SNPs in KIT and KITLG genes may be associated with oligospermia in Chinese population

Abstract

KIT/KITLG signaling system is crucial for spermatogenesis, which suggests that KIT and KITLG genes may be involved in spermatogenesis impairment and male infertility. To explore the possible association of KIT and KITLG genes with male infertility having spermatogenesis impairment, polymorphism distributions of SNP rs3819392 in KIT gene as well as rs995030 and rs4474514 in KITLG gene were investigated in 372 patients with idiopathic azoospermia or oligospermia and 205 fertile controls. As a result, the significant differences in polymorphism distributions of SNP rs3819392 in KIT gene and rs4474514 in KITLG gene were observed between the patients with oligospermia and controls. The frequencies of allele G (94.2% versus 90.0% p?=?0.022) and genotype GG (89.2% versus 82.0% p?=?0.042) in patients with oligospermia were significantly higher than those in controls at rs3819392 locus in KIT gene. In addition, the genotype CC of rs4474514 in KITLG (8.2% versus 3.4%, p?=?0.034) also significantly increased in oligospermic patients in comparison to controls. These findings indicated that SNP rs3819392 in KIT gene and rs4474514 in KITLG gene may be associated with oligospermia, suggesting that polymorphism of KIT and KITLG genes may play a role in oligospermia.     

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Seven novel KIT mutations in horses with white coat colour phenotypes

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.     

Molecular genetics of coat colour variations in White Galloway and White Park cattle

White Galloway cattle exhibit three different white coat colour phenotypes, that is, well marked, strongly marked and mismarked. However, mating of individuals with the preferred well or strongly marked phenotype also results in offspring with the undesired mismarked and/or even fully black coat colour. To elucidate the genetic background of the coat colour variations in White Galloway cattle, we analysed four coat colour relevant genes: mast/stem cell growth factor receptor (KIT), KIT ligand (KITLG), melanocortin 1 receptor (MC1R) and tyrosinase (TYR). Here, we show that the coat colour variations in White Galloway cattle and White Park cattle are caused by a KIT gene (chromosome 6) duplication and aberrant insertion on chromosome 29 (Cs₂₉) as recently described for colour‐sided Belgian Blue. Homozygous (Cs₂₉/Cs₂₉) White Galloway cattle and White Park cattle exhibit the mismarked phenotype, whereas heterozygous (Cs₂₉/wt₂₉) individuals are either well or strongly marked. In contrast, fully black individuals are characterised by the wild‐type chromosome 29. As known for other cattle breeds, mutations in the MC1R gene determine the red colouring. Our data suggest that the white coat colour variations in White Galloway cattle and White Park cattle are caused by a dose‐dependent effect based on the ploidy of aberrant insertions and inheritance of the KIT gene on chromosome 29.     

Allelic Mutations of KITLG,Encoding KIT Ligand,Cause Asymmetric and Unilateral Hearing Loss and Waardenburg Syndrome Type 2

Linkage analysis combined with whole-exome sequencing in a large family with congenital and stable non-syndromic unilateral and asymmetric hearing loss (NS-UHL/AHL) revealed a heterozygous truncating mutation, c.286_303delinsT (p.Ser96Ter), in KITLG. This mutation co-segregated with NS-UHL/AHL as a dominant trait with reduced penetrance. By screening a panel of probands with NS-UHL/AHL, we found an additional mutation, c.200_202del (p.His67_Cys68delinsArg). In vitro studies revealed that the p.His67_Cys68delinsArg transmembrane isoform of KITLG is not detectable at the cell membrane, supporting pathogenicity. KITLG encodes a ligand for the KIT receptor. Also, KITLG-KIT signaling and MITF are suggested to mutually interact in melanocyte development. Because mutations in MITF are causative of Waardenburg syndrome type 2 (WS2), we screened KITLG in suspected WS2-affected probands. A heterozygous missense mutation, c.310C>G (p.Leu104Val), that segregated with WS2 was identified in a small family. In vitro studies revealed that the p.Leu104Val transmembrane isoform of KITLG is located at the cell membrane, as is wild-type KITLG. However, in culture media of transfected cells, the p.Leu104Val soluble isoform of KITLG was reduced, and no soluble p.His67_Cys68delinsArg and p.Ser96Ter KITLG could be detected. These data suggest that mutations in KITLG associated with NS-UHL/AHL have a loss-of-function effect. We speculate that the mechanism of the mutation underlying WS2 and leading to membrane incorporation and reduced secretion of KITLG occurs via a dominant-negative or gain-of-function effect. Our study unveils different phenotypes associated with KITLG, previously associated with pigmentation abnormalities, and will thereby improve the genetic counseling given to individuals with KITLG variants.     

Molecular characterization, expression and mapping of porcine LMP2 and MECL-1 genes.

Low molecular mass polypeptides 2 (LMP2) and Multicatalytic endopeptidase complex subunit (MECL-1) are two of three catalytic beta-type subunits of the 20s proteasome and upon interferon gamma-induction. LMP2 is critical for the production of major histocompatibility complex (MHC) class I ligand and T-lymphocytes. The LMP2 gene is located in the MHC region, but MECL-1 is located outside the MHC region. They are involved in the antigen presentation and are important candidate genes for an initial exploration of relationships between the antigen processing genes and disease resistance. In this report, the porcine LMP2 and MECL-1 cDNA were cloned and A 5099 bp LMP2 genomic DNA structure was identified, then two single nucleotide polymorphisms were detected in the exon2 and exon5 of LMP2 gene in 367 individuals. The LMP2 and MECL-1 genes putative protein included 219,274 amino acids, respectively. Alignment and phylogenetic of predicted porcine LMP2 and MECL-1 amino acid sequence with their homologies were analyzed. Tissues expression of LMP2 and MECL-1 mRNA were observed by real time quantitative PCR (Q-PCR) method, the results revealed MECL-1 expressed widely in all tissues, but LMP2 was not detected in muscle. The porcine MECL-1 gene was mapped to chromosome 6, closely linked to microsatellite SW1108 (LOD = 4.09, 84cR) by radiation hybrid panel.     

KIT Proto-oncogene Exon 8 Deletions at Codon 419 are Highly Frequent in Acute Myeloid Leukaemia with Inv(16) in Indian Population

The KIT gene is a receptor tyrosine kinase class III expressed by early hematopoietic progenitor cells and plays a significant role in hematopoietic stem cell proliferation, differentiation and survival which is considered to be a remarkable feature in the course of growth of acute myeloid leukaemia (AML). Owing to insufficient study of mutations in the KIT gene, the diagnosis and rate of recurrence of these mutations with divergent subtypes in AML cases in India is of concern. In order to find out the frequency of mutations of KIT gene exon 8 in 109 AML cases, we have performed polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) followed by DNA sequencing and have identified 24 mutations in exon 8 in 13 cases, including deletions at codon 418 (n = 3), 419 (n = 11) and 420 (n = 5) as well as point mutations at codon 417 (n = 1) and 421 (n = 4). In eleven AML cases, exon 8 deletion and point mutations involved the loss at codon Asp419 immoderately conserved cross species placed in the receptor extracellular domain. Frequency elevation of the KIT proto-oncogene exon 8 deletion and point mutations in AML cases allude a crucial function for this region of the receptor extracellular domain. Thus, we report the incidence of acquired mutations in exon 8, with consistent loss at codon Asp419, in 10.09 % of AML cases in a selected Indian population.     

cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans

Dramatic pigmentation changes have evolved within most vertebrate groups, including fish and humans. Here we use genetic crosses in sticklebacks to investigate the parallel origin of pigmentation changes in natural populations. High-resolution mapping and expression experiments show that light gills and light ventrums map to a divergent regulatory allele of the Kit ligand (Kitlg) gene. The divergent allele reduces expression in gill and skin tissue and is shared by multiple derived freshwater populations with reduced pigmentation. In humans, Europeans and East Asians also share derived alleles at the KITLG locus. Strong signatures of selection map to regulatory regions surrounding the gene, and admixture mapping shows that the KITLG genomic region has a significant effect on human skin color. These experiments suggest that regulatory changes in Kitlg contribute to natural variation in vertebrate pigmentation, and that similar genetic mechanisms may underlie rapid evolutionary change in fish and humans.     

Le cancer du testicule : facteurs de risque génétiques et environnementaux

The incidence of testicular cancer (TC) has a distinct geographic distribution but is increasing in most countries, including France. The most likely origin of TC is a lack of normal germ cell differentiation in the foetal testis. The maintained immature germ cells could proliferate after puberty to induce testicular tumor. Because of its frequent association with cryptorchidism and infertility, TC could be part of the testicular dysgenesis syndrome. The role of genetic factors is suggested by the frequency of familial TC cases, but no responsible gene has been clearly identified until now. Among the various studied genes, those regulating the KITLG/KIT pathway involved in primordial germ cell proliferation seem to play an important role. Studies made in immigrants and twins suggest the influence of environmental factors on the origin of TC. Hormonal deregulation occurring during prenatal life or puberty could facilitate the development of TC. However, the role of exogenous substances acting as endocrine disruptors has not been demonstrated yet.     

Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR–RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.     

Linked markers exclude KIT as the gene responsible for appaloosa coat colour spotting patterns in horses

The appaloosa coat colour pattern of the horse is similar to that caused by the rump-white (Rw) gene in the mouse. In the mouse Rw colour pattern is the result of an inversion involving the proto-oncogene c-kit (KIT). Therefore, we investigated KIT as a candidate gene that encodes the appaloosa coat colour gene (Lp) in horses. KIT plays a critical role in haematopoiesis, gametogenesis, and melanogenesis and encodes a transmembrane tyrosine kinase receptor that belongs to the PDGF/CSF-1/c-KIT receptor subfamily. Half-sib families segregating for Lp were uninformative for a reported polymorphism in KIT. However, KIT is located on horse chromosome 3 close to albumin (ALB), serum carboxylesterase (ES), vitamin D-binding protein (GC) and microsatellite markers ASB23, LEX007, LEX57, and UCDEQ437. Indeed, KIT and ASB23 were localized to ECA3q21-22.1 and 3q22.1-22.3, respectively, by fluorescent in situ hybridization. Family studies were conducted to investigate linkage of Lp to these markers using eight half-sib families in which Appaloosa stallions were mated to solid coloured mares. Linkage of Lp to the chromosome region containing ES, ALB, GC, ASB23, UCDEQ437, LEX57, and LEX007 was investigated by a multipoint linkage analysis using the computer program GENEHUNTER. LOD scores over the interval under investigation ranged from -4.28 to -12.48, with a score of -12.48 at the location for ASB23. Therefore, it was concluded that appaloosa (Lp) is not linked to any of the tested markers on ECA3, and thus Lp is unlikely to be the product of KIT.     

FES kinase participates in KIT-ligand induced chemotaxis

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.     

Gastrointestinal stromal tumors: overview of pathologic features, molecular biology, and therapy with imatinib mesylate

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. These tumors develop at any site but are most commonly reported in the stomach. They originate from the neoplastic transformation of the intestinal pacemaker cell, the interstitial cell of Cajal. GISTs strongly express the receptor tyrosine kinase KIT and have mutations in the KIT gene, most frequently in exon 11 encoding the intracellular juxtamembranous region. Expression of KIT is seen in almost all GISTs, regardless of the site of origin, histologic appearance, or biologic behavior, and is therefore regarded as one of the key diagnostic markers. Distinction from smooth muscle tumors, such as leiomyosarcomas, and other mesenchymal tumors is very important because of prognostic differences and therapeutic strategies. Predicting the biologic behavior of GISTs is often difficult by conventional pathologic examination; tumor size and mitotic rate are the most important prognostic indicators. The prognostic significance of KIT mutations is controversial and thus far has not been clearly linked with biologic behavior. KIT mutations are associated with tumor development, and cytogenetic aberrations are associated with tumor progression. The pathogenesis of GISTs involves a gain-of-function mutation in the KIT proto-oncogene, leading to ligand-independent constitutive activation of the KIT receptor. KIT-wild-type GISTs have shown mutually exclusive platelet-derived growth factor receptor (PDGFR) mutation and activation. The use of imatinib mesylate (also known as Gleevec or STI-571) has greatly increased the therapeutic efficacy for this otherwise chemotherapy-resistant tumor. GISTs with very low levels of KIT expression may respond to imatinib mesylate therapy if the receptors are activated by specific mechanisms. KIT-activating mutations fall into two groups: the regulatory type and the enzymatic site type. The regulatory type of mutation is conserved at the imatinib binding site, whereas the enzymatic site mutation has a structurally changed drug-binding site, resulting in drug resistance. Resistance to the drug is the major cause of treatment failure in cancer therapy, emphasizing the need for researchers to understand KIT signaling pathways so as to identify new therapeutic targets. This review summarizes the pathologic features of GISTs, recent advances in understanding their molecular and biologic features, and therapy with imatinib mesylate.