DETECTION OF ATP7B MUTATIONS IN WILSON DISEASE BY DNA SEQUENCING
Hoàng Anh Vũ, Phan Thị Xinh
Purpose: Wilson disease is a disorder of copper metabolism manifesting as neurological symptoms and liver damage. Mutation of the ATP7B gene is a major criteria for definite diagnosis of Wilson disease, especially important in cases with differential diagnosis in clinical practice. We aim to establish to a procedure of DNA sequencing for promoter and coding region of ATP7B and use for investigating ATP7B mutations in clinical Wilson’s disease samples.
Methods: Genomic DNA was extracted from blood samples of 18 patients suspected of Wilson disease, including one with neurological symptoms and the others with liver manifestations. We designed specific primers for amplification of promoter and coding region of ATP7B by polymerase chain reaction (PCR). PCR products were sequenced bidirectionally using Sanger’s technique on an ABI 3130 Genetic Analyzer system.
Result: All 21 exons and exon – intron boundaries were amplified by 11 pairs of specific primers, with PCR products range from 265 bp to 2502 bp. The amplified promoter fragment was 1494 bp. ATP7B mutations were found in 8 patients including 2 with homozygous, 1 with single heterozygous and 5 with compound heterozygous mutation status. We did not detect any mutation in promoter. The mutation sites were scattered in exon 2 (c.525_526insA and c.314C>A), exon 8 (c.2333G>T and c.2304_2305insC), exon 11 (c.2705T>C), exon 13 (c.2975C>T), exon 14 (c.3079G>C), and exon 20 (c.4112T>C). Among these, the c.2705T>C and c.3079G>C are novel mutations. Several single nucleotide polymorphisms were detected in patients with and without mutations.
Conclusion: We reported here a procedure for DNA sequencing that can use for detection of ATP7B gene mutations, facilitating diagnosis of Wilson disease in clinical practice.
Key words: Wilson disease, ATP7B gene, gene mutation, DNA sequencing.
DETECTION OF FIP1L1-PDGFRA FUSION GENE IN HYPEREOSINOPHILIC SYNDROME
Phan Thị Xinh, Hoàng Anh Vũ
Purpose: Hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by persistence of eosinophilia in association with damage to multiple organs. There are approximately 10-15% of patients with HES expressing FIP1L1-PDGFRA fusion gene and these patients have good response with low dose imatinib mesylate. We conducted this study to set up RT-PCR procedure for detecting FIP1L1-PDGFRA fusion gene in patients diagnosed with HES.
Methods: We designed one forward primer on exon 8 of the FIP1L1 gene and 1 reverse primer located on exon 14 of the PDGFRA gene to amplify the FIP1L1-PDGFRA fusion gene from 37 patients who were diagnosed with HES using RT-PCR.
Results: We showed here that 5 patients expressing FIP1L1-PDGFRA fusion gene in a total of 37 patients with HES, accounting for 13.5%. Four of the 5 patients with the expression of FIP1L1-PDGFRA responded well to low-dose imatinib (100 mg/day) in terms of clinical, hematological and molecular criteria.
Conclusion: RT-PCR can detect FIP1L1-PDGFRA fusion gene in patients with HES, helping clinicians to select more effective treatments other than corticoid regimen in some cases with increased eosinophils.
Key words: Hypereosinophilic syndrome, FIP1L1-PDGFRA fusion gen, imatinib.
DETERMINATION OF HLA-B*1502 WITH DNA SEQUENCING
Hoàng Anh Vũ, Phan Thị Xinh
Purpose: HLA-B*1502 allele is a valuable predictive biomarker for carbamazepine-induced severe skin reactions in Asian populations. We conducted this study to establish a procedure of DNA sequencing for the HLA-B gene and apply the technique for investigating HLA-B*1502 allelic frequency from peripheral blood samples.
Methods: From June 2014 to February 2015, genomic DNA was extracted from blood samples of 89 epileptic patients who were requested for HLA-B*1502 testing before carbamazepine prescription. A fragment of the HLA-B gene harboring exons 2 and 3 was amplified with polymerase chain reaction using a pair of specific primers. PCR products were sequenced bidirectionally by Sanger method on an ABI 3130 Genetic Analyzer. Electropherograms were analyzed by comparing to known HLA-B reference sequences to identify the HLA-B*1502 allele.
Result: We successfully determined the HLA-B*1502 allelic status from PCR products for all 89 samples. Out of 27 samples positive for HLA-B*1502 allele (30.3%), 9 were found homogeneous and the other 18 were heterogeneous.
Conclusion: DNA sequencing method can determine HLA-B*1502 allele correctly, which might be useful for prediction of risk for severe skin reaction to carbamazepine.
Key words: HLA-B gene, DNA sequencing, carbamazepine, severe skin reaction.
DETECTION OF IDH1 GENE MUTATION (p.R132H) IN GLIOMAS BY ASO-PCR
Hoàng Anh Vũ, Đỗ Phú Quang, Trần Minh Thông
Objectives: IDH gene mutational status is one of the important prognostic factors in gliomas. The p.R132H mutation in IDH1 gene due to a substitution from guanine to adenine at nucleotide 395 is the most common of these mutations. We establish an ASO-PCR procedure to identify this mutation from paraffin blocks of gliomas.
Materials and Methods: The study was performed with 33 glioma samples collected from Cho Ray Hospital. Samples were DNA extracted using FFPE gDNA ReliaprepTM Miniprep System kit from Promega, USA. Some first samples were investigated by DNA sequencing technique to select positive control carrying p.R132H mutation. Specific primer for mutant allele was designed and ASO-PCR conditions with positive control were optimized and applied to verify the mutation in the remaining samples.
Results: ASO-PCR using 3 primers in one reaction allows amplifying specifically p.R132H mutant samples, confirmed by DNA sequencing. We detected 11 mutations in 28 cases of WHO grade II and III gliomas (39.3%), but not any mutation in 5 cases of WHO grade I and IV gliomas (glioblastoma).
Conclusion: ASO-PCR can be used in the screening step of IDH1 gene mutations (p.R132H) in gliomas.
Keywords: gliomas, IDH1 gene, mutations, ASO-PCR.
SPECTRUM OF RB1 GENE MUTATIONS IN RETINIBLASTOMA
Hoang Anh Vu1,6, Le Thai Khuong2, Nguyen The Vinh1, Nguyen Huynh Minh Quan1,
Phan Thi Xinh3, Nguyen Ngoc Chau Trang4, Nguyen Thanh Luan5, Cao Mong Phi An6, Nguyen Cong Kiet5
Backgroud: Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intra-ocular malignancy in children caused by mutations in the RB1 tumor suppressor gene. An early diagnosis is critical for survival and eye preservation, thus identification of RB1 mutations is important for unequivocal diagnosis of hereditary retinoblastoma and risk assessment in relatives. We aim to describe the spectrum of RB1 mutations in Vietnamese patients.
Materials and Methods: Using Sanger DNA sequencing, we investigated mutations in RB1 exons 1 – 27 and promoter region from 51 patients diagnosed with retinoblastoma at the Ho Chi Minh City Eye Hospital. Genomic DNA and total RNA were extracted from fresh tumor tissue and peripheral blood samples, followed by PCR amplification and sequencing at Center for Molecular Biomedicine, University of Medicine and Pharmacy, Ho Chi Minh City.
Results: The study cohort included 13 bilateral and 38 unilateral retinoblastoma probands (20 female and 31 male, aged from 1 month to 66 months). RB1 mutations were detected in 38 out of 51 tumor tissue samples (74.5%), of which 2 samples only carried mutations at RNA level. In total, 37 different types of RB1 mutations were found in tumors, including 14 nonsense mutations, 11 frameshift mutations, 9 splicing mutations, 2 missense mutations, and 1 promoter mutation. Among 34 patients tested for mutations in blood, there were 17 individuals with germline mutations, including 8 mutations in 9 bilateral retinoblastoma probands (88.9%) and 9 mutations in 25 unilateral retinoblastoma probands (36%).
Conclusion: Considerable percentage of individuals with sporadic unilateral RB1 carried germline mutations, indicating the importance of genetic testing for all children with retinoblastoma. Our findings help to characterize the spectrum of mutations present in the Vietnamese population and can improve genetic diagnosis of retinoblastoma.
Keywords: Retinoblastoma, RB1, mutation, sequencing.
MUTATIONS OF THE WAS GENE IN WISKOTT - ALDRICH SYNDROME
Phan Thị Xinh, Hoàng Anh Vũ
Purpose: Wiskott – Aldrich syndrome is a rare X-linked primary immunodeficiency disorder characterized by the triad of eczema, thrombocytopenia, and severe and often recurrent infection. Mutations in the WAS gene cause this syndrome. We conducted this study to set up a DNA sequencing procedure for investigating WAS mutations in Vietnamese patients.
Methods: We designed specific primers for amplification of 12 WAS exons and exon – intron boundaries from blood of 7 infant patients with Wiskott – Aldrich syndrome. PCR products were sequenced using Sanger’s technique on an ABI 3130 Genetic Analyzer system and analyzed with the CLC Main Workbench software.
Results: We successfully amplified the coding region of WAS gene by 2 sets of primer pair, generating PCR products of 2459 bp and 4603 bp. Sequencing analyses detected 5 mutations in patient’s samples, including 3 in exon 4 (c.397G>A, c.436C>T, and c.372_375insGG), 1 in exon 7 (c.665delC), and 1 in exon 10 (c.1267_1271insG)
Conclusion: DNA sequencing can detect different types of WAS mutation which facilitates confirmation of Wiskott – Aldrich syndrome in Vietnamese childhood patients.
Key words: Wiskott – Aldrich syndrome, WAS gene, exon, mutation.