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Background: The RHD gene is one of the most complex blood group genes. The molecular background of the RHD gene in RhD-negative and RhD-positive individuals varies within and among different populations. Knowing the molecular basis of the RHD gene in a specific population is required to establish effective genotyping methods. While the molecular basis has been revealed in many ethnicities, such as Caucasians and Black Africans, it still requires elucidation in Arabs. Objectives: The aim of this study was to gain insights into the molecular basis of RhD-positive and RhD-negative phenotypes in Saudi donors. Materials and Methods: Conventional serological tests were used to determine the Rh phenotypes in 136 Saudi donors by typing D, C, c, E, and e antigens. Multiplex-PCR and Single Specific Primer-PCR were used to detect the presence of exons 3, 4, and 7 and the hybrid Rhesus box gene, respectively, in RhD-negative and/or RhD-positive samples. Results: Of the 136 samples, 70 were RhD positive and 66 were RhD negative. None of the RhD-negative donors had any of the three tested exons, whereas the hybrid Rhesus box gene was detected in all, indicating the zygosity status of the RHD deletion allele. The hybrid Rhesus box gene was detected in 79% of the RhD-positive individuals, suggesting high frequencies of RHD-negative haplotypes. Conclusions: The study findings indicate that Saudis with the RhD-negative phenotype are likely to have an entire RHD deletion in the homozygous state. However, a more comprehensive analysis of variant RHD alleles in the Saudi population is required to implement effective and dedicated molecular RHD typing strategies. [ABSTRACT FROM AUTHOR]

Copyright of Pamukkale Medical Journal is the property of Pamukkale Journal of Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: The Taiwan Oncology Society (TOS) and the Taiwan Society of Pathology (TSP) have collaborated to present a joint position paper on the molecular tumor boards (MTBs) within the medical institutions of Taiwan. Materials and Methods: To raise awareness of MTBs among health-care professionals, policymakers, and the public, a total of 20 experts from TOS and TSP formulated a joint consensus statement through a voting process. Results: The joint statement proposes key recommendations: (1) MTB discussions encompass diverse molecular analyses including next-generation sequencing (NGS), RNA sequencing, whole-exon sequencing, and whole-genomic sequencing addressing relevant genomic changes, tumor mutation burden, microsatellite instability, and specific biomarkers for certain cancers. (2) MTB meetings should involve multidisciplinary participants who receive regular updates on NGS-related clinical trials. (3) Prioritize discussing cases with unique clinical needs, gene alterations lacking treatments, untreatable neoplasms, or oncogenes unresponsive to targeted therapies. (4) Base MTB discussions on comprehensive patient data, including genetics, pathology, timing of specimen collection, and NGS outcomes. (5) MTBs offer treatment recommendations: standard therapies, off-label use, clinical trials, genetic counseling, and multidisciplinary reviews. (6) MTB effectiveness can be gauged by member composition, case reviews, treatment suggestions, and patient outcomes. Encourage government incentives for MTB engagement. Conclusion: The primary aim of this initiative is to promote the advancement of precision oncology in Taiwan. [ABSTRACT FROM AUTHOR]

The diagnosis of maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus caused by a mutation in a single gene, is often uncertain until genetic testing is performed. We report a 13-yr-old Korean boy who was initially diagnosed with type 2 diabetes (T2DM). MODY was suspected because of his nonobese body habitus and family history of multiple affected members. Targeted panel sequencing of all MODYrelated genes was performed using the NextSeq 550Dx platform (Illumina). Sanger sequencing was performed using blood samples from the parents, siblings, and other relatives. A frameshift variant in the 3' region of the last exon of PDX1 was detected in the patient and his family members with diabetes. PP1_Moderate criterion was applied and this variant was confirmed to be the genetic cause of diabetes in the family and classified as likely pathogenic. The study highlights the importance of genetic testing for nonobese, early-onset diabetic patients with multiple affected family members. Increased awareness and aggressive genetic testing for MODY are needed. [ABSTRACT FROM AUTHOR]

Context: Limited information is available concerning the genetic spectrum of pheochromocytoma and paraganglioma (PPGL) patients in South America. Germline SDHB large deletions are very rare worldwide, but most of the individuals harboring the SDHB exon 1 deletion originated from the Iberian Peninsula. Objective: Our aim was to investigate the spectrum of SDHB genetic defects in a large cohort of Brazilian patients with PPGLs. Methods: Genetic investigation of 155 index PPGL patients was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification, and/or target next-generation sequencing panel. Common ancestrality was investigated by microsatellite genotyping with haplotype reconstruction, and analysis of deletion breakpoint. Results: Among 155 index patients, heterozygous germline SDHB pathogenic or likely pathogenic variants were identified in 22 cases (14.2%). The heterozygous SDHB exon 1 complete deletion was the most frequent genetic defect in SDHB, identified in 8 out of 22 (36%) of patients. Haplotype analysis of 5 SDHB flanking microsatellite markers demonstrated a significant difference in haplotype frequencies in a case-control permutation test (P=0.03). More precisely, 3 closer/informative microsatellites were shared by 6 out of 8 apparently unrelated cases (75%) (SDHB-GATA29A05-D1S2826-D1S2644 | SDHB-186-130-213), which was observed in only 1 chromosome (1/42) without SDHB exon 1 deletion (X2 = 29.43; P<0.001). Moreover, all cases with SDHB exon 1 deletion had the same gene breakpoint pattern of a 15 678 bp deletion previously described in the Iberian Peninsula, indicating a common origin. Conclusion: The germline heterozygous SDHB exon 1 deletion was the most frequent genetic defect in the Brazilian PPGL cohort. Our findings demonstrated a founder effect for the SDHB exon 1 deletion in Brazilian patients with paragangliomas. [ABSTRACT FROM AUTHOR]

Copyright of Iraq Journal of Market Research & Consumer Protection / Al-Mağallaẗ al-ʿIrāqiyyaẗ li-Buḥūṯ al-Sūq wa-Ḥimāyaẗ al-Mustahlik is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region. Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs). Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs. Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants. [ABSTRACT FROM AUTHOR]

Small-cell lung cancer (SCLC) transformation from EGFR mutant adenocarcinoma is a rare entity that is considered to be a new phenotype of SCLC. While transformation from adenocarcinoma (ADC) with EGFR exon 19 deletions and exon 21 L858R point mutations has been described, to our knowledge, no cases of transformation to SCLC from exon-18-mutated ADC have been reported. We reported a clinical case of a patient with exon-18-EGFR-transformed SCLC, and we performed a systematic review of the literature. [ABSTRACT FROM AUTHOR]

Somatic MET exon 14 skipping mutations (MET ex14) are targetable driver mutations for non-small cell lung cancer (NSCLC), responsive to MET inhibitors. Objective: This study seeks to further characterize the clinicopathologic features and mutational profile of MET ex14 variant NSCLC. Design: Retrospective review of all MET ex14 tested NSCLC. Testing for selected BRAF, EGFR, HER2, KRAS, and MET mutations was performed using a clinically validated NGS assay, followed by MiSeq sequencing. Variants were classified as significant (Tier1/2) or variants of uncertain significance (VUS) per 2017 AMP/ASCO/CAP Joint Consensus Guidelines. PD-L1 expression was assessed by immunohistochemistry. Results: Of 2296 NSCLCs tested between 2017-7/2019, MET ex14 variants were present in 44 (1.9%). A total of 32 of 44 variants were MET exon 14 skipping, while the other 12 mutations were significant missense (3) or VUS (9). Of nine VUS, five were adjacent to the canonical splice site and likely to impact splicing. Four cases had concomitant mutations. Of 35 cases with known clinical staging, stage 1-2 = 20 (57%), stage 3 = 3 (9%), and stage 4 = 12 (34%). Of 19 resected NSCLSs, histological types and growth pattern included 7 lepidic pattern-predominant. A high percentage of tumors with MET ex14 mutations are positive for PD-L1, and the percentage of cases with PD-L1 expression >50% trends higher in more advanced disease. Conclusions: Most MET variants identified in our cohort (73%) are MET ex14 skipping. The prevalence of MET ex14 variants is 1.9%, and a large percentage of tumors has lower clinical stage and less aggressive pathologic features. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junctionÐproximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes. [ABSTRACT FROM AUTHOR]

Background and aims: Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main reason for the incidence of PKU. To date, more than 1180 variants have been detected in the PAH gene. Given that the distribution pattern of mutations in the PAH gene is specific to each population, the present study was conducted to detect exon 4 mutations and adjacent flanking regions of the PAH gene in northern Iran. Methods: This is a descriptive cross-sectional study, in which 24 unrelated PKU patients in Taleghani Hospital in Gorgan were enrolled for a one-year period. After extraction of genomic DNA from leukocytes, identification of exon 4 mutations and adjacent flanking regions was performed using polymerase chain reaction (PCR) and sequencing techniques. Results: In this study, IVS4+1G>A mutation was detected in one allele (2.08%) among 48 alleles. Moreover, IVS4+47C>T and IVS3-22C>T polymorphisms were observed in 12 alleles (25%) and eight alleles (16.7%), respectively. Conclusion: In the present study, IVS4+1G>A mutation was only found in 2% of chromosomes. Hence, different mutations are responsible for PKU disease in the north of Iran, and further studies are recommended to identify all mutations in the PAH gene in the region. [ABSTRACT FROM AUTHOR]

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy. [ABSTRACT FROM AUTHOR]

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA) signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3-0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the major oncological problems in Poland. Pembrolizumab monotherapy can be applied as first-line treatment in patients with advanced non-small-cell lung cancer (NSCLC) with the expression of programmed death ligand 1 (PD-L1) in = 50% of tumor cells. The article presents a case report of a female patient with advanced lung adenocarcinoma and high PD-L1 expression and an additional MET exon 14 skipping mutation. Despite the advanced stage of the disease, the patient benefited spectacularly from pembrolizumab administered following stereotactic radiotherapy for central nervous system (CNS) metastases. Partial remission followed by long-term stabilization of the disease was achieved. Unfortunately, the therapy was discontinued due to grade-3 pulmonary toxicity observed after 3 years of treatment. Despite the discontinuation of the pembrolizumab therapy, the disease has currently been stabilized and inflammatory changes have slowly resolved upon administration of corticosteroid. [ABSTRACT FROM AUTHOR]

Objective: The secretion of salivary immune factors and the accumulation of nonimmune elements represent the first line of oral immunity defense against cariogenesis. As the interplay between genetic and environmental predisposing factors in inducing dental caries is controversial, we investigated the association between three genetic polymorphisms in Egyptian children.Method: Our genetic network analysis demonstrated that AMELX, ENAM, LTF, MEPE, BMP2, BMP4, IFNG, IL6, TNF, PROM1, TUFT1, and CDKN1A play a critical role in promoting an anti-cariogenic effect in different ethnic groups. Enamelin, lactotransferrin, and amelogenin-X (AMELX) exon-intron boundaries were sequenced in 148 patients with extensive caries (n = 74) or caries-free patients (n = 74) from different Egyptian hospitals.Results: The presence of specific genetic polymorphisms, namely ENAM rs3796703, AMELX rs946252, and LTF rs1126478, showed a significant association with increased susceptibility to dental caries within the caries-susceptible group. These particular polymorphisms exhibited a noteworthy presence or occur- rence rate in individuals who were more prone to developing dental caries.Conclusion: ENAM rs3796703, AMELX rs946252 were frequently detected in the caries-susceptible group, suggesting the possible correlation between the defect in these proteins and the incidence of developing dental caries. The polymorphisms in LTF rs1126478 were related to a high DMFT index (≥7) among Egyptian children. [ABSTRACT FROM AUTHOR]

This study examined the relationship between GHE5 polymorphisms and growth and carcass characteristics of meat-type sheep breeds reared in Turkey. A total of 202 lambs were tested, consisting of five breeds. By SSCP analysis and nucleotide sequencing, 14 nucleotide changes (12 substitutions and two deletions) were identified in four variants of GHE5. In the coding region of GHE5, five substitutions occur, including c.1588C>Y(C/T) (Ala160Val), c.1603A>M(A/C), c.1604G>S(G/C) (Lys165Thr), c.1606A>W(A/T) (Gln166Leu), and c.1664C>Y(C/T). P3 female and P1 male lambs had the highest rump height at weaning, whereas P3 females and P2 males had the highest chest depth (p < 0.05). At yearling, P1 variant lambs have longer body length (BL; p < 0.05), wider leg circumferences, and thinner cannon bone perimeter (CBP) (p > 0.01), in contrast to P2 variant lambs, which have a shorter BL and thicker CBP. Furthermore, P2 had a greater percentage of neck, shoulder, and leg, P1 had a greater percentage of loin, and P3 had a greater percentage of rack, but there was no significant difference between them. A marker-assisted selection approach can be used to improve sheep carcass quality traits by taking advantage of the nucleotide substitutions found on GHE5 and the detected differences between variants. [ABSTRACT FROM AUTHOR]

Causative to the neurodegenerative Huntington’s disease (HD), a mutational huntingtin (HTT) protein consists of an unusual expansion on the poly-glutamine (polyQ) region in the first exon (exon-1) domain. Despite its significance on HD progression, the structural role of the exon-1 with the polyQ region is still elusive. As HTT is an intrinsically disordered protein (IDP), a large ensemble of various conformations (instead of a mostly single native conformation) is required to characterize its structural properties and to infer biological functions, which is challenging even for the most state-of-the-art experimental techniques. For this reason, molecular dynamics (MD) simulations with enhanced sampling techniques are ideal to compliment experiment on collecting such a large ensemble of thermodynamically accessible structures. Here, we performed large-scale temperature replica-exchange MD (T-REMD) simulations on the exon-1 with an illustration on the necessity of using T-REMD instead of unbiased regular MD. By comparing T-REMD data and unbiased MD data, we discovered that (1) the dynamics of polyQ regions are extremely sluggish and glassy at the room temperature and the relaxation of the system cannot be achieved within a reasonable amount of time without utilizing an enhanced sampling method and (2) an ensemble of protein structures containing the surprising cis-peptide bonds in the proline-rich domain can be obtained at much elevated temperatures. Our results may provide valuable insights for future studies on the HTT as well as other IDPs using the T-REMD method. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS) is a childhood sarcoma composed of myoblast-like cells, which suggests a defect in terminal skeletal muscle differentiation. To explore potential defects in the differentiation program, we searched for mRNA splicing variants in genes encoding transcription factors driving skeletal muscle lineage commitment and differentiation. We studied two RMS cases and identified altered splicing resulting in “skipping” the second of three exons in MYOD1. RNA-seq data from 42 tumors and additional RMS cell lines revealed exon 2 skipping in both MYOD1 and MYF5 but not in MYF6 or MYOG. Complementary molecular analysis of MYOD1 mRNA found evidence for exon skipping in five additional RMS cases. Functional studies showed that so-called MYODΔEx2 protein failed to robustly induce muscle-specific genes, and its ectopic expression conferred a selective advantage in cultured fibroblasts and an RMS xenograft. In summary, we present previously unrecognized exon skipping within MYOD1 and MYF5 in RMS, and we propose that alternative splicing can represent a mechanism to alter the function of these two transcription factors in RMS. [ABSTRACT FROM AUTHOR]

The multidrug and toxic compound extrusion (MATE) family plays pivotal roles in the detoxification process in plants, while no information has been provided for this gene family in melon (Cucumis melo L.) thus far, limiting our understanding of its functions in melon acclimation to stressful environments. In this study, a total of 39 MATEs (CmMATE1-CmMATE39) were observed in the melon genome; these were unevenly distributed in all chromosomes, with the most on Chromosome 1. Based on their orthologous relationship with those from Arabidopsis, rice, and sorghum, melon MATEs were clustered into three subfamilies of Clades I, II, and III, wherein 23, 9, and 7 members were included, respectively. Variable exon number was observed in CmMATEs, and the most were harbored by CmMATE8. Gene ontology (GO) term and cis-regulatory element (CRE) analyses pointed to the potential roles of CmMATEs in both the regulation of melon development and acclimation to various abiotic and biotic stressors. The RNA-seq and qRT-PCR (quantitative real-time PCR) results demonstrated that under normal growth conditions, CmMATEs were expressed in a tissue- and development-specific manner, while their abundance apparently varied in a stress-dependent manner when melon plants were exposed to unfavorable environmental conditions. Altogether, these observations could expand our knowledge about the plant MATE family and benefit functional genomics analysis for CmMATEs in the future. [ABSTRACT FROM AUTHOR]

Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multicenter experience with two novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (<18 years) admitted in the period between 2012 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complementary immune workup. We identified 5 patients (3 males) from four different families harboring two novel mutations in the complement components C6–C8. Genetic mutations were identified by whole-exome sequencing or by sequencing of the coding exons of a single gene based on the findings in the immune workup. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune workup demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. Diagnosis delay ranged between 0 and 30 years. Conclusion: Awareness of risk factors for primary complement deficiencies, even at the first infectious episode, should facilitate prompt immune and genetic workup, commencing diagnosis and proper treatment for the patient and family. What is Known: • Deficiencies in the classical terminal complement components increase susceptibility to invasive meningococcal infections. • Recurrent meningococcal infections mandate a diagnostic workup of the complement system. What is New: • Genetic workup can be utilized for prompt diagnosis of complement deficiencies. • High rates of consanguinity, even in the presence of a single meningococcal infection, should promote immune and genetic workups. [ABSTRACT FROM AUTHOR]

A number of genomic mutations that are thought to be strongly involved in the development of schizophrenia (SCZ) and autism spectrum disorder (ASD) have been identified. Abnormalities involving oligodendrocytes have been reported in SCZ, and as a related gene, oligodendrocyte lineage transcription factor 2 (OLIG2) has been reported to be strongly associated with SCZ. In this study, based on the common disease-rare variant hypothesis, target sequencing of candidate genes was performed to identify rare mutations with a high effect size and the possibility that the identified mutations may increase the risks of SCZ and ASD in the Japanese population. In this study, the exon region of OLIG2 was targeted; 370 patients with SCZ and 192 with ASD were subjected to next-generation sequencing. As a result, one rare missense mutation (A33T) was detected. We used the Sanger method to validate this missense mutation with a low frequency (<1%), and then carried out a genetic association analysis involving 3299 unrelated individuals (1447 with SCZ, 380 with ASD, and 1472 healthy controls) to clarify whether A33T was associated with SCZ or ASD. A33T was not found in either case group, and in only one control. We did not find evidence that p.A33T is involved in the onset of ASD or SCZ; however, associations with this variant need to be evaluated in larger samples to confirm our results. [ABSTRACT FROM AUTHOR]

Objectives: It is believed that there are still unclear areas in the formation mechanism of leiomyomas. In our study, it was aimed to investigate the formation mechanisms of leiomyomas due to local MED 12 gene exon 2 mutation and local microRNA-124 expression in a Turkish population. Material and methods: Thirty patients who underwent hysterectomy for leiomyoma uteri at Gaziantep University between January 2013 and January 2016 were included in our study. In the pathology specimens of these patients, the patient's myometrium tissue and her own leiomyoma tissue were analysed via quantitative Realtime PCR in association with MED 12 exon 2 mutation and microRNA-124 expression. Results: The average age of the 30 patients included in our study is 46.67 ± 5.42 and 13 patients had single leiomyoma; 17 patients had more than one leiomyoma. There were significantly higher c.130G>T (p.G44C) mutation and c.131G>A (p.G44A) mutation of MED 12 gene exon in leiomyoma tissues than healthy myometrium tissues of same patients. There was a 3.7-fold decrease in the expression of microRNA-124 in leiomyoma tissues compared to intact eutopic myometrium tissues, but this difference was not statistically significant. Conclusions: In recent studies, it has been suggested that MED 12 gene may play an active role in the formation of fibroids. MED12 and β-catenin/Wnt pathway were emphasized, and alternative genetic pathways are sought in fibroid formation. Also, tumour suppressor and oncogenesis effects of microRNAs have been demonstrated in many different studies. Since it is involved in the Wnt pathway, microRNA-124 has been blamed by some previous studies for the formation of fibroids. This study demonstrates that MED12 exon 2 mutations and probably microRNA-124 gene expressions might contribute to uterine leiomyoma pathology. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the clinical significance of prenatal diagnosis and prognostic evaluation of fetal intestinal malrotation with midgut volvulus via ultrasonography. Material and methods: Ultrasonographic findings and clinical outcomes of fetal intestinal malrotation with midgut volvulus prenatally diagnosed via ultrasound at the Zhangzhou Hospital Affiliated of Fujian Medical University from January 2013 to May 2020 were summarised and analyzed. Results: Eleven cases of fetal intestinal malrotation with midgut volvulus were accurately prenatally diagnosed according to the specific ultrasound signs, such as 'whirlpool sign' and 'twining sign'. Indirect and nonspecific ultrasonographic signs included ascites in four cases and echogenic bowel in nine, all of which were accompanied by intestinal dilatation at various degrees. Among all cases, two were complicated with other system abnormalities, and one had a chromosome abnormality. Three pregnant women chose termination of pregnancy, and eight neonates were transferred to the pediatric department for surgery after obtaining written informed consent. An accurate prenatal diagnosis was confirmed postoperatively, and the fetus recovered well postoperatively. Conclusions: Fetal intestinal malrotation with midgut volvulus has typical sonographic features, and ultrasonography is the method of choice for prenatal diagnosis of this disease. Fetal intestinal malrotation with midgut volvulus is a non-fatal congenital malformation that can be treated. Prenatal diagnosis is essential for early postnatal treatment, and early surgery can often obtain good efficacy and prognosis. [ABSTRACT FROM AUTHOR]

Background and aims: In the phenylalanine hydroxylase (PAH) gene, various mutations are mainly responsible for phenylketonuria (PKU). After thalassemia, PKU is considered as the most common autosomal recessive disease among Iranian population. Therefore, identifying the mutations that cause the disease in this population is of great importance. This study aimed to identify exon 5 mutations of PAH gene in PKU patients from Golestan, the northern province of Iran, and compare these mutations with those onesa found by studies carried out in other parts of the country. Methods: During a one-year period, 25 unrelated PKU patients aged 1-23 years and from different parts of Golestan province were included in the study. Then, their genomic DNAs were extracted from their blood samples and PCR-sequencing method was used to identify the mutations. The sequencing results were analyzed using Chromas and CLC Main Workbench v3.5 software. Results: In this study, R158Q mutation was detected with a frequency of 6%. This mutation was homozygous in one PKU patient, but it was heterozygous in another one. These patients had cPKU phenotype. Conclusion: Evaluation of mutations proved to be a useful technique for molecular diagnosis of PKU and identification of disease carriers in the given population. Taking into account the fact that only one exon of PAH gene was explored in this study, however, it is recommended that further studies be conducted to investigate other exons in order for obtaining the complete mutation spectrum of this gene in PKU patients in Golestan province. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a debilitating neurodegenerative pediatric disease characterized by low levels of the survival motor protein (SMN). Humans have two SMN genes that produce identical SMN proteins, but they differ at a key nucleotide in exon 7 that induces differential mRNA splicing. SMN1 primarily produces full-length SMN protein, but due to the spliceosome's inability to efficiently recognize exon 7, SMN2 transcripts are often truncated. SMA occurs primarily through mutations or deletions in the SMN1 gene; therefore, current therapies use antisense oligonucleotides (ASOs) to target exon 7 inclusion in SMN2 mRNA and promote full-length SMN protein production. Here, we explore additional methods that can target SMN splicing and therapeutically increase full-length SMN protein. We demonstrate that in vitro heat treatment of cells increases exon 7 inclusion and relative abundance of full-length SMN2 mRNA and protein, a response that is modulated through the upregulation of the positive splicing factor TRA2 beta. We also observe that HSP90, but not HSP40 or HSP70, in the heat shock response is essential for SMN2 exon 7 splicing under hyperthermic conditions. Finally, we show that pulsatile heat treatments for one hour in vitro and in vivo are effective in increasing full-length SMN2 levels. These findings suggest that timed interval treatments could be a therapeutic alternative for SMA patients who do not respond to current ASO-based therapies or require a unique combination regimen. [ABSTRACT FROM AUTHOR]

Background: Cryptic exons are typically characterised as deleterious splicing aberrations caused by deep intronic mutations. However, low‐level splicing of cryptic exons is sometimes observed in the absence of any pathogenic mutation. Five recent reports have described how low‐level splicing of cryptic exons can be modulated by common single‐nucleotide polymorphisms (SNPs), resulting in phenotypic differences amongst different genotypes. Methods: We sought to investigate whether additional 'SNPtic' exons may exist, and whether these could provide an explanatory mechanism for some of the genotype–phenotype correlations revealed by genome‐wide association studies. We thoroughly searched the literature for reported cryptic exons, cross‐referenced their genomic coordinates against the dbSNP database of common SNPs, then screened out SNPs with no reported phenotype associations. Results: This method discovered five probable SNPtic exons in the genes APC, FGB, GHRL, MYPBC3 and OTC. For four of these five exons, we observed that the phenotype associated with the SNP was compatible with the predicted splicing effect of the nucleotide change, whilst the fifth (in GHRL) likely had a more complex splice‐switching effect. Conclusion: Application of our search methods could augment the knowledge value of future cryptic exon reports and aid in generating better hypotheses for genome‐wide association studies. [ABSTRACT FROM AUTHOR]

In the male germline, the machinery to repress retrotransposons that threaten genomic integrity via the piRNA pathway is established in gonocytes. It has been reported that disruption of the piRNA pathway leads to activation of retrotransposons and arrests spermatogenesis before it enters the second meiosis; however, its effects on gonocytes have not been fully elucidated. In this study, we analyzed the effects of Asz1 deletion, which is a crucial component of the piRNA pathway, on the gonocyte transcriptome. In Asz1-null gonocytes, MIWI2, which is responsible for introducing DNA methylation to retrotransposons in a piRNA-dependent manner, disappeared from the nuclei of fetal gonocytes. Transcriptome analysis revealed that retrotransposons targeted by the piRNA pathway and non-annotated transcript variants were upregulated in gonocytes from neonatal Asz1-/- mice. These non-annotated transcript variants were chimeras generated by joining exons transcribed from retrotransposons and canonical genes. DNA methylation analysis showed that retrotransposons that induce the expression of aberrant chimeric transcripts are not fully methylated. This was consistent with the impaired nuclear localization of MIWI2 in Asz1-null gonocytes. Furthermore, heterogeneity of DNA methylation status in retrotransposons was observed in both gonocytes and their descendants. This suggests that the piRNA system in gonocytes can potentially prevent spermatogenic cell populations bearing aberrant chimeric transcripts from propagating later in spermatogenesis. In conclusion, Asz1 is required to repress retrotransposons and retrotransposon-driven aberrant chimeric transcripts in gonocytes through the piRNA pathway. [ABSTRACT FROM AUTHOR]

The article provides an overview of the results of modern genetic studies of human cognitive abilities. Finding genetic factors, associated with cognitive abilities, will have far-reaching ramifications at all levels of understanding from DNA to brain and to behavior. Despite its complexity, cognitive ability is a reasonable candidate for molecular genetic research because it is one of the most heritable features of behavior. The first attempts to find genetic factors, associated with cognitive abilities, focused on genes, involved in brain development and function, but this direction proved to be unproductive, as it turned out that there are about 18.000 genes, and it was too difficult to detect among them those genes that are involved in cognitive processes. In addition, a considerable number of genetic factors of human traits are single-nucleotide polymorphisms (SNPs) which are in non-coding DNA regions rather than in traditional genes. The effect of each separate SNP is unimportant, and a clear expression of the general cognitive ability is noticeable only if all the associated SNPs are involved. Currently, over 11,000 such SNPs have been identified, which are uneven in different functional regions of the genome: over 60 % in gene introns, almost 30 % in intergenic DNA regions, about 5 % in gene exons, and about 5 % in transcribed regions (downstream, upstream) and frame regions (UTR’5, UTR’3) of genes. Also there are found 74 SNPs, associated with school achievements. These SNPs are disproportionately located in genes that regulate transcription and alternative splicing of other genes, which are expressed in nerve tissues of the brain during its prenatal development. Finding genetic factors that explain the inheritance of cognitive abilities is important for both science and society. Information about these factors can be used in other fields of human science – human genetics and medicine. It will open up new scientific horizons for education too owing to understanding of the genetic aspects of learning and memory. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), one of the most common progressive and severely disabling neuromuscular diseases in children, can be largely attributed to the loss of function of the DMD gene on chromosome Xp21.2-p21.1. This paper describes the case of a 10-year-old boy diagnosed with DMD. Whole exome sequencing confirmed the hypothesized large partial exonic deletion of c.7310-11543_7359del (chrX:g.31792260_31803852del) spanning exon 51 and intron 50 in DMD. This large deletion was verified to be de novo by PCR, and the two breakpoints were further confirmed by Sanger sequencing and long-read whole-genome sequencing. Notably, this partial exonic deletion was the only complex variation in the deep intron regions or intron–exon junction regions in DMD. In addition, the case study demonstrates the clinical importance of using multiple molecular genetic testing methods for the diagnosis of rare diseases. [ABSTRACT FROM AUTHOR]

Although large exons cannot be readily recognized by the spliceosome, many are evolutionarily conserved and constitutively spliced for inclusion in the processed transcript. Furthermore, whether large exons may be enriched in a certain subset of proteins, or mediate specific functions, has remained unclear. Here, we identify a set of nearly 3,000 SRSF3‐dependent large constitutive exons (S3‐LCEs) in human and mouse cells. These exons are enriched for cytidine‐rich sequence motifs, which bind and recruit the splicing factors hnRNP K and SRSF3. We find that hnRNP K suppresses S3‐LCE splicing, an effect that is mitigated by SRSF3 to thus achieve constitutive splicing of S3‐LCEs. S3‐LCEs are enriched in genes for components of transcription machineries, including mediator and BAF complexes, and frequently contain intrinsically disordered regions (IDRs). In a subset of analyzed S3‐LCE‐containing transcription factors, SRSF3 depletion leads to deletion of the IDRs due to S3‐LCE exon skipping, thereby disrupting phase‐separated assemblies of these factors. Cytidine enrichment in large exons introduces proline/serine codon bias in intrinsically disordered regions and appears to have been evolutionarily acquired in vertebrates. We propose that layered splicing regulation by hnRNP K and SRSF3 ensures proper phase‐separation of these S3‐LCE‐containing transcription factors in vertebrates. Synopsis: Although their recognition poses a challenge to spliceosomes, large exons are often conserved and spliced. Here, this is linked to their nucleotide composition, which drives splicing factor recruitment and endows encoded proteins with specific properties. Analysis of ˜ 3,000 SRSF3‐dependent large exons reveals enrichment for cytidine nucleotidesCytidine enrichment favors recruitment of splicing factors SRSF3 and hnRNP KInclusion of cytidine‐rich large exons encoding serine/proline‐rich intrinsically disordered regions (IDRs) is found in vertebrate transcription factorsLoss of IDR inclusion upon SRSF3 depletion‐induced exon‐skipping disrupts transcription factor phase separation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the genetic characteristics of one of the MEFV gene variants, p.Glu148Gln (E148Q), in patients with familial Mediterranean fever (FMF) and examine its significance in Japanese patients with recurrent fever. Methods: The clinical phenotype and genomic variants of systemic autoinflammatory diseases (SAIDs), including MEFV, were analyzed in 211 Japanese patients with recurrent fever. Genetic analysis was performed via next-generation sequencing of exons, including exon-intron boundaries. Results: Twelve patients met the diagnostic criteria for SAIDs other than FMF. Considering 199 patients with recurrent fever, 137 cases (68.8%) were clinically diagnosed with FMF. Although Bonferroni-adjusted p-value did not reach significance level, the group containing heterozygous E148Q and other variants tended to be at higher risk of developing the FMF phenotype (nominal p¼.036) than the group with heterozygous E148Q only. Comparison between the group with heterozygous E148Q and other variants and the heterozygous group containing non-E148Q showed no statistically significant difference in FMF phenotype expression (nominal p¼1.00). Conclusion: Patients with heterozygous E148Q and other variants exhibited higher expression of FMF phenotype than those with heterozygous E148Q only, and suggested that other variants than E148Q as well as exon 10 variants might contribute to the FMF phenotype. [ABSTRACT FROM AUTHOR]

Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPIα) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0–20 µM MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPIα (activity) levels. [ABSTRACT FROM AUTHOR]

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (50-donor, lariat branch point element or 30-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s). [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most common malignancies in the world. It's estimated about 1.8 M new CRC cases worldwide per year. A somatic mutation in the BRAF gene in the tumor is a negative prognostic factor. This work is aimed at studying the clinical and genetic characteristics of Russian CRC patients with the BRAF mutation. The BRAF mutations were studied by Sanger sequencing and digital droplet PCR in 489 patients and found in 34 (7%) cases. The most common mutation was p.V600E (82%). Also, rare variants were found: p.K601E, p.N581I, p.G596R, and p.D594N. All the patients with rare mutations were characterized by an unfavorable prognosis of the disease. The clinical features of the patients with BRAF mutations in the study include the predominant primary tumor site in the rectum, in addition to the right colon. Then, most of the cases were diagnosed in the advanced stages of the disease and were represented by high-grade adenocarcinomas. This article demonstrates the feasibility of analysis of the entire exon 15 of BRAF gene in CRC patients regardless of tumor localization. [ABSTRACT FROM AUTHOR]

TP53 is one of the most frequently altered genes in cancer; it can be inactivated by a number of different mechanisms. NM_000546.6 (ENST00000269305.9) is by far the predominant TP53 isoform, however a few other alternative isoforms have been described to be expressed at much lower levels. To better understand patterns of TP53 alternative isoforms expression in cancer and normal samples we performed exon-exon junction reads based analysis of TP53 isoforms using RNA-seq data from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTEx) project. TP53 C-terminal alternative isoforms have abolished or severely decreased tumor suppressor activity, and therefore, an increase in fraction of TP53 C-terminal alternative isoforms may be expected in tumors with wild type TP53. Despite our expectation that there would be increase of fraction of TP53 C-terminal alternative isoforms, we observed no substantial increase in fraction of TP53 C-terminal alternative isoforms in TCGA tumors and CCLE cancer cell lines with wild type TP53, likely indicating that TP53 C-terminal alternative isoforms expression cannot be reliably selected for during tumor progression. [ABSTRACT FROM AUTHOR]

Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon's excluded segment. [ABSTRACT FROM AUTHOR]

Background: Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. Results: Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns' overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. Conclusions: Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q [ABSTRACT FROM AUTHOR]

The genus Hoya is highly diverse and many of its species are popular ornamental plants. However, the relationships between Hoya and related genera (the Hoya group) are not fully resolved. In this study, we report 20 newly sequenced plastomes of species in the Hoya group. The complete plastomes vary in length from 175,405 to 178,525 bp while the LSCs vary from 90,248 to 92,364 bp and the complete SSCs vary from 2,285 to 2,304 bp, making the SSC in the Hoya group one of the shortest known in the angiosperms. The plastome structure in the Hoya group is characterised by a massive increase in the size of the inverted repeats as compared to the outgroups. In all ingroup species, the IR/SSC boundary moved from ycf1 to ndhF while this was not observed in outgroup taxa, making it a synapomorphy for the Hoya group. We have also assembled the mitogenome of Hoya lithophytica, which, at 718,734 bp, is the longest reported in the family. The phylogenetic analysis using exons from 42 taxa in the Hoya group and three outgoups confirms that the earliest divergent genus in the Hoya group is Papuahoya, followed by Dischidia. The relationship between Dischidia and the clade which includes all Hoya and Oreosparte taxa, is not fully supported. Oreosparte is nested in Hoya making it paraphyletic unless Clemensiella is recognised as a separate genus. [ABSTRACT FROM AUTHOR]

Whole-exome sequencing is an attractive alternative to microarray analysis because of the low cost and potential ability to detect copy number variations (CNV) of various sizes (from 1–2 exons to several Mb). Previous comparison of the most popular CNV calling tools showed a high portion of false-positive calls. Moreover, due to a lack of a gold standard CNV set, the results are limited and incomparable. Here, we aimed to perform a comprehensive analysis of tools capable of germline CNV calling available at the moment using a single CNV standard and reference sample set. Compiling variants from previous studies with Bayesian estimation approach, we constructed an internal standard for NA12878 sample (pilot National Institute of Standards and Technology Reference Material) including 110,050 CNV or non-CNV exons. The standard was used to evaluate the performance of 16 germline CNV calling tools on the NA12878 sample and 10 correlated exomes as a reference set with respect to length distribution, concordance, and efficiency. Each algorithm had a certain range of detected lengths and showed low concordance with other tools. Most tools are focused on detection of a limited number of CNVs one to seven exons long with a false-positive rate below 50%. EXCAVATOR2, exomeCopy, and FishingCNV focused on detection of a wide range of variations but showed low precision. Upon unified comparison, the tools were not equivalent. The analysis performed allows choosing algorithms or ensembles of algorithms most suitable for a specific goal, e.g. population studies or medical genetics. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development. [ABSTRACT FROM AUTHOR]

Objective: Dystrophinopathies are the most frequently researched neuromuscular disease group due to their characteristic and diverse clinical and genetic spectrum. This study aims to evaluate the deletion and duplication profile of the dystrophin gene in Turkey by investigating data from a tertiary center. Material and Methods: Dystrophin MLPA and microarray results of 53 patients, 49 with a dystrophinopathy and 4 with a neurogenetic and syndromic disorder pre-diagnosis, who were referred to the Medical Genetics Clinic of Ankara City Hospital between February 2019-December 2020 were retrospectively evaluated. Results: Of the 53 patients, 4 had various exon duplications and 49 had deletions. 33 of these mutations caused frame-shift (62.3%), while 20 caused in-frame (37.7%) changes. Fifty (94.3%) patients underwent maternal studies and 14 (26.4%) of these had de novo mutations. Mutations were observed most frequently in the central rod domain (69.7%) followed by the actin-binding domain (7.5%) of the dystrophin gene and 12 of 33 patients with frameshift mutation (36%) patients were found to be candidates for the exon skipping treatments that are still subject to clinical research. Conclusion: This study has shed light on the incidence of dystrophin deletion/duplication mutations in our population and has revealed that a majority of patients are suitable candidates for treatments which are still not in routine use. Considering ever-growing number of dystrophin gene-based treatment options, data on population-specific mutation types is of great importance. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy. [ABSTRACT FROM AUTHOR]

Mobile element insertions (MEIs) typically exceed the read lengths of short-read sequencing technologies and are therefore frequently missed. Recently, a founder Alu insertion in exon 4 of RP1 has been detected in Japanese patients with macular dystrophy by PCR and gel electrophoresis. We aimed to develop a grep search program for the detection of the Alu insertion in exon 4 of RP1 using unprocessed short reads. Among 494 unrelated Korean patients with inherited eye diseases, 273 patients with specific retinal phenotypes who were previously genotyped by targeted panel or whole exome sequencing were selected. Five probands had a single heterozygous truncating RP1 variant, and one of their unaffected parents also carry this variant. To find a hidden genetic variant, whole genome sequencing was performed in two patients, and it revealed AluY c.4052_4053ins328/p.(Tyr1352Alafs*9) insertion in RP1 exon 4. This AluY insertion was additionally identified in other 3 families, which was confirmed by PCR and gel electrophoresis. We developed simplified grep search program to detect this AluY insertion in RP1 exon 4. The simple grep search revealed a median variant allele frequency of 0.282 (interquartile range, 0.232–0.383), with no false-positive results using 120 control samples. The MEI in RP1 exon 4 was a common founder mutation in Korean, occurring in 1.8% of our cohort. The RP1-Alu grep program efficiently detected the AluY insertion, without the preprocessing of raw data or complex installation processes. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression. [ABSTRACT FROM AUTHOR]

Age-related hearing impairment (ARHI) is the most common sensory disorder in older adults. We conducted a genome-wide association meta-analysis of 121,934 ARHI cases and 591,699 controls from Iceland and the UK. We identified 21 novel sequence variants, of which 13 are rare, under either additive or recessive models. Of special interest are a missense variant in LOXHD1 (MAF = 1.96%) and a tandem duplication in FBF1 covering 4 exons (MAF = 0.22%) associating with ARHI (OR = 3.7 for homozygotes, P = 1.7 × 10−22 and OR = 4.2 for heterozygotes, P = 5.7 × 10−27, respectively). We constructed an ARHI genetic risk score (GRS) using common variants and showed that a common variant GRS can identify individuals at risk comparable to carriers of rare high penetrance variants. Furthermore, we found that ARHI and tinnitus share genetic causes. This study sheds a new light on the genetic architecture of ARHI, through several rare variants in both Mendelian deafness genes and genes not previously linked to hearing. Erna Ivarsdottir et al. report a genome-wide association meta-analysis for age-related hearing loss in the Icelandic and UK populations. They identify 21 novel variants, 13 of which are rare, and reveal a genetic correlation between age-related hearing loss and tinnitus. [ABSTRACT FROM AUTHOR]

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