Frequency of pharmacogenomic variants affecting safety and efficacy of immunomodulators and biologics in a South Asian population from Sri Lanka

Abstract

Background

Immunomodulators are important for management of autoimmune diseases and hematological malignancies. Significant inter-individual variation in drug response/reactions exists due to genetic polymorphisms. We describe frequency of identified genetic polymorphisms among Sri Lankans.

Methods

Sri Lankan data were obtained from an anonymized database of 670 participants. Data on variants and global distribution of Minor Allele frequency (MAF) of other populations (South Asian, Ashkenazi-Jewish, East-Asian, European-Finnish, European-non-Finnish, Latino-American, African/African-American) were obtained from pharmGKB online database.

Results

SLC19A1 (rs1051266) variant had a MAF (95% CI) of 63.3% (60.7–65.9). Other common variants included FCGR3A (rs396991), MTHFR (rs1801133), ITPA (rs1127354), CYP2C9*3 (rs1057910) and NUD15*3 (rs116855232), with MAFs of 35.3% (32.7–37.9), 12.2% (10.4–13.9), 10.9% (9.2–12.6), 9.8% (8.2–11.4), 8.3% (6.8–9.8) respectively. Less commonly present variants included CYP2C9*2 (rs1799853) (2.5%[1.7–3.4]), TPMT*3C (rs1142345) (1.9%[1.1–2.6]), TPMT*3B (rs1800460) (0.2%[0–0.5]), CYP3A5*6 (rs10264272) (0.2%[0–0.4]) and CYP3A4*18 (rs28371759) (0.1%[0–0.2]). The SLC19A1 (rs1051266), NUD15*3 (rs116855232), CYP2C9*3 (rs1057910), FCGR3A (rs396991), and ITPA (rs1127354) showed significantly higher frequencies in Sri Lankans compared to many other populations, exceptions include FCGR3A in Ashkenazi-Jewish and ITPA in East-Asians. Conversely, MTHFR (rs1801133), TPMT*3B (rs1800460), and CYP2C9*2 (rs1799853) were significantly less prevalent among Sri Lankans than in  many other populations. Sri Lankans exhibited lower prevalence of TPMT*3C (rs1142345) compared to European-non-Finnish, Latino-Americans, and African/African-Americans; CYP3A4*18 (rs28371759) compared to East-Asians; and CYP3A5*6 (rs10264272) compared to African/African-Americans and Latino-Americans.

Conclusion

Sri Lankans exhibit higher frequencies in variants reducing methotrexate efficacy (SLC19A1), increasing azathioprine myelotoxicity (NUDT15), and lower frequencies in variants linked to increased azathioprine toxicity (TPMT*3B, TPMT*3C), reduced tacrolimus efficacy (CYP3A4*18), and methotrexate toxicity risk (MTHFR). Beneficial variants enhancing rituximab efficacy (FCGR3A) are more prevalent, while those reducing tacrolimus dosage (CYP3A5*6) are less common. This highlights need for targeted medication strategies to improve treatment outcomes.

Background

The advent of immune-modulatory therapies has transformed the management of many inflammatory and immune-mediated diseases including autoimmune inflammatory rheumatic diseases (e.g. systemic lupus erythematosus, vasculitis and rheumatoid arthritis), inflammatory neurological disorders (e.g. transverse myelitis, multiple sclerosis and myasthenia gravis) and inflammatory gastrointestinal diseases (e.g. bowel disease and autoimmune hepatitis) [1, 2]. They are vital for remission induction and maintenance of disease control in many of these conditions. In addition, immune-modulatory therapies have proven efficacy in the treatment of certain hematological malignancies, and they are the corner stone in prevention of solid organ transplant rejection [3]. Conventional immune suppressants such as calcineurin inhibitors, azathioprine, sulfasalazine, and mycophenolate mofetil are small molecules which inhibit cellular signaling. These either switch off, silence or modify immune pathways [2]. Biologics include monoclonal antibodies targeting pro-inflammatory cytokines, their receptors, or adhesion molecules in immune cell types (e.g. TNF alpha inhibitors and anti-CD 20 antibodies) and those that mimic, replace or augment endogenous cytokines (e.g. interferons) [4].

With the evolving understanding of pathophysiology of inflammatory and autoimmune diseases, therapies are aimed at achieving disease remission or low disease activity to curtail disease progression and damage accrual [5]. Hence, these immune modulators are now being used much earlier in the disease course and with greater intensity. However, there is significant inter-individual variability in response to therapy and patients may require successive trials of different therapies [6]. For example, some studies indicate that genetic factors can account for 20% to 95% of the variability in drug response depending on the specific drug and condition being treated [7]. This in turn leads to delays in achieving therapeutic targets, poorer outcomes and high direct and indirect cost due to cost of treatment and increased morbidity. When patients do not respond to initial therapies, successive trials of different drugs are required to identify an effective therapy, increasing the overall cost of treatment. For example, a study estimated that the direct annual cost of treating a patient with rheumatoid arthritis using biologic therapies, including those who had to switch drugs due to lack of response, ranged from $19,016 to $39,420 per patient, depending on the number of drug switches required [8]. In addition, other factors that increase cost include, the requirement for more frequent monitoring, additional diagnostic tests, and possibly higher doses or combinations of drugs to achieve the desired therapeutic effect in such patients with poor response. Moreover, indirect cost due to factors such as lost productivity and long term disability due to poor disease control is also significant. For example, in rheumatoid arthritis, this indirect cost can range from 1260 to 37,994 Euros annually per patient [9]. Furthermore, both conventional and biologic immune modulators are known to cause serious or sometimes fatal adverse drug reactions (ADRs) [4]. The incidence and severity of these ADRs also vary among patient groups. These variations in response to treatment and ADRs is due to the heterogeneity of disease as well as genetic variations resulting in pharmacokinetic and pharmacodynamic differences among individuals [6]. For instance, when considering azathioprine, myelotoxicity can occur in 2–7% of patients, with the risk and severity being higher in those with genetic polymorphisms such as TPMT deficiency, being nearly 100% and severe for homogenous carriers of variants with no enzyme activity, and varying from 35 to 50% in those who are heterozygous carriers [10].

The commonest genetic variations seen in the human genome are single nucleotide variants (SNVs) [11]. Over the last couple of decades pharmacogenetic studies have investigated and identified a number of SNVs that contribute to these inter-individual differences in drug efficacy and ADRs [12]. For instance, recent studies have identified several SNVs in the TPMT (thiopurine-S-methyltransferase) gene, encoding the main enzyme metabolizing azathioprine [13]. Genetic variants in TPMT gene are prevalent in approximately 10% of Caucasians, leading to variation in TPMT activity [14]. In addition, there is an increased likelihood of bone marrow toxicity from azathioprine in TPMT deficient individuals [15]. Therefore, testing for TPMT activity or TPMT genetic variants is recommended prior to commencing azathioprine in populations with a high prevalence of these variants. Similarly, allelic variants have been found in enzymes metabolizing immunosuppressants such as tacrolimus affecting its blood levels and dose requirements among patients [16]. These are some examples where genetic variants are known to affect blood levels, ADRs and therapeutic response to immunosuppressants. Understanding these pharmacogenomics variations allow individualized treatment according to a patient’s genetic makeup in order to achieve maximum efficacy with minimal adverse effects, which improves patient outcomes and reduces healthcare cost [17]. However, there is significant inter-ethnic heterogeneity in the prevalence of such genetic variations and the variant alleles [18]. Therefore, cost effective pre-treatment testing strategies need to be guided by local data on prevalence of these genetic variants.

Recent studies have shown that variants affecting metabolism and response to certain drugs such as warfarin in Sri Lankans vary from those described in Europeans and other non-South Asian populations [19, 20]. However, treatment of diseases using immunosuppressants and biologics in Sri Lanka are largely based on international or regional guidelines which are based on evidence from non-South Asian populations. Therefore, this study aims to explore the prevalence of identified genetic variants known to affect metabolism, adverse effects or response to treatment of selected immune modulators among Sri Lankans. Furthermore, since Sri Lankans are known to be genetically homogenous with other South Asian populations, findings from the present study will have wider implications.

Materials and methods

This study aims to explore the prevalence of identified genetic variants known to affect metabolism, adverse effects or response to treatment of selected immune modulators among Sri Lankans. The data regarding genes and variants related to safety and efficacy of immunomodulators and biologics were accessed through the freely available online database PharmGKB (Pharmacogenomics Knowledge Base) (www.pharmgkb.org). This platform provides evidence-based information on variant annotations, clinical guideline annotations and FDA (Food and Drug Administration) drug label annotations, which are freely available for clinical and research purposes. Each annotation is accompanied by the level of evidence, from Level 1 (highest) to Level 4 (unsupported) (15).

For the purpose of this study, data related to selected immunomodulators, and biologics were filtered out from this database. Information regarding their respective gene, gene variants/haplotypes, SNV ID, wild type/variant allele and the level of evidence were also obtained. This was further filtered based on the level of evidence where only the genes and variants with evidence levels of 1 (A and B) or 2 (A and B) were selected for the study (Table 1). Ethics approval for the study was obtained from the Faculty of Medicine, University of Colombo (EC-22-012).

Table 1 Pharmacologically important genes and variants associated with safety and efficacy of immunomodulators and biologics

Genotype and data analysis

The Centre for Genetics and Genomics at the Faculty of Medicine, University of Colombo maintains an anonymized database of genetic variants of Sri Lankans who underwent whole exome sequencing to diagnose the genetic aetiology of rare disorders and inherited cancers using the Illumina Next-generation sequencer platforms during the period from January 2015 to December 2023. An in-house bioinformatics pipeline was used for the variant analysis. The resulted variants were interpreted according to the standard American College of Medical Genetics (ACMG) guidelines. Prior ethics approval had been obtained for use of this genomic data for pharmacogenomics related studies in the Sri Lankan population [EC-22-012]. The genotypic data of a total of 670 Sri Lankans related to the selected genes and variants involved in the metabolism of immunomodulators and biologics was filtered out from this database and analyzed retrospectively. Homozygous and heterozygous allele counts for each genotype were obtained and the genotypic frequencies and the minor allele frequencies (MAFs) for each variant tested for the Hardy–Weinberg equilibrium and reported with a 95% confidence interval. Ethnicity (Sinhalese, Tamil or Moor) was assigned on the basis of self-reported ethnicity.

To compare the frequencies with other populations, the MAFs of East Asian, South Asian, Ashkenazi Jewish, Latino/Admixed American, African/African-American, European (Finnish) and European (non-Finnish) populations were obtained from the Genome Aggregation Database (https://gnomad.broadinstitute.org/). Afterwards, Chi-square test was used to compare the differences in allele frequencies between Sri Lankans and the above-mentioned population groups, considering a p-value less than 0.05 as significant.

Results

The immunomodulators and biologics affected by the genomic variants included azathioprine, methotrexate, peginterferon alfa-2b, rituximab, siponimod and tacrolimus. Accordingly, the genes related to the above drugs; CYP2C9 (cytochrome P450 family 2 subfamily C member 9), CYP3A4 (cytochrome P450 family 3 subfamily A member 4), CYP3A5 (cytochrome P450 family 3 subfamily A member 5), ITPA (inosine triphosphatase), MTHFR (methylenetetrahydrofolate reductase), SLC19A1 (solute carrier family 19 member 1), FCGR3A (Fc gamma receptor IIIa), TPMT and NUDT15 (nudix hydrolase 15) genes were studied and analyzed. The observed genotype frequencies of each variant in the Sri Lankan population were consistent with the Hardy–Weinberg equilibrium and are elaborated below. They are shown in Table 2 along with the MAFs for each variant. The study population consisted of 670 Sri Lankans, of which 44.8% were males (n = 300). Majority were Sinhalese in ethnicity (82.4%; n = 552), followed by Moors (8.2%; n = 55) and Tamils (4.0%; n = 27), while details regarding ethnicity were not available for 36 participants (5.4%). The MAFs of the variants in the population are shown in Table 2, whilst the MAFs in the different ethnicities are depicted in Table 3.

Table 2 Genotype and minor allele frequencies of pharmacogenomic variants associated with selected immunomodulators and biologic

Table 3 Genotype and minor allele frequencies of pharmacogenomic variants associated with selected immunomodulators and biologics in ethnic sub-populations

CYP2C9 gene

Thirty-two individuals (4.8%; 95% CI 3.2–6.4) and 115 individuals (17.2%; 95% CI 14.3–20.2) were heterozygous for the CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) variants, respectively. The MAFs of the CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) variants were 2.5% (95% CI 1.7–3.4) and 9.8% (95% CI 8.2–11.4), respectively with no statistically significant difference in the MAFs in the ethnic sub-population analysis (Table 3). The MAF of the CYP2C9*2 (rs1799853) variant was lower in the Sri Lankan population when compared with Ashkenazi Jews, Europeans (Finnish and non-Finnish) and Latino Americans, while being higher than in East Asians. In contrast, the CYP2C9*3 (rs1057910) variant was more frequent among Sri Lankans than East Asians, Europeans (Finnish and non-Finnish), Latino Americans and African/African Americans (Table 4).

Table 4 Minor allele frequencies of genetic variants associated with immunomodulator and biologics in different populations compared with the Sri Lankan population

CYP3A4 gene

One (0.2%; 95% CI 0.0–0.4) individual was heterozygous for CYP3A4*18 variant (rs28371759), which had a MAF of 0.1% (95% CI 0.0–0.2) among Sri Lankans (Table 2). The variant was significantly less frequent among Sri Lankans in comparison to East Asians (Table 4).

CYP3A5 gene

For the CYP3A5*6 (rs10264272) variant, only two individuals (0.3%; 95% CI 0.0–0.7) were heterozygous, with no homozygous individuals. The MAF of CYP3A5*6 (rs10264272) variant was 0.2% (95% CI 0.0–0.4) (Table 2). The CYP3A5*6 (rs10264272) variant was significantly less frequent in Sri Lankans in comparison to Latino Americans and African/African Americans (Table 4).

ITPA gene

The rs1127354 variant of the ITPA gene had 122 heterozygous individuals (18.2%; 95% CI 15.3–21.1) whereas 12 individuals (1.8%; 95% CI 0.8–2.8) were homozygous for the variant. The MAF was 10.9% (95% CI 9.2–12.6). In the ethnic sub-population analysis, there was no statistically significant difference in the MAFs of the ITPA gene rs1127354 variant between ethnicities (Table 3). The presence of the ITPA rs1127354 variant was significantly higher among Sri Lankans, than Europeans (Finnish and non-Finnish), Latino Americans and African/African Americans, whilst being lower than among East Asians (Table 4).

MTHFR gene

22.5% (95% CI 19.4–25.7) were heterozygous and 0.9% of the individuals (0.2–1.6) were homozygous for the rs1801133 variant of the MTHFR gene among Sri Lankans. The MAF for this variant was 12.2% (95% CI 10.4–13.9), with no significant difference in the MAFs between the different sub-populations (Table 3). When compared with other populations the rs1801133 variant of the MTHFR gene was less frequent among Sri Lankans, than in Ashkenazi Jews, East Asians, Europeans (Finnish and non-Finnish), and Latino Americans.

SLC19A1 gene

The MAF of the rs1051266 variant of the SLC19A1 gene was 63.3% (95% CI 60.7–65.9) (heterozygous: n = 288 [43.0%; 95% CI 39.2–46.7] and variant homozygous: n = 280 [41.8%; 95% CI 38.1–45.5]). There was no significant difference in the MAFs in the different ethnic sub-populations in Sri Lanka (Table 3). The variant was significantly more frequent among Sri Lankans than in individuals from East Asia, Europe (Finnish and non-Finnish), Latino America and Africa (Table 4).

FCGR3A gene

The rs396991 variant of the FCGR3A had a MAF of 35.3% (95% CI 32.7–37.9), with 313 individuals (46.7%; 95% CI 42.9–50.5) heterozygous and 80 individuals (11.9%; 95% CI 9.5–14.4) homozygous for the variant allele. Although the MAF was higher in the Tamil population in the ethnic sub-population analysis, this difference was not statistically significant (Table 3). Sri Lankans had a significantly lower frequency of the variant in comparison to Ashkenazi Jews, while having a significantly higher frequency than Europeans (Finnish) and Latino Americans.

TPMT gene

TPMT*3B (rs1800460) and TPMT*3C (rs1142345) variants had 3 (0.4%; 95% CI 0–1.0) and 23 (3.4%; 95% CI 2.1–4.8) individuals, respectively that were heterozygous. There were no homozygous individuals for the TPMT*3B (rs1800460) variant, while 1 individual (0.2%; 95% CI 0–0.4) was homozygous for the TPMT*3C (rs1142345) variant. Their MAFs were 0.2% (95% CI 0–0.5) and 1.9% (95% CI 1.1–2.6), respectively. There was no difference in MAFs among different Sri Lankan ethnicities for the TPMT*3B (rs1800460) or TPMT*3C (rs1142345) variants. The MAF of the TPMT*3B (rs1800460) variant was significantly lower in the Sri Lankan population when compared with Ashkenazi Jews, Europeans (Finnish and non-Finnish) and Latino Americans, while the TPMT*3C (rs1142345) variant was also less frequent among Sri Lankans than Europeans (non-Finnish), Latino Americans and African/African Americans.

NUDT15 gene

One hundred and one individuals (15.1%; 95% CI 12.4–17.8) were heterozygous for the NUD15*3 (rs116855232) variant, whereas 5 individuals (0.8%; 95% CI 0.1–1.4) were homozygous, with a MAF of 8.3% (95% CI 6.8–9.8). There was no statistically significant difference in the MAF of the variant in the different ethnic sub-populations of Sri Lanka (Table 3). However, the MAF of the variant was significantly higher in the Sri Lanka population, in comparison to Europeans (Finnish and non-Finnish), Latino Americans, African/African Americans, and Ashkenazi Jews (Table 4).

Discussion

Although much of the inter-individual variability observed in treatment response and adverse effects of immunomodulators has been attributed to genetic variants affecting pharmacodynamics/pharmacokinetics of these medicines, at present there is no evidence from Sri Lanka, with limited evidence in other South Asian ethnicities. The present study examines the MAFs of variants with level 1 or 2 evidence (PharmGKB) indicating likely causation of drug phenotype of selected immunomodulators. Of these, frequencies of variants CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), MTHFR (s1801133), CYP3A5 (rs776746), CYP3A4 (rs2740574), and ITPA (rs1127354) have been described by Chan et al. in a previous study with a smaller study population (n = 197)[20], showing comparable results. The present study also described additional variants with likely clinical implications including CYP3A5*6 (rs10264272), NUD15*3 (rs116855232), FCGR3A (rs396991), SLC19A1 (rs1051266), TPMT*3B (rs1800460) and TPMT*3C (rs1142345).

Currently there are no evidence-based country specific guidelines for management of autoimmune and autoinflammatory conditions or malignancies in Sri Lanka. Therefore, treatment of these diseases are based on recommendations in international guidelines which are based on evidence from European and other Caucasian populations. However, the present study demonstrates that some genetic variants which affect treatment response and ADRs of certain immunomodulators vary significantly between Sri Lankans/South Asians and other populations. Of these, CYP3A5*6 (rs10264272) and CYP2C9*2 (rs1799853) variants which affect metabolism of tacrolimus and siponimod, respectively are likely of no clinical significance in Sri Lanka due to their very low frequency.

Siponimod is a sphingosine 1-phosphate receptor (S1PR) modulator licensed for treatment of secondary progressive multiple sclerosis [21]. It induces a dose-dependent functional antagonism at S1PR1, resulting in a reduction in peripheral lymphocytes. This reduces the circulating lymphocytes and prevent T cells from entering the central nervous system, limiting ensuing inflammation. Similar to its therapeutic effect, serious ADRs of siponimod such as cardiac adverse effects and liver transamination elevations are also dose dependent [22, 23]. CYP2C9*3 (rs1057910) is a no function allele, which reduces the metabolism of siponimod in the liver and results in higher plasma levels. This variant has shown to be significantly more frequent among Sri Lankans, compared to Latino/admixed American (< 0.001), African/African American (< 0.001), and European populations (< 0.001), with a MAF of 9.8%. According to current recommendations siponimod is contra-indicated in patients homozygous for CYP2C9*3 (rs1057910) genotype and the dose is halved for heterozygous individuals [24]. Therefore, based on the frequency of the variant observed, and its noted association of increased plasma levels of siponimod, Sri Lankans are more likely to experience adverse effects and dose titration based on genetic phenotypes could be beneficial.

Pegylated interferon alfa-2b (PEG-IFN α2b) is FDA approved for the treatment of chronic hepatitis C in combination with ribavirin (RBV). Although PEG-IFN α2b has now gone out of use in many countries with the advent of directly acting antiviral (DAA), it is still used in resource poor settings. ITPA (rs1127354) allele encodes inosine triphosphate pyrophosphohydrolase, which reduces the risk of anaemia during combination therapy for hepatitis C [25]. Studies have shown patients with normal ITPase activity require more frequent RBV dose reductions and were more likely to be given erythropoietin [26]. Our results show that this variant is more frequent among Sri Lankans and other Asian populations compared to European, African and Latin American populations, indicating that Asians are less likely to develop anaemia with PEG-IFN α2b and RBV therapy.

Methotrexate has been in use for treatment of malignancies as well as a number of autoimmune and autoinflammatory diseases [27]. Genes that contribute to this inter-individual variability in therapeutic response and adverse effects of methotrexate primarily fall under folate pathway genes (e.g. MTHFR, DHFR[Dihydrofolate reductase], MTR[Methyltetrahydrofolate-Homocysteine Methyltransferase]) or transporter genes (e.g. OAT1[Organic Anion Transporter 1], SLCO1B1[Solute Carrier Organic Anion Transporter Family Member 1B1], SLC19A1) [28]. SLC19A1 encodes the Reduced Folate Carrier 1 (RFC 1) which almost exclusively transports methotrexate into cells [29]. A number of SNVs have been described to affect its function. The SLC19A1 rs1051266 (80 T > C) variant CC genotype has been shown to have lower intracellular methotrexate polyglutamate levels and poor response to methotrexate compared to individuals who are heterozygous or homozygous for the wild type allele [30]. This effect has been observed with both low dose therapy for rheumatological diseases and high dose therapy for malignancies [31, 32]. In addition, the CC genotype is postulated to be associated with increased mucositis, liver toxicity and bone marrow toxicity [33,34,35]. The present study shows that SLC19A1 (rs1051266) C allele is more frequent among Sri Lankans with a MAF of 63.3%, a frequency higher than East Asian, European, Latino American and African populations. However, being one of the affordable and widely available DMARDs, methotrexate is drug of choice for many conditions including rheumatoid arthritis, particularly in a resource limited setting such as Sri Lanka. Therefore, further larger studies to examine the clinical effect of this variant in Sri Lanka is warranted. Conversely, the MAF of MTHFR (rs1801133) variant is significantly much less among Sri Lankans (12.2%) compared to East Asians, Europeans and Latino Americans. Evidence indicates that the presence of the MTHFR (rs1801133) variant (AA and AG phenotypes) leads to increased risk of ADRs of methotrexate including elevation of liver transaminases and alopecia [36, 37]. In Sri Lanka, only 0.9% are homozygous for MTHFR (rs1801133) variant, with a MAF of 12.2% and therefore, its contribution to methotrexate toxicity seen among Sri Lankans is likely to be lower in comparison to other populations with higher MAFs.

Rituximab, which is licensed for the treatment of a variety of B cell malignancies, as well as autoimmune diseases, is a chimeric IgG1 monoclonal antibody, which binds to CD20 protein primarily expressed on B cells [38]. The coated B cells undergo cell death by antibody-dependent cytotoxicity (ADCC) resulting from receptors for IgG on natural killer cells binding to rituximab coated B cells [39]. rs396991 variant of the Fc fragment of IgG type 3A (FCG3A) has been shown to increase the affinity of IgG to rituximab in an allele-dose-dependent manner [40]. Consequently, large cohort studies have demonstrated that the presence of the FCGR3A gene rs396991 variant is associated with better response to rituximab in patients with rheumatoid arthritis and systemic lupus erythematosus [41, 42]. The present study shows that this variant is more prevalent among Sri Lankans compared to European (Finnish) and Latino American populations and less frequent than in Ashkenazi Jewish population, with a MAF of 35.3% and 11.9% being homozygous for the rs396991 C allele. However, there was no significant difference with other populations. The approved dose of rituximab for rheumatoid arthritis is two doses of 1 g given two weeks apart every 24 weeks or based on clinical evaluation [43]. However, since its licensing, randomized controlled trials have shown that low dose regimes are equally efficacious with better clinical and laboratory safety profile compared to the approved dose [44, 45]. Interestingly, most published studies of Finnish and Latin American populations in which the MAF of FCGR3A rs396991 variant is lowest, are for the standard dose with very limited data on the use of low dose rituximab [46, 47]. Therefore, further studies to determine whether ultra-low pharmacogenomic based dosing of rituximab provides comparable disease control, whilst reducing ADRs could be beneficial.

Azathioprine dose adjustments based on pharmacogenomics is one of its most successful applications of genomics in clinical practice [48]. Azathioprine is rapidly converted to its active form, 6-mercaptopurine which is then transported into cells. 6-mercaptopurine is converted into active metabolites 6-thioguanine nucleotides (6-TGNs), through a series of steps. Elevated levels of 6-TGNs, are known to be toxic and cause bone marrow suppression [49]. There are a number of SNVs identified in the gene encoding thiopurine methyltransferase (TPMT) enzyme, which is responsible for inactivating intermediate compounds during the metabolism of thiopurines [49]. Of these, 90% of the poor metabolizer phenotypes is due to three variants: TPMT*2, *3A, *3C [50]. Furthermore, active metabolites are hydrolyzed into inactive forms by the Nudix hydrolase 15 (NUDT15). NUD15*3 (rs116855232) is a no function allele, which leads to accumulation of active metabolites of azathioprine, resulting in increased risk of myelosuppression [51].

Asians are known to have much lower frequency of TPMT variants, as shown in the present study as well with TPMT*3B (rs1800460) and TPMT*3C (rs1142345) variants compared to Caucasians and African American populations. However, incidence of leukopenia is relatively higher among Asians (9–12%) compared to Caucasians (approximately 3–5%) [52]. This is partly attributed to the higher prevalence of NUD15*3 (rs116855232) no function allele among both South and East Asians populations compared others [51]. Presently the Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines and the US FDA recommends pharmacogenetic testing for NUDT15 phenotype prior to starting azathioprine [53]. It is recommended to use an alternative therapy for homozygous NUDT15 deficiency (TT genotype), and to reduce dosages by 20–50% from the normal starting dose in patients with CT genotype. This strategy has been shown to be cost effective or cost saving [54]. In Sri Lanka, careful consideration of the cost-effectiveness is important where 15.1% of the population having the CT genotype and 0.8% having the TT genotype, if NUDT15 genotype testing it to be made available routinely.

Important limitations in the present study need to be highlighted. The primary ethnic sub-population is Sinhalese (82.4%; n = 552), with lesser representation from other ethnicities. According to the Department of Census and Statistics, Sri Lanka (2022), the ethnic composition of the country includes Sinhalese (74.9%), Sri Lankan Tamils (11.2%), Indian Tamils (4.1%), and Sri Lankan Moors (9.3%) [55]. The predominance of Sinhalese participants in our study reflects this demographic distribution. However, although the analysis does not reveal a significant difference in the MAFs (Table 3) between the different ethnicities, the smaller representation in other sub-populations precludes a detailed comparison. In addition, the population includes individuals who underwent whole exome sequencing for various other conditions, and presently not on immunomodulator/biologic therapy, thereby preventing direct clinical correlations. However, given the paucity of data on the topic among South Asians, these results help to bridge an important gap in the present knowledge and understanding of the impact of pharmacogenomic variants on efficacy and safety of these medicines. In addition, it is important to note that the analysis is limited to variants located within the exome, and for variants relevant to immunomodulators and biologics. In the future, we aim to expand our research to include pharmacogenomic variants relevant to other therapeutic areas such as, psychiatry, and cardiology, to provide a more comprehensive analysis.

Conclusions

Sri Lankans exhibit higher frequencies in variants reducing methotrexate efficacy (SLC19A1), increasing azathioprine myelotoxicity (NUDT15), and lower frequencies in variants linked to increased azathioprine toxicity (TPMT*3B, TPMT*3C), reduced tacrolimus efficacy (CYP3A4*18), and methotrexate toxicity risk (MTHFR). Beneficial variants enhancing rituximab efficacy (FCGR3A) are more prevalent, while those reducing tacrolimus dosage (CYP3A5*6) are less common. This highlights need for targeted medication strategies to improve treatment outcomes.