Effect of PNPLA3, TM6SF2 and MBOAT7 Genetic Variants on Non-alcoholic Fatty Liver Disease Therapeutic Outcome.
Condition: Non-Alcoholic Fatty Liver Disease; Insulin Resistance
MeSH terms: Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease; Insulin Resistance
Study type: Interventional

Summary: 
      Patatin-like phospholipase domain-containing protein-3 (PNPLA3), the transmembrane 6
      superfamily member 2 protein (TM6SF2) and membrane bound O-acyltransferase domain containing
      7 (MBOAT7) genes are involved in non-alcoholic fatty liver disease (NAFLD) development and
      worsening. Following the actual scientific knowledge, some studies have identified the
      genetic background surrounding NAFLD, counting up to forty different genetic variants that
      seem to exert also a crucial role in the disease evolution, according to the natural history,
      until hepatocellular carcinoma onset. However, few data exist regarding their influence on
      the treatment response. The aim was to explore the effect of 303 mg of silybin-phospholipids
      complex, 10 mg of vitamin-D and 15 mg of vitamin-E twice a day for six months in NAFLD
      patients carrying PNPLA3-rs738409, TM6SF2-rs58542926 and MBOAT7-rs641738 genetic variants.
      The assessed mutations are independently associated with no response to a silybin/vitamin
      D-based therapy and could be useful therapeutic predictive markers in this context.
    

Detailed description: 
      The aim of this study was to evaluate if the PNPLA3 rs738409, TM6SF2 rs58542926 and MBOAT7
      rs641738 could influence the response of NAFLD patients regarding metabolic parameters, liver
      damage and hepatic fat accumulation ones, to a treatment with 303 mg of silybin-phospholipids
      complex, 10 mg of vitamin D, and 15 mg of vitamin E twice a day for six months.

      The investigators performed a baseline comparison of Weight, waist-to-height ratio (WHtR),
      blood pressure measurement, body mass index (BMI), Blood glucose and insulin, the homeostatic
      model for insulin resistance assessment (HOMA-IR), aspartate and alanine aminotransferase
      (AST, ALT), gamma-glutamyl transferase (GGT), blood count, C reactive protein (CRP), the
      Thiobarbituric Acid Reactive Substance (TBARS), liver stiffness and controlled attenuation
      parameter (CAP) among the three study groups: NAFLD wild type control group (n. 30), NAFLD
      treated wild type group (n. 30), NAFLD treated mutated group (n. 32). The block randomization
      method was used to randomize the 60 not mutated patients in the NAFLD wild-type control group
      and NAFLD treated wild type one, using the online randomization software
      http://www.graphpad.com/quickcalcs/index.cfm.

      The wild-type control group was composed by NAFLD patients without PNPLA3, TM6SF2 and MBOAT7
      mutations that did not receive any type of treatment during the study period.

      The patients inserted in the NAFLD treated wild type and NAFLD treated mutated groups were
      undergone to an oral administration of 303mg of silybin-phospholipid complex, 10mg of vitamin
      D, and 15mg of vitamin E, twice a day for six months. None of the enrolled patients
      dropped-out the study.

      At the end of treatment, the assessment of WHtR, blood pressure measurement, BMI, Blood
      glucose and insulin, HOMA-IR, AST, ALT, GGT, blood count, CRP, TBARS, liver stiffness and CAP
      was re-performed. During the experimental observation, patients were on free diet on the
      basis of dietary habits before the enrollment and any type of physical exercise was
      recommended during the study period. Food intake was evaluated both at baseline and end of
      treatment using a computerized software. The investigators recorded, with a diet diary, the
      food intake of a complete week, including working days and the weekend. On the basis of the
      quantities and qualities of food consumed, the soft elaborates the daily energy intake and
      the percentage/caloric amount of macronutrients. For the physical exercise assessment, the
      investigators submitted a specific questionnaire at baseline and end of treatment with some
      simple questions: Are the participant doing or have the participant ever done (in the last
      two years) sport in a continuative and regular way? Have the participant changed his/her
      daily physical activity in the last six months? 1: no, 2: yes; if yes, has the physical
      activity enhanced or worsened? Alcohol consumption was assessed at the beginning and at the
      end of the treatment. One hundred two patients with histological diagnosis of NAFLD followed
      by the Hepatogastroenterology Division of the University of Campania "Luigi Vanvitelli,
      between January and October 2017 were screened, after signing an informed consent, for the
      PNPLA3 rs738409, TM6SF2 rs58542926 and MBOAT7 rs641738 genetic variants and thirty-two met
      the inclusion criteria for the study and showed at least one among PNPLA3 I148I/M, I148M/M,
      TM6SF2 167E/K, 167K/K and MBOAT7 TMC4C/T or TMC4T/T genetic variants, were enrolled together
      with sixty patients without the mutations. Ten patients were excluded from the enrollment due
      to the coexistence of several comorbidities and/or advanced stages liver disease such as
      cirrhosis and/or hepatocellular carcinoma (HCC).

      The definition of the presence/absence of NAFLD and the staging were assessed by performing a
      liver biopsy, serological tests and collecting clinical data. Medical history, alcohol
      consumption (AUDIT-C), medications, drug abuse, smoking habits, were also investigated. Blood
      pressure, weight, height were directly measured and WHtR was calculated. BMI was also
      calculated by dividing the weight (kg) by the square of height (m). The patients were
      undergone after 12 hours fast to peripheral venous blood samples collection in order to
      evaluate some biochemical parameters and to perform the genetic analysis.

      Insulin, GGT, CRP, levels were measured enzymatically using commercially available kits, AST,
      ALT and glucose using colorimetric assay kit (Amplite 13801/13803 and Thermo Fisher
      Scientific EIAGLUC). HOMA-IR was also calculated using the formula: fasting insulin (μU/mL) ×
      plasma glucose (mmol/L)/22.5.

      FibroScan® transient elastography (TE) was performed using the FibroScan® version 502
      (Echosens, Paris, France) with standard probes (M and XL probes. The extra large (XL) probe
      was used when the distance from the skin to the liver capsule, assessed by ultrasonography,
      exceeded 2.5 cm and/or when BMI was >30. FibroScan® was performed by an expert physician
      obtaining ten acceptable measurements, defined as a successful liver stiffness (LS)
      measurement, with the maximum number of attempts set at 20. The criteria proposed by Boursier
      et al. were used to consider the measurement "very reliable" (IQR/M ≤ 0:1), "reliable" (0:1 <
      IQR/M ≤ 0:3 or IQR/M > 0:3 with LS median < 7:1 kPa), or "poorly reliable" (IQR/M > 0:3 with
      LS median ≥ 7:1 kPa). On the basis of CAP these scores, the investigators classified the
      enrolled patients in S0, no steatosis (0%-10% fat; 0-237 dB/m); S1, mild steatosis (11%-33%
      fat; 238-259 dB/m); S2, moderate steatosis (34%-66% fat; 260-292 dB/m); and S3, severe
      steatosis (>67% fat; ≥293 dB/m) in accordance with calculation of the attenuation of
      ultrasonic signals used for TE.

      TBARS assay was performed using 10 μl of serum. The cromogen TBARS was quantified using a
      spectrophotometer at a wavelength of 532nm with 1,1,3,3-tetramethoxyprophane as a standard.
      The amount of TBARS was expressed as nmol/μg of protein. Presented data are the mean ±
      standard deviation, resulting from three independent experiments.

      The genomic DNA extraction from peripheral blood samples was performed by using the
      extraction kit PureLink Genomic DNA Kit (Invitrogen by Life Technologies, USA). The DNA
      amount of each sample was assessed by spectrophotometer (NanoDrop Thermo Fisher Scientific)
      using a wave length of 260 nm. The presence of sample contamination was assessed by using a
      280 nm wave length evaluation and all the sample showed good degree of purity because of the
      260/280 ratio was between 1.8 and 2. The DNA extracted was then stored in -20°C freezer until
      the analysis of the polymorphisms.

      The single nucleotide polymorphisms (SNPs) analysis, using the access code to the data bank
      TM6SF2 rs58542926 (SNP 1), MBOAT7 rs641738 (SNP2), PNPLA3 rs738409 (SNP3), was performed
      using the DNA genotyping RealTime PCR with TaqMan (Applied Biosystem) c_89463510_10 for SNP
      1, c_8716820_10 for SNP 2, c_7241_10 for SNP 3 probes. All the evaluations were done in three
      phases: DNA PCR amplification, allelic identification and end point analysis with melting
      curve.

      The first phase was done by using the AmpliTaq Gold® DNA polymerase contained in the TaqMan
      Universal PCR Master Mix amplifying the target sequence by specific primers contained in the
      SNP Genotyping Assay 40X together with TaqMan® MGB probes: one marked with fluorochrome VIC®
      that recognize the sequence of the allele 1 and another probe marked with fluorochrome FAM™
      that recognize the sequence of the allele 2. For each experiment a 48 well plate was used, in
      which, a part of the biologic samples of unknown genotype for the SNP analysis, three
      different negative controls were analyzed in order to avoid possible errors due to the
      contamination of samples. Each experiment was done in triplicate. The amplifications were
      performed using the StepOne™ Real Time PCR System (Applied Biosystems). The genotyping
      assessment is based on the allelic discrimination thanks to a different fluorescence of the
      specific gene primer, using two MGB TaqMan® probes, marked at 5' with a different
      fluorochrome: SNP1 in reverse G in VIC® and A in FAM™; SNIP2 in forward C in VIC® and T in
      FAM™; SNIP3 in forward C in VIC® e G in FAM™. For the results analysis the software
      StepOne™2.0 was used. After the differentiation of the fluorescence made from the probes to
      the background in each well, it measures the normalized signal intensities (Rn) projecting
      the results in allelic discrimination plot. The software gives to the samples a specific
      genotype based on the fluorescence signal position: horizontal axis (allele one), vertical
      axis (allele 2) diagonal axis (both allele one and two). The prevalence of a specific
      fluorescence on the other one identified the homozygosis genotype, on the contrary the
      presence of both the heterozygosis.

      Statistical Analysis The number of patients (30 in the NAFLD wild-type control group, 30 in
      the NAFLD treated wild type and 32 in NAFLD treated mutated ones) was calculated using the
      power and sample size calculation function of STATA-14® for mac-OS, on the basis of an
      expected difference among the study groups in the response of the HOMA-IR to the therapy,
      assuming a double amount of patient responder to the therapy in wild type group in comparison
      to the mutated one. Specifically, the investigators considered the patients responder if at
      least one of the following criteria was addressed: normalization of the HOMA-IR (<2.5) at the
      end of treatment starting from baseline values greater than 2.5; reduction of the HOMA-IR ≥ 2
      points at the end of treatment in comparison to baseline. On the basis of this difference,
      the investigators estimated 29 patients per arm as the correct sample size of subjects to be
      investigate maintaining an 0.01 alpha error and a 90% statistical power in a two-sided test
      with a 95% Confidence Interval. A Kolgoromov-Smirnov for normality was performed to evaluate
      if parametric or non-parametric analysis should be applied. Wilcoxon signed ranks test,
      Mann-Whitney U test and t-test for dependent or independent groups were performed in order to
      compare continuous variables. The Kruskal-Wallis test or ANOVA test with post-hoc Bonferroni
      analysis, in case of non-normal or normal distribution respectively, were performed to
      compare the continuous variables among three groups. Pearson's or Kendall Tau-b correlations
      as well as linear regression were applied to test the associations among variables. Multiple
      logistic regression analysis was performed to assess the relationship between the genotype of
      patients (NAFLD treated wild type vs mutated patients) and the therapeutic outcome on:
      insulin, HOMA-IR, ALT, CRP and TBARS. The investigators identified in the following values
      the specific cut-offs to consider the parameter improved: insulin normalization (< 24
      micro-IU/ml), normalization of the HOMA-IR (<2.5) at the end of treatment starting from
      baseline values greater than 2.5 and/or reduction of the HOMA-IR ≥ 2 points at the end of
      treatment in comparison to baseline, ALT normalization (<45 IU/L), CRP normalization (<0.6
      mg/dL) or reduction of at least 1 mg/dL, TBARS reduction of at least 10 nmol/μg. The
      abovementioned parameters were chosen in relation to the main therapeutic effect of silybin
      in this context, considering NAFLD as a systemic disease. The relative risk (RR) of a useful
      therapeutic outcome considering the genotype of the patients was calculated considering the
      confounding variables (age, sex, comorbidities, medications, liver stiffness and CAP).
      Statistical significance was defined as p<0.05 in a two-tailed test with a 95% Confidence
      Interval.

      Statistical analyses were performed using Statistical Program for Social Sciences (SPSS®)
      vs.18.0.
    

Intervention name(s): Nutraceutical therapy
Intervention type(s): Drug
Intervention description(s): Oral administration of 303mg of silybin-phospholipid complex, 10mg of vitamin D, and 15mg of vitamin E, twice a day for six months
Other intervention name(s): Silybin-phospholipid, Vitamin D and vitamin E complex
Intervention MeSH terms: Vitamin D; Vitamins; Vitamin E; Silybin
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: The 92 enrolled patients were divided in three groups: NAFLD wild type control group (n. 30), NAFLD treated wild type group (n. 30), NAFLD treated mutated group (n. 32). The block randomization method was used to randomize the 60 not mutated patients in the NAFLD wild-type control group and NAFLD treated wild type one.
The patients inserted in the NAFLD treated wild type and NAFLD treated mutated groups were undergone to an oral administration of 303mg of silybin-phospholipid complex, 10mg of vitamin D, and 15mg of vitamin E, twice a day for six months. At the baseline and end of treatment, the assessment of WHtR, blood pressure measurement, BMI, Blood glucose and insulin, HOMA-IR, AST, ALT, GGT, blood count, CRP, TBARS, liver stiffness and CAP was performed. During the experimental observation, patients were on free diet on the basis of dietary habits before the enrollment and any type of physical exercise was recommended during the study period.
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: non-alcoholic fat ty liver disease; genetics; insulin resistance; nutraceutics