Dyslipidemia: Genetics, lipoprotein lipase and HindIII polymorphism

The direct link between lipid metabolism alterations and the increase of cardiovascular risk are well documented. Dyslipidemias, including isolated high LDL-c or mixed dyslipidemia, such as those seen in diabetes (hypertriglyceridemia, high LDL-c or low HDL-c), correlate with a significant risk of cardiovascular and cerebrovascular disease worldwide. This review analyzes the current knowledge concerning the genetic basis of lipid metabolism alterations, emphasizing lipoprotein lipase gene mutations and the HindIII polymorphism, which are associated with decreased levels of triglycerides and LDL-c, as well as higher levels of HDL-c. These patterns would be associated with decreased global morbidity and mortality, providing protection against cardiovascular and cerebrovascular diseases.

The relationship between dyslipidemia and atherosclerosis continues to be an area of active research, since the prevalence of atherosclerosis and associated cardiovascular complications continue to increase in the industrialized world 1 . Cardiovascular disease (CVD) constitutes the greatest cause of morbidity and mortality globally with a high incidence in countries of all economic categories 2 . Evidence supporting a causal relationship between lipid profile abnormalities and the risk of coronary artery disease (CAD) is overwhelming, confirming that hypercholesterolemia is an independent risk factor for CVD 3-5 . In addition, hypertriglyceridemia and mixed dyslipidemias have been associated with the aggregation of metabolic risk factors, like hypertension (HTN) 6 and obesity 7 .
The worldwide prevalence of dyslipidemia varies between different individuals, depending on race, age, socio-economic and cultural factors, lifestyle and genetics. This prevalence has increased significantly in growing cities with economic growth 9 . The factors are undoubtedly related to high calorie intake described by other Western nations 10 ; while lower prevalence has been reported for these pathologies in Canada and South Korea where 45% and 44.1% of their respective populations present evidence of dyslipidemia 11,12 . The difference of prevalence in these nations is indisputably tied to lifestyle with various publications highlighting diets rich in fiber and low in fats and refined sugars 13,14 .
In Brazil, de Souza et al. 2003. 15 reported the most frequent dyslipidemias in their 1039 sample population were isolated low HDL-C (18.3%), hypertriglyceridemia (17.1%), and isolated hypercholesterolemia (4.2%). These results differ from those reported in the CARMELA study from Mexico between 2003 and 2005 16 , where the incidence of dyslipidemia in 1722 person sample size reported 16.4% with hypercholesterolemia and 32.5% with hypertriglyceridemia -slightly higher than those reported in Brazil. The differences could be related to the food preferences within Mexico, which include greater amounts of fat, simple sugars, and processed ingredients 17 . It must also be noted that these studies are over 10 years old as no more recent publications in Latin America were found. With the continued rise in dyslipidemias and obesity, these numbers do not necessarily reflect the current reality of these pathologies in the studied populations.
In Venezuela, the CARMELA study evaluated the prevalence of these lipid metabolism disorders in the city of Barquisimeto.

Dyslipidemia genetics
The association between family history of dyslipidemia and the risk of CVD is supported by a large body of evidence 18-22 . Additionally, the great advancement in DNA analysis techniques has aided research surrounding CVD and related genetics and epigenetics. Understanding gene mutations or polymorphisms involved in the synthesis, transport, and metabolism of lipoproteins allows recognition of potential therapeutic targets and alternative treatments through identification of new molecules 1,3,20 .
Dyslipidemia is one of the most well characterized cardiovascular risk factors 19,20 . This not only depends on diet, but also on the synthesis and metabolism of lipoproteins conditioned by gene expression. Given the importance and the great diversity of proteins that participate in lipid metabolism, one might expect that a single defect in any step of gene expression would affect the quantity or quality of the product and potentially predispose to dyslipidemias and CVD 19 .

Amendments from Version 1
We made several modifications based on the reviewer input. In the "Dyslipidema: The Current Status" section the last four paragraphs were reworded and updated. This allows for improved reading comprehension and better supporting evidence. Per Reviewer 2 request, we added information about ABCA1 into our manuscript under the "Dyslipidemia Genetics" section. The written portion of the other genes was also reworded for improved comprehension and flow. In the "LPL Polymorphism" section we added more details about Ser447x and reworded both the PvuII and HinIII details. Finally, the "HindIII (rs320) Polymorphism" section received additional details in the first, penultimate, and last paragraphs. The authors feel this is a much stronger rendition of the manuscript that achieves the requested changes.

REVISED
One genetic abnormalities associated with low HDL-c and increased CVD risk is the Taq IB polymorphism located in chromosome 16q21. This gene alters cholesteryl ester protein transferase (CEPT), which decreases HDL-c concentration 23 . Some deletions, inversions, and substitutions of the APO AI-IV, CII, and CIII genes are also associated with both premature CVD and low HDL-c 24,25 . Total deficiency of lecithin cholesterol acyl transferase (LCAT) can be seen after transition of C→T in codon 147 of exon 4 (W147R), G→A in codon 293 of exon 6 (M293I), as well as partial deficiencies of LCAT due to transition of C→T. Additionally, the substitution of threonine for isoleucine in codon 123 (T123I) causes decreased HDL-c and higher cholesterol in the intima of arterial vessels 26,27 .
Below, some of the genetic alterations associated with low levels of HDL-c and a higher risk of CVD are highlighted: • CETP: This mediates the exchange of lipids between lipoproteins. With high levels of CETP, HDL are transitioned into triglycerides (TGs), becoming the substrate for hepatic lipase where TGs are hydrolyzed. Apoproteína (Apo) A-1 is degraded in tubular renal cells and diminishes the amount of HDL-C -increasing the atherogenic potential. The polymorphism rs1801706 (c.*84G>A) of the CETP gene is associated with CAD 27 .
• Familial hypoalphalipoproteinemia and HDL-C deficiency: Approximately 50% of the HDL-C alterations are explained by polygenic defects in various chromosomal loci that control apolipoprotein expression (A-I, A-II, C-II, C-III y A-IV). Multiple genetic variations such as deletions, inversions, and substitutions of gene coding for apolipoproteins are associated with severe CAD 28,29 .
• LCAT: This liver-synthesized enzyme circulates in plasma forming complexes with HDL and participating in the inverse transport of cholesterol. LCAT deficiencies cause an accumulation of free cholesterol in tissues. One of the most recent described gene alterations is the P-274-S polymorphism that afects biogenesis of HDL-C 30 and favors development of CVD.
• ABCA1: This protein mediates the transport of cholesterol and phospholipids from the cells to LDL. The C-69-T polymorphism of the gene codifies this protein and alterations can result in lower levels of HDL-C with higher levels of TG in obese children 31 . Additionally, the rs2515602, rs2275542, rs1800976, and rs4149313 polymorphisms are associated with obesity and can negatively affect one's lipid profile base on one study performed on 535 Chinese patients 32 .
• FTO: This includes a group of 45 genes related to obesity that were grouped together during phylogenetic analysis 33 and perform an important function in the regulation of food ingestion. FTO mutations are associated with obesity, metabolic syndrome and CAD 34 . The literature does not specify which lipid metabolism genes affect FTO, but the rs9939609 polymorphism is associated with low HDL-C levels.
The following are some genetic alterations associated with hypercholesterolemia and hypertriglyceridemia, including their relationship with increased cardiovascular risk:  43 . Recent work has encountered differences in the distribution of the diverse alleles of ApoA among patients with atherosclerosis and the isoforms of low weight molecular B, S1, and S2. These are found most frequently in carriers of coronary insufficiency who also show elevated levels of Lp(a) 44 . This suggests that the short alleles of ApoA contribute to atherogenesis, increasing the plasma concentration of Lp(a).
• HL gene -hepatic lipase and phenotype of combined familial hyperlipidemia. Combined familial hyperlipidemia is a genetic lipid disorder that accounts for 10-20% of premature CAD worldwide.  This information justifies the use of genetic markers for early diagnosis and cardiovascular risk assessment, especially in children and adolescents, in order to adopt early nutritional or pharmacologic interventions with the aim to mitigate atherosclerotic artery disease.

Lipoprotein lipase
The LPL gene is located on the short arm of chromosome 8, on the region 21.3 (8p21.3). It is formed of 10 exons and 9 introns (Figure 1) Around 100 mutations have been described on the LPL gene. The most frequent are Asp9sn, Gly188Glu and Asn291Ser. The mutations in the homozygous form are associated with hyperlipoproteinemia type I (familial chylomicronemia). Heterozygous mutations have a significant incidence in the general population (3-7%) and leads to up to a 50% decreased activity of LPL, causing an increase in TG and a decrease in HDL-c. All these lipid profile patterns increase the risk of CVD 61 .

LPL gene polymorphisms
Genetic studies have revealed around 100 mutations and polymorphisms in simple nucleotides on the LPL gene, some are protective, which others are deleterious: 1. Ser447x (rs328) polymorphism is located in exon 9, where cytosine is substituted by guanine on position 1959. This polymorphism leads to the suppression of both final amino acids, serine and glycine on position 447 of the protein that codifies a LPL protein prematurely truncated, which has increased lipolytic activity and increased levels of post-heparin LPL activity in X447 carriers. This is associated with the variant Ser447X, with low levels of TG, small increases of HLD-C levels, and a moderate CVD risk reduction 62 . These results differ from those of Emamian et al. 61 who studied 271 obese individuals and reported elevated TGs in carriers of this polymorphism. In studies of postprandial lipids, it has been reported that the aforementioned carriers present with elevated blood glucose and TGs than non-carriers 62 .
Thes reports clearly indicate that the benefit of this mutation are limited in patients of normal weight under the evaluated conditions.
2. PvuII (rs285) polymorphism, located on intron 6, is located 1.57 kb from the Splicing Acceptor (SA) site. This polymorphism is the product of a change of cytosine for thymine. The region containing the PvuII site is similar to the splice location. This suggests that a change at C497-T may interfere with the correct splicing of mRNA. Even though the physiological role associated with this polymorphism is not completely clear, it has been associated with high TG and low HDL-C levels 63 . A meta analysis revealed that this polymorphism reduces the risk of suffering from an MI 64 and, therefore, appears to have a protective effect against CVA.
3. HindIII (rs320) polymorphism is one of the most common polymorphisms of LPL gene (see below).

HindIII (rs320) polymorphism
HindIII is a transition of intronic bases of thymine (T) to guanine (G) on position 495 of intron 8 of the LPL gene, which eliminates the restriction site for the HindIII enzyme (Figure 2 and Figure 3). Sequential analysis has determined that this HINDIII recognition site corresponds with the binding consensus sequence for the transcription factors Sp1, GATA, C/EBP, and TBP. The first three are implicated in the regulation of the gene transcription involved in lipid metabolism 65 The allele H+ (presence of thymine "T" or restriction site of HindIII enzyme) results in a cut on the base pair sequence in two bands of 217pb and 139pb. This is associated with a decrease in the activity of LPL in comparison with the allele H-(presence of "G" or absence of the enzymatic restriction site or presence of HindIII polymorphism). With 137pb, in which there is no cut in the LPL gene intron 8 sequence, maintaining a unique sequence of 356pb (Figure 4) 69 , leading to both alterations in lipidic metabolism and cardiovascular risk profile modifications in these populations.
Some studies have demonstrated that the common allele (T or H+) is associated with lower levels of HDL-c in contrast with the uncommon allele (G or H-) 70,71 . In addition, those individuals with H+/H+ genotype had a higher concentration of serum levels of TG when compared with homozygous genotype H-/H-66,67, 70,72 . Similarly, there have been reports of high serum levels of LDL-c 71 and a higher global cardiovascular risk in patients who carry the common allele (T or H+), see Table 1. Some studies had reported a significant drop in the LPL activity among carriers of the uncommon G allele when compared with the more common allele T 57 .
LPL expressed by macrophages and other cells contained in the vascular walls is involved in the early atherogenic process and is associated with increased atherosclerosis. Overexpression of LPL is also associated with insulin resistance and HTN by increased sodium retention, inflammation, vascular remodeling, sympathetic nervous system activation, oxidative stress and vasoconstriction [73][74][75] .
On the other hand, HTN (mostly systolic) has been shown to be associated with the polymorphism HindIII in the Mexican population in studies by Muñoz-Barrios et al. 76 . Similarly, the homozygous genotype for the common allele (H+) was associated with a higher risk of myocardial infarction in       80 .
In other studies, Imeni et al. 86 evaluated the relationship between CAD risk and the distribution of HindIII polymorphism genotypes and found no statistical significant differences between healthy Irani individuals and those with CAD history.
Ahmadi et al. 87 also showed no significant association between the gene and CAD in the 115 Swiss subjects evaluated. These findings are in contrary to the expected improved cardio-cerebral function expected and leave this line of research open for future investigations.
This polymorphism has not only been associated with HA, rather also with insulin resistance. This is best demonstrated with a study of 110 Asian females with gestational diabetes who were found to have a reduced resistance to insulin than carriers of this rare allele 88 .
From a neurologic point of view, there is scant data associating homozygous common genotype (H+/H+) with the development of Alzheimer's disease of late appearance. This is founded on the LPL function in regulation cognitive function, mediated by cholesterol and Vitamin E transport to neuronal cells on the hippocampus and other brain areas 64 .
These investigations suggest that the presence of the HindIII polymorphism exerts a positive influence in lipid metabolism in patients with normal BMI. Future studies should focus in more detail on the protective function of this genetic factor in the general population.

Conclusions
Dyslipidemias are independent risk factors for atherosclerotic artery disease. High TC, TAG and LDL-C, as well as decreased serum HDL-C, are frequently associated with low physical activity and poor eating habits, but there is a large number of mutations and single nucleotide polymorphism related to a specific protein dysfunction within major lipoprotein metabolism pathways like CETP, ApoA, LCAT, LDL receptor, Apo B-100 and LPL.
In this regard, the LPL gene HindIII polymorphism (rare allele H-) poses a protective function through its role in producing an improved lipid profile (low TG and LDL-c and high HDL-c). On the other hand, the presence of common allele (T or H+) is associated with pro-atherogenic dyslipidemias and raised cardiovascular risk. The uncommon allele (G or H-) with an absence of restriction HindIII enzyme exhibits a lower prevalence of at least 20% according to the current available literature.
There are no studies in Venezuela that allows us to know the true prevalence of the HindIII polymorphism, nor to corroborate the association with changes in the lipid profile or an increased risk for cardiovascular diseases, so we suggest performing a national populational genetic study in search for this lipidic disorders with the aim to has a better understanding of the cardiovascular risk factors in Latin America.

Data availability
All data underlying the results are available as part of the article and no additional source data are required.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. entire intron 8 sequence along with full-length human LPL promoter were carried out in COS-1 and human vascular smooth muscle cells. The mutant allele was associated with significantly decreased luciferase expression level compared to the wild type allele in both the muscle (3.394 ± 0.022 vs. 4.184 ± 0.028; P=4.7 × 10 ) and COS-1 (11.603 ± 0.409 vs. 14.373 ± 1.096; P<0.0001) cells. This study demonstrates for the first time that the polymorphic III site in the LPL gene is functional because it affects the Hind binding of a transcription factor and it also has an impact on LPL expression.