Detection of genomic structure variants associated with wrinkled skin in Xiang pig by next generation sequencing

Introduction

Skin is a complex organ covering the entire surface of animal body. The skin aging processes are affected by intrinsic or extrinsic factors [1]. One of clinical manifestation of skin aging is wrinkles [2]. Whether caused by internal or external factors, wrinkles are inseparable from cutaneous aging. In the action of intrinsic factors, the proliferation of cells is reduced, and the senescence process of cells are increased, resulting in structural and physiological disorders, and forming a thin and atrophic, finely wrinkled skin [1]. Under the stimulation of external factors (ultraviolet rays), the reactive oxygen species (ROS) accumulate, causing cell proliferation impaired, metalloproteinase expression increased, and dermal extracellular matrix degraded, then taking deep and laxity wrinkles [3, 4].

Over past decades, research has gained crucial insight into underlying genetic factors of skin aging, especially the contribution of genetic variation. Such as single nucleotide polymorphisms. The mutation c.1824C > T of Lamin A/C gene (LMNA) is confirmed to be the cause of Hutchinson-Gilford Progeria Syndrome (HGPS) [5]. The rs185146, rs12203592, and rs4268748 polymorphisms near SLC45A2, IRF4, and MC1R, respectively, are associated with pigmentation and skin aging [6]. The insertion/deletion (InDel) variations are also related with skin aging. A recessive c.1030_1033delCTGT deletion in FOXN1 caused skin wrinkles and hairlessness in Barman cats [7]. Similarly, the gain or loss of copy number may also be involved in skin aging. The gaining copy number of the HAS2 gene is associated with a strong selection for wrinkles phenotypes in the Chinese Shar-Pei dogs [8]. Importantly, the contribution of structural variations (SVs) to complex phenotypes accounted for 83.6% percent of the total genetic variation [9]. For example, a translocation or a deletion that removes the C-terminus of ELN gene can cause cutis laxa [10]. Large scale deletion of mitochondrial genome leads to wrinkles phenotypes, such as the 4,977 bp common deletion [11]. In addition, gene expression, microRNA regulation and epigenetic factors also play an important role in skin aging [12–14]. Skin aging seems to involve multiple genes.

The Xiang pig is a miniature pig breed that originated in the mountainous area of Guizhou Province in China. Some Chinese indigenous pigs have obvious wrinkles on the forehead, such as Chinese Erhualian and Meishan. It’s reported that the G allele in GRM4 is beneficial to increase Erhualian pig facial wrinkles by using genome-wide association analysis [15]. Evidence of selective sweeps in the genome of Meishan pig reveals that strong selective sweep signals of NFKB1 may have resulted in the wrinkled skin and face [16]. Xiang pigs also have characteristic facial wrinkles like other native pig breeds. But a few individuals displayed dense wrinkles on the back and buttock. This is a special systemic skin wrinkle phenotype, and it can be inherited, with an incidence of about 1-3%. So far, the cause of the systemic skin wrinkles in Xiang pig is still enigmatic. In the present study, we employed histology methods to characterize the structure of the skin in Xiang pigs with skin wrinkled, and we performed whole-genome resequencing using Illumina HiSeq2500 to identify genomic structure variants in these pigs. The aim of this study is to uncover genetic variants associated with skin wrinkles of Xiang pig. It might help us to better understand the underlying molecular mechanism of skin aging, which probably was the genetic mechanisms for the mutant phenotype of Xiang pigs with systemic wrinkle.

Results

Discussion

Under natural conditions, young mammals rarely have systemic skin wrinkles. Skin wrinkles generally appear in adulthood and increase with age. But there are a few special cases in some mammals, such as Chinese Shar-Pei dogs and Birman cats, show systemic skin wrinkles at very young age or at birth. In Xiang pigs, the systemic skin wrinkle phenotype was found in a few of individuals, and appeared in two months after birth and became more and more obvious with age. Originally, the mutated phenotype was observed in a boar and among his offspring, accounting for about 1-3% of the entire populations. The trait of WXP characterizes autosomal recessive heredity, and its genetic mechanism is unknown.

Skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. Skin aging is associated with structural and functional changes in both of epidermis and dermis. Morphological alterations become apparent in epidermis, including structural changes within the basal membrane and a decrease in cell proliferation [17]. Dermal collagen and elastic fibrils of aging skin are fragmented and disorganized, which impairs the structural integrity and mechanical properties of the skin [18]. In our study, the observation of histological structure indicated that the wrinkle skin of Xiang pig was abnormally thicken, which is similar to aging skins. The epidermal layer proliferated and its thickness was not uniform in skin of WXP. Epidermal hyperplasia hinders the excretory function of skin, which is prone to parasites infection and inflammation in sunken skin. At the same time, we observed that the fibrous structure of the dermis layer was disordered with fiber breakage and decrease of the fiber bundle. Furthermore, subcutaneous fat layer became thinner. Our observation suggested that the aging probably was the cause of the skin wrinkling in Xiang pig.

To uncover molecular mechanism of the skin wrinkle in Xiang pigs, we performed genome resequencing by NGS technology and identified 32,308 SVs from genomes of Xiang pigs and European pigs. Among the identified SVs, 965 SVs were screened from the genome of Xiang pig with wrinkle skin. After annotation, the 965 specific SVs covered 553 Ensembl genes, including protein-coding genes, pseudogenes, lncRNA and snRNA genes. The protein-coding genes were enriched in several KEGG pathways related to skin aging, including RNA transport, Notch signaling pathway, Wnt signaling pathway, mTOR signaling pathway. It indicated that these SV events might mediate the skin phenotypic difference between WXP and XP. RNA transport from the nucleus to the cytoplasm is fundamental for gene expression. In the RNA transport pathway, nuclear pore complexes (NPCs) and translation initiation factors (eIFs) were affected by SV. Particularly, missense mutations in some eIFs induce aging [19]. Notch signaling is directly involved in differentiation in the skin, and impacts on inflammatory processes in the skin [20]. The Wnt signaling pathway is significantly down-regulated in elderly skin [21]. The nuclear lamina was one of significantly enriched GO term. Mutations in the nuclear lamina genes lead to the loss of its function, resulted in a wide range of human degenerative and premature aging diseases, such as Hutchinson–Gilford progeria syndrome (HGPS), where the skin shows premature aging [22]. The wrinkled Xiang pigs appeared skin aging at 2 months old, which may be related to this SV in nuclear lamina. At the same time, changes in nuclear lamina also affect genomic stability, and the genomic instability is known to be one of hallmarks of aging [23]. Nuclear lamina is closely related to structure and function of nuclear membrane, chromatin and nuclear pore complex. In addition, many involved genes have been found to response to external biotic stimulus and immune system regulation. The skin of wrinkled Xiang pigs is rubefaction, which is manifested as low-grade inflammation. This rubefaction is reported to link the immune system: known as inflammaging or inflamm-ageing, seems to be the common biological factor responsible for the decline and the onset of disease in the elderly [24]. Aging cause disorders of multiple components of the immune system, such as increased sensitivity to infections [25]. Our results suggested that the skin wrinkling of Xiang pig was accompanied by changes in many aspects, such as blocked of some RNA transport and translation process, unstable of nuclear lamina, imbalance of the immune system.

In our study, we finally found that 65 SVs in 59 protein genes were probably concerned in the wrinkled skin of Xiang pig. We mapped these genes to NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/?term=aging) and found that 22 genes have been reported to be related to aging. Among them, 13 SVs in nine genes (CHD3, NOLC, PPP2R1A, EIF4G2, LMNA, TNXB, RAB1A, RBPJ, PABPC1) could cause the variations of the coding sequences and intron, which resulted in the loss of stop codon or frameshift mutation and aberrant alternative splicing. For example, the deletion of 239 bp among exon 38 and 3'UTR made the CHD3 loss of 40 coding codons and stop codon, which resulted in truncated polypeptide. CHD3 (chromodomain helicase DNA binding protein 3) is one of NURD components (a ubiquitous chromatin remodeling complex). NURD has been implicated in transcriptional repression at specific promoters and been shown to associate with pericentromeric heterochromatin [26]. Low expression of the protein or loss of the activity of several NURD components including HDAC1 and CHD3 in normal cells are sufficient to recapitulate age-dependent chromatin defects [27]. Our results suggested that the mutant gene might cause CHD3 dysfunctions and age-dependent chromatin defects in Xiang pig cells. An 89 bp deletion was found among intron 6 and exon 7 of PPP2R1A gene in WXP, which resulted in frameshift mutation and early termination. PPP2R1A (protein phosphatase 2 scaffold subunit A alpha) is a scaffolding subunit of PP2A (protein phosphatase 2A). PP2A regulates a variety of cellular functions, including DNA replication, transcription, translation, cell apoptosis and so on. Moreover, PP2A is predominantly regarded as a tumor suppressor. Mutations in PPP2R1A frequently occur in cancer, such as lung, breast, and melanoma [28]. PPP2R1A is associated with Alzheimer's disease (AD) and specific cognitive domains, and its mRNA and protein levels are elevated in the patient’s brain [29]. Therefore, PPP2R1A is a potential target related to the skin aging process of pig. EIF4G2 is one of the components of eukaryotic initiation factor 4F (EIF4F). The deletions variations among exons/intron 7, 10, 11, and 16 of EIF4G2 were potential abnormal replicating variables. We examined the impact of two SVs in exons/introns on EIF4G2 mRNA splicing (Figure 6). Sequencing showed that it did not violate the normal processing of EIF4G2 mRNA. However, the deletion of introns 10 and 11 may affect gene expression. More and more evidences show that the destruction of the translation mechanism greatly promotes the development and progression of cancer [30]. Down-regulation of EIF4G2 expression will decrease translation and cell proliferation and induce cellular senescence [31]. The LMNA gene encodes nuclear lamins, and gives rise to lamin A and lamin C through alternative splicing. Mutations in LMNA cause multiple degenerative disorders, such as HGPS, caused by a point mutation in the 5'-terminus site of exon 11 [32]. The product of this abnormal splicing transcript, defined as progerin, is mainly found in skin fibroblasts and undifferentiated keratinocytes [33]. Remarkably, we found the deletion of 93 bp in intron 10 and 11 exon of LMNA gene, and the first two bases of exon 11 were ablated, which resulted in frameshift mutations of the gene and a short peptide of 478 aa. LMNA gene mutations elicit genomic instability and limit cellular proliferative capacity [34]. In peoples carrying the mutation, a severe premature senility occurs during childhood [5]. In addition to being a nuclear localization signal binding protein, the nucleolus and coiled-body phosphoprotein 1 (NOLC1) can also be used as a partner to shuttle between the nucleolus and the cytoplasm. Overexpression of NOLC1 induced cell cycle arrest and apoptosis by disturbing the organization of nucleolus [35]. But when NOLC1 expression is down-regulated under the interference of shNOLC, the expression of MDM2 proto-oncogene is inhibited while the expression of the apoptosis-related genes (such as TNF-α) were up-regulated in nasopharyngeal carcinoma (NPC) cells [36]. Our study found a 63 bp insertion in exon 10 of NOLC1 gene of WXP. This resulted in the retention of intron 10 of NOLC1 gene and the addition of 11 amino acids to the encoded protein. And the expression of mutant genes was significantly higher than that of normal genes. When the expression of NOLC1 was up-regulated, it may promote the excessive proliferation of epidermal cells and form a thicker epidermis. Moreover, we found that the 153 bp duplication of intron 26 and exon 26 in TNXB gene caused frameshift mutation and truncated or aberrant splicing transcripts. Furthermore, the duplication of 153 bp in exon 26 of TNXB resulted in truncated peptide (2823 aa). TNXB encode the large extracellular matrix glycoprotein tenascin XB. Tenascin XB is reported to regulate collagen deposition by dermal fibroblasts [37]. In human, variants in TNXB cause a rare monogenic autosomal recessive subtype of Ehlers-Danlos syndromes (EDSs) with hyperextensible and fragile skin [38].

Furthermore, other 46 SVs among 44 genes of Xiang pig mainly caused intron variant. Introns are those sequences which are transcribed and subsequently excised from the primary transcript by splicing to produce the mature RNA. Pre-mRNA splicing depends on the recognition of splice acceptor / donor, exon-intron boundaries, and splicing regulatory sequences. The regulatory elements include donor / acceptor sites, exon and intron splicing enhancers (ESE and ISE) and exon and intron splicing silencers (ESS and ISS). Specific splicing activator and repressors (trans-acting elements) correctly recognize these elements and help to perform the splicing process appropriately. Lose or insertion of the sequence containing these elements may lead to incorrect recognition of exons and introns, and can cause the production of abnormal transcripts of mutated genes, which are the main reason of many diseases [39, 40]. And deregulation of pre-mRNA splicing is related to cellular senescence and the aging phenotype [41]. In our study, by using the 46 SV sequences from the introns, we applied for identification of splicing regulatory sequences by software RegRNA 2.0 online. We found that 42 SVs contained one or more regulatory elements, including ESE, ISE, ESS, MBE (Musashi binding element), and PAS (polyadenylation signal). The splice-site mutations of some genes in WXP may affect its senescence phenotype, such as XYLT1 and FUT8. A 262 bp deletion was found from intron 3 of XYLT1, which included ESE, SXL binding site, C-to-U RNA editing sites. Xylosyltransferase 1 (XYLT1) catalyzes the first step of adding glycosaminoglycan (GAG) chains to the proteoglycan core protein. Glycosylation modification is essential for normal proteoglycan (PG) function. Mutations in XYLT1 cause skeletal dysplasia and severe growth retardation [42]. In cultured fibroblasts, UV-caused downregulation of XYLTs results incomplete glycosylation and secretion of protein [43]. The 262 bp deletion fragment in XYLT1 gene contained 14 trans-acting factor binding sites, which bind to SRp40, SC35 and Sxl proteins (Supplementary Figure 3). Studies have shown that the destruction of ESE lead to exon skipping and expresses low levels of functional protein [44]. In WXP, the expression of homozygous deletion type of XYLT1 in the skin was significantly lower than that of normal genotype (P < 0.05). Loss of ESE in intron 3 might down-regulate the transcription of XYLT1. When the expression of XYLT1 is insufficient, intact form of PG protein and core protein form were reduced, which affects the structure and space filling of skin. And 858 bp deletion was found from intron 8 of FUT8, which contained acceptor, ESE, ESS, Polyadenylation sites, MBE, PAS, GU-rich destabilization elements (Supplementary Figure 3). The core protein requires glycosylation to have a function. For example, TGFβ1 lacking the core fucosylation causes a significant imbalance in TGFβ1 receptor activation and signal transduction, resulting in an increase in matrix metalloproteinases (MMPs) [45]. The main molecular mechanism of the skin aging process is attributed to the loss of mature collagen and increased expression of MMPs [46]. Studies have demonstrated that glycosaminoglycans are reduced in aging skin and core proteoglycans are damaged [47]. Point mutations at ESE or ESS can cause aberrant splicing such as exon skipping or intron retention, and produce abnormal transcripts [48]. Loss of ESE, ESS in intron 8 of FUT8 gene of WXP disrupted splicing regulatory sequences, created probably new ones, or activated the cryptic ones. Alternative polyadenylation sites will produce different C-terminal of protein isoforms, called the coding region-APA (CR-APA) [49]. It is reported that intron polyadenylation of the Pdgfra gene leads to increased expression of shorter transcription variants with truncated kinase domains, thereby reducing tissue fibrosis of muscles [50]. Lack of polyadenylation in intron 8 probably resulted in the loss of truncated transcripts of FUT8 gene. The Musashi binding element inhibits mRNA translation by binding to the Musashi protein family and competing with eIF4G [51]. When the binding site is missing, it will promote translation. The loss of splicing regulatory elements in intron 8 was estimated to be related to FUT8 gene expression. In fact, the expression of FUT8 in homozygous deletion mutant samples was much lower than that of normal type of Xiang pig. Insufficient expression of glycosyltransferase will cause glycosylation defects, affect the normal function of proteoglycan, and eventually lead to structural disorder of the extracellular matrix (ECM). Glycosylation defects was likely to a potential factor for skin aging of WXP.

Moreover, deletions were detected in the upstream of four genes (LMNB1, CCL5, RNF14, EIF6). After analysis of the deletion sequences, it was found that they all contained transcription factor binding sites. Transcription factors (TFs) are a family of DNA-binding proteins and recognized as the master regulators of gene expression. Evidence has shown that deletion of the conserved DNA binding site of transcription factor upstream of the gene, affects the transcription activity of the promoter and cause the decrease of gene expression [52]. Notably, the four genes are more or less related with skin aging [53–56]. Finally, the insertion of 88 bp among 3' UTR made the elongated 3' tail of MDM2 gene. Mdm2 is the key negative regulator of the tumour suppressor p53 and as a chromatin modifier [57]. It's reported that activation of endogenous p53 by ablation of Mdm2 can induce accelerated aging phenotypes in mice skin [58]. The 88 bp insertion mutation in the MDM2 gene might affect its localization and translation efficiency. Studies in human cell lines show that the lengths of 3' UTRs differentially regulate the localization of membrane proteins [59]. And the 3' UTR maintains the proper stability of mRNA and ensure normal mRNA nuclear export and translation efficiency [60]. In our work, the expression level of the insertion mutant transcript of MDM2 gene was higher than that of normal. Elevated MDM2 expression is exacerbated chromosome instability in aging mice [61]. This will be a new direction for our in-depth research on the skin wrinkle of Xiang pigs.

In summary, we reported a new skin phenotype in Xiang pigs, which is characterized by systemic wrinkles, thickened skin and wispy hair. We sequenced the genome of the phenotypic variant individuals of WXPs and comparison with data from XP and EUP, and identified 965 SVs specific in WXP, covering 514 protein-coding genes. According to gene function and enrichment analysis, we found that 65 candidate SVs in 59 protein genes were probably related with the systemic wrinkle of WXP. The insertion/deletion and duplication variations in these genes resulted in the loss of stop codon or frameshift mutations, and aberrant alternative splicing of transcripts. These genes are involved in cell lamin filament, intermediate filament cytoskeleton, supramolecular complex, cell differentiation and regulation of macromolecule metabolic process etc. Our study suggested that the loss of function or aberrant expression of these genes lead to structural disorder of nuclear and the extracellular matrix (ECM) in skin cells of Xiang pig, which probably was the genetic mechanisms for the mutant phenotype of Xiang pigs with systemic wrinkle.

Materials and Methods

Supplementary Materials

Supplementary Figures

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