Prognostic Value of NTCP p.Ser267Phe Variant in Patients with Chronic Hepatitis B

Bülent ÇAKAL; Alp ATASOY; Mehveş PODA; Bilger ÇAVUŞ; Mesut BULAKÇI; Mine GÜLLÜOĞLU; Filiz AKYÜZ

doi:10.4274/nkmj.galenos.2024.26121

ABSTRACT

Aim:

In this study, the aim is to detect polymorphisms in the gene encoding the sodium taurocholate cotransporting polypeptide (NTCP), the functional receptor for hepatitis B virus (HBV) and hepatitis D virus.

Materials and Methods:

The study included a total of 293 patients, with 150 diagnosed with chronic hepatitis B (CHB) and 143 undergoing liver parenchyma biopsy procedures due to different clinical indications. Total DNA was isolated from liver biopsy samples. The TaqMan SNP genotyping method was used to determine the rs2296651 polymorphism in the SLC10A1 gene, which leads to the NTCP S267F variation.

Results:

In patients with CHB and the control group, the NTCP-interacting domain was highly conserved, and no variation of the SNP rs2296651 in the SLC10A1 gene leading to the NTCP S267F variation was detected in any of the patients.

Conclusion:

It was thought that in patients with CHB, the impact of the NTCP S267F variation on the progression of HBV-associated diseases and its influence on the therapeutic efficacy of anti-viral agents targeting NTCP blockade may be limited.

Keywords:

Hepatitis B virus, chronic hepatitis B, single nucleotide polymorphism, sodium taurocholate co-transporting polypeptide

INTRODUCTION

Although the development of cirrhosis and liver cancer, which may be related to hepatitis B virus (HBV), can be prevented with current treatments, it remains an important public health problem because there is no treatment that will ensure complete viral clearance and it may cause approximately one million people to lose their lives worldwide every year¹. Chronic HBV (CHB) infections are a dynamic process shaped by the interaction of viral factors belonging to the virus, host factors belonging to the infected individual and environmental factors. Therefore, viral factors such as genotype and mutations, genetic and immunologic factors of the infected individual and environmental factors are critical determinants of the natural course of chronic viral hepatitis, the development and prognosis of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC), and treatment responses^2,3.

Sodium taurocholate cotransporter polypeptide (NTCP) is a functional receptor for human HBV and its satellite virus hepatitis D virus (HDV)⁴. NTCP is a cell surface glycoprotein localized on chromosome 14, encoded by the SLC10A1 gene, the number one member of the solute carrier family 10 (sodium/bile acid cotransporter family, SLC10) and involved in the enterohepatic circulation of bile salts expressed by hepatocytes⁵. In this respect, the entry step involving the interaction of the viral surface protein (surface) PreS1 with the PreS1-specific receptor NTCP on the hepatocyte surface and subsequent transfer into the cell via endocytosis is of critical importance for HBV infections. Therefore, NTCP constitutes one of the new therapeutic targets for preventing the binding and entry of the virus into hepatocytes for anti-HBV treatment^6-8.

On the other hand, it has been reported that the p.Ser267Phe (S267F) polymorphism (single nucleotide polymorphism; SNP) detected on the NTCP gene, especially p.Ser267Phe (S267F) polymorphism, may cause changes in the physiological function of NTCP, leading to a decrease in HBV entry into the cell and infection load. Therefore, it is hypothesized that these genetic variants of NTCP may be associated with resistance to HBV infection and the risk of developing HBV infection-related liver cirrhosis and HCC. It is also suggested that sequence differences in the NTCP interaction domain (HBV PreS1) may negatively affect treatment responses to HBV entry inhibitors designed for anti-HBV therapy by causing changes in NTCP binding affinity^9-15.

Therefore, the aim of this study was to identify polymorphisms in the gene encoding the HBV and HDV functional receptor NTCP and to evaluate the effects of variation of the NTCP S267 polymorphism on the clinical outcomes of patients and the prognostic impact of new therapeutics targeting NTCP in predicting treatment efficacy.

GİRİŞ

Hepatit B virüs (HBV) ilişkili olabildiği siroz ve karaciğer kanseri gelişimi, mevcut tedaviler ile önlenebilir olmasına karşın, tamamen viral klerensini sağlayacak bir tedavisinin henüz bulunmaması ve dünya genelinde her yıl yaklaşık bir milyon insanın yaşamını yitirmesine neden olabilmesi nedeniyle önemli bir halk sağlığı sorunu olarak güncelliğini korumaktadır¹. Kronik HBV (KHB) enfeksiyonları virüse ait viral föktörler ile enfekte bireye ait konak faktörleri ve ayrıca çevresel faktörlerin karşılıklı etkileşimi ile şekillenen dinamik bir süreçtir. Dolayısıyla kronik viral hepatitlerin doğal seyri, siroz ve hepatosellüler karsinoma (HSK) gibi son dönem karaciğer hastalıklarının gelişmesi ve prognozu ile tedavi yanıtlarının şekillenmesinde genotip ve mutasyonlar gibi viral faktörler ile enfekte bireyin genetik ve immünolojik faktörleri ile çevresel faktörler kritik belirleyicidir^2,3.

Sodyum taurokolat kotransporter polipeptiti (NTCP) insan HBV ve onun satellit virüsü olan hepatit D virüs (HDV) için fonksiyonel bir reseptördür⁴. NTCP kromozom 14’de lokalize, çözünmüş madde taşıyıcı aile 10’un (sodium/bile acid cotransporter family, SLC10) bir numaralı üyesi SLC10A1 geni tarafından kodlanan, hepatositler tarafından eksprese edilen safra tuzlarının enterohepatik sirkülasyonunda görev alan bir hücre yüzey glikoproteinidir⁵. Bu açıdan HBV enfeksiyonları için viral yüzey proteini (surface) PreS1 ile hepatosit yüzeyindeki PreS1’e spesifik reseptörü NTCP aracılığı etkileşimi ve sonrası endositoz yolu ile hücre içine transferini kapsayan giriş basamağı kritik öneme sahiptir. Dolayısıyla NTCP, anti-HBV tedavisi amacıyla virüsün hepatositlere bağlanması ve girişinin engellenmesine yönelik yeni terapötik hedeflerden birini oluşturmaktadır^6-8.

Buna karşın NTCP geni üzerinde saptanan özellikle p.Ser267Phe (S267F) polimorfizminin (single nucleotide polymorphism; SNP), NTCP’nin fizyolojik fonksiyonunda değişime neden olarak, HBV’nin hücre içine girişi ve enfeksiyon yükünün azalmasına neden olabildiği bildirilmiştir. Dolayısıyla NTCP’nin bu genetik varyantlarının HBV enfeksiyonuna direnç ve HBV enfeksiyonu ile ilişkili karaciğer sirozu ve HSK gelişme riski ile ilişkili olabileceği hipotez edilmektedir. Ayrıca NTCP etkileşim domainindeki (HBV PreS1) sekans farklılıklarının NTCP’ye bağlanma afinitesinde değişikliğe neden olarak anti-HBV tedavisi amacıyla tasarlanan HBV giriş inhibitörlerine karşı tedavi yanıtlarını olumsuz yönde etkileyebileceği de öne sürülmektedir^9-15.

Dolayısıyla bu çalışmanın amacı HBV ve HDV fonksiyonel reseptörü NTCP kodlayan gendeki polimorfizmleri saptamak ve NTCP S267 polimorfizminin varyasyonunun hastaların klinik sonuçları üzerindeki etkileri ile NTCP’yi hedef alan yeni terapötiklerin tedavi etkinliğinin öngörülmesinde prognostik etkisini değerlendirmektir.

MATERIALS AND METHODS

This study was supported by the Scientific and Technical Research Council of Turkey (TÜBİTAK) project number 218S769. Ethical approval of the study was granted by the Ethics Committee of İstanbul University, İstanbul Faculty of Medicine (no: 2018/1251, date: 14.09.2018).

Selection and Identification of the Cases

Patient Study Group

In this study, 150 treatment-naive patients with CHB who were diagnosed with CHB infection due to HBsAg seropositivity for more than 6 months and who were followed up by the Gastroenterohepatology Clinic of İstanbul University, İstanbul Medical Faculty, and in whom liver parenchymal biopsy was planned for histologic and clinical evaluation were included (Table 1).

Control Group

This study included 63 patients diagnosed with chronic hepatitis C infection and 80 patients who underwent liver parenchymal biopsy for different kinetic indications other than viral hepatitis agents (non-viral) by the Gastroenterohepatology Clinic of İstanbul University, İstanbul Medical Faculty (Table 1).

Clinical Material

Liver biopsy samples were obtained from the patients. Liver biopsy procedures were performed in the Gastroenterohepatology Clinic and interventional radiology units of İstanbul Medical Faculty. Total DNA samples obtained from liver biopsy specimens using a commercial kit (QIAamp DNA Mini kit, Qiagen GmbH, Hilden, Germany) were stored at -20 °C for SNP genotyping and sequence analysis to detect viral mutations.

SNP Genotyping (SLC10A1/rs2296651)

Genotype Analysis

Diagnosis and analysis of SNPs from human DNA samples isolated intrahepatically from patients’ liver tissue samples were performed using a real-time PCR (RT-PCR) device and method provided by the manufacturer (Applied Biosystems, Foster City, CA, USA). TaqMan SNP genotyping method was used for genotyping the NTCP S267F variant (c.800G>A, SLC10A1/rs2296651). For SNP analyses, StepOne Software programs were used for amplification curves obtained from TaqMan SNP genotyping assay and allelic discrimination.

Demographic and clinical laboratory data: Demographic, other clinical laboratory and pathologic data were obtained from patient files and/or the electronic data recording system of the hospital.

Statistical Analysis

Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) (16.0 software, SPSS Inc., Chicago, IL). Chi-square test and/or Fisher’s tests were used to compare categorical variables between data, and Mann-Whitney U and one-way ANOVA tests were used to compare non-categorical data. Results were expressed as mean and standard deviation. P<0.05 values were accepted as statistically significant differences.

RESULTS

Characteristics of Patients

Demographic, clinical and histological data of the patients included in the study are summarized in Table 1. In summary, 150 CHB patients, 78 males and 72 females, with a mean age of 41.63±13.17 years, were included in this study. The mean necroinflammatory activity (grade) level was 4.96±2.8 and the mean fibrosis stage (stage) level was 2.04±1.30 according to the histologic activity index obtained from the histologic evaluation of liver biopsy specimens. These data indicate that the patients included in the study group generally had limited inflammatory activity and fibrosis consistent with active chronic hepatitis.

Demographic, clinical, clinical laboratory, virologic and histopathologic data of 143 control group patients, including 63 patients with chronic hepatitis C infection and 80 patients with non-viral liver disease, 58 males and 85 females, are summarized in Table 1. The histologic grade of inflammation was 6.00±2.10 and the stage of fibrosis was 2.59±1.13 in patients with chronic hepatitis C. Of the patients who underwent liver biopsy for clinical indications other than viral hepatitis, 32 patients (40%) had fatty liver disease.

SLC10A1/rs2296651 Polymorphism (S267F, NTCP Variant)

In this study, S267F (G/A) genotype and polymorphism in SLC10A1/rs2296651 gene were investigated in total human DNA obtained from the liver tissue of 150 CHB patients and 143 patients in the control group. No S267F (G/A) polymorphism was found in any of the patients in both CHB and control groups. In the molecular analysis of all samples in the patient and control groups, the allele in the codon encoding the 267^th amino acid (AA) of the SLC10A1/rs2296651 gene was homozygous as G/G. G/A heterozygous or A/A homozygous alleles were not detected in any patient (Table 2).

DISCUSSION

In recent years, detection of genetic variations in the SLC10A1 gene encoding NTCP and investigation of their effects on the HBV-associated receptor function of NTCP is a current approach. The most common NTCP-associated genetic variant is reported to be the S267F polymorphism in scientific studies conducted predominantly in Asian populations to detect genetic variations in the SLC10A1 gene. In some of the scientific studies conducted for this purpose, it has been reported that the S267F polymorphism detected on the SLC10A1 gene may cause a change in the physiological function of NTCP, leading to a decrease in HBV entry into the cell and infection load. Therefore, it is hypothesized that NTCP S267F genetic variant may be associated with resistance to HBV infection and the risk of development of liver cirrhosis and HCC associated with HBV infection^10-13,16,17.

It has also been suggested that the NTCP S267F (c.800G>A, rs2296651) variant, which is located in the 4^th exon of the NTCP gene and causes missense mutation, causes a decrease in the receptor function of HBV, which may result in loss of HBV binding to the receptor, intracellular entry and decreased replication capacity⁹.

In a study conducted to examine the association of the NTCP S267F variant with HBV clearance, HBV-associated cirrhosis, HCC and resistance to HBV infection, the S267F (A allele) variant was detected at a higher rate in the healthy group than in HBV-infected patients. Therefore, it was reported that the S267F variant may be associated with a decreased risk of developing HBV-related cirrhosis and HCC and disease progression, but may not be associated with spontaneous HBV clearance. It has been reported that the S267F variant is generally protective against HBV infection and related diseases; however, this variant does not prevent the progression of cirrhosis towards HCC¹⁰.

In a study involving an Asian (Chinese) population of approximately 2000 patients and 2000 healthy controls investigating the effect of NTCP variations on resistance to CHB infection and clinical outcomes, it was reported that the NTCP Ser267Phe variant was associated with resistance to CHB and a decrease in the incidence of HBV-related liver diseases¹².

Today, Bulevirtide, a synthetic lipoprotein based on the first 47 aa of HBV Pre-S1, has been synthesized and pre-clinical and clinical studies are ongoing to test its therapeutic efficacy^19-21. Therefore, sequence differences in the NTCP interaction domain (HBV Pres1) and identification of single nucleotide polymorphisms (SNP) in NTCP may be useful in predicting the therapeutic efficacy of HBV cell entry inhibitors planned for clinical use in the near future for the treatment of HBV infections.

In a large-scale study in Taiwan, in which the association of the NTCP Ser267Phe variant with the serostatus of CHB infection and the risk of HBV-associated cirrhosis and HCC development was examined in 3801 people with CHB and 3801 HBsAg-negative people as the control group, the S267F variant was found in 18.5% of the control group, 17.2% in cirrhotic cases and 13.2% in non-cirrhotic HCC patients. It was reported that the risk of developing HCC was 25 times higher in individuals with GG genotype compared to GA and AA genotypes, and AA genotype was statistically significantly associated with HBsAg seronegativity. In conclusion, it was reported that the S267F variant was associated with resistance to CHB infection, HBV-related cirrhosis and decreased risk of development of HCC, and it was also predicted that analyzing the detection of this variant in patients together with HBV DNA levels may be useful in identifying patients with low risk of HBV-related HCC²².

On the other hand, the data of some scientific studies investigating the effects of these genetic variants on the HBV-related receptor function of NTCP do not confirm the data in this study^23-25. In a study examining the relationship between HBV infection and S267F (rs2296651) variation, it was reported that rs2296651 variant was not detected in either the infected or control groups and as a result, this SNP may be specific for the Asian population²⁶. Therefore, the role of these variants on resistance to HBV infection and development of CHB-related cirrhosis, HCC and anti-HBV therapies is still controversial. Moreover, the prevalence of NTCP SNP rs229665 may vary according to ethnicity, geography and HBV endemicity.

In our study, the SNP rs2296651, which causes the S267F variation in the SLC10A1 gene encoding the functional receptor NTCP of HBV, was not detected in any of the patients. Therefore, the effects of S267F variation on the clinical and histologic features of the patients could not be evaluated in this study. Therefore, the data obtained in this study suggest that the utility of SLC10A variants as a novel biomarker for identifying patients with CHB with a low risk of CHB-associated cirrhosis and HCC is limited, and that their role in reducing the efficacy of anti-viral therapeutics targeting NTCP blockade may be quite limited. Therefore, the prognostic value of the SNP rs2296651, which causes the S267F variation in the SLC10A1 gene, remains unclear.

Study Limitations

The limitations of this study were the small number of patients and samples included in this study and the detection of the S267F variation in the SLC10A1 gene at the mRNA level.

TARTIŞMA

Son yıllarda NTCP’yi kodlayan SLC10A1 gendeki genetik varyasyonların saptanması ve bunların NTCP’nin HBV ilişkili reseptör fonksiyonu üzerindeki etkilerinin araştırılması güncel bir yaklaşımdır. SLC10A1 genindeki genetik varyasyonların tespiti amacıyla ağırlıklı olarak Asya populasyonu üzerinde yapılan bilimsel çalışmalarda en sık rastlanan NTCP ilişkili genetik varyantın; S267F polimorfizmi olduğu rapor edilmektedir. Bu amaçla gerçekleştirilmiş bilimsel çalışmaların bir kısmında SLC10A1 geni üzerinde saptanan S267F polimorfizminin, NTCP’nin fizyolojik fonksiyonunda değişime neden olarak, HBV’nin hücre içine girişinin ve enfeksiyon yükünün azalmasına neden olabildiği bildirilmiştir. Dolayısıyla NTCP S267F genetik varyantının HBV enfeksiyonuna direnç ve HBV enfeksiyonu ile ilişkili karaciğer sirozu ve HSK gelişme riski ile ilişkili olabileceği hipotez edilmektedir^10-13,16,17.

NTCP geninin 4. ekzonunda yer alan ve yanlış anlamlı mutasyona (missense) sebep olan NTCP S267F(c.800G>A, rs2296651) varyantının, HBV’nin reseptör fonksiyonunda azalmaya neden olduğu bunun da HBV’nin reseptöre bağlanma kaybına, hücre içine girişe ve replikasyon kapasitesinin azalması ile sonuçlanabildiği de öne sürülmektedir⁹.

NTCP S267F varyantının HBV klerensi, HBV ilişkili siroz, HSK ve HBV enfeksiyonuna direnç ile ilişkisinin irdelenmesi amacıyla yapılan bir çalışmada S267F (A allel) varyantı sağlıklı grupta HBV ile enfekte hastalara göre daha yüksek oranda tespit edilmiştir. Dolayısıyla S267F varyantının HBV ilişkili siroz ve HSK gelişme riski ile hastalık progresyonunda azalma ile ilişkili olabileceği buna karşın spontan HBV klerensi ile ilişkili olmayabileceği rapor edilmiştir. S267F varyantının genel olarak HBV enfeksiyonu ve ilişkili hastalıklardan koruyucu niteliğe sahip olduğu, bununla birlikte bu varyantın sirozun HSK yönünde progresyonununu ise engellemediği belirtilmektedir¹⁰.

NTCP varyasyonlarının varyasyonların KHB enfeksiyonuna direnç ve klinik sonuçlarına etkisinin araştırıldığı Asya (Çin) populasyonunu kapsayan yaklaşık 2000 hasta ve 2000 sağlıklı kontrol grubu içeren bir çalışmada, NTCP Ser267Phe varyantının KHB’ye direnç ve HBV ilişkili karaciğer hastalıklarının insidansında azalmayla ilişkili olduğu rapor edilmiştir¹².

Ayrıca NTCP, HBV ve HDV enfeksiyonlarının tedavisi amacıyla virüsün hepatositlere bağlanması ve girişinin engellenmesine yönelik yeni terapötik hedeflerden biridir. NTCP’nin reseptör fonksiyonun engellenmesi amacıyla HBV PreS1 domaininin NTCP bağlanma motifini içeren N-terminal bölgesindeki ilk 47 aa’nin (2-48 aa) ucuna (2. aa, lizin) miristik asitin (tetradekanoik asit) eklenmesi (Miristilasyon; Protein N-myristoylation) ile elde edilen sentetik bir lipoproteinin tedavide kullanılması planlanmaktadır^6,18. Günümüzde HBV Pre-S1’in ilk 47 aa’ni baz alarak elde edilen sentetik bir lipoprotein olan Bulevirtide’nin (BLV) sentez edilmiş ve terapötik etkinliğinin test edilmesine yönelik pre-klinik ve klinik çalışmalar devam etmektedir^19-21. Bu nedenle NTCP etkileşim domainindeki (HBV Pres1) sekans farklılıkları ve NTCP’deki tek nükleotid polimorfizmlerin (Single nucleotide polymorphism, SNP) tanımlanması, HBV enfeksiyonlarının tedavisi amacıyla yakın gelecekte klinik kullanımı planlanan HBV hücre giriş inhibitörlerinin tedavi etkinliğinin öngörülmesinde yararlı olabilir.

Tayvanda NTCP Ser267Phe varyantının KHB enfeksiyonunun serostatüsü, HBV ilişkili siroz ve HSK gelişme riski ile ilişkisinin irdelendiği 3801’i KHB ve kontrol grubu olarak HBsAg negatif 3801 kişiyi kapsayan geniş çaplı bir çalışmada S267F varyantı kontrol grubunda %18,5, sirozlu olgularda %17,2, non-sirotik HSK’lılarda ise %13,2 oranında tespit edilmiştir. GG genotipli bireylerde GA ve AA genotipine göre HSK gelişme riskinin 25 kat daha yüksek olduğu, AA genotipinin istatistiksel olarak anlamlı oranda HBsAg seronegatifliği ile ilişkili olduğu rapor edilmiş. Sonuç olarak S267F varyantının KHB enfeksiyonuna direnç, HBV ilişkili siroz ve HSK gelişme riskinde azalma ile ilişkili olduğu bildirilmiş, ayrıca hastalarda bu varyantın tespitinin HBV DNA düzeyleri ile birlikte analiz edilmesinin HBV ilişkili HSK riski düşük hastaları tanımlamada faydalı olabileceği öngörülmüştür²².

Buna karşın saptanan bu genetik varyantların NTCP’nin HBV ilişkili reseptör fonksiyonu üzerindeki etkilerinin araştırıldığı bazı bilimsel çalışmaların verileri ise bu çalışmadaki verileri doğrulamamaktadır^23-25. HBV enfeksiyonu ile S267F (rs2296651) varyasyonu arasındaki ilişkinin irdelendiği bir çalışmada da gerek enfeksiyonlu gerekse kontrol gruplarının hiçbirinde rs2296651 varyantının tespit edilemediği, sonuç olarak bu SNP’nin Asya popülasyonuna spesifik olabileceği belirtilmiştir²⁶. Dolayısıyla bu varyantların HBV enfeksiyonuna direnç ve KHB ile ilişkili siroz, HSK gelişimi ayrıca anti-HBV tedavileri üzerindeki rolleri halen tartışmalıdır. Ayrıca NTCP SNP rs229665’nin prevelansı etnisite, coğrafya ve HBV endemisitesine göre farklılıklar gösterebilmektedir.

Bizim çalışmamızda hastaların hiçbirinde HBV’nin fonksiyonel reseptörü NTCP’yi kodlayan SLC10A1 genindeki S267F varyasyona sebep olan SNP rs2296651 tespit edilmedi. Bu nedenle bu çalışmada S267F varyasyonunun hastaların KHB’li özellikleri üzerindeki etkileri değerlendirilemedi. Dolayısıyla bu çalışmadan elde edilen veriler, SLC10A varyantlarının KHB iliskili siroz ve HSK riski düşük kronik B hepatitli hastaların tanımlanmasında yardımcı yeni bir biyolojik belirteç olarak kullanılabilirliğinin sınırlı olduğuna işaret ettiği gibi, NTCP blokajını hedef alan anti-viral terapötiklerin tedavi etkinliğini azaltması yönündeki rolünün de oldukça sınırlı olabileceğini düşündürmektedir. Bu yüzden SLC10A1 genindeki S267F varyasyonuna neden olan SNP rs2296651 saptanmasının prognostik değerliliği konusu belirsizliğini sürdürmektedir.

Çalışmanın Kısıtlılıkları

Bu çalışmaya dahil edilen hasta ve örneklem sayısının az olması ile SLC10A1 genindeki S267F varyasyonun mRNA düzeyinde tespit edilemesi çalışmanın kısıtlılıklarını oluşturmaktaydı.

CONCLUSION

In conclusion, the data obtained from this study suggest that the NTCP interaction domain is highly conserved in patients with CHB, and therefore the potential negative impact of S267F variations in the SLC10A1 gene encoding NTCP on the progression of HBV-associated liver damage and antiviral therapeutics targeting NTCP may be quite limited.

Ethics

Ethics Committee Approval: Ethical approval of the study was granted by the Ethics Committee of İstanbul University, İstanbul Faculty of Medicine (no: 2018/1251, date: 14.09.2018).

Informed Consent: Retrospective study.

Authorship Contributions

Surgical and Medical Practices: B.Ç., A.A., B.Ç., M.B., F.A., Concept: B.Ç., F.A., Design: B.Ç., F.A., Data Collection or Processing: B.Ç., A.A., M.P., B.Çav., M.B., M.G., F.A., Analysis or Interpretation: B.Ç., M.P., M.G., F.A., Literature Search: B.Ç., F.A., Writing: B.Ç., F.A.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was supported by the Scientific and Technical Research Council of Turkey (TÜBİTAK) project number 218S769.

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