Russian Medical Inquiry
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Molecular genetic markers and metabolic disorders in non-alcoholic fatty liver disease

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DOI: 10.32364/2587-6821-2022-6-5-206-212

A.B. Krivosheev1, V.N. Maksimov1,2, K.Yu. Boyko3, E.E. Levykina3, E.S. Mikhaylova1,4, N.A. Varaksin5, M.A. Kondratova1, I.A. Krivosheeva1,3, A.I. Autenshlyus1,4

1Novosibirsk State Medical University, Novosibirsk, Russian Federation

2Research Institute for Therapy and Preventive Medicine — Branch of the Federal Research Center Institute of Cytology and Genetics of the Siberian Branch of the RAS, Novosibirsk, Russian Federation

3City Clinical Hospital No. 1, Novosibirsk, Russian Federation

4Federal Research Center for Fundamental and Translational Medicine, Novosibirsk, Russian Federation

5JSC "Vektor-Best", Novosibirsk, Russian Federation

Aim: to assess the probability of unfavorable outcomes of non-alcoholic fatty liver disease (NAFLD) based on clinical, biochemical, and molecular genetic parameters.

Patients and Methods: 440 individuals were examined. Among them, 115 patients (84 men and 31 women) aged 23–69 (mean 49.3±1.1 years) were diagnosed with NAFLD and 325 healthy volunteers (172 men and 153 women) aged 25–67 (mean age 47.9±0.6 years) were controls. Molecular genetic testing for TCF7L2 (ТС, СС, and ТТ genotypes) was performed in all participants. The rates of Glu342Lys (PIZ) and Glu264Val (PIS) mutations of α1-antitrypsin gene (SERPINA1) and 282Y and 63D alleles of hemochromatosis (HFE) gene were evaluated. Standard liver function tests (ASAT, ALAT, bilirubin), lipid metabolism (total cholesterol, triglycerides, HDL, LDL), excretory porphyrin metabolism (porphyrin precursors [δ-aminolaevulinic acid and porphobilinogen] and fractions [uroporphyrin and coproporphyrin]), and cytokine profile (interleukins 1β, 6, 8, 10, and 1Ra, tumor necrosis factor/TNF α) were assessed.

Results: the rates of TCF7L2 genotype, 282Y and 63D HFE gene alleles were similar in NAFLD patients and healthy controls. Meanwhile, Glu342Lys (PIZ) and Glu264Val (PIS) SERPINA1 gene polymorphisms were significantly more common in NAFLD patients vs. general population. The odds ratio (OR) has demonstrated that Glu342Lys (PIZ) genotype occurrence is 3.9 times greater in the NAFLD group than in healthy controls (NZ + ZZ vs. NN: OR=3.90, 95% CI 1.5–10.5, p=0.007), while Glu264Vol (PIS) genotype occurrence is 6.6 times greater in the NAFLD group than in healthy controls (NS vs. NN: OR=6.6, 95 CI 2.4–18.3, p<0.001). Abnormalities of porphyrin metabolism and cytokine profile were detected in most participants (71.3% and 82.6%, respectively). Unfavorable NAFLD outcomes were reported in 30 patients (26.1%).

Conclusions: molecular genetic testing and specific blood biochemistry allows for predicting NAFLD outcome. Describing metabolic disorders allows for assessing the risk of unfavorable outcome.

Keywords: non-alcoholic fatty liver disease, molecular genetic testing, TCF7L2 gene, α1-antitrypsin gene (SERPINA1), hemochromatosis gene (HFE), lipid metabolism, porphyrin metabolism, cytokine profile, unfavorable outcome.

For citation: Krivosheev A.B., Maksimov V.N., Boyko K.Yu. et al. Molecular genetic markers and metabolic disorders in non-alcoholic fatty liver disease. Russian Medical Inquiry. 2022;6(5):206–212 (in Russ.). DOI: 10.32364/2587-6821-2022-6-5-206-212.


Background

In the last decade, detection rate of non-alcoholic fatty liver disease (NAFLD) in the Russian Federation has increased significantly. According to Russian multicenter observational epidemiological studies DIREG_L_0193 (2007) [1] and DIREG 2 (2014) [2], NAFLD detection rate increased from 26.1% to 33.3%. This trend is accounted for by not simply improved diagnosis of the disease. For a long time, a universal pathophysiological syndrome, insulin resistance (IR) and hyperinsulinemia (which develop even at the very early stages of NAFLD) was considered to play a key role in the pathogenesis of NAFLD. Moreover, this pattern remains so far [3]. Meanwhile, the role of molecular genetic tests and specific techniques is now increasing, thereby enabling more accurate diagnosis and prediction of the course of internal diseases (including chronic liver diseases and NAFLD), and prediction of the risk of complications and unfavorable outcomes [4–6]. The role of various candidate genes whose polymorphisms are significant for the development and progression of NAFLD is actively studied. In particular, the association with the G allele of the rs666089 ADIPOR1 gene and PNPLA3/148M gene polymorphism is discussed [5, 7].

The aim of this study was to assess the probability of unfavorable outcomes of NAFLD based on clinical, biochemical, and molecular genetic parameters.

Patients and Methods

One hundred fifteen patients (84 men and 31 women) aged 23–69 (mean 49.3±1.1 years) with established NAFLD were examined. The diagnosis was established by complex examination according to the Russian Liver Society and Russian Gastroenterological Association clinical guidelines on NAFLD diagnosis and management [8]. To verify hepatic steatosis in NAFLD patients, hepatic steatosis index (HIS) was calculated: HIS = 8 × (AST/ALT) + BMI + 2 (if female) + 2 (if diabetes). HIS values of 36 and above indicate that NAFLD positive diagnosis is highly likely (sensitivity 93.1%, specificity 92.4%) [9]. HIS was higher in the study group (41.4±1.3) compared to the control group, thereby establishing hepatic steatosis. Hepatotropic viral infections and chronic alcoholism were ruled out [10].

The control group included 325 healthy volunteers (172 men and 153 women) aged 25–67 (mean age 47.9±0.6 years). This age-matched group was randomly sampled from permanent residents of the city of Novosibirsk at a ratio of 1:3 (1 patient per 3 controls). These individuals were examined as a part of the MONICA [11] and HAPIEE [12] programs, the WHO international epidemiological programs on studying morbidity and mortality from cardiovascular diseases and lipid disorders in various regions and populations.

All participants underwent molecular genetic testing. Genotyping for the presence/absence of Glu342Lys (PIZ) andGlu264Val (PIS) mutations of α1-antitrypsin gene (SERPINA1) and 282Y and 63D alleles of hemochromatosis (HFE) gene, TCF7L2 gene, was performed.

Study group patients underwent complex examination, i.e., standard liver function tests, lipid metabolism, and cytokine profile (interleukin/IL-1β, IL-6, IL-8, IL-10, and IL-1Ra, tumor necrosis factor/TNF α) were assessed. Their levels increase in viral hepatitis, cirrhosis, autoimmune hepatitis, alcoholic liver disease, hepatocellular carcinoma, and other digestive diseases [13–15]. These parameters were measured using standard immunoassay reagents. Porphyrin precursors (δ-aminolaevulinic acid and porphobilinogen) and fractions (uroporphyrin and coproporphyrin) were evaluated by chromatography–spectrophotometry.

All individuals examined were followed up for 5 years. The rates of adverse events, i.e., death, acute vascular events (myocardial infarction, transient ischemic attack, stroke), and the development of cirrhosis, were recorded.

The results of clinical and lab tests were analyzed using variance estimation methods. Continuous measures were compared after testing for the normal distribution using the Kolmogorov-Smirnov test. For normally distributed values, one-way ANOVA was applied. For non-normally distributed values, the Mann–Whitney test for two independent samples was applied. Differences between the mean values were considered significant at p<0.05. Differences between the occurrences of qualitative variables were assessed using the χ2 test or Pearson’s correlation coefficient. When establishing the reliability of associations, the χ2 distribution table was used (results were considered reliable at p<0.05 at appropriate degrees of freedom (n1). Association of two qualitative parameters was assessed using fourfold contingency tables with calculating odds ratio (OR) and confidence intervals (95% CI).

Results and Discussion

In the NAFLD group, 342Lys (PIZ) and 264Val (PIS) alleles of the SERPINA1 gene were identified in 22 patients (19.1%). 264Val (PIS) mutation occurred slightly more often (see Table 1). Meanwhile, in the control group, 342Lys (PIZ) and 264Vol (PIS) alleles of the SERPINA1 gene were identified in 14 individuals (4.3%). Therefore, 342Lys (PIZ) and Glu264Val (PIS) SERPINA1 gene polymorphisms were significantly more common in NAFLD patients. The chance of detecting 342Lys (PIZ) genotype in the NAFLD group was 3.9 times greater than in healthy controls (NZ + ZZ vs. NN: OR=3.90, 95% CI 1.5–10.5, p=0.007), while the chance of detecting 264Vol (PIS) genotype in the NAFLD group was 6.6 times greater than in healthy controls (NS vs. NN: OR=6.6, 95 CI 2.4–18.3, p<0.001).

Таблица 1. Частота носительства аллелей Glu342Lys (PIZ) и Glu264Vol (PIS) гена SERPINA1 и аллелей 282Y и 63D гена HFE в основной и популяционной группах Table 1. The occurrence of Glu342Lys (PIZ) and Glu264Vol (PIS) SERPINA1 gene alleles and 282Y and 63D

Meanwhile, 282Y and 63D HFE gene alleles were identified in 36 (31.3%) patients with NAFLD. 63D was detected in 30 patients (26.1%), while 282Y was detected in 6 patients (5.2%). In the comparison group, 282Y and 63D HFE gene alleles were found in 115 individuals (35.4%). 63D allele occurred much more often that 282Y allele (by 4.5 times). No significant differences in the rates of 282Y and 63D HFE gene alleles between the groups were reported (χ2 =0.63, p>0.5 at n´=3).

Comparative evaluation of carbohydrate metabolism was performed in 115 NAFLD patients based on the clinical guidelines “Algorithms for specialized medical care in diabetes” [16] (see Table 2). All patients were divided into three groups based on the TCF7L2 genotypes. Group 1 included 58 patients with the TC genotype (50.4%). Group 2 included 50 patients with the CC genotype (43.5%). Group 3 included 7 patients with the TT genotype (6.1%). The TCF7L2 gene is considered the key gene in terms of the development of pancreatic β-cell dysfunction and carbohydrate metabolism disorders, and diabetes manifestation [13, 17]. Abnormalities in carbohydrate metabolism were reported in 84 patients with NAFLD (73.0%). Prediabetes was diagnosed in 21 patients (mostly with TC genotype). The largest group included patients with manifest type 2 diabetes with TC (n = 34, 58.6%) and CC (n = 26, 52.0%) genotypes. During the examination, diabetes was first diagnosed in 7 patients with the TC genotype and 5 patients with the CC genotype (n = 12, 10.4%).

Таблица 2. Характеристика углеводного обмена у больных НАЖБП с учетом генотипирования по гену TCF7L2 Table 2. Carbohydrate metabolism in NAFLD patients with different TCF7L2 genotypes

Abnormalities of porphyrin metabolism were detected in most participants (n = 82, 71.3%) and were significantly more common in patients with the TC genotype (n = 45, 39,1%). Meanwhile, no significant differences were revealed between CC and TT genotypes (χ2 = 0.57, р >0.5 at n´=4). In patients with Glu324Lys (PIZ) and Glu246Vol (PIS) alleles of the SERPINA1 gene and 282Y and 63D alleles of the HFE gene, the rates of porphyrin metabolism abnormalities were almost similar (72.7% and 80.6%, respectively; see Table 3).

Таблица 3. Частота нарушений порфиринового обмена по результатам молекулярно-генетического тестирования по генам TCF7L2, SERPINA1 и HFE у больных НАЖБП Table 3. The occurrence of porphyrin metabolism abnormalities based on molecular genetic testing (TCF7L 

The analysis of cytokine profile has demonstrated that IL-1β, IL-6, and IL-1Ra were predominant irrespective of the TCF7L2 genotype (see Table 4). A similar trend was reported in patients with Glu324Lys (PIZ) and Glu246Vol (PIS) alleles of the SERPINA1 gene and 282Y and 63D alleles of the HFE gene.

Таблица 4. Частота нарушений показателей цитокинового спектра по результатам молекулярно-генетического тестиро- вания по генам TCF7L2, SERPINA1 и HFE у больных НАЖБП Table 4. The occurrence of cytokine profile abnormalities based on molecular genetic test

Liver function tests (ALT, AST, total bilirubin) were within normal ranges. No significant differences were reported in patients with and without SERPINA1 and HFE gene mutations (see Table 5). In contrast, all lipid spectrum parameters were significantly higher than normal ranges. Moreover, high- and low-density lipoprotein cholesterol levels were significantly worse in patients with SERPINA1 and HFE gene mutations. These mutations significantly (p<0.02 to p<0.05) contributed to the worsening of porphyrin metabolism parameters and an increase in IL-6 and IL-1Ra levels.

Таблица 5. Состояние метаболических показателей с учетом результатов молекулярно-генетического обследования при НАЖБП Table 5. Metabolic parameters based on molecular genetic testing in NAFLD patients

Окончание таблицы 5 Table 5 (continued)

In 2016–2020, unfavorable NAFLD outcomes were reported in 30 patients (26.1%). They included death (n = 17, 56.7%), acute vascular events, i.e., myocardial infarction, stroke, and transient ischemic attack (n = 8, 26.7%), and cirrhosis (n = 5, 16.6%).

Conclusions

Molecular genetic testing for the TCF7L2 gene in patients with NAFLD demonstrates that unfavorable outcomes are most common in the CC genotype (n = 15, 50.0%), more rarely, in the TC genotype (n = 13, 43.3%), and, much less often, in the TT genotype (n = 2, 6.7%). In 50% of patients with unfavorable outcomes, 282Y and 63D alleles of the HFE gene and 342Lys (PIZ) and 264Val (PIS) alleles of the SERPINA1 gene were reported. The analysis of risk factors for unfavorable outcomes of NAFLD shows that cytokine (IL-1β, IL-6, IL-1Ra) activity is reported to be more common (n = 27, 90%). Porphyrin metabolism disorders were detected also common (n = 22, 73.3%). The most relevant clinical parameter was abdominal obesity. Molecular genetic testing and specific blood biochemistry allows for predicting unfavorable NAFLD outcome.


About the authors:

Aleksandr B. Krivosheev — Dr. Sc. (Med.), professor of the Prof. G.D. Zalesskiy Department of Faculty Therapy, Novosibirsk State Medical University; 52, Krasnyi av., Novosibirsk, 630091, Russian Federation; ORCID iD 0000-0002-4845-8753.

Vladimir N. Maksimov — Dr. Sc. (Med.), Professor, Head of the Laboratory of Molecular Genetic Testing of Therapeutic Diseases, Research Institute for Therapy and Preventive Medicine — Branch of the Federal Research Center Institute of Cytology and Genetics of the Siberian Branch of the RAS; 175/1, B. Bogatkov str., Novosibirsk, 630089, Russian Federation; professor of the Department of Biology and Medical Genetics, Novosibirsk State Medical University; 52, Krasnyi av., Novosibirsk, 630091, Russian Federation; ORCID iD 0000-0002-7165-4496.

Konstantin Yu. Boyko — resident of the Department of Endocrinology, City Clinical Hospital No. 1; 6 build. 4, Zalesskiy str., Novosibirsk, 630047, Russian Federation; ORCID iD 0000-0003-3293-0061.

Elena E. Levykina — resident of the Department of Gastroenterology, City Clinical Hospital No. 1; 6 build. 4, Zalesskiy str., Novosibirsk, 630047, Russian Federation; ORCID iD 0000-0002-2029-0557.

Elena S. Mikhailova — researcher of the Central Research Laboratory, Novosibirsk State Medical University; 52, Krasnyi prospect, Novosibirsk, 630091, Russian Federation; researcher, Federal Research Center for Fundamental and Translational Medicine; 2, Timakov str., Novosibirsk, 630060, Russian Federation; ORCID iD 0000-0001-8576-3717.

Nikolay A. Varaksin — Head of the Laboratory of Cytokines, JSC "Vektor-Best"; build. 36, working settlement Koltsovo, Research and Production Zone, Novosibirsk, 630559, Russian Federation; ORCID iD 0000-0002-0733-7787.

Maria A. Kondratova — C. Sc. (Med.), assistant of the Prof. G.D. Zalesskiy Department of Faculty Therapy, Novosibirsk State Medical University; 52, Krasnyi av., Novosibirsk, 630091, Russian Federation; ORCID iD 0000-0002-7971-6479.

Inga A. Krivosheeva — C. Sc. (Med.), assistant of the Department of Occupational Diseases with the Course of Endocrinology, Novosibirsk State Medical University; 52, Krasnyi av., Novosibirsk, 630091, Russian Federation; Head of the Department of Endocrinology, City Clinical Hospital No. 1; 6 build. 4, Zalesskiy str., Novosibirsk, 630047, Russian Federation; ORCID iD 0000-0002-3575-4983.

Aleksandr I. Autenshlyus — Dr. Sc. (Biol.), Head of the Central Research Laboratory, Novosibirsk State Medical University; 52, Krasnyi av., Novosibirsk, 630091, Russian Federation; leading researcher, Federal Research Center for Fundamental and Translational Medicine; 2, Timakov str., Novosibirsk, 630060, Russian Federation; ORCID iD 0000-0002-6538-0089.

Contact information: Aleksandr B. Krivosheev, e-mail: krivosheev-ab@narod.ru.

Financial Disclosure: no authors have a financial or property interest in any material or method mentioned.

There is no conflict of interests.

Received 27.03.2022.

Revised 19.04.2022.

Accepted 18.05.2022.

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