BMPR1B Research Articles

PCTAIRE Protein Kinase 1 (PCTK1) Suppresses Proliferation, Stemness, and Chemoresistance in Colorectal Cancer through the BMPR1B-Smad1/5/8 Signaling Pathway.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality worldwide. Even with advances in therapy, CRC mortality remains high. Therefore, there is an urgent need to develop effective therapeutics for CRC. PCTAIRE protein kinase 1 (PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family, and the function of PCTK1 in CRC is poorly understood. In this study, we found that patients with elevated PCTK1 levels had a better overall survival rate in CRC based on the TCGA dataset. Functional analysis also showed that PCTK1 suppressed cancer stemness and cell proliferation by using PCTK1 knockdown (PCTK1-KD) or knockout (PCTK1-KO) and PCTK1 overexpression (PCTK1-over) CRC cell lines. Furthermore, overexpression of PCTK1 decreased xenograft tumor growth and knockout of PCTK1 significantly increased in vivo tumor growth. Moreover, knockout of PCTK1 was observed to increase the resistance of CRC cells to both irinotecan (CPT-11) alone and in combination with 5-fluorouracil (5-FU). Additionally, the fold change of the anti-apoptotic molecules (Bcl-2 and Bcl-xL) and the proapoptotic molecules (Bax, c-PARP, p53, and c-caspase3) was reflected in the chemoresistance of PCTK1-KO CRC cells. PCTK1 signaling in the regulation of cancer progression and chemoresponse was analyzed using RNA sequencing and gene set enrichment analysis (GSEA). Furthermore, PCTK1 and Bone Morphogenetic Protein Receptor Type 1B (BMPR1B) in CRC tumors were negatively correlated in CRC patients from the Timer2.0 and cBioPortal database. We also found that BMPR1B was negatively correlated with PCTK1 in CRC cells, and BMPR1B expression was upregulated in PCTK1-KO cells and xenograft tumor tissues. Finally, BMPR1B-KD partially reversed cell proliferation, cancer stemness, and chemoresistance in PCTK1-KO cells. Moreover, the nuclear translocation of Smad1/5/8, a downstream molecule of BMPR1B, was increased in PCTK1-KO cells. Pharmacological inhibition of Smad1/5/8 also suppressed the malignant progression of CRC. Taken together, our results indicated that PCTK1 suppresses proliferation and cancer stemness and increases the chemoresponse of CRC through the BMPR1B-Smad1/5/8 signaling pathway.

Polymorphisms Analysis of BMP15, GDF9 and BMPR1B in Tibetan Cashmere Goat (Capra hircus).

The Tibetan cashmere goat is a prolific goat breed in China. In sheep breeds, natural mutations have demonstrated that the transforming growth factor beta (TGF-β) super family ligands, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor (BMPR1B), are essential for ovulation and increasing litter size. In this study, 216 female Tibetan cashmere goats were sampled, and candidate genes with fecundity traits were detected via restriction fragment length polymorphism (RFLP) and sequenced. Four polymorphic loci were found in specific amplification fragments of BMP15 and GDF9. Two SNP sites of the BMP15 gene were discovered, namely G732A and C805G. The G732A mutation did not cause the change in amino acids, and the frequencies of each genotype were 0.695 for the GG type, 0.282 for the GA type and 0.023 for the AA type. The C805G mutation caused amino acids to change from glutamine to glutamate. The genotype frequencies were 0.620 for the CC type, 0.320 for the CG type and 0.320 for the CG type. For the GG type 0.060, the G3 and G4 mutations of the GDF9 gene were all homozygous mutations. Two known SNP sites, C719T and G1189A, were detected in the Tibetan cashmere goat GDF9 gene, of which the C719T mutation caused a change of alanine to valine, with a genotype frequency of 0.944 for the CC type and 0.056 for the CT type, whereas no TT type was found. The G1189A mutation caused valine to become isoleucine, and the frequencies of each genotype were 0.579 for the GG type, 0.305 for the GA type and 0.116 for the AA type; G1, B2, B3, B4, FecXH, FecXI, FecXL, G2, G5, G6, G7, G8, FecGE, FecTT and FecB mutations were not found in Tibetan cashmere goats. The results of this study provide a data basis for future studies of BMP15, GDF9 and BMPR1B gene mutations in goats.

Single-Nucleotide Polymorphisms Identified within Exon 2 of Fertility-Associated Bone Morphogenetic Protein (BMP15) Gene in Three Romanian Sheep Breeds

The improvement of the reproductive traits of animals is of great interest for livestock production. Due to its positive impact on the sheep industry’s profitability, prolificacy is one of the most economically significant biological traits, showing variation between and within breeds of domestic sheep (Ovis aries). Different mutations in BMPR-1B, BMP15 and GDF9 genes coding for the transforming growth factor-β (TGFβ) superfamily have been shown to influence the ovulation rate and litter size. Numerous single-nucleotide polymorphisms (SNPs) in the bone morphogenetic protein 15 (BMP15) gene have been linked to ewe fecundity. Using targeted PCR amplification and Sanger sequencing, we were able to identify heterozygous SNPs in exon 2 of BMP15 in three sheep breeds reared in Romania: Tsigai, Cluj Merino and Tsurcana. The sequence analysis revealed three previously documented mutations, namely the missense mutation c.755T>C (L252P), which is predicted to change the tertiary structure of the BMP15 protein, and two silent mutations, c.747T>C (P249P) and c.1047G>A (V349V). In addition, we also identified one novel silent mutation, c.825G>A (S275S). Based on our findings and publicly available data, we indicate four putative mutational hotspots within exon 2 of BMP15 that could be considered for improving the indigenous sheep breeds through targeted gene editing and SNP genotyping strategies.

High frequency of heterozygous rare variants of the SLC34A1 and SLC9A3R1 genes in patients with atypical femur fracture

Atypical femur fractures (AFFs) are rare fragility fractures originating at the lateral cortex of the femur, affecting the subtrochanteric or diaphyseal area of thebone with a transverse morphology. Occurrence of AFF is specifically associated with a small number of rare monogenic congenital metabolic bone disorders, such as hypophosphatasia, and with long-term treatment with antiresorptiondrugs. The exact pathogenesis of these fractures remains poorly understood and, except for cases of diagnosed HPP or other AFF-causing bone diseases, it is not possible to assess which patients are at higher riskof developing AFFs as a consequence of anti-resorption therapy. We genetically screened 25 unrelated patients who had developed at least one AFF. Genetic screening was performed through a nextgeneration sequencing analysis with a customized panel containing 76 human genes involved in the regulation of the mineralization processWe genetically screened 25 unrelated patients who had developed at least one AFF. We found a relatively high frequency (32.0%) of heterozygous rare variants inthe SLC34A1 and SLC9A3R1 genes, two genes whose heterozygous inactivating mutations have been respectively associated with autosomal dominant hypophosphatemic nephrolithiasis/osteoporosis types 1 and 2 (NPHLOP1and NPHLOP2). Other heterozygous rare variants were found in the BMPR1B, CYP27B1, FBN1, MEPE, PIGO, and PHOSPHO1 genes, each in a single AFF case (4.0%). Our findings suggest that rarevariants of SLC34A1 and SLC9A3R1 could represent a possible genetic risk factor for the occurrence of AFFs. On the other hand, AFFs could represent an unsuspected clinical manifestation and/or an anti-resorption therapycorrelatedadverse event in patients with NPHLOP disorders.

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Ovarian expression analysis of fecundity candidate genes in Goat breeds differing in prolificacy

To understand the genetics of goat fertility, goat breeds differing in prolificacy were used for exploring candidate genes using quantitative polymerase chain reaction. Ovaries (both left and right) of Black Bengal, Osmanabadi and Sirohi goats (n=3 from each breed) were collected from the local abattoir located in their respective breeding tracts. The RNA isolated from antral follicles was converted into cDNA. The β-Actin (ACTB) was used as a reference gene for quantifying the expression of BMP15, GDF9, BMPR1B, KISS1 and GPR54 mRNAs in ovaries. The results showed that BMPR1B, GDF9 and BMP15 genes were highly expressed whereas KISS1 and GPR54 were slightly expressed in the ovaries. Significant differences (P<0.05) were recorded in the gene expression of GDF9 and BMP15. Expression of these two genes varied in prolificacy being highest in Black Bengal followed by Osmanabadi and least in Sirohi. BMPR1B expression did not vary significantly among the goat breeds. In conclusion, the study provides baseline data and contributes toward the understanding of the molecular basis of prolificacy in goat breeds.

Integrated Proteotranscriptomics of the Hypothalamus Reveals Altered Regulation Associated with the FecB Mutation in the BMPR1B Gene That Affects Prolificacy in Small Tail Han Sheep.

The litter size and ovulation rate are different among ewes of different FecB genotypes in Small Tail Han sheep. These variants in reproductive phenotypes may be regulated by hormones released by the hypothalamic-pituitary-ovarian axis. However, there have been few reports on the hypothalamus regarding regulating an increase in ovulation in sheep with FecB mutation at different estrous stages. Thus, we examined the abundance of hypothalamus tissue protein profiles of six FecB mutant homozygous (BB) and six wild-type (WW) ewes at the luteal and follicular phases. We determined this abundance by tandem mass tag-based quantitative analysis and parallel reaction monitoring methods. Furthermore, an integrated proteotranscriptomic analysis was performed by the Data Integration Analysis for Biomarker discovery using the latent variable approaches for Omics studies (DIABLO) framework to examine biological processes and pathway alterations by the FecB mutant. The abundance of 154 proteins was different between the two estrous stages. Growth hormone and prolactin were particularly enriched in the neuroactive ligand-receptor interactions, the prolactin signaling pathway, and the PI3K-Akt signaling pathway which are related to hypothalamic function and reproduction. We combined proteome and transcriptome data from different estrous stages and genotypes. There is a high correlation (Pearson correlation coefficient = 0.99) between the two datasets in the first two components. We applied the traditional single-omic multivariate approach to obtain differentially abundant proteins and differentially expressed genes. The major fertility related biomarkers were selected using the two approaches mentioned above. Several key pathways (GABAergic synapse, neuroactive ligand-receptor interaction, estrogen and MAPK signaling pathways) were enriched, which are central to gonadotrophin-releasing hormone (GnRH) secretion and reproduction. A higher level of gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2) expression was observed in BB ewes as compared to WW ewes. This finding suggested that a greater production of GnRH during follicular development in BB ewes may explain the higher mature follicle number in mutant ewes. FKBP prolyl isomerase 1A (FKBP1A), which was a major feature factor in the proteome selected by DIABLO, was an important switch for activating the transforming growth factor beta (TGFβ) pathway, and its expression was higher in the WW ewes than in the BB ewes. We suggest that BB sheep maintain TGFβ pathway activity by reducing FKBP1A protein abundance. This innovative data integration in the hypothalamus may provide fresh insight into the mechanisms by which the FecB mutation affects sheep fertility, while providing novel biomarkers related to reproductive endocrinology in sheep breeding.

A unique 15-bp InDel in the first intron of BMPR1B regulates its expression in Taihu pigs

BackgroundBMPR1B (Bone morphogenetic protein receptor type-1B) is a receptor in the bone morphogenetic protein (BMP) family and has been identified as a candidate gene for reproductive traits in pigs. Our previous study in Taihu pigs found a specific estrogen response element (ERE) in the first intron of the BMPR1B gene that is associated with the number born alive trait. However, little is known about the mechanism by which the ERE regulates the expression of BMPR1B in the endometrium.ResultsHere, a 15-bp InDel (insertion/deletion) (AGCCAGAAAGGAGGA) was identified as a unique variation in Taihu pigs, and was shown to be responsible for the binding of the type I receptor of estrogen (ESR1) to the ERE using dual-luciferase assays. Four BMPR1B transcripts (T1, T2, T3, and T4) were identified by 5′ RACE in endometrial tissue. Expression of T3 and T4 in the endometrium of Meishan pigs was significantly higher than in Duroc pigs during pregnancy. Luciferase assays showed that three distinct BMPR1B promoters may drive expression of T1, T3, and T4. Interestingly, ERE-mediated enhancement of T4 promoter activity significantly increased expression of Transcript T4 in the endometrium of Taihu pigs (P < 0.05). In contrast, the ERE inhibited activity of the T3 promoter and decreased expression of the T3 transcript in the Duroc background (P < 0.05). In summary, we identified a 15-bp InDel in the Taihu ERE that can be used as a molecular marker for the number born alive trait, characterized the 5′ untranslated regions (UTRs) of BMPR1B transcripts in the endometrium, and determined how the transcripts are processed by alternative splicing events.ConclusionsOur results provide a foundation for understanding the transcriptional regulation of BMPR1B and its contributions to the unique breeding prolificacy characteristics of Taihu pigs.

Two Retrotransposon Elements in Intron of Porcine BMPR1B Is Associated with Phenotypic Variation.

It has been established that through binding to bone morphogenetic proteins (BMPs), bone morphogenetic protein receptor I B (BMPR1B) can mediate transforming growth factor β (TGF-β) signal transduction, and is involved in the regulation of several biological processes, such as bone and muscle formation and homeostasis, as well as folliculogenesis. Also known as FecB, BMPR1B has been reported as the major gene for sheep prolificacy. A number of previous studies have analyzed the relationship between single nucleotide polymorphisms (SNPs) in this gene and its related performance. In recent years, with the illustration of the effect of retrotransposon insertion on the expression of the proximal genes or phenotypic variation, retrotransposon insertion polymorphisms (RIPs) have been used as a novel type of molecular marker in the evaluation of evolution, population structure and breeding of plant and domestic animals. In this study, the RIPs in porcine BMPR1B gene were excavated, and thereafter verified using a comparative genome and polymerase chain reaction (PCR). The potential effects of phenotype, gene expression and functions related to RIPs were also explored. The results showed that 13 distinct RIPs were identified in introns of porcine BMPR1B. Among these, only BMPR1B-SINE-RIP9 and BMPR1B-LINE-RIP13 displayed a close relationship with the growth traits of Large White pigs. Moreover, the total number of BMPR1B-SINE+/+-RIP9 individuals born was found to be significantly higher than that of SINE−/− (p < 0.05). These two RIPs showed an obvious distribution pattern among Chinese indigenous breeds and Western commercial breeds. The expression of BMPR1B in ovaries of adult BMPR1B-SINE+/+-RIP9 Sushan pigs was found to be significantly higher in comparison to those of BMPR1B-SINE−/−-RIP9 (p < 0.05). SINE insertion of BMPR1B-SINE-RIP9 and LINE insertion of BMPR1B-LINE-RIP13 were observed to significantly increase the activity of Octamer binding transcription factor 4 (OCT4) minipromoter in CHO and C2C12 cells (p < 0.01). Therefore, these two RIPs could serve as useful molecular markers for modulating the growth or reproductive traits in assisted selection of pig breeding, while the mechanisms of the insertion function should be studied further.

First genetic characteristics of Polish patients with idiopathic pulmonary arterial hypertension

Abstract Introduction Pulmonary arterial hypertension (PAH) is a rare, severe disorder of multifactorial origin. Genetic alterations in BMP/SMAD pathways were previously associated with disease development, however the exact role of other genetic factors from BMP/SMAD signalling is still unclear. Purpose We aimed to search for known PAH-associated mutations and potential novel variants responsible for disease onset in Polish PAH patients. Methods We prospectively recruited 93 consecutive idiopathic PAH patients from a single pulmonary hypertension reference centre between years 2009 and 2020. Eligible patients had pre-capillary pulmonary hypertension with pulmonary vascular resistance &amp;gt;3 Wood units in the absence of other causes of pre-capillary pulmonary hypertension. The presence of large gene rearrangements was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) reactions in the ENG, ACVRL1 and BMPR2 genes (SALSA MLPA Kit, MRC-Holland). We have further sequenced a panel of selected 48 genes from BMP/SMAD and related pathways using next-generation sequencing (SureSelect XT library preparation Kit, Agilent; NextSeq 500 sequencer, Illumina) and assessed causative potential of rare variants (minor allele frequency in non-finish Europeans below 1%) with Mutation Taster web-based tool. Results We identified at least one likely-causative variant of investigated genes in 51 (54.8%) subjects. Large genomic rearrangements were found by MLPA in 2 patients in ENG and ACVRL1 (ALK1) genes. We have found mutations in BMPR2 gene in 12 patients (12.5%) from our cohort with most variants present in one sample and 3 variants present in 2 samples. Mutations were located mostly in protein kinase domain (6 variants) and Activin type I and II receptor domain (2 variants). We have found 5 likely pathogenic variants in 6 patients in genes previously associated with PAH other than BMPR2 (SMAD9, KCNA5, KCNK3, EIF2AK4). In total, potentially disease causing mutations in literature-known genes, were present in 18 patients, which accounted for 18.75% of tested cohort. Medium impact variants were also present in KCNA5, TGFBR2, AQP7, BMP6, EIF2AK4, FBP1, NOTCH1, NOTCH3, SMAD7, TBX4, TGFB2, TOPBP1, GDF5, IL6, PPARA, RXRA, KCNK3, KLF4, BMPR1B, ILK, TGFBR1, SMAD9, PPARD and TGFB3 genes. Additionally, in one patient with clinical diagnosis of pulmonary venoocclusive disease we identified an unknown homozygous frameshift variant (p.Phe1523fs/c.4567_4570delTTTG) in exon 35 of EIF2AK4 gene. Conclusions We found potentially disease causing mutations in 18,75% Polish patients diagnosed with IPAH most of them were present in BMPR2 gene which is in line with data from other European cohorts. Additionally we found an unknown mutation in the EIF2AK4 gene. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Narodowe Centrum Nauki - National Science Centre

MiR-1306 induces cell apoptosis by targeting BMPR1B gene in the ovine granulosa cells.

Bone morphogenetic protein receptor type-1B (BMPR1B) is one of the major gene for sheep prolificacy. However, few studies investigated its regulatory region. In this study, we reported that miR-1306 is a direct inhibitor of BMPR1B gene in the ovine granulosa cells (ovine GCs). We detected a miRNA response element of miR-1306 in the 3’ untranslated region of the ovine BMPR1B gene. Luciferase assay showed that the ovine BMPR1B gene is a direct target of miR-1306. qPCR and western blotting revealed that miR-1306 reduces the expression of BMPR1B mRNA and protein in the ovine granulosa cells. Furthermore, miR-1306 promoted cell apoptosis by suppressing BMPR1B expression in the ovine granulosa cells. Overall, our results suggest that miR-1306 is an epigenetic regulator of BMPR1B, and may serve as a potential target to improve the fecundity of sheep.

Evaluation of the BMPR-1B gene functional polymorphisms and their association with litter size in Qinghai Tibetan sheep

The Bone morphogenetic protein receptor-1B (BMPR-1B) was one of the most major candidate gene for sheep follicular development and litter size trait. The BMPR-1B gene encoded a structural and functional highly conserved transmembrane protein related to sheep reproductive performance. To further validate its genetic effect, genotype-phenotype associations were performed in Tibetan sheep. Through DNA sequencing of the entire coding region of the BMPR-1B gene using DNA of 30 unrelated Tibetan ewes (10 Singletons Tibetan ewes, and 20 Twins Tibetan ewes). And genotyping the target SNPs loci using PCR-RFLP (or PCR-SSCP) combined with DNA sequencing and analyzing their association with lambing traits in 510 Tibetan ewes. One mutation in 3′UTR (A1354G) and three mutations in exons (G192A, A746G, T864C) were found to be strongly associated with lambing traits in Tibetan ewes in this study. χ2 suitability test showed that all of SNPs were Hardy-Weinberg equilibrium. After independence test, A746G, T864C and A1354G of BMPR-1B gene polymorphisms were significantly correlated with litter size in all-types of Tibetan sheep. These results indicate that the variations in the BMPR-1B genes and their corresponding genotypes may be considered as marker-assisted selection (MAS) for litter traits in Tibetan sheep breeding.

Validation Study of c.746A&gt;G SNP of BMPR1B Gene Associated with Litter Size in Sheep Genetic Resources of Bangladesh

The BMPR1B gene is one of the major fecundity genes that have been investigated in different sheep populations worldwide for its association with prolificacy traits. The present study was performed to validate the association of c.746A&gt;G SNP of BMPR1B gene with litter size trait in different sheep populations of Bangladesh. A total of 192 blood samples were collected from ewes of both farmers’ and institutional flocks comprising sheep populations of Jamuna River Basin (JRB), Barendra (BAT), Coastal (COR), Garole (GAR) and Muzaffarnagari (MUZ). Genotyping of the individuals was performed using PCR-RFLP method and single marker association analysis was carried out to evaluate the relationships between resultant genotypes and litter size trait using agricolae and pastecs packages of R software. The prolificacy attributed homozygous FecBBB genotype frequencies were 66, 50 and 55%, respectively in JRB, BAT and GAR populations whereas it was only 0-2% in MUZ and COR populations. Association analysis revealed highly significant (P&lt;0.001) association of litter size trait with genotypes and populations. The mean litter sizes of JRB, BAT, GAR, COR and MUZ were 2.17±0.15, 1.88±0.07 1.90±0.10, 1.12±0.03 and 1.02±0.02, respectively. The prolific JRB, BAT, GAR ewes produced 0.67 to 0.83 more lambs per lambing than their wild type FecB++ counterparts. Thus, this study validated the potential contribution of the investigated c.746A&gt;G SNP that could be applied in marker assisted selection (MAS) program for identifying high prolific ewes in order to improve litter size trait at the population level

Genetic variability in 5' untranslated region of BMPR1B gene from prolific Avishaan strain of sheep

Untranslated regions (UTRs) of the mRNAs have significant contribution towards gene regulation and expression. FecB gene is a member of transforming growth factor-p superfamily. A mutation in exon 8 of gene gives the FecB phenotype in sheep. In the present study 5′UTR region (992 bp encompassing 5′UTR and upstream sequences) and exon 8 region (712 bp encompassing exon 8 and partial sequences of upstream and downstream introns) of FecB gene were sequenced and characterized in prolific Avishaan sheep and its parental stock i.e. Garole, Malpura and Patanwadi. Sequence analysis of 992 bp fragment revealed 14 SNPs and one non-synonymous mutation (rs402422707) was observed in exon 1. Promoter was located in nucleotide 40 to 90 in amplified 5′ UTR sequence. Transcription factor binding site prediction identified 65 segments in the amplified 5′UTR and 5′ upstream sequence as potential binding sites for different transcription factors (TFs). In all 37 TFs binding sites were located on these sequences and majority of these sites were identified for SP-1 (12 segments), c/EBPalp (9 segments), NF-1 (4 segments), 2 segments each of RAP-1, HNF-1, c-Ets-1, TEC1, and Pit1a. These potential binding sites for TFs would be helpful in identifying site of interaction to control gene expression in Avishaan sheep.

Polymorphism of Prolificacy Genes (BMP15, BMPR 1B and GDF9), in the Native Goat (Capra hircus) of Cameroon

The main objective was to contribute to a better understanding of molecular characteristics of the local goat in order to improve its productivity and specifically to: analyse genetic polymorphism of three prolificacy genes (BMP15, BMPR1B, and GDF9) and test the association of genetic polymorphism and prolificacy of local goats. Tissue samples were collected from 446 animals, and 24 representative female goats were selected to analyse the genetic polymorphism of the prolificacy genes. The selected goats were divided into two groups of 12 females for high prolificacy (more than three kids consecutively in four parity) and 12 females for low prolificacy (less than two kids consecutively in four parity). Chi-square was used to test the association between genetic polymorphism and prolificacy of local goat. The main results showed that BMP15 gene is monomorphic, whereas the two other genes (BMPR1B and GDF9) display polymorphism. For BMPR1B gene, the ten mutations found did not change the corresponding amino acid. Allelic and genotypes frequencies of mutations of this gene varied from one mutation to another and between the two groups of females (high and low prolificacy). Chi-square test of the polymorphism of this gene shows that C34T and A120G mutations of exon 3 are significantly associated (p &lt; 0.05) with prolificacy and can be considered as potential genetic markers for improving prolificacy in the native goat. For the GDF9 gene, three mutations were detected in exon 1 with alleles A and G1 of frequency 0.261 and 0.130 for A35G; G2 and C1 of frequency 0.696 and 0.304 for G81C; then G3 and C2 of frequency 0.696 and 0.304 for G255C. The mutations G81C and G255C appeared under BLAST and were missense mutations P27A and A85G respectively while A35G is located in the non-translated 5’ region of the gene. Chi-square test between each genotype for any site and the prolificacy was not significant (P &gt; 0.01) suggesting that these two characters are not associated. Two mutations were detected in exon 2 at C881T and A1160G sites with C and T and A and G alleles respectively. The two mutations changed the corresponding amino acid from Alanine to Valine at the position 273 in the protein and from Valine to Isoleucine at the position 397 in the protein respectively. Allelic and genotypes frequencies of mutations varied from one mutation to another and between the two groups of females (high and low prolificacy). Chi-square test of the polymorphism shows that, although C881T and A1160G mutations were not significantly associated (P &gt; 0.05) with prolificacy, the alleles responsible for the variation of amino acid increased the litter size. Therefore, further studies with increased sample size will help to verify the results.

PSXII-2 Identification of candidate SNPs associated with high prolificacy in Romanov sheep

Abstract Prolificacy is a very important trait in sheep. Romanov sheep, the well-known Russian sheep breed, are characterized by very high prolificacy; however, the genetic basis of this unique property of Romanov sheep is still unknown. It was reported that Ovine BMPR1B gene, located on the OAR6, is associated with prolificacy in several sheep breeds. The aim of our study was to identify candidate SNPs within BMPR1B gene, related to prolificacy. To achieve this goal, using NGS technology, we sequenced ovine BMPR1B gene in Romanov sheep (n = 6), which are characterized by high prolificacy (about 270 lambs per 100 ewes). The sequences of BMPR1B gene of Noire du Velay, Tan, Southdown and Australian Horned Merino sheep breeds as well as Asiatic mouflon (n = 1), which are characterized by significantly lower prolificacy (from 110 to 180 lambs per 100 ewes) were derived from publicly available sources and used for comparison. FST analysis performed in PLINK 1.9 program revealed 10 SNPs with values higher than 0.8. The majority of candidate SNPs under putative selection were localized in the region from 29,382,098 to 29,430,387 on OAR6 of Ovine reference genome (Oar_v3.1 (Ensembl release 98). Thus, we can suggest, that this region of the BMPR1B gene can be considered as the putative region, associated with high prolificacy of Romanov sheep. Additional studies will be needed to confirm the effect of identified candidate SNPs on prolificacy traits. The research results will be useful for artificial selection of sheep with higher prolific capacity, including the introduction of desired alleles in sheep populations using genome editing technologies. This work was supported by the Russian Ministry of Science and Higher Education No. 0445-2019-0024 and RFBR No. 20-516-56002.

Association between novel variants in BMPR1B gene and litter size in Mongolia and Ujimqin sheep breeds.

Prolificacy is an important trait of animals, specifically for sheep. The Bone morphogenetic protein receptor 1B (BMPR1B) is a major gene affecting the litter size of many sheep breeds. The well-known FecB mutation (Q249R) was associated fully with the hyper prolific phenotype of Booroola Merino. However, the identification of variation in all exonic regions of BMPR1B was rare. In this study, we sequenced all exonic regions of BMPR1B gene of Mongolia sheep breed, and ten novel variants were detected by direct sequencing. Among them, the litter size of the Mongolia ewes with the CC genotype was significantly higher (0.34 additional lambs, p<.05) than those with the TT genotype of the g.29346567C>T single nucleotide polymorphism (SNP). The litter size of the Mongolia ewes with the TT genotype was significantly higher (0.19 additional lambs, p<.05 and .31 additional lambs, p<.01, respectively) than those with the GT and GG genotypes of the c.1470G>T SNP. The silent c.1470G>T mutation is predicted to increase the stability of the mRNA secondary structure through reducing minimum free energy and is predicted to change the mRNA secondary structure of BMPR1B. Our findings may give potentially useful genetic markers for increasing litter size in sheep.

An investigation of the effects of BMPR1B, BMP15, and GDF9 genes on litter size in Ramlıç and Dağlıç sheep.

This study was carried out to determine the presence of polymorphisms in genes affecting litter size. The SNPs in bone morphogenetic protein receptor type 1B (BMPR1B), bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) genes were detected in 60 uniparous and 60 multiparous ewes from Ramlıç and Dağlıç breeds. The ewes are maintained in nine public herds at the breeding station of the Afyonkarahisar Sheep and Goats Breeders' Association and lambed in two consecutive breeding seasons. PCR and DNA sequencing analyses were conducted, and 36, 4, and 11 SNPs in Ramlıç and 40, 3, and 11 SNPs in Dağlıç were detected in BMPR1B, BMP15, and GDF9 genes, respectively. A total of 16 SNPs in Ramlıç and 10 SNPs in Dağlıç breeds for three genes were found to be significant (). The resulting analyses showed that four SNPs (g.49496G>A, c.1658A>C, c.2037C>T, c.2053C>T) of the BMPR1B gene and one deletion mutation (c.28_30delCTT) in the BMP15 gene of the Ramlıç breed as well as five SNPs (c.1487C>A, c.2492C>T, c.2523G>A, c.2880A>G, and c.2763G>A) of the BMPR1B gene of the Dağlıç breed have significant positive regression coefficients in the desired direction of the rare allele. The observed mutations have potential to be used as genetic markers in the selection of prolific animals for both breeds.

The expression and mutation of &amp;lt;i&amp;gt;BMPR1B&amp;lt;/i&amp;gt; and its association with litter size in small-tail Han sheep (&amp;lt;i&amp;gt;Ovis aries&amp;lt;/i&amp;gt;)

Previous studies have shown that BMPR1B promotes follicular development and ovarian granulosa cell proliferation, thereby affecting ovulation in mammals. In this study, the expression and polymorphism of the BMPR1B gene associated with litter size in small-tail Han (STH) sheep were determined. The expression of BMPR1B was detected in 14 tissues of STH sheep during the follicular phase as well as in the hypothalamic–pituitary–gonadal (HPG) axis of monotocous and polytocous STH sheep during the follicular and luteal phases using quantitative polymerase chain reaction (qPCR). Sequenom MassARRAY® single nucleotide polymorphism (SNP) technology was also used to detect the polymorphism of SNPs in seven sheep breeds. Here, BMPR1B was highly expressed in hypothalamus, ovary, uterus, and oviduct tissue during the follicular phase, and BMPR1B was expressed significantly more in the hypothalamus of polytocous ewes than in monotocous ewes during both the follicular and luteal phases (). For genotyping, we found that genotype and allele frequencies of three loci of the BMPR1B gene were extremely significantly different () between the monotocous and polytocous groups. Association analysis results showed that the g.29380965AG locus had significant negative effects on the litter size of STH sheep, and the combination of g.29380965AG and FecB (Fec – fecundity and B – Booroola; A746G) at the BMPR1B gene showed that the litter size of AG–GG, AA–GG, and GG–GG genotypes was significantly higher compared with other genotypes (). This is the first study to find a new molecular marker affecting litter size and to systematically analyze the expression of BMPR1B in different fecundity and physiological periods of STH sheep.

Association of BMPR-1B Gene 3’-UTR Region Polymorphism with Litter Size in Tibetan Sheep

Association of BMPR-1B Gene 3’-UTR Region Polymorphism with Litter Size in Tibetan Sheep