Six Novel Mutations Identified in the Glycoproteins Ib Alpha, Ib Beta and IX Genes Among Twenty-Two Unrelated Patients with Bernard-Soulier Syndrome in Brazil

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1156-1156
Author(s):  
Lucia Helena de Siqueira ◽  
Miriam P Beltrame ◽  
Fernanda P G Cunha ◽  
Marina P Colella ◽  
Joyce M Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Genetic Diagnosis ◽  
Restriction Enzymes ◽  
Bleeding Disorder ◽  
Site Directed Mutagenesis ◽  
Novel Mutations ◽  
Index Cases ◽  
Bernard Soulier Syndrome

Abstract Abstract 1156 Introduction: Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection and Analysis of Gene Mutations in Patients with Glanzmann's Thrombasthenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2373-2373
Author(s):  
Boyang Sun ◽  
Donglei Zhang ◽  
Huiyuan Li ◽  
Xueqing Dou ◽  
Renchi Yang
Keyword(s):  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Adenosine Diphosphate ◽  
Severe Bleeding ◽  
Missense Mutations ◽  
Novel Mutations ◽  
Genetic Characteristics ◽  
Pathogenic Variants ◽  
Standards And Guidelines

Background: Glanzmann thrombasthenia (GT) is a rare inherited disorder of bleeding, and it is characterized by the impaired or absent platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid(AA), but normal reaction to ristocetin. There is qualitative or quantitative defect in platelet integrin αIIbβ3(GPIIb/IIIa). Pathogenic variants of either αIIb or β3 unit could cause GT. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org); it totally lists 236 variants of ITGA2B gene and 170 variants of ITGB3 gene. Aim: To characterize the clinical manifestation and molecular basis of GT patients in China, and update the pathogenic variants database. Method: Clinical features are evaluated in 104 patients with GT. New generation sequencing was performed with a custom designed panel for the bleeding and platelet disease involving 76 genes, while ITGA2B and ITGB3 were enrolled. Result: The initial bleeding occurred before 1 age in most patients. Incidence of consanguinity is 12.5%. Symptoms lessened with age in about 30% patients. Female patients suffered more severe bleeding than male patients. Fifty different mutations were detected, among which 15 were novel. Most patients were compound heterozygotes and most mutations detected were missense mutations. Among 15 novel mutations, there were 7 missense mutations, 2 nonsense mutations, 2 splicing mutations, 4 frameshift mutations. Pathogenicity of all novel mutations were evaluated according to the standards and guidelines of ACMG. All variants detected were pathogenic or likely pathogenic. Furthermore, c.1750C>T [p.R584X] and c.2333A>C [p.Q778P] in ITGA2B were detected in 10 and 16 unrelated families, strongly suggesting a founder effect. Conclusion: Our study reports the largest cohort of GT in China, describing the clinical, laboratory and genetic characteristics of 104 patients. We found 15 novel pathogenic mutations in ITGA2B and ITGB3 causing GT. Theses novel findings expand the GT mutation spectrum. Disclosures No relevant conflicts of interest to declare.

Genetic Diagnosis of Inherited Bleeding Disorders in 1250 Probands From India: A Single Centre Experience

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1127-1127
Author(s):  
Giridhara R Jayandharan ◽  
Govindanattar Sankari Devi ◽  
S RajKumar ◽  
Everette Nelson ◽  
Ramachandran V Shaji ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Hemophilia A ◽  
Genetic Diagnosis ◽  
Molecular Data ◽  
Bleeding Disorder ◽  
Factor V ◽  
Bleeding Disorders ◽  
Single Centre ◽  
Glanzmann Thrombasthenia ◽  
Bernard Soulier Syndrome

Abstract Abstract 1127 Congenital defects of platelets or plasma proteins involved in haemostasis generally lead to bleeding disorders. Von Willebrand disease (VWD) and hemophilia A and B are the most common while defects in other plasma coagulation proteins and platelet factors are relatively rare with an incidence of <1: 1–2 million. Despite significant advancements in the world, state of the art care is inaccessible to a vast majority of patients in developing countries. Genetic diagnosis aimed at offering carrier detection and prenatal diagnoses thus remains an important part of the management of these patients. From the year 2000, we have systematically established cost-effective genetic diagnoses services for not only hemophilia A and B but for other rare bleeding disorders [Factor (F) 1, F2, F5F8, F7, F10, F11 and F13] and platelet disorders [Glanzmann Thrombasthenia, Bernard Soulier Syndrome, Wiskott Aldrich Syndrome] as well. Our approach for molecular diagnosis includes screening the target genes for common mutations first, followed by point mutation screening by PCR and conformation sensitive gel electrophoresis and DNA sequencing, as needed, after that. For large genes such as F8 (186kb), ITG2B and ITG3A (65kb), we employ multiplex amplification of the coding regions to simplify the mutation detection protocols. Alternately, linkage analysis has also been established for some diseases such as hemophilia A, hemophilia B and Glanzmann Thrombasthenia to assist in those families where no mutation is found. Using these validated protocols, we have compiled genetic mutations in 1246 probands from 803 families with various bleeding disorders (table 1). These does not include patients with VWD for which we have only now initiated genetic evaluation. Mutations were identified in 762 families (95%). Of these, 358 were unique mutations, of which 103 (29%) are novel. We were not able to characterize disease causing mutation in a proportion (5 %, n=41/803) of families which is similar to the published literature. The frequency and distribution of the various mutations (Table 1) for all the disorders is similar to the spectrum reported in the human genome mutation database. Based on the availability of this molecular data, we have successfully offered carrier diagnosis to 334 probands from 271 unrelated families while prenatal diagnosis was carried out on 124 probands from 113 unrelated families. The development of efficient diagnostic algorithms for each of the bleeding disorder has helped to limit the turnaround time for genetic diagnoses to an average of 3–7 days at a cost of about $100-150 per sample for reagents and disposables. In conclusion, this report is the largest single-centre molecular data from a cohort of patients with bleeding disorders. The availability of such vast amount of genetic information and robust techniques for molecular diagnosis for different disorders of hemostasis contributes significantly to the effective management of these patients with bleeding disorders in India. Table 1. Bleeding disorder Total samples analyzed Unique families analyzed Total reported mutations Novel mutations Intron22 inversion Intron1 inversion Missense Nonsense Frame shift Splice site Major deletions No mutations identified Factor VIII deficiency 856 479 99 53 47% (n=227) 3% (n=13%) 16% (n=76) 10% (n=46) 12% (n=57) 3% (n=12) 2% (n=10) 6% (n=27) Factor IX deficiency 153 120 47 31 66% (n=79) 7% (n=8) 8% (n=9) 3% (n=3) 10% (n=12) 2% (n=2) Glanzmann Thrombasthenia 60 58 32 0 57 (n=34) 14% (n=8) 17% (n=10) 5% (n=3) 0 6.9% (n=4) Wiskott Aldrich Syndrome 48 29 12 1 10% (n=3) 24% (n=7) 24% (n=7) 14% (n=4) 3 % (n=1) 31% (n=9) Bernard-Soulier syndrome 28 27 2 11 56% (n=15) 4% (n=1) 0 48% (13) 0 0 Fibrinogen deficiency 28 26 2 13 15% (n=4) 0 31% (n=8) 58% (n=15) 0 0 Factor VII deficiency 23 14 11 2 50% (n=7) 29% (n=4) 14% (n=2) 14% (n=2) 0 0 Factor XI deficiency 12 12 5 1 100% (n=12) 0 0 17% (n=2) 0 0 Factor XIII deficiency 9 9 11 0 36% (n=4) 9% (n=1) 18% (n=2) 36% (n=4) 0 0 Combined Factor V+VIII deficiency 9 9 4 0 11% (n=1) 0 56% (n=5) 33% (n=3) 0 0 Factor II deficiency 8 8 7 2 88% (n=7) 0 0 25% (n=2) 0 0 Factor × deficiency 7 7 8 0 100% (n=7) 0 0 14 (n=1) 0 0 Factor V deficiency 5 5 4 0 40% (n=2) 0 0 60% (n=3) 0 0 Total 1246 803 255 103 251 75 100 57 23 41 Disclosures: No relevant conflicts of interest to declare.

Identification of Two Novel Mutations in PKHD1 Gene from Two Families with Polycystic Kidney Disease by Whole Exome Sequencing

2021 ◽  
Vol 22 ◽  
Author(s):  
Masoud Heidari ◽  
Hamid Gharshasbi ◽  
Alireza Isazadeh ◽  
Morteza Soleyman-Nejad ◽  
Mohammad Hossein Taskhiri ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Exome Sequencing ◽  
Polycystic Kidney Disease ◽  
Whole Exome Sequencing ◽  
Novel Mutation ◽  
Genetic Diagnosis ◽  
Genetic Alterations ◽  
Polycystic Kidney ◽  
Novel Mutations ◽  
Whole Exome

Background:: Polycystic kidney disease (PKD) is an autosomal recessive disorder resulting from mutations in the PKHD1 gene on chromosome 6 (6p12), a large gene spanning 470 kb of genomic DNA. Objective: The aim of the present study was to report newly identified mutations in the PKHD1 gene in two Iranian families with PKD. Materials and Methods: Genetic alterations of a 3-month-old boy and a 27-year-old girl with PKD were evaluated using whole-exome sequencing. The PCR direct sequencing was performed to analyse the co-segregation of the variants with the disease in the family. Finally, the molecular function of the identified novel mutations was evaluated by in silico study. Results: In the 3 month-old boy, a novel homozygous frameshift mutation was detected in the PKHD1 gene, which can cause PKD. Moreover, we identified three novel heterozygous missense mutations in ATIC, VPS13B, and TP53RK genes. In the 27-year-old woman, with two recurrent abortions history and two infant mortalities at early weeks due to metabolic and/or renal disease, we detected a novel missense mutation on PKHD1 gene and a novel mutation in ETFDH gene. Conclusion: In general, we have identified two novel mutations in the PKHD1 gene. These molecular findings can help accurately correlate genotype and phenotype in families with such disease in order to reduce patient births through preoperative genetic diagnosis or better management of disorders.

Characterising the phenotype and mode of inheritance of patients with inherited peripheral neuropathies carrying MME mutations

2018 ◽  
Vol 55 (12) ◽  
pp. 814-823 ◽  
Author(s):  
Vincenzo Lupo ◽  
Marina Frasquet ◽  
Ana Sánchez-Monteagudo ◽  
Ana Lara Pelayo-Negro ◽  
Tania García-Sobrino ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Late Onset ◽  
Axonal Neuropathy ◽  
Genetic Diagnosis ◽  
Hereditary Neuropathy ◽  
Motor Neuropathy ◽  
Charcot Marie Tooth Disease ◽  
Hereditary Motor Neuropathy ◽  
Index Cases ◽  
Mode Of Inheritance

BackgroundMutations in the metalloendopeptidase (MME) gene were initially identified as a cause of autosomal recessive Charcot-Marie-Tooth disease type 2 (CMT2). Subsequently, variants in MME were linked to other late-onset autosomal dominant polyneuropathies. Thus, our goal was to define the phenotype and mode of inheritance of patients carrying changes in MME.MethodsWe screened 197 index cases with a hereditary neuropathy of the CMT type or distal hereditary motor neuropathy (dHMN) and 10 probands with familial amyotrophic lateral sclerosis (fALS) using a custom panel of 119 genes. In addition to the index case subjects, we also studied other clinically and/or genetically affected and unaffected family members.ResultsWe found 17 variants in MME in a total of 20 index cases, with biallelic MME mutations detected in 13 cases from nine families (three in homozygosis and six in compound heterozygosis) and heterozygous variants found in 11 families. All patients with biallelic variants had a similar phenotype, consistent with late-onset axonal neuropathy. Conversely, the phenotype of patients carrying heterozygous mutations was highly variable [CMT type 1 (CMT1), CMT2, dHMN and fALS] and mutations did not segregate with the disease.ConclusionMME mutations that segregate in an autosomal recessive pattern are associated with a late-onset CMT2 phenotype, yet we could not demonstrate that MME variants in heterozygosis cause neuropathy. Our data highlight the importance of establishing an accurate genetic diagnosis in patients carrying MME mutations, especially with a view to genetic counselling.

scholarly journals Erythrocyte Pyruvate Kinase Deficiency in Non-spherocytic Hemolytic Anemia: A System of Multiple Genetic Markers?

Blood ◽  
1968 ◽  
Vol 32 (1) ◽  
pp. 33-48 ◽  
Author(s):  
WOLF W. ZUELZER ◽  
ABNER R. ROBINSON ◽  
THERESA H. J. HSU
Keyword(s):  
Hemolytic Anemia ◽  
Genetic Diagnosis ◽  
Glucose Consumption ◽  
Normal Glucose ◽  
Red Cell Survival ◽  
Severe Type ◽  
Subordinate Role ◽  
Index Cases ◽  
2,3 Dpg

Abstract Extreme intrafamilial differences between PK-deficient phenotypes regarding hemolysis, ATP stability, and glucose consumption were observed in two pedigrees in which the index cases had severe nonspherocytic hemolytic anemia. Genetic analysis was consistent with heterozygosity for two distinct interacting mutants in minimally affected relatives of severely anemic homozygotes. Neither the mature erythrocytes of the former nor the reticulocyte-rich cell populations of the latter showed accumulation of glycolytic intermediates, but 2,3-DPG was elevated in both. Despite severe PK deficiency, red cell survival in the minimal type was near normal, glucose consumption was unaffected in three of four subjects tested, and ATP maintenance in vitro was adequate, in contrast to the severe type in which these parameters were grossly depressed. The genetic and pathophysiologic implications of these findings are discussed. The possibility is considered that defective glycolysis may play a subordinate role in the hemolytic process associated with PK deficiency and that the enzyme defect may be a genetic marker for as yet unknown erythrocytic abnormalities involving an increase of 2,3-DPG and possibly primary membrane lesions creating excessive demands on the energy metabolism of the erythrocytes. Regardless of the mode of gene action, it is concluded that the nonspherocytic hemolytic anemias associated with PK deficiency are genetically and phenotypically heterogeneous, and that the genetic diagnosis cannot rest on PK assay alone.

scholarly journals CYP2E1-DEPENDENT VARIATIONS IN HEPATOCYTES DAMAGE DURING TREATMENT OF TUBERCULOSIS

2019 ◽  
Vol 16 (3) ◽  
pp. 20-26
Author(s):  
L.V. Natrus ◽  
L.V. Gayova ◽  
O.O. Gorkunenko ◽  
P.A. Chernovol ◽  
M.V. Zelinska
Keyword(s):  
Gene Polymorphism ◽  
Maintenance Therapy ◽  
Genomic Dna ◽  
Clinical Laboratory ◽  
Intensive Therapy ◽  
Venous Blood ◽  
Control Group ◽  
Drug Induced ◽  
Cyp2e1 Gene ◽  
Tb Treatment

Relevance. Investigation of polymorphism in a locus of CYP2E1 as the prognostic factor of drug-induced hepatotoxicity at anti-TB therapy is significant due to the influence of CYP2E1 on drug metabolism. The objective of the investigation is to analyze the association of rs2070676 СYP2E1 gene polymorphism with drug-induced hepatotoxicity by means of the clinical-laboratory values of serum transaminases at anti-TB treatment. Materials and methods. The study involved 47 patients with drug-susceptible tuberculosis first time discovered. 58 healthy volunteers comprised a control group. Laboratory indices were determined in venous blood three times: before the treatment as baseline; in 2 months of intensive therapy (isoniazid, rifampicin, ethambutol, pyrazinamide), then in 4 months of maintenance therapy (isoniazid, rifampicin). Serum activities of enzymes ALT, AST, and GGT were measured by standard algorithm on automatic analyzer BS-300. Analysis of rs2070676 polymorphism of CYP2E1 gene was performed by polymerase chain reaction using standard PureLink® Genomic DNA Kit for Purification of Genomic DNA; Manufacturer of INVITROGEN (USA). For statistical processing, IBM SPSS Statistics 23 was applied. Results. Investigation of serum ALT and AST in patients with major genotype CYP2E1 (C/C) showed the lower baseline ALT and AST levels comparing to the control group, which might be caused by suppression of hepatocytes functions at the development of the disease. Anti-TB treatment caused an increase in ALT and AST levels comparing to the baseline in patients with major CYP2E1 (C/C) genotype. In the group with C/G polymorphism, the baseline ALT level didn’t differ much from the baseline of the control group; it showed a decrease after intensive therapy and returned back to the initial level at maintenance therapy. This might be related to the certain protective property of СYP2E1 gene polymorphism. The AST level was increased after intensive therapy (to a smaller extent than for the patients with major C/C genotype) and remained on the same level at maintenance therapy. A study of GGT showed a gradual increase regardless of genotype. Conclusion. According to the data of the experiment, the status of hepatocytes in patients with tuberculosis at baseline and during treatment was different depending on the CYP2E1 genotype. The results of the experiment indicate that the CYP2E1 gene polymorphism has a certain protecting role. It reduces the level of drug metabolites and hepatotoxicity which causes mitochondrial dysfunction.

Genetic diagnosis of Hailey–Hailey disease in two Chinese families: novel mutations in theATP2C1gene

2009 ◽  
Vol 34 (8) ◽  
pp. e968-e971 ◽  
Author(s):  
Y. G. Ding ◽  
H. Fang ◽  
L. M. Lao ◽  
X. J. Jiang ◽  
H. C. Chen
Keyword(s):  
Genetic Diagnosis ◽  
Novel Mutations ◽  
Chinese Families

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

scholarly journals Genetic Landscape of Congenital Myasthenic Syndromes From Turkey: Novel Mutations and Clinical Insights

2017 ◽  
Vol 32 (8) ◽  
pp. 759-765 ◽  
Author(s):  
Uluç Yiş ◽  
Kerstin Becker ◽  
Semra Hız Kurul ◽  
Gökhan Uyanik ◽  
Erhan Bayram ◽  
...  
Keyword(s):  
Cholinesterase Inhibitors ◽  
Neuromuscular Disorders ◽  
Genetic Diagnosis ◽  
Common Type ◽  
Child Neurology ◽  
Novel Mutations ◽  
Congenital Myasthenic Syndromes ◽  
Genetic Landscape ◽  
Prompt Diagnosis ◽  
Myasthenic Syndromes

Congenital myasthenic syndromes are clinically and genetically heterogeneous disorders of neuromuscular transmission. Most are treatable, but certain subtypes worsen with cholinesterase inhibitors. This underlines the importance of genetic diagnosis. Here, the authors report on cases with genetically proven congenital myasthenic syndromes from Turkey. The authors retrospectively reviewed their experience of all patients with congenital myasthenic syndromes, referred over a 5-year period (2011-2016) to the Child Neurology Department of Dokuz Eylül University, Izmir, Turkey. In addition, PubMed was searched for published cases of genetically proven congenital myasthenic syndromes originating from Turkey. In total, the authors identified 43 (8 new patients, 35 recently published patients) cases. Defects in the acetylcholine receptor (n = 15; 35%) were the most common type, followed by synaptic basal-lamina associated (n = 14; 33%) and presynaptic syndromes (n = 10; 23%). The authors had only 3 cases (7%) who had defects in endplate development. One patient had mutation GFPT1 gene (n = 1; 2%). Knowledge on congenital myasthenic syndromes and related genes in Turkey will lead to prompt diagnosis and treatment of these rare neuromuscular disorders.

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