Splice Site Of Exon Research Articles

Generation of non-human primate models of Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Despite the considerable research efforts, exact pathomechanisms still remain unknown, and no effective treatment is available. AD research field have mainly utilized mouse models for decades, but species differences between rodents and primates may constrain us from understanding the precise disease mechanisms. We utilized Transcription Activator-Like Effector Nuclease (TALEN) to delete the 3' splicing site (SS) of exon 9 of the presenilin 1-encoding gene (PSEN1) in the common marmoset (Callithrix jacchus), a small new world primate. We successfully deleted the 3' SS of the PSEN1 gene (PSEN1-ΔE9) in the marmoset embryos by microinjection of TALEN, and obtained mutant PSEN1 neonates. Sequence analysis of mRNA in these embryos and neonates confirmed skipping of exon 9 as expected, which has been reported as a pathogenic mutation causing early onset familial AD. Whole genome sequencing illustrated an inclusive absence of off-target effects. Quantitative analysis of amyloid-β (Aβ) in the cultured medium of primary fibroblasts revealed elevation of Aβ42/Aβ40 ratio in PSEN1-ΔE9 fibroblasts. WB analysis detected uncleaved full-length PS1 protein as well as N- and C-terminal fragments of PS1 protein in the PSEN1-ΔE9 fibroblasts. To our knowledge, we have successfully generated the word's first AD model of non-human primates. These animals are expected to show early amyloid pathology in the brain and to contribute to cutting-edge research to elucidate primate-specific mechanism in AD. In addition, we will make the mutant animals available to researchers worldwide in the fight against AD in the future.

A Presumed Synonymous Mutation of PKD2 Caused Autosomal Dominant Polycystic Kidney Disease in a Chinese Family.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by the pathogenic mutation of PKD1 or PKD2 gene and usually affects bilateral kidneys. Synonymous mutations are generally assumed to be neutral as they do not alter amino acids. Herein, we described an extremely rare ADPKD child caused by a heterozygous synonymous mutation of PKD2 gene accompanied by massive proteinuria and congenital solitary kidney. Clinical characteristics of the patients were summarized. Whole-exome sequencing was performed to screen the disease-causing gene mutation, and reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were applied to analyze the impact of the identified mutation on gene transcription and splicing. Polycystic changes were found in the solitary kidney of a girl initially presented with nephrotic-range proteinuria. Thereafter her mother and 2 other family members were diagnosed to be ADPKD. Whole-exome sequencing of the proband identified a heterozygous synonymous mutation (c.1716G>A, p.Lys572=) located in the splicing site of exon 7 in PKD2 gene, which was co-segregated with the PKD phenotype in the family. RT-PCR and direct sequencing of amplified products revealed that this heterozygous synonymous mutation led to exon7 skipping in PKD2 gene. We reported an extremely rare child case of ADPKD2 in combination with solitary kidney and nephrotic-range proteinuria, and firstly confirmed the pathogenicity of a heterozygous synonymous mutation (c.1716G>A) in PKD2 gene. The results indicate that synonymous mutations should not be excluded from disease-causing if they are located in splicing site of an exon.

A Robust Ex-Vivo Erythropoiesis System For Studying Transcriptional Regulation Of Globin Genes In Thalassaemia

Ex-vivo differentiation of hematopoietic stem cells and progenitors to erythroid cells helped tremendously in our understanding of transcriptional and epigenetic regulation of normal erythroid differentiation as large number of nucleated cells can be generated by these protocols. However, to study the disease mechanisms in haematological diseases, there are practical difficulties in obtaining mobilized CD34+ cells from the patients. Alternatively, peripheral blood mononuclear cells (PBMNCs), which have a small fraction of haematopoietic progenitors, have also been used to obtain cultured erythroid cells (cErCs). Though PBMNCs is an alternative source, the current protocols using these cells do not generate sufficient number of cErCs, which is a major hurdle for performing transcriptional and epigenetic studies in erythroid cells from patients with β thalassaemia and sickle cell disease. We established a modified two phase culture protocol using a serum free basal medium with IL-3, IL-6, Flt3, SCF in phase I and Epo, SCF, IL-3 and iron compounds in phase II and could generate up to 108 cells from PBMNCs isolated from 5-10ml blood of patients with homozygous β thalassaemia and those who were compound heterozygous for β thalassaemia and HPFH or δβ thalassaemia. Real time quantitative PCR (qPCR) analysis showed steady increase in the expression of α and β globin genes in different stages of erythropoiesis. As we obtained a large number of nucleated cells using this protocol, we could perform chromatin immunoprecipitation (ChIP) to study the binding of RNA Polymerase II (RNAPII), transcription cofactors, CBP and p300, and histone modifications at the regulatory sequences of β globin cluster. High levels of RNAPII was found at the coding sequences of β globin gene in the cErCs from both normal individuals and patients with splice site mutations suggesting that these mutations do not cause significant reduction in the transcription rate. Both CBP and p300 were found to bind at the HS3 and HS2 regions of locus control region in both normal and β thalassaemia cultures though they were not found at the promoter sequences of actively transcribed genes in the β globin cluster. In the patients compound heterozygous for β thalassaemia and HPFH-3 or (Aγδβ)° deletion/Inversion, we found increased γ globin gene expression compared to the normal controls and ChIP also showed high levels of RNAPII occupancy at the coding sequences of γ globin gene. As it has been shown earlier that alternative splicing is a transcriptional regulatory mechanism for cell specific expression of genes, we performed experiments to identify the alternatively spliced transcripts of β globin gene and their co-ordinated expression in human erythropoiesis. RT-PCRs and sequencing of β globin cDNA using various sets of primers that bind at the upstream, downstream and coding sequences showed 3 aberrantly spliced transcripts formed by the cryptic splice sites in exon1 and 4 novel transcripts by the activation of splice sites at the 5'UTR. Interestingly, 5' splice site mutations in intron 1 did not activate cryptic splice sites in the coding sequences and instead, they activated specific cryptic splice sites upstream b globin gene. Q-PCR with chimeric primers for the exact quantitation of the splice variants showed that the splice site mutations also cause significant reduction (> 90%) in expression of normally spliced transcripts suggesting that nonsense mediated decay is not the major mechanism for the reduced β globin production by these mutations. We found high levels of one of the aberrantly spliced transcripts with 5' splice site in exon 1 (∼20%) in normal individuals. All the splice variants showed steady increase in their expression from early to late stages of erythropoiesis. Our study shows that ex-vivo erythropoiesis system is an invaluable tool for studying the mechanism of globin gene regulation and switching. Identification of specific splice variants in cErCs of normal individuals and in patients with β thalassaemia could define the molecular mechanisms by which splice site mutations cause β-thalassaemia. The presence of abundant splice variants in normal controls suggests that β globin gene is regulated by multiple promoters and other regulatory elements and identification of these sequences will help in understanding the molecular basis of human globin gene regulation. Disclosures:No relevant conflicts of interest to declare.

High-Resolution SNP-Array Profiling of “Ultra-High Risk” CLL Cases Reveals New Recurrent Genomic Alterations

High-Resolution SNP-Array Profiling of “Ultra-High Risk” CLL Cases Reveals New Recurrent Genomic Alterations

Phenotypic implications of pathogenic variant types in Pompe disease.

Newborn screening and therapies for Pompe disease (glycogen storage disease type II, acid maltase deficiency) will continue to expand in the future. It is thus important to determine whether enzyme activity or type of pathogenic genetic variant in GAA can best predict phenotypic severity, particularly the presence of infantile-onset Pompe disease (IOPD) versus late-onset Pompe disease (LOPD). We performed a retrospective analysis of 23 participants with genetically-confirmed cases of Pompe disease. The following data were collected: clinical details including presence or absence of cardiomyopathy, enzyme activity levels, and features of GAA variants including exon versus intron location and splice site versus non-splice site. Several combinations of GAA variant types for individual participants had significant associations with disease subtype, cardiomyopathy, age at diagnosis, gross motor function scale (GMFS), and stability of body weight. The presence of at least one splice site variant (c.546 G > C/p.T182 = , c.1076-22 T > G, c.2646 + 2 T > A, and the classic c.-32-13T > G variant) was associated with LOPD, while the presence of non-splice site variants on both alleles was associated with IOPD. Enzyme activity levels in isolation were not sufficient to predict disease subtype or other major clinical features. To extend the findings of prior studies, we found that multiple types of splice site variants beyond the classic c.-32-13T > G variant are often associated with a milder phenotype. Enzyme activity levels continue to have utility for supporting the diagnosis when the genetic variants are ambiguous. It is important for newly diagnosed patients with Pompe disease to have complete genetic, cardiac, and neurological evaluations.

Two SMARCAD1 Variants Causing Basan Syndrome in a Canadian and a Dutch Family

Basan syndrome is an autosomal dominant genodermatosis characterized by congenital adermatoglyphia, transient congenital facial milia, neonatal acral bullae, and absent or reduced sweating. Basan syndrome is rare and has been reported in only 10 kindreds worldwide. It is caused by variants in the skin-specific isoform of SMARCAD1, which starts with an alternative exon 1. All reported variants, except for one large deletion, are point mutations within the donor splice site of the alternative exon 1. In this paper, we report two families with Basan syndrome and describe two SMARCAD1 variants. In one family, we have identified a complex structural variant (a deletion and a nontandem inverted duplication) using whole-genome optical mapping and whole-genome sequencing. Although this variant results in the removal of the first nine exons of SMARCAD1 and exon 1 of the skin-specific isoform, it manifested in the typical Basan phenotype. This suggests that unlike the skin-specific isoform, a single copy of full-length SMARCAD1 is sufficient for its respective function. In the second family, whole-exome sequencing revealed a deletion of 12 base pairs spanning the exon‒intron junction of the alternative exon 1 of the skin-specific SMARCAD1 isoform. In conclusion, we report two additional families with Basan syndrome and describe two SMARCAD1 pathogenic variants.

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Complete CSN1S2 Characterization, Novel Allele Identification and Association With Milk Fatty Acid Composition in River Buffalo.

The αs2-casein is one of the phosphoproteins secreted in all ruminants' milk, and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterized in detail in buffaloes with only two alleles detected (reported as alleles A and B), and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realized at the donor splice site of exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, six in the promoter, and three SNPs in the coding region (g.11072C>T, g.12803A>T, and g.14067A>G) with two of them responsible for amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at the donor splice site of exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E, and F) responsible for seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form, and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, and B2 are deleted alleles because all are characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes, and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acid composition was carried out. The SNP g.14067A>G showed a significant association (P < 0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker-assisted selection programs aiming for the improvement of buffalo milk fatty acid composition.

Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes

Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p.Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance.

Generation of nonhuman primate models of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is the leading cause of dementia worldwide. Despite the considerable research efforts, exact pathomechanisms still remain unknown, and no effective treatment is available. AD research field have mainly utilized mouse models for decades, but species differences between rodents and primates may constrain us from understanding the precise disease mechanisms.MethodWe utilized Transcription Activator‐Like Effector Nuclease (TALEN) to delete the 3’ splicing site (SS) of exon 9 of the presenilin 1‐encoding gene (PSEN1) in the common marmoset (Callithrix jacchus), a small new world primate.ResultWe successfully deleted the 3’ SS of the PSEN1 gene (PSEN1‐ΔE9) in the marmoset embryos by microinjection of TALEN, and obtained mutant PSEN1 neonates. Sequence analysis of mRNA in these embryos and neonates confirmed skipping of exon 9 as expected, which has been reported as a pathogenic mutation causing familial AD. Quantitative analysis of amyloid‐β (Aβ) in the cultured medium of primary fibroblasts revealed elevation of Aβ42/Aβ40 ratio in PSEN1‐ΔE9 fibroblasts. WB analysis detected un‐cleaved full‐length PS1 protein as well as N‐ and C‐terminal fragments of PS1 protein in the PSEN1‐ΔE9 fibroblasts.ConclusionTo our knowledge, we have successfully generated the word’s first AD model of non‐human primates. These animals are expected to show early amyloid pathology in the brain and to contribute to cutting‐edge research to elucidate primate‐specific mechanism in AD. In addition, we will make the mutant animals available to researchers worldwide in the fight against AD in the future.

Nonsense-associated altered splicing of MAP3K1 in two siblings with 46,XY disorders of sex development

Although splicing errors due to single nucleotide variants represent a common cause of monogenic disorders, only a few variants have been shown to create new splice sites in exons. Here, we report an MAP3K1 splice variant identified in two siblings with 46,XY disorder of sex development. The patients carried a maternally derived c.2254C>T variant. The variant was initially recognized as a nonsense substitution leading to nonsense-mediated mRNA decay (p.Gln752Ter); however, RT-PCR for lymphoblastoid cell lines showed that this variant created a new splice donor site and caused 39 amino acid deletion (p.Gln752_Arg790del). All transcripts from the variant allele appeared to undergo altered splicing. The two patients exhibited undermasculinized genitalia with and without hypergonadotropism. Testosterone enanthate injections and dihydrotestosterone ointment applications yielded only slight increase in their penile length. Dihydrotestosterone-induced APOD transactivation was less significant in patients’ genital skin fibroblasts compared with that in control samples. This study provides an example of nonsense-associated altered splicing, in which a highly potent exonic splice site was created. Furthermore, our data, in conjunction with the previous data indicating the association between MAP3K1 and androgen receptor signaling, imply that the combination of testicular dysgenesis and androgen insensitivity may be a unique phenotype of MAP3K1 abnormalities.

Phylogenetic Analyses of Glycosyl Hydrolase Family 6 Genes in Tunicates: Possible Horizontal Transfer.

Horizontal gene transfer (HGT) is the movement of genetic material between different species. Although HGT is less frequent in eukaryotes than in bacteria, several instances of HGT have apparently shaped animal evolution. One well-known example is the tunicate cellulose synthase gene, CesA, in which a gene, probably transferred from bacteria, greatly impacted tunicate evolution. A Glycosyl Hydrolase Family 6 (GH6) hydrolase-like domain exists at the C-terminus of tunicate CesA, but not in cellulose synthases of other organisms. The recent discovery of another GH6 hydrolase-like gene (GH6-1) in tunicate genomes further raises the question of how tunicates acquired GH6. To examine the probable origin of these genes, we analyzed the phylogenetic relationship of GH6 proteins in tunicates and other organisms. Our analyses show that tunicate GH6s, the GH6-1 gene, and the GH6 part of the CesA gene, form two independent, monophyletic gene groups. We also compared their sequence signatures and exon splice sites. All tunicate species examined have shared splice sites in GH6-containing genes, implying ancient intron acquisitions. It is likely that the tunicate CesA and GH6-1 genes existed in the common ancestor of all extant tunicates.

A novel missense mutation of RPGR identified from retinitis pigmentosa affects splicing of the ORF15 region and causes loss of transcript heterogeneity

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP), in which exon open reading frame 15 (ORF15) of RPGR has been implicated to play a substantial role. We identified a novel hemizygous missense mutation E585K of RPGR from whole-exome sequencing of RP. RNA-Seq analysis and functional study were conducted to investigate the underlying pathogenic mechanism of the mutation. Our results showed that the mutation actually affected RPGR ORF15 splicing. RNA-Seq analysis of the human retina followed by validation in cells revealed a complex splicing pattern near the 3′ boundary of RPGR exon 14 in the ORF15 region, resulting from a variety of alternative splicing events (ASEs). The wildtype RPGR mini-gene expressed in human 293T cells confirmed these ASEs in vitro. In contrast, without new RNA species detected, the mutant mini-gene disrupted the splicing pattern of the ORF15 region, and caused loss of RPGR transcript heterogeneity. The RNA species derived from the mutant mini-gene were predominated by a minor out-of-frame transcript that was also observed in wildtype RPGR, resulting from an upstream alternative 5′ splice site in exon 14. Our findings therefore provide insights into the influence of RPGR exonic mutations on alternative splicing of the ORF15 region, and the underlying molecular mechanism of RP.

Two novel intragenic variants in the FMR1 gene in patients with suspect clinical diagnosis of Fragile X syndrome and no CGG repeat expansion

The major and most well-studied genetic cause of Fragile-X syndrome (FXS) is expansion of a CGG repeat in the 5′-UTR of the FMR1 gene. Routine testing for this expansion is performed globally. Overall, there is a paucity of intragenic variants explaining FXS, a fact which is being addressed by a more systematic application of whole exome (WES) and whole genome (WGS) sequencing, even in the diagnostic setting. Here we report two families comprising probands with a clinical suspicion of FXS and no CGG repeat expansions. Using WES/WGS we identified deleterious variants within the coding region of FMR1 in both families. In a family from Finland we identified a complex indel c.1021-1028delinsTATTGG in exon 11 of FMR1 which gives rise to a frameshift and a premature termination codon (PTC), p.Asn341Tyrfs*7. Follow-up mRNA and protein studies on a cell line from the proband revealed that although the mRNA levels of FMR1 were not altered, Fragile X Mental Retardation 1 Protein (FMRP) was undetectable. Additionally, we identified a variant, c.881-1G > T, affecting the canonical acceptor splice site of exon 10 of FMR1 in an Australian family. Our findings reinforce the importance of intragenic FMR1 variant testing, particularly in cases with clinical features of FXS and no CGG repeat expansions identified.

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.

Activation of cryptic splicing in bovine WDR19 is associated with reduced semen quality and male fertility.

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.

SUN-078 Clinical, Hormonal, Psychosexual Aspects, Gonadal Tumors and Genetic Background of an Androgen Insensitivity Syndrome Cohort

Introduction: Androgen Insensitivity Syndrome (AIS) is the most common cause of Differences of Sexual Development (DSD) in 46, XY individuals. It is a X-linked genetic disease caused by allelic variants in the Androgen Receptor Gene (Xq11-12), leading to 3 different phenotypes: Complete (CAIS), Partial (PAIS) or Mild (MAIS). Methods: Patients with clinical suspicion of AIS (familial history, atypical genitalia, primary amenorrhea and/or inguinal hernia) performed hormonal serum measurements (LH, FSH, estradiol, testosterone) and molecular sequencing of the ARgene, including all exonic regions (8 exons) and the 5’UTR region. Psychosexual variables (gender identity, gender role and sexual orientation) were evaluated through questionnaires. Gender identity was also evaluated through projective psychological test (HTP test). A histopathological study and immunostainining of CD240 and OCT3/4 were carried out for all individuals submitted to gonadectomy.Results: This cohort is made up of 64 individuals: CAIS (n=26) and PAIS (n=38), from 46 different families (24 PAIS; 22 CAIS). Inguinal hernia was the first clinical presentation in 35% of CAIS. Among the PAIS, 20 (52%) were assigned as female at birth, while 18 (48%) as male. Among In the group of PAIS, external genitalia virilization (Sinnecker score) influenced sex assignment (p<0.01). Final height and weight were similar between PAIS and CAIS. Furthermore, gender identity at adulthood, gender role at childhood and sexual orientation were in agreement with sex assignment in virtually all cases of both PAIS and CAIS. No gender change was observed. Molecular diagnosis was obtained in 96% of CAIS and in 87% of PAIS. There were 10 novel AR allelic variants (4 in CAIS - 2 small deletions, 1 missenseand 1 at splicing site and PAIS - 5 missenseand 2 synonymous variants (both causing a new exonic splicing site leading to a short AR protein). LH ranged from 9 to 48 UI/L (mean 19), testosterone from 190 to 1500 ng/dL (mean 438), without phenotype differences. Seminoma was identified in 2 out 24 (8%) individuals with CAIS (at 19 and 20 years of age). This rate was higher taking into account only those who underwent gonadectomy after puberty (16 years old or later: 2 out 17 (12%). Among PAIS there were 2 cases of NICG (at 3 and 19 years of age) and none of seminoma. Conclusion: Hormonal levels did not enable us to differ PAIS and CAIS. Inguinal hernia is a common clinical presentation of AIS. External genitalia appearance in PAIS influenced sex assignment. The psychosexual development in AIS usually complies with sex assignment. No gender change was observed. There is a risk of seminoma in CAIS, especially after puberty, which is not low enough to be ignored.

Identification and functional characterization of a novel splicing variant in the F8 coagulation gene causing severe hemophilia A

BackgroundWe have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. ObjectivesTo determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. MethodsPatient peripheral blood mononuclear cells were isolated and differentiated into monocyte‐derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site‐directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA‐mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti‐FVIII antibodies and FVIII antigen, by ELISA. ResultsHA‐MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti‐FVIII antibodies. ConclusionsHerein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.

HnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3' Splice Sites.

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3′ splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.