Manifestation of obesity and its comorbidities starts from early childhood onwards 1. Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity of obesity. Both excessive BMI and visceral obesity are recognized risk factors for NAFLD. In patients with severe obesity undergoing bariatric surgery, the prevalence of NAFLD can exceed 90%, and up to 5% of patients may have unsuspected cirrhosis 2.
Hepatic steatosis differs among different ethnic groups, being highest among Hispanics followed by Caucasians. Several gene variants have been identified so far by genome-wide association studies (GWAS) or by a candidate gene approach to be associated with fatty liver disease. The PNPLA3 rs738409 and the GCKR rs1260326 are the strongest variants associated with fatty liver in pediatrics 3.
Our study aimed to assess the prevalence of rs 1260326 polymorphism within the glucokinase regulatory protein (GCKR) gene in NAFLD and nonalcoholic steatohepatitis (NASH) among a sample of obese Egyptian children.
Patients and methods
This study included 100 obese children who were followed-up at the obesity clinic of Ain Shams University Children’s Hospital. Fifty patients with NAFLD and 50 without NAFLD were included.
Study candidates were further subclassified into three groups:
- Group I: 50 obese children with ‘no hepatomegaly’ and normal liver enzymes.
- Group II: 30 obese children with ‘hepatomegaly’ and normal liver enzymes.
- Group III: 20 obese children with ‘hepatomegaly and elevated liver enzymes’(NASH) 4.
In addition, 50 healthy children matched for age and sex were included as the control group (group IV) for the genetic study.
Obese children were diagnosed according to Centers for Disease Control and Prevention criteria 5. However, all obese children with liver disease other than NAFLD were excluded from the study.
Medical records of all study candidates were reviewed for full medical history with special emphasis on onset, course, duration of obesity, and evidence of liver disease. All study candidates underwent thorough clinical examinations including anthropometric measures such as weight, height, and BMI. Abdominal examination, especially for organomegaly, and chest and cardiovascular examinations were carried out. Blood tests were carried out for full blood count with differential count, lipid profile, random blood sugar, HbA1c, and liver enzymes (alanine transaminase, aspartate transaminase, alkaline phosphatase). In addition, all study candidates underwent abdominal ultrasound for detecting bright liver.
The polymorphic rs1260326 variant of the GCKR gene was studied in patients with NAFLD (groups II and III) and the control group (group IV) (in accordance with the Helsinki declaration).
Studying the rs1260326 functional variant of the glucokinase regulatory protein gene polymorphism
DNA was extracted from peripheral blood lymphocytes by spin column method using a GeneJET Genomic DNA purification kit (#K072, Pure Extreme Fermentas Life Sciences; Thermo Scientific, Vilnius, Lithuania). The eluted DNA was stored at −20°C until further application. Genotyping was performed for the single nucleotide polymorphisms (SNPs) across the GCKR gene. The fragment bearing the rs1260326 functionally relevant SNP consisted of a C–T substitution coding for a proline-to-leucine substitution at position 446 (P446L).The following primers were used – forward, 5′-TGC AGA CTA TAG TGG AGC CG-3′ and reverse, 5′-CAT CAC ATG GCC ACT GCT TT-3′6. Genotyping was performed following amplification of ∼200 ng of template genomic DNA in 20-μl volume using 10 pmol each of the two primers, 1.5-mM MgCl2, 400 μmol/l of each dNTP, 1-U hot start Taq polymerase, and 1× PCR buffer. The PCR (S24 Quanta Biotech; Surrey, England, UK) consisted of the following: predenaturation at 96°C for 3 min, followed by 35 cycles of denaturation at 96°C for 30 s, annealing at 60°C for 30 s, primer extension for 1 min at 72°C, and final extension at 72°C for 5 min. The amplicon (231 bp) was digested by HpaII restriction endonuclease (Fermentas, Thermo Scientific). The digestion of the 231-bp amplicon of the rs1260326 CC genotype resulted in 18-, 63-, 150-bp fragments, the TT genotype resulted in 18- and 213-bp fragments, and the heterozygous genotype consisted of 18-, 63-, 150-, 213-bp fragments.
The genotypes of the digested products were determined by electrophoresis on 2% agarose gels stained with ethidium bromide in 1× Tris-EDTA-borate buffer against a 50-bp ladder molecular weight GeneRuler 50 bp DNA ladder (Fermentas, #SM0373;Thermo Scientific/Fermentas). The PCR products and digested fragments were all documented using Gel Documentation System (InGenius Syngene; InGeniusSyngeneTM, Cambridge, UK).
The distributions of polymorphisms, genotypes, and allele frequencies were all statistically compared between patients and healthy controls.
Collected data were reviewed, coded, and statistical analyzed using SPSS program, version 21.0 (IBMSPSS Inc.; Chicago, Illinois, USA) (statistical package of the social science). For descriptive statistics, mean values and their SDs were calculated to measure central tendency and dispersion of quantitative data. Frequency of occurrence was calculated to measure qualitative data. For analytic statistics, comparison between groups was performed using the χ2-test one-way analysis of variance. The level of significance was considered at a P-value of greater than 0.05.
Our study included 50 obese children with NAFLD (groups II and III). They were 29 males and 21 females, with a mean age of 8.5±4.4 years and mean duration of obesity of 4.2±2 years. Their average BMI was 36.13±6.30 kg/m2. Thirty (60%) of the NAFLD patients suffered from hepatomegaly but had no elevated liver enzymes (group II), whereas the other 40% had hepatomegaly and elevated liver enzymes (group III).
Table 1 shows that there is a highly significant statistical difference between groups II, III, and I regarding duration of obesity, weight, BMI, hip circumference, and waist circumference and a significant statistical difference regarding height. A nonsignificant statistical difference regarding sex was present.
The lipid profiles of the three groups are shown in Table 2, with a significantly elevated lipid profile among group III. In addition, liver enzymes as well as random blood sugar were markedly higher in patients with NASH.
Table 3 demonstrates that the most prevalent GCKR genotype among obese children with hepatomegaly was CC (52%), followed by CT heterozygosity (34%), and then TT(14%), with a highly significant difference when compared with the control group (P=0.001). The most prevalent genotype in the control group was heterozygosity for CT allele. The most frequently segregated allele among patients was C (69%) followed by T (31%), whereas it was equally distributed among the control group (50%). The most prevalent genotype in group II was CC (60%), followed by CT heterozygosity (30%), and then TT (10%). However, the difference was statistically insignificant (P=0.344), and C, T allele segregation showed a nonsignificant distribution. The most frequently segregated allele in group II was C (75%) followed by T (25%). Similarly, in group III, the most frequently segregated allele was C (60%) followed by T (40%) (Table 4).
There was no significant distribution of either genotypes or alleles compared with different laboratory and clinical parameters among both groups II and III (Tables 5 and 6).
This study aimed to assess the prevalence of rs1260326 SNP in the GCKR in NAFLD and NASH among obese Egyptian children.
We found a highly significant statistical difference between obese children without hepatomegaly, those with hepatomegaly but no elevated enzymes, and those with NASH regarding duration of obesity, weight, BMI, hip circumference, and waist circumference. This is in agreement with Assy et al.7 who conducted their study on a group of children with NAFLD with normal liver enzymes compared with a group of children with NASH. They found that children with NASH had higher anthropometric measures (BMI, height, weight, waist, and hip parameters) and had a tendency to be older than children with NAFLD.
Triglyceride (TG) accumulation in the liver is the earliest hallmark of NAFLD 8,9. In our study, it was noted that laboratory lipid profile was significantly higher among obese children with NASH in comparison with those with hepatomegaly but without elevated liver enzymes and the control group. Korsten-Reck et al.10, showed that obese children are at risk of dyslipoproteinemia and related diseases. ‘Children with the highest BMI and lowest physical fitness have the lowest HDL-C values and increased TG, indicating a higher risk for the metabolic syndrome’. Another study by Neuschwander-Tetri et al.11 showed that patients with NASH had significantly higher levels of liver transaminases (aspartate transaminase, alanine transaminase), γ-glutamyltransferase, TGs, and cholesterol compared with those without definite NASH.
GWAS have revealed an association between certain SNPs of the GCKR gene and hypertriglyceridemia in patients with diabetes mellitus type 2 12.
Glucokinase is a phosphorylating enzyme that regulates the metabolism of hepatic glucose and stimulates hepatic lipogenesis. Two SNPs in the GCKR gene, rs1260326 and rs780094, were found to be related to the development of NAFLD 13.
The most prevalent genotype among our sample of Egyptian obese children with hepatomegaly was CC (52%) followed by CT heterozygosity (34%); therefore, the most frequently segregated allele among this group (NAFLD and NASH) was C. The most prevalent genotype among group II alone was CC as well (60%) followed by CT heterozygosity (30%). In the NASH group, the genotype CC was (40%) equal to CT heterozygosity (40%). However, the most prevalent genotype in the control group was CT heterozygosity (64%). Recent GWAS have demonstrated an association between SNPs in the GCKR gene with hepatic steatosis. SNPs characterized by a C-to-T substitution on genes encoding proteins of the lipogenesis pathways have been associated with NAFLD 14.
GWAS have focused on a SNP (rs1260326) in the GCKR gene, previously associated with TG levels. It has been found that this SNP is associated with increased hepatic fat accumulation along with higher very low density lipoprotein and TG levels 6. Santoro et al.6 stated that the GCKR SNP rs1260326 minor allele (T) frequency was 0.446 in Caucasians, 0.129 in African-Americans, and 0.355 in Hispanics. They found that the minor allele (T) of the GCKR rs1260326 is associated with fatty liver and with higher serum TGs and large very low density lipoprotein levels in obese adolescents. This association was evident in all ethnic groups studied, and it was independent of age, sex, z-score BMI, and glucose tolerance.
In addition, Tan et al.14 reported a higher frequency of risk-allele T among NAFLD patients compared with controls. Therefore, they suggested that the risk-allele T of the rs1260326 is associated with predisposition to NAFLD and NASH with significant fibrosis.
To conclude, in our study of Egyptian obese children, we found that the C allele of the rs1260326 variant was the most prevalent among obese children with NAFLD including those with NASH.
Conflicts of interest
There are no conflicts of interest.
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