Single Nucleotide Polymorphisms (SNPs) in Exon 6 of Lecithin Cholesterol Acyltransferase (LCAT) Gene in Indonesian Local Sheep

Lecithin cholesterol acyltransferase (LCAT) is a soluble enzyme that converts cholesterol and lecithin to cholesteryl esters and lysolecithins on the surface of high density lipoprotein and plays an important role in lipoprotein metabolism. The research was aimed to explore single nucleotide polymorphisms of LCAT gene in Indonesian local sheep. A total of 118 genomic DNA of Indonesian local sheep were used in this research, consisted of Sumatera Thin Tail (43 heads), Garut (19 heads), Javanese Thin Tail (17 heads), Javanese Fat Tail (6 heads), Rote Island (7 heads), Kissar (7 heads), Sumbawa (10 heads), and Lembah Palu (9 heads). Polymerase chain reaction was used to amplify genomic DNA for exon 6 (250 bp) and direct sequencing method was used to identify polymorphism sequences. The sequences were analyzed with BioEdit and MEGA 5.2 software. The BLAST sequence was obtained from Gene Bank GQ 150556.1. The results showed three novel SNPs, i.e. c.742C>T, c.770 T>A and c.882C>T. Substitution of cytosine to thymine c.742 is a synonymous mutation; thymine to adenine c.770 and cytosine to thymine c.882 are non-synonymous mutations. Polymorphisms of LCAT gene exon 6 was found in Sumatera Thin Tail, Javanese Thin Tail, Javanese Fat Tail, Garut, Lembah Palu, and Rote Island.


INTRODUCTION
Indonesia has a wide variety of sheep that are scattered in various areas with good breeding and establish certain morphological characters. According to habitat and morphometric characters, Indonesian local sheep was grouped into 5 clusters, i.e. Garut, Javanese Thin Tail (Jonggol), Fat Tail (Kissar, Rote Island, and Sumbawa), Donggala and Javanese Fat Tail (Indramayu and Madura) (Sumantri et al., 2007). Sheep is an economically important animal in Indonesia, especially provide fresh meat, sacrificial animals (Inounu, 2011), social, culture and source of genes for use in the improvement of local sheep through crosses between them and or exotic sheep (Sumantri et al., 2007). According to FAO (2002), local livestock is important to be protected because they adapt to local environment and low quality feed and are more resistant to local diseases and parasites. Indonesian local sheep had a high diversity based on morphological characters (Sumantri et al., 2007), and microsatellite analysis (Sumantri et al., 2008a;Jakaria et al., 2012).
Sheep is one of the meat-producing livestocks in Indonesia. However lamb is relatively less attractive to some communities in Indonesia. Lamb consumption is still very low, around 5% that is equivalent to 0.24 g/capita/year (Inounu, 2011). The low consumption of lamb is caused by a relatively high price, a distinctive odor that is difficult to remove, and the public perceptions that lamb has high cholesterol and saturated fatty acid. They are concerned by health factors, due to largely statements from the medical profession that lamb may contain too much saturated fatty acid (SFA) and trans mono unsaturated fatty acids (MUFA), which can be major risks for the development of coronary heart disease. The composition of the SFA, trans fatty acids, and dietary cholesterol were main factor to mortality rate determining differences on heart disease in seven countries (Kromhout et al., 1995).
The content of cholesterol is an important assessment of the consumer to consume lamb because it can affect health status. Meat cholesterol and SFA: poly unsaturated fatty acid (PUFA) ratio, are measures for providing "ASUH food" and to increase competitiveness to fulfill domestic market. Throughout the life of livestock, body fat was increased and the fatty acid composition also changed (Wood et al., 2008). SFA and MUFA contents were increased faster than PUFA due to increased of fat content, caused PUFA/SFA ratio would be decreased (De Smeet et al., 2004). Cholesterol content of meat sheep is not effected by diet, breed (Costa et al., 2009) and increased weight and age at slaughter (Wheeler et al., 1987).
Previous studies showed that cholesterol and marbling quality are regulated by functional genes. Deposition of fat in the muscle was determined by a balance of between anabolic and catabolic processes such as lipogenic and lipolytic, as well as transport of fatty acid and total of fatty acid used. The balanced of the processes was determined by the amount of fat consumed, de novo fat synthesis, triacylglycerol synthesis, lipids degradation and fatty acid transport (Zhao et al., 2010).
Lecithin cholesterol acyltransferase (LCAT) is a central enzyme in the extracellular metabolism of blood lipoproteins. This enzyme is synthesized in the liver Crisa et al., 2010), brain and testes (Reinshagen et al., 2009) and then secreted into the blood plasma. This enzyme converts cholesterol and lecithin into cholesterol ester and lysolecithin on the surface of high density lipoprotein (HDL), especially in the process of reverse cholesterol transport Crisa et al., 2010). The absence of this enzyme causes the accumulation of free cholesterol in the blood and tissues . The enzyme is encoded by LCAT gene that was located in choromosome 14 th in Ovis aries. This gene consists of 6 exons and 5 introns and exon 6 is the longest exon with the length of 559 bp (Gene Bank with Accession No.GQ.150556.1).
Single Nucleotide Polymorphism (SNP) is a DNA sequence variation that arises when a single nucleotide (A, T, C or G) is different from the sequences generally. Dunner, et al. (2013) stated that SNP within candidate gene have been tested for predictive value for carcass traits and some commercial tests based on SNP marker panels are being proposed to breeders for genotype animals. The genomic selection currently used to estimate breeding values for quantitative traits due to small population sizes and lack of high accuracy estimated breeding values (EBV). Therefore, genomic selection can be improved by extending the panel of SNP in the candidate loci and better estimating SNP effects in different populations. In human, SNPs was associated with human genetic diseases (Lin & Zeng, 2006).
Several previous studies have reported polymorphisms of LCAT gene in pig Qiao et al., 2010;Chalupova et al., 2012); dairy cow's (Loor et al., 2007) and sheep (Crisa et al., 2010;Moioli et al., 2012). Crisa et al. (2010) found one SNP in intron 2 g.181 T>C and 2 SNPs in exon 6 c.806 G>A and c.1075 T>C. Mutation in this region negatively correlated to the fatty acid C 18:2 and positively correlated to milk production and stearic fatty acids affect the diversity of sheep's milk (Crisa et al., 2010;Moioli et al., 2012). Exploration of variability of this gene in Indonesian local sheeps has never been conducted. Therefore, exploration of LCAT gene on Indonesian local sheep was conducted to find Marker Assisted Selection (MAS) to produce and develop local sheep in the future.

Materials
Fifty eight blood samples of Indonesian local sheep consisted of 44 samples from Padang (West Sumatera) and 14 samples from Garut (West Java) were collected by using venoject 3 mL from jugular vein. The blood samples were preserved in ethanol absolute 96% with ratio of 1:1 and kept in room temperature for laboratory analysis. Other extracted DNAs deposited at the Genetics and Animal Breeding Laboratory were used in this study, i.e. Garut, West Java (Garut-Margawati meat type 5 heads), Javanese Thin Tail, West Java (Jonggol, 7 heads; MT Farm Bogor, 10 heads); Javanese Fat Tail, East  Java (Situbundo, 6 heads), Rote Island (7 heads), Kissar-South West Molucas (7 heads), Sumbawa-West Nusa Tenggara (10 heads), and Donggala, Central Sulawesi (9 heads). A total of 118 samples of Indonesian local sheeps were used in this study.

DNA Extraction
Genomic DNA was extracted by using Phenol-chloroform technique (Sambrook et al., 1989) and modified by using buffer lysis cell (250 µL 1 x STE, 40 µL SDS and 10 µL proteinase-K). The DNA was purified by adding 40 µL 5M NaCl, 400 µL phenol chloroform and 400 µL CIAA (Chloroform Iso Amyl Alcohol) and precipitated by using 40 µL 5M NaCl and 800 µL ethanol absolute. The precipitation was washed once by adding 800 µL of 70% ethanol and centrifuged with the speed of 12.000 rpm for 5 min. The ethanol was discarded and evaporated, then the precipitated DNA was disolved in 100 µL of 80% TE (Elution buffer).

DNA Amplification and Direct Sequencing Method
The DNA was amplified with Polymerase Chain Reaction (PCR). Each PCR reaction was made with cocktail 50 ng (2-3 µL) DNA template, 0.25 µM primer forward and reverse, 12.5 µL Dream Tag Green Master Mix from Thermo Scientific #K 1081 and dH 2 O up to 25 µL. The forward primer sequence was F'5-GAGCAGCGCATGACGACAACG-3' and reverse primer sequence was F'5-AGGTGCTAGGAGTGGGCAGGC-3'. The position of primer forward and primer reverse in PCR product of LCAT gene were shown in Figure 1 and the length of PCR product yield was 250 bp ( Figure   2). Samples were initially denaturated at 95 o C for five minutes and followed by 35 cycles of denaturation at 95 o C for 45 s, annealing at 62 o C for 45 s and extension at 72 o C for one min. Final extension was at 72 o C for 5 min.
GeneAmp PCR system 9700 and Master Cycler Personal 22331 Eppendorf were used for PCR amplification. PCR products were then separated on 1.5% agarose/0.5 x TBE, stained with 2.5 µL of ethidhium bromide (EtBr) and calibrated with 100 bp ladder marker. Electrophoresis chamber was run on 100 volt power supply for thirty minutes. Finally, the gel was visualized under UV transilluminator ( Figure 2). The PCR product samples were then subjected to direct sequence analysis by dideoxy sequencing in ABI 3730 XL automated DNA sequencer at the 1 st base laboratory Singapore.

Data Analysis
The results of sequence fragment of LCAT gene exon 6 th were analyzed with BioEdit (Hall, 2011), MEGA version 5.2 (Kumar et al., 2004) and POP GENE ver.1.31 (Yeh et al., 1999) software. The BLAST sequence was obtained from Gene Bank with accession number of GQ 150556.1. The analyzed sequence was required to ensure the fragment of LCAT gene of sheep and to find out the existence of mutation in LCAT gene and polymorphism in the sequence.
Frequency of gene was estimated by Nei & Kumar (2000): where: x i = frequency of i gene x j = frequency of j gene Frequency of genotypes was measured as by Nei & Kumar (2000):

X ii = (N ii /N) x 100%; X ij = (N ij /N) x 100% X jj = (N jj /N) x 100%
where: X ii = frequency of ii genotype X jj = frequency of jj genotype X ij = frequency of ij genotype Deviation frequency genotype from Hardy -Weinberg equilibrium was analyzed by chi square test (X 2 test) as follows Nei & Kumar (2000); where: X 2 = chi square test O = the observed numbers of frequency

E = the expected numbers of frequency
The observed heterozygosity (H o ) and excepted heterozygosity (H e ) were estimated with POPGENE 32 version 1.31 software (Yeh et al., 1999); where: H o = the observed heterozigosity H e = the expected heterozigosity w k = size population relatively X kij = frequency of genotype AiAj, the k-population

Discovery of Single Nucleotide Polymorphisms of LCAT Gene
One hundred and eighteen (118) (Figure 4c). Substitution of cytosine to thymine which was detected at c.742 was synonymous mutation, because it did not change amino acid (Ala>Ala). This mutation found in H 2 H 2 (4 heads) and H 4 H 10 (1 head), a total of 5 individuals dispersed on the Sumatera Thin Tail (2 heads); Garut (2 heads) and Lembah Palu (1 head). Heterozygous genotype (CT) found in H1H2 and H1H4 were found on Sumatera Thin Tail (1 head) and Javanese Thin Tail (4 heads). Synonymous SNPs could encode sequences with the same amino acid composition but structure and function changed (Komar, 2007).
Substitution of thymine to adenine at base c.770 and cytosine to thymine at base c.882 were non-synonymous mutation, because they changed phenylalanine to isoleucine (Phe>Ile) and alanine to valine (Ala>Val). Non Synonymous SNPs (nsSNPs) caused protein product changed and in humans, most associated with inherited diseases (Bao & Cui, 2005).
SNP at base c.882 displayed three genotypes (CC, CT, and TT). CC genotype was found in 106 individuals that were scattered on the 5 diplotypes, i.e.: H1H1, H1H2, H1H3, H1H4, and H2H2. Genotype CT was found in 3 diplotypes, i.e.: H1H5, H3H5, and H4H10, while the TT genotype was found only in diplotipe H5H6. According to Nei & Kumar (2000), in most nucleotide sequences there were more nucleotide sites that potentially produced synonymous and nonsynonymous sites vary from gene to gene.
The results showed that the spread of SNPs in Indonesian local sheep found in 6 sub-populations, except Kissar and Sumbawa were indicated monomorphic. High gene diversity was found in thin tail group (Sumatera Thin Tail and Javanese Thin Tail), followed by Garut and fat tail group. Crissa et al. (2010) reported 3 new SNPs in the LCAT gene of 3 breed sheeps (Altamurana, Gentile di Puglia, and Sarda) i.e.: g.181T>C, c.806G>A and c.1075T>C, with diversity gene that was relatively low.Each mutations that arise could affect molecular function through change to protein sta-  , with diversity gene that was relatively low. The mutation at c.806 was a non-synonymous mutation (Asp to Asn) that has a negative effect on the ratio fatty acid C18:2 and C18:3 in milk of sheep (Crissa et al., 2010;Moioli et al., 2011). Qiao et al. (2010), showed that SNP at g.266 G>C in intron 1 was significantly associated with lean fat ratio, leaf fat weight or carcass length of three breed pigs and cholesterol level in blood plasma .
LCAT reaction on lipoproteins consists of several steps; 1) binding lipoprotein/lipid surface by enzyme, 2) the activation of LCAT by apo lipoproteins 3) binding of lipid substrates 4) catalytic and finally 5) generate the lipid products (Jonas, 2000;Kaysen, 2007). The decrease of LCAT activity was implicated to cow's fertility decrease and fatty liver diseases (Uchida et al., 1995). Cholesterol esterification by LCAT is important for its transport from liver to peripheral tissues, such as corpus luteum. In addition, cholesterol serves as a substrate for progesterone synthesis in the other organ. The results of in vitro studies indicate the activation of LCAT by Apo A-I, but the exact mechanism is not known (Rousset et al., 2010).
Insertion of adenine results in a frameshift mutation at base g.214 in human, altering a large portion of LCAT enzyme, including both protein regions with putative lipase activity (Bender et al., 2007). Two mutations were detected in human LCAT that the Tyr 83 (frameshift mutation) resulted in the synthesis of truncated 82 amino acid enzyme, which would be, if secreted, most likely non functional and the Tyr 156 (Asn substitution) was formed an amphipathic helix, a residue within the hydrophobic phase of the helix resulting in a lower pH (Klein et al., 1993). LCAT enzyme was played on formation and maturation HDL and reverse cholesterol transport proses in intravascular (Savel et al., 2012). The absence of LCAT was resulted in the accumulation of free cholesterol in the blood and tissues (Qiao et al., 1997).

The Frequencies of Gene, Genotype and Heterozygosity
Frequency of gene or frequency of allele is a measure of the relative frequency of a particular gene/allele in a population (Nei & Kumar, 2000). Genetic diversity within a population can be used as a parameter in studying the population and evolutionary genetics, identifiying genes that control the diversity of economic nature by the detection of positive alleles at locus having economic values to produce desirable traits .
The results showed a high gene diversity at locus c.742 found in Sumatera Thin Tail, Javanese Thin Tail, Garut, and Lembah Palu. The frequency of C gene was the predominat around 88.2%-98.8%, and the frequency of T gene was around 1.2%-11.8% and those were indicated as a polymorphic loci ( Table 2). The average frequency of genotype CC (94.10%) was higher than CT (3.2%) and TT (2.7%) in all population. The SNPs loci were in Hardy-Weinberg disequilibrium in the Sumatera Thin Tail, Garut and Lembah Palu (P<0.01), except in The Javanese Thin Tail was in Hardy-Weinberg equilibrium. Genotype frequency deviations that can arise due to mutation, migration, selective mating and selection, could rapidly change genotype equilibrium that appears in a population. A population are in a Hardy-Weinberg equilibrium if genotype frequency and gene frequency constant from one generation to the next generation (Nei & Kumar, 2000).
At the base c.770, it was found two types of genotypes (AT and TT), except on Kissar and Sumbawa only one genotype (TT). The frequency of TT genotype was higher than AT genotype on all population. The high frequency of TT genotype in the population resulted in a high frequency of T (76.5%-94.2%). The genotype frequencies at this SNP was fitted with Hardy-Weinberg equilibrium (P>0.05) on all population and the observation heterozygosity and expectation heterozygosity were relatively similar (Table 3).
Mutation at base c.882 C>T was found on Sumatera Thin Tail, Javanese Thin Tail, Garut, and Rote Island. The frequency of CC genotype (89.6%) was higher than Sub population genes of Indonesian Local Sheeps were reported in calpastatin gene (Sumantri et al., 2008b;Dagong et al., 2011) and no polymorphism was detected in myostatin gene .

CONCLUSION
Three SNPs in the LCAT gene were detected at the c.742 C>T; c.770 T >A and c.882 C>T. The combination of these three SNPs formed nine diplotypes. Substitution of cytosine to thymine c.742 is synonymous mutation (alanine>alanine); thymine to adenine c.770 and cytosine to thymine c.882 are non-synonymous mutation that change phenylalanine>isoleucine and valine>alanine. Polymorphisms of LCAT gene exon 6 was found in Sumatera Thin Tail, Javanese Thin Tail, Javanese Fat Tail, Garut, Lembah Palu, and Rote Island. Further research needs to be done to determine the expression of LCAT gene diversity in the three new SNPs and its relationship to the meat fat quality.