Intronic Site Research Articles

Profiling human pathogenic repeat expansion regions by synergistic and multi-level impacts on molecular connections.

Whilst DNA repeat expansions cause numerous heritable human disorders, their origins and underlying pathological mechanisms are often unclear. We collated a dataset comprising 224 human repeat expansions encompassing 203 different genes, and performed a systematic analysis with respect to key topological features at the DNA, RNA and protein levels. Comparison with controls without known pathogenicity and genomic regions lacking repeats, allowed the construction of the first tool to discriminate repeat regions harboring pathogenic repeat expansions (DPREx). At the DNA level, pathogenic repeat expansions exhibited stronger signals for DNA regulatory factors (e.g. H3K4me3, transcription factor-binding sites) in exons, promoters, 5'UTRs and 5'genes but were not significantly different from controls in introns, 3'UTRs and 3'genes. Additionally, pathogenic repeat expansions were also found to be enriched in non-B DNA structures. At the RNA level, pathogenic repeat expansions were characterized by lower free energy for forming RNA secondary structure and were closer to splice sites in introns, exons, promoters and 5'genes than controls. At the protein level, pathogenic repeat expansions exhibited a preference to form coil rather than other types of secondary structure, and tended to encode surface-located protein domains. Guided by these features, DPREx ( http://biomed.nscc-gz.cn/zhaolab/geneprediction/# ) achieved an Area Under the Curve (AUC) value of 0.88 in a test on an independent dataset. Pathogenic repeat expansions are thus located such that they exert a synergistic influence on the gene expression pathway involving inter-molecular connections at the DNA, RNA and protein levels.

ODP398 A Novel NR5A1 Gene Mutation Causing 46, XY DSD Without Adrenal Insufficiency in an Immigrant Boy from Dominican Republic

Abstract Background The NR5A1 gene encodes steroidogenic factor-1 (SF1), a key transcription factor involved in adrenal and gonadal development. We present a 12-year old boy with a novel, dominant, heterozygous, and probably pathogenic variant of this gene (c.102+1G>C) resulting in incomplete masculinization [46, XY disorder of sex development (DSD)]. Clinical case: This 12-year-old boy, who had immigrated from the Dominican Republic to the United States, was referred at 8 years of age with a diagnosis of congenital adrenal hyperplasia (CAH). He received hCG injections as a neonate and had been prescribed hydrocortisone 7 mg/m 2 /day and fludrocortisone 0.1 mg/day orally for CAH, although compliance was poor. After migrating to the United States, his supply of medications depleted, and he was taken to a local emergency room with fatigue and abdominal pain. Vital signs and electrolyte levels were normal. There was no history of adrenal crisis, testes were not palpable, and inguinal testicles measuring 2.8×1.1 cm on the right and 2.2×1 cm on the left were reported on MRI. When he presented to the pediatric endocrinology clinic, his mother denied history of consanguinity, PCOS, or use of hormones during pregnancy. She reported a history of two spontaneous miscarriages with father of patient and a younger healthy son from another partner. Physical exam showed ambiguous genitalia with underdeveloped scrotum, mild hyperpigmentation, bilateral non-palpable testicles within scrotum, severe penoscrotal hypospadias, stretched penile length (SPL) of 2cm×1.5 cm, and chordee. He had axillary hair and a Tanner stage 2 for pubic hair. A high-dose ACTH stimulation study (250 mcg IV cosyntropin) showed a peak cortisol level at 19.6 μg/dL, peak 17-hydroxyprogesterone of 100 ng/dL, and baseline and peak pregnenolone levels of 6 ng/dL and 43 ng/dL, respectively, with low to normal values of other precursors ruling out CAH. Gonadotropin levels were prepubertal (LH < 0.3 mIU/mL, FSH 2.9 mIU/mL), testosterone <4 ng/dL, and dihydrotestosterone 0.73 ng/dL. Chromosomal studies confirmed 46, XY. With a clinical diagnosis of 46,XY DSD (undervirilized male) and male gender identity, a 2-stage orchiopexy was completed. After a six-month course of monthly 50 mg cypionate testosterone, SPL improved to 4 cm and pubic hair progressed to Tanner stage 3. Surgical repair of penoscrotal hypospadias and chordee were then completed. A 46, XY DSD/complete gonadal dysgenesis DNA panel sequencing identified a novel, dominant, heterozygous, likely pathogenic variant of the NR5A1 gene [c.102+1G>C]. This is a canonical splice donor site of intron 2. Conclusion A novel sequence change in the NR5A1 gene causing a severe phenotype of 46,XY undervirilized male is presented here in a boy originally misdiagnosed with CAH. This case emphasizes the need for complete evaluation with molecular genetic screening for those patients with 46,XY DSDs. Presentation: No date and time listed

Upgrading an intronic TMEM67 variant of unknown significance to likely pathogenic through RNA studies and community data sharing.

A couple of Ashkenazi Jewish descent was referred for an early anatomy scan at 14+2weeks of gestation following a previous pregnancy termination due to posterior encephalocele and enlarged kidneys. The index pregnancy was also positive for several fetal abnormalities, including enlarged kidneys with cystic dysplasia and abnormal cerebellar morphology highly suggestive of Joubert syndrome. Trio exome sequencing revealed compound heterozygosity for variants in the TMEM67 gene: a known pathogenic maternally inherited variant found in trans with a paternal intronic variant of unknown significance. RNA analysis revealed that the intronic variant creates a cryptic acceptor splice site in intron 12, leading to the insertion of 22 bp and causing a frameshift with a premature stop codon. This analysis enabled the reclassification of the intronic variant to likely pathogenic. This information empowered the couple to make informed reproductive choices and opt for preimplantation genetic testing (PGT) for future pregnancies.

Evaluation of Mean Percentage of Full-Length SMN Transcripts as a Molecular Biomarker of Spinal Muscular Atrophy.

The elevation of SMN transcript and protein level remains the principal aim of SMA therapy. Still, there is no standard molecular biomarker for the assessment of its efficacy. In the current study, we tested three methods of SMN transcript level measurement using real-time RT-PCR, quantitative fluorescent RT-PCR, and a semiquantitative RT-PCR gel densitometric assay. We examined several potential mRNA-based biomarkers and examined their sensitivity and reliability by comparing the obtained values in peripheral blood mononuclear cells of SMA patients, SMA carriers, and healthy individuals. We found that the mean percentage of full-length (FL-SMN) transcripts relative to the total sum of FL-SMN and exon 7-deleted (Δ7 SMN) transcripts detected by semiquantitative and quantitative fluorescence RT-PCR differed significantly between the three analyzed groups. The relevance of this biomarker was proven in an SMN2-targeting therapeutic experiment. We showed that the values of the biomarker changed significantly in SMA fibroblast cell cultures after treatment with therapeutic antisense oligonucleotides targeting the ISS-N1 site in intron 7 of the SMN2 gene. The obtained results indicate the convenience of using the mean percentage of FL-SMN transcripts determined by semiquantitative and quantitative fluorescence RT-PCR as a putative biomarker for the assessment of SMA therapy efficacy in vitro.

A splicing silencer in SMN2 intron 6 is critical in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disease caused by homozygous deletions or mutations of the SMN1 gene. SMN2 is a paralogous gene of SMN1 and a modifying gene of SMA. A better understanding of how SMN2 exon 7 splicing is regulated helps discover new therapeutic targets for SMA therapy. Based on an antisense walk method to map exonic and intronic splicing silencers (ESSs and ISSs) in SMN2 exon 7 and the proximal regions of its flanking introns, we identified one ISS (ISS6-KH) at upstream of the branch point site in intron 6. By using mutagenesis-coupled RT-PCR with SMN1/2 minigenes, immunochromatography, overexpression and siRNA-knockdown, we found this ISS consists of a bipartite hnRNP A1 binding cis-element and a poly-U sequence located between the proximal hnRNP A1 binding site (UAGCUA) and the branch site. Both HuR and hnRNP C1 proteins promote exon 7 skipping through the poly-U stretch. Mutations or deletions of these motifs lead to efficient SMN2 exon 7 inclusion comparable to SMN1 gene. Furthermore, we identified an optimal antisense oligonucleotide that binds the intron six ISS and causes striking exon 7 inclusion in the SMN2 gene in patient fibroblasts and SMA mouse model. Our findings demonstrate that this novel ISS plays an important role in SMN2 exon 7 skipping and highlight a new therapeutic target for SMA therapy.

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.

P.10 Compound CACNA1S heterozygosity resulting in a novel phenotype of congenital myopathy and early onset periodic paralysis: report of two probands

Skeletal muscle contraction is mediated by excitation-contraction coupling (ECC), a process involving the rapid elevation of cytosolic calcium. L-type voltage gated calcium channels located on T-tubule membranes are the voltage sensors of ECC. The dihydropyridine receptor (DHPR) contains the pore forming basic functional elements of these calcium channels and is encoded by the <i>CACNA1S</i> gene. Recessive mutations in the <i>CACNA1S</i> gene have been recently reported with a severe congenital myopathy and facial involvement. We describe two sisters with compound heterozygous mutations involving the <i>CACNA1S</i> gene. Both had neonatal hypotonia, muscle weakness, and delayed motor milestones. Prior to one year of age, periodic paralysis was evident as episodic flaccidity and loss of postural control. Periodic paralysis episodes continued to occur most notably with cold exposure, lasting around 10 minutes each. Lab evaluation revealed normal CPK. Electrophysiological evaluation revealed normal motor and sensory nerve conduction studies, and normal long exercise testing. Limb cooling and cold provocation led to clinical weakness and a drop in motor amplitude. Magnetic resonance imaging of the older sister showed symmetrical tigroid pattern fat replacement and atrophy of bilateral gluteus maximus muscles. Targeted next generation sequencing revealed two variants in the <i>CACNA1S</i> gene: both c.3526-2A>G and c.2831G>A (p.Cys944Tyr) are not described in the gnomAD database. Both variants co-segregated into affected sisters and Sanger sequencing revealed that c.3526-2A>G was inherited from the unaffected mother and c.2831G>A from the unaffected father, thus revealing a recessive pattern of inheritance. The c.3526-2A>G is expected to disrupt the acceptor splice site of intron 27 by <i>in silico</i> analysis, whereas the c.2831G>A variant replaces a highly conserved cysteine residue with tyrosine at codon 944 in the DIII-S5 of the Cav1.1 protein. This novel phenotype of a milder congenital recessive myopathy and early onset periodic paralysis expands the phenotypic spectrum of <i>CACNA1S</i> gene mutations.

HvGBSSI mutation at the splicing receptor site affected RNA splicing and decreased amylose content in barley.

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy (Wx-1) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1 mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. There were two single-base mutations G1086A and A2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation is located at the 3′ splicing receptor (AG) site of the fourth intron, resulting in an abnormal RNA splicing. The A2424G mutation was a synonymous mutation in the ninth intron. The pre-mRNA of Wx-1 was incorrectly spliced and transcribed into two abnormal transcripts. The type I transcript had a 6 bp deletion in the 5′ of fifth exon, leading to a translated HvGBSSI protein lacking two amino acids with a decreased starch-binding capacity. In the type II transcript, the fourth intron was incorrectly cleaved and retained, resulting in the premature termination of the barley Wx-1 gene. The mutations in the Wx-1 decreased the enzymatic activity of the HvGBSSI enzyme and resulted in a decreased level in amylose content. This work sheds light on a new Wx-1 gene inaction mechanism.

PSVIII-B-15 Potential Involvement of DNA Methylation of First Intron in Transcriptional Regulation During Bovine Subclinical Mastitis Caused by Staphylococcus Aureus

Abstract DNA methylation involvement in the regulation of mammary gland inflammatory defense against pathogens, including Staphylococcus aureus(SA) has been documented. However, the mechanisms are not clear. The possible influence of DNA methylation of first introns (DNA-MeFI) on transcriptional activities during subclinical mastitis was studied by profiling the methylome (whole genome methylation sequencing) and transcriptome (RNA-sequencing) of milk somatic cells from cows(n=15) with SA subclinical mastitis (SACs) and healthy cows (HCs)(n=13), followed by bioinformatics processing with standard tools. The DNA-MeFI was calculated, for each of 19411 genes found as expressed in the samples, by averaging the methylation levels of all CpG sites in the first intron. At genome-wide, the DNA-MeFIs was inversely correlated with gene expression (GE) (Pearson’s r=-0.173, p=1.60×10-122)(Figure 1-A). Similarly, the difference in the DNA-MeFI and GE between SACs and HCs was also inversely correlated (Pearson’s r=-0.149, p=4.25×10-97)(Figure 1-B). Application of machine learning by Gaussian Mixed model (GMM) using scikit-learn in python to the changes of DNA-MeFI and GE between SACs and HCs revealed 2866 outliers (GMM’s p&amp;lt; 0.005) that showed significant changes in the DNA-MeFI and/or GE. 644 genes with &amp;gt;10% difference in DNA-MeFI and |log2FC| &amp;gt;1 in GE were selected and referred to as differentially methylated and expressed genes (DME-genes). 644 DME-genes were significantly enriched in 5 GO terms and 11 KEGG pathways related to diseases and immune functions, including Staphylococcus aureus infection and Natural killer cell mediated cytotoxicity (Table 1). Furthermore, 74.84% DME-genes showed inverse changes in DNA-MeFI and GE. Besides, the DNA-MeFI of 264 DME-genes were found to correlate significantly with their GE (|rho| &amp;gt;0.3, FDR&amp;lt;0.05), including 225 DME-genes(85.23%) with inverse correlations(rho&amp;lt; -0.3, FDR&amp;lt; 0.05). 39 DME-genes with positive correlations were significantly enriched in 21 GO terms mainly related to metabolic processes. In conclusion, the DNA-MeFI possibly participate in the regulation of gene expression during bovine subclinical mastitis caused by SA.

The ATRX splicing variant c.21-1G>A is asymptomatic

The ATRX variant c.21-1G>A was detected by an exome analysis of a patient with Cockayne syndrome without alpha thalassemia X-linked intellectual disability syndrome (ATR-XS). In addition, variants in ERCC6 were detected. ATRX c.21-1G>A is localized at the splicing acceptor site of intron 1. This splicing event, NM_000489.6: c.21_133del p.S7Rfs*1, induces exon 2 deletion and early termination. The start codon in exon 3 of ATRX is presumed to produce a slightly shorter but functional ATRX protein.

Intraspecific Variability and Distribution Difference within the Ribosomal Introns of the Discrete Plasmodiophora brassicae Group in Japan: A Case Study for Complex Dynamics of Intron Evolution

Analysis of the ribosomal introns of Plasmodiophora brassicae populations infecting the cruciferous weed Cardamine occulta revealed the complex dynamics of size, intraspecific variability, and distribution. The results showed that P. brassicae populations from the weed have lost multiple introns in the small and large subunits of the ribosomal RNA genes. Moreover, the retained introns, despite a largely mutual share of conserved parts with the cosmopolitan strains, contained numerous novel structures. These structural differences comprise a high level of polymorphisms, such as transversion point mutations occurring at sites involving the intronic splicing sites or insertions/deletions at the binding sites. Two geographical P. brassicae populations from C. occulta carried a lengthy intron-encoded ORF and putative mobile elements established in the large subunit. A few P. brassicae populations from the Brassica crops also harbored polymorphic introns that shared common mutated motifs with the weed-affecting group. The diversity of ribosomal introns observed from those investigated populations demonstrated the genetic distinction of the P. brassicae populations from C. occulta. The genetic variations might play a key role in the adaptability of the weed-infecting populations and are more likely related to the process of pathogenesis for the cosmopolitan P. brassicae infecting the Brassica crops.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Heterologous expression of hAID in E. coli leads to the production of a splice isoform of AID: hAIDδC, a mystery to be explored

Activation-induced cytidine deaminase (AID) is a key player that initiates antibody diversification in activated B-cell. AID mediates somatic hypermutation (SHM) and class switch recombination (CSR) via the deamination of cytosine to uracil at the Ig locus, resulting in the production of high-affinity antibodies. AID is predominantly restricted to Ig genes, whereas off-targeting of AID leads to lymphocyte-related malignancies. Interestingly, apart from FL-AID other splice isoforms of AID are highly expressed in the lymphocyte malignancies. In our study, we found that the heterologous expression of hAID-FL in E. coli cells produced two induced bands of hAID as demonstrated by SDS-PAGE and western blotting. Remarkably, peptide mapping data predicted that one band is hAID-FL and the other is its splice isoform, hAIDδE4a. To get an insight into why E. coli cells expressed hAID-FL and hAID variant, we mutated the 5′ and 3’ splice site of a putative intron of hAID, but it failed to produce only hAID-FL. Incidentally, hAID expressed with fusion partners also displayed two bands, and peptide mapping data strongly suggest that besides hAID-FL, the lower band showed a significant number of amino acids missing towards the C-terminal domain (named as hAIDδC). Our results are the first report to show that expression of recombinant hAID alone or irrespective of solubilization tags in E. coli cells produced hAID-FL and hAIDδC. It will be fascinating to explore the potential mechanism underlying the expression of hAIDδC from recombinant hAID plasmid in E. coli cells.

A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa.

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Discovery of a DFR gene that controls anthocyanin accumulation in the spiny Solanum group: roles of a natural promoter variant and alternative splicing.

Anthocyanins are important pigments that impart color in plants. In Solanum, different species display various fruit or flower colors due to varying degrees of anthocyanin accumulation. Here we identified two anthocyanin-free mutants from an ethylmethane sulfonate-induced mutant library and naturally occurring mutants in Solanum melongena, with mutations in the 5' splicing site of the second intron of dihydroflavonol-4-reductase (DFR) - leading to altered splicing. Further study revealed that alternative splicing of the second intron was closely related to anthocyanin accumulation in 17 accessions from three cultivated species: S. melongena, Solanum macrocarpon and Solanum aethiopicum, and their wild related species. Analysis of natural variations of DFR, using an expanded population including 282 accessions belonging to the spiny Solanum group, identified a single-nucleotide polymorphism in the MYB recognition site in the promoter region, which causes differential expression of DFR and affects anthocyanin accumulation in fruits of the detected accessions. Our study suggests that, owing to years of domestication, the natural variation in the DFR promoter region and the alternative splicing of the DFR gene account for altered anthocyanin accumulation during spiny Solanum domestication.

3'UTR-Seq analysis of chicken abdominal adipose tissue reveals widespread intron retention in 3'UTR and provides insight into molecular basis of feed efficiency.

Feed efficiency (FE) is an important trait in the broiler industry due to its direct correlation to efficient muscle growth instead of fat deposition. The present study characterized and compared gene expression profiles in abdominal fat from broiler chickens of different FE levels to enhance the understanding of FE biology. Specifically, traditional whole-transcript RNA-sequencing (RNA-seq) and 3’ UTR-sequencing (3’ UTR-seq) were applied to 22 and 61 samples, respectively. Overall, these two sequencing techniques shared a high correlation (0.76) between normalized counts, although 3’ UTR-seq showed a higher variance in sequencing and mapping performance statistics across samples and a lower rate of uniquely mapped reads. A higher percentage of 3’ UTR-seq reads mapped to introns suggested the frequent presence of cleavage sites in introns, thus warranting future research to study its regulatory function. Differential expression analysis identified 1198 differentially expressed genes (DEGs) between high FE (HFE) and intermediate FE (IFE) chickens with False Discovery Rate < 0.05 and fold change > 1.2. The processes that were significantly enriched by the DEGs included extracellular matrix remodeling and mechanisms impacting gene expression at the transcriptional and translational levels. Gene ontology enrichment analysis suggested that the divergence in fat deposition and FE in broiler chickens could be associated with peroxisome and lipid metabolism possibly regulated by G0/G1 switch gene 2 (G0S2).

A Founder Mutation in the POMC 5'-UTR Causes Proopiomelanocortin Deficiency Through Splicing-Mediated Decrease of mRNA.

The syndrome of adrenal insufficiency, obesity, and red hair is a rare autosomal recessive disorder. The majority of disease-causing variants associated with the syndrome are located in the coding region of the POMC gene. This work describes 7 unrelated patients who shared a novel homozygous mutation in the 5'-untranslated region (UTR) of the POMC gene and functionally characterize this novel variant. Whole-exome sequencing (WES) with autozygosity mapping, Sanger sequencing, model expression system studies, and RNA sequencing were used for identification of the disease-causing variant and its subsequent functional characterization. Seven unrelated patients of the Perm Tatar ethnic group presented with hypoglycemia and excessive weight gain, low plasma adrenocorticotropin, and cortisol. Five of 7 children had red hair; 6 of 7 patients also showed signs of bronchial obstruction. WES showed shared autozygosity regions overlapping the POMC gene. Sanger sequencing of the POMC 5'-UTR detected a homozygous variant chr2:25391366C > T (hg19) at the splice donor site of intron 1. As demonstrated by the model expression system, the variant led to a significant decrease in the POMC messenger RNA level. Analyses of the patients' haplotypes were suggestive of the founder effect. We estimate that the mutation must have occurred at least 4.27 generations ago (95% CI, 0.86-7.67). This report presents a new molecular mechanism of POMC deficiency and contributes to the information on phenotypic variability in patients with this disorder.

Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

Genetic variants of Pompe disease among a sample of Egyptian pediatric patients

Pompe disease is an autosomal recessive disorder initiated by the deficiency of lysosomal acid α-glucosidase enzyme (GAA) due to disease associated variants of the Alpha glucosidase (GAA) gene. We aimed at genetic screening of Egyptian pediatric patients diagnosed as Pompe disease, hence identifying GAA gene variants, and highlighting the significance of molecular testing for this fatal disease. A cross-sectional cohort study recruiting thirteen unrelated pediatric patients was performed. Confirmation of clinically suspect Pompe disease was accomplished by assessing GAA enzyme level using fluorometric technique, followed by molecular genetic analysis using Sanger sequencing method of GAA gene. Genetic diagnosis of 11/13 (84.6%) of the studied cohort was achieved through detecting the culprit disease causing variant in homozygous pattern in their GAA gene. According to the American College of Medical Genetics and Genomics (ACMG) classification evidence; 10/11 (90.9%) of those variants were classified as pathogenic, while 1/11 (9.1%) c.1979G>A (p. Arg660His) was classified as likely pathogenic. A novel variant g.11734 G>C c.956-1 G>C was identified in homozygous pattern in intron 5 of GAA gene in 1/11(9.1%) patient, using Mutation taster and Human splicing finder in-silico prediction tools to evaluate its pathogenicity, it was recognized that it is a disease-causing one. It results in alteration of the acceptor splicing site of intron 5 with consequent possible skipping of exon 6. Molecular analysis of GAA gene is of immense importance not only for confirming Pompe disease diagnosis but also for determination of treatment options, lifestyle, genetic counseling, and prenatal diagnosis.