Bp Of Exon Research Articles

Genetic analysis of platelet-derived growth factor receptor-like gene (PDGFRL) polymorphism and melanin traits in Lanping black-boned sheep (Ovis aries)

Abstract. In the intricate tapestry of Yunnan Province's biodiversity, the Lanping black-boned sheep (LPBB) emerges as a captivating enigma, distinguished by its profound melanin pigmentation adorning both its skin and its internal organs. Initially cataloged in the 1950s within the confines of Lanping County, this exceptional mammalian species presents a scarcity and uniqueness that extends beyond its geographic origins. Here, we collected 100 blood samples from Lanping black-boned sheep along with 50 samples each from Lanping normal sheep (LPN) and Huize normal sheep (HZN), all sourced from Yunnan Province. Our investigation focused on the association between the platelet-derived growth factor receptor-like gene (PDGFRL) polymorphism and the distinctive melanin characteristics observed in Lanping black-boned sheep. Utilizing UV–visible spectrophotometry, we assessed the melanin indexes present, such as tyrosinase activity and true melanin in the sheep blood, and the results demonstrated a significant elevation in melanin indexes for Lanping black-boned sheep compared to the control group (P<0.05). We also identified three synonymous mutation sites within a partial 1128 bp exon fragment of the gene-encoding PDGFRL (EX2-G408A, EX5-T184C, and EX5-G222T). Notably, Lanping black-boned sheep, harboring genotypes GG, TT, and GG at these specific sites, showcased a pronounced surge in tyrosinase activity, eumelanin / total melanin ratios, and plasma colorimetric values when contrasted with the control group (P<0.05). The discernment of GG, TT, and GG as the prevailing genotypes at their respective genetic loci in Lanping black-boned sheep heralds a breakthrough in our understanding of the genetic markers associated with black pigmentation. However, all three loci are silent mutations and do not alter the phenotypic changes. Whether they affect changes in melanin content through other metabolic pathways requires further study. In conclusion, the PDGFRL gene was silenced by mutations in our study and affected blood melanin levels. However, the gene did not undergo a missense mutation that altered the phenotypic changes, and the exact channel through which the changes in melanin content were affected needs to be further verified.

Allele mining for Granule Bound Starch Synthase1 (GBSS1) gene governing amylose content in aromatic rice (Oryza sativa L.) germplasm

The current research investigated sequence polymorphism and allelic variation in the GBSS1 gene responsible for determining the apparent amylose content (AAC) in an aromatic rice germplasm panel. Analysis of 271 germplasm accessions from India revealed a wide range of variation in AAC, from 4.81% (Manipur Black Rice) to 35.88% (IGSR -3-1-5). Re-sequencing and sequence analysis of a representative subset of 28 accessions unveiled a nucleotide diversity (π) of 0.00374, identifying 72 sequence variants. Two novel non-synonymous SNPs were identified: a G/A transition in exon 2, resulting in the substitution of glutamic acid at the 91st position with lysine, and a C/T transition in exon 11, substituting alanine at the 468thposition with valine. Additionally, two previously reported SNPs were validated: A/C in exon 6 (Tyr224Ser) and C/T in exon 10 (Pro415Ser). Furthermore, a 23 bp exon 2 duplication was found in Manipur Black Rice (4.81%), Bhogali Bora (6.20%), and NJ-72 (7.36%), indicating the prevalence of this allele in the very low AAC category (3–9%) in addition to the glutinous or waxy rice category with 0–2% AAC. These findings are significant for manipulating starch content in aromatic rice germplasm.

Fishing for the gonadotropin releasing hormone from the genome of Salminus brasiliensis (Characiformes: Bryconidae).

The technological development of tools that enable the spawning of different native species is paramount to enable ex situ conservation initiatives, as well as providing means for commercial hatchery of threatened fish which, in turn, relieve fisheries pressure over wild stocks. Neotropical migratory freshwater fish depend on hormonal induction for spawning in hatcheries, through expensive methods of limited efficiency. Salminus brasiliensis is one of the largest Neotropical freshwater fish, a piscivorous top-predator, prized in angling, highly valued in the market, and appreciated in gastronomy. Teleost fish have either, two or three GnRH paralogous genes: GnRH1, GnRH2 and the GnRH3. The expression products of these paralogous isoforms consist of a larger prepro-GnRH polypeptide, which undergoes post-translational proteolytic processing to yield the active decapeptide hormone. There is increasing interest in characterizing and understanding these neuropeptides, because of its practical application in hatchery spawning. We present the characterization of GnRH1's coding sequence for the prepro-GnRH1 polypeptide of S. brasiliensis. An annotation from a genomic assembly was used for searching for GnRH paralogues, based on data from anonymous predicted transcripts. The sequence retrieved for GnRH1 was then used as a query for searching the uncharacterized GnRH paralogues from full genomes of Characiformes deposited at NCBI. The S. brasiliensis GnRH1 gene sequence retrieved was targeted for PCR and submitted to Sanger sequencing, allowing for its confirmation. It spans 423 bp (exon 1: 128 bp; intron: 161 bp; and exon 2: 1134 bp), with open reading frames coding for 264 and 88 amino acids, respectively. The different variants retrieved for the prepro-GnRH (1, 2 and 3) from Characiformes genomes and deposited sequences from NCBI grouped in three distinct clades in a neighbor joining tree, each forming a monophyletic branch and with the S. brasiliensis sequences nested within the expected groups. Here we observed a variation at a proteolytic site (GKR→GRR), reported as highly conserved in vertebrates up to now, that can potentially alter the cleavage site and modify the peptide topology. This work has characterized, for the first time, the sequence of the GnRH1 coding for its prepro-GnRH peptide, for a member of the Charaficormes order. This will help to promote research and development of tools for broodstock spawning and environmental management of S. brasiliensis and related migratory fish.

Structure characteristics of mutation sites in two waxy alleles from Yunnan waxy maize (Zea mays L. var. certaina Kulesh) landraces.

A large number of waxy maize landraces are distributed in Yunnan and surrounding areas, and abundant waxy alleles of different types are distributed in these landraces. The identification of waxy alleles is helpful to the protection and utilization of these waxy landraces. This study introduced structure characteristics of waxy genes from two specific landraces of Yunnan, Zinuoyumi and Myanmar Four-Row Wax. Zinuoyumi has two waxy alleles wx-Cin4 and wx-Cin4-2; Myanmar Four-Row Wax has three waxy alleles wx-D10, wx-Reina and wx-D11. The wx-Cin4-2 and wx-D11 are two types of waxy alleles first reported in this study. The wx-Cin4-2 has two mutation sites, deletion of 30 bp in exon 10, insertion of a 1,267 bp non-long terminal repeat (non-LTR) retrotransposon Cin4 in intron 10, and 13 bp extra sequence were found at 5' end of the Cin4; the mutation site of wx-D11 is a 1,082 bp deletion from exons 11 to 14 of the waxy gene and is replaced with a 72 bp filler sequence. This study enriched the type of waxy allele from Yunnan waxy maize landraces and further discussed the molecular basis for the formation of mutation sites of wx-Cin4-2 and wx-D11.

Sequencing and Characterization of αs2-Casein Gene (CSN1S2) in the Old-World Camels Have Proven Genetic Variations Useful for the Understanding of Species Diversification.

The CSN1S2 gene encodes αs2-casein, the third most abundant protein in camel milk. Despite its importance in foals, human nutrition, and dairy processing, the CSN1S2 gene in camels has received little attention. This study presents the first complete characterization of the CSN1S2 gene sequence in Old-World camels (Camelus bactrianus and Camelus dromedarius). Additionally, the gene promoter, consisting of 752 bp upstream of exon 1, was analyzed. The entire gene comprises 17 exons, ranging in length from 24 bp (exons 4, 8, 11, and 13) to 280 bp (exon 17). Interesting was the identification of the exon 12 in both species. The promoter analysis revealed 24 putative binding sites in the Bactrian camel and 22 in dromedary camel. Most of these sites were typical elements associated with milk protein, such as C/EBP-α, C/EBP-β, Oct-1, and AP1. The SNP discovery showed relatively high genetic diversity compared to other camel casein genes (CSN1S1, CSN2, and CSN3), with a total of 34 polymorphic sites across the two species. Particularly noteworthy is the transition g.311G>A in the CSN1S2 promoter, creating a new putative consensus binding site for a C/EBP-β in the Bactrian camel. At the exon level, two novel variants were found. One was detected in exon 6 of the Bactrian camel (g.3639C>G), resulting in an amino acid replacement, p.36Ile>Met. The second variant was found in noncoding exon 17 of dromedary CSN1S2 (g.1511G>T). Although this mutation occurs in the 3'-UnTranslated Region, it represents the first example of exonic polymorphism in the CSN1S2 for this species. This SNP also affects the binding sites of different microRNAs, including the seed sequence of the miRNA 4662a-3p, highlighting its role as a regulatory factor for CSN1S2 gene. A PCR-RFLP was set up for genotyping a dromedary Tunisian population (n = 157), and the minor allele frequency was found to be 0.27 for the G allele, indicating a potential yield improvement margin. The interspersed elements (INEs) analysis revealed 10 INEs covering 7.34% and 8.14% of the CSN1S2 sequence in the Bactrian and dromedary camels, respectively. Furthermore, six elements (A, B, F, H, I, and L) are shared among cattle and camels and are partially found in other ruminants, suggesting a common ancestral origin of these retrotransposons. Conversely, elements C, D, E, and G are specific to camels.

Clinical report and genetic analysis of a neonate with genitourinary and/or brain malformation syndrome caused by a non-coding sequence variant of PPP1R12A.

Genitourinary and/or brain malformation syndrome (GUBS) is a recently discovered syndrome involving abnormalities of the neurological or urogenital system. PPP1R12A may be the pathological gene causing this syndrome. Currently, to our knowledge, there is only one study related to GUBS in the world. Here, we report a clinical case of a Chinese newborn with congenital micropenis caused by a non-coding sequence pathogenic variant of PPP1R12A, providing additional evidence on genetic causes of genital malformation. The genetic cause of the patient's malformation was detected using trio-whole exome sequencing and Sanger sequencing, and reverse transcription-PCR analysis was performed by constructing the minigene mutant plasmid in vitro. Genetic testing revealed a novel heterozygous variant, c.2666+3A>G, of the PPP1R12A gene of the patient. The parents at this site were wild-type, indicating that this might be a de novo variant. The minigene experiment showed that the c.2666+3A>G plasmid led to the deletion of 17 bp in exon 20, and a new mRNA product c.2650_2666del (p.Thr884IleTer2) with skipping of exon 20 was produced. This may lead to PPP1R12A haploinsufficiency and cause biological harm. To our knowledge, this is the first clinical study on a rare variant of PPP1R12A in the Chinese population. The c.2666+3A>G may lead to external genitalia malformation, such as congenital micropenis in male neonates. The results of this study further verified the correlation between GUBS and PPP1R12A haploinsufficiency and revealed the important role of a non-coding sequence variant in the pathogenesis of the disease.

Association of polymorphic variants of the mstn gene with live weight in kalmyk curled sheep and their crosses

The article provides a rationale for the feasibility of genetic marking of productivity at an early age. The phenotypic effect of genotypes of the polymorphic myostatin gene on live weight was studied in sheep of the Kalmyk curly breed and their crossbreeds with the Charollais and Dorper breeds. To achieve this, the following tasks were addressed: genotyping and recording of performance indicators of animals of the Kalmyk curly breed (KC), Charollais (C), Dorper-Kalmyk crossbred ewes (½D×½KC), purebred and crossbred young animals; determination of the influence of MSTN gene genotypes on live weight and average daily gain. The research subjects were KC breeding rams (n=6), C breeding rams (n=2), KC ewes (n=40); ½D×½KC crossbred ewes (n=40). The number and gender distribution of the offspring obtained were as follows: KC (n=26); ½C×½KC (n=32), ½C×¼D×¼KC (n=50). DNA extraction was performed from whole blood of the sheep. Animal genotyping was conducted using the PCR-RFLP method. A 337 bp exon 3 fragments of the MSTN gene was amplified using primers, and the results were analyzed using HRM analysis technology. It was found that among KC breeding rams, carriers of the M allele were more frequently identified, while among C breeding rams, carriers of the N allele were predominant, and among purebred and crossbred ewes, the prevalence of the M allele carriers was observed. Among purebred KC young animals and ½C×¼D×¼KC crossbreeds, carriers of the N allele predominated, whereas among ½C×½KC crossbreeds, carriers of the M allele were predominant. Population genetic analysis revealed a significant redistribution in favor of an increase in the frequency of the heterozygous MN genotype among the three-breed ½C×¼D×¼KC young animals, while among the two-breed ½C×½KC, the NN genotype was prevalent.

Contribution of the intron retained in the <i>Nxf1</i> gene transcript to the phylogeny of the order <i>Chiroptera</i>

This work is devoted to the possibilities of using a specific intron-containing block of the Nxf1 gene in phylogenetic studies.
 Our attention was drawn to the conservative intron of the Nxf1 (nuclear export factor) gene. This intron is a part of an evolutionarily conserved block formed with flanking 110 bp and 37 bp exons, similar in representatives of various taxonomic groups. This evolutionary conservative block in our previous works was designated cassette intron. The Nxf1 genes are found in all representatives of Opisthokonta, and may be a convenient object for phylogenetic studies.
 The Nxf1 gene sequences of seventeen representatives of the order Chiroptera obtained from publicly available databases (ensembl, ncbi). Alignment algorithm: MUSCLE. Programs: MEGA-X version 10.1.7, IQTree, Mesquite, MrBayes, and FigTree.v1.4.4. Estimation methods: Maximum Likelihood and Bayes Inference.
 The use of Nxf1 gene sequences that include only exons or only introns leads to unequal loss of accuracy in establishing evolutionary relationships in comparison with the model based on the complete gene sequence. Sequences involving all exons plus a cassette intron give the same result as the complete Nxf1 gene sequence.
 The obtained results indicate the importance of the cassette intron in the evolution of the Nxf1 gene of Chiroptera.

459 Regulation of the keratinocyte progenitor to differentiation switch by alternative mRNA splicing

Alternative splicing (AS) of messenger RNAs diversifies gene function by generating protein isoforms with different biological properties. Over 95% of human genes are alternatively spliced, but the functional consequences of most AS events are still not known. Here, using long-read RNA sequencing, we identified 5,826 AS events associated with human epidermal differentiation (percent spliced index >10%, FDR <0.01). AS events were enriched in genes associated with histone modification and phosphorylation (p < 0.001). The most common type of AS was cassette exon splicing, in which an intervening exon between two other exons is included or skipped in the mRNA sequence. We functionally characterized a cassette exon in mitogen-activated protein kinase (MAP3K7), which had not been previously studied in human epidermis. Basal epidermal progenitors preferentially express a shorter MAP3K7 isoform but switch to the longer isoform in suprabasal, differentiated epidermis with inclusion of an additional 81 bp exon. Using isoform-specific knockdown and overexpression, we demonstrate that inclusion of a single exon in MAP3K7 is necessary and sufficient to transition human epidermal keratinocytes from a replicating progenitor state with high clonogenic capacity into a post-mitotic differentiated state. We also performed binding motif analysis of sequences adjacent to epidermal AS events which identified 26 RNA binding proteins predicted to mediate mRNA splicing in epidermal keratinocytes, including ten with previously unstudied roles in the skin. In summary, our study defines the repertoire of alternative mRNA splicing events in the human epidermis and demonstrates the potential of this resource to identify functional AS events and splicing RBPs that regulate epidermal differentiation.

Phenotype-Based Genetic Analysis Reveals Missing Heritability of ABCA4-Related Retinopathy: Deep Intronic Variants and Copy Number Variations

PurposeTo identify the missing heritability of ABCA4-related retinopathy in a Chinese cohort.MethodsWe recruited 33 unrelated patients with ABCA4-related retinopathy carrying a monoallelic variant in ABCA4. All patients underwent ophthalmic examinations. Next-generation sequencing of the whole ABCA4 sequence, including coding and noncoding regions, was performed to detect deep intronic variants (DIVs) and copy number variations (CNVs).ResultsWe identified eight missing pathogenic ABCA4 variants in 60.6% of the patients (20/33), which comprised five DIVs and three CNVs. The five DIVs, including four novel (c.1555-816T>G, c.2919-169T>G, c.2919-884G>T, and c.5461-1321A>G) and one reported (c.4539+1100A>G), accounted for the missing alleles in 51.5% of the patients. Minigene assays showed that four novel DIVs activated cryptic splice sites leading to the insertions of pseudoexons. The three novel CNVs consisted of one gross deletion of 1273 bp (exon 2) and two gross duplications covering 25.2 kb (exons 28–43) and 9.4 kb (exons 38–44). The microhomology domains were identified at the breakpoints and revealed the potential mechanisms of CNV formation.ConclusionsDIVs and CNVs explained approximately two-thirds of the unresolved Chinese cases with ABCA4-related retinopathy. Combining results from phenotypic-directed screening, targeting the whole ABCA4 sequencing and in silico tools can help to identify the missing heritability.

Association of POU1F1 gene polymorphism with pre-pubertal body weight in Attappady Black goats of India

The present study was carried out to identify single nucleotide variations in Pituitary specific Transcription factor – 1 (POU1F1) gene and evaluate their association with body weight at different stages of growth before the onset of puberty in meat-type Attappady Black goats. A total of 100 Attappady goats were screened for polymorphism using PCR-SSCP. Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. Four fragments of POU1F1, 170 bp (exon 1), 234 bp (exon 3), 200 bp (exon 4) and 232 bp (exon 6) were analyzed. The 170 bp and 200 bp fragments were found to be monomorphic. The 234 bp fragment showed two diplotypes (GG and GA) with one synonymous single nucleotide polymorphism (g 0.42 C > T) in exon 3. The GA heterozygotes had significantly higher (p ≤ 0.05) body weight than GG homozygotes. at six months of age. The 232 bp fragment showed two diplotypes (CC and TC) with one novel synonymous single nucleotide polymorphism (g 0.180 C > T) in exon 6. The TC variants had significantly higher (p ≤ 0.05) body weight at six and nine months of age compared to the CC variants. The POU1F1 polymorphisms in the present study significantly contributed to variation in body weights at the onset of puberty at six and nine months of age indicating the role of POU1F1-mediated transcriptional regulation of genes coding for the various hormones of the neuro-endocrine system influencing growth and sexual maturity. The study suggests the possibility of using POU1F1 polymorphisms as markers for the selection of goats for higher pre-pubertal body weights in the genetic improvement programmes.

CD55-deficiency in Jews of Bukharan descent is caused by the Cromer blood type Dr(a-) variant.

The complement system regulator CD55 was initially found to carry the Cromer blood group system antigens, and its complete loss of function was subsequently revealed to cause a severe monogenic gastrointestinal syndrome characterized by protein-losing enteropathy and susceptibility to venous thrombosis. Here we present homozygosity to the CD55 c.596C>T; p.Ser199Leu variant, which was previously described as the Cromer Dr(a−) genotype, in two Bukharan Jewish CD55-deficiency patients with variable disease severity. We confirm that this missense variant causes aberrant splicing and deletion of 44 bp in exon 5, leading to premature termination and low expression of the CD55 protein. Furthermore, Patient 1 exhibited a mildly abnormal B cell immunophenotyping profile. By population screening we established that this variant is highly prevalent in the Bukharan Jewish population, with a carrier frequency of 1:17, suggesting that many similar patients are un- or mis-diagnosed. The phenotypic variability, ranging from abdominal pain when eating a high-fat diet to the full CD55-deficiency phenotype, is likely related to modifiers affecting the proportion of the variant that is able to escape aberrant splicing. Establishing the diagnosis of CD55-deficiency in a timely manner, even in patients with milder symptoms, may have a critical effect on their management and quality-of-life since treatment with the complement inhibitor eculizumab is highly effective in ameliorating disease manifestations. Awareness of founder mutations within certain populations can further guide genetic testing and prevent a diagnostic odyssey, by placing this CD55 variant high on the differential diagnosis.

Polymorphism detection of DGAT1 and Lep genes in Anatolian water buffalo (Bubalus bubalis) populations in Turkey.

Acyl-CoA: diacylglycerol–acyltransferase 1 (DGAT1) enzyme plays a key role in controlling the synthesis rate triglyceride from diacylglycerol. Leptin (LP, OB, obese) is an important hormone that synthesizes mostly from adipose tissue and regulates glucose metabolism and homeostasis. DGAT1 and Lep genes are closely related to reproduction, growth, milk yield and composition in water buffalo breeds. This study aimed to identify genetic variation in the DGAT1 and Lep gene regions in 150 water buffalo individuals from five different provinces of Turkey using DNA sequencing. A total of 38 nucleotide variations and indels have identified 761 bp long partial intron 2 and exon 3 and 5 UTR regions of the Lep gene in Anatolian water buffalo populations; 422 bp long partial exon 7–9 and exon 8 regions of DGAT1 gene were amplified and two mutations were defined in the point of 155 and 275 nucleotide that is three genotypes for S allele and Y allele of DGAT1 gene in intron 7 in Anatolian buffalo populations, respectively. These SNPs may have an effect on reproduction, growth, milk yield and composition in water buffalo populations and may prove to be useful for water buffalo breeding.

Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus).

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies.

Targeted Deep Sequencing of GATA1 more Sensitively Identifies Transient Myeloproliferative Disorder in Neonates with Down Syndrome

Down syndrome (DS) is caused by a constitutional trisomy 21 and up to 10% of neonates with DS develop a temporary accumulation of leukemic blasts in the peripheral blood, called transient myeloproliferative disorder (TMD). In most cases, TMD is a self-limiting disorder, however 20% of children develop serious symptoms and treatment with low dose cytarabine is then necessary. TMD is caused by a mutation in the GATA1 gene, occurring in cells with trisomy 21. Around 20% of children with TMD will develop a myeloid leukemia (ML) before the age of 4 years. It is a megakaryoblastic leukemia characterized by a GATA1 mutation identical to the mutation these children had in their TMD blast cells. The primary aim of this study was to define a population based frequency of TMD in DS neonates.In this nation-wide study we prospectively screened 368 peripheral blood (PB) samples from DS neonates (< 4 weeks old) in the Netherlands between January 1st 2008 and January 1st 2013 for TMD using morphology and immunophenotyping (IPT) (Dutch Trial Registry number NTR1667). TMD was defined as >5% myeloblasts in the PB. Patients with less than 5% of cells in the PB consistent with TMD using IPT were also included. PB samples of TMD positive patients were tested for GATA1 exon 2 mutations using Sanger sequencing. Patients with TMD were followed at least until the age of 4 years. TMD positive patients without a GATA1 exon 2 mutation using Sanger sequencing and TMD negative patients were retrospectively screened for GATA1 exon 2 and 3 mutations using targeted deep sequencing. Using this technique we could detect mutations with a frequency of 0.1%.In the prospective part of the study, TMD was diagnosed in 45 of the 368 cases screened (12.2%). In 41 of 45 neonates with TMD, GATA1 mutation analysis was performed. In 4 cases no GATA1 PCR was performed. In 28 cases a GATA1 exon 2 mutation was detected (28/41=68%), while in 13 cases no GATA1 mutation was detected.It was recently shown that there is a subgroup of DS neonates who do not have TMD by conventional criteria, but when more sensitively screened do harbour a GATA1 mutation (Roberts et al, Blood 2013). Therefore using targeted deep sequencing we retrospectively screened the 13 TMD cases without a GATA1 mutation by Sanger sequencing and 2 of the 4 cases that were not screened at TMD diagnosis. No GATA1 exon 3 mutations were detected. In 9 out of 15 patients a GATA1 mutation was detected using targeted deep sequencing. Both cases not prospectively Sanger sequenced were positive. 7 of 13 TMD samples tested negative using Sanger sequencing, using targeted deep sequencing were positive for a GATA1 mutation (median mutational frequency 6,8%). Overall, 43 TMD patient samples were screened using Sanger sequencing and/or targeted deep sequencing and 38 of these tested GATA1 positive (88%).In addition, we retrospectively screened 319 of the 323 peripheral blood samples of the TMD negative DS neonates for GATA1 using targeted deep sequencing. No GATA1 exon 3 mutations were detected. In 11 out of 319 TMD negative cases we detected a GATA1 exon 2 mutation with a median mutational frequency of 0,8%.14 children with DS born in the Netherlands between 1-1-2008 and 31-12-2013 were diagnosed with ML DS (follow-up until 31st of dec 2016). Of these 14 children, 7 were screened neonatally and in 6 cases TMD was diagnosed. Therefore 6 out of 45 TMD patients identified using conventional screening developed ML DS (13.3%). Out of the 11 ‘subclinical’ TMD cases only detected by targeted deep sequencing GATA1 screening, one patient was later diagnosed with ML DS (1/11=9.1%). The patient's neonatal blood sample harboured two different GATA1 exon 2 mutations (14 base pair (bp) insertion in 0,24% of cells and a 1 bp insertion in 2,6% of cells). In the ML DS cells of this patient the identical 1 bp exon 2 insertion in GATA1 was detected.In conclusion, conventional screening for TMD using immunophenotyping en morphology identified TMD in 12% of the screened DS neonates. An additional 3.4% can be detected using targeted deep sequencing. This is clinically relevant as these children are at risk of developing ML DS. Therefore, for future clinical studies we are developing targeted deep sequencing methods screening for GATA 1 in neonatal DS PB samples. DisclosuresKaspers:Janssen-Cilag: Research Funding.

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases

Microexons are small sized (≤51 bp) exons which undergo extensive alternative splicing in neurons, microglia, embryonic stem cells, and cancer cells, giving rise to cell type specific protein isoforms. Due to their small sizes, microexons provide a unique challenge for the splicing machinery. They frequently lack exon splicer enhancers/repressors and require specialized neighboring trans-regulatory and cis-regulatory elements bound by RNA binding proteins (RBPs) for their inclusion. The functional consequences of including microexons within mRNAs have been extensively documented in the central nervous system (CNS) and aberrations in their inclusion have been observed to lead to abnormal processes. Despite the increasing evidence for microexons impacting cellular physiology within CNS, mechanistic details illustrating their functional importance in diseases of the CNS is still limited. In this review, we discuss the unique characteristics of microexons, and how RBPs participate in regulating their inclusion and exclusion during splicing. We consider recent findings of microexon alternative splicing and their implication for regulating the function of small GTPases in the context of the microglia, and we extrapolate these findings to what is known in neurons. We further discuss the emerging evidence for dysregulation of the Rho GTPase pathway in CNS diseases and the consequences contributed by the mis-splicing of microexons. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

Association of inhibin alpha gene polymorphism with litter size and growth in Malabari goats of India

Abstract The present study was carried out to identify single nucleotide variations in inhibin alpha (INHA)geneto evaluate its association with production traits in Malabari goats. A total of 251 Malabari adult goats were screened for polymorphism using PCR-SSCP.Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. Five fragments, 274 bp, 220 bp, 160 bp, 287 bp and 253 bp covering the two exons of INHA were analyzed. The 274 bp fragment showed two diplotypes (AA and AB) and sequencing revealed one synonymous SNP (c.129 G > A) in exon 1. The AA genotype showed significant (p ≤ 0.05) association with body length. Similar monomorphic band pattern was observed for 220 bp fragment of exon 2. The 160 bp fragment exhibited two genotypes DD and DE with a synonymous SNP c.567 G > A in exon 2. Goats carrying DD genotype had significantly (p ≤ 0.05) higher adult body weight and body length compared to DE genotype. Three diplotypes, HH, HJ and JJ with a synonymous SNP, c.651 G > A were observed at 287 bp fragment. Significantly (p C. Genotypes of INHA 253 bp (exon 2) locus had significant (p ≤ 0.05) influence on litter size, body weight and trunk circumference. The PP and PQ showed a significantly higher litter size and trunk circumference (p ≤ 0.05) compared to QQ genotypes.Present study indicated the potential role of INHA gene on reproduction and growth, hence it can be used as a candidate gene in the improvement of Malabari goats.

Comparative Functional Analysis in vitro of 2 COL4A5 Splicing Mutations at the Same Site in 2 Unrelated Alport Syndrome Chinese Families

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.

Molecular cloning and variant analysis of the TSHR gene in goose (Anser cygnoides)

ABSTRACT 1. The thyroid-stimulating hormone receptor (TSHR) is a glycoprotein hormone receptor which has a pivotal role in metabolic regulation and photoperiod control during reproduction in birds and mammals. However, the molecular characterisation of TSHR in goose is unknown. 2. The goose TSHR cDNA (TSHR-1) is 2334 bp in length and encodes a protein of 763 amino acids. This trial identified another three novel splice variants of goose TSHR, TSHR-2 (lacking the exon 3 in TSHR-1 transcript), TSHR-3 (lacking the exon 6 in the TSHR-1 transcript) and TSHR-4 (lacking 12 bp of exon 8 and the entire exon 9 in the TSHR-1 transcript). 3. Bioinformatics analysis indicated that all the deduced TSHR amino acid sequences contained seven putative transmembrane domains, and the TSHR-3 protein lacked one potential N-linked glycosylation site (N-E-S) compared to the other three deduced proteins. 4. A phylogenetic tree based on amino acid sequences showed that the goose TSHR protein was closely related to those of other avian species, especially duck and chickens. 5. One microsatellite and three single nucleotide polymorphisms (SNPs) were identified. For the c1109A/G locus, AA and GA genotypes were found in the Zhedong-White goose population, GG and GA genotypes were detected in the Landes goose population, but the AA genotype was only detected in the other four goose populations. 6. All the information derived from this study can facilitate further studies on the functions of the goose TSHR gene.

The Polymorphic Gene of Single Nucleotide Polymorphism (SNP) of Phytoene Synthase (PSY) to Characterize Carotenoids Yellow Root Cassava

Cassava (Manihot esculenta Crantz.) is a carbohydrate sources containing a limited amount of micronutrients, but some genotypes contain β-carotene as the precursor of vitamin A in the storage roots and leaves. Improvement of β-caroteneand minerals such as Fe / Zn content of cassava’s nutrition is mostly through by biofortification program. The storage root of β-carotene recognized by a yellow or yellowish color while the apical shoots with red to purplish. β-carotenein carotenoid biosynthetic pathway is an expression of the phytoene synthase (PSY) gene. The MePSY2 gene, one of the three MePSY family is the key gene to characterize carotenoids related gene in cassava. In this study, sequencing of the two cassava fulllenght PSY genomic DNA was carried out in conserved areas in the PSY gene region (PSY1 and PSY2) from the DNA of the cassava leaves. Adira1, Carvita25 and Ubi Kuning are yellow root storage genotypes (K1, K2 and K3) while Adira4 and Menti are white root storage genotypes (P1 and P2). Carvita25 is induced somaclonal variant of the Adira4 genotype. Contiq and consensus of nucleotide base sequences from the five cassava genotypes and CM3306-4 cultivars (acc GU111715.1) as references were analysed using the lasergene DNASTAR sequence analysis program. The results of the alignment of the base sequence constituent of the MePSY2 gene showed that the PSY2 gene with amplified genome length was 2,380 base pairs (bp) consisting of 1,140 bp exon region and 1,240 bp intron region. In the conserved coding region, there was a difference of one nucleotide base, that is, base C in two white tuber cassava genotypes replaced with A in three yellow tuber cassava genotypes in the 1.485 base (C1.485A). The SNP converts the amino acid (aa) alanine (A) to aspartic acid (D) at the 191th (A191D). Single Nucleotide polymorphism in conserved coding region can be used further as carotenoid marker for plant breeding of yellow root cassava. Keywords: β carotene, PSY gene, polymorphic gene SNP, yellow root cassava.