Molecular and structural studies of Japanese patients with sialidosis type 1

To gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of ⁶⁴⁹G-to-A and ⁷²⁷G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course. Received: March 21, 2000 / Accepted: May 10, 2000     

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation‐sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease. Hum Mutat 23:32–39, 2004. © 2003 Wiley‐Liss, Inc.     

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R, and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions. Received: Stptember 21, 2001 / Accepted: November 2, 2001     

Combined sialidase (neuraminidase) and β-galactosidase deficiency. Clinical, morphological and enzymological observations in a patient

A patient with combined deficiency of sialidase and beta-galactosidase is described. This now 39-year-old man, who is of Japanese origin, showed gradually progressive clinical features from the age of six years. Many of these features are commonly found in sialidosis type 2 or in GM1-gangliosidosis. Both sialidase and beta-galactosidase activities were deficient in leucocytes and cultured fibroblasts. Leucocytes of his mother showed activities of both enzymes in the lower limit of the control range. Morphologically, the pattern of storage products in a skin biopsy resembled in many respects that seen in GM1-gangliosidosis. Moreover, storage products which could be typical of sialidosis were also observed. Since the patient showed angiokeratomata, the morphological findings were compared with those specific to Fabry's disease, but no similarities were found. An enzymological diagnosis of the disease is most reliable on cultured fibroblasts, discriminating it from sialidosis type 2 and GM1-gangliosidosis. In view of recent findings, leucocytes seem to be less suitable for the establishment of the diagnosis galactosialidosis.     

Molecular pathology of galactosialidosis in a patient affected with two new frameshift mutations in the cathepsin A/protective protein gene

Galactosialidosis is a recessively inherited lysosomal storage disease characterized by the combined deficiency of neuraminidase and β-galactosidase secondary to the genetic deficiency of cathepsin A/protective protein. In lysosomes, cathepsin A forms a high-molecular-weight complex with β-galactosidase and neuraminidase that protects these enzymes against intralysosomal proteolysis. In a patient affected with late infantile form of galactosialidosis, we found two new cathepsin A mutations, a two-nucleotide deletion, c517delTT and an intronic mutation, IVS8+9C→G resulting in abnormal splicing and a five-nucleotide insertion in the cathepsin A cDNA. Both mutations cause frameshifts and result in the synthesis of truncated cathepsin A proteins, which, as suggested by structural modeling, are incapable of dimerization, complex formation, and catalysis. However, enzymatic assays, gel-filtration, and Western blot analysis of the patient's cultured skin fibroblast extracts showed the presence of a small amount of normal-size, catalytically active cathepsin A and cathepsin A-β-galactosidase 680 kDa complex, suggesting that a low amount of cathepsin A mRNA is spliced normally and produces the wild-type protein. This may contribute to the relatively mild phenotype of the patient and illustrates the importance of critically comparing molecular results with clinical and biochemical phenotypes. Hum Mutat 11:461–469, 1998. © 1998 Wiley-Liss, Inc.     

Multiple clusters of A(H1N1)pdm09 virus circulating in severe cases of influenza during the 2010–2011 season: A phylogenetic and molecular analysis of the neuraminidase gene

The molecular characterization of circulating influenza A viruses is crucial to detect mutations potentially involved in increased virulence, drug resistance and immune escape. A molecular and phylogenetic analysis of A(H1N1)pdm09 neuraminidase (NA) gene sequences from different patient categories defined according to the severity of influenza infection were analyzed. A total of 126 influenza A(H1N1)pdm09 positive samples from patients with severe infections in comparison with those with moderate and mild infections was performed in Lombardy (Northern Italy, nearly 10 million inhabitants) during the 2010–2011 season. NA sequences included in this study segregated into five distinct clusters. Nineteen amino acid substitutions were detected exclusively in NA sequences of viruses identified in patients with severe or moderate influenza infection. Three of them (F74S, S79P, E287K) were observed in virus strains with the 222G/N hemagglutinin mutation. None of NA sequences under study had mutations related to the resistance to the NA inhibitors. Four out of 126 (3.2%) NA sequences from patients with severe infection lost a N‐linked glycosylation site due to the change from N to K at residue 386. Two additional N‐linked glycosylation sites in the NA stalk region (residues 42 and 44) were found in 12 (9.5%) NA sequences. Sporadic NA mutations were detected in NA viral sequences from critically ill patients, and no variants with reduced sensitivity to NA inhibitors were observed either in treated or untreated patients. J. Med. Virol. 85: 944–952, 2013. © 2013 Wiley Periodicals, Inc.     

Molecular study of Nucleophosmin 1(NPM1) gene in acute myeloid leukemia in Kurdish population

BackgroundIn patients with Acute Myeloid Leukemia (AML) the most frequent acquired molecular abnormalities and important prognostic indicators is nucleophosmin-1 (NPM1) mutations. Our study aims was molecular study of Nucleophosmin -1 gene in Acute Myeloid Leukemia in Kurdish population.Patients &MethodsA total of 50 patients with AML, (36) of them attended Nanakaly Hospital and (14) attended Hiwa Hospital and 30 healthy subjects as control were selected randomly, all were matched of age and gender. Polymerase chain reaction (PCR) was used for detection of NPM1 gene mutation. Three samples of PCR product for NPM1 gene mutations were sequenced, and mutations were determined by comparison with the normal NPM1 sequence NCBI (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_002520","term_id":"1838744830"}}NM_002520).ResultsOut of 50 patients with AML, 5 (10%) of them were NPM1 gene mutation positive, and 45 (90%) were negative. The mutation were a base substitution (C to A), (G to C), (G to T), transversion mutation in addition of frame shift mutation and all mutated cases were heterozygous and retained a wild type allele.ConclusionIdentification of NPM1 mutations in AML are important for prognostication, treatment decision and optimization of patient care.     

子宫内膜异位症与细胞色素P450 1A1基因A4889G突变的相关性

目的探讨细胞色素P450 1A1(cytochrome P450 1A1,CYP1A1)基因A4889G突变与子宫内膜异位症(endometriosis,EM)的遗传易感性的关系.方法采用等位基因特异性聚合酶链反应的方法,研究了76例EM患者和80名正常对照CYP1A1基因A4889G位点碱基突变.两组均为广东籍汉族妇女.结果 CYP1A1基因A4889G位点等位基因A、G在EM组和对照组分布的差异有显著性(χ2=7.498,P<0.01),其中等位基因G使患EM的相对危险度提高了1.957倍.CYP1A1基因A4889G位点各基因型分布在两组间差异有显著性(χ2=6.915,P<0.05),GG基因型与AA基因型相比患EM的危险度高3.437倍(χ2=5.430,P<0.05).结论 CYP1A1基因A4889G突变等位基因与EM有一定的关联,突变基因型增加了EM的发病风险.     

2009H1N1流感病毒神经氨酸酶基因克隆与表达

目的制备2009H1N1流感病毒神经氨酸酶（NA）重组蛋白,为建立H1N1快速检测方法和神经氨酸酶抑制剂筛选模型奠定基础。方法采用MDCK细胞方法分离2009H1N1流感病毒,提取病毒RNA,RT-PCR生成NA基因,构建原核表达载体PET-102/D-TOPO-NA,IPTG诱导表达重组蛋白,SDS-PAGE凝胶电泳、WersternBlotting鉴定重组蛋白。结果成功分离2009H1N1流感病毒,NA蛋白119、152、275、292位氨基酸分别为Glu、Arg、His和Cys。SDS-PAGE凝胶电泳蛋白分子相对分子质量约为64000,与预期一致。WesternBlotting证实该蛋白具有神经氨酸酶抗原活性。结论 IPTG诱导原核表达神经氨酸蛋白的最适浓度为0.1mmol/L。实验得到的蛋白尚需进一步纯化。     

未结合型高胆红素血症患者UGT1A1基因的突变分析

目的 分析未结合型高胆红素血症患者尿苷二磷酸葡糖醛酸基转移酶1A1(uridine diphosphate glucuronosyltransferase 1A1,UGT1A1)基因的突变情况,并探讨突变与血清总胆红素的关系.方法 抽提患者外周血基因组DNA,扩增UGT1A1基因的编码序列及启动子区,对扩增产物进行测序以确定基因突变.结果 在61例未结合型高胆红素血症患者中,共发现46例存在UGT1A1基因的突变.突变可分为5种,依据频率高低依次为211G→A、TATAA盒TA插入、686C→A、1091C→T以及1352C→T.携带单一位点纯合突变合并其它位点杂合突变患者的血清胆红素水平明显高于单位点纯合突变或多位点杂合突变患者(P＜0.05).综合UGT1A1基因突变与血清总胆红素水平,44例未结合型高胆红素血症患者被诊断为Gilbert综合征,2例被诊断为Ⅱ型Crigler-Najjar综合征.结论 患者血清总胆红素水平不仅与UGT1A1基因突变数量有关,而且与突变的杂合或纯合状态有关.     

Identification of the minimal combination of clinical features in probands for efficient mutation detection in the FBN1 gene

Mutations identified in the fibrillin-1 (FBN1) gene have been associated with Marfan syndrome (MFS). Molecular analysis of the gene is classically performed in probands with MFS to offer diagnosis for at-risk relatives and in children highly suspected of MFS. However, FBN1 gene mutations are found in an ill-defined group of diseases termed ‘type I fibrillinopathies'', which are associated with an increased risk of aortic dilatation and dissection. Thus, there is growing awareness of the need to identify these non-MFS probands, for which FBN1 gene screening should be performed. To answer this need we compiled the molecular data obtained from the screening of the FBN1 gene in 586 probands, which had been addressed to our laboratory for molecular diagnosis. In this group, the efficacy of FBN1 gene screening was high in classical MFS probands (72.5%,), low (58%) in those referred for incomplete MFS and only slight (14.3%) for patients referred as possible MFS. Using recursive partitioning, we found that the best predictor of the identification of a mutation in the FBN1 gene was the presence of features in at least three organ systems, combining one major, and various minor criteria. We also show that our original recommendation of two systems involved with at least one with major criterion represents the minimal criteria because in probands not meeting these criteria, the yield of mutation identification drastically falls. This recommendation should help clinicians and biologists in identifying probands with a high probability of carrying a FBN1 gene mutation, and thus optimize biological resources.     

弥散峰度成像预测直肠腺癌UGT1A1*28基因突变的价值

目的:评估弥散峰度成像（DKI）对直肠腺癌尿苷二磷酸葡萄糖醛酸基转移酶1A1（UGT1A1）*28基因突变的预测价值。方法:回顾性研究。纳入山西省肿瘤医院2016年11月—2020年8月167例直肠腺癌患者的临床资料,其中男98例、女69例,年龄29~89岁、中位年龄为62岁。患者术前均行MR常规序列和DKI序列检查,...     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

一个中国耳聋家系的SLC26A4基因分析

目的 确定一个非综合征型耳聋家系的致病基因。方法 应用聚合酶链反应-直接测序方法，对溶质转运蛋白家族26，成员4（solute carrier family 26，member 4；SLC26A4）基因的所有外显子及其与内含子交界处进行测序寻找突变。结果 在该家系先证者发现SLC26A4基因的N392Y、S448X复合杂合突变，其父亲为S448X的杂合突变，其母亲为N392Y的杂合突变。结论 SLC26A4基因的N392Y、S448X复合杂合突变是导致该先证者耳聋发生的原因。     

Investigation of the CACNA1A gene as a candidate for typical migraine susceptibility

Typical migraine is a complex neurological disorder comprised of two main subtypes: migraine with (MA) and without aura (MO). The disease etiology is still unclear, but family studies provide strong evidence that defective genes play an important role. Familial hemiplegic migraine (FHM) is a very rare and severe subtype of MA. It has been proposed that FHM and MA may have a similar genetic etiology. Therefore, genetic studies on FHM provide a useful model for investigating the more prevalent types of typical migraine. FHM in some families has been shown to be caused by mutations in a brain‐specific P/Q‐type calcium channel α1 subunit gene (CACNA1A) on chromosome 19p13. There has also been a report of a CACNA1A mutation being associated with MA in a patient from a family with predominant FHM. We have previously demonstrated suggestive linkage of typical migraine in a large Australian family to the FHM region on chromosome 19p13. These findings suggest that CACNA1A may also be implicated in the etiology of typical migraine in this pedigree. To investigate this possibility, we sequenced two patients carrying the critical susceptibility haplotype surrounding CACNA1A. No disease‐causing mutations or polymorphisms were revealed in any of the 47 exons screened. To determine whether the CACNA1A gene was implicated in typical migraine susceptibility in the general Caucasian population, we also analyzed 82 independent pedigrees and a large case control group. We did not detect any linkage or association in these groups and conclude that if CACNA1A plays a role in typical migraine, it does not confer a major effect on the disease. © 2001 Wiley‐Liss, Inc.     

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.     

Molecular and functional analysis of SUMF1 mutations in multiple sulfatase deficiency

Multiple sulfatase deficiency (MSD) is a rare disorder characterized by impaired activity of all known sulfatases. The gene mutated in this disease is SUMF1, which encodes a protein involved in a post-translational modification at the catalytic site of all sulfatases that is necessary for their function. SUMF1 strongly enhances the activity of sulfatases when coexpressed with sulfatase in Cos-7 cells. We performed a mutational analysis of SUMF1 in 20 MSD patients of different ethnic origin. The clinical presentation of these patients was variable, ranging from severe neonatal forms to mild phenotypes showing mild neurological involvement. A total of 22 SUMF1 mutations were identified, including missense, nonsense, microdeletion, and splicing mutations. We expressed all missense mutations in culture to study their ability to enhance the activity of sulfatases. Of the predicted amino acid changes, 11 (p.R349W, p.R224W, p.L20F, p.A348P, p.S155P, p.C218Y, p.N259I, p.A279V, p.R349Q, p.C336R, p.A177P) resulted in severely impaired sulfatase-enhancing activity. Two (p.R345C and p.P266L) showed a high residual activity on some, but not all, of the nine sulfatases tested, suggesting that some SUMF1 mutations may have variable effects on the activity of each sulfatase. This study compares, for the first time, clinical, biochemical, and molecular data in MSD patients. Our results show lack of a direct correlation between the type of molecular defect and the severity of phenotype.