Reinforce revealing missing heritability in the gene of inherited retinal diseases / 中华眼底病杂志

Inherited retinal diseases (IRD) are a group of genetic disorders with high genetic and clinical heterogeneity. Genetic diagnosis has become one essential method for patients with IRD in their clinical management. So far, about 30% of the patients with IRD cannot get molecular diagnosis (no pathogenic variant detected or only mono-allele variant identified in AR genes) using target or whole exome sequencing. Most missing heritability or variants for these patients were variants located in no-coding regions (deep intron or promoter regions) and structure variants of the known IRD genes. It is more challenge to reveal this kind of missing variants, which need using whole genome sequencing combined with other cellular or molecular assays.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at the Bact state, an active spliceosome but not catalytically primed, which depends on Smad Nuclear Interacting Protein 1 (SNIP1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing, and that its misregulation contributes to neurodegeneration.

Group Ⅱ introns and the application in biotechnology: a review / 生物工程学报

Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.

Correlation between type Ⅱ CD36 deficiency and intron polymorphism / 中国输血杂志

【Objective】 To study the correlation between type ⅡCD36 deficiency and the polymorphism of intron sequence. 【Methods】 A total of 516 random healthy platelet donors from Liaoning Blood Center were involved: 241 of them were tested for CD36 antigen and CD36 gene sequence; the remaining 275 cases were sequenced only. CD36 antigen was detected by flow cytometry, and gene sequence was analyzed by PCRamplification and sequencing. 【Results】 Among the 241 samples, 1 case of type Ⅰ deficiency and 4 cases of type Ⅱ deficiency were detected, with frequencies of 0.41% and 1.66%, respectively. There was no nucleotide change in the coding region of 3 cases of typeⅡdeficiency. All individuals with type Ⅱ deficiency shared a common polymorphism in the intron 3, that is, carried (TG) 11 and 12 linked variants, and both were located in the same allele. The gene frequency of (TG)11 in the 516 random population was only 11.72%, which was much lower than 30.43% of (TG)13. The gene frequency of 12 linked variants in the random population was 8.81%. Almost all 12 linked variants occurred simultaneously with (TG)11, but only about 72.7% of (TG)11 were tandem with 12 linked variants. Flow cytometry showed that the expression of CD36 antigen on platelet in samples carrying only (TG)11 was comparable to that of normal samples, while the vast majority of samples carrying both (TG)11 and 12 linked variants had significantly lower CD36 antigen levels. 【Conclusion】 The (TG)11 in the intron 3 region is not platelet-specific silent allele, but there are some indirect correlations. There may be multiple platelet-specific silent allele.

Three cases of cerebraltendinous xanthomatosis caused by compound heterozygous mutation of CYP27A1 gene / 中华神经科杂志

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive genetic disease caused by mutation of CYP27A1 gene. This article reported three cases with clinical phenotypes of CTX and CYP27A1 gene mutation and analyzed the pedigree with a literature review. All the three CTX cases had c.379C>T (p.Arg127Trp) missense mutation on exon 2 of CYP27A1 gene. They all had compound heterozygous mutation and two cases had new type of exon and intron compound mutation. This article enriched the types of CYP27A1 gene mutations in CTX patients. The primers of CYP27A1 gene should also cover more gene sequences including intron regions.

Conservation of gene architecture and domains amidst sequence divergence in the hsrω lncRNA gene across the Drosophila genus: an in silico analysis

The developmentally active and cell-stress responsive hsrx locus in Drosophila melanogaster carries two exons, one omega intron, one short translatable open reading frame (ORFx), long stretch of unique tandem repeats and an overlapping mir-4951 near its 30 end. It produces multiple long noncoding RNAs (lncRNAs) using two transcription start and four termination sites. Earlier cytogenetic studies revealed functional conservation of hsrx in several Drosophila species. However, sequence analysis in three species showed poor conservation for ORFx, tandem repeat and other regions while the 16 nt at 50 and 60 nt at 30 splice junctions of the omega intron, respectively, were found to be ultra-conserved. The present bioinformatic study using the splice-junction landmarks in D. melanogaster hsrx identified orthologues in publicly available 34 Drosophila species genomes. Each orthologue carries a short ORFx, ultra-conserved splice junctions of omega intron, repeat region, conserved 30 -end located at mir-4951, and syntenic neighbours. Multiple copies of conserved nonamer motifs are seen in the tandem repeat region, despite a high variability in the repeat sequences. Intriguingly, only the omega intron sequences in different species show evolutionary relationships matching the general phylogenetic history in the genus. Search in other known insect genomes did not reveal sequence homology although a locus with similar functional properties is suggested in Chironomus and Ceratitis genera. Amidst the high sequence divergence, the conserved organization of exons, ORFx and omega intron in this gene’s proximal part and tandem repeats in distal part across the Drosophila genus is remarkable and possibly reflects functional importance of higher order structure of hsrx lncRNAs and the small omega peptide.

Comparative study of the SBP-box gene family in rice siblings

SBP-box genes are a class of plant-specific transcription factors which have a common DNA-binding domain(SBP-domain) with an unusual zinc-finger architecture. Many of the genes in this class are thought to play adevelopmental role and a few are involved in the determination of plant architecture. We have made acomparative study of these genes in the genomes of rice (Oryza sativa japonica and Oryza sativa indica) andits nine siblings using a recently proposed hybrid method for orthology and paralogy detection (HyPPO).According to HyPPO, the SBP-box proteins of rice siblings could be divided into twenty primary orthologousgroups on the basis of their overall sequence features. This contrasts with a much less number of groups foundin earlier work with other plant genomes using phylogenetic analysis of the SBP-domains only. The orthologous groups reported by HyPPO showed close correspondence in exon–intron structure and motif conservation. Comparison between different Oryza species revealed disparity in the maintenance of orthologousgenes which may result in their different developmental characteristics. Inclusion of the SBP-box proteins fromA. thaliana did not result in any change in the orthologous groups except for the A. thaliana proteins beingadded to some of the existing groups. The closer correspondence between the proteins in the primaryorthologous clusters is expected to help in a more reliable prediction of their functions. It is also expected toprovide better insight into the evolutionary history of this class of plant-specific proteins.

The relationship between bronchopulmonary dysplasia and SP-B intron 4 gene polymorphism in Mongolian and Han newborns in Inner Mongolia / 中国新生儿科杂志

Objective To study the relationship between pulmonary surfactant protein B (SP-B) intron 4 gene polymorphism and bronchopulmonary dysplasia (BPD) in premature infants.Method From January 2016 to January 2019,premature infants diagnosed with BPD in our hospital were selected as the BPD group,and non-BPD premature infants of the same ethnic group were selected as the control group.The genotype and allele distribution of SP-B intron 4 were analyzed using polymerase chain reaction (PCR)method.Result A total of 74 infants with BPD were included,including 30 Mongolian infants and 44 Han infants.A total of 134 cases were in the control group,including 56 Mongolian infants and 78 Han infants.Wild type and variant type (including insertion and deletion) could be detected in SP-B intron 4 gene in both Mongolian and Han infants.The frequencies of wild and variant genotypes and alleles in Mongolian BPD infants were similar with the control group [36.7％ (11/30) vs.19.6％ (11/56),21.7％ (13/60) vs.12.5％ (14/112)] (P ＞ 0.05).The frequencies of wild and variant genotypes and alleles in Han infants with BPD were significantly different from the control group [31.8 ％ (14/44) vs.12.8 ％ (10/78),20.5 ％(18/88)vs.7.1％(11/156)] (P＜0.05).Conclusion The variation of intron 4 gene in SP-B may be related with the genetic susceptibility of Han infants with BPD in Inner Mongolia.

Insights into the evolution of extracellular leucine-rich repeats in metazoans with special reference to Toll-like receptor 4

The importance of the widely spread leucine-rich repeat (LRR) motif was studied considering TLRs, the LRR-containingprotein involved in animal immune response. The protein connects intracellular signalling with a chain of molecularinteractions through the presence of LRRs in the ectodomain and TIR in the endodomain. Domain analyses with humanTLR1-9 reported ectodomain with tandem repeats, transmembrane domain and TIR domain. The repeat number variedacross members of TLR and remained characteristic to a particular member. Analysis of gene structure revealed absence ofcodon interruption with TLR3 and TLR4 as exceptions. Extensive study with TLR4 from metazoans confirmed thepresence of 23 LRRs in tandem. Distinct clade formation using coding and amino acid sequence of individual repeatsillustrated independent evolution. Although ectodomain and endodomain exhibited differential selection pressure, withinthe ectodomain, however, the individual repeats displayed positive, negative and neutral selection pressure depending ontheir structural and functional significance.

Enrichment of rare alleles within epigenetic chromatin marks in the first intron

In previous studies, we demonstrated that some sites in the first intron likely regulate gene expression. In the present work, we sought to further confirm the functional relevance of first intron sites by estimating the quantity of rare alleles in the first intron. A basic hypothesis posited herein is that genomic regions carrying more functionally important sites will have a higher proportion of rare alleles. We estimated the proportions of rare single nucleotide polymorphisms with a minor allele frequency < 0.01 located in several histone marks in the first introns of various genes, and compared them with those in other introns and those in 2-kb upstream regions. As expected, rare alleles were found to be significantly enriched in most of the regulatory sites located in the first introns. Meanwhile, transcription factor binding sites were significantly more enriched in the 2-kb upstream regions (i.e., the regions of putative promoters of genes) than in the first introns. These results strongly support our proposal that the first intron sites of genes may have important regulatory functions in gene expression independent of promoters.

Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions / 生物工程学报

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.

Intron mutations of argininosuccinate lyase (ASL) gene exist in a family with argininosuccinic aciduria / 临床检验杂志

Objective@#To perform gene detection and gene mutation analysis in a family with inherited metabolic diseases characterized as increased citrulline (Cit) by the MS/MS assay. @*Methods@#The peripheral blood samples were collected from the family members, and genomic DNA was extracted for gene diagnosis, which was performed by the whole exon sequencing method. The novel mutation gene was cloned into pcDNA3.1(+) vector, and its pathogenicity was verified by the Mini-gene assay in cultured cells in vitro. @*Results@#The clinical diagnosis of the proband as argininosuccinic aciduria (ASA) was clear. Two pathogenic mutations, c.281G＞T (p.Arg94Leu) and c.208-15 T＞A, were detected in the argininosuccinate lyase (ASL) gene, and they were not reported previously. The Mini-gene expression in vitro confirmed that c.208-15 T＞A could cause aberrant splicing, resulting in the retention of 13 bp in intron 2. @*Conclusion@#Two new pathogenic mutations of ASL gene, c.208-15 T＞A and c.281G＞T, are found in an ASA family, which enriches the mutation profile of ASL gene. The Mini-gene assay is a simple and effective tool for the research of intron mutations.

The identification of Sect. Cruciata (Gentiana) species using mtDNA nad1/b-c and nad5/d-e fragments / 药学学报

italic>Gentiana section Cruciata (Gentianaceae) is a medicinally important section of herbs, including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji. Here, we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data. A total of 144 nad1/b-c and nad5/d-e sequences from 11 species within Gentianaceae were obtained, including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica (outgroup). The results showed that mtDNA nad1/b-c has species- level resolution within the section of Cruciata, i.e. the variable in the position 45 “C” could be used as a stable marker locus to distinguish G. robusta from other taxa; the variable in the position 352 and 353 “GA” could distinguish G. crassicaulis and G. tibetica from other taxa within the section. Intraspecies genotype variability was detected in nad1/b-c sequences of G. officinalis and G. siphonantha, respectively. These genotypes could be used as potential DNA barcode. In addition, intraspecies genotype variability was detected in nad5/d-e sequences of G. macrophylla, G. officinalis and G. siphonantha, respectively. Based on the stable marker locus, a species-specific PCR protocol was developed using the primer PF to identifying G. robusta in the section. This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana, and provide the essence for the species identification within Gentiana section Cruciata.

Correlation between bronchopulmonary dysplasia and polymorphism of intron 5 gene of surfactant pro-tein B in neonates in Inner Mongolia / 中国小儿急救医学

Objective To study the correlation between polymorphism of intron 5 gene of surfactant protein B(SP-B)and bronchopulmonary dysplasia(BPD) in neonates and to explore the relevance of the pathogenesis of BPD at the genetic level.Methods Fifty infants with BPD admitted to the NICU in the Hos-pital of Inner Mongolia Medical University from November 2016 to November 2017 were selected as the BPD group.Among them,13 were Mongolian nationality,37 were Han nationality. Fifty newborn without BPD during the same period were assigned to the control group in this study,among them,15 were Mongolian na-tionality,35 were Han nationality.PCR technique was used to detect SP-B gene intron 5 polymorphism and its genotype and allele distribution. Results Regardless of Mongolian or Han nationality,three genotypes of SP-B intron 5 genotypes were detected:wild type,insert type and deletion type.The frequencies of the three genotypes were 73.0%(27/37),10.8%(4/37) and 16.2%(6/37) respectively,and the allele frequencies were 77.0%(57/74),9.5%(7/74) and 13.5%(10/74) respectively in the Han nationality BPD group.The frequencies of the three genotypes in the Han nationality control group were 82.9%(29/35),11.4%(4/35) and 5.7%(2/35)respectively;the allele frequencies were 85.7%(60/70),8.6%(6/70) and 5.7%(4/70) respectively.In the Mongolian BPD group,the frequencies of the above three genotypes were 53.8%(7/13), 15.4%(2/13) and 30.8%(4/13) respectively,and the allele frequencies were 61.5%(16/26),15.4%(4/26) and 23.1%(6/26) respectively.The frequencies of the above three genotypes in the Mongolian con-trol group were 53.3%(8/15),26.7%(4/15) and 20.0%(3/15),the allele frequencies were 66.7%(20/30),20.0%(6/30),13.3%(4/30) respectively.There were no significant differences in the allele fre-quencies and the genotypes of SP-B intron 5 between the Han nationality BPD group and the Han control group,the Mongolian BPD group and the Mongolian control group(all P>0.05).Conclusion No poly-morphisms of SP-B intron 5 and BPD in newborns of the Han nationality and Mongolian nationnality in Inner Mongolia are found.

Development of indel markers for molecular authentication of Panax ginseng and P. quinquefolius / 中国中药杂志

Panax ginseng and P. quinquefolius are two kinds of important medicinal herbs. They are morphologically similar but have different pharmacological effects. Therefore, botanical origin authentication of these two ginsengs is of great importance for ensuring pharmaceutical efficacy and food safety. Based on the fact that intron position in orthologous genes is highly conserved across plant species, intron length polymorphisms were exploited from unigenes of ginseng. Specific primers were respectively designed for these two species based on their insertion/deletion sequences of cytochrome P450 and glyceraldehyde 3-phosphate dehydrogenase, and multiplex PCR was conducted for molecular authentication of P.ginseng and P. quinquefolius. The results showed that the developed multiplex PCR assay was effective for molecular authentication of P.ginseng and P. quinquefolius without strict PCR condition and the optimization of reaction system.This study provides a preferred ideal marker system for molecular authentication of ginseng,and the presented method can be employed in origin authentication of other herbal preparations.

Introns in Cryptococcus

In Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.

Estudio molecular de la inversión de los intrones 1 y 22 del factor VIII de la coagulación en niños con hemofilia A severa utilizando técnica de PCR de larga distancia / Molecular study in children with hemophilia A in Colombia: analysis of Intron 1 and 22 inversion using long-distance PCR technique

Resumen Introducción. La hemofilia A es una enfermedad recesiva ligada al cromosoma X, con una incidencia de 1 en 5 000 a 10 000 varones y es el trastorno hemostático congénito más frecuente en varones. En pacientes con fenotipo severo, las inversiones de los intrones 22 y 1 son las mutaciones más comunes con una prevalencia del 45% a 50% y del 1% al 5% de los pacientes, respectivamente. Objetivo. Determinar la frecuencia de la inversión de los intrones 1 y 22 del gen del factor VIII de la coagulación en menores de 18 años con hemofilia A severa en Bogotá D.C. Materiales y métodos. Estudio descriptivo y transversal. La identificación de la inversión de los intrones 1 y 22 del gen del factor VIII se realizó mediante técnicas de reacción en cadena de polimerasa de larga distancia. Resultados. Se estudiaron 30 pacientes y se encontró inversión del intrón 22 en 12 pacientes (40%) e inversión 1 en 3 pacientes, cifras similares a las observadas en otros estudios. Conclusiones. Se encontraron las inversiones de los intrones 1 y 22 en la mitad de los pacientes evaluados. Los resultados son reproducibles, por lo que constituyen una herramienta útil para la identificación de las dos mutaciones más frecuentes en hemofilia A severa.

Abstract Introduction: Hemophilia A is an X-linked recessive disease with an incidence of 1 in 5 000 to 10 000 males. It is the most common congenital hemostatic disorder in men. The inversion of introns 1 and 22 in patients with a severe phenotype is considered the most frequent abnormality, with a prevalence of 1 to 5% and 45 to 50%, respectively. Objective: To determine the frequency of introns 1 and 22 inversions in factor VIII gene in children under 18 years with severe hemophilia A in Bogotá. Materials and methods: This is a non-experimental, descriptive, transverse study. The inversions of introns 1 and 22 for factor VIII gene were identified using long-distance polymerase chain reaction techniques in pediatric patients with severe Hemophilia A treated in different centers of Bogotá, Colombia. Results: Thirty patients were analyzed. Inversion of intron 22 was found in 12 patients (40%), while inversion of intron 1 was observed in 3 patients. These findings are similar to other studies. Conclusions: Inversions of intron 22 and 1 were found in half of this group of patients. These results are reproducible and useful to identify the two most frequent mutations in severe hemophilia A patients.

Identification of processed Chinese medicinal materials using DNA mini-barcoding / 中国天然药物

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psbA-trnH, rbcL, matK, trnL (UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL (UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%-20% of the processed samples, while the amplification rates of the trnL (UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL (UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.

Association of Single Nucleotide Polymorphisms of Autophagy-related Genes Atg7 Site rs11706903 and Parkin-son's Disease / 中国康复理论与实践

Objective To investigate the relationship between the single nucleotide polymorphisms(SNP)of rs11706903,intron region of Atg7,and Parkinson's disease(PD).Methods From January,2013 to March,2017,130 PD patients and 109 healthy subjects were recruit-ed and collected the blood samples.SNPs of rs11706903 were detected with polymerase chain reaction restriction fragment length polymor-phism.Results For the patients,the genotype frequencies of rs11706903 were AA 10.00%,AC 52.31% and CC 37.69%;and allele frequen-cies were A 36.15% and C 63.85%.For the healthy controls,the genotype frequencies of rs11706903 were AA 7.34%,AC 49.54% and CC 43.12%;and allele frequencies were A 32.11% and C 67.89%.There was no significant difference in frequencies of genetypes and alleles be-teen two groups(χ 2<1.001,P>0.05).Conclusion There might be no relationship between SNPs of Atg7 intron region rs11706903 and PD.

The association of the polymorphism of the 5-HTT gene with alcohol use disorders / 中华行为医学与脑科学杂志

Objective To investigate the possible associations of two polymorphisms (5-HTTLPR and STin2VNTR)of the serotonin transporter gene with alcohol use disorders (AUD).Methods 281 AUD cases (AUDIT score≥ 10) and 277 healthy controls (AUDIT score ≤5) were recruited in this study.All participants were genotyped using the PCR technique.Results The frequency of the L-allele of the 5-HTTLPR was 39.01％,and the 10-allele of STin2VNTR was 8.42％ in this population,the allele frequencies of both polymorphisms were consistent with Asian normal populations.No significant association was observed between 5-HTTLPR and AUD,but the genotypic and allele frequencies of the STin2VNTR were significant different between two groups even after Bonferroni adjustment,the 12 repeat allele of the STin2VNTR was significantly associated with the risk effect for AUD.Haplotype analysis for those two polymorphisms revealed no association between 4 haplotype combinations and AUD.Conclusion There is no relationship between 5-HTTLPR and AUD.The STin2VNTR polymorphism of 5-HTT may play a role in the pathogenesis of AUD.