REVIEW

OFFICIAL JOURNAL Clinical Applications and Implications of Common and Founder Mutations in Indian Subpopulations

www.hgvs.org Arunkanth Ankala,1∗ † Parag M. Tamhankar,2 † C. Alexander Valencia,3,4 Krishna K. Rayam,5 Manisha M. Kumar,5 and Madhuri R. Hegde1 1Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia; 2ICMR Genetic Research Center, National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India; 3Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; 4Department of Pediatrics, University of Cincinnati Medical School, Cincinnati, Ohio; 5Department of Biosciences, CMR Institute of Management Studies, Bangalore, Karnataka, India Communicated by Arupa Ganguly Received 24 October 2013; accepted revised manuscript 16 September 2014. Published online 27 November 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22704

that include a lack of widespread awareness about genetic disorders in the general population and the scarcity of specialized medical ABSTRACT: South Asian Indians represent a sixth of the world’s population and are a racially, geographically, and professionals and affordable genetic tests. Seeking a molecular di- genetically diverse people. Their unique anthropological agnosis and understanding the risk estimates are critical to making structure, prevailing caste system, and ancient religious sound reproductive choices, especially in families with an affected practices have all impacted the genetic composition of most individual. Adding to this adversity is the absence of a properly func- of the current-day Indian population. With the evolving tioning social health care system and insufficient encouragement socio-religious and economic activities of the subsects and of individual health insurance by the government. Consequently, castes, endogamous and consanguineous marriages became even interested and informed individuals cannot afford to seek a a commonplace. Consequently, the frequency of founder molecular diagnosis or genetic services. The World Health Organi- mutations and the burden of heritable genetic disorders zation (WHO) has appropriately stressed the need for early diag- rose significantly. Specifically, the incidence of certain nosis, prevention, and management of genetic disorders in devel- autosomal-recessive disorders is relatively high in select oping countries and released detailed guidelines [Christianson and Indian subpopulations and communities that share com- Modell, 2004]. However, to practice these guidelines and reduce mon recent ancestry. Although today clinical genetics and disease burden, there must be efforts to improvise and make diag- molecular diagnostic services are making inroads in India, nostic tests affordable. In a vast country like India, where the disease the high costs associated with the technology and the tests burden is tremendous and resources are limited, low-cost diagnostic often keep patients from an exact molecular diagnosis, tests that target specific common mutations or founder mutations in making more customized and tailored tests, such as those select communities or subpopulations hold significance. Rightly so, interrogating the most common and founder mutations studies aimed at identifying many more common as well as founder or those that cater to select sects within the population, mutations are expected to further the development of low-cost and highly attractive. These tests offer a quick first-hand af- targeted assays, such as those discussed here. Here, we make an effort fordable diagnostic and carrier screening tool. Here, we to outline the significance of such studies by providing examples of provide a comprehensive catalog of known common mu- recently identified founder mutations. We also discuss the common tations and founder mutations in the Indian population genetic disorders in India and provide a thorough catalog of com- and discuss them from a molecular, clinical, and historical mon and founder mutations, along with their clinical applications perspective. and significance. Hum Mutat 36:1–10, 2015. C 2014 Wiley Periodicals, Inc. KEY WORDS: founder mutations; clinical genetics; India; Current State of Clinical Genetics Services in India common variants; molecular testing With the efforts of national agencies and funding bodies, such as the Indian Council of Medical Research (ICMR) founded by the Ministry of Health and Family Welfare of India, the pace of research and expanded clinical services in genetics has picked up Background in India in recent years. ICMR has institutionalized the Genetic Rough estimates are that more than 50 million individuals in Research Center (GRC) to regulate genetic research in the coun- India are affected with single-gene disorders (monogenic disorders) try and offer clinical services to diagnose genetic disorders. In ad- [Singh et al., 2010]. This number is expected to rise due to factors dition to the national agencies, private organizations such as the Organization for Rare Diseases India (ORDI) are making signifi- cant efforts to extend genetic services and address the challenges Additional Supporting Information may be found in the online version of this article. and concerns of rare diseases in India [Rajasimha et al., 2014]. †These authors contributed equally to the manuscript. Currently, there are only 68 accredited centers in India offering ge- ∗Correspondence to: Arunkanth Ankala, PhD, Department of Human Genetics, netic testing and/or counseling services (http://geneticsindia.org); Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322. E-mail: most of these clinical laboratories only offer diagnostic assays that [email protected] target either a single more recurrent mutation or a small set of

C 2014 WILEY PERIODICALS, INC. Table 1. Founder Mutations in Agrawals

Mutation Allele Disease Gene OMIM# Mutation frequency (%) count Region/sect Reference

Hallervorden–Spatz syndrome PANK2 606157 c.216dupA Founder (58.3) 7/12 Agrawals Aggarwal et al. (2010) ∗ Heme oxygenase 1 deficiency HMOX1 141250 c.130C>T (p.R44 ) Founder (100) 2/2a Agrawals Radhakrishnan et al. (2011) LGMD2A CAPN3 114240 c.2338G>C (p.D780H) Founder (50) 9/18 Agarwals Ankala et al. (2013) LGMD2A CAPN3 114240 c.2051–1G>T Founder (50) 9/18 Agarwals Ankala et al. (2013) Megalencephalic leukodystrophy MLC1 605908 c.135dupC Founder (100) 62/62 Agarwals of Gorospe et al. (2004) with cysts North India aFounder effect of the mutation was established by haplotype studies and detection of the mutation in multiple affected individuals (personal communication by IC Verma). Allele count, number of alleles with mutation/number of alleles tested. well-established mutations within a specific gene. Moreover, there disorders in Indian populations (Table 1). In addition to founder ef- are only two established genetic counseling programs in India, and fects, cultural and religious practices in India are such that intracom- as few as 25 trained (formally or informally) genetic counselors munal and intracaste arranged marriages are frequent, further rais- across the length and breadth of the country [Elackatt, 2013]. These ing the risk of carriers of the same mutation or disease being united. limitations call into serious question the amount of counseling (if Identifying and cataloging founder mutations in Indian populations any) and the testing options available or offered to a couple making will allow for carrier and prenatal testing options, regardless of fam- reproductive decisions. ily history, holding out the promise of controlling disease burden.

Disease Burden and Control Measures in Practice in India Founder Mutations and Their Contribution to Disease While most developed countries have an established and closely Prevalence monitored newborn screening program (NSP) to identify new- In general, founder mutations are defined as recurrent mutations borns with selected genetic disorders (e.g., endocrinopathies and observed on a single haplotype background in a particular popula- metabolic disorders) and provide early intervention, there is no tion. They help us understand the evolutionary aspects of human such program in place yet in India. With several pilot newborn disease [Drayna, 2005]. As a special category of mutation, founder screening studies reporting a high incidence of a selected group of mutations enable scientists to trace the ancestry, migration, and disorders, the overall burden on the nation is becoming clear, and growth of specific human populations over time [Vernengo et al., there is growing urgency to implement a national NSP [Kapoor 2011]. Apart from these anthropological traits, founder mutations et al., 2013]. Even though a NSP allows for the early identification also hold medical significance in that they cause the high frequencies of and intervention for a disease in affected individuals, the cost of recessive diseases in certain populations, such as the AJs men- and discomfort of continuous treatment and management are sig- tioned earlier [Slatkin, 2004], the Helsinki population of Finland nificant, both for an individual’s family and the nation. A second [Lao et al., 2008], as well as several Indian subpopulations, includ- and more broadly applicable (to more diseases) protocol that com- ing the Agrawal community [Ankala et al., 2013]. In fact, founder plements the NSP effort and helps meet the objective of reducing mutations are believed to contribute more to the burden of reces- disease burden is the carrier screening option, which can directly sive diseases than consanguinity alone in certain populations like reduce disease incidence. India’s [Reich et al., 2009]. This is a consequence of the shared de- Carrier screening for a genetic disease is commonly defined as the scent of most Indian subpopulations [IGVC, 2008]. Evidence from testing of asymptomatic individuals for heterozygous mutations in population genetics studies dating back to over 30–100 generations the disease-associated gene (or genes). This is usually performed for in Indo-European and Dravidian-speaking groups suggests that en- recessive disorders to assess the risk of the offspring being affected dogamy prevents founder events from being erased by gene flow with the disease. This further allows for prenatal testing and provides [Reich et al., 2009]. The traditional practice of intracaste endogamy, an option to terminate affected pregnancies. While the probability which has continued for thousands of years, has contributed signif- of couples both being carriers for a single disease is rare for most icantly to the excessive cases of recessive disease in India. Haldane diseases, this is not always the case for certain diseases in certain pop- (1964) appropriately stated that recessive characteristics will become ulations. For example, individuals of Ashkenazi Jewish (AJ) descent rarer in India when intercaste marriages become common. are several times more likely (one in 18) to be carriers for certain diseases, like Gaucher disease type I, cystic fibrosis (CF), and Tay- Sachs disease, than they are for most other diseases [ACOG Com- Clinical Significance of Founder Mutations mittee on Genetics, 2009]. Consequently, the American Congress of Obstetricians and Gynecologists (ACOG) and American College Over the years, research has uncovered many more of these of Medical Genetics and Genomics (ACMGG) recommend carrier founder and common mutations in Indian subpopulations asso- testing for a common panel of mutations in all AJ individuals who ciated with a variety of recessive diseases. A few studies, such as are considering pregnancy [Gross et al., 2008]. Most of these mu- the one we recently reported, have also explored the clinical utility tations represent founder mutations in the AJ population that have of these mutations [Ankala et al., 2013]. We reported a cohort of been inherited over several generations. Similar recommendations Indian patients from the Agrawal community, who all had a clin- for carrier screening have been made for hemoglobinopathies in ical diagnosis of limb-girdle muscular dystrophy (LGMD). These individuals of African, Southeast Asian, and Mediterranean descent patients from several unrelated families were referred for clinical [ACOG Committee on Obstetrics, 2007]. Even though the diseases evaluation and enrolled in the study over a period of 10 years. Dur- might be different, similarly high incidences and carrier frequen- ing this period, the patients underwent invasive muscle biopsies, cies of one in 20 to one in 50 have been reported for various genetic immunohistochemical and immunoblotting studies for subtype

2 HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 Table 2. Incidences and Carrier Rates of Common Genetic Disor- Similar smaller panels and targeted testing assays are currently avail- ders in India able, such as the rhabdomyolysis panel for Caucasians, which detects 71%–97% of carriers by interrogating just the seven common mu- Disorder Incidence Estimated carrier frequency tations in the three genes ACADM, PYGM,andAMPD1 [Van Adel Congenital hypothyroidism 1:1,700 One in 21 and Tarnopolsky, 2009]. More recently, pan-ethnic carrier screening Galactosemia 1:10,300 One in 51 panels for over 90 genes and 500 mutations have become available G-6-P deficiency (South India) 1:2,200 One in 24 thanks to the vast technological advancements in SNP genotyping Congenital adrenal hyperplasia 1:2,600 One in 26 ß-Thalassemia 1:3,500 One in 30 [Srinivasan et al., 2010]. It was the desire to understand these po- α-Thalassemia 1:1,500 One in 20 tential applications of founder and common mutations that drove Phenylketonuria 1:18,300 One in 68 the present study. Amino acid disorders 1:3,600 One in 30

The carrier rates and incidences for each disorder reported in this table are estimates Cataloging Common and Founder Mutations in Indian from several pilot newborn screening studies [Devi and Naushad, 2004; Morton and Nance, 2006; Kapoor and Kabra, 2010]. Subpopulations Lately, central authorities and senior investigators in India have recognized the significance of generating massive sequence data characterization, and molecular diagnosis. Due to inconclusive for various subpopulations and maintaining variant frequency protein studies, panel-based sequencing of all known LGMD- databases. As a result, efforts such as the Indian Genome Vari- associated genes was performed. Two founder mutations, a mis- ation Data Base (IGVDB) have been initiated by the Indian sense (c.2338G>C; p.D780H) and a splice-site (c.2051–1G>T) mu- Genome Variation Consortium to sequence known clinically sig- tation, were identified in CAPN3 among all families included in the nificant genes in the more than 50 subpopulations identified study. Subsequently, presymptomatic and carrier testing in immedi- nationwide (http://www.igvdb.res.in/). Simultaneously, a second ate family members revealed other affected individuals and carriers. major database, namely, the Indian Genetic Disease Database As a result, targeted mutation testing for the founder mutations (http://www.igdd.iicb.res.in/), was also launched to collect and was launched, and several additional unrelated LGMD families have maintain a comprehensive database of disease-causing mutations been uncovered since the test became available in India (Rashna Das- specific to Indian subpopulations [Pradhan et al., 2011]. While this tur, personal communication). The evaluation and establishment database is an important initial source of various mutations specific of the founder effect of the above CAPN3 mutations have helped to Indian subpopulations, it does not categorize or emphasize the several families avoid the diagnostic odyssey of various tests and significance of common and founder mutations and their clinical spending a substantial amount of money. Similarly, a founder mu- applications. Therefore, in this study, we assembled a comprehen- tation, c.135 136insC in the MLC1 gene, was reported to be causative sive catalog of the more frequent and founder mutations, along with of megalencephalic leukodystrophy in all 31 patients of the Indian the frequencies and subpopulations or communities they have been Agrawal community included in the study [Gorospe et al., 2004]. reported in. We reviewed the literature for all known common and A few others include c.135 136insC in MLC1, causative of cystic founder mutations reported for various genetic disorders in Indian megalencephalic leukodystrophy [Gorospe et al., 2004], c.130C>T populations, searching through NCBI PubMed and Google Scholar (p.R44X) in HMOX1, causative of heme oxygernase-1 deficiency for all studies on founder and common mutations reported in In- (I.C. Varma, personal communication), c.215 216insA in PANK2, dian populations up until May, 2014. The clinical application and causative of Hallervorden-Spatz disease [Aggarwal et al., 2010], significance of such catalogs of common disease-causing mutations and c.2338G>C (p.D780H) c.2051–1G>TinCAPN3, causative of is well appreciated, as in the case of CF [Bobadilla et al., 2002a, LGMD2A [Ankala et al., 2013] (Table 2). Further details includ- 2002b; Castellani et al., 2008]. In fact, Castellani et al. (2008) pub- ing mutation frequencies and references for each of these and other lished a comprehensive catalog of frequent mutations in the CFTR founder and common mutations discussed in this study are included gene observed in various ethnicities and populations worldwide, in the Supporting Information (Supp. Table S1). which has been revolutionary and a very dependable resource for a The next step to take advantage of these founder and common large number of laboratories and clinicians diagnosing CF [Castel- mutations will be the development of a carrier or targeted muta- lani et al., 2008]. We believe the common and founder mutations tion panel, like the one developed for the AJ population [Leib et al., listed here will be equally useful for clinical laboratories and ge- 2005]; several clinical laboratories around the world now offer a car- neticists in India and overseas since many Indian subgroups have rier screening panel for the more frequent and founder mutations immigrated and settled throughout the world. associated with a variety of disorders in this population. These pan- els currently include about 17–20 different diseases, among them CF, storage disorders like Gaucher disease, Tay-Sachs disease, and nema- Recurrent and Founder Mutations Associated with line myopathy. The test involves targeting analysis for the founder Metabolic Disorders mutations in the genes associated with these diseases known to have a carrier frequency as high as one in 14 (Gaucher disease) to one Inborn errors of metabolism comprise a large group of genetic in 100 (Bloom syndrome). Knowledge of these founder mutations diseases characterized by a clinically significant block in a metabolic not only allows for a carrier screening panel that reduces the dis- pathway and resultant accumulation of a substrate and/or deficiency ease carrier risk by more than 100–200-fold, but also allows for the of a corresponding product. With the availability of enzymatic and use of quicker and cheaper methods of testing. Instead of having to other biochemical testing options, diseases that are clinically ac- sequence the entire gene or specific exon, a more variant-targeted tionable, such as phenylketonuria, and storage diseases, such as approach can be adopted, such as allele-specific PCR, SNP arrays, Tay-Sachs disease, Gaucher disease, and mucopolysaccharidoses, and pyrosequencing. With these approaches, the presence or absence are increasingly being diagnosed and characterized. Consequently, of the specific variant here, a founder mutation is specifically ana- this has led to phenotypic characterization and identification of a lyzed without having to sequence the entire exon or PCR product. mutation spectrum, including recurrent and founder mutations in

HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 3 Table 3. Common and Founder Mutations in Metabolic Disorders

Mutation frequency Allele Disease Gene OMIM# Mutation (%) count Region/sect Reference

∗ Wilson disease ATP7B 606882 c.813C>A (p.C271 ) 18.54 23/124 East India Gupta et al. (2005) Wilson disease ATP7B 606882 c.1216T>G (p.S406A) 18.54 23/124 East India Gupta et al. (2005) Wilson disease ATP7B 606882 c.1708–1G>C 9.67 12/124 East India Gupta et al. (2005) Wilson disease ATP7B 606882 c.448 452delGAGGG 5.64 7/124 East India Gupta et al. (2005) 5 Alpha reductase deficiency SRD5A2 607306 c.737G>A (p.R246Q) 100 4/4 Uttar Pradesh Sahu et al. (2009) (5RD) Werner syndrome WRN 277700 c.561A>G(p.(= )) Founder (67) 4/6 Kerala, Tamil Saha et al. (2013) Nadu Gaucher disease GBA 606463 c.1448T>C (p.L483P) Most common NK/48 Not known Bisariya et al. (2011) Gaucher disease GBA 606463 c.1342G>C (p.D448H) Second most common NK/48 Not known Bisariya et al. (2011) Tay Sachs disease HEXA 606869 c.1385A>T (p.E462V) Founder (40) 12/30 Gujarat Mistri et al. (2012) Primary hyperoxaluria type I AGXT 604285 c.302T>C (p.L101P) 100 6/6 North India Chanchlani et al. (2012) G6PD deficiency G6PD 305900 c.131C>G (p.A44G) 13 22/167 Scheduled Sukumar et al. (2004) tribes, Marathas and Brahmins G6PD deficiency G6PD 305900 c.477G>C (p.M159I) 21 6/29 All parts of Sarkar et al. (2010) India G6PD deficiency G6PD 305900 c.592C>T (p.R198C) 21 6/29 All parts of Sarkar et al. (2010) India G6PD deficiency G6PD 305900 c.563C>T (p.S188F) 60.40 101/167 Nation wide Sukumar et al. (2004) G6PD deficiency G6PD 305900 c.949G>A (p.E317K) 24.50% 41/167 Nation wide Sukumar et al. (2004)

The DNA mutation numbering used in this entire manuscript corresponds to cDNA sequence number. Nucleotide number 1 corresponds to the “A” of the translation initiation codon ATG of the reference sequence (the cDNA sequence used for each gene is provided in the Supp. Table S1.) Allele count, number of alleles with mutation/number of alleles tested; NK, not known; founder, founder mutations. various populations. Tay-Sachs disease is a classic lysosomal stor- (>88%) [Carney et al., 2009]. In fact, based on the origin and evo- age disease caused by mutations in the HEXA gene. The infantile lution of the Duarte-2 (D2) allele as laid out by Carney et al. (2009) variety of the disease is a progressive neurodegenerative condition and the high frequency of the D314 allele, along with the 5 GTCA that usually leads to death by 2–3 years of age. Common or fre- repeat contraction (4-bp deletion) [Singh et al., 2012a], it appears quent mutations in this gene have been reported for different pop- that carrier and homozygote frequencies of the D2 allele might be ulations, including the AJ population, where c.1277 1278insTATC, significantly high among Indians, despite the fact that they may not c.1421+1G>C, and c.805G>A (p.G269S) [Kaback et al., 1993] ac- present with classic galactosemia. Combined, these studies suggest count for around 95% of cases; the French-Canadian population that mutations contributing to a majority of classic galactosemia has a common 7.6-kb deletion [Myerowitz and Hogikyan, 1986], cases in India have yet to be identified. On the other hand is Gaucher and the Japanese population has a splice mutation c.571–1G>Tac- disease, which is caused by mutations in the GBA gene and associated counting for 80% of cases [Tanaka et al., 1999]. Whether there are with hepatosplenomegaly, thrombocytopenia, and CNS manifesta- similar common mutations for Indian populations is not fully clear. tions due to the accumulation of toxic glucocerebrosides. According A recent study in individuals with Tay-Sachs from six unrelated to the International Collaborative Gaucher Group (ICGG) registry Gujarati families of India identified a founder mutation c.1385A>T that collects patient data from more than 60 countries, four common (p.E462V) [Mistri et al., 2012] (Table 3). Subsequent carrier screen- mutations in the GBA gene account for about 90% of all reported ing by amplification refractory mutation screening (ARMS) in 500 Gaucher cases. Recent studies in the Indian population identified healthy individuals identified two additional carriers belonging to the same common mutations, c.1448T>C (p.L444P), c.1226A>G different communities [Mistri et al., 2012]. This and other sim- (p.N370S), c.115+1G>A, c.1342G>C (p.D409H), and c.1263del55, ilar studies indicate that certain common mutations are specific to account for about 50% of the Gaucher cases [Bisariya et al., to ethnicity, race, and community, and therefore recommend ini- 2011; Ankleshwaria et al., 2014]. In contrast to Tay-Sachs and galac- tial first-hand screening for community-specific or region-specific tosemia, a more universal molecular screening strategy may be ap- founder mutations. plied for Gaucher disease in Indian populations. These findings Another similar example is the GALT gene, which is associated highlight the significance of characterizing and understanding the with galactosemia. While the classic GALT mutation, c.563A>G mutation spectrum of each disease in each individual population. (p.Q188R), accounts for over 70% of classic galactosemia cases of Northern European descent [Tyfield et al., 1999], this mutation Mutations Associated with Coagulopathies and Blood is probably not that common in the Indian subcontinent, where Disorders the reported frequency is around 3.6% in individuals with classic galactosemia [Singh et al., 2012a, 2012b]. In fact, the mutation is β-Thalassemia is a major hereditary anemia resulting from mu- believed to have arisen in central Europe in the last 20,000 years tations in the β-globin gene HBB and the defective hemoglobin [Flanagan et al., 2010]. Moreover, the variant c.940A>G(referredto production that results [Weatherall et al., 1988]. Both α-andβ- as N314D allele), long considered a mutation accounting for over thalassemias together with sickle cell anemia account for a dis- 40% of Duarte galactosemia in India, is rather a polymorphism, ease frequency of about one in 400 live births [Angastiniotis and with D314 being the ancient allele and D314N being the more Modell, 1998]. Disease and carrier frequencies of thalassemias are recent polymorphic allele common in the European population significantly higher in the African and Indian-Asian subcontinents

4 HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 Table 4. Common Mutations of Coagulopathies and Blood Disorders

Mutation frequency Disease Gene OMIM# Mutation (%) Allele count Region/sect Reference

Bernard–Soulier syndrome GP1BB 138720 c.124 145del22 37.03 20/54 Kerala Sumitha et al. (2011) Bernard–Soulier syndrome GP9 173515 c.70T>C (p.C24R) 22.22 12/54 Kerala Sumitha et al. (2011) Von Willebrand disease 613160 c.2908delC Founder (100) 24/24 Kachi Modh Ghanchi Kasatkar et al. (2013) (community), Gujarat, West India ∗ Von Willebrand disease VWF 613160 c.5335C>T (p.R1779 ) Founder (90) 9/10 Uttar Pradesh (Gaderia Kasatkar et al. (2013) community) B-thalassaemia HBB 141900 c.92+5G>C 54.70 4,652/8,505 North, South, East, and Sinha et al. (2009) West India B-thalassaemia HBB 141900 619-bp deletion 10 850/8,505 North, South, East, and Sinha et al. (2009) West India B-thalassaemia HBB 141900 c.92+1G>T 6.20 527/8,505 North, South, East, and Colah et al. (2004) West India B-thalassaemia HBB 141900 c.124 127delTCTT 6.10 519/8,505 North, South, East, and Colah et al. (2004) West India B-thalassaemia HBB 141900 c.27dupG 5.50 468/8,505 North, South, East, and Colah et al. (2004) West India ∗ B-thalassaemia HBB 141900 c.47G>A(p.W16 ) 5.50 468/8,505 North, South, East, and Colah et al. (2004) West India B-thalassaemia HBB 141900 c.92G>C (p.R31T) 2.60 221/8,505 North, South, East, and Sinha et al. (2009) West India B-thalassaemia HBB 141900 c.-138C>T 46.5 41/88 Jat Sikhs of Punjab Garewal et al. (2005) Haemophilia A F8 300841 Intron 22 inversion 51 51/101 Not known Jayandharan et al. (2005) Haemophilia A F8 300841 Intron 1 inversion 2 2/101 Not known Jayandharan et al. (2005) Haemophilia B F9 300746 c.316G>A (p.G106S) Founder in mild 24/24 Gujarat Quadros et al. (2009) cases (100)

Allele count, number of alleles with mutation/number of alleles tested. due to the selective advantage over malaria [Angastiniotis and Mod- for appropriate treatment options, such as prophylactic factor infu- ell, 1998]. A thorough study done by Sinha et al. (2009) demon- sions and gene therapies that are either currently available or being strates that a common intronic mutation c.92+5G>C(IVS1–5G>C) explored [Ponder, 2011]. accounts for more than 50% of the mutations in Indians [Sinha et al., 2009]. Although more than 100 different mutations have been identified along the length of the gene, the six most common muta- CF and Mutations Common in Indian Subpopulations tions alone account for more than 90% of the thalassemia cases in India (Table 4). As such, most clinical laboratories across the coun- CF, caused by mutations in the CFTR gene, is a complex, multi- try offer an inexpensive targeted test for this handful of mutations, system disease affecting epithelia of the pancreas, intestine, respira- even though HBB is a very small gene with only three exons. tory tract, male genital tract, hepatobiliary system, and the exocrine Hemophilia A is an X-linked recessive bleeding disorder caused sweat glands [Zielenski, 2000]. CF is the most common life-limiting by highly heterogeneous mutations in the coagulation factor VIII autosomal-recessive disorder in the Caucasian population, with a gene, F8. The birth prevalence of hemophilia A is approximately heterozygote frequency of one in 25 [Castellani et al., 2008]. Of over 1:4,000 to 1:5,000 live male births worldwide among most countries 900 known mutations, p.F508del (࢞F508) is the most common, and races [Tuddenham, 1994; Stonebraker et al., 2010a; Stonebraker accounting for about 30%–80% of mutant alleles depending on et al., 2010b]. While several mutations have been described in the the ethnic group [Bobadilla et al., 2002b]. The 23-mutation panel F8 gene, an intron 22 inversion is the most frequent mutation, ac- recommended by ACMG and the extended 39-mutation panel of- counting for nearly half of all reported cases of severe hemophilia fered for carrier testing detect 65%–90% of carriers in the United A, including cases in India [Jayandharan et al., 2005; Bagnall et al., States [Watson et al., 2004]. Until recently, CF was thought to be 2006] (Table4). Hemophilia B, caused by factor IX deficiency, is phe- uncommon in Indians, but more and more cases are being diag- notypically indistinguishable from hemophilia A, but is less frequent nosed. Studies comparing CF mutation frequencies in Indians are (one in 25,000 live male births) worldwide [Bolton-Maggs and Pasi, also being reported, showing that similar to other ethnicities, the 2003]. Mild-to-moderate hemophilia B is most often caused by mis- ࢞F508 mutation is the most common mutation, accounting for as sense mutations rather than the gross gene alterations, frameshift, high as 54% of cases seen in one study [Ashavaid et al., 2005] (Supp. splice-site, or nonsense mutations that are common to the more se- Table S1). The clinical implications and diagnostic applications of vere phenotype. Approximately half of the missense mutations are common and founder mutations in a disease are best illustrated by recurrent, with some clearly representing a founder effect. A single this one disease, the disease frequency and burden of which have missense mutation c.10430G>A (p.G60S) has been associated with been tremendously reduced by the recommended carrier screening all moderately severe-to-mild Indian hemophilia B patients included panel. A similar outcome is expected for all diseases overall, with the in a study (i.e., 22 unrelated individuals) [Quadros et al., 2007]. implementation of a comprehensive carrier panel of the common Since molecular testing not only distinguishes the two hemophilias, or founder mutations compiled in this study. Although implemen- but also allows for carrier and prenatal testing, knowing founder tation of a NSP in India may be imminent, whether diseases like and frequent mutations is beneficial. A confirmed diagnosis allows CF should be included in the screen is still being argued; this makes

HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 5 Table 5. Common Mutations of Ear and Eye Disorders

Mutation Disease Gene OMIM# Mutation frequency (%) Allele count Region/sect Reference

Nonsyndromic hearing loss GJB2 121011 c.71G>A 87 52/60 Not known Padma et al. (2009) (p.W24∗) 73 187/256 Shankar Mani et al. (2009) Nonsyndromic hearing loss GJB2 121011 c.35delG 13.04 3/23 Karnataka, Tamil Maheshwari et al. (2003) Nadu, and Delhi Nonsyndromic hearing loss GJB2 121011 c.235delC 3.30 2/60 Not known Padma et al. (2009) Oculocutaneous albinism TYR 606933 c.832C>T Founder (100) 52/52 Tili group of East Chaki et al. (2005) (OCA1) (p.R278∗) India, West Bengal Primary congenital glaucoma CYP1B1 601771 c.1103G>A 15.41 45/292 Not known Reddy et al. (2003) (p.R368H) Primary congenital glaucoma CYP1B1 601771 c.685G>A 2.39 7/292 Not known Reddy et al. (2003) (p.E229K) Primary congenital glaucoma CYP1B1 601771 c.29 30insA 18 8/46 North India Tanwar et al. (2009) Primary congenital glaucoma CYP1B1 601771 c.1168C>T 16 7/46 North India Tanwar et al. (2009) (p.R390C), c.1168C>A (p.R390S), c.1169G>A (p.R390H) Primary open-angle MYOC 601652 c.1109C>T Multiple reports Reported in Kanyakumari Rose et al. (2007); Bayat glaucoma (p.P370L) various district, South et al. (2008) ethnicities India around the world Primary open-angle MYOC 601562 c.144G>T 44 4/9 South, East, West, Chakrabarti et al. (2005) glaucoma and PCG (p.Q48H) and North India Lattice corneal dystrophy TGFB1 190180 c.417C>T 57 8/14 Not known Chakravarthi et al. (2005) (p.R124C) Granular corneal dystrophy TGFB1 190180 c.1710C>T 94.70 18/19 Not known Chakravarthi et al. (2005) (p.R555W)

Allele count, number of alleles with mutation/number of alleles tested. offering a carrier screening panel for these disorders even more atrophy, anomalously deep anterior chamber, and structurally nor- crucial [Kapoor and Kabra, 2010]. mal posterior segment, except for progressive glaucomatous optic atrophy [Azmanov et al., 2011]. CYP1B1, the gene encoding cy- tochrome P450 1B1, and LTBP2, which encodes latent-transforming Mutations in Ear and Eye Disorders growth factor beta-binding protein 2, are the only currently known Hearing loss is the most common sensory disorder in humans. genes with mutations that cause PCG [Stoilov et al., 2002]. Two other The severity of hearing loss can be mild to profound, affecting loci, GLC3B on 1p36 and GLC3C on 14q24.3, have been linked to anywhere from low-to-high frequencies. One in 500 newborns is af- PCG, but the causative genes are not known [Stoilov et al., 2002; fected with bilateral permanent sensorineural hearing loss 40 dB or Firasat et al., 2008]. While more than 100 different mutations have greater, and the frequency increases sevenfold among adolescents, to been reported in the CYP1B1 and LTBP2 genes, some mutations seven in 500 [Morton and Nance, 2006]. About two-thirds of hear- are more common and specific to certain ethnic groups, such as ing loss is due to genetic factors, with the remaining third caused by those of the Roma Slovakian, Saudi Arabian, and Gypsy popula- tions [Bejjani et al., 1998; Ali et al., 2009]. Interestingly, the common environmental factors [Hilgert et al., 2009; Raviv et al., 2010]. Muta- ∗ LTBP2 mutations, c.412delG, c.895C>T (p.R299 ), c.1243–1256del, tions in the GJB2 gene encoding connexin 26, which cause DFNB1, ∗ are the most common cause of hearing loss and account for about and c.331C>T (p.Q111 ), reported in the Gypsy populations, the 50% of cases with autosomal-recessive hearing loss in many popu- founders of which are known to have migrated out of the Indian lations [Kochhar et al., 2007; Cohen and Phillips, 2012]. Mutation subcontinent centuries ago, have been reported in individuals from frequencies for the various GJB2 mutations in the Indian popula- Pakistan but not India, to date [Ali et al., 2009]. On the contrary, tion are listed in Table 5. Primary congenital glaucoma (PCG) is completely different mutations contribute to the majority of PCG the most common type of childhood glaucoma, with autosomal- cases in India (Table 5). In addition, a single mutation c.1149G>A recessive inheritance and an incidence ranging from one in 30,000 (p.R368H) accounts for about 18% of individuals with PCG in In- to one in 1,250. Specifically, the birth prevalence is one in 5,000– dia. Likewise, other frequent and well-known mutations include the 22,000 in Western countries, one in 2,500 in the Middle East, one c.144G>T mutation in MYOC that causes both primary open-angle in 1,250 in the Roma (Gypsy) population of Slovakia, and one in glaucoma and PCG [Chakrabarti et al., 2006] (see Table 5). 3,300 in the Indian state of Andhra Pradesh, where the disease ac- counts for approximately 4.2% of all childhood blindness [Dandona Mutations in Other Disorders et al., 2001; Dandona and Dandona, 2001]. PCG is characterized by elevated intraocular pressure, enlargement of the globe (buphthal- LGMDs are characterized by wide genetic and clinical heterogene- mos), edema, opacification of the cornea with rupture of Descemet’s ity. Because of the heterogeneity of LGMD and the lack of diagnostic membrane (Haabs striae), thinning of the anterior sclera and iris specificity, there are few reports on its prevalence. Establishing the

6 HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 Table 6. Common Mutations in Other Disorders

Disease Gene OMIM# Mutation Mutation frequency (%) Allele count Region/sect Reference

∗ Megaloblastic SLC19A2 603941 c.196G>T(p.E66 ) 62 16/26 Kashmir and Punjab Ricketts et al. (2006) anemia syndrome Hydatidiform mole NLRP7 609661 c.2078G>C (p.R693P) Founder (64) 9/14 North India Slim et al. (2009) Hydatidiform mole NLRP7 609661 c.2738A>G (p.N913S) Founder (36) 5/14 North West India Slim et al. (2009) LGMD2C SGCG 608896 c.525delT 43.70 7/16 Mumbai, West India Khadilkar et al. (2009) LGMD2C SGCG 608896 c.848G>A (p.C283Y) 100 36/36 Gypsies migrated Piccolo et al. (1996) from India Ovarian cancer BRCA1 113705 c.68 69delAG 16.40 10/61 Kerala, Karnataka, Vaidyanathan et al. (2009) Andhra Pradesh, and Tamil Nadu Hypohidrotic EDAR 604095 c.1144G>A (p.G382S) Founder (42) 10/24 Not known Bashyam et al. (2012) ectodermal dysplasia ∗ Tricho-Hepato- TTC37 614589 c.2808G>A (p.W936 ) Founder (100) 4/4 Gujarati Kotecha et al. (2012) Enteric syndrome Progressive pseu- WISP3 603400 c.1010G>A (p.C337Y) Founder (40) 20/50 South India Dalal et al. (2012) dorheumatoid dysplasia Combined pituitary PROP1 601538 c.112 124 del13 Founder (9.8) 15/153 Indian subcontinent Turton et al. (2005) hormone deficiency Haim–Munk CTSC 602365 c.2127A>G (p.Q286R) Founder (100) 100/100 Jews from Cochin Hart et al. (2000) syndrome and immigrated to Papillon–Lefevre Israel syndrome Congenital adrenal CYP21A2 613815 c.293–13A/C>G. 38.7 48/124 Delhi Marumudi et al. (2012) hyperplasia 28.2 13/46 Mathur et al. (2001) Congenital adrenal CYP21A2 613815 c.515T>A (p.I172N) 21 26/124 Delhi Marumudi et al. (2012) hyperplasia 31.8 15/46 Mathur et al. (2001) ∗ Congenital adrenal CYP21A2 613815 c.952C>T (p.Q318 ) 28.2 35/124 Delhi Marumudi et al. (2012) hyperplasia 22.7 10/46 Mathur et al. (2001) ∗ Growth hormone GHRHR 139191 c.214G>T(p.E72 ) Founder (35) 34/97 West India Desai et al. (2013) deficiency

Allele count, number of alleles with mutation/number of alleles tested. type of LGMD can be useful when discussing the clinical course of be used for carrier screening of 500 mutations, or even more [Tanner the disease; however, this can be very expensive given the number of et al., 2014]. Targeted mutation assays, because they are targeted, can genes involved, the clinical overlap of the disease subtypes, and the be much better than whole-exome sequencing or whole-genome se- lack of common mutations for targeted testing. A handful of com- quencing methodologies for overcoming several issues, such as low mon and founder mutations have been reported for LGMDs, which coverage, high turnaround times, data storage and processing chal- include the c.525delT in the γ -sarcoglycan (SGCG) gene [Khadilkar lenges, and incidental findings. While many smaller laboratories et al., 2009] (Table 6) and c.2338G>C (p.D780H) and c.2051–1G>T may not be able to afford to offer these high-throughput assays, in the CAPN3 gene (Table 2). The founder effect and clinical impact affordable low-cost tests based on techniques such as PCR-RFLP, of these founder mutations in CAPN3 were discussed earlier. In addi- ARMS-PCR, and allele-specific oligonucleotide blot analysis may tion, some recent studies have suggested that Indian subpopulations be widely offered. Currently, multiple research laboratories in In- harbor certain unique variants in genes including DOK5, MSTN, dia each working on a particular gene of interest offer these tests CLOCK,andPPARA, and analysis of these variants may predict an individually. More comprehensive panel tests are available through individual’s predisposition to complex metabolic disorders, such as only a handful of laboratories in the country. In most laborato- type 2 diabetes and lipid metabolism disorders [Metspalu et al., ries in India, sequencing an entire gene for detection of sequence 2011]. Thus, on the one hand, there are all the founder mutations variants is not offered routinely, both due to limitations in test de- associated with just certain small subpopulations, but there are also velopment and interpretation, as well as for the cost associated with these variants with selective advantage that have been evolutionarily performing the test. While analysis of a single variant (such as a retained and so are common among the different subpopulations known founder mutation) costs about 1,500 INR (about 25–30 US in the country. dollars), the price of sequencing a gene as small as 10 exons long can be more than three times that price. In addition, the turnaround Test Development and Clinical Applications time can vary from a week for the known mutation detection to 4–5 weeks for a complete gene. Other traditional assays, such as the With the rapid advances in sequencing methodologies and next- PCR-RFLP and ARMS-PCR that can be applicable for detecting the generation sequencing capabilities, more affordable and targeted presence or absence of a certain variant, can provide even cheaper tests can be designed and offered clinically. High-throughput auto- screening tests, thereby underscoring the clinical significance of re- mated testing protocols, such as microarray-based chip assays, can current and founder mutations. Providing a catalog of founder and

HUMAN MUTATION, Vol. 36, No. 1, 1–10, 2015 7 common mutations in the Indian population and their contribution ACOG Committee on Genetics. 2009. ACOG Committee Opinion No. 442: preconcep- to associated diseases will shed light on their clinical significance and tion and prenatal carrier screening for genetic diseases in individuals of Eastern provoke development of a carrier screening test, eventually reducing European Jewish descent. Obstet Gynecol 114:950–953. Aggarwal A, Schneider SA, Houlden H, Silverdale M, Paudel R, Paisan-Ruiz C, Desai S, disease burden. Munshi M, Sanghvi D, Hardy J, Bhatia KP, Bhatt M. 2010. Indian-subcontinent NBIA: unusual phenotypes, novel PANK2 mutations, and undetermined genetic forms. Mov Disord 25:1424–1431. Implications of Founder Mutations Ali M, McKibbin M, Booth A, Parry DA, Jain P, Riazuddin SA, Hejtmancik JF, Khan SN, Firasat S, Shires M, Gilmour DF, Towns K, et al. 2009. Null muta- Founder populations have always been an invaluable resource tions in LTBP2 cause primary congenital glaucoma. Am J Hum Genet 84:664– for molecular geneticists, enabling them to determine the molecu- 671. lar basis of Mendelian disorders [Risch et al., 1995; Arcos-Burgos Angastiniotis M, Modell B. 1998. Global epidemiology of hemoglobin disorders. Ann N Y Acad Sci 850:251–269. and Muenke, 2002]. Several founder populations have been stud- Ankala A, Kohn JN, Dastur R, Gaitonde P, Khadilkar SV, Hegde MR. 2013. Ancestral ied extensively to explore this potential, as well as to understand founder mutations in calpain-3 in the Indian Agarwal community: historical, the implications of founder events and mutations, such as in the AJ clinical, and molecular perspective. 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