Ignite Creation Date:
2024-05-06 @ 3:28 PM
Last Modification Date:
2024-10-26 @ 1:50 PM
Study NCT ID:
NCT04640324
Status:
COMPLETED
Last Update Posted:
2020-11-24
First Post:
2020-11-17
Brief Title:
Effect of PNPLA3 TM6SF2 and MBOAT7 Genetic Variants on Non-alcoholic Fatty Liver Disease Therapeutic Outcome
Sponsor:
University of Campania Luigi Vanvitelli
Organization:
University of Campania Luigi Vanvitelli
Study Overview
Official Title:
The Role of the PNPLA3 TM6SF2 and MBOAT7 Genetic Variants in the Response to Silybin-phospholipid Complex Vitamin D and Vitamin E Based Therapy for Non-alcoholic Fatty Liver Disease Patients
Status:
COMPLETED
Status Verified Date:
2020-11
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief 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 silybinvitamin 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 httpwwwgraphpadcomquickcalcsindexcfm
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 percentagecaloric 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 hisher 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 I148IM I148MM TM6SF2 167EK 167KK and MBOAT7 TMC4CT or TMC4TT 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 andor advanced stages liver disease such as cirrhosis andor hepatocellular carcinoma HCC
The definition of the presenceabsence 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 1380113803 and Thermo Fisher Scientific EIAGLUC HOMA-IR was also calculated using the formula fasting insulin μUmL plasma glucose mmolL225
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 25 cm andor 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 IQRM 01 reliable 01 IQRM 03 or IQRM 03 with LS median 71 kPa or poorly reliable IQRM 03 with LS median 71 kPa On the basis of CAP these scores the investigators classified the enrolled patients in S0 no steatosis 0-10 fat 0-237 dBm S1 mild steatosis 11-33 fat 238-259 dBm S2 moderate steatosis 34-66 fat 260-292 dBm and S3 severe steatosis 67 fat 293 dBm 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 1133-tetramethoxyprophane as a standard The amount of TBARS was expressed as nmolμg 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 260280 ratio was between 18 and 2 The DNA extracted was then stored in -20C 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 StepOne20 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 25 at the end of treatment starting from baseline values greater than 25 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 001 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 Pearsons 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-IUml normalization of the HOMA-IR 25 at the end of treatment starting from baseline values greater than 25 andor reduction of the HOMA-IR 2 points at the end of treatment in comparison to baseline ALT normalization 45 IUL CRP normalization 06 mgdL or reduction of at least 1 mgdL TBARS reduction of at least 10 nmolμg 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 p005 in a two-tailed test with a 95 Confidence Interval
Statistical analyses were performed using Statistical Program for Social Sciences SPSS vs180
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 httpwwwgraphpadcomquickcalcsindexcfm
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 percentagecaloric 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 hisher 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 I148IM I148MM TM6SF2 167EK 167KK and MBOAT7 TMC4CT or TMC4TT 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 andor advanced stages liver disease such as cirrhosis andor hepatocellular carcinoma HCC
The definition of the presenceabsence 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 1380113803 and Thermo Fisher Scientific EIAGLUC HOMA-IR was also calculated using the formula fasting insulin μUmL plasma glucose mmolL225
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 25 cm andor 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 IQRM 01 reliable 01 IQRM 03 or IQRM 03 with LS median 71 kPa or poorly reliable IQRM 03 with LS median 71 kPa On the basis of CAP these scores the investigators classified the enrolled patients in S0 no steatosis 0-10 fat 0-237 dBm S1 mild steatosis 11-33 fat 238-259 dBm S2 moderate steatosis 34-66 fat 260-292 dBm and S3 severe steatosis 67 fat 293 dBm 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 1133-tetramethoxyprophane as a standard The amount of TBARS was expressed as nmolμg 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 260280 ratio was between 18 and 2 The DNA extracted was then stored in -20C 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 StepOne20 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 25 at the end of treatment starting from baseline values greater than 25 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 001 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 Pearsons 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-IUml normalization of the HOMA-IR 25 at the end of treatment starting from baseline values greater than 25 andor reduction of the HOMA-IR 2 points at the end of treatment in comparison to baseline ALT normalization 45 IUL CRP normalization 06 mgdL or reduction of at least 1 mgdL TBARS reduction of at least 10 nmolμg 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 p005 in a two-tailed test with a 95 Confidence Interval
Statistical analyses were performed using Statistical Program for Social Sciences SPSS vs180
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None