip between cases and controls that kept LD preserved in both the groups however with their distinct alleles. 3 SNPs based risk haplotype distribution among Non-expanded G4C2 repeats

To evaluate the significance of block 5 SNPs (Table S3), we had selected three SNPs (rs3849942, rs774359 and rs2453554) for haplotype association with respect to various G4C2 repeat alleles in G4C2-Ex negative ALS cases and healthy controls. The three marker haplotype data showed a skewed occurrence of TCT risk haplotype (72% and 70%) in normal intermediate alleles (≥7) over CTC non-risk haplotype (28% and 29%) in G4C2 negative ALS cases and controls, respectively as evident from Figure-2C, Table-S6. The allelic and genotype frequencies of the 3 marker SNPs obtained for all cases and controls is elaborated in Table-S7. Although TCT haplotype was also observed with normal G4C2 alleles (2-6) in a small proportion representing 4-6%, CTC was the major haplotype structure for normal short (2-6) G4C2 alleles. Out of other five G4C2 positive cases (not included in Genome wide genotyping), four were carriers of TCT while one case was carrier of CTC haplotype. Hence of the 19 identified G4C2 positive cases 15 had Finnish risk allele T (rs3849942) associated haplotype while remaining had non-Finnish haplotype Taken together, these results indicate the presence of two lineages for G4C2 expansion, the common Finnish haplotype (haplo A) and a second lineage (Non-Finnish, haplo B) which may have arisen from the TCT haplotype background. This inference was further supported with the LD pattern observed in this region (Figure S2). Principal component analysis of G4C2 expansion carriers On PCA, clustering of our cases and controls (based on whole genome wide markers) was seen with SAS subjects from 1000 genome population (Figure 3). Further analysis of case:controls with SAS population showed no major difference in clustering pattern and a heterogeneous dispersion of all cases [carrying Finnish (haplo-A) or Non-Finnish (haplo-B)] and controls were seen. We further did PCA of our study subjects with comparative populations (1000g) w.r.t. only 1MB region flanking G4C2 and interestingly, distinct patterns of case control clusters were seen as expected (similar to haplotype analysis, mentioned above). Cases with Haplo-A clustered among both EUR-Finnish and SAS population subjects whereas cases with Haplo-B clustered within SAS group of subjects. Controls subjects had a distinctive cluster from cases among SAS and EUR subjects (Figure 2).

DISCUSSION Various studies have proposed the existence of distinct genetic architecture among European and Asian ALS population (Cruts et al., 2013;Majounie et al., 2012;van der et al., 2013;Zou et al., 2017). Even among Asian population, there is limited genetic data on ALS cases from the Indian subcontinent. Moreover, there is

striking difference in the presentation of ALS cases when compared amongst cases of varied ethnicity (Garcia-Redondo et al., 2013). This prompted us to involve a large and variable cohort of ALS patients from different geographical regions of India to provide a better understanding of the genetic basis of ALS as well as to discover the existing pathogenicity of C9orf72 repeat expansion. This is apparently the first study from India employing approximately 600 ALS cases and 362 controls. The selection of samples for this study was entirely random as it was dependent on the number of samples received from various hospitals across India for genetic screening. This in turn added variability to the number of samples included from different geographical regions. In the current patient cohort, 10.7% of f-ALS cases and 2.8% of s-ALS cases were found positive for C9orf72 expansion. A meta-analysis study by Zou et al., involving several population-based studies showed that the frequency of occurrence of C9orf72 pathogenic expansion is 2.3% in f-ALS and 0.3% in s-ALS for Asian population, whereas in the European population, the estimates are 33.7% and 5.1% for f-ALS and sALS respectively (Zou et al., 2017). The pattern observed for C9orf72 positive cases from India is relatively different from other Asians and neither does it coincide with Europeans, thus reflecting the genetic heterogeneity existing amongst Indians of different geographical origin. Furthermore, it is known that SOD1 is the most common causative gene for f-ALS in the Asian population (Majounie et al., 2012;Zou et al., 2017). Screening of 138 s-ALS cases and all f-ALS cases for SOD1 showed only one positive implying that the SOD1 mutation is not a causative factor for f-ALS cases in the Indian population. This inference is supported by the observation made by (Zou et al., 2017), regarding the presence of north-south gradient with respect to the frequency of SOD1 mutation across Asia with highest frequency in Koreans followed by Japanese, Iranians, Chinese and least in Indians (Zou et al., 2017). The major phenotypic characteristics of C9orf72 positive cases from India are partially distinct when compared with the cases worldwide. Majority of repeat expansion carriers had limb onset ALS with few cases of bulbar onset, contrary to other studies which show bulbar onset for C9orf72 positive ALS cases (Gijselinck et al., 2018). Also, minor cognitive abnormalities not accounting to FTD have been observed in 2 positive cases which is a clinical outcome observed in ALS patients globally (Byrne et al., 2012). Highlighting some of the significant findings of this study, we report that the age of onset of C9orf72 positive cases has been found to be highly variable ranging from 34-65 years, where 10 cases have the age of onset in the third and fourth decade. This is in corroboration with the finding that the age of onset of ALS is a decade earlier in the Indian Population (Nalini et al., 2008), whereas, in contrast, western populations show an older age of ALS onset (Chio et al., 2013). Several factors, genetic as well as environmental might be

responsible for early onset of ALS in this fraction of world population which needs to be investigated deeply. Another significant observation is the predominance of expansion positive cases from the eastern region of India. Of particular relevance is the observation that the maximum number of ALS cases included in the study had come from Southern India followed by northern > eastern and lastly western. However, nearly half of the positive cases belonged to the eastern geographical region of India. An epidemiological study on ALS patients from a hospital in eastern region of India had already reported a high prevalence of early onset ALS in West Bengal, one of the eastern states of India (Das et al., 2012). Our result is also supported by the incidence data of ALS from different geographical regions reported through various studies as shown in Table-S8. Henceforth, this study provides evidence for the pathogenic C9orf72 expansion being the dominant causative factor for ALS from eastern India. It has been reported that lower incidence of pathogenic C9orf72 expansions in Asian cohorts is due to shorter G4C2 repeat lengths which confers lower risk for instability (Ng and Tan, 2017). The most common allele in all populations has been found to be 2 followed by 6, 7 and 8 in Chinese and 5 and 8 in Europeans (Wang et al., 2015). Among Indians, the most frequently observed allele was 2 followed by a near equal representation of 4, 5, 6, 7 and 8 alleles in both cases and controls, signifying a different genetic architecture existing in the Indian population. Moreover, number of samples carrying normal intermediate alleles did not show significant variation between patients and controls, as reported earlier through diverse studies (Itzcovich et al., 2016;Mok et al., 2012;Ng and Tan, 2017). A remarkable finding was the significant presence of normal intermediate alleles in both non-expansion ALS cases as well as controls. It can be plausible that the presence of normal intermediate C9orf72 alleles in healthy individuals may give rise to sporadic ALS cases over many subsequent generations owing to the instability of repeat regions (Jones et al., 2013). Similarly, the lowest occurrence of normal intermediate alleles in the expansion negative ALS cases from southern India when compared with other regions (Table-S9), can be a contributing factor for the absence of greater number of cases showing positive pathogenic C9orf72 expansion in this group. Further studies need to be carried out to validate this hypothesis. The pathogenic G4C2 expansion has been derived on a common founder risk haplotype of Finnish origin (Majounie et al., 2012;Mok et al., 2012). This 82-SNP risk haplotype (232kbp) segregating with G4C2 repeat allele (fully or partially) has been found in majority of C9orf72 associated ALS cases among various populations. The European G4C2-Ex carriers share a 110kbp region with the Finnish founder haplotype (Laaksovirta et al., 2010;van der et al., 2013). This possibly suggests that recombination events in the past has broken the core haplotype (Finnish) structure in different populations. Additionally, few reports have

suggested the existence of other haplotypes as well. Chiang et al., had deliberated that two risk haplotypes exist in the Swedish population segregating with G4C2-Ex (Chiang et al., 2017). Two studies, one from China and the other from Australia have particularly reported C9orf72 positive cases (2 and 1 respectively) with a haplotype background containing non-risk allele (CC) for rs3849942, a surrogate marker for G4C2-Ex (Chen et al., 2016); Dobson-Stone et al., 2012). Our dataset indicates two haplotypes existing in the Indian population. Haplo A which is a derivation of G4C2 haplotype of Finnish origin found majorly (11/14) in Indian C9orf72 mutation carriers, further strengthening the global presence of common lineage for C9orf72 repeat expansion. However, the length of the conserved block is ≈90kbp (chr9:27484911-27574515), which corresponds to a small part of the Finnish founder haplotype. The second haplotype, Haplo B, carries the Non-Finnish alleles at rs3849942 and other associated SNPs. Of note is the observation that majority of the alleles of risk haplotype were non-ancestral alleles (non-chimp) whereas the ancestral alleles were largely associated with normal G4C2 allele. This led us to hypothesize that there are two lineages of haplotype for G4C2 expansion, one European and the other Asian as the second haplotype is supported by one previous study from China (Chen et al., 2016). Another explanation could be that the expansion arose as one major event in the past (≈6000yrs ago) on the Finnish haplotype background and subsequently, due to recent admixtures between Europeans and Asians might have created breaks in LD due to recombination events. But the absence of such haplotype carrying Non-Finnish allele at rs3849942 in C9orf72 positive populations other than Asians might confer a distinct haplotype of Asian origin. This is supported by the findings of PCA which has shown a distinctive clustering pattern for Haplo A cases with EUR-Finnish and SAS population and Haplo B cases with SAS subjects only when viewed over 1MB region flanking G4C2 repeat region. The presence of ATNX2 intermediate allele (29-33) as a genetic modifier in ALS cases, is a well-documented fact across different studies (Corrado et al., 2011;Daoud et al., 2011;Elden et al., 2010). In our study, the correlation existing between intermediate CAG repeats in ATNX2 allele and ALS is apparent only when the cut-off for its association with ALS has been taken as 30-33 repeats rather than 29 repeats for ATXN2. This finding is in concordance with previous study of European ALS patients and controls where only repeats >30 were significantly different between the two groups (Lee et al., 2011). Nonetheless, the intermediate CAG range for ALS might vary in different studies due to ethnicity (Wang et al., 2014). Another aspect where ethnicity has played a significant role is the near absence of C9orf72 expansion in LOCA and HD phenocopies since only one positive case out of 480 was detected from India implying that C9orf72 expansion might not be present in neurodegenerative disorders other than ALS and FTD. Future studies should be necessitated in this direction.

Conclusion This study establishes the prevalence of C9orf72 expansion in ALS cases from India. The predicted geographical variability will help in providing a better understanding of the origin of the disease as well as, can be accounted for while performing appropriate genetic testing. Moreover, the presence of two haplotypes in the Indian population needs further evidence to support the theory that expansion may have occurred on different haplotype backgrounds. Furthermore, the high frequency of risk associated haplotype in control samples can contribute to the manifestation of the pathogenic expansion in future generations by conferring instability to the repeat region. We thus present an effective support to interpret future burden of ALS cases in India. Acknowledgement: We are grateful to the patients for participating in this study. This work was financially supported by Council of Scientific and Industrial Research (CSIR)-funded GOMED project (MLP1601 and MLP1802) Disclosure statement: Nothing to report for any author.

Reference List

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Legends to Figures: Figure 1: Frequency distribution of G4C2 allele in C9orf72 negative ALS cases and controls showing no statistical significant differences (p value 0.97). Results for repeats 9-24 is magnified in upper right of graph. Figure 2: Haplotype analysis of G4C2 expansion carriers. A) Schematic showing reported Finnish haploblock markers in the region of ~230 kbp region (Laaksovirta et al. 2010). The haplotype analysis in Indian G4C2 expansion carriers showing two haplotypic lineages based on a core haplotype region in 31 kbp region. Majority of G4C2 carriers had shared region of finnish at-risk markers while three carrier chromosome had other allele at each position. B) the haplotype frequency distribution comparison between G4C2 expansion carriers and Non-G4C2 expansion carriers. C) The 3 markers haplotype based analysis of Non-G4C2 alleles in Normal and short intermediate range showing predominance of at risk Finnish haplotypes with short intermediate range G4C2 alleles. Figure 3: Principal component analysis using genome wide loci of G4C2 positive cases and controls and their relatedness with 1000 genome populations. Upper-left panel showing relation of Indian G4C2 cases/controls with SAS population of 1000 genome dataset. Left panel showing PCA of SAS subjects and G4C2 case/controls showing clustering of G4cases among converging zone of PCA where representation of all the SAS population can be found, however it is closer to SAS-BEB population. The lower panel shows further PCA analysis of G4C2 cases with SAS and EUR sub-populations of 1KG. This shows PCA w.r.t. SNPs from a 500kbp upstream and downstream of G4C2 repeat region itself and it was observed that G4C2 cases appeared in an intermediate region where SAS and EUR population shows an overlap.

Table 1: Demographic and clinical examination findings of ALS patients and G4C2 expansion positive cases. Parameters

Total ALS Cases (n=593)

C9orf72

Positive

Cases

(n=19) D-ALS (n=514); P-ALS (n=62); D-ALS (n=18); FTD-ALS FTD-ALS (n=17) Male 396 (66.8)

(n=1) 10 (52.6)

Female 197 (33.2)

9 (47.4)

Eastern Region 189 (31.8)

9 (47.4)

Geographical Origin n (%)

Western Region 12 (2.0)

2 (10.5)

Northern Region 137 (23.1)

4 (21.5)

Southern Region 229(38.6)

4 (21.5)

Central Region 11 (1.9)

0 (0)

North-East Region 15 (2.5)

0 (0)

Familial ALS n (%)

28 (4.7)

3 (15.8)

Sporadic ALS n (%)

565 (95.3)

16 (84.2)

Site of Onset n (%) Bulbar 152 (29.1)

5 (31.6)

Limb 363 (69.5)

12 (57.9)

Undefined 7 (1.3)

2 (10.5)

First Clinical symptom n (%) Dysarthria 100 (19.1) Swallowing Difficulty 6 (1.1) Memory Disturbance 2 (0.38) UE; LE; UE+LE 159 (30.5); 129 (24.7); 26 (5) Undefined 100 (19.1) Age at Diagnosis [mean (SD)], 51.0 ± 12.5

4(21.1) 0(0) 1(5.2) 6(31.6); 6(31.6); 0(0) 2(10.5) 47.6± 12.6

years Age at Onset [mean (SD)], years

49.1 ± 12.8

46.2 ± 13.2

Duration of Illness [mean (SD)], 19.9 ± 29.8

15.5 ± 11.3

months Other neurological disease n (%)

5 (0.9)

(missing clinical data),n

71

2 (10.5) 2

D-ALS= Definitive ALS; P-ALS=Probable ALS; ALS-FTD=ALS with Frontotemporal dementia. sporadic ALS; UE=Upper Extremity; LE=Lower Extremity

f-ALS= Familial ALS; s-ALS=

Highlights Hexanucleotide repeat(G4C2) expansion in C9orf72 is a major causative factor for familial Amyotrophic Lateral Sclerosis (ALS). Shamim et al. have described 3.2% G4C2 hexanucleotide expansion positive Indian ALS cases from screening of larger number of patients (n=593). Two lineages of G4C2 expansion associated haplotypes were observed in Indian ALS subjects.

The work Described in C9orf72 Hexanucleotide repeat expansion in Indian ALS patients: A common founder and its geographical predilection. has not been published previously (except in the form of an abstract, a published lecture or academic thesis, see 'Multiple, redundant or concurrent publication' for more information), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.