Inversión de los intrones 1 y 22 del F8 en pacientes con hemofilia A severa y portadoras del noreste de Uruguay / F8 intron 1 and 22 inversions in patients with severe haemophilia A and in carriers of the northeastern region of Uruguay / Inversão dos introns 1 e 22 do F8 em pacientes com hemofilia A severa e portadoras do nordeste de Uruguai

Resumen: La hemofilia A (HA) es la coagulopatía ligada al cromosoma X más frecuente. Es causada por mutaciones en el gen del factor VIII (FVIII) de coagulación (F8). La HA puede ser severa cuando la actividad del FVIII es menor a 1% (FVIII: C<1IU/dL). Casi la mitad de las HA severas son producidas por inversiones del F8, como la del intrón 1 (Inv1) y del intrón 22 (Inv22). Los pacientes con HA severa experimentan sus primeros sangrados generales entre los 9,7 - 10,9 meses, ocurriendo principalmente en las articulaciones. Se investigó la presencia de la Inv1 e Inv22 en la región noreste de Uruguay (departamentos de Tacuarembó, Rivera y Cerro Largo) para estimar su frecuencia y detectar la presencia de portadoras. Fueron estudiados 14 individuos (ocho pacientes con HA severa, cuatro madres y dos hermanas de pacientes) de cinco familias. La investigación de las inversiones se realizó aplicando las pruebas de inverse shifting-PCR (IS-PCR). La Inv1 se encontró en dos pacientes (hermanos) de Tacuarembó, en su hermana y madre (portadoras), mientras que un paciente de Rivera y su madre (portadora) resultaron positivos para la Inv22. Preliminarmente, en conjunto, la Inv1 y la Inv22 representan la causa de la HA severa en el 40% de las familias del noreste de Uruguay, valor menor a lo esperado; sin embargo, debido a la reducida población estudiada, la Inv1 muestra una frecuencia preliminar (20%, 1/5 familias, 25%, 2/8 pacientes) considerablemente mayor a estudios previos. Estos datos permiten caracterizar la etiología genética de la hemofilia, la detección de las portadoras, conocer la distribución geográfica de las mutaciones y el asesoramiento genético.

Summary: Hemophilia A (HA) is the most common X-linked coagulopathy, it is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). HA can be severe when the FVIII activity is less than 1% (FVIII: C <1IU / dL). Almost a half of the severe HAs are produced by inversions of F8, the intron 1 (Inv1) and intron 22 (Inv22). Patients with severe HA show their first general bleeding between 9.7 - 10.9 months, mainly in the joints. We researched the presence of Inv1 and Inv22 in the Northeast region of Uruguay (Departments: Tacuarembó, Rivera and Cerro Largo) to estimate their frequency and detect the presence of carriers. We studied 14 individuals in 5 different families (8 patients with severe HA, 4 mothers and 2 sisters of patients). The inversion study was carried out using inverse shifting-PCR (IS-PCR) tests. Inv1 was found in 2 patients (siblings) from Tacuarembó, in their sister and mother (carriers). A patient from Rivera and his mother (carrier) were positive for Inv22. Inv1 and Inv22 are the cause of severe HA in 40% of the patients in North East of Uruguay, less than expected; however, due to the reduced population studied, Inv1 shows a considerably higher frequency than previous studies. These data enable us to characterize the genetic etiology of hemophilia, to adequately monitor patients, detect carriers, the geographical distribution of mutations and the corresponding genetic counseling for families.

Resumo: A hemofilia A (HA) é a coagulopatia ligada ao cromossomo X mais frequente, causada por mutações no gene do fator VIII (FVIII) de coagulação (FVIII) (F8). A HA pode ser grave quando a atividade do FVIII é menor que 1% (FVIII: C <1IU / dL). Quase metade da HA grave é produzida por inversões de F8, como a do Íntron 1 (Inv1) e do Íntron 22 (Inv22). Pacientes com HA grave experimentam seu primeiro sangramento geral entre 9,7 e 10,9 meses, principalmente nas articulações. A presença de Inv1 e Inv22 na região nordeste do Uruguai (departamentos: Tacuarembó, Rivera e Cerro Largo) foi investigada para estimar a sua frequência e detectar a presença de portadora. Foram estudados 14 indivíduos (8 pacientes com HA grave, 4 mães e 2 irmãs de pacientes) de 5 famílias. A pesquisa das inversões foi realizada aplicando os testes de inverse shifting -PCR (IS-PCR). Encontramos Inv1 em 2 pacientes (irmãos) de Tacuarembó, na sua irmã e mãe (portadoras), enquanto 1 paciente de Rivera e sua mãe (transportadora) foram positivos para Inv22. Preliminarmente, Inv1 e Inv22 juntos representam a causa de HA grave em 40% das famílias do nordeste do Uruguai, valor inferior ao esperado, no entanto, devido à pequena população estudada, Inv1 mostra uma frequência preliminar (20%, 1/5 famílias, 25%, 2/8 pacientes) consideravelmente mais alta que os estudos anteriores. Esses dados permitem-nos caracterizar a etiologia genética da hemofilia, detectar aos portadores, conhecer a distribuição geográfica das mutações e realizar aconselhamento genético.

ABC genotyping and putative virulence factors of Candida albicans clinical isolates

Aims@#Candida albicans is a diploid yeast which interacts with the host in a complex nature involving several fungal virulence factors and host’s response. In this study, we investigated the different ABC genotypes of 26 clinical C. albicans isolates which is based on the presence of absence of transposable intron in the 25S rDNA, and the phenotypic expression of their virulence factors: phospholipase production, esterase production, haemolytic activities, biofilm formation, and white-opaque switching. @*Methodology and results@#In this study, we investigated the ABC genotypes of 26 clinical C. albicans isolates, and the phenotypic expression of their virulence factors. The C. albicans isolates were tested for their in vitro abilities in exhibiting the following virulence factors: phospholipase, biofilm, esterase, hemolysin and phenotypic switching. Phospholipase activities and biofilm formation were detected in 57.7% and 65.38% of the isolates, respectively. All of the isolates showed phenotypic white-type colony, while none showed esterase and hemolytic activities. ABC genotyping revealed that 50% of the isolates were grouped under Genotype A, followed by Genotype C (42.3%), and B (7.69%). @*Conclusion, significance and impact of study@#This study provides information in regard to virulence potential and the ABC genotype of C. albicans from the Philippines.

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.

Introns: The Functional Benefits of Introns in Genomes

The intron has been a big biological mystery since it was first discovered in several aspects. First, all of the completely sequenced eukaryotes harbor introns in the genomic structure, whereas no prokaryotes identified so far carry introns. Second, the amount of total introns varies in different species. Third, the length and number of introns vary in different genes, even within the same species genome. Fourth, all introns are copied into RNAs by transcription and DNAs by replication processes, but intron sequences do not participate in protein-coding sequences. The existence of introns in the genome should be a burden to some cells, because cells have to consume a great deal of energy to copy and excise them exactly at the correct positions with the help of complicated spliceosomal machineries. The existence throughout the long evolutionary history is explained, only if selective advantages of carrying introns are assumed to be given to cells to overcome the negative effect of introns. In that regard, we summarize previous research about the functional roles or benefits of introns. Additionally, several other studies strongly suggesting that introns should not be junk will be introduced.

Gene polymorphism in intron 4 of surfactant protein-B in bronchopulmonary dysplasia infants / 中华围产医学杂志

ObjectiveTo investigate the change of gene polymorphorism of surfactant protein-B (SP-B) intron 4 in infants with bronchopulmonary dysplasia (BPD).MethodsForty-five infants with BPD (BPD group) and ninety-nine infants without lung diseases (control group) who admitted into Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from July 2008 to July 2011 were selected into this study.Genotyping for fragment length polymorphism of SP-B intron 4 was performed by polymerase chain reaction (PCR),agarose gel electrophoresis,cloning and sequencing methods in both groups.Differences of allele frequencies (invariant allele and variant allele) and genotype frequencies (invariant genotype and variant genotype) between BPD group and control group were analyzed.The differences of gestational age and birth weight between the two groups were compared with Independent-Samples t test.The gender composition and differences of allele or genotype frequencies between the two groups were compared with Chi-square test.Results Invariant allele frequencies in BPD group and control group were 83.3％ (75/90) and 92.0％ (182/198),and variant allele frequencies were 16.7％ (15/90,including eight insertion alleles and seven deletion alleles) and 8.1％ (16/198,including eight insertion alleles and eight deletion alleles).There were significant differences between the two groups (x2 =4.75,P =0.029).In BPD group,there were 32 cases (71.1 ％,32/45) invariant genotypes and 13 cases (28.9 ％,13/45,including seven cases insertions and six cases deletions) variant genotypes; in the control group,there were 85 cases invariant genotypes (85.8％,85/99) and 14 cases (14.1％,14/99,six insertions and eight deletions) variant genotypes.Significant difference was found between the two groups (x2=4.42,P＜0.036). ConclusionsVariations of SP-B intron 4 were more in BPD infants,and the variation of SP-B intron 4 might be associated with BPD.

Identification of a Novel Intronic Transcript Containing 11q13.5 HERV-W gag Sequence / 天津医药

Objective:To identify the novel intronic transcripts containing 11q13.5 HERV-W gag sequence, and explore the modulation of the transcripts on the alternative splicing of host gene PTD015. Methods:Half-nested PCR and touchdown PCR were used to amplify the target transcripts. The transcripts were cloned and sequenced. The plasmids inserted with the target transcripts were transfected into JEG3 cells and the alternative spliced mRNA levels of PTD015 were measured with real time PCR. Results:A 1 739 bp novel intronic transcript containing 755 bp 11q13.5 HERV-W gag sequence, 527 bp 5′ long terminal repeat and 457 bp sequence on the 5′-end of 11q13.5 HERV-W was identified. The intronic antisense transcripts significantly down-regulated the alternative spliced mRNA levels of PTD015. Conclusion:The intronic antisense transcripts originating from the second intron of gene PTD015 could modulate the alternative splicing of the host gene PTD015.

Simultaneous mutations in exon and intron of a novel human leukocyte antigen-B*3818 allele revealed by genomic sequence analysis / 中华检验医学杂志

Objective To analysis the genomic sequence of a novel human leukocyte antigen (HLA)-B*3818 allele.Methods Full length genomic sequence of an unknown HLA-B allele was cloned,followed by bi-directional sequencing and the specificity of the antigen coded by this novel allele was defined by microcytotoxicity assay.The frequency and haplotype of this novel allele was acquired by population census and parentage analysis.Results The full length genomic sequence of this novel HLA-B*3818 allele with accession number FJ561482 differs from HLA-B*380201 by two nucleotide changes in exon 4 and intron 5,respectively.One change is located at nt 660 in exon 4 where C→A alternation,which results in an amino acid substitution from Asp(GAC)to Glu(GAA)at codon 196.This alternation is a new single nucleotide polymorphism compared with all other HLA-B alleles.Another is located at genomic position 2133 in intron 5(A→C).Except for this substitution,the intron sequences of HLA-B*3818 allele are identical to those of other HLA-B*38 alleles including HLA-B*380101,B*380201 and B*3814.The serological specificity of HLA-B*3818 is B38 and the frequency of this new allele is less than 0.000 5 in Chinese Han population.The parentage analysis showed the haplotype of novel allele is A*030101-Cw*010201-B*3818-DRB1*1312-DOB1*060101.Conclusion The simultaneous mutations in exon and intron were found in the Hovel HLA-B*3818 allele,and so it can present more sequence information for studies and applications associated with HIA genes by analyzing the genomic sequences of novel HLA alleles.

Analysis of single nucleotide polymorphisms of CRYGA and CRYGB genes in control population of western Indian origin.

Aim: Polymorphisms in gamma-crystallins ( CRYG ) can serve as markers for lens differentiation and eye disorders leading to cataract. Several investigators have reported the presence of sequence variations within crystallin genes, with or without apparent effects on the function of the proteins both in mice and humans. Delineation of these polymorphic sites may explain the differences observed in the susceptibility to cataract observed among various ethnic groups. An easier Restriction Fragment Length Polymorphism (RFLP)-based method has been used to detect the frequency of four single nucleotide polymorphisms (SNPs) in CRYGA / CRYGB genes in control subjects of western Indian origin. Materials and Methods: A total of 137 healthy volunteers from western India were studied. Examination was performed to exclude volunteers with any ocular defects. Polymerase chain reaction (PCR)-RFLP based method was developed for genotyping of G198A (Intron A), T196C (Exon 3) of CRYGA and T47C (Promoter), G449T (Exon 2) of CRYGB genes. Results: The exonic SNPs in CRYGA and CRYGB were found to have an allele frequency 0.03 and 1.00 for ancestral allele respectively, while frequency of non-coding SNP in CRYGA was 0.72. Allele frequency of T90C of CRYGB varied significantly ( P = 0.02) among different age groups. An in-silico analysis reveals that this sequence variation in CRYGB promoter impacts the binding of two transcription factors, ACE2 (Member of CLB2 cluster) and Progesterone Receptor (PR) which may impact the expression of CRYGB gene. Conclusions: This study establishes baseline frequency data for four SNPs in CRYGA and CRYGB genes for future case control studies on the role of these SNPs in the genetic basis of cataract.

Advances in research on intronic miRNAs feedback regulation of their host genes / 军事医学科学院院刊

miRNAs were discovered less than a decade ago, and have emerged as important regulators of gene expression in mammals. A large number of miRNAs have been identified to be located within the intronic regions of protein-encoding genes(host genes) and called intronic miRNAs. The intronic miRNAs may play a key role in regulating the expression and function of their host genes due to the fact that most of them are co-expressed with the host genes. In this paper, the recent advances on the research on potential relationship between intronic miRNAs and their host genes are reviewed.

Effect of human factor Ⅸ intron 1 inserted in forward orientation into retroviral vectors on expression of factor Ⅸ in muscle cells / 第二军医大学学报

Objective:To investigate the effects of human factor Ⅸ (FⅨ) intron 1 inserted in forward orientation into retroviral vector on the expression of FⅨ in muscle cells. Methods: Two FⅨ mini-genes, FⅨm1 and FⅨm2, were inserted in forward orientation into retroviral vector backbone, LⅨSN, constructing 2 vectors named LⅨm1SN and LⅨm2SN, containing shortened intron of 1.4 and 0.3 kb, respectively. Retrovirus was made by transfecting PA317 packaging cells. Transient and stable expression of FⅨ was tested in SCID mouse muscle cells (SCID-MB). Results: The vector titers and expression levels of LⅨm1SN and LⅨm2SN in SCID-MB were 1 to 2 folds higher than that of LⅨSN. Some SCID-MB colonies transfected with LⅨm2SN contained intact FⅨm2 inton elements, suggesting that splicing of virus mRNA may not be complete in packaging cells. Conclusion: FⅨ intron 1 inserted in forward orientation in retroviral vectors can increase retroviral titer and the expression level of FⅨ in muscle cells.