Lastly, we compared of the frequency of PGx biomarkers between the patients according to their age. However, no significant differences in PGx variant frequencies were observed between pediatric (n = 60) and adult (n = 16) subgroups (p > 0.05 for all major variants) (Supplementary Table 1).
PGx is a rapidly growing field in pharmacotherapy, aiming to explain inter-individual variability in drug response and to guide personalized treatment strategies. In Jordan, PGx testing is still in its early stages, with limited implementation in clinical practice. In this study, we identified the most frequent genetic variants with actionable clinical recommendations related to drug selection, dosage adjustment, and the avoidance of serious adverse effects among a sample of Jordanian individuals with genetic diseases.
These findings can serve as a valuable reference for the distribution of pharmacogenes within Jordanians suffering of genetic diseases.
Previous PGx studies in Jordanians primarily utilized conventional genotyping techniques such as polymerase chain reaction (PCR), restriction fragment length polymorphism, and allele-specific PCR [20, 21]. In contrast, the current study employed whole genome sequencing (WGS), a more comprehensive and accurate method for detecting genetic variation [22]. Therefore, it can be suggested that our results are more reliable than those obtained through previous PCR-based methods.
This study included patients with diverse genetic disorders. The study's aim was not to correlate PGx variants with specific diagnoses of genetic disorder, but to assess the overall burden of actionable PGx variants in a population at high risk for drug-related complications due to polypharmacy. Future studies with larger sample size can allow up to analysis the correlation between PGx biomarkers with a specific genetic disorder.
The CYP450 enzyme family plays a key role in drug pharmacokinetics, and genetic variants in CYP450 genes can significantly influence drug metabolism and therapeutic outcomes. In this study, the most frequently identified genetic alleles were CYP2C19*2, CYP2C19*17, and CYP2D6*10, all of which are known to impact the metabolism of antidepressants such as selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) [23]. These variants are classified as Level A evidence in the PharmGKB database, indicating strong clinical significance, and are included in PGx guidelines for dose adjustment of antidepressants [24]. CYP2C19*2 is associated with a poor metabolizer phenotype, often requiring up to a 50% dose reduction of SSRIs depending on the overall genotype, while CYP2C19*17 leads to ultra-rapid metabolism, which may cause subtherapeutic drug levels and necessitate higher doses [25]. The CYP2D6*10 allele reduces enzymatic activity and slows the metabolism of drugs like paroxetine and fluoxetine, potentially increasing plasma concentrations and the risk of side effects, especially in *10/*10 carriers [26]. Additionally, CYP2C19*2 and CYP2C19*17 also affect the response to the antiplatelet drug clopidogrel; CYP2C19*2 carriers may experience clopidogrel resistance and require an alternative such as prasugrel, whereas CYP2C19*17 carriers are at increased risk of bleeding due to enhanced drug activation [27]. Given that approximately 15% of study participants carried at least one of these variants, a similar proportion of patients may be at risk of altered response to antidepressants or clopidogrel. Notably, variability in antidepressant efficacy and clopidogrel resistance is highly noticed by physicians in Jordan, suggesting that genetic background may contribute to these outcomes. Therefore, the findings of this study support the implementation of PGx testing to optimize prescribing of antidepressants and clopidogrel in Jordanian patients with genetic diseases.
The CYP2B6 gene encodes an enzyme involved in the metabolism of several anesthetic and analgesic drugs, including propofol, ketamine, and methadone [28]. Genetic variants such as CYP2B6*2, 3, 5, 6, and 7 can alter enzyme activity and significantly impact drug response [29]. Among these, CYP2B*6/*6 is the most extensively studied and is associated with reduced enzyme activity, leading to slower metabolism and prolonged drug effects, especially with agents like ketamine, due to higher and prolonged drug plasma concentrations [30]. The previous study done by Alrabayeh et al. [31] in Jordan screened CYP2B6*2, 3, 5, 6, and 7 and found that it affect the dose and response of propofol [31]. According to our results, it is speculated that the response toward anesthetic drugs will be affected by the CYP2B6 alleles among 18% of the participants. This high percentage makes us to strongly take into consideration the CYP2B6 genotyping among patients with genetic diseases before the administration of anesthetic drugs.
The UGT1A1*28 variant is located in the 5' untranslated region of the UGT1A1 gene. The UGT1A1 gene encodes the enzyme UGT1A1 enzyme which plays a major role in the elimination of SN-38, the active metabolite of the chemotherapeutic agent irinotecan. The UGT1A1*28 variant is associated with reduced UGT1A1 expression and activity, leading to decreased clearance of SN-38. As a result, patients carrying this variant may experience increased systemic exposure to SN-38, which significantly raises the risk of dose-dependent toxicities such as severe neutropenia and diarrhea [32]. The Dutch Pharmacogenomics Working Group strongly recommend gentyping of UGT1A1 before administration of irinotecan to cancer patients [33]. This is the first report in Jordan reporting the frequency of UGT1A1*28 variant. Accordingly, it can be predicted that 8% of the patients with genetic disorders are at the risk of irinotecan-induced toxicity due to the presence of the UGT1A1*28 allele. This may indicate the importance of UGT1A1*28 genotyping before administration of irinotecan.
The identified genetic variants in the DPYD gene in this study, DPYD*4, DPYD*5, DPYD*6, DPYD*9 A, DPYD*48, and the HapB3 haplotype have been associated with reduced enzymatic activity, leading to accumulation of the anticancer active fluoropyrimidine metabolites and an increased risk of severe, potentially life-threatening toxicities, including myelosuppression, mucositis, diarrhea, and neurotoxicity [34]. AlKhateeb et al. studied previously the influence of limited DPYD variants among colorectal cancer patients in Jordan, but he missed the genotyping of other clinically relevant DPYD alleles, including DPYD*4, *5, *6, *9, *48 and the HapB3 haplotype that have been linked to clinically relevant decreases in DPD function [35], and are recognized in pharmacogenetic guidelines for dose adjustment of fluoropyrimidines [36].
The non-synonymous G6PD Mediterranean variant (rs5030868) is a severe deficiency allele of the G6PD gene, commonly found in individuals of Middle Eastern descent. This variant significantly reduces the activity of the G6PD enzyme, which is essential for protecting red blood cells against oxidative damage. As a result, carriers are at high risk of developing acute hemolytic anemia when exposed to oxidative drugs such as the antimicrobial primaquine, dapsone, and sulfonamides [37]. Although its frequency is very low (3.9%) among the studied sample, caution should be taken into consideration for prevention of serious complications induced due to the G6PD Mediterranean variant and accordingly, genotyping of G6PD Mediterranean variant testing is recommended before prescribing these medications.
The HLA-B*57:01 allele is a genetic marker strongly associated with abacavir-induced hypersensitivity reactions in patients with Human immune deficiency Virus (HIV). This reaction can be severe and potentially life-threatening, manifesting with symptoms like fever, rash, gastrointestinal distress, and respiratory issues [38]. The presence of HLA-B*57:01 has a high predictive value for abacavir HSR, and therefore, screening for this allele is mandatory prior to initiating abacavir therapy. We found in this study that only one participant carried the HLA-B*57:01 variant and hence this patient might be at high risk of abacavir-induced hypersensitivity reactions.
Genetic variations in the SLCO1B1 gene, which encodes the hepatic transporter OATP1B1, play a crucial role in the hepatic uptake and clearance of various drugs, particularly statins such as simvastatin. Among the key variants, rs4149056 (c.521 T > C) is the most clinically significant and is associated with reduced transporter function. This leads to increased plasma concentrations of statins and a heightened risk of statin-induced myopathy, especially in individuals with homozygous or compound heterozygous genotypes [39]. Additionally, Alhawari et al., found that heterozygous SLCO1B1*1/*37 genotype is significantly associated with atorvastatin hepatotoxicity and efficiency among diabetic patients in Jordan [10]. The current study added to our knowledge that other clinically significant SLCO1B1 variants *5, *14, *15, and *20 are present among patients with genetic disorders in Jordan in a comparable frequencies to SLCO1B1 variants, and hence may contribute to the high inter-individual variation in the statin response among Jordanians.
The CYP4F2 V433M variant affects the function of the CYP4F2 enzyme, which is involved in vitamin K metabolism and the regulation of blood clotting. The V433M variant is associated with reduced enzyme activity, leading to altered vitamin K oxidation and a potential increase in vitamin K levels [40]. This has clinical significance in warfarin therapy, where carriers of this variant often require higher warfarin doses to achieve optimal anticoagulation [6]. We found in this study that 42% of the patients carried The V433M variant which is in agreement with what was reported previously among diabetic [41] and healthy Jordanians [42]. This high prevalence of the V433M variant among Jordanians emphasize the need to incorporate the CYP4F2 V433M variant in the clinical practice in prediction of warfarin dose.
The VKORC1 -1639G > A (rs9923231) variant affects the expression of the VKORC1 gene, encoding the vitamin K epoxide reductase complex subunit 1, the primary target of warfarin. The A allele is associated with lower VKORC1 expression, resulting in increased warfarin sensitivity and a need for lower maintenance doses [43]. In the current study, 34 individuals carry the heterozygous while six carried the homozygous VKORC1 -1639G > A. In similar to the CYP4F2 V433M variant, both variants affect warfarin response and both are highly frequent among the patients. In addition, both variants have A Level with a high prediction of the dose of warfarin. It can be concluded that the high frequency of CYP4F2 V433M variant and VKORC1 -1639G > A variants could explain the high inter-individual variation in the drug response and genotyping of these variants is highly recommended in Jordan to avoid the excess bleeding that can occur due to high warfarin dose.
Notable differences exist in PGx biomarkers between the Jordanian population and other major global groups. For instance, Jordanians have a higher frequency of the CYP2D6*10 allele than Polish individuals (13.2 vs. 5%) but a lower frequency of CYP2C19*17 (18.4 vs. 36%) [44]. In contrast, Southeast Asian populations exhibit a much higher prevalence of UGT1A1*28 (22.3 vs. 5.3% in Jordanians) [45]. However, Brazilian populations show frequencies for biomarkers related to warfarin and antidepressant metabolism that are closer to those in Jordanians [46].
Additionally, the results of allele frequency of PGx biomarkers found in this study match with what was reported among neighboring Middle Eastern populations. The frequency of CYP2D6*10 (13.4%) is in parallel with Middle Eastern populations (15-20%) [7, 9]. These collective comparisons suggest that dosing guidelines established in other ethnic groups should not be extrapolated to Jordanians without caution.
It is important to note that the PGx variants analyzed in this study are germline and not linked to specific diseases. This study primarily included patients with rare genetic disorders, some of whom may have higher consanguinity rates. Although the observed variant frequencies align with previous reports in healthy Jordanian populations, these findings should be interpreted with caution when extrapolating to the broader Jordanian population.
Findings of this study support the implementation of PGx testing in Jordan. However, some challenges remain against the implementation of PGx testing. These include limited access to PGx information and low awareness among healthcare providers [47]. Furthermore, regulatory policies regarding genetic testing and patient data privacy are still under development.
Although this study reached it aim, there are some limitations that can be acknowledged. The relatively small sample size in this study resulted in wide confidence intervals for some rare variants, while adequately powered for common alleles (20% frequency ± 10% precision) [16]. This limitation is due to the rarity of patients with confirmed genetic disorders and limited biobank resources. However, the results of this study can be considered as a primary reference for most relevant PGx biomarkers among genetic patients in Jordan and larger multi-center studies are warranted for more estimates.
Additionally, this study did not include clinical drug response data or treatment outcomes, as its primary focus was on identifying the frequency of clinically actionable PGx variants in a genetically high-risk population. While the absence of phenotype-genotype correlation limits the immediate assessment of clinical impact, the identified variants are well-established in CPIC guidelines and have documented effects on drug efficacy and safety in other populations.
Lastly, this study did not gather detailed data on drug usage. However, medical records of INNOVIA Biobank Center show that many patients are prescribed long-term medications metabolized by enzymes like CYP2D6, CYP2C19, and CYP3A4. Given the high frequency of actionable PGx variants identified in this study, personalized pharmacotherapy strategies could significantly enhance treatment safety and efficacy, particularly in this population where polypharmacy is common.