Single nucleotide polymorphisms in LCAT may contribute to dyslipidaemia in HIV-infected individuals on HAART in a Ghanaian population

Highly active antiretroviral therapy (HAART) is known to cause lipid abnormalities such as dyslipidaemia in HIV-infected individuals. Yet, dyslipidaemia may not independently occur as it may be worsened by single nucleotide polymorphisms (SNPs) in lecithin cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL). This case–control study was conducted in three-selected hospitals in the Northern part of Ghana. The study constituted a total of 118 HIV-infected participants aged 19–71 years, who had been on HAART for 6–24 months. Dyslipidaemia was defined based on the NCEP-ATP III criteria. HIV-infected individuals on HAART with dyslipidaemia were classified as cases while those without dyslipidaemia were grouped as controls. Lipid profile was measured using an automatic clinical chemistry analyzer and genomic DNA was extracted for PCR (GeneAmp PCR System 2700). Overall, the prevalence of dyslipidaemia was 39.0% (46/118). High levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and reduced levels of high-density lipoprotein cholesterol (HDL-C) were observed in all cases. A total of 256 selected PCR amplicons comprising 137 LPL (exons 3, 5 and 6) and 119 LCAT (exons 1, 4, and 6) were sequenced in 46 samples (Inqaba Biotech). Six (6) clinically significant SNPs were identified in exons 1 and 4 for LCAT whereas 25 non-clinically significant SNPs were identified for LPL in exons 5 and 6. At position 97 for LCAT exon 1, there was a deletion of the nucleotide, ‘A’ in 32.5% (13/40) of the sampled population while 67.5% (27/40) of the sample population retained the nucleotide, ‘A’ which was significantly associated with dyslipidaemic outcomes in the study population (p = 0.0004). A total of 25 SNPs were identified in exons 5 and 6 of LPL; 22 were substitutions, and 3 were insertions. However, none of the 25 SNPs identified in LPL exon 5 and 6 were statistically significant. SNPs in LCAT may independently contribute to dyslipidaemia among Ghanaian HIV-infected individuals on HAART, thus, allowing genetic and/or functional differential diagnosis of dyslipidaemia and creating an opportunity for potentially preventive options.

Inclusion and exclusion criteria. Long-term use of ART has been demonstrated to exponentiate the development of dyslipidaemia among HIV infected individual. To investigate the role of SNPs in dyslipidaemia among HIV infected individuals while minimizing the effect of duration on HAART for dyslipidaemia, we included individuals who has been on HAART for at most two years. A total of 118 HIV-infected individuals who had been on HAART for 6-24 months were included in the final analysis for this study (Fig. 1) 24 patients who had been of HAART for less than 6 months and 158 who were on lipid lowering drugs, had neurological disorder, were pregnant women, and had AIDS were excluded from the study.
Biochemical analysis. Venous blood sample (5 mL) was taken from these participants, 4 mL was dispensed into a gel vacutainer tube for the estimation of serum lipids using a fully automatic chemistry analyzer (Vital Scientific Selectra Flexor XL, UK) while 1 mL was dispensed into EDTA vacutainer tube for DNA extraction for PCR analysis. The Friedewald's formula, LDL-C = TC-(HDL-C-TG/2.2) was used for calculating LDL-C. Friedewald's equation over miscalculates LDL-c when serum triglycerides (TG) are high, therefore subjects with TG ≥ 4.2 mmol/L were excluded from the study. This is to avoid potential bias affecting the association of LDL-C with dyslipidaemia.
Dyslipidaemia was defined in this study as the presence of at least two lipid abnormalities (i) hypertriglyceridaemia-fasting serum triglyceride level ≥ 1.7 mmol/L (150 mg/dL), (ii) Reduced high density lipoprotein (HDL)-cholesterol, (iii) serum HDL-cholesterol ≤ 1.03 mmol/L (40 mg/dL) (males) or 1.29 mmol/L (≤ 50 mg/ dL) (females), (iv) high serum low density lipoprotein (LDL)-cholesterol ≥ 3.37 mmol/L (130 mg/dL) and (v)   Figure 2 shows the various primer amplicons on agarose gel, along the DNA ladder (1000 bp). We designed primers that targeted exons 1, 4 and 6 in LCAT and exons 3, 5 and 6 in LPL, This full-length gel shows that, LCAT exon 6 is bigger than exon 1 and exon 1 is bigger than exon 4, since the smallest amplicon runs fasted (Fig. 2). DNA sequencing. A total of 256 selected PCR amplicons comprising 137 LPL (exons 3, 5 and 6) and 119 LCAT (exons 1, 4, and 6) samples were successfully sequenced at Inqaba Biotech, Pretoria, South Africa. DNA sequencing was done by the automated Sanger sequencing method 26 . The PCR amplified products were purified and sequenced under standard conditions on the ABI 3500XL Genetic Analyzer (POP7, Big Dye 3.1). The sequencing data was analyzed using Variant Reporter version 1.0 (Applied Biosystems, Foster City, CA) and Sequencher version 4.8 (Gene Codes Corporation, Ann Arbor, MI) according to Inqaba Biotech, Pretoria, South Africa. The sequence results were received and analysed using Chromas, Technelysium DNA sequencing software at https ://techn elysi um.com.au/wp/chrom as and multiple sequence alignment carried out using Clustalw at https ://www.genom e.jp/tools -bin/clust alw. Protein modelling was carried out using the ExPASy Bioinformatics Resource Portal at https ://web.expas y.org/trans late (Supplementary Figs. S1-S4).
Statistical analysis. Data was entered into Microsoft excel 2016 and exported to GraphPad Prism version 6.0 (www.graph pad.com) for analysis. Data was presented as numbers, percentages, means, and 95% confidence interval and, median and interquartile range. The Kolmogorov-Smirnov test was used to check the normality of the continuous variables. Parametric continuous were compared between groups using student paired t-test and expressed as mean (95% CI) while non-parametric variables were performed with the Mann-Whitney test and expressed as median (inter-quartile range). Logistic regression models were fitted to test for associations between dyslipidaemia and selected single SNP. After the sequencing and alignment, some participants had DNA sequence same as the reference sequence and were classified as such. Other participants had DNA sequences different from the reference sequence and were classified as the SNP population. A P < 0.05 was considered statistically significant.  Table 2 shows a comparison between case and control subjects regarding demographic characteristics, weight measurement, haemodynamic and lipid parameters, and dyslipidaemic indices. In all, 39.0% (46/118) of the study population developed dyslipidaemia based on NCEP-ATPII definition and were classified as cases, while 61.0 (72/118) were without dyslipidaemia and were classified as controls. There were more females than males (99 vs. 19). The mean duration of ART drugs was 16 months. The case-subjects were significantly weightier than the control-subjects [65.5 vs. 59.3 p = 0.0071]. Higher levels of triglyceride (p = 0.0439), TC (p < 0.0001), LDL-C (p < 0.0001), non-HDL-C (p < 0.0001) and VLDL-C (p = 0.0449) were found in case-subjects than the control-subjects. Compared to the case-subjects, control-subjects recorded significantly higher levels of HDL-C [0.7 vs. 1.5, p < 0.0001]. Figure 3 shows the distribution duration of HAART. The majority of the subjects had been on HAART between 18-24 months (45.8%) followed by 12-17 months (37.3%) and the least being 6-11 months (16.9%) respectively (Fig. 3).

Single nucleotide polymorphism identified in LCAT . The study identified 5 substitutional SNPs in
LCAT exon 1. At position 86 of LCAT exon 1, A was substituted for C in 12.5% (5/40) of the sample population while 87.5% (35/40) retained a C in the same position. This C → A nucleotide change did not significantly influence dyslipidaemia in the study population (p = 0.091). At position 89 of LCAT exon 1, A is substituted for G in 12.5% (5/40) of the sampled population while 87.5% (35/40) of the sampled population retained the G. This G → A SNP was not significantly associated with dyslipidaemia outcomes in HIV-infected individuals on HAART (p = 0.342). At position 97 of LCAT exon 1, there was a deletion of A in 32.5% (13/40) of the sampled population while 67.5% (27/40) of the sample population retained the A. This SNP was significantly associated with dyslipidaemic outcomes in the study population (p = 0.0004). At position 111, A was substituted for C in 7.5% (3/40) of the sampled population, while 92.5% (37/40) of the population retained C. This C → A nucleotide change was not significantly associated with dyslipidaemia in the study population (p = 0.231). Similarly, at position 121, 12.5% (5/40) of the study population expressed A while 87.5% (35/40) of the study population expressed C, this SNP was not significantly associated with dyslipidaemia in the study population (p = 0.342). Another SNP, a deletion of A was identified at position 8 of LCAT exon 4 in 91.3% (42/46) of the sampled population while 8.7% (4/46) of the study population retained an A at the same position. The deletion of A at position 8 of LCAT exon 4 was not significantly associated with dyslipidaemia outcomes in the sampled population (p = 0.990). No SNP was identified in LCAT exon 6 ( Table 3). Table 4 shows the types of nucleotide changes identified in LPL and their associations with dyslipidaemia in the study population.   (Table 4).     www.nature.com/scientificreports/ study population. There was no statistically significant difference between the SNP and the reference population for the lipid parameters (p > 0.05) ( Table 5).

Discussion
HAART administration interferes with triglyceride-rich lipoprotein hydrolysis by interfering with its binding to lipoprotein lipase, thereby hindering normal chylomicron, LDL, VLDL catabolism, trapping of fatty acids in peripheral adipose tissues and use by muscles 27 . HAART also interferes with the degradation of the nuclear transcriptionally active factor SREBP1 (nSREBP1), which is the master transcription control protein involved in plasma lipid synthesis 27,28 . The nSREBP1, therefore, lingers in the nucleus and continuously stimulate the transcription and translation of genes involved in the lipid biosynthesis pathway 29,30 . The prevalence of dyslipidaemia observed in the present study was 39.0%, which is consistent with cross-sectional studies and case-control studies in Ghana and other populations 24,[31][32][33] . In the present study, dyslipidaemia was defined by increased plasma total cholesterol, LDL-C and decreased HDL concentrations, which is similar to the criteria used in cross-sectional studies by Hu et al. 34 , and Chattopadhyay and Aldous 35 . The latter studies demonstrated that hypercholesterolaemia, hypertriglyceridaemia, and hypoalphalipoproteinaemia together with lipodystrophy were principal indices for dyslipidaemia among HIV-infected individuals after HAART exposure 35 Supplementary Figs. S2, S3, and S4, https ://www.expas y.org/prote omics /prote in_struc ture). This results in a change in the first 5 N-terminal amino acid sequences of the mature mutant LCAT . The 4 to 8 N-terminal amino acids located in the membrane-binding domain of the normal LCAT enzyme play a critical role in LCAT's recognition, specificity, selectivity, and binding to apolipoprotein A-I (ApoA-I) in HDL particles 36,37 .
A change in the first 5 N-terminal amino acids, therefore, impairs the function of the membrane-binding domain of the mutant LCAT's, affecting its ability to specifically recognize, select and bind to its substrate (HDL). This change in the type and sequence of the first five amino acids in the mutant LCAT's structure severely impairs activation and catalytic activity of the mutant enzyme 38 accounting for the reduction in the rate of cholesterol esterification and reverse transport to the liver for clearance from the body. The cholesterol therefore Table 5. Comparison of lipid profile results amongst reference sequence and SNP populations. Unpaired t-test comparison of lipid profile parameters. † Denotes a significant comparison between reference population and SNP population when P-value is < 0.05, ++- † † † Denotes a significant comparison between reference population and SNP population when P-value is < 0.001. SEQ sequence, LDL low density lipoprotein, HDL high density lipoprotein, TRIG triglycerides, VLDL very low-density lipoprotein, CR coronary risk. FREQ frequency, ∆ change, LCAT lecithin cholesteryl acyl transferase, Data are presented as means ± SD, discrete, fractions and percentages. Reference population were defined as individuals with 'normal' nucleotide sequenced Whereas SNP population were those with nucleotide changes. www.nature.com/scientificreports/ accumulated in the body, leading to dyslipidaemia observed among the present study participants with this SNP. This is consistent with the findings of Francone and Fielding 39 who documented that, LCAT has amino (N) and carboxyl (C) terminal extensions that are not predicted to have significant secondary structure. However, the LCAT N-terminus amino acid residues 2 to 5 of the mature protein is known to be important for LCAT activity, mediating contacts with apolipoprotein A-I (ApoA-I) in HDL particles. The amino acid residues 2-5 represent the macromolecular interaction site for HDL particles 39 . Murray et al. 40 and Vanloo et al. 41 documented in separate site-directed mutagenesis and antibody-binding experiments that, amino acid residues in the N-terminal region of LCAT play a structural role and ApoA-1's binding to this site leads to the activation of LCAT. This study's findings also agree with the findings of Manthei et al. 37 and Glukhova et al. 36 who also documented that truncations in the N-terminus are critical for LCAT activity on HDL as it contains residues that are critical components of the HDL membrane-binding domain. Manthei et al. 37 and Glukhova et al. 36 also concluded that amino acid residues 4-8 have backbone amines that are critical for mediating interaction with ApoA-1 on HDL particles, substrate recognition, specificity, and selectivity.
Schindler et al. 42 and Dube et al. 43 also reported that LCAT glycoprotein has 4 N-glycosylation (Asn 20, 84, 272, and 384) and 2 sites of O-glycosylation sites (Thr407 and Ser409). The carbohydrate component constitutes 25% of LCAT's total mass, the majority being N-linked [44][45][46] . Neuraminidase removal of the carbohydrate moiety of human LCAT led to a 60% decrease in the activity of the enzyme 42,[45][46][47] . Their finding supports the finding of this study that a change in a functional domain of LCAT affects the activity of the enzyme. The change in the first 5 amino acids decreases the activity of the mutant LCAT without affecting LCAT protein synthesis and secretion 42,44,48 .
The influence of this SNP identified at position 97 of the mutant LCAT on plasma HDL cholesterol concentration is evidenced by the findings that hypoalphalipoproteinemia is one of the highest prevalent form of dyslipidemia present in this study. This shows the significant clinical influence of this SNP in inducing dyslipidaemia in HIV-infected individuals on HAART. In this study, 5 other SNPs (4 in LCAT exons 1 and 1 in exon 4) identified in LCAT do not have any significant effect on the dyslipidaemic outcomes in the study population. Twenty-five (25) SNPs were identified in LPL but none of these SNPs had a significant effect on the dyslipidaemic outcomes in the study population. This explains the observation that plasma triglycerides and VLDL concentrations did not change significantly after HAART.
Despite these findings, there were some limitations such as the sample size of the study was small, and SNPs present in other enzymes involved in the metabolism of lipids in the body were not sequenced. Also, the study did not longitudinally assess lipid parameters for the HIV-infected individuals which would have substantiated the association of SNPs presence and dyslipidaemia at baseline. Notwithstanding our observed findings are in line with previous reports.

Conclusion
The study has demonstrated the existence of SNPs in LCAT and LPL among HIV-infected individuals with dyslipidaemia in Ghana. The presence of SNPs at position 97 of LCAT exon 1 leads to the formation of a mutant LCAT which potentially reduces the rate of cholesterol esterification and reverse transportation to the liver for excretion. This supports the observation that reduced HDL-C was one of the commonest types of lipid abnormalities among the study population. The SNPs identified in LPL did not significantly affect TG levels, supporting the observation that total triglycerides and VLDL concentrations did not change significantly after HAART. This study recommends the use of genotyping to prospectively identify individuals with SNPs that contribute to dyslipidaemia. Further longitudinal studies are required in larger HIV cohort to ascertain the findings in the present study.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.