Novel missense variant L46Q of fatty acid synthase gene and fatty acids content in Awassi sheep

ABSTRACT.

This study was conducted to investigate the association between the polymorphism of the FASN gene with fatty acid content in Awassi sheep. A total of 100 male Awassi sheep between the ages of one and two and a half years old were used in this study. Phenotypic measurement was recorded at slaughter, and from each animal, the longissimus dorsi (LD) muscle samples were taken to analyze the fatty acid profile. Genotyping, sequencing reactions, and in silico tools were performed to confirm the variants in amplified fragments. The result of genotyping revealed two genotypes (AA and AB) of the ovine FASN gene (exon 3). Novel SNP (L46Q) was discovered only within the FASN gene (AB genotype). All utilized in silico tools revealed remarkably deleterious effects for the L46Q on the mutant protein structure, function, and stability. Association analysis revealed that the AB genotype has significantly (p < 0.05) higher levels of animal length and monounsaturated fatty acids (MUFA) with lower amounts of saturated fatty acids (SFA) content than the AA genotype. In conclusion, novel SNP (L46Q) was discovered within the FASN gene (AB genotype), made the animals that has the AB genotype associated with good meat quality traits and this polymorphism may serve as markers for meat quality.

Keywords:
fatty acids composition; FASN gene; meat quality; rams

Introduction

Small ruminants, especially native breed types, play an important role in the livelihoods of a considerable part of the human population in the tropics from socio-economic aspects (Ebrahimi, Mohammadabadi, & Esmailizadeh, 2017). Therefore, integrated attempt in terms of management and genetic improvement to enhance production is of crucial importance (Moghadaszadeh, Mohammadabadi, & Esmailizadeh, 2015). Economical and biological efficiency of sheep production enterprises generally improves by increasing productivity and reproductive performance of ewes (Mohammadabadi, 2016; Al-Thuwaini, 2022). Awassi breed is the most predominant sheep in the Middle East areas that are characterized by a high ability to cope with hardy conditions (Al-Thuwaini, 2021a), with high meat and milk production than the other surrounding breeds in the Middle East (Jawasreh, Al-Amareen, & Aad, 2019; Ajafar, Kadhim, & AL-Thuwaini, 2022a). Several genes of lipid metabolism regulate the fatty acid content of livestock meat (Quiñones, Bravo, Calvo Lacosta, & Sepúlveda, 2017; Ajafar, AL-Thuwaini, & Dakhel, 2022b). Among them, the fatty acid synthase (FASN) gene is used to improve fatty acid components (Shi et al., 2019). The FASN gene mapped on chromosome 11 in sheep and on chromosome 19 in cattle (Oztabak et al., 2014), which encodes enzymes responsible for fatty acid synthesis, elongation, and desaturation (Quiñones et al., 2017). Fatty acid synthase (FAS or FASN) is a multifunctional enzyme complex that organizes de novo biosynthesis of long-chain fatty acids (Raza et al., 2018) and synthesizes saturated and unsaturated fatty acids (Pećina & Ivanković, 2021). There are associates of genetic polymorphism with meat quality traits (Zalewska, Puppel, & Sakowski, 2021). The polymorphism of the TE-FASN gene in sheep revealed the impact of the genotypes on the fatty acid content (Esteves et al., 2019). Several variants in the bovine FASN gene correlated to the fat content and the composition of fatty acids in both milk and meat (Ciecierska et al., 2013). The g. 17924GG genotype in FASN led to lower saturated fatty acid (SFA) including Myristic and palmitic acids and higher levels of oleic acid as main monounsaturated fatty acids (MUFA) amounts in Korean cattle (Cho et al., 2010). In Korean and Angus cattle, the GG genotype of the FASN gene is associated with higher levels of MUFA and lower levels of SFA (Ciecierska et al., 2013). On the other hand, determination of gene polymorphism is important in farm animals breeding (Shamsalddini, Mohammadabadi, & Esmailizadeh, 2016; Gholamhoseini, Mohammadabadi, & Asadi Fozi, 2018; Gooki, Mohammadabadi, Fozi, & Soflaei, 2019) to define genotypes of animals and their associations with productive, reproductive and economic traits (Pasandideh, Mohammadabadi, Esmailizadeh, & Tarang, 2015). Based on the above consideration, no research yet on the association of the FASN gene with the fatty acid content has been reported in Awassi sheep. Thus, the current study was conducted to evaluate the association of the FASN gene polymorphism and fatty acids content in Awassi sheep.

Material and methods

Animals

This study was performed according to regulations of the international recommendations for the care and use of animals under Al-Qasim Green University's approval (Agri, No. 015,3,12), at the College of Agriculture /Department of Animal Production for the period from October /2017 to June / 2018. A total of 100 male Awassi sheep between the ages of one and two and a half years old and weight between 25 to 40 kg were included in this study. The referred animals were randomly selected from three flocks in the middle Euphrates regions of Iraq. In each studied flock, 10-12 rams were randomly allocated to mate with about 20-25 ewes per ram, with male identification recorded. Animals were kept on seasonal grass during spring and autumn, while in winter, animals were kept indoors and fed concentrated food. Animals were slaughtered at abattoirs of Babylon, and from each animal, the longissimus dorsi (LD) muscle samples (~100 g) were taken between the 12 and 13^th ribs at 45 min. post mortem, collected, and fractionated to analyze the fatty acid content. HPLC technique was used and the different fatty acids contents were calculated using the method of Salimon, Omar, and Salih (2017). The fatty acid composition was determined using the GS solution 2.42 software. Phenotypic measurements including body weight (BW), back fat thickness (BFT), body length (BL), abdominal fat (AF), and fat tail weight (FTW) were recorded at slaughter according to Al-Thuwaini et al. (2022).

DNA isolation and PCR amplification

The high salt method of Al-Shuhaib (2017) was conducted to isolate the genomic DNA from the whole blood. The primers used to amplify the ovine gene were designed using the Primer-BLAST online server (Ye et al., 2012) according to the sequence of the FASN gene (exon 3) for ovine (GenBank accession numbers NC_019468.2). The sequence of the used primer in this study was: F: 5'-AGGTCAGAGAATTAA AGCT-3', R: 5' GGAAGTGACAGTGGTTTT-3'. PCR experiments were conducted as follow: initial denaturation for 5 min, followed by 30 cycles for 30 sec of denaturation (95°C), annealing (56.7°C), and extension (72°C), with a final extension (72°C) for 5 min. The specificity of PCR amplicons was verified by electrophoresis on agarose gel then submitted to SSCP protocols (Al-Thuwaini, 2021b).

Genotyping and sequencing analysis

The SSCP experiment was performed according to Imran, Al-Thuwaini and Al-Shuhaib (2020) protocol. For single-stranded conformation polymorphism (SSCP) analysis, 10 µL of each amplification product was mixed with 10 µL of SSCP denaturing buffer heated for 7 min. at 95°C and then chilled on ice for 7 min. SSCP analysis was conducted in 10% polyacrylamide gels (37.5:1) at 200 V for 4h in TBE (0.5×) buffer at a constant temperature of 20°C. The silver staining of SSCP patterns on the gels was visualized by methods described by Byun, Fang, Zhou and Hickford (2009). For each genotype, the PCR products were sent for purification and sequencing of multiple sequence alignment programs, according to DNA Star, EditSeq. / ClustalW, with the sequences published in the GenBank database taken as a reference to identify the polymorphisms. The observed mutations were visualized and checked the novelty by SnapGene Viewer, ver. 4.0.4. (GSL. Biotech. LLC) and the ensemble genome browser 96 (https://asia.ensembl.org/index.html).

In silico prediction

Many computational tools were utilized to assess the consequences of the observed missense variants on the resulting mutant protein structures, functions, and stability, namely SIFT, PolyPhen-2, Provean, SNAP2, and I-Mutant2.0 (Imran et al., 2020).

Statistical analysis

The genetic diversity of the FASN gene was analyzed using PopGen32 software, v. 1.31 (Yeh & Yang, 1999). Association analysis was analyzed using SPSS v23.0 (IBM Corp, 2015). The significant effect of genotype on the various phenotypic parameters was performed using General linear mixed-effects models (GLMMs) with the following model;

Yijk =µ + Gi + αj + eijk

Where: Yijkis the phenotypic trait, μ is the overall mean, Gi is the fixed effect associated with the ith SNP genotype(i= AA, AB),αj is the random effect of the jth sire and eij= random error with assumed NID (0, σ2e). Normality was tested using the Kolmogorov-Smirnov test. Preliminary statistical analyses indicated that age, season, and nutrition were not found to affect phenotypic characteristics and thus they were not included in the model.

Results and discussion

Genotyping analysis of FASN gene

The SSCP analysis revealed two variations within the DNA samples that amplified by the ovine FASN (exon 3) specific primer pair (Figure 1).

Figure 1
SSCP non-denaturing polyacrylamide gel electrophoresis of ovine FASN gene (exon 3) PCR fragments, which showed two SSCP banding patterns in Awassi sheep.

Results of genetic diversity and Hardy-Weinberg test for FASN (exon 3) gene were presented in Table 1. The genetic analysis showed that the predominant genotype was AA with a genotype frequency of 60%. The X ² test indicated that the polymorphism of FASN (exon 3) in Awassi sheep was not at Hardy-Weinberg equilibrium (Table 1).

Table 1
Genetic diversity parameters for the FASN gene in Awassi breed.

	Genotype frequencies (n)		Allele frequencies		Ho	He	Ne	X 2
	AA	AB	A	B	0.4000	0.321	1.470	6.07
Awassi	0.60 (60)	0.40 (40)	0.80	0.20

• Abbreviations: (n) - samples number, X ² - chi-square, Ho - observed heterozygosity, He - Expected heterozygosity, Ne- effective allele number. Chi-square tests have one degree of freedom within the significance level p < 0.05.

Sequence and in silico analysis of FASN gene

The SSCP analysis reported two different genotypes in the studied animals. Sequence analysis of the ovine FASN locus identified four SNPs, between the two resolved genotypes and the FASN (exon 3) NCBI reference sequences (Figure 2) which confirmed the results of the SSCP analysis. The pattern of each SNP that discovered by sequencing was listed in (Table 2). Several SNPs were discovered in FASN (exon 3) reference in comparison with two genotypes AA and AB (Table 2).

Table 2
The differences of nucleic acids and the amino acid patterns between genotype AA and genotype AB in exon 3 of Ovis aries of the FASN gene.

No.	Wild type	Mutant	Genotype	Position in the PCR fragment	Position in the genome	Type of SNP	Amino acid change
1	T	A	AB	84	49890901	Exonic (L47)	L47Q
2	T	C	AA & AB	131	49890948	Exonic (S63)	S63P
3	G	A	AB	247	49891064	Intronic variant	-
4	T	G	AB	281	49891098	Intronic variant	-

Figure 2
Sequences and SNP positions of two genotypes AA and AB in the Awassi sheep FASN (exon 3) gene.

Postulated three-dimensional structure of Ovis aries FASN protein was performed using protein homology/analogy recognition engine (Phere2), ver 2.0 and PyMOL-v1, 7.0.1 software (www.shrodinger.com) (Figure 3).

Figure 3
Virtual 3-D structure of ovine FASN. A) Reference type protein (Before mutation), B) mutant protein (in AA genotype), C) mutated protein (in AB genotype).

Consequences of the observed missense variants on the resulting altered protein structures, functions, and stability were evaluated and their results were shown in Table 3. Many bioinformatics tools were used to evaluate the consequences of the observed missense variants on the resulting altered protein structures, functions, and stability including SIFT, PolyPhen-2, Provean, SNAP2, and I-Mutant2.0 (Table 3).

Table 3
The in silico analysis of the observed nonsynonymous SNPs in FASN.

SNP	SIFT		PolyPhen-2		PROVEAN		SNAP2		I-Mutant2
	score	prediction	score	prediction	score	prediction	score	prediction	score	prediction
L47Q	0.01	Affect protein function	0.924	Probably damaging	-5.700	Deleterious	67	Effect	-1.00 (DDG-kcal mol-1)	Decrease Stability
S63P	0.27	Tolerated	0.891	Probably damaging	-3.331	Deleterious	63	Effect	-3.14 (DDG-kcal mol-1)	Decrease Stability

Two missense mutation was found in two genotype AA and AB genotype. Only L47Q was found in AB genotype with an entire deleterious consequence on protein function and stability according to in silico tools (Table 3) and was predicted by the missense 3D server (Figure 3). Thus, it was found that there was an alteration between AA and AB genotypes regarding altered disordered positions, which may be responsible for this alteration in fatty acids content made AB genotype has good meat quality traits. This result is in concord with the study of Hayakawa et al. (2015) that studied the association between g.841G>C SNP in the FASN gene and associated with the fatty acid content of Japanese Black cattle. Bartoň, Bureš, Kott, & Řehák, (2016) identified one SNP in the FASN gene of Holstein cattle and revealed a significant relationship with fatty acid content in the longissimus dorsi muscle.

Association analysis

FASN (exon 3) gene polymorphism and animal traits

Table 4 refers to the effect of FASN gene polymorphism (exon3) on animal traits. Significant differences (p < 0.05) in the length of the animal for genotypes AA and AB of FASN (exon 3) gene showed that animals with AB genotype had a greater body length compared with AA genotypes (p ˂0.05). While there was no significant association (p > 0.05) in the other animal traits among the genotypes (Table 4).

Table 4
Relationship between FASN gene polymorphism (exon 3) and animal traits in Awassi sheep.

Animal traits	Genotypes (LSM ± SE)		p-value
	AA (60)	AB (40)
live body weight (kg)	29.32 ± 0.24	33.52 ± 0.31	0.13
body length (cm)	70.30 ± 1.45 b	82.53 ± 1.15 a	0.04 *
Fat tail (kg)	2.11 ± 0.05	2.43 ± 0.02	0.57
Abdominal fat (kg)	1.63 ± 0.03	1.45 ± 0.01	0.82
Back fat (kg)	1.42 ± 0.10	1.61 ± 0.01	0.29
Carcass weight (kg)	23.53 ± 0.37	25.11 ± 0.22	0.13

• LSM, Least square mean ± SE standard error; * (p < 0.05); Different superscript in the same row refers to significant differences (p < 0.05).

Matsuhashi et al. (2011) that reveal polymorphisms in the FASN gene showed no effect on any animal traits that support our result. In contrast, another study reveals the three SNPs in the FASN gene that correlated to the subcutaneous fat thickness and growth traits in cattle (Souza et al., 2012). The TT genotype at g. 13232 C > T is correlated with higher intramuscular fat in Qinchuan cattle (Raza et al., 2018).

FASN gene polymorphism (exon 3) and lipid profile

The results of the current study showed significant differences (p ˂ 0.05) in the lipid profile level between FASN (exon 3) genotypes. AA genotype had significantly higher (p <0.05) levels of LDL concentration than AB genotype (72.68 ± 1.68 and 66.72 ± 0.93 (mg dL^-1) respectively, while no significant differences (p > 0.05) in other lipid profile concentrations among genotypes (Table 5).

Table 5
Association analysis between FASN polymorphism (exon 3) and lipid profile in Awassi sheep.

Indices	Genotypes (LSM ± SE)		p-value
	AA(60)	AB(40)
Cholesterol (mg dL-1)	115.32 ± 1.21	111.17 ± 2.31	0.54
Triglyceride (mg dL-1)	77.53 ± 1.63	75.81 ± 1.01	0.23
High density lipoprotein (mg dL-1)	26.37 ± 0.79	26.54 ± 0.25	0.46
Very low density lipoprotein (mg dL-1)	15.79 ± 0.83	15.01 ± 0.77	0.22
Low density lipoprotein (mg dL-1)	72.68 ± 1.64 a	66.72 ± 0.93 b	0.01*

• LSM, Least square mean ± SE standard error; * (p < 0.05); Different superscript in the same row refers to significant differences (p < 0.05).

Alter cellular cholesterol concentrations belong to the function of the FASN enzyme by increased mitochondrial oxidation of fatty acids (Carroll et al., 2018). Mahmoud, Mohammad, and Ezat (2016) refer to a significant association between FASN level with triglyceride and LDL concentrations.

FASN gene polymorphism (exon 3) and fatty acid composition

Statistical analysis for the fatty acid content of intramuscular in the longissimus dorsi muscle and FASN (exon 3) genotypes, shown in Table 6. The AB genotype had the lowest content of Capric acid (2.14 ± 0.04), Myristic C14:0 (0.56 ± 0.04) and Stearic C18:0 (0.001 ± 0.0001), while highest content of Oleic C18:1n9 (2.11 ± 0.05) than AA genotype. No significant association of the FASN genotypes was observed in other fatty acid composition.

Table 6
The relationship between FASN polymorphism (exon 3) and fatty acids composition in Awassi sheep.

Fatty acids composition	Genotypes (LSM ± SE)		p-value
	AA(60)	AB(40)
SFA
Caprylic	2.53 ± 0.01	1.35 ± 0.02	0.15
Capric	6.33 ± 0.02b	2.14 ± 0.04a	0.01*
Lauric	0.53 ± 0.05	0.47 ± 0.03	0.42
Myristic	1.53 ± 0.01b	0.56 ± 0.04a	0.02*
Palmitic	0.83 ± 0.02	0.94 ± 0.05	0.71
Stearic	0.02±0.003b	0.001±0.0001a	0.01*
Phytanic	0.84 ± 0.03	0.75 ± 0.04	0.59
MUFA
Ricinoleic	0.45 ± 0.01	0.39 ± 0.02	0.31
Ricinelaidic	0.56 ± 0.03	0.70 ± 0.03	0.63
β-linolenic	1.99 ± 0.06	2.53 ± 0.04	0.25
α-linoleic	0.73 ± 0.07	0.78 ± 0.01	0.40
Oleic	1.32 ± 0.02b	2.11 ± 0.05a	0.03*
Petroselinic	0.73 ± 0.05	0.90 ± 0.01	0.61
Elaidic	0.11 ± 0.001	0.18 ± 0.004	0.42
Vaccenic	0.04 ± 0.0001	0.07 ± 0.002	0.78
PUFA
α-linolenic	0.51 ± 0.03	0.52 ± 0.01	0.22

• LSM, Least square mean ± SE standard error; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; * (p < 0.05); Different superscript in the same row refers to significant

Significant impacts reported of the FASN polymorphism on fatty acids content of the intramuscular in Japanese black cattle might indicate that this polymorphism might influence the β-ketoacyl reductase domain function in the FASN gene (Matsuhashi et al., 2011). A significant association is reported between fatty acid content and g.13232C > T SNP in the FASN gene in Qinchuan cattle (Raza et al., 2018). This suggested that the SNP g. 17924 G>A may change the activity in the TE domain of the FASN gene resulting in differences in fatty acids content between genotypes (Oztabak et al., 2014).

Conclusion

Novel SNP (L46Q) was discovered within the FASN gene (AB genotype), made the animals that have the AB genotype associated with good meat quality traits with the lowest content of SFA and the highest content of MUFA of Awassi sheep.

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Publication Dates

• Publication in this collection
  12 Aug 2022
• Date of issue
  2022

History

• Received
  20 Oct 2020
• Accepted
  16 Feb 2021