Prokaryotic expression and activity detection of insulin-degrading enzyme mutant T142A / 中国生物制品学杂志

@#Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate cleavage sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min~(-1) and the Michaelis constant(K_m) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_m was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

Analysis on clinical and genetic characteristics of children with ATP-sensitive potassium channel congenital hyperinsulinism / 中华胰腺病杂志

Objective:To analyze clinical characteristics and genetic characteristics of children with ATP sensitive potassium passage (K ATP-HI). Methods:Forty-five children with genetically confirmed K ATP-HI and their families admitted to Beijing Children′s Hospital of Capital Medical University between February 2002 and December 2018 were selected as the study subjects. A detailed retrospective analysis of the patient's clinical characteristics, diagnosis and treatment process, disease-causing gene carrying status and later follow-up data was performed. ABCC8/KCNJ11 gene was sequenced by second-generation sequencing technology. Results:Among 45 children with K ATP-HI, 34 cases (75.6%) were neonatal onset, the first symptoms of 21 cases (46.7%) were convulsions. 39 cases had been treated with diazoxide, including 12 cases (30.8%) with good efficacy, 16 cases (41%) with poor efficacy and 11 cases with uncertain efficacy. Octreotide was further applied in 18 patients with uncertain or ineffective efficacy after diazoxide treatment, and 13 cases (72.2%) were effective, 3 cases were ineffective, and 2 cases were uncertain. 10 CHI patients who were ineffective to drug treatment or had clearly focal lesions confirmed by 18F-dopa positron emission by computed tomography ( 18F-DOPA PET) scans had undergone surgical treatment, 8 of which underwent partial pancreatectomy and blood glucose returned to normal after the operation; the other 2 cases underwent subtotal pancreatectomy and both had secondary diabetes after operation. Among 45 children with K ATP-HI, 1 case carried both ABCC8 and KCNJ11 mutations, 10 cases carried ABCC8 compound heterozygous mutations, and the remaining 34 cases carried ABCC8/KCNJ11 single genetic mutation. Among them, 21 cases had paternal inheritance, and 3 cases had maternal inheritance, 6 cases were identified with de novo mutations. Conclusions:Diazoxide treatment was ineffective for most K ATP-HI children, but octreotide had a higher effective rate. Partial pancreatectomy for focal type patients had a higher cure rate, and there was a risk of secondary diabetes after subproximal pancreatectomy, so it was very important to clarify the histological type of children before surgery. ABCC8 gene mutations and KCNJ11 gene mutations were the main pathogenic genes of K ATP-HI. Among patients carrying mutations in single ABCC8 or KCNJ11 gene mutation, K ATP-HI inherited by paternity were the majority. Some K ATP-HI children can relieve the hypoglycemia symptoms by themselves.

Construction of Pnpla3 I148M and Tm6sf2 E167K double mutant mouse model / 临床肝胆病杂志

Objective To construct a Pnpla3 148M/M Tm6sf2 167K/K double mutant mouse model by crossbreeding Pnpla3 148M/M homozygous mice and Tm6sf2 167K/K homozygous mice. Methods Pnpla3 148I/M Tm6sf2 167E/K heterozygous mice were bred by hybridization of Pnpla3 148M/M Tm6sf2 167E/E and Pnpla3 148I/I Tm6sf2 167K/K homozygous mice, and the Pnpla3 148M/M Tm6sf2 167K/K mice were obtained by the self-crossbreeding of Pnpla3 148I/M Tm6sf2 167E/K mice. Male mice of Pnpla3 148M/M Tm6sf2 167K/K ( n =6), Pnpla3 148M/M Tm6sf2 167E/E ( n =6), and Pnpla3 148I/I Tm6sf2 167K/K ( n =6) genotypes and Wt mice ( n =6) were fed with normal diet for 8 weeks, and then the glucose and lipid metabolism indices were measured. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t -test was used for further comparison bewteen two groups. Results Agarose gel electrophoresis and nucleic acid sequencing results showed that the Pnpla3 148M/M Tm6sf2 167K/K double mutant mouse model was successfully constructed. There were no significant difference in body weight between the Pnpla3 148M/M Tm6sf2 167K/K mice and the Pnpla3 148M/M Tm6sf2 167E/E , Pnpla3 148I/I Tm6sf2 167K/K , and Wt mice (all P > 0.05). The Pnpla3 148M/M Tm6sf2 167K/K mice had a significantly higher liver wet weight than the Wt mice ( P 0.05). Also, there were no significant differences in the serum levels of biochemical indices between the Pnpla3 148M/M Tm6sf2 167K/K mice and the Pnpla3 148M/M Tm6sf2 167E/E , Pnpla3 148I/I Tm6sf2 167K/K , and Wt mice (all P > 0.05). Oil red O staining of the liver showed that more lipid accumulation was observed in the Pnpla3 148M/M Tm6sf2 167K/K mice than in the Pnpla3 148M/M Tm6sf2 167E/E and Wt mice. Conclusion The Pnpla3 148M/M Tm6sf2 167K/K double mutant mouse model was successfully constructed. Pnpla3 Ⅰ 148M and Tm6sf2 E 167K double mutations can cause abnormal glucose metabolism in mice.

Investigation on the distribution frequency of Rh and Kell blood group antigens in the Uyghur population in Xinjiang and the molecular mechanism of Kell positive individuals / 中国输血杂志

【Objective】 To investigate the distribution frequency and characteristics of Rh and Kell erythrocyte blood group antigens in Uygur population in Xinjiang, and to explore the molecular mechanism of K gene positive patients, so as to build a local rare blood group bank and improve the ability of clinical blood security. 【Methods】 From June 2018 to February 2020, blood samples of 4 000 unrelated Uygur healthy individuals from the Medical Examination Center of our hospital and other cooperative hospitals across the autonomous region were selected. Rh and Kell blood group antigens were detected using K/Rh antigen microcolumn gel cards. The exons of Kell gene were amplified by PCR and then subjected to electrophoresis and direct sequencing to investigate the molecular mechanism. 【Results】 In Xinjiang Uygur healthy population, 1) The RhD negative rate was 5.675% (227/4 000), including 5 phenotypes; RhD positive rate was 94.325% (3 773/4 000), including 9 phenotypes, which were in line with Hardy-Weinberg equilibrium distribution. The C/E antigen frequency in RhD negative and positive patients was 13.216%/4.185% vs 52.876%/25.788% (PC, g. 412A>G, exon 6, g. 133C>T, and g. 189T>C, respectively, two of which caused changes in amino acid sequence: alanine at position 193 to methionine (p.Ala193Met) and alanine 423 to valine (p.al423Val). The prediction of RNA secondary structure and protein conformation after mutation using relevant biological information software found that the mutation caused changes in RNA secondary structure, free energy, protein conformation and function. 【Conclusion】 The frequency of RhD antigen negative in Xinjiang Uygur population was higher than that in other ethnic groups, and the distribution of C/E antigen was different in D antigen negative/positive patients. The distribution of K antigen in Kell blood group system was higher than that in other ethnic groups (P<0.05). The primary and secondary structure changes of nascent peptide chain caused by a single point mutation in Kell gene may be one of the molecular mechanisms of K antigen positivity.

MELAS syndrome caused by A3252G mutation in MT-TL1 gene: One case report and literature review / 中华内分泌代谢杂志

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes(MELAS) is one of the most common inherited mitochondrial diseases. This paper reports a rare mutation associated with MELAS syndrome, the m. 3252 A>G mutation in the MT-TL1 gene encoding the mitochondrial tRNALeu(UUR). The 6-year-old girl suffered from recurrent convulsion and lactic acidemia. The mtDNA sequencing detected a variant m. 3252A>G(MT-TL1 gene) in the proband and her maternal relatives. The heteroplasmic levels in peripheral blood and urine sediment were 66.53% and 97.42%, respectively, which were obviously higher than those of her maternal relatives. Together with 3 previously reported cases, the variant m. 3252A>G could be classified pathogenic. All the reported pathogenic variants in MT-TL1 gene were reviewed to explore the genotype-phenotype correlations of pathogenic variants in MT-TL1 gene.

Estructura genética de cepas de Mycobacterium tuberculosis farmacorresistentes en Perú con base en haplotipos obtenidos de un ensayo de sonda lineal / Genetic structure of drug-resistant strains of Mycobacterium tuberculosis in Peru based on haplotypes obtained from a linear probe assay

RESUMEN Objetivo. Determinar la estructura genética de las cepas drogorresistentes de Mycobacterium tuberculosis que circularon en todo el Perú durante los años 2011-2015 a través de haplotipos obtenidos de un ensayo con sondas en línea. Materiales y métodos. Se analizaron 6589 muestras que ingresaron al Instituto Nacional de Salud para el diagnóstico rutinario mediante el ensayo GenoType® MTBDRplus v2, durante el periodo de estudio. Se crearon haplotipos resistentes mediante la concatenación de 21 sitios polimórficos de los genes evaluados por el ensayo con sondas en línea, y se realizó el análisis de asociación con fenotipos obtenidos por el método de proporciones agar 7H10. Resultados. Las mutaciones de mayores frecuencias fueron: rpoB S531L (55,4%) y rpoB D516V (18,5%) para la resistencia a rifampicina, y katG S315T (59,5%) e inhA c-15t (25,7%) para la resistencia a isoniacida. Se obtuvieron 13 haplotipos representativos (87,8% de muestras analizadas) de los cuales seis correspondieron al genotipo multidrogorresistente, cuatro al genotipo monorresistente a isoniacida y tres al genotipo monorresistente a rifampicina. Dieciocho departamentos, y la provincia del Callao, presentaron una alta diversidad haplotípica; cuatro presentaron moderada diversidad y dos presentaron baja diversidad. Conclusiones. Existe una alta diversidad haplotípica en la mayoría de los departamentos, además de una concentración de las cepas de Mycobacterium tuberculosis drogorresistentes en las ciudades de Lima y Callao. Asimismo, las cepas de Mycobacterium tuberculosis con perfil drogorresistente que circulan en el Perú contienen principalmente los marcadores genéticos de mayor prevalencia a nivel mundial asociados con la resistencia frente a rifampicina e isoniacida.

ABSTRACT Objective. To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. Materials and methods. A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. Results. The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. Conclusions. Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

Mutação de novo no gene DEAF1 e sua relação com o autismo: estudo de caso / De novo mutation in the DEAF1 gene and its relation to autism: a case study / Mutación de nuevo en el gen DEAF1 y su relación con el autismo: estudio de caso

Objetivo: Analisar as implicações, impactos e o desenvolvimento de um indivíduo diagnosticado com TEA e portador da mutação de novo no gene DEAF1, a partir das várias perspectivas de intervenções realizadas. Método: Trata-se de um estudo descritivo com histórico dos tratamentos, resultados laboratoriais e genéticos mais recentes do paciente. Resultados: Sintomas notados aos 2 anos e diagnóstico específico aos 5. Aos 8 anos teve a primeira crise convulsiva tônico-clônica e o Eletroencefalograma alterado. Após obteve o diagnóstico molecular confirmado. Possuía epilepsia refratária de difícil controle, que houve piora com uma tentativa do uso de derivados canabinoides em conjunto com estimulação elétrica transcraniana. No momento, com os tratamentos, atendimentos multidisciplinares, dieta de exclusão de alérgenos e medicações de controle individual, diminuíram a intensidade das crises epiléticas e houve melhor controle do seu estado geral. Conclusão: Este estudo descreve como a mutação de novo no gene DEAF1 está relacionada com o TEA e com o comprometimento do desenvolvimento neurocognitivo. As terapias e métodos devem respeitar cada paciente na sua individualidade.

Objective: To analyze the implications, impacts and development of an individual diagnosed with ASD and carrying a de novo mutation in the DEAF1 gene, from the various perspectives of interventions performed. Method: This is a descriptive study, with the patient's history of treatments, and most recent laboratory and genetic results.Results: Symptoms were noticed at 2 years old and specific diagnosis at 5. At 8 years old he had his first tonic-clonic seizure and the electroencephalogram was altered. After, it was obtained the confirmed molecular diagnosis. He had refractory epilepsy that was difficult to control and aggravated with an attempt to use cannabinoid derivatives in conjunction with transcranial electrical stimulation. At the moment, treatments, multidisciplinary care, allergen exclusion diet and individual control medications, reduced the intensity of epileptic seizures and there was better control of his general condition. Conclusion: This study describes how the de novo mutation in the DEAF1 gene is related to ASD and neurocognitive development impairment. Therapies and methods must respect each patient in their individuality.

Objetivo: Analizar las implicaciones, impactos y desarrollo de un individuo diagnosticado de TEA y portador de una mutación de novo en el gen DEAF1, desde las distintas perspectivas de las intervenciones realizadas. Método: Este es un estudio descriptivo, con el historico de tratamientos del paciente y los resultados genéticos y de laboratorio más recientes. Resultados: Los síntomas se notaron a los 2 años y el diagnóstico específico a los 5. A los 8 años tuvo su primera crisis tónico-clónica y se alteró el electroencefalograma. Posteriormente se obtuvo el diagnóstico molecular confirmado. Tenía epilepsia refractaria que era difícil de controlar y se agravaba con un intento de utilizar derivados cannabinoides junto con estimulación eléctrica transcraneal. En el momento, los tratamientos, la atención multidisciplinar, la dieta de exclusión de alérgenos y los medicamentos de control individual, redujeron la intensidad de las crisis epilépticas y hubo un mejor control de su estado general. Conclusión: Este estudio describe cómo la mutación de novo en el gen DEAF1 se relaciona con el TEA y el deterioro del desarrollo neurocognitivo. Las terapias y los métodos deben respetar a cada paciente en su individualidad.

Association of caspase recruitment domain protein 9 gene polymorphism with the risk of acute pancreatitis / 中华胰腺病杂志

Objective:To evaluate the correlation between Card 9 gene polymorphism and acute pancreatitis(AP).Methods:70 AP patients and 70 healthy subjects from Shanghai Songjiang District Central Hospital from June 2019 to February 2020 were enrolled. TaqMan probe method was used to assay genotype distributions of the Card 9 polymorphisms rs10870077, rs4077515, rs10781499, rs141992399, rs139265120. Real-time quantitative PCR was used to determine the level of Card 9 mRNA, electrochemiluminescence immunoassay was applied to assay IL-6 and procalcitonin, and nephelometry was performed to measure the C-reactive protein(CRP).Results:Compared with the control group, the genotype and allele frequency of Card 9 gene rs10870077 C>G were significantly elevated in AP patients with statistically significant difference (31.4% vs 50.0%, P<0.05). There was no statistically significant difference on the allele frequency of Card 9 rs4077515AG and rs10781499AG, especially on rs141992399 C>G and rs139265120 A>G. C>Gpolymorphism in Card 9 rs10870077 resulted in an obvious increase of serum Card 9 mRNA expression in AP patients from 3.90±1.96 to 6.20±2.82, and the difference was statistically significant ( P<0.05), but there was no statistically significant difference on the Card 9 mRNA between AP patients with Card 9 rs4077515AG and rs10781499AG and the controls. IL level in AP patients with Card 9 rs10870077CG was greatly higher than that in those with Card 9 rs10870077 CC and GG [(614.7±1531.8 ng/L vs (372.5±1127.9)and (385.5±598.7)ng/L]. But compared with GG genotype, CRP level was obviously decreased [(34.84±50.64)mg/L vs (55.30±87.02)mg/L], and the procalcitonin was obviously increased [(1.98±4.70)μg/L vs (0.77±1.12)μg/L], and all the differences were statistically significant (all P value<0.05). Conclusions:Card 9 rs10870077 C>G mutation could upregulate the expression of Card 9 mRNA and increase IL-6 level, which may be a high risk for the occurrence of AP patients.

Association of gene mutations in the pre-C, C, and basic core promoter regions of hepatitis B virus with traditional Chinese medicine syndrome types / 临床肝胆病杂志

ObjectiveTo investigate the association of gene mutations in the pre-C, C, and basic core promoter (BCP) regions of hepatitis B virus (HBV) with traditional Chinese medicine (TCM) syndrome types in patients with chronic hepatitis B (CHB). MethodsA retrospective analysis was performed for the clinical data of CHB patients who were diagnosed and treated at the outpatient service and ward of Spleen, Stomach, and Hepatobiliary Department, The First Affiliated Hospital of Henan University of Chinese Medicine, from November 2014 to June 2018. Related clinical data were collected and recorded, including general information, HBV serological markers, HBV gene mutations, and information obtained by four TCM diagnostic methods. Syndrome differentiation and typing were performed for each patient with reference to the criteria for TCM syndrome differentiation of viral hepatitis, and the association of gene mutation in the pre-C, C, and BCP regions of HBV with TCM syndrome types was analyzed. The chi-square test was used for comparison of categorical data between groups, and the Kruskal-Wallis H test was used for comparison of continuous data between multiple or two groups. ResultsA total of 235 patients with CHB were enrolled, among whom 101 had internal retention of damp-heat, 88 had stagnation of liver Qi and spleen deficiency, 17 had blood stasis obstructing the collaterals, 19 had liver-kidney Yin deficiency, and 10 had spleen-kidney Yang deficiency. There were significant differences in sex, age, and course of disease between the patients with different TCM syndrome types (χ2=17.389, H=173.280, H=86.520, all P＜0.01), and there was a significant difference in age between the CHB patients with gene mutations in the pre-C, C and BCP regions of HBV (H=30.150, P＜0.001). There was a significant difference in the distribution of TCM syndrome types between the CHB patients with gene mutations in the pre-C, C and BCP regions of HBV (χ2=58.117, P＜0.001), and internal retention of damp-heat and stagnation of liver Qi and spleen deficiency were major TCM syndrome types accounting for 80.43%. The patients with internal retention of damp-heat tended to have A1762T and G1764A mutations, and those with stagnation of liver Qi and spleen deficiency tended to have G1896A, A1762T, and G1764A mutations; G1764A mutation was often observed in the patients with blood stasis obstructing the collaterals or liver-kidney Yin deficiency, and I97L mutation was often observed in the patients with spleen-kidney Yang deficiency. ConclusionGene mutations in the pre-C, C, and BCP regions of HBV are associated with TCM syndrome types in CHB patients, and internal retention of damp-heat and stagnation of liver Qi and spleen deficiency are the most common TCM syndrome types. I97L mutation is often observed in patients with spleen-kidney Yang deficiency.

Construction of point mutation rabbits using CRISPR/Cas9 / 浙江大学学报·医学版

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.

Methyl-CpG-binding protein 2 gene mutations and its association with epilepsy: a single centre study from the Indian subcontinent

Rett syndrome (RTT) is an X-linked disorder caused by mutations in MECP2 in majority of cases. It is characterized by arrested development between 6 and 18 months of age, regression of acquired hand skills and speech, stereotypic hand movements, gait abnormalities and seizures. There are a very few studies in India which illustrates mutation spectrum in RTT. None of the studies have correlated seizures with the genotype. This study describes the phenotype and genotype spectrum in children with RTT syndrome and analyses the association of epilepsy with various clinical features and molecular findings. All children with RTT in our cohort had global developmental delay. Genetic diagnosis identified mutations of the MECP2 in all 25 children where RTT was suspected. We have identified point mutations in 20 patients, one insertion and four deletions by Sanger sequencing, namely c.1164_1207 (44 bp), c.1165_1207 (43 bp), c.1157_1197 (41 bp) del and c.1157_1188 (32 bp). Clinically, none of the patients with deletion had seizures. We identified one novel insertion variant c.337_338 (p.S113Ffs*9). All the deletions were located in the C-terminal region. Majority of the mutations (22/25) were identified in exon 4 which comprised of nonsense and missense types. Screening of hotspot mutations in exon 4 should be the first line evaluation in diagnosis of RTT. Molecular testing could help in specific management of seizures in RTT.

A case of ichthyosis follicularis, atrichia and photophobia syndrome caused by a novel mutation in the MBTPS2 gene / 中华皮肤科杂志

A case of ichthyosis follicularis,alopecia and photophobia syndrome caused by a novel mutation c.1165C＞T in the membrane-bound transcription factor protease site 2 (MBTPS2) gene was firstly reported.The proband presented with dry skin,congenital hairlessness,follicular keratotic papules,photophobia,epilepsy,and mental and motor retardation.Next-generation and Sanger sequencing analysis confirmed that the proband and his mother both had a c.1165C＞T (p.pro389Ser) mutation in exon 9 of the MBTPS2 gene.According to the clinical manifestations of the patient and genetic characteristics of the MBTPS2 gene mutation,the patient was diagnosed with ichthyosis follicularis,alopecia and photophobia syndrome.

Analysis of clinical phenotype of a α2-globin gene mutation -IVS-Ⅱ-55 (T→G) / 临床检验杂志

Objective@#To identify a α-globin gene mutation-IVS-Ⅱ-55 (T→A) and analyze hematological characteristics of IVS-Ⅱ-55 (T→G) carriers. @*Methods@#The peripheral blood samples were collected from the members of five family and three sporadic IVS-Ⅱ-55(T→G) carriers for the analysis of RBC parameters and hemoglobin electrophoresis. Gap-PCR, PCR-RDB (reverse dot blot) and DNA sequencing were carried out for the identification of gene deletion and mutation of α-globin and β-globin. @*Results@#The results of RBC parameters of five infant probands which presented with microcytic hypochromic anemia were below the normal reference interval. One of the adult carriers of IVS-Ⅱ-55 (T→G) heterozygote alone presented with microcytic hypochromic anemia, and the others showed normal RBC parameters. The hematological phenotype index (MCV, MCH and HbA 2 ) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and βCD27-28M/N were 65.0 fL, 20.3 pg and 5.8% respectively. The hematological phenotype index (MCV, MCH, HbA 2 and HbF) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and SEA-HPFH were 81.9 fL, 26.5 pg, 3.0% and 29.0% respectively. The HbA 2 levels of all carriers of IVS-Ⅱ-55 (T→G) heterozygote alone were in normal range. No abnormal hemoglobin band was detectable on hemoglobin electrophoresis for all the carries. @*Conclusion@#The carriers of IVS-Ⅱ-55(T→G) heterozygote alone were asymptomatic. The phenotype of compound heterozygote for β-thalassemia was similar to that of β-thalassemia alone.

Research progress on structural modification of α1-adrenergic receptor influencing its biological activities and its physiological function / 中国药理学通报

The α1-adrenergic receptor (α1-AR) belongs to G-protein coupled receptors (GPCRs) and contains three subtypes, namely α1A, α1B and α1D. Studies have shown that structural changes in the α1-ARs receptor can lead to changes in their physiological functions. In the present study, we summarize the structural changes in four aspects of α1-ARs receptors, including carboxy-terminal truncation, dimerization, allosteric effects, and point mutations that lead to endocytosis, phosphorylation, desensitization, and affinity for agonists.

Novel DPY19L2 variants in globozoospermic patients and the overcoming this male infertility / 亚洲男科学杂志(英文版)

Globozoospermia has been reported to be a rare but severe causation of male infertility, which results from the failure of acrosome biogenesis and sperm head shaping. Variants of dpy-19-like 2 (DPY19L2) are highly related to globozoospermia, but related investigations have been mainly performed in patients from Western countries. Here, we performed a screening of DPY19L2 variants in a cohort of Chinese globozoospermic patients and found that five of nine patients carried DPY19L2 deletions and the other four patients contained novel DPY19L2 point mutations, as revealed by whole-exome sequencing. Patient 3 (P3) contained a heterozygous variant (c.2126+5G>A), P6 contained a homozygous nonsense mutation (c.1720C>T, p.Arg574∗), P8 contained compound heterozygous variants (c.1182-1184delATC, p.Leu394-Ser395delinsPhe; c.368A>T, p.His123Arg), and P9 contained a heterozygous variant (c.1182-1184delATCTT, frameshift). We also reported intracytoplasmic sperm injection (ICSI) outcomes in the related patients, finding that ICSI followed by assisted oocyte activation (AOA) with calcium ionophore achieved high rates of live births. In summary, the infertility of these patients results from DPY19L2 dysfunction and can be treated by ICSI together with AOA.

A novel TNS4 mutant in prostate cancer cells and its mechanism / 肿瘤

Objective: To investigate the expression and mutation status of tensin family member 4 (TNS4) gene in various prostate cancer cell lines, and to explore its potential molecular mechanism. Methods: The expression of TNS4 gene in several prostate cancer cell lines was detected by RT-PCR. The full-length cDNA of TNS4 gene was amplified for the restriction endonuclease hydrolysis and DNA sequencing analysis. Meantime, the recombinant plasmids carrying wild- or mutant-type TNS4 gene were constructed and transfected into prostate cancer PC-3 cells, then the effects of TNS4 over-expression on the mRNA and protein expressions as well as the localization of signal transducer and activator of transcription 1 (STAT1) were detected by RT-PCR, Western blotting and immunofluorescence microscopy, respectively. Results: As compared with benign prostatic hyperplasia BPH1 cells, TNS4 gene was lowly expressed in prostate cancer cell lines DU145, LNCaP and 22Rv1 (all P 0.05). Additionally, the over-expression of mutant TNS4 significantly promoted nuclear translocation of STAT1 protein (P < 0.01). Conclusion: TNS4 gene is lowly expressed in prostate cancer cell lines, and there is a novel TNS4 mutant (S143P). TNS4 over-expression can up-regulate the expression of STAT1 protein, furthermore the mutant TNS4 can alter the location of STAT1 protein.

Evaluation of Consistency in detection of epidermal growth factor receptor gene T790M mutation in plasma and tumor specimens of patients with lung adenocarcinoma / 中华肿瘤杂志

Objective@#To evaluate the consistency in detection of T790M mutation of epidermal growth factor receptor gene (EGFR) in plasma and tumor samples of patients with lung adenocarcinoma.@*Methods@#The tumor tissues or cytological specimens of 12 patients with operable lung adenocarcinoma(stage Ⅰ-ⅢA) and 100 patients with advanced stage ⅢB-Ⅳ lung adenocarcinoma were collected, among which 11 patients showed acquired resistance for gefitinib (11/100). In the same period, peripheral blood samples were collected from all patients and 50 healthy volunteers. Amplification refractory mutation system (ARMS) was used to detect EGFR mutations in tumor specimens. Next Generation Sequencing(NGS) based circulating single-molecule amplification and resequencing technology (cSMART)was performed to quantitatively detect the EGFR mutations in circulating tumor DNA (ctDNA) from plasma specimens.@*Results@#The sensitivity, specificity and concordance rate of EGFR T790M mutation between plasma and tissue specimens from 100 advanced stage patients were 50.0%, 72.9% and 72.0%, respectively. For L858R mutation and exon 19 deletion mutations, the above mentioned sensitivity, specificity and concordance rate were 91.7%, 100.0%, and 98.0%, as well as 79.2%, 100.0% and 95.0%, respectively. The L858R mutation and exon 19 deletion mutations were not detected in plasma of 50 healthy volunteers, whereasT790M mutation(1.0±0.0 copies) was found in 7 individuals(7/50, 14.0%). Similarly, in 12 resectable patients, 4 (4/12, 33.3%) T790M mutations were found in plasma (1.2±0.2 copies), but no L858R mutation and 19 exon deletion mutations. In comparison, 28.0% of patients with advanced lung adenocarcinoma (28/100)had detectable T790M mutation in plasma with copy numbers (34.0±22.7 copies). Furthermore, the copy numbers of T790M were 268.2±119.9 in plasma of 5 cases with acquired gefitinib-resistance.@*Conclusions@#In patients with advanced stages of lung adenocarcinoma, the detection of T790M mutation in plasma and tumor specimens is low. The T790M mutation also exists in the plasma of some healthy controls, suggesting that T790M mutation participates in EGFR signaling pathway and it might function in healthy population.

Factor Ⅴ Leiden mutation is not associated with ischemic stroke in Chinese young adults / 国际脑血管病杂志

Objective To study the correlation between coagulation factor Ⅴ Leiden mutation and ischemic stroke in Chinese young adults. Methods From July 2016 to January 2018, 80 young patients with ischemic stroke (18-45 years) admitted to the Department of Neurology, the First Affiliated Hospital of Shantou University Medical College and 80 controls were enrolled prospectively. The demographic data, vascular risk factors were documented. Sanger sequencing was used to detect factor Ⅴ Leiden mutation. Results The 1691 sites of factor Ⅴ genes in the case group and control group were all wild types. Multivariate logistic regression analysis suggested that hypertension ( odds ratio [ OR ] 4. 308, 95％ confidence interval [CI] 3. 321-5. 067; P = 0. 001), hyperlipidemia (OR 2. 734, 95％ CI 2. 214-3. 378; P = 0. 005), and smoking (OR 5. 293, 95％ CI 3. 003-6. 180; P = 0. 010) were the independent risk factors for ischemic stroke in young adults, and high-density lipoprotein cholesterol (OR 0. 611, 95％ CI 0. 457-0. 709; P = 0. 027) was its independent protective factor. Conclusion Factor Ⅴ Leiden mutation is not associated with ischemic stroke in Chinese young adults. Hypertension, dyslipidemia and smoking are still the main risk factors for ischemic stroke in young adults.

Novel FOXG1 mutation in a patient with congenital Rett variant: a case report / 天津医药

@#Objective To summarize the clinical features of children with congenital Rett variant caused by mutation of FOXG1 and provide the reference for the diagnosis and treatment of the disease. Methods The clinical data of a patient diagnosed as congenital Rett syndrome variant type were summarized. The DNA samples of peripheral blood from the patient and her parents were extracted. The targeted high-throughput sequencing technology was used to detect the sequence of targeted genes, which were associated with the symptoms of the child. Genes were then verified by sanger sequencing. Chromosomal microarray analysis was performed to detect chromosome microdeletions and microduplications. Results The child carried the c.506dupG, p.G169Gfs＊ 286 heterozygous mutations on FOXG1 gene, which located in 14q12, and her parents were wild-type. After querying the HGMD, Clinvar and dbSNP databases, we found that it was not reported. This case was clearly diagnosed as congenital Rett syndrome variant type. We confirmed that the mutation locus was a new mutation. Conclusion For cases with congenital Rett variant manifestations, FOXG1 gene mutation examination is recommended, and preventive treatment of partially predictable dysfunction should be carried out.

Ferritin light chain gene mutations in two Brazilian families with hereditary hyperferritinemia-cataract syndrome / Mutações no gene da cadeia leve da ferritina em duas famílias brasileiras com síndrome hereditária hiperferritinemia-catarata

ABSTRACT Hereditary hyperferritinemia-cataract syndrome is an autosomal dominant genetic disorder associated with mutations in the 5'UTR region of the ferritin light chain gene. These mutations cause the ferritin levels to increase even in the absence of iron overload. Patients also develop bilateral cataract early due to accumulation of ferritin in the lens, and many are misdiagnosed as having hemochromatosis and thus not properly treated. The first cases were described in 1995 and several mutations have already been identified. However, this syndrome is still a poorly understood. We report two cases of unrelated Brazilian families with clinical suspicion of the syndrome, which were treated in our department. For the definitive diagnosis, the affected patients, their parents and siblings were submitted to Sanger sequencing of the 5'UTR region for detection of the ferritin light gene mutation. Single nucleotide polymorphism-like mutations were found in the affected patients, previously described. The test assisted in making the accurate diagnosis of the disease, and its description is important so that the test can be incorporated into clinical practice.

RESUMO A síndrome hereditária hiperferritinemia-catarata é uma doença genética autossômica dominante associada a mutações na região 5'UTR do gene da cadeia leve da ferritina. Estas mutações elevam os níveis de ferritina, mesmo na ausência de sobrecarga de ferro. Os pacientes também desenvolvem catarata bilateral precocemente, devido ao acúmulo de ferritina no cristalino, e muitos são erroneamente diagnosticados como portadores de hemocromatose, sendo tratados de maneira inadequada. Os primeiros casos foram descritos em 1995, e diversas mutações já foram identificadas. Entretanto, essa síndrome ainda é pouco conhecida. Relatamos dois casos de famílias brasileiras, não relacionadas, com suspeita clínica da síndrome, que foram atendidas em nosso serviço. Para o diagnóstico definitivo, os pacientes afetados, seus pais e irmãos foram submetidos à pesquisa de mutação do gene ferritina, por sequenciamento de Sanger da região 5'UTR. Foram encontradas mutações do tipo polimorfismo de nucleotídeo único nos pacientes afetados, já descritas anteriormente. O teste auxiliou no diagnóstico preciso da doença e é importante ser divulgado, para ser incorporado na prática clínica.