GDF9 Research Articles

A Review on the Genetic Basis of Growth and Prolificacy Traits in Sheep

The performance of an animal for a particular trait is the result of its genetic merit and the effects of the environment where it exists. To set up genetic improvement in sheep, the genetic component attributed to the trait of interest need to be defined. The aim of this review was to describe major candidate genes influencing growth traits and prolificacy in sheep. Although growth and prolificacy are quantitative traits and are expected to be influenced by many genes with individual genes contributing small effects, there are major genes that have been identified with significant influence on growth and prolificacy. The CLPG, GDF8 and CAST genes are some of the major genes that have strong influence on sheep growth and carcass quality. The CLPG mutation can cause pronounced effect in the muscle found in the hindquarter and is responsible for the muscular hypertrophy phenotype in sheep. The GDF8 gene also play important role in increasing muscle depth due to mutation in the regulatory region and coding sequences. The CAST gene is an endogenous and specific inhibitor of calpain enzyme and thereby regulates the rate and extent of muscle tenderization following slaughtering. For prolificacy, BMP15, GDF9 and BMPR1B have been shown to exert significant influence on ovulation rate and litter size in various sheep breeds in the world. Both of the three genes are member of the TGF-beta family protein that encode protein product responsible for growth, differentiation and proliferation of ovarian follicles. The mechanism of action for such major genes are associated with the existence of mutation in the coding sequence resulting amino acid change as well as in the regulatory region that vary the expression level and inheritance of the genes. Up to now, better attempts have been made to describe the genetic basis of growth and prolificacy in sheep. However, more works are needed to characterize other genes influencing these traits. More importantly, making use of the identified genes in sheep breeding program through marker assisted and genomic selection should receive due attentions.

Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFβ receptor ActRIIb, the TGFβ signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

PO-007 Association of six gene polymorphism with tendon injuries in Chinese athletes

Objective In recent years, more and more studies have shown that gene polymorphism is associated with susceptibility and recovery of sports injury. We select Collagen type I alpha 1 gene(COL1A1 ),Collagen type V alpha 1 gene (COL5A1),Collagen type XII alpha 1 gene (COL12A1),1ollagen type XIV alpha 1 gene(COL14A1),Tenascin C gene(TNC), Growth/differentiation factor-5 gene(GDF-5) polymorphic loci to study their relationship with tendon injuries in Chinese athletes.
 Methods A case-control experiment was designed to analyze the distribution characteristics of six gene polymorphism loci in 65 chinese athlete injured group and 115 control group. These six polymorphic loci were detected by PCR-RFLP.
 Results The distributions of COL1A1 TT genotype , COL5A1 CC genotype and GDF-5 CC genotype were decreased in injured group compared with the control group. The COL12A1,COL14A1, TNC gene polymorphic loci showed no significant difference between two groups. The COL1A1, COL5A1 and GDF-5 genes were involved in encoding for collagen, matrix metallopeptidase, tenascin and growth factors which protect the athletic from the musculoskeletal injuries, particularly in tendon and ligament tissues.
 Conclusions The genetic loci will help to identify individuals with advantageous physical performance and a lower chance of suffering from injuries.

Expression Analysis of the Prolific Candidate Genes, BMPR1B, BMP15, and GDF9 in Small Tail Han Ewes with Three Fecundity (FecB Gene) Genotypes.

Simple SummaryAs important prolific candidate genes, BMPR1B, BMP15, and GDF9 may affect the lambing performance of sheep. Therefore, regarding the three FecB genotypes of Small Tail Han (STH) sheep (FecB BB, FecB B+, and FecB ++), this study explored the gene expression characteristics of different tissues using reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR). The results showed that BMPR1B, BMP15, and GDF9 expression differed between the selected tissues, with all being highly expressed in the ovaries. Further analysis indicated that there was no significant difference in BMPR1B expression among the three FecB genotypes, but both GDF9 and BMP15 had the highest expression in FecB B+. As for other non-ovarian tissues, expression also varied. This study is relevant to understanding the high prolificacy of the STH breed.The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.

Key elements involved in Epstein\u2013Barr virus-associated gastric cancer and their network regulation

BackgroundThe molecular mechanism of Epstein–Barr virus (EBV)-associated gastric cancer (EBVaGC) remains elusive. A collection of molecular regulators including transcription factor and noncoding RNA (ncRNAs) may affect the carcinogenesis of EBVaGC by regulating the expression and function of key genes. In this study, integration of multi-level expression data and bioinformatics approach was used to identify key elements and their interactions involved in mechanism of EBVaGC and their network regulation.MethodsData of the gene expression profiling data sets (GSE51575) was downloaded from GEO database. Differentially expressed genes between EBVaGC and normal samples were identified by GEO2R. Gene ontology and pathway enrichment analyses were performed using R packages Cluster profiler. STRING database was used to find interacting proteins between different genes. Transcription factors in differentially expressed genes were obtained from TF Checkpoint database. Using Cytoscape, we built transcription factor regulation network. miRNAs involved in the gene-interacting proteins and the miRNA-targeted lncRNA were predicted through miRWalk. Using ViRBase, EBV related miRNA regulation network was built. Overlapping genes and regulators of the above three networks were further identified, and the cross network was constructed using Cytoscape software. Moreover, the differential expressions of the target genes and transcription factors in the cross network were explored in different molecular subtypes of GC using cBioPortal. By histological verification, the expression of two main target genes in the cross network were further analyzed.ResultsA total of 104 genes showed differential expressions between EBVaGC and normal tissues, which were associated with digestion, G-protein coupled receptor binding, gastric acid secretion, etc. Pathway analysis showed that the differentially expressed genes were mainly enriched in gastric acid secretion and protein digestion and absorption. Using STRING dataset, a total of 54 proteins interacted with each other. Based on the transcription factor network, the hub transcription factors IRX3, NKX6-2, PTGER3 and SMAD5 were identified to regulate their target genes SST and GDF5, etc. After screening and matching in miRwalk datasets, a ceRNA network was established, in which the top five miRNAs were hsa-miR-4446-3p, hsa-miR-5787, hsa-miR-1915-3p, hsa-miR-335-3p and hsa-miR-6877-3p, and the top two lncRNAs were RP5-1039K5.19 and TP73-AS1. According to the EBV related miRNA regulation network, CXCL10 and SMAD5 were found to be regulated by EBV-miR-BART1-3p and EBV-mir-BART22, respectively. By overlapping the three networks, CXCL10, GDF5, PTGER3, SMAD5, miR-6877-3p, RP5-1039K5.19, TP73-AS1, EBV-miR-BART1-3p and EBV-mir-BART22 were found to be key elements of regulation mechanism of EBVaGC. CXCL10, GDF5, PTGER3 and SMAD5 were also differentially expressed among the four molecular subtypes of GC. The histological verification experiment showed differential expressions of the two main target genes GDF5 and CXCL10 between EBVaGC and non-tumor tissues as well as EBVnGC.ConclusionIn the current study, our results revealed key elements and their interactions involved in EBVaGC. Some hub transcription factors, miRNAs, lncRNAs and EBV related miRNAs were observed to regulate their target genes. Overlapping genes and regulators were observed in diverse regulation networks, such as CXCL10, GDF5, PTGER3, SMAD5, miR-6877-3p, RP5-1039K5.19, TP73-AS1, EBV-miR-BART1-3p and EBV-mir-BART22. Moreover, CXCL10, GDF5, PTGER3 and SMAD5 were also differentially expressed among the four molecular subtypes of GC. The histological verification experiment showed differential expressions of the two main target genes GDF5 and CXCL10 between EBVaGC and non-tumor tissues as well as EBVnGC. Therefore, the identified key elements and their network regulation may be specifically involved in EBVaGC mechanisms.

Expresión génica del factor de crecimiento BMP15, GDF9, FGF2 y sus receptores en células foliculares bovinas

Introducción. El crecimiento y la maduración folicular implican una serie de transformaciones de diversos componentes del folículo, como el ovocito, las células de la granulosa y de la teca. Varios factores de crecimiento, incluyendo factor de crecimiento de diferenciación 9 (GDF9), proteína morfogénica ósea 15 (BMP15) y factor de crecimiento de fibroblastos básico (FGF2) son importantes para el desarrollo folicular y maduración de ovocitos, por su capacidad de aumentar la proliferación de las células de la granulosa, teca y el estroma ovárico. Objetivo. Evaluar la expresión de los factores de crecimiento GDF9, BMP15, FGF2 y sus principales receptores: el receptor 1 del factor de crecimiento transformante beta (TGFβ-R1), el receptor de la proteína morfogénica del hueso tipo IB (BMPR-IB) y el receptor del factor de crecimiento fibroblástico 2 (FGFR2) en células foliculares bovinas. Materiales y métodos. Se realizó la extracción de ARN total de pooles de ovocitos (OOs) y células del cumulus (CCs) de complejos cumulus-ovocito (COCs) y del pellet de células foliculares (PCs) provenientes de 70 ovarios obtenidos de 96 vaquillonas para carne, colectados en un frigorífico local. Los patrones de expresión de los factores de crecimiento y sus receptores en las células foliculares bovinas fueron evaluados por retro-transcripción seguida de la reacción en cadena de la polimerasa (RT-PCR). Resultados. La presencia de los transcriptos de ARNm de los genes GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB y FGFR2 fue detectada por RT-PCR en todas las células estudiadas. Es la primera vez que se reporta en ovocitos bovinos la expresión de los receptores TGFβ-R1 y BMPR-IB. Conclusiones. La presencia de transcriptos de factores de crecimiento y sus receptores en las células estudiadas, indica que estos factores podrían actuar como reguladores paracrinos y autocrinos de la foliculogénesis.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Detection of single nucleotide polymorphisms at major prolificacy genes in the Mehraban sheep and association with litter size

Abstract The present study aimed to investigate the presence of polymorphisms at four known genes controlling ovine prolificacy i.e. BMP15, GDF9, BMPR1B and B4GALNT2 in a sample of 115 Iranian Mehraban ewes and their association with litter size (LS) and lambs’ birth weight (BW) traits. Using Sanger sequencing of exons and polymorphism specific genotyping, ten SNPs (Single Nucleotide Polymorphisms) were observed in only two genes, GDF9 and BMPR1B. Seven SNPs were found in the GDF9 gene on the chromosome 5. Among them, six were already described in the coding sequence, and a new one (g.41840985C&gt;T) was found in the 3’UTR. In the BMPR1B gene on the chromosome 6, three novel SNPs were detected in the exon 7 (g.29382184G&gt;A; g.29382337G&gt;A and g.29382340G&gt;A). Allelic frequencies were established for six SNPs among the ten identified and they were in Hardy-Weinberg equilibrium. A significant association was found between the novel SNPs found in the exon 7 of BMPR1B and LS. Present results indicate the potential role of the BMPR1B locus in controlling prolificacy of Mehraban sheep and provide genetic markers for further exploitation in selection to improve reproductive efficiency.

"Evaluation of four genes associated with primary ovarian insufficiency in a cohort of Mexican women".

Primary ovarian insufficiency (POI) is a clinical condition observed in women younger than 40years of age, characterized by amenorrhea, hypoestrogenism, high levels of follicle-stimulating hormone (FSH), and infertility. Mutations in some master regulators of the development, maturation, and maintenance of ovarian follicles such as BMP15, FSHR, FOXL2, and GDF9 have been suggested as etiological factors in the development of POI. The aim of this study, the first in the Mexican population, is to evaluate the presence of mutations or polymorphisms in these four candidate genes. In a sample of 20 Mexican patients with idiopathic POI, we looked for and analyzed genetic variants in BMP15, FSHR, FOXL2, and GDF9 genes. We observed two polymorphisms: a coding change, c.919G>A (p.Ala307Thr), in the FSHR gene and a synonymous variant, c.447C>T (p.Thr149Thr), in the GDF9 gene. These two variants have been reported previously as polymorphisms (rs6165 and rs254286, respectively). We observed no significant difference associated with POI in the patients when compared with a healthy control group (p > 0.05). Also, no exonic variants were found for the genes BMP15 and FOXL2 in the individuals tested. The lack of association of the evaluated genes in this sample of Mexican women is consistent with the complex genetic etiology of POI that is observed across cohorts studied thus far.

Genetic characterization of three fertility genes in Egyptian sheep and goat breeds

One of the effective approaches for genetic improvement of productivity traits in farm animals is markerassisted selection (MAS) depending on the genetic markers that are associated with superior productivity traits. The improvement of fertility trait is one of the main targets in small ruminant breeding programs. This work aimed to identify RFLPs and SNPs variations among three fertility genes in Egyptian sheep and goat breeds. RFLP analysis of the amplified fragments at 462-bp from exon 1 of GDF9 using HpaII endonuclease showed the presence of two genotypes GG and AG. Depending on the presence of the restriction site of TaqI endonuclease (T^CGA) in the 348-bp amplified fragment from exon 5 of GPR54 gene, the results showed the presence of two alleles, C and T with three genotypes, viz. CC, TT and CT. The PCR amplified fragments of 190-bp from FecB gene were digested with AvaII restriction enzyme and the results showed that all tested animals had the same homozygous non-carrier genotype (++). It was concluded that the identification of genetic structure and nucleotide sequences of GDF9, GPR54 and FecB genes is considered the first step towards the genetic improvements of fertility trait in Egyptian small ruminants where these genes are associated with different fertility traits parameter like ovulation rate, ovarian follicular development, puberty and litter size in small ruminant breeds.

Association of the polymorphisms FecXR, FecGH, and FecGI and non-genetic factors that affect the prolificacy of Colombian creole sheep

Objective: To determine the effect of the FecXR, FecGH and FecGI genetic polymorphisms and other non-genetic effects on the natural prolificacy of Colombian creole hair sheep. Materials: The birth number of the mother, the date of service (season and year), the male used and the prolificacy of 50 OPC females were recorded. The FecXR, FecGI and FecGH loci were genotyped with direct PCR and PCR-RFLP. The allelic and genotypic frequencies, the observed (Ho) and expected heterozygosity (He), the fixation index and the deviations from the Hardy-Weinberg equilibrium (EHW) were calculated. The polymorphisms and non-genetic factors were associated with prolificacy using the GLM procedure. Findings: The FecXR and FecGH loci were monomorphic without the mutated allele, which is why the association analysis with prolificacy was not carried out. The FecGI locus of the GDF9 gene was polymorphic with frequencies of G = 0.89 and A = 0.11. The homozygous AA genotype was not found, and the GG and GA genotypes had frequencies of 0.78 and 0.22, respectively. The Ho was greater than the He; this excess of heterozygotes was not significant and neither were the deviations of the EHW (p>0.05). The average prolificacy was 1.30 ± 0.30. The genotype in the FecGI locus did not significantly affect the prolificacy (p = 0.087) but did so more so in the heterozygotes. The non-genetic effects analyzed, such as the number of the mother’s birth, the season and year of conception, and the father, did not affect the natural prolificacy of the OPC (p> 0.05). Application: The FecGI and FecGH loci were monomorphic, and FecGI locus was polymorphic; however, the genetic variation and non-genetic factors were not associated with the prolificacy. Keywords: Bone Morphogenetic Protein-15 Gene, Growth Differentiation Factor-9 Gene, Reproduction

Polymorphism of GDF9 and BMPR1B genes and their association with litter size in Markhoz goats.

The main objective of this study was to investigate the polymorphism of GDF9 and BMPR1B genes and their relationship with litter size in Markhoz goats. The polymorphism of GDF9 and BMPR1B genes as well-documented genes regarding fecundity in sheep and goat was investigated using RFLP-PCR and a tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR) in Markhoz goats. The 164 blood samples were collected from the raised goats in Sanandaj Markhoz goat Performance Testing Station. The DNA extraction was carried out by salting-out procedure, and then, PCR was performed using four and two pairs of primers to detect polymorphism in GDF9 and BMPR1B genes, respectively. To disclose GDF9 loci polymorphism, PCR products were digested with SspI (G3288A), PvuII (G423A), MvaI (A959C) and MspI (G1189A) restriction enzymes. The results showed that these mutations are available in tested animals. Parity had no significant effect on litter size. Also, the effects of different genotypes of GDF9 and BMPR1B had no significant effect on litter size. Further studies with a high number of animals with minimum relatedness for testing the association of these SNPs and others in the fecundity genes with reproductive traits may be worthwhile.

Can Genetics Predict Sports Injury? The Association of the Genes GDF5, AMPD1, COL5A1 and IGF2 on Soccer Player Injury Occurrence.

Genetics plays an integral role in athletic performance and is increasingly becoming recognised as an important risk factor for injury. Ankle and knee injuries are the most common injuries sustained by soccer players. Often these injuries result in players missing training and matches, which can incur significant costs to clubs. This study aimed to identify genotypes associated with ankle and knee injuries in soccer players and how these impacted the number of matches played. 289 soccer players, including 46 professional, 98 semi-professional and 145 amateur players, were genetically tested. Ankle and knee injuries and the number of matches played were recorded during the 2014/15 season. Four genes were assessed in relation to injury. Genotypes found to be associated with injury included the TT (nucleobase) genotype of the GDF5 gene, TT and CT (nucleobase) genotypes of AMPD1 gene, TT genotype of COL5A1 and GG (nucleobase) genotype of IGF2 gene. These genes were also associated with a decrease in the number of matches played.

Anti-Müllerian hormone and antral follicle count are more effective for selecting ewes with good potential for in vivo embryo production than the presence of FecGE mutation or eCG pre-selection tests

This study aims to compare four different methods for selecting high responding sheep donors for in vivo embryo production. These methods include a pre-selection eCG test (eCG), antral follicle count (AFC), plasma anti-Müllerian hormone measurement (AMH) and genotyping for the presence of the FecGE mutation (a polymorphism in the GDF9 gene associated with increased ovulation rate). Santa Ines ewe lambs (n = 25) underwent superovulation (SOV) with 800 IU equine chorionic gonadotropin (eCG) and the corpus luteum (CL) count was recorded by laparoscopy after eight days. At the D0eCG, blood samples for AMH and genotyping analysis were collected. Twenty-one days after the end of the eCG test, the same animals underwent SOV with 200 mg of FSH, administered in six decreasing doses, and then naturally mated. Immediately before the beginning of the FSH protocol (D0FSH), and at the moment of the first FSH dose (D9FSH), the AFC was assessed. Plasma AMH was again determined at the D9FSH. After each screening process, animals were classified as having a high (HR), or low (LR), potential of response (using specific thresholds for each method). Then, the ewes' response to SOV and embryo yield for each screening method, classified as HR or LR, were compared. Animals classified as HR by AFC (HRAFC) and by AMH concentration (HRAMH) at the D9FSH, produced more viable embryos than those classified as LRAFC and LRAMH (HRAFC 6.2 ± 3.2 vs LRAFC 2.8 ± 3.0 and HRAMH 6.6 ± 3.6 vs LRAMH 3.0 ± 2.9). Pre-selection tests with eCG and different FecGE genotypes, either heterozygous (+/E) or wild type (+/+), were unable to discriminate HR or LR animals. A tendency (P = 0.06) to have lower plasma AMH was observed in heterozygous FecGE (+/E) ewes. In conclusion, both AFC and plasma AMH can be used to select donor ewes with a higher potential of response for in vivo embryo production.

Brachdactyly Instigated as a Result of Mutation in GDF5 and NOG Genes in Pakistani Population.

Objectives:Brachdactyly a genetic disorder associated with the abnormal development of metacarpals, phalanges or both which results in the shortening of hands and feet. Mutations in the contributing genes has been recognized with the majority of the investigated syndromic form of brachdactyly. The current study was proposed to examine mutation in NOG and GDF5 genes in a Pakistani family.Methods:Poly Acrylamide Gel Electrophoresis and Polymerase Chain Reaction was used for the genomic screening and linkage analysis to observe the mutation in genes. The samples were collected from Luckki Marwat district, KPK, while the research study was conducted in the department of Biochemistry, Quaid-I-Azam University, Islamabad, Pakistan.Results:After survey, family was identified with brachdactyly type A2 and investigated a heterozygous arginine to glutamine exchange in the growth demarcation factor 5 in all the victim persons. Different types of skeletal dysplasia resulted due to mutation in the GDF5 genes. Novel GDF5 genes mutations were reported with distinct limb malformation and sequencing of coding region revealed that the mildly affected individuals were heterozygous while the harshly affected individuals were homozygous.Conclusion:The current study reported the genetic variability and concluded that the Brachdacytyly type A2 and type B2 resulted due to mutation in GDF5 and NOG genes respectively. A new subtype of brachydactyly (BDB2) was instigated as a result of novel mutations in NOG. The mutation has been reported for the first time in Pakistani population and especially in Pushtoon ethnic population.

Identification of the first homozygous 1‐bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing

Targeted massively parallel sequencing (TMPS) has been used in genetic diagnosis for Mendelian disorders. In the past few years, the TMPS has identified new and already described genes associated with primary ovarian insufficiency (POI) phenotype. Here, we performed a targeted gene sequencing to find a genetic diagnosis in idiopathic cases of Brazilian POI cohort. A custom SureSelectXT DNA target enrichment panel was designed and the sequencing was performed on Illumina NextSeq sequencer. We identified 1 homozygous 1-bp deletion variant (c.783delC) in the GDF9 gene in 1 patient with POI. The variant was confirmed and segregated using Sanger sequencing. The c.783delC GDF9 variant changed an amino acid creating a premature termination codon (p.Ser262Hisfs*2). This variant was not present in all public databases (ExAC/gnomAD, NHLBI/EVS and 1000Genomes). Moreover, it was absent in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The patient's mother and her unaffected sister carried the c.783delC variant in a heterozygous state, as expected for an autosomal recessive inheritance. Here, the TMPS identified the first homozygous 1-bp deletion variant in GDF9. This finding reveals a novel inheritance pattern of pathogenic variant in GDF9 associated with POI, thus improving the genetic diagnosis of this disorder.

GDF9 gene polymorphism and its association with litter size in two Russian sheep breeds

The purpose of this work was to study the GDF9 gene polymorphism in two sheep breeds raised in Russia and to identify its association with litter size. A variety of allelic variants of the GDF9 gene (GDF9/G1 and GDF9/G4 sites) in Salsk and Volgograd sheep breeds was studied. Mutations of the GDF9 gene that lead to improvement of reproductive traits in these sheep breeds were revealed. The obtained results of the alleles and genotypes frequencies for the GDF9 gene showed a low level of polymorphism at G1 and G4 sites. The studied populations were found to have high frequencies of G allele and GG genotype at G1 site and of A allele and AA genotype at G4 site of the GDF9 gene. Herewith all the individuals heterozygous at G1 site were also heterozygous at G4 site. Homozygous AA (G1) and GG (G4) genotypes in the study population were not observed. In the Salsk sheep population the individuals with AG genotype at G1 site had the highest fertility. A similar pattern was observed at G4 site. The litter size of Salsk sheep with GG genotype (G1) was 1.13. Among the Volgograd sheep, the individuals with AG genotype were also characterized by the highest fertility; the litter size of the individuals with the GG genotype (G1) was 1.22. A similar pattern was observed at G4 site. The litter size of animals with AG genotype was 1.88 and the AA genotype − 1.22. Thus, the positive and significant relation between the heterozygous AG/GDF9 genotype and litter size of animals has been established.

The genetic polymorphisms of TGFβ superfamily genes are associated with litter size in a Chinese indigenous sheep breed (Hu sheep)

Litter size, which is a complex economic trait in sheep, is affected by polygenes. Hu sheep are a prolific native breed in China and also an ideal resource for studying the genetic mechanism that controls litter size in sheep. In this study, we investigated the genetic effects of candidate genes on litter size in a large experimental population (n = 2021) of Hu sheep. A total of 23 single nucleotide polymorphisms (SNPs) including six reported major mutations (FecB, FecXI, FecXB, FecXH, FecGI, and FecGH) and 17 novel SNPs in 10 candidate genes involved in reproduction were genotyped using KASPar technology. Genotyping showed that Hu sheep carry the FecB mutation, but not the FecXI, FecXB, FecXH, FecGH, and FecGI mutations. Among the remaining 18 SNPs, 16 tagged SNPs were selected based on the HAPLOVIEW program. Analysis of single marker association indicated a significant association between litter size in Hu sheep and three mutations in the TGFβ superfamily (FecB, GDF9 and TGFBR2 genes). Quantitative trait modes (QTMs) analysis revealed that the FecB and GDF9 mutations exerted an additive effect, while the mutation located within TGFBR2 gene was dominant. Linear regression analysis of the association between multiple markers and litter size indicated a correlation between homozygous ewes with the GG/AA/TT genotype and larger litter size than any of the other genotypes. In conclusion, the FecB, GDF9, and TGFBR2 polymorphisms were implicated as genetic markers of potential importance in marker-assisted selection (MAS) strategies to improve litter size in Hu sheep.

Detection of Polymorphism in Exon 2 of GDF9 gene in Pure and Crossbred of Pakistani Sheep

Detection of Polymorphism in Exon 2 of GDF9 gene in Pure and Crossbred of Pakistani Sheep

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.