BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos 10.3892/br.2018.1123 BR-0-0-1123 Articles Distribution of the ATP-binding cassette transporter ABCG8 IVS1-2A>G genotype and clinical characteristics of gallbladder patients in Northeastern Mexico: A pilot study FrigerioPamela 1 Cepeda-NietoAna Cecilia 2 Marcos-MoralesSelim 1 Peña-VelázquezAbel 1 Dávila-FloresSergio 3 Salinas-SantanderMauricio 2 Facultad de Medicina Unidad Saltillo, Universidad Autónoma de Coahuila, Saltillo, Coahuila 25000, Mexico Departamento de Investigación, Facultad de Medicina Unidad Saltillo, Universidad Autónoma de Coahuila, Saltillo, Coahuila 25000, Mexico Departamento de Cirugía, Hospital Universitario de Saltillo ‘Dr. Gonzalo Valdés Valdés’, Universidad Autónoma de Coahuila, Saltillo, Coahuila 2500, Mexico Correspondence to: Dr Mauricio Salinas-Santander, Departamento de Investigación, Facultad de Medicina Unidad Saltillo, Universidad Autónoma de Coahuila, 205 Calle Francisco Murguía Sur, Zona Centro, Saltillo, Coahuila 25000, Mexico, E-mail: msalinsa@yahoo.com 09 2018 03 07 2018 9 3 266 270 13032018 29062018 Copyright © 2018, Spandidos Publications 2018

Biliary lithiasis is a multifactorial pathology determined by the interaction of genes and the environment, characterized by alterations in cholesterol homeostasis and in the metabolism of bile salts. A number of gene polymorphisms and mutations have been identified in the ATP-dependent cholesterol transporter (ABCG8) associated with lithiasis disease. The aim of the present study was to evaluate the association of the ABCG8 gene mutation IVS1-2A>G with cholecystolithiasis in patients from Northeast Mexico. This was a pilot study including 90 Mexican subjects diagnosed by ultrasonography, 57.8% of which presented gallstones. The studied parameters included: Lipid profile, total protein in plasma and polymerase chain reaction-restriction fragment length polymorphism genotyping. Significant differences were identified in total plasma protein, weight and BMI values, with these being these higher in subjects with gallstones (P<0.05). The presence of the mutant allele IVS1-2G was not detected, and the IVS1-2A wild-type allele was present in 100% of the population. Therefore, no association was apparent between the presence of the splice site mutation in ABCG8 (IVS1-2A>G) and the presence of gallstones in the evaluated subjects.

 ATP-binding cassette transporter G8 IVS1-2A>G mutation biliary lithiasis gallstones Introduction

Cholecystolithiasis is an established health problem with medical, social and economic implications due to its typically high rate of occurrence and associated complications. It is a chronic and frequent pathology of the digestive system, and is generally treated by cholecystectomy (1). Cholecystolithiasis is among the five main reasons prompting surgical intervention worldwide, and the primary reason in Mexico and other countries in Latin America (1), and thus represents the digestive disease with greatest economic burden in Western countries (2). In modern times, biliary lithiasis has been described as a multifactorial pathology, determined by the interaction of genes and the environment and characterized by alterations in cholesterol homeostasis and in the metabolism of bile salts (3). In this regard, the ATP-binding cassette gene subfamily G member 8 (ABCG8), which transports cholesterol to the canalicular membrane of hepatocytes and the brush border of enterocytes, has been described as a major genetic determinant for the development of gallstones in humans (3), and is among the most studied of ABC transporters at clinical and experimental levels (4–6).

In 2002, it was reported that Sitosterolemia may be caused by mutations in exon 5 of the ABCG8 gene (c.584T>A; Leu195Gln) in a German population (7). In fact, mutations and polymorphisms in ABCG5 and ABCG8 genes were identified in these patients (7). In other studies, the involvement of ABCG8 gene polymorphisms D19H and Y54C/T400K have been associated with the development of gallstone disease in German, Romanian, Scandinavian, Chinese and Indian populations, among others (8,9). Recently, a genome-wide association study meta-analysis conducted by Joshi et al (10) revealed the association of two ABCG8 gene polymorphisms, rs11887534 and rs4245791, with the risk of developing gallstones.

Furthermore, a study conducted by Hubacek et al (11) identified an ABCG8 gene mutation in the splicing acceptor of intron 1 (IVS1-2A>G) in patients with Sitosterolemia, inducing alternative splicing. However, to the best of our knowledge there have been no new studies describing the participation of IVS1-2A>G gene mutation in the development of Sitosterolemia and gallstone disease to date.

As gallstone lithiasis is a common pathology in Mexico, and there is a lack of published Mexican studies determining the existence of clinical, biochemical and molecular markers for prognostic utility, the present study aimed to analyze the association between altered clinical parameters, including biochemical factors, lipid profile and body mass index, and the ABCG8 gene mutation IVS1-2A>G in the development of cholecystolithiasis in patients from Northeast Mexico, in order to identify possible therapeutic targets. To this end a comparative cross-sectional in-hospital pilot study was performed.

 Materials and methods <sec> <title>Selection of participants

A total of 90 Mexican patients recruited between January 2016 and July 2017 from the Department of Surgery of the University Hospital of Saltillo, Mexico, were organized into two groups. The first group consisted of patients presenting with cholesterol gallstones, while the second group consisted of control subjects without gallstones. Approval for the use of human subjects in clinical research was obtained in December 2015 from the Ethics Committee of the University Hospital of Saltillo. Informed consent was obtained from all included subjects following explanation of the study protocol.

The inclusion criteria for the first and second groups were as follows: Subjects of both sex and of any age who attended the University Hospital of Saltillo, with (patient group) and without (control group) diagnosis of cholelithiasis. Patients with any genetic diseases including familial hypercholesterolemia, with gallbladder cancer or with non-adequate (hemolyzed) blood samples were excluded from both groups. The patient were selected following upper abdominal ultrasonography (ACUSONX300; Siemens Medical Solutions, Erlangen, Germany). The subjects were weighed and measured for height using a BAME scale with a stadiometer (model MMBAD02; Bame Industries, Puebla, Mexico). BMI was calculated as bodyweight divided by the square of the height (kg/m2). Peripheral venous blood samples (5 ml) were collected into two 6 ml tubes, one without anticoagulant, which was processed for lipid profile with a Vitros 250 autoanalyzer (GMI Inc., Ramsey, MN, USA) using a ready-made dry slide chemistry kit (Lipid Panel; Johnson & Johnson, New Brunswick, NJ, USA) and the other with EDTA anticoagulant for DNA extraction. The lipid profile assessed total cholesterol, triglycerides, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and very low-density lipoprotein (VLDL)-cholesterol. The atherogenic index of the plasma was determined as the ratio of LDL to HDL. Reference values were according those in the Guidelines of the Mexican Official Standard (‘Norma Oficial Mexicana NOM-037-SSA2-2012, for the prevention, treatment and control of dyslipidemias’) (12).

 DNA extraction

The peripheral venous blood samples in the EDTA tubes were centrifuged at 1,800 × g for 5 min at room temperature, and the buffy-coat was processed for high molecular weight genomic DNA isolation with phenol-chloroform, precipitated in ethanol and suspended in Tris-EDTA (pH 7.8).

 Genotyping the splice site mutation in ABCG8 (IVS1-2A>G)

The ABCG8 gene was genotyped by polymerase chain reaction (PCR), followed by restriction analysis using the enzyme NlaIV (New England Biolabs, Ipswich, MA, USA) following the protocol described by Hubacek et al (11). PCR was performed using an MJ Mini PTC1148 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a volume of 25 µl as follows: 250 ng genomic DNA, 0.5 µM primers (forward, 5′-TGTCTTCTCCTATGTTCTCAGCAG-3′ and reverse primer, 5′-TGCTCCTCTCCCCTTGAAC-3′), 0.2 mM dNTPs, 1.5 mM MgCl2 and 2.5 units Taq DNA polymerase (Promega Corporation, Madison, WI, USA). The PCR program consisted of 30 cycles at 94°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec.

The IVS1-2A allele results in the gain of a restriction site for the NlaIV enzyme. When the IVS1-2A allele is present, the 199 bp PCR amplicon is digested with the enzyme NlaIV, generating two fragments of 164 and 136 bp (11). The IVS1-2G allele was detected by the lack of cleavage by the NlaIV enzyme, leaving the 199 bp PCR amplicon intact.

 Statistical analysis

Demographic, clinical, biochemical and molecular data was collected from the patients and organized into a database. All statistical calculations were performed using SPSS software version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). Quantitative variables were expressed as means ± standard deviation and the qualitative values as percentages. Descriptive statistics were performed for the demographic, clinical and biochemical data of each subject. Any difference in the quantitative values obtained between clinical, biochemical and molecular parameters was determined through Student's t-test for variables with normal distribution and Mann-Whitney U test for variables with non-normal distribution. The possible association between genotype and categorical variables of biological and clinical parameters was assessed using contingency tables and χ2 statistics. P<0.05 was considered to indicate statistical significance.

 Results <sec> <title>Clinical characteristics of the participants

A total of 90 subjects were analyzed, of which 67 were women (74.4%) and 23 were men (25.6%). Overall 57.8% of the study subjects had gallstones [15 men (16.7%) and 37 women (41.1%)], leaving 42.2% without [8 men (8.9%) and 30 women (33.3%)].

The most frequent pathologies observed in the included subjects were diabetes mellitus type 2 (8.9%, n=8; in 3 subjects with normal gallbladder and 5 with gallstones), systemic arterial hypertension (4.4%, n=4; in 1 subject with normal gallbladder and 3 with gallstones), and both diabetes and hypertension (4.4%, n=4; in 1 subject with normal gallbladder and 3 with gallstones).

Regarding biochemical characteristics (Table I), 73 patients (81.1%) exhibited normal cholesterol levels (50–200 mg/dl) whereas 17 (18.9%) exhibited high cholesterol levels (>200 mg/dl) and thus were hypercholesterolemic; 41 patients (45.6%) presented normal triglyceride levels (50–150 mg/dl) while 47 (52.2%) presented high levels (>150 mg/dl; hypertriglyceridemic) and 2 patients (2.2%) exhibited values <50 mg/dl. HDL values ranged from 40–60 mg/dl in 42 patients (46.7%) to <40 mg/dl in 41 patients (45.6%) and >60 mg/dl in 7 patients (7.8%). LDL values were optimal, between 0–130 mg/dl, in 82 patients (91.1%) and high, >130 mg/dl, in 8 patients (8.9%). VLDL values were normal, between 2–30 mg/dl, in 45 patients (50%) and high, >30 mg/dl, in 45 patients. The atherogenic index was estimated to be 0.7–3.9 mg/dl in 81 patients (90.0%), >3.9 mg/dl in 5 patients (5.6%) and <0.7 mg/dl in 4 patients (4.4%).

Concerning the group of 52 patients with cholecystolithiasis, 12 (23.1%) exhibited hypercholesterolemia, 28 (53.8%) exhibited hypertriglyceridemia, 27 (51.9%) exhibited low HDL, 27 (51.9%) exhibited elevated VLDL and 6 (11.5%) exhibited elevated LDL levels. In the control group, hypercholesterolemia was determined in 5 (13.2%) of 38 subjects, as well as hypertriglyceridemia in 16 (50.0%), low HDL levels in 14 (36.8%), elevated VLDL in 18 (47.4%) and elevated LDL in 2 (5.3%) of these subjects.

Regarding the distribution of biochemical parameters, sex and diagnosis of gallstones, no significant differences were identified between the groups (Table I).

When comparing the mean values for the clinical and biochemical variables considered, a significant difference between the normal gallbladder and gallstone groups was identified in total protein in plasma (6.23±1.25 vs. 6.75±0.97 mg/dl; P=0.028), weight (72.4±13.9 vs. 78.3±14.3 kg; P=0.022) and BMI (27.1±5.74 vs. 29.0±5.29; P=0.038); these parameters were significantly higher in the group with gallstones (Table II).

 Allele and genotype analysis

The analysis by PCR-restriction fragment length polymorphism assay revealed that the wild-type allele A (IVS1-2A) was present in 100% of the population (IVS1-2A/IVS1-2A genotype), whereas the variant allele G (IVS1-2G) was absent. For this reason, a causal relationship between the frequency of the mutation and the clinical and biochemical data could not be determined.

 Discussion

Gallstones are a condition occurring globally though exhibits a variable prevalence according to the demographics of the analyzed population. A prevalence of 20.0% has been reported in the United Kingdom (13), whereas in Mexican-American women a prevalence of 26.7% has been reported (14). Regarding the latter, however, necropsy studies performed in Mexico have reported a lower prevalence of 14%, with a higher prevalence in females (20.4%) than in males (8.5%) (15). Meanwhile, indigenous populations from Chile and Peru appear highly susceptible to lithiasis, presenting a nearly 100% risk of disease development in the female population (13). These studies are in accord with results in the present study, where gallstone prevalence (57.8% overall) was higher in women (41.1%) than in men (16.7%).

The present study aimed to analyze the association between the ABCG8 gene mutation IVS1-2A>G and cholelithiasis, mainly because this gene mutation has been previously implicated with an increased hepatic secretion of cholesterol and consequent predisposition to gallstones (11).

Studies in mice have revealed that the stimulation of ABCG8 by Niemann-Pick C2, a cholesterol-binding protein secreted by the biliary system, resulted in the positive regulation of biliary cholesterol secretion (16). A study of more than 500,000 genotyped single nucleotide polymorphisms suggested that the His19 variant of the ABCG8 gene could be associated with more efficient cholesterol transport into bile (17). Meanwhile, a study conducted in mice revealed that the ABCG5/G8-independent pathway serves an important role in the regulation of biliary cholesterol secretion, in the transport of HDL-derived cholesterol from plasma to bile, and in gallstone formation, separate from the ABCG5/G8 pathway (18). A previous meta-analysis demonstrated a marked association between the D19H polymorphism and gallstone disease. In this same study, the T400K and Y54C polymorphisms also appeared associated with gallstone disease, though to a lesser extent (9,19).

Although association of the ABCG8 gene mutation with an increased risk of developing gallstones has been reported previously (7), the present study identified no apparent association between the IVS1-2A>G mutation with the development of gallstones in a Mexican population; since 100% of the analyzed population presented the wild-type allele A (IVS1-2A). However, it is noteworthy that the sample size was small (n=90), and thus potentially compromised the conclusions drawn from this study, since a sample size of at least 196 subjects would be required for a sufficient statistical power (n of Saltillo population = 807,537, prevalence of 15 and 95% confidence level); nevertheless, this was enough to reflect the genotypic distribution of the splice site mutation in ABCG8 (IVS1-2A>G) in the study population.

The presence of gallstones in the analyzed population was significantly associated with higher values of total protein in plasma (6.75 g/dl; P=0.028), as well as with higher weight (78.3 kg; P=0.022) and BMI (29.0 kg/m2; P=0.038) when compared with the subjects without gallstones. Regarding total protein in plasma, it is noteworthy that the average protein value determined in patients with and without gallstones was within the normal range (6.5–8.1 g/dl) (20); however, the difference in values between the groups was identified to be significant and therefore associated with the presence of gallstones. These results have not been reported previously in similar studies.

Concerning weight and BMI, previous study has not established a direct correlation between overweight or obese phenotype and the development of symptomatic cholelithiasis, mainly because it is also observed in subjects with normal BMI (21); regardless, the risk of symptomatic cholelithiasis appears to increase with higher BMI (21). This association was also apparent in the present study, where BMI within the overweight range (25–30 kg/m2) (22) was associated with the presence of gallstones. Furthermore, there may be a direct correlation between increased percentage of general and abdominal fat and increased risk of developing gallbladder disease, with previous studies indicating an almost two-fold increased risk even within the range of normal BMI (23).

Other clinical and biochemical variables analyzed in this study, including cholesterol, Triglycerides, HDL, LDL and VLDL levels, atherogenic index, height and sex, were not determined as significant risk factors for the development of gallstones in the analyzed population.

In conclusion, no association was apparent between the presence of the splice site mutation in ABCG8 (IVS1-2A>G) and the presence of gallstones in the evaluated subjects. However, alterations were observed in the level of total protein, weight and BMI, which were significantly higher in the subjects presenting with gallstones.

 Acknowledgements

The authors would like to thank Dr Daniel Diaz, former researcher at the Department of Clinical Biochemistry of the University Hospital of Charles University, Hradec Kralove, Czech Republic, for his assistance in proofreading the manuscript.

 Funding

The present study was funded by the Research Department of the Facultad de Medicina Unidad Saltillo and the Hospital Universitario de Saltillo ‘Dr. Gonzalo Valdes’, Universidad Autónoma de Coahuila, Saltillo, Mexico.

 Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

 Authors' contributions

PF, SMM, SDF and MSS conceived the study. PF, ACCN, SMM, MSS and APV designed the study. PF and SMM were responsible for patient enrollment. PF, SMM and MSS performed the data analyses. PF, SMM, and MSS interpreted the data. PF, SMM, ACCN, MSS, APV drafted the manuscript. PF, SMM, ACCN, MSS and APV critically revised the manuscript. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

The Institutional Review Board of the Hospital Universitario de Saltillo, Saltillo, Coahuila, Mexico, approved and registered the study, December 2015. Informed consent for participation was obtained from all included subjects following explanation of the study protocol.

 Consent for publication

All included subjects provided written informed consent allowing the publication of relevant data on the terms of data anonymization.

 Competing interests

The authors declare that they have no competing interests.

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Clinical and biochemical parameters.

Clinical/biochemical parameter Variable Normal gallbladder (n=38) Gallstones (n=52) χ2 P-value
Sex, n Male 8 15 0.701 0.402
Female 30 37
Cholesterol, mg/dl 50–200 33 40 1.410 0.235
>200 5 12
Low-density lipoprotein, mg/dl 0–130 36 46 0.433 0.510
>130 2 6
Very low-density lipoprotein, mg/dl 2–30 20 25 0.182 0.670
>30 18 27
Triglycerides, mg/dl <50 2 0 2.809 0.246
50–150 17 24
>150 19 28
High-density lipoprotein, mg/dl <40 14 27 3.408 0.182
40–60 22 20
>60 2 5
Atherogenic index <0.7 1 3 0.560 0.756
0.7–3.9 35 46
>3.9 2 3

Clinical and biochemical parameters associated with gallstones.

Clinical/biochemical parameter Normal gallbladder (n=38) Gallstones (n=52) P-value
Cholesterol, mg/dl 159.5±46.8 166.9±51.3 0.482
Triglycerides, mg/dl 163.7±81.0 174.1±98.2 0.597
HDL, mg/dl 42.2±14.6 44.2±17.9 0.560
LDL, mg/dl 86.2±34.4 87.1±39.4 0.910
VLDL, mg/dl 32.7±16.2 34.8±19.7 0.584
Atherogenic index 2.21±0.89 2.14±1.00 0.722
Total plasma protein, mg/dl 6.23±1.25 6.75±0.97 0.028
Weight, kg 72.4±13.9 78.3±14.3 0.022
Height, m 1.64±0.08 1.64±0.08 0.755
Body mass 27.1±5.74 29.0±5.29 0.038
index, kg/m2

Data shown are the mean ± standard deviation.