The Mutant Project: Inside the Global Race to Genetically Modify Humansby Eben Kirksey. St. Martins Press, November 2020. Excerpt previously published by Black Inc.

Surreal artwork in the hotel lobbya gorilla peeking out of a peeled orange, smoking a cigarette; an astronaut riding a cyborg giraffewas the backdrop for bombshell news rocking the world. In November 2018, Hong Kongs Le Mridien Cyberport hotel became the epicenter of controversy about Jiankui He, a Chinese researcher who was staying there when a journalist revealed he had created the worlds first edited babies. Select experts were gathering in the hotel for the Second International Summit on Human Genome Editinga meeting that had been called to deliberate about the future of the human species. As CNN called the experiment monstrous, as heated discussions took place in labs and living rooms around the globe, He sat uncomfortably on a couch in the lobby.

He was trying to explain himself to Jennifer Doudna, the chemist at UC Berkeley, who is one of the pioneers behind CRISPR, a new genetic-engineering tool. Doudna had predicted that CRISPR would be used to direct the evolution of our species,* writing, We possess the ability to edit not only the DNA of every living human but also the DNA of future generations. As He went through his laboratory protocol, describing how he had manipulated the genes of freshly fertilized human eggs with CRISPR, Doudna shook her head. She knew that this moment might be coming someday, but she imagined that it would be in the far future. Amid the bustle of hotel guests, science fiction began to settle into the realm of established fact.

St. Martins Publishing Group

I was checking in to Le Mridien as the story broke and first heard rumors about Hes babies while chatting in the elevator with other summit delegates. We had come to Hong Kong to discuss the science, ethics, and governance of CRISPR and an assortment of lesser-known tools for tinkering with DNA. Struggling to overcome intense jet lagfresh off planes from Europe, the United States, and other parts of Asiawe listened to speculation in the hotels hallways while swimming through reality, caught between waking and dreaming.

Opening the door to my hotel room, a luxury suite courtesy of the U.S. National Academy of Sciences, I hunted for reliable sources of information online. I had been invited to speak on the research ethics panel, after Jiankui He, so I needed to play catch-up, fast. I found YouTube videos posted by Hes lab just hours before, offering details of the experiment. Posing in front of his laboratory equipment, with a broad smile on his face, He announced to the world: Two beautiful little Chinese girls, named Lulu and Nana, came crying into this world as healthy as any other babies a few weeks ago. The experiment aimed to delete a single gene with CRISPR. This new technique of genetic surgery, He claimed, could produce children who were resistant to the HIV virus.

Hunched over the glowing screen of my laptop, I perused the opinions that were just starting to form. Chinese media pundits suggested that a Nobel Prize might be in the making, saying that He was following in the footsteps of scientists who produced the first controversial test-tube baby in 1978. A raucous debate was taking place on WeiboChinas prominent social media platformas 1.9 billion people viewed the hashtag # (#FirstGeneEditedHIVImmuneBabies). Some Chinese influencers were praising Jiankui He as a national scientific hero. Others condemned him, saying that it was shameful to treat children like guinea pigs. Journalists were starting to discover Dr. Hes ties to biotechnology companiesone reportedly worth US$312 millionand alleged that there were serious financial conflicts of interest.

Anyone who follows the news knows the basic story. Over the next few days, Jiankui He experienced a meteoric rise to fame, followed by a dramatic fall from grace. Eventually, he lost his university job and was thrown in jail. A district court in China sentenced him to three years in prison for practicing medicine without a license, denouncing his pursuit of personal fame and profit.

Dr. Hes story is a gateway into a much bigger enterprise: the tale of CRISPR and the emergence of genetic medicine. The gala was quietly abuzz with news of other efforts to genetically modify humans. Experiments were already underway in England, the United States, and many other labs in mainland China. As billionaires and Wall Street investors were getting in on the action, as scientists and doctors were making careers out of CRISPR, I wondered: Who counts as a visionary, and who becomes a pariah?

He spoke about his gene-editing experiment that led to the birth of twin girls while at a summit in Hong Kong in 2018. VOAIris Tong/Wikimedia Commons

He was not alone in the pursuit of fame and fortune. It seemed like none of the scientists at the gala were innocent of financial conflicts of interest. Collectively, these enterprising biologists had already raised hundreds of millionsfrom venture capitalists, big pharma companies, and the stock marketfor genetic engineering experiments in human patients. I overheard excited chatter about new investment opportunities. The first gene therapy, a cancer treatment, had recently been approved in the United Stateswith a US$475,000 price tag. While the scientists gushed about the CRISPR revolution, I was quietly thinking about how genetic medicine is producing other upheavals in society. Profit-driven ventures in research and medicine were producing a new era of dramatic medical inequality.

As market forces propelled CRISPR into the clinic, I set out to answer basic questions about science and justice: Who is gaining access to cutting-edge genetic medicine? Are there creative ways to democratize the field? Panning out, I also explored questions that could have profound implications for the future of our species: Should parents be allowed to choose the genetic makeup of their children? How much can we actually change about the human condition by tinkering with DNA?

As a cultural anthropologist, I have often found myself opposing biologists in debates about human nature. Ever since Margaret Mead wrote her 1928 classicComing of Age in Samoa, anthropologists have argued that a persons life is shaped by the social environment in which each is born and raised rather than genetic heredity alone.Anthropologists have recently joined other progressive thinkers to imagine how science has enabled new experimental possibilities for human beings.Now we are studying how the human social environment has been shaped by synthetic chemistry, smartphones, the internet, and biotechnology.

My goal has been to map how genetic engineering will transform humanity. Rather than limit my research to a single culture, I followed CRISPR around the globe. I tracked the impact of this gene-editing tool as it traveled from media reports to laboratories, through artificial intelligence algorithms, and into the cells of embryos and the bodies of living people. Using an anthropological lens, I examined new forms of power as scientists, corporate lobbyists, medical doctors, and biotechnology entrepreneurs worked to redesign life itself.

I will offer you a mosaic portrait. This is a story of people and concerns on either side of the dynamics of power that has emerged with CRISPR. I moved among the powerful in their native habitats: conferences, fancy hotels, restaurants, corporate offices, and cluttered labs. To understand how social inequality is changing in this brave new world, I also interviewed chronically ill patients, disabled scholars, and hackers. From the power centers to the margins, I went where I could find answers. Very old conflicts were playing out even as new technologies transformed science and medicine.

An exhibit on reproductive technologies at the China National GeneBank envisions a future where robots rear human embryos. Eben Kirksey

When I set out to meet some of the first genetically modified people, I f
ound activists who were battling insurance agents and biotechnology companies for potentially lifesaving treatments. Nearly a decade before Dr. He stirred up controversy in China, a small group of HIV-positive gay men in the United States quietly participated in a clinical trial dubbed the first-in-man gene-editing experiment. Researchers aimed to delete a gene from these menthe same DNA sequence later targeted by Hein hopes of engineering resistance to the virus and repairing damage to their immune systems from AIDS. One veteran HIV activist who participated in this study, Matt Sharp, convinced me that having his DNA altered wasnt a big deal and that genetic engineering does indeed have real medical promise. Sharp also confirmed my suspicions: Biotech companies are putting profits ahead of human health as they search for lucrative applications of gene editing in the clinic.

Gene editing is not a particularly good metaphor for explaining the science of CRISPR. With a computer, I can easily cut and paste text from one application to another, or make clean deletionsletter by letter, line by line. But CRISPR does not have these precise editorial functions. CRISPR is more like a tiny Reaper drone that can produce targeted damage to DNA. Sometimes it makes a precision missile strike, destroying the target. It can also produce serious collateral damage, like a drone attack that accidentally takes out a wedding party instead of the intended target. Scientists often accidentally blast away big chunks of DNA as they try to improve the code of life. CRISPR can also go astray when the preprogrammed coordinates are ambiguous, like a rogue drone that automatically strikes the friends, neighbors, and relatives of suspected terrorists. CRISPR can persist in cells for weeks, bouncing around the chromosomes, producing damage to DNA over and over again every time it finds a near match to the intended target.

How much can we actually change about the human condition by tinkering with DNA?

It is important to signal a sense of risk or a need for caution in using CRISPR. Other metaphorslike genetic surgery or DNA hackinghave been proposed to replace the idea of editing. The idea of genetic surgery suggests that there can be a slip of the surgeons knife, creating an unintended injury. Each of these imagesthe targeted missile, the surgeons scalpel, the hackers codeoffers a perspective on how CRISPR works, even while concealing messy cellular dynamics. In the absence of a perfect metaphor, ultimately, I think that technical language describes it best: CRISPR is an enzyme that produces targeted mutagenesis.

In other words, CRISPR generates mutants.

Strictly speaking, we are all mutants. At a molecular level, each of us is unique. Each of us starts life with 4080 new mutations that were not found in our parents. From birth, each of us has around 20 inactive genes from loss-of-function mutations. During the course of a normal human life, we also accumulate mutations in our bodies, even in our brains. By the time we reach age 60, a single skin cell will contain between 4,000 and 40,000 mutations, according to a study in theProceedings of the National Academy of Sciences. These genetic changes are the result of mistakes made each time our DNA is copied during cell division or when cells are damaged by radiation, ultraviolet rays, or toxic chemicals. Generally, mutations arent good or bad, just different.

Mutants in popular culture play important roles in our high-tech myths. Some cartoons simply celebrate mutation as whimsical possibility. The pizza-eating Teenage Mutant Ninja Turtles are known for fighting crime in support of established law and order. Darker speculative fiction uses mutants to illustrate the hypocrisy and inhumanity of the scientific establishment. Violent experiments on children who were born with special abilities feature in recent Netflix series likeStranger Things. Horror flicks and video games featuring mindless zombies and flesh-eating mutants have a common theme: Science could create monsters that cannot be controlled.

Reporters who sounded the alarm about Lulu and Nanas birthcalling them freaky CRISPR Frankenbabiesclearly had not done their literary homework. Frankensteins monster is now popularly imagined as a dimwitted giant with electrodes in his neckfollowing imagery from the first black-and-white film, put out by Universal Pictures in 1931. The originalFrankenstein, Mary Shelleys gothic novel from 1818, described a superhuman creature that was driven by the desire to be loved. The highly intelligent, articulate, and high-minded creature only turned violent when he was shunned by human society. Amid the controversy about Dr. Hes experiment, a political theorist and literary scholar named Eileen Hunt Botting defended the rights of genetically modified children to live, love, and flourish. Flipping the mainstream script, she wrote an essay for TheWashington Postsuggesting that Frankenstein is an apt cautionary tale about the possibility of devastating discrimination against a bioengineered child.

Some media reports on Lulu and Nana, the first known gene-edited human babies, referenced the science-fiction character Frankenstein (shown here from the film by that name). Universal Pictures/Wikimedia Commons

During my international adventures in the world of CRISPR research, I kept science fiction classics close at hand. The rich archive of speculative fiction has helped me understand the perils and potential of experiments that are remaking the human species.

Scientists have identified some geneslike those associated with eye and skin colorthat would be relatively easy to manipulate. One Russian American gene-editing expert, Fyodor Urnov, intimated that it should be biologically possible to engineer soldiers or athletes with enhanced endurance, speed, and muscle mass. Genetic enhancements come with serious health risks, but military leaders have a long history of ignoring the health and well-being of their soldiers. Fertility clinics also have a bad track record as profit-driven enterprises, ready to sell couples expensive and scientifically unproven treatments. The New Hope Fertility Center in Manhattan is already advertising a new technique: Couples could soon have the opportunity to create designer babies with CRISPR.As scientists speculate about post-racial futures and nightmare military scenarios, as market forces bring new genetic technologies into the clinic at a dizzying speed, it is time to slow down and establish some clear rules for the road. Misguided attempts to improve the human species have already produced atrocitieslike the Nazi death camps that systematically eliminated homosexuals and Jews from the population. In the wrong hands, CRISPR could have devastating consequences for humanity.

This excerpt has been edited slightly for style and length.

* Clarification: This quote comes from A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution, written by Jennifer Doudna and Samuel Sternberg.

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CRISPR Mutants - The Dawn of CRISPR Mutants - SAPIENS - SAPIENS

Genetically modified crops could provide a solution to the world hunger problem, but how serious are the risks for our ecosystems?pixnio.com

Just over 20 years ago, agroup of environmental activistsdestroyed experimental GM maize being grown on a Norfolk farm in a landmark act of protest, which brought genetically-engineered crops into the public eye, and was followed by global demonstrations and the adoption of severely restrictive legislature by the EU. Whilst some of the major food-producing countries of the world have become more open to genetically-engineered crops, public attitudes still remain largely hostile. In the UK, 40% of adults surveyed in 2012 believed the government should not be endorsing the use of genetically-engineered crops. These expressions of distrust largely stem from a lack of understanding surrounding genetically-engineered crops asurvey in 2019 found that only 32% of UK adults felt informed about GM crops, and misinformation spread by anti-GMO campaigns has done nothing to alleviate this.

In reality, the facts of genetic engineering are far simpler than such campaigns would make them appear.

In reality, the facts of genetic engineering are far simpler than such campaigns would make them appear. Earlier efforts mainly relied on the use of the bacterium Agrobacterium tumefaciens to introduce foreign DNA into the genome of a plant embryo, and the use of antibiotic-resistance marker genes to select transformed plants. This initially gave rise to fears of spreading antibiotic resistance through genetic engineering, although these marker genes have generally been replaced by plant-derived markers in the transformation process.

With the advent of CRISPR-Cas9 technology, however, engineering of plant genomes has become significantly easier. CRISPR-Cas9 utilises a mechanism found in prokaryotic immune systems, in which characteristic DNA sequences of potentially harmful bacteria are stored in a cluster of sequences, known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). These sequences can be transcribed and used to guide the DNA-cleaving activity of the Cas9 protein in genetic engineering, guide RNA for a locus in the plant genome is used to target cuts. CRISPR-Cas9 technology is proving crucial in genetic engineering, thanks to the ease with which endogenous genes can be edited without inserting foreign DNA, which helps the public image of genetically-engineered crops.

But whilst health risks of genetically-engineered crops on the market have been rigorously examined and disproved, these crops are not without their faults. One of the greatest risks posed by transgenic crops is the potential for transgene flow into wild crop relatives, potentially conferring pest or herbicide resistance. Whilst experimental crops are isolated to reduce this risk, this is often not possible for commercial crops, and evidence suggests some small-scale spread of transgenic traits occurring around fields of transgenic crops. The difficulty in preventing transgene spread, however, is that the methods used for instance, pollen sterility can prevent farmers from harvesting and replanting seeds, forcing them to repeatedly buy expensive seed from the developers. This may create a financial barrier to the benefits of such crops for those who might need them most.

Yet with the world population set to hit 8.1 billion by 2025, global solutions are now required to meet the challenges of feeding the growing population in an increasingly adverse climate. Given that roughly 37% of habitable land area is already used in agriculture, the capacity for further expansion is limited, and so increasing the efficacy of crop growth is therefore needed to meet demand. This will likely require the rapid improvement of crops through genetic engineering, with advances in adapting existing plant responses to abiotic stress for instance, increasing the production of osmoprotectants that protect protein structure in drought conditions likely to prove crucial in improving crop productivity whilst minimising strain on land and water resources.

Despite the risks, the improving reliability of transgeniccrop isolation and the benefits of genetically-engineered crops make a compelling case for extending their use. This is especially true for countries experiencing massive population growth, which often also bear the brunt of climate change so what is hindering this?

You dont have to look any further than the case of Golden Rice for the answers. The poster child of the genetically-engineered crop movement, Golden Rice was initially developed in the early 2000s as a transgenic rice strain with aVitamin A content sufficient to provide 80-100% of the RDI in a single cup of rice. This was a solution developed to combat the lack of the vitamin in the diets of many developing countries, with a third of children worldwide estimated to be Vitamin A deficient, leaving them at high risk of death or blindness. Given repeated testing proving both the efficacy and the safety of the rice, it would seem a foregone conclusion that its use in filling the coverage gaps in vitamin supplement distribution would be widely approved. Yet to this day, not a single crop of Golden Rice has been grown outside of experimental trials.

The reasons for this can be traced back to the legislation governing genetically-engineered crops, such as the Cartagena Protocol, which prevents the introduction of new biotechnology should it pose a risk to human or environmental health. Despite very low rates of gene flow from cultivated rice to wild species, and limited evidence to suggest the transgene would persist in wild populations, this protocol was used to ban the introduction of Golden Rice in the EU, which, in conjunction with Greenpeace campaigns, fed fears surrounding the unsafe nature of the crop. However, rulings in recent years appear to be turning the tide; earlier approval from the health authorities of the US, Australia, New Zealand and Canada has been followed by approval in the Philippines and impending approval in Bangladesh, which hopefully signals the start of Golden Rice growth in countries affected by Vitamin A deficiency.

The challenge for the future lies mainly in the general publics understanding and perception of genetic engineering

Although progress is being made in the introduction of genetically-engineered crops, the future of research and development in crop engineering is looking dim. With recent reclassification of GM crops by the EU to include gene-edited crops, those edited using CRISPR-Cas9 are now as severely restricted as transgenic crops. This comes at a time when effective solutions for food production are needed more than ever, and so immediate action is needed if genetically-engineered crop development is to continue. The challenge for the future lies mainly in the general publics understanding and perception of genetic engineering; if improved, this could have considerable influence in producing a more considered approach to GM crop legislation cutting the red tape and allowing the benefits of genetically-engineered crops to reach those most in need.

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Introduction

Heterogeneous drug response is the major hurdle in the successful treatment of diseases, which is due to genetic variations in the drug metabolizing enzyme genes. Knowledge of allelic frequency distribution of drug metabolizing enzymes within populations can be useful to identify risk groups for adverse drug reaction and to optimize drug doses. It can be utilized to select representative populations in clinical trials. The cytochrome P450 (CYP) family is an important enzyme of ADME (related to absorption, distribution, metabolism and excretion of drug) genes, of which CYP2C9 is the major constituent of CYP2C subfamily in the human liver. It metabolizes a wide range of drugs including anticoagulant (warfarin), nonsteroidal anti-inflammatory (celecoxib, diclofenac), antidiabetic (nateglinide, tolbutamide), antihypertensive (irbesartan, losartan) and anti-epileptic (phenytoin).1 Several variations in CYP2C9 have been reported, which affect metabolism of the drug. Most notable variations are CYP2C9*2 (R144C) and CYP2C9*3 (I359L), which significantly decreases enzyme activity.2 Interestingly, these variations are highly heterogeneous among world population; (1) 819% and 3.316.3% in Caucasian; (2) 00.1% and 1.13.6% in Asian; (3) 2.9% and 2.0% in African-American; and (4) 04.3% and 02.3% in Black/African, respectively.3 In addition, other rare and functionally relevant variations were also reported in various populations, which includes; (1) CYP2C9*6, 0.6% frequency in African-Americans;4 (2) CYP2C9*4, 0.5% in African-Americans and 6% in Caucasians;2,5 and (3) CYP2C9*13, 0.190.45% in Asian.6 Dai et al reported several rare variants in the Han Chinese population.7

Several studies have been performed on CYP2C9 in Indian populations. However, most of studies have focused only on CYP2C9*3 and CYP2C9*2 variants. Grik et al observed CYP2C9*3 only in the Indo-European population (0.381.85%), whereas it was absent in Dravidian, Austroasiatic and Tibeto-Burman populations.8 Indian populations are well known for their genetic diversity and practice of endogamy, hence they are expected to have high frequency of homozygous allele9. Many studies have shown that the variations in CYP2C9 are associated with therapeutic heterogeneity in Indian populations. CYP2C9*2 and *3 has been reported with less hydroxylation (or metabolism) of phenytoin in vivo in South Indian populations,10 compared to wild type CYP2C9*1. Ramasamy et al reported phenytoin toxicity in a patient with normal dose of 300 mg/day, who had CYP2C9*3/*3 genotype.11 The same symptoms were also reported by Thakkar et al in South Indian populations.12 Both of these drugs are metabolized by CYP2C9. Some of the drugs, metabolized by CYP2C9 have narrow therapeutic index eg warfarin, phenytoin, and tolbutamide. This is the reason that small change in the metabolizing activity of CYP2C9 may cause major changes in an individuals response against a drug. Considering this, we explored genetic diversity of functionally relevant variations of CYP2C9 within the Indian subcontinent and compared with other world populations. The outcome of this study may be useful to understand heterogeneous therapeutic response and development of personalized therapy for the populations of Indian subcontinent. Moreover, identification of South Asian-specific putative functional variants and associated haplotypes will open opportunity for further study.

A total of 1278 samples from 36 diverse Indian populations, in terms of ethnicity, linguistic and geographical locations, were included in this study (Table 1).9,13 Furthermore, 210 samples of South Asian origin were selected from our collection of whole genome/exome datasets. For comparison, 489 and 598 samples of South Asian origin were selected from the 1000 Genomes Project and GenomeAsia 100K Project, respectively.14,15 This work has been approved by the Institutional Ethical Committee of CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India. Informed written consent has been obtained from all the participants. The present study is conducted in accordance with the Declaration of Helsinki.

Ten milliliter intravenous blood samples of subjects were collected in an EDTA vacutainer, after obtaining informed written consent. Genomic DNA was extracted from whole blood, using the protocol described previously.16 These steps were followed for all samples which were subjected to either Sanger sequencing or next-generation sequencing (exome/genome).

All the nine exons, their respective intron-exon boundary, 3 and 5 UTR of CYP2C9 have been re-sequenced. For designing of primer, DNA sequence of ENST00000260682 from Ensembl (v75) has been used. Out of 3 mRNA of CYP2C9, only ENST00000260682 translate to protein. Primer3.0 web-based tool (http://simgene.com/Primer3) was used for designing the primers and further primers specificity were checked with NCBI-primer blast. The details of primer sequences are given in Supplementary Table 1. Polymerase chain reaction (PCR) was performed in 10.0 L volume, which contains 5.0 L of 2 EmeraldAmp GT PCR master mix, 10.0 ng of genomic DNA and 0.1 p mole (final concentration) of each primer. Thermal cycling conditions used are as follows: initial denaturation step of five minutes at 94C, followed by 35 cycles of denaturation step of 30 seconds. at 94C, annealing step of 30 seconds. at 55C, extension step of two minutes at 72C, followed by single step of final extension of seven minutes at 72C. PCR products were cleaned with Exo-SAP-IT (USB, Affymetrix, USA) with recommended protocol of the manufacturer. Cleaned PCR products (1.0 L) were subjected to sequencing using BigDye terminator (v3.1) cycle sequencing kit (Thermo Fisher Scientific, USA) and analyzed using ABI 3730XL DNA Analyzer. Sequences were edited and assembled using AutoAssembler (v1.0) software. Statistical analysis was performed using R packages. Gap package was used to calculate HWE equilibrium. The 95% confidence interval of allelic and genotypic percentage was calculated with ClopperPearson and SisonGlanz method using DescTools package of R. Surfer trial version (18.1.186) was used to interpolate frequency spectrum with Kriging gridding method and plots were generated using maps and spaMM package of R.

For whole genome and exome sequencing, libraries were prepared as per manufacturers protocol using Illumina Nextera DNA Flex Library Prep kit and Illumina TrueSeq DNA LP for enrichment kit, respectively. Sequencing of above library was performed on Illumina NovaSeq 6000 system. On an average of 30 and 100 coverage was generated for the whole genome and exome, respectively.

The sequencing data from all the samples was trimmed for adapters using Cutadapt (v2.7). The whole-genome datasets were aligned and processed to call variants using the pipeline of DRAGEN (v3.6.3), a Bio-IT platform for genome sequence data analysis. In case of whole-exome datasets, reads were aligned using the BWA tool (v0.7.10) and variants were called using the recommended pipeline of GATK4. The human reference genome version GRCh38 was used for the alignments of reads. The BCF tool was used to extract variants present in the CYP2C9. In the next step, all VCF files were combined with option CombineGVCFs of GATK. Variants were annotated using Variant Effect Predictor tool of Ensembl (v95.3). For phasing of the variants, PopgenPipeline Platform (PPP) was used with PHASE algorithm of BEAGLE. Novel haplotypes obtained in the current study are deposited to PharmVar (https://www.pharmvar.org/).

The A>C (rs1057910/CYP2C9*3) is a non-synonymous mutation, which replace isoleucine with leucine (ATT>CTT; Ile359Leu) and decreases enzyme activity. To explore the C allele frequency in Indian populations, initially we confirmed HardyWeinberg equilibrium (HWE). It was observed that 11 populations were not in HWE (p-value <0.01), which include one Indo-European population, Haryana Pandit (p-value=4.41106), one Austroasiatic, Gond (p-value=7.24108) and nine Dravidian populations; Mudaliar and Nadar from Tamil Nadu (p-val ue=1.971011 and 2.071012, respectively), Gawali from Karnataka (p-value=2.33105), Kurumba from Kerala (p-value=7.74106) and Thoti, Chenchu, Patkar and Vaddera from Andhra Pradesh (p-value=5.32104, 7.24108, 5.73107, 1.3105 and 4.67103, respectively) (Table 1).

Initially, we excluded those samples, which were not in HWE and estimated 9.51% (133 out of 1398) C allele in Indian populations, similar (p-value=0.286 and 0.2425) to South Asian populations of the 1000 Genomes Project (107 out of 978) and the GenomeAsia 100K Project (158 out of 1448) (Figure 1A). Further, we categorized samples on the basis of their linguistic affiliation and observed that Tibeto-Burman have lowest percentage of C allele (6.12%; 6 out of 98). Moreover, we observed 9.82% (44 out of 448), 8.41% (32 out of 380) and 9.88% (51 out of 516) of C allele frequency in Austro-Asiatic, Dravidian and Indo-European populations, respectively (Table 1). Interestingly, Tibeto-Burmans are insignificantly different (p-value=0.1127) from East Asians (27 out of 1001). Adi Dravidiars (scheduled caste) of Tamil Nadu, Ho (scheduled tribe) of Jharkhand and Baiswar (caste) of Uttar Pradesh have 17.857%, 15.385% and 16.176% of CYP2C9*3, respectively, which are higher in their respective linguistic group; while C allele is completely absent in Bhil of Gujarat, Raj-Gond of Madhya Pradesh and Chakesang Naga of Nagaland (Table 1). Our findings suggest that a high level of local heterogeneity exists in Indian subcontinent and we did not find any correlation with geographical distance (Figure 1B and Table 1). It is evident in the allele frequency map that Indian populations have a high frequency of CYP2C9*3, compared to other world populations (Figure 1A and Table 1). We observed a decreasing gradient of C allele frequency from the Indian subcontinent to Europeans (Figure 1A).

Figure 1 Geospatial frequency distribution of CYP2C9*3 and CYP2C9*3/*3. Genotypic and allelic frequency was interpolated with kriging method, and density map generated to explore geospatial frequency distribution. (A and C) represents the allelic (CYP2C9*3) and genotypic (CYP2C9*3/*3) distribution in world-wide population, while (B and D) represents distribution within South Asian populations. In (B and D), all samples from current study and the 1000 Genomes Project, present in HWE, were used in interpolation and represented as triangular and circle, respectively. It is evident in geospatial frequency map that South Asian populations have a high frequency of CYP2C9*3 and show high heterogeneity within the subcontinent. The same is true for CYP2C9*3/*3.

On the basis of founder events and longtime practice of endogamy, we have already predicted a high frequency of homozygous alleles in Indian populations.9,17 Since CYP2C9*3/*3 significantly decreases metabolic activity of enzymes compared to both CYP2C9*1/*3 and CYP2C9*1/*1, it would be interesting to explore genotype frequencies also in Indian populations. As expected, we observed a higher percentage (<5%) of CYP2C9*3/*3 among Indians, comparative to other world populations, who have 01% (Figure 1C and Table 2). Out of 21 populations of the 1000 Genomes Project, who lived outside the Indian subcontinent, only TSI (Italian populations) and CHS (South Chinese populations) have homozygous genotype (0.9 and 1%), while out of five populations who are living in the Indian subcontinent, three (PJL, ITU, and GIH) have 1% of CYP2C9*3/*3 (Table 2). Moreover, 1.25% South Asian samples of the GenomeAsia 100K project, were homozygous for the CYP2C9*3 allele. In the present study, we observed 05% CYP2C9*3/*3, of which Bhilala of Madhya Pradesh and Ho of Jharkhand have 5% and 3%, respectively; higher in Indo-Europeans and Austro-Asiatic linguistic groups (Table 2 and Figure 1D). We did not observe homozygous genotype CYP2C9*3/*3 in Tibeto-Burman as well as in Dravidian populations after excluding the populations, which were not in HWE (Figure 1D). In the NGS data repository, C allele was observed in 14.28% (60 out of 420). Out of 210 subjects, five (2.39%) and 50 (23.81%) were homozygous and heterozygous for the C allele, respectively.

Table 2 Distribution of CYP2C9*1 and *3 Genotype in Different Ethnic Populations.

A few rare nonsynonymous variants have also been observed in the current study. In 1278 samples, nonsynonymous C>T variant (rs28371685) which replaces the amino acid arginine with tryptophane (p.Arg335Trp) and determines the CYP2C9*11 haplogroup was found in three samples (one each in Chenchu, Telagas of Andhra Pradesh, and Mudliar of Tamil Nadu). Besides this, other functional variants rs1799853 (p.Arg144Cys) and rs72558189 (p.Arg335Trp) were observed in 10 and six samples of NGS data repository, respectively. These variants are associated with CYP2C9*2 and *14 haplotypes (Table 3).

Table 3 Rare Putative Functional Variants and Associated CYP2C9 Haplotypes

In total, eight rare and putative functional variants were not present in any reported CYP2C9 haplotypes. To determine the haplotypes, variants present within 3000 base-pair upstream and 250 base-pair downstream of CYP2C9 were utilized. In total, eight haplotypes were identified and annotation was obtained from PharmVar consortium (Table 3, Figure 2A and B). The haplotype CYP2C9*69 was identified in two subjects, CYP2C9*66 was identified in three subjects while other haplotypes were observed in only one subject. The nonsynonymous variants present in CYP2C9*63, *64, *65, *67 and *69 are predicted to be deleterious in both SIFT and Polyphen predictions. The p.Leu362Val present within CYP2C9*66 is predicted to be tolerated/benign. The Leu362 is present within hydrophobic substrate binding pocket of CYP2C9 and conversion from leucine to valine can affect assess of drug to the heme group of active site.24 A rare splice-site donor variant rs542577750 is present within CYP2C9*68 which can affect splicing of intron-7 (Figure 2B).

Figure 2 Distribution of variants in CYP2C9. (A) Rare and common putative functional variants observed in the current study. In total, 11 variants were nonsynonymous and one was splice donor variant. Other upstream and synonymous variants were used to determine haplotype of subjects. (B) Novel CYP2C9 haplotypes observed in current study.

In the Genome Aggregation Database project (gnomAD), rs578144976 and rs542577750 is reported only in South Asian samples (allele frequency=0.00085 and 0.00049). Moreover, the c.839C>G, c.978G>T, c.572A>G and c.1325G>T was not observed in any subjects of the gnomAD project. Besides South Asian subjects, the rs141489852 and rs776908257 was observed in American and non-Finnish European populations also. It suggests that CYP2C9*64, *65, *66, *68, *69 and *70 haplotypes are South Asian-specific.

CYP2C9 is highly expressed in the human liver and metabolizes a wide range of drugs. Several nonsynonymous mutations have been associated with less catalytic activity of CYP2C9 and intrinsic clearance of drugs. The CYP2C9*3 allele has been reported with hypersensitive reaction against phenytoin in epilepsy patients,18 and decreased metabolism of celecoxib.19 It was also reported with high incidence of response rate against sulfonamides, and urea derivatives.20 The in vitro studies suggest that CYP2C9*2 and CYP2C9*3 alleles reduce enzyme activity 2994% and 7191%, respectively, clearance rate of many drugs, which includes S-warfarin, tolbutamide, fluvastatin, glimepiride, tenoxicam, candesartan, celecoxib and phenytoin.21 Of which, S-warfarin, phenytoin and tolbutamide have a narrow therapeutic index and patients need the right amount of drug depending upon age, gender, and genetic make-up for successful treatment of disease. Moreover, homozygous mutations have more effect compared to heterozygous. The CYP2C9*3/*3 reduces 95% compared to 64% clearance rate by CYP2C9*1/*3.22 Considering the higher level of evidence of association between CYP2C9*3 and drug response, CPIC (Clinical Pharmacogenomics Implementation Consortium) categorized CYP2C9*3 under level-1A.23

Many studies have shown that the variations in CYP2C9 are assoc
iated with therapeutic heterogeneity in Indian populations. CYP2C9*2 and *3 have been reported with less hydroxylation (or metabolism) of phenytoin in vivo in South Indian populations,10 compared to wild type CYP2C9*1. Ramasamy et al reported phenytoin toxicity in a patient with normal dose of 300 mg/day, who had CYP2C9*3/*3 genotype.11 The same symptoms were also reported by Thakkar et al in South Indian populations.12 South Asians have a unique evolutionary history and have been practicing endogamy for many centuries, hence the high frequency of homozygous CYP2C9*3/*3 identified in the current study is not surprising. A similar trend was also observed in samples of the 1000 Genomes Project in which South Asians have high allelic and genotypic frequency of CYP2C9*3. Since CYP2C9*3/*3 has a more pronounced effect, we predict heterogeneous drug response in South Asians compared to other world populations. It would be interesting to find out if all South Asian populations have a high frequency of CYP2C9*3 and *3/*3 alleles. We explored the frequency distribution, but did not find any correlation with linguistic or geographical location. Some of the populations have a high frequency of CYP2C9*3, eg 35.7% of individuals from the Adi Dravidars have the CYP2C9*3 allele, while some of the populations have a low frequency of the CYP2C9*3 allele. Approximately 1428%, 036%, 032%, and 019% of individuals speaking Austro-Asiatic, Dravidian, Indo-European and Tibeto-Burman languages had the CYP2C9*3 allele. This suggests that South Asians are highly heterogeneous for this locus. Moreover, patients from Vysya, Mahli, Warli, Medari, Reddy, Ho, Baiswar, and Adi Dravidar populations, who have >20% individuals with CYP2C9*3 allele, should be genotyped for better treatment of disease. But this approach must be established first and its efficacy must be evaluated. We also find other rare haplotypes. Of which, three were already reported and eight were novel. Out of eight novel haplotypes, CYP2C9*64, *65, *66, *68, *69*70 and haplotypes are South Asian-specific as variants present within these haplotypes are reported only in South Asian subjects of the gnomAD project. All of the novel haplotypes are predicted to be deleterious and may have effects on protein function. It would be interesting to explore the effects of these novel haplotypes on the metabolic activity of CYP2C9 and find genetic association with therapeutic response in large samples.

In conclusion, we identified high genetic heterogeneity in CYP2C9 locus among South Asian populations. We observed higher frequency of CYP2C9*3 and CYP2C9*3/*3 alleles among South Asian populations, compared to populations from the rest of the world. The CYP2C9*3 has been associated with therapeutic response. Moreover, in the in vitro studies, the effect of CYP2C9*3/*3 allele was seen more pronounced compared to heterozygous and wild type homozygous genotype. As South Asians have a high frequency of CYP2C9*3, it would be interesting to explore the potential of CYP2C9*3 as marker for personalized therapy. Furthermore, it would be interesting to compare frequency of responder and nonresponder patients among populations and to find correlation with frequency spectrum of pharmacologically important variations. We also observed several nonsynonymous rare variants and novel haplotypes (CYP2C9*63-*70) in the present study. Of which, CYP2C9*64, *65, *66, *68, *69 and *70 haplotypes are South Asian-specific. The SIFT and PolyPhen algorithm predicts that these variants are deleterious and damaging. Therefore, individuals having CYP2C9 haplotypes with deleterious variants may have different metabolic activity compared to wild type. Collectively, our data provide fundamental knowledge of CYP2C9 genetic polymorphisms in South Asia, which could be relevant to further CYP2C9-related functional research and for personalized medicine.

We express our deepest condolence on the passing away of Mr Saurav Sharma. This work was supported by Council of Scientific and Industrial Research (CSIR), Government of India. Sheikh Nizamuddin was supported by ICMR JRF-SRF research fellowship. KT was supported by J C Bose Fellowship from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (GAP0542). We thank Prof. Andrea Gaedigk for her help in submission of haplotypes to the PharmVar consortium.

The authors report no conflicts of interest in this work.

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