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The association of type-2 diabetes mellitus (T2DM) and cataract with mtDNA mutation has been reported before. Despite the high prevalence of DM and cataract in Indonesia, a study of the mtDNA variants in Indonesia in correlation with the two diseases is still limited. MT.3243A>G is one of the hotspots mutations for mitochondrial diseases, but the explanation for its occurrence in patients with pure cataract is still elusive. Therefore, the objective of this study was to analyze the mitochondrial genome variants from T2DM and cataract patients in Indonesia using the direct sequencing method. The homology analysis of the genome to the Cambridge reference sequence resulted in 86 variants, including 20 variants that cause amino acid substitutions. Based on the Mitomap data, 17 of the 20 variants were novel. Upon comparison with the 12 normal variant genomes, 11 of 17 variants were suggested to be associated with T2DM and cataract diseases since they code the protein in complex-I (ND4L, ND5, and ND6), complex-III (cytb), and complex-V (ATP6) of the respiratory complex. Interestingly, MT.3316G>A, for the first time, is shown in a pure cataract patient. In addition, the novel phenotype of MT.5460G>A and MT.10398A>G were revealed, which are T2DM and cataract in one patient. Based on our study, these diseases might be related to the disruption of the ATP metabolism due to the structure and function changes of proteins involved in the respiratory complex. This discovery is expected to offer an understanding of the origins of gene-level clinical differences, particularly in Indonesia. [ABSTRACT FROM AUTHOR]

Background & Objective: Epithelial ovarian cancer (EOC) is the most prevalent type of ovarian cancer. Previous studies have elucidated different pathways for the progression of this malignancy. The mutation in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene, a member of the MAPK/ERK signaling pathway, plays a role in the development of EOC. The current study aimed to determine the frequency of the BRAF V600E mutation in ovarian serous and mucinous tumors, including borderline and carcinoma subtypes. Methods: A total of 57 formalin-fixed paraffin-embedded samples, including serous borderline tumors (SBTs), low-grade serous carcinomas (LGSCs), high-grade serous carcinomas (HGSCs), mucinous borderline tumors (MBTs), and mucinous carcinomas, and 57 normal ovarian tissues were collected. The BRAF V600E mutation was analyzed using polymerase chain reaction (PCR) and sequencing. Results: While 40% of the SBT harbor BRAF mutation, we found no BRAF mutation in the invasive serous carcinoma (P=0.017). Also, there was only 1 BRAF mutation in MBT and no mutation in mucinous carcinomas. In addition, we found no mutation in the control group. Conclusion: The BRAF mutation is most frequent in borderline tumors but not in invasive serous carcinomas. It seems that 2 different pathways exist for the development of ovarian epithelial neoplasms: one for borderline tumors and the other for high-grade invasive carcinomas. Our study supports this hypothesis. The BRAF mutation is rare in mucinous neoplasms. [ABSTRACT FROM AUTHOR]

Abstract Objective The nucleoside diphosphate linked moiety X (Nudix)-Type motif 15 (NUDT15) enzyme is involved in thiopurine metabolism. Genetic variants in the NUDT15 gene result in decreased NUDT15 activity, which in addition to decreased thiopurine S-methyltransferase (TPMT) activity, contributes to thiopurine toxicity. Current standard approaches of NUDT15 genetic analysis have mainly been targeting several common variants. We aimed to develop a clinical-grade DNA-based assay for genetic analysis of the NUDT15 gene using Sanger di-deoxy sequencing. Results Sanger sequencing results were fully concordant with the expected NUDT15 genotype in all 17 cell line samples with known NUDT15 variants (accuracy = 100%; 95% CI 80.49 to 100.00%). Precision studies showed 100% intra-run repeatability and 100% inter-run reproducibility, respectively. Genetic analysis of the NUDT15 gene was performed for 80 patients of Asian ethnicity with wildtype TPMT. 76% (N = 61) of the studied individuals had NUDT15 *1/*1 diplotype. 25% (N = 14) of Chinese and 36% (N = 5) of Malays were found to carry at least 1 non-functional NUDT15 allele. Our study confirmed a high frequency of NUDT15 c.415C>T and c.55_56insGAGTCG variants in the Chinese and Malay ethnic groups in Singapore, highlighting the importance of determining NUDT15 genotype prior to thiopurine dosing.

Introduction: Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the androgen receptor (AR), through ACE2 receptor and TMPRSS2, to enter nasal and upper airways epithelial cells. Genetic analyses revealed that HSD3B1 P1245C polymorphic variant increases dihydrotestosterone production and upregulation of TMPRSS2 with respect to P1245A variant, thus possibly influencing SARS-CoV-2 infection. Our aim was to characterize the HSD3B1 polymorphism status and its potential association with clinical outcomes in hospitalized patients with COVID-19 in Southern Switzerland.Materials and Methods: The cohort included 400 patients hospitalized for COVID-19 during the first wave between February and May 2020 in two different hospitals of Canton Ticino. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue blocks, and HSD3B1 gene polymorphism was evaluated by Sanger sequencing. Statistical associations were verified using different test.Results:HSD3B1 polymorphic variants were not associated with a single classical factor related to worse clinical prognosis in hospitalized patients with SARS-CoV-2. However, in specific subgroups, HSD3B1 variants played a clinical role: intensive care unit admission was more probable in patients with P1245C diabetes compared with P1245A individuals without this comorbidity and death was more associated with hypertensive P1245A>C cases than patients with P1245A diabetes without hypertension.Discussion: This is the first study showing that HSD3B1 gene status may influence the severity of SARS-CoV-2 infection. If confirmed, our results could lead to the introduction of HSD3B1 gene status analysis in patients infected with SARS-CoV-2 to predict clinical outcome.

Objective: The nucleoside diphosphate linked moiety X (Nudix)-Type motif 15 (NUDT15) enzyme is involved in thiopurine metabolism. Genetic variants in the NUDT15 gene result in decreased NUDT15 activity, which in addition to decreased thiopurine S-methyltransferase (TPMT) activity, contributes to thiopurine toxicity. Current standard approaches of NUDT15 genetic analysis have mainly been targeting several common variants. We aimed to develop a clinical-grade DNA-based assay for genetic analysis of the NUDT15 gene using Sanger di-deoxy sequencing. Results: Sanger sequencing results were fully concordant with the expected NUDT15 genotype in all 17 cell line samples with known NUDT15 variants (accuracy = 100%; 95% CI 80.49 to 100.00%). Precision studies showed 100% intra-run repeatability and 100% inter-run reproducibility, respectively. Genetic analysis of the NUDT15 gene was performed for 80 patients of Asian ethnicity with wildtype TPMT. 76% (N = 61) of the studied individuals had NUDT15 *1/*1 diplotype. 25% (N = 14) of Chinese and 36% (N = 5) of Malays were found to carry at least 1 non-functional NUDT15 allele. Our study confirmed a high frequency of NUDT15 c.415C>T and c.55_56insGAGTCG variants in the Chinese and Malay ethnic groups in Singapore, highlighting the importance of determining NUDT15 genotype prior to thiopurine dosing. [ABSTRACT FROM AUTHOR]

Introduction: The object of this study is to identify the frequency isocitrate dehydrogenase 1 gene (IDH1) mutations and 10q23/PTEN locus alterations in Turkish patients with glioblastoma multiforme (GBM). Methods: For this purpose, DNAs obtained from paraffin-embedded archival materials belong to 54 cases diagnosed as GBM were applied direct sequencing. In addition, overall 25 cases and 10 non-neoplastic brain tissues which were used as controls were analyzed by fluorescent in situ hybridization (FISH). Results: We detected 5 heterozygous IDH1 c.395G>A mutations (9.3%) with wild-type arginine 132 replaced by histidine (R132H). Although there is no statistical significance between the age of the cases and IDH1 p.R132H mutation, the patients harboring the p.R132H mutation were younger (median age; 38) than patients without this mutation (median age; 52). The median survival time of the patients with this mutation was calculated at 7 months, while it was 5 months for the patients with no mutation; however, this finding was not statistically significant. According to our FISH results, we found a total of 16 10q23/PTEN alterations, of which 8 were hemizygous deletion (32%) and 8 were monosomy (32%). Discussion and Conclusion: Understanding the prevalence of IDH1 mutations and 10q23/PTEN alterations may have importance in terms of the diagnosis, prognosis, and treatment of GBM patients. [ABSTRACT FROM AUTHOR]

Abstract Background It is very rare for clear cell sarcomas (CCS) to arise in the bone. During diagnosis, it is important to distinguish primary CCS of bone from bone metastasis of melanoma because this difference fundamentally changes the therapeutic options. Recently, characteristic fusion genes of CCS have been detected using reverse transcription polymerase chain reaction (RT-PCR) or direct sequencing which allowed to distinguish CCS from melanoma. However, there was no study applying these analyses with positive results. In this case, we describe the use of fusion gene analysis to diagnose a primary CCS of the bone. Case presentation A 36-year-old male presented with a four-months history of left knee pain. Magnetic resonance imaging showed a lesion in the left femoral medial epicondyle. Histological examination of the biopsy specimen revealed proliferating oval or rounded cells. These cells had clear cytoplasm arranged in fascicles or compact nests with frequent deposits of brown pigment. Furthermore, immunohistochemistry analysis revealed that tumor cells were positive for S-100 protein, HMB-45, Melan-A, and SOX10. It stained negative for CD34 and BRAF v600e. Conclusively, detection of the EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing confirmed that the lesion was a primary CCS of the bone. Wide-margin resection and reconstruction with a tumor endoprosthesis were performed. Conclusions Herein, we diagnosed a rare case of primary CCS of the bone by detecting EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing. Since fluorescence-in situ hybridization (FISH) and RT-PCR could show false positive by mainly due to technical problems, it is better to perform direct sequencing to confidently diagnose the tumor as a primary CCS especially at very rare site such as bone.

Summary: We developed a high‐resolution and comprehensive typing method for swine leukocyte antigen 3 (SLA‐3), an MHC class I gene, employing locus‐specific genomic PCR followed by subsequent direct sequencing. A total of 292 individuals from nine pure, one cross‐breed and six cell lines were successfully typed. A total of 21 SLA‐3 alleles were identified, of which four were found to be novel alleles. However, the allelic diversity of SLA‐3 was lower than that of previously reported class I genes, SLA‐1 and ‐2. More SLA‐3 alleles were observed in the Landrace and Yorkshire breeds than the other breeds. SLA‐3*04:01 was identified in seven out of nine breeds and was the most widely distributed allele across all breeds. Therefore, the typing method reported in this study completes our efforts to develop high‐resolution typing methods for major SLA molecules, facilitating the combined analysis of major SLA genes from field samples, which is important to understand the relationship between the adaptive immune responses against pathogens and the immunogenetic makeup of an individual. [ABSTRACT FROM AUTHOR]

Background. Rhesus phenotype has been determined in 404 persons which have problems with blood groups identification. Genetic typing of antigen RhD variants was performed in 73 individuals. Objective of the work was to give molecular and serological characteristics of the antigen RhD weak types.Materials and methods. Method of rhesus phenotype determination in direct agglutination test on plane by using of anti-D, anti-C, anti-c, anti-Cw, anti-E and anti-e monoclonal antibodies; gel method of rhesus phenotype determination; methods of genetic typing of RhD; methods of antigen RhD determination in the classic indirect antiglobulin test and in the gel indirect antiglobulin test; method of antigen RhD determination in the saline agglutination test.Results. Serological methods identified 73 red blood samples with the weakened expression of RhD antigen. Molecular methods showed the reasons of weakness of antigen expression. Three RHD*D weak types which are common in Russians (RHD*D weak type 1–3) were identified and for the first time 3 types were found – RHD*D weak type 67, RHD(G255R) and RHD(JVS5-38del4). Serological characteristic of RhD weak types was given. It was shown that combined using of monoclonal antibodies in direct agglutination test and in gel is the most effective serological method of the antigen variants detection. Red blood cells with weak RhD antigens can be recognized by weakness or absence of agglutination with monoclonal antibodies on plane if agglutination in gel was 3+4+.Conclusion. Concrete weak RhD variants can be determined only by genetic typing. Serologically weak antigen variants can be detected by using of at least two series of monoclonal antibodies or by using of two different methods (it is preferable).

The contribution of PAX8 genetic variants to congenital hypothyroidism (CH) is not well understood. We aimed to study the genetic variability of exons 3 and 5 of PAX8 gene among a cohort of children with congenital hypothyroidism in correspondence to their clinical aspect. Blood samples were collected from 117 children (63 girls and 54 boys) with CH and enrolled as cases (Group I). All cases underwent biochemical confirmation with low FT4 and high TSH levels and thyroid gland imaging, along with equal number of matched apparently healthy individuals who served as controls (Group II). Genomic materials for exons 3 and 5 of PAX8 gene were extracted, amplified by PCR, detected by electrophoresis, purified, and sequenced by the Sanger technique through the application of ABI 3730x1 DNA Sequencer. Out of 117 cases, eight different effective PAX8 mutations were detected in exon 3 (G23D, V35I, I34T, Q40P, p.R31C, p.R31H, p.R31A, and p.I47T) in 14 patients with their sonographic findings ranged from normal, hypoplastic to thyroid agenesis. Besides the reported mutations, one novel mutation; R31A was detected in 1 euotopic case. Exon 5 analysis revealed no detected mutations elsewhere. In contrast, all healthy control children showed no mutation and normal sonographic findings. Mutations in exon 3 of PAX8 gene, implies its important role in thyroid development and function, as a first estimate of PA8 mutation rate in Egyptian patients with CH having normal and dysgenetic gland. Using ultrasound is mandatory for diagnosis and guiding the treatment of children with CH. [ABSTRACT FROM AUTHOR]

Background and Aims: Metabolic abnormalities in T2DM (Type 2 diabetes mellitus) include classic manifestations such as impaired insulin secretion, synthesis and peripheral insulin resistance. The intronic variants rs7903146 and rs12255372 of the TCF7L2 (transcription factor 7-like 2) gene are strongly associated with risk of incidence of T2DM and impaired β-cell functions. Studies addressing the early T2DM onset, and early insulin dependence in T2DM patients of south Tamil Nadu are still lacking, and hence the present study focuses in determining the influence of the TCF7L2 polymorphisms in the incidence and disease course in the T2DM patients of south Tamil Nadu. Methods: Anthropometric measurements and biochemical parameters were carried out in early onset (Group A), early onset insulin dependent T2DM patients (Group B) and non-insulin dependent T2DM patients (Group C). PCR, allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the genotype and the impact of the TCF7L2 SNPs in the T2DM disease course. Results: Female T2DM patients with the CT/TT rs7903146 genotype (P = 0.005) and the rs12255372 GT/TT genotype (P = 0.036) exhibited a significantly low mean age for T2DM incidence. Correlation/regression analysis in the T2DM patients revealed that rs12255372 (P = 0.042) is associated with early onset in the Group C patients and the rs7903146 (P = 0.018), rs12255372 (P = 0.026) are associated with insulin dependence in the group B patients. Conclusion: Screening for the TCF7L2 polymorphisms will prevent T2DM incidence and enable life style changes, appropriate therapeutic strategies that would help combat the accelerated disease course in the T2DM patients.

Background: It is very rare for clear cell sarcomas (CCS) to arise in the bone. During diagnosis, it is important to distinguish primary CCS of bone from bone metastasis of melanoma because this difference fundamentally changes the therapeutic options. Recently, characteristic fusion genes of CCS have been detected using reverse transcription polymerase chain reaction (RT-PCR) or direct sequencing which allowed to distinguish CCS from melanoma. However, there was no study applying these analyses with positive results. In this case, we describe the use of fusion gene analysis to diagnose a primary CCS of the bone.Case Presentation: A 36-year-old male presented with a four-months history of left knee pain. Magnetic resonance imaging showed a lesion in the left femoral medial epicondyle. Histological examination of the biopsy specimen revealed proliferating oval or rounded cells. These cells had clear cytoplasm arranged in fascicles or compact nests with frequent deposits of brown pigment. Furthermore, immunohistochemistry analysis revealed that tumor cells were positive for S-100 protein, HMB-45, Melan-A, and SOX10. It stained negative for CD34 and BRAF v600e. Conclusively, detection of the EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing confirmed that the lesion was a primary CCS of the bone. Wide-margin resection and reconstruction with a tumor endoprosthesis were performed.Conclusions: Herein, we diagnosed a rare case of primary CCS of the bone by detecting EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing. Since fluorescence-in situ hybridization (FISH) and RT-PCR could show false positive by mainly due to technical problems, it is better to perform direct sequencing to confidently diagnose the tumor as a primary CCS especially at very rare site such as bone. [ABSTRACT FROM AUTHOR]

Background: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with several underlying genetic abnormalities. Several studies have tried to elucidate the prognostic significance of cytokine receptor-like factor 2 (CRLF2) overexpression in pediatric B-cell precursor (BCP)-ALL; however, it is still controversial. Methods: CRLF2 expression was assessed by flow cytometry in 87 newly diagnosed BCP-ALL pediatric patients, and 80 age and gender-matched control group. Janus Kinase2 (JAK2) (R683) mutation analysis was also performed in those identified to have CRLF2 overexpression with adequate DNA samples by direct sequencing. Results: CRLF2 overexpression was identified in 26/87 (29.9%) of our patients with cutoff set at mean fluorescence intensity (MFI = 3.8) using the Receiver Operating Characteristic (ROC) curve. There were no significant differences in the clinical and laboratory features between patients with high and low-CRLF2 expression, apart from thrombocytopenia which showed statistically significant association with the low-expression group (p = 0.041). Sequence analysis of samples with high CRLF2 expression (n = 23) revealed that 2/23 (8.7%) cases harbored the mutation JAK2 (R683). CRLF2 levels did not have a significant impact on either overall survival (OS) or disease free survival (DFS) (p = 0.601; p = 0.212, respectively). Conclusions: CRLF2 overexpression was not an adverse parameter in pediatric BCP-ALL patients. However, patients with CRLF2 overexpression may harbor the JAK2 mutation presenting a group that can benefit from targeted therapy by kinase inhibitors. The usage of CRLF2 expression to monitor minimal residual disease of BCPALL would be an area of interest for further evaluation. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the resistance of fluoroquinolones (FQs) and the effects of mutations in the resistance gene in clinical isolates of P. aeruginosa isolated from different sources in Al-Hussein Hospital, Al-Samawah city, Iraq. The basic mechanism of the resistant of fluoroquinolones in P. aeruginosa is via mutations occurring in the basic bacterial gyrA gene encoding-subunit A of DNA gyrase . Forty clinical isolates from various sourced (burn 7 (17.5 %), wound 7 (17.5 %), ear 2 (5 %), operation room 12 (30 %), urine 3 (7.5 %), and industrial dialysis center 9 (22.5 %)) were isolated based on bacteriological methods confirmed by 16s rRNA gene using PCR technique. A sensitivity test was conducted to all isolates by Kirby-Pour method using 7 antibiotics of fluoroquinolones. Amongst the 40 clinical isolates, 10 were resistant and 3 were sensitive to all tested antibiotics, while 27 were intermediate, resistant and sensitive to two or more of tested antibiotics, with the resistance being confirmed by the minimum inhibitor concentration (MIC) test. The ten resistant isolates were used to examine the mutations in gyrA gene. A direct sequence method was used and revealed eight mutations in gyrA gene at different positions. In addition, we found that fluoroquinolone activity in the sensitive isolates, after sequencing for these isolates, is a bacteriostatic activity. The results of this study showed the gyrA mutations resulting from the excessive use of antibiotics are one of the mechanisms may be that leading to fluoroquinolone resistance. [ABSTRACT FROM AUTHOR]

Background: Sitosterolemia (STSL), also known as phytosterolemia, is a rare autosomal recessive hereditary disease caused by mutations in the ABCG5 or ABCG8 genes. The disease is a result of disorders in lipoprotein metabolism, and is characterized by tendinous and tuberous xanthomas, elevated plasma cholesterol and phytosterol levels, and thrombocytopenia and hemolytic anemia in several patients. The manifestations of STSL are diverse and can easily be misdiagnosed. In recent years, cases of this disease in children have been reported in succession. There is therefore a need for clinicians to improve identification of STSL and perform early intervention. Methods: We evaluated four children with STSL caused by genetic mutations in ABCG5 or ABCG8, as well as their family members, by analyzing their clinical characteristics and performing Trio‐whole exome sequencing. The biological consequences of the mutations were analyzed using various bioinformatics software. We also analyzed the consequences of a mutation commonly observed in STSL patients on the structure of the protein involved. Results: We identified five previously unreported pathogenic mutations of different phenotypes of STSL: ABCG5 NM_022436:c.1337G>A; ABCG8 NM_022437:c.965‐1G>A, c.323‐1G>C, c.1418C>G and c.1534G>A. We also report the structural changes brought about by a mutation common in STSL patients, as well as the possible consequences of these changes. Conclusions: Our findings further broaden the genotypic and phenotypic profiles of the onset of STSL in the pediatric population and provide information for the diagnosis and treatment of this disease. [ABSTRACT FROM AUTHOR]

Background: The development of whole‐exome sequencing (WES) and whole‐genome sequencing (WGS) for clinical purposes now allows the identification of multiple pathogenic variants in patients with a rare disease. This occurs even when a single causative gene was initially suspected. We report the case of an 8‐year‐old patient with global developmental delays and dysmorphic features, with a possibly pathogenic variant in three distinct genes. Methods: Trio‐based exome sequencing was performed by IntegraGen SA (Evry, France), on an Illumina HiSeq4000 (Illumina, San Diego, CA, USA). Sanger sequencing was performed to confirm the variants that were found. Results: WES showed the presence of three possibly deleterious variants: KMT2A: c.9068delA;p.Gln3023Argfs*3 de novo, PAX3: c.530C>G;p.Ala177Gly de novo and DLG3: c.127delG;p.Asp43Metfs*22 hemizygous inherited from the mother. KMT2A pathogenic variants are involved in Wiedemann‐Steiner syndrome, and PAX3 is the gene responsible for Waardenburg syndrome. DLG3 variants have been described in a non‐syndromic X‐related intellectual disability. Conclusions: Considering the dysmorphic features and intellectual disability presented by this patient, these three variants were imputed as pathogenic and their association was considered responsible for his phenotype. Dual molecular diagnoses have already been found by WES in several cohorts with an average of diagnostic yield of 7%. This case demonstrates and reminds us of the importance of analyzing exomes rigorously and exhaustively because, in some cases (< 10%), it can explain superimposed traits or blended phenotypes. [ABSTRACT FROM AUTHOR]

We present two unrelated Japanese pedigrees with achondrogenesis type 1b (ACG1B), characterized by prenatally lethal fetal hydrops and severe micromelia. The affected members in these pedigrees carried a common homozygous missense point mutation in solute carrier family 26 member 2 (SLC26A2), a gene associated with ACG1B (NM_000112:c.1987G>A). This loss‐of‐function point mutation causes substitution of glycine 663 with arginine in a highly conserved loop domain of SLC26A2. Interestingly, only a few cases of this mutation have been registered in Japanese genomic databases, and there are no reports of this mutation in any major genomic databases outside Japan. Furthermore, we confirmed the presence of a homozygous stretch of approximately 75 kb surrounding the pathogenic variant. Our findings suggest that this missense point mutation in SLC26A2, which is likely the cause of the ACG1B phenotypes in these unrelated fetuses, is distributed exclusively in Japan. [ABSTRACT FROM AUTHOR]

Environment around Chernobyl recoveres from nuclear accident in 1986 and provides a base for plant adaptation research. Here, the initial platform was established for molecular screening of flax genome affected by radioactive contamination. Flax (Linum usitatissimum L.) cultivated in the radio-contaminated, and remediated experimental field near Chernobyl was investigated. Specifically, two gene isoforms of FAD3 gene, FAD3A and FAD3B, were evaluated using the restriction analysis, and direct sequencing. The aim of the presented study was to identify mutations, and single nucleotide polymorphisms within the FAD3A and FAD3B genes. The investigation was performed more than 25 years after Chernobyl accident. Therefore, the effect of chronic low dose rate irradiation on flax plants was considered. [ABSTRACT FROM AUTHOR]

Objective To develop a sequence-specific polymerase chain reaction (SS-PCR) method for detection of single nucleotide polymorphism (SNP) in GC rich region and analyze the C729T SNP in the exon 3 of estrogen receptor α gene of endometrial cancer (EC). Methods We selected 22 EC and 42 controls. A new GC rich region SS-PCR was developed. The two internal specific primers, identical to the two single strands of double strand DNA, were designed, and the 3' end of the primer coincided with the SNP locus. The PCR extension was controlled by the 3' end primer and the SNP was determined according to the bands of the extension product. The specificity of the extension reaction was increased by introducing a mismatched base at the third position upstream in the 3' end of the SNP specific primer. This method was used to confirm C729T genotypes in the exon 3 SNP, followed by direct sequencing. Results The results showed that the SS-PCR was appropriate for genotyping C729T SNP of exon 3 in human ERα gene. Conclusion A GC rich region SS-PCR method developed can be successfully applied in C729T SNP determination in the exon 3 of estrogen receptor α gene of endometrial cancer. [ABSTRACT FROM AUTHOR]

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Abstract Background Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. Findings In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. Conclusions The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

Background and Aims: Metabolic abnormalities in T2DM (Type 2 diabetes mellitus) include classic manifestations such as impaired insulin secretion, synthesis and peripheral insulin resistance. The intronic variants rs7903146 and rs12255372 of the TCF7L2 (transcription factor 7-like 2) gene are strongly associated with risk of incidence of T2DM and impaired β-cell functions. Studies addressing the early T2DM onset, and early insulin dependence in T2DM patients of south Tamil Nadu are still lacking, and hence the present study focuses in determining the influence of the TCF7L2 polymorphisms in the incidence and disease course in the T2DM patients of south Tamil Nadu. Methods: Anthropometric measurements and biochemical parameters were carried out in early onset (Group A), early onset insulin dependent T2DM patients (Group B) and non-insulin dependent T2DM patients (Group C). PCR, allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the genotype and the impact of the TCF7L2 SNPs in the T2DM disease course. Results: Female T2DM patients with the CT/TT rs7903146 genotype (P = 0.005) and the rs12255372 GT/TT genotype (P = 0.036) exhibited a significantly low mean age for T2DM incidence. Correlation/regression analysis in the T2DM patients revealed that rs12255372 (P = 0.042) is associated with early onset in the Group C patients and the rs7903146 (P = 0.018), rs12255372 (P = 0.026) are associated with insulin dependence in the group B patients. Conclusion: Screening for the TCF7L2 polymorphisms will prevent T2DM incidence and enable life style changes, appropriate therapeutic strategies that would help combat the accelerated disease course in the T2DM patients. [ABSTRACT FROM AUTHOR]

High resolution melting (HRM) assay is a novel technology for the fast, high-throughput, sensitive, post-PCR analysis of genetic mutations. Myeloid differentiation primary response 88 (MYD88) mutations are frequently reported in chronic lymphocytic leukemia (CLL) and confer a worse prognosis. The objective of the present study was to assess the value of HRM analysis for the rapid screening of MYD88 mutations in patients with CLL. Genomic DNA samples were extracted from the bone marrow of 129 newly diagnosed patients with CLL. A plasmid with an MYD88-L265P mutation was constructed, and the p.L265P substitution, which is the predominant MYD88 mutation in CLL, was detected using HRM analysis and direct sequencing. The plasmid pCMV-MYD88-L265P-Mu was successfully constructed as a positive control, and was verified by direct sequencing. The normalized and shifted melting curves of 6/129 (4.65%) samples were clearly different from those of other patients by HRM analysis. In addition, the 794T>C mutation in MYD88 was identified in 6 (4.65%) patients by direct sequencing. Sensitivity evaluation revealed that the HRM assay had a higher sensitivity (to 1% dilution) than direct sequencing, in addition to being convenient and time-saving. The MYD88 p.L256P mutation has been implicated to be associated with adverse prognosis in CLL. HRM analysis has the potential to be a routine prescreening technique to identify the MYD88 p.L256P mutation and may facilitate the clinical treatment of CLL. [ABSTRACT FROM AUTHOR]

Objectives Rett syndrome is an X linked dominant neurodevelopmental disorder which almost exclusively affects females. The syndrome is usually caused by mutations in MECP2 gene, which is a nuclear protein that selectively binds CpG dinucleotides in the genome. Materials & Methods To provide further insights into the distribution of mutations in MECP2 gene, we investigated 24 females with clinical characters of Rett syndrome referred to Alzahra University Hospital in Isfahan, Iran during 2015-2017. We sequenced the entire MECP2 coding region and splice sites for detection of point mutations in this gene. Freely available programs including JALVIEW, SIFT, and PolyPhen were used to find out the damaging effects of unknown mutations. Results Direct sequencing revealed MECP2 mutations in 13 of the 24 patients. We identified in 13 patients, 10 different mutations in MECP2 gene. Three of these mutations have not been reported elsewhere and are most likely pathogenic. Conclusion Defects in MECP2 gene play an important role in pathogenesis of Rett syndrome. Mutations in MECP2 gene can be found in the majority of Iranian RTT patients. We failed to identify mutations in MECP2 gene in 46% of our patients. For these patients, further molecular analysis might be necessary. [ABSTRACT FROM AUTHOR]

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells. [ABSTRACT FROM AUTHOR]

Background: Nontuberculosis Mycobacterium (NTM) is the most common bacterium transferred to human from cow milk and products. The presence of these bacteria in the cow's milk stands as a public health concern particularly among those individuals consuming raw milk and dairy products. In this study the determination of 16S rRNA gene sequence method was used in order to evaluate the amount of Non tuberculosis Mycobacterium incidence and determine its types in milk samples gathered from Isfahan province. Methods: The cetyltrimethylammonium bromide (CTAB) and phenol chloroform were used for DNA extraction in isolates recognized as Non tuberculosis Mycobacterium, then using designed primers, 16S rRNA polymerase chain reaction was done and the types were determined after sequencing. Findings: 9 cultures, out of 119 samples gathered from Isfahan farms, were recognized positive in which 8 samples had M. fortuitum and one of them was M. gordonae. Conclusion: NTM incidence was 8.6% in evaluated milk samples. By using polymerase chain reaction (PCR) with 16SrRNA and sequencing, the fortuitum isolate type I incidence was 88.9% and Gordonae type III incidence was 11.1%. PCR method and sequencing of 16S rRNA gene which were used in this study are able to recognize isolates up to 100%.

Dyskeratosis congenita (DKC) is a rare inherited disease that is characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. DKC is caused by an abnormality in a component of the telomerase and shelterin complexes. TINF2 encodes a protein in the shelterin complex and TERC encodes a component of the telomerase complex. Mutations of both genes have been associated with DKC. This study examined mutations in TINF2 and TERC by direct DNA sequencing in a Thai patient with DKC. We identified a novel mutation (c.845G>T) that is located in exon 6 of TINF2 and changes an arginine to leucine (Arg282Leu). This identified mutation could be applied for molecular genetic diagnosis and genetic counseling of patients with DKC.

Exploring the genetic basis for idiopathic congenital nystagmus is critical for improving our understanding of its molecular pathogenesis. In the present study, direct sequencing using gene specific primers was performed in order to identify the causative mutations in two brothers from a Chinese family who had been diagnosed with idiopathic congenital nystagmus. A comprehensive ophthalmological examination, including eye movement recordings, fundus examination, and retinal optical coherence tomography imaging was also conducted, to characterize the disease phenotype. The results revealed that the two brothers exhibited clear signs of nystagmus without any other ocular anomalies. Direct sequencing revealed a G to T transition (c.886G>T) in exon 9 of the four-point-one, ezrin, radixin, moesin domain-containing 7 (FRMD7) gene, which resulted in a conservative substitution of glycine to cysteine at codon 296 (p.G296C), leading to idiopathic congenital nystagmus in the two affected brothers. c.886G>T is a novel idiopathic congenital nystagmus-inducing mutation in the FRMD7 gene. This finding expands the spectrum of known gene mutations in idiopathic congenital nystagmus, and may be useful for faster gene diagnosis, prenatal testing, the development of potential gene therapies, and for improving the understanding of the molecular pathogenesis of idiopathic congenital nystagmus. [ABSTRACT FROM AUTHOR]

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos. [ABSTRACT FROM AUTHOR]

Background and objective Epidermal growth factor receptor (EGFR) inhibitors are mainly used for the targeted therapy of non-small cell lung cancer (NSCLC). Therefore, EGFR mutations should be detected to treat lung cancer. The aim of this study is to determine the detection rate of NSCLC in patients with EGFR gene mutations by conducting direct sequencing and ARMS assay. Methods A total of 451 patients who were diagnosed with NSCLC between April 2012 and June 2013 participated in this study. Gene mutation was detected in the exon of EGFR 18 to 21 by direct sequencing and ARMS assay. Results All of the 451 cases of NSCLC were subjected to direct sequencing and ARMS assay. Using both techniques, we detected the same EGFR mutation in 127 cases and different EGFR mutations in 5 cases, but no mutations were detected in 186 cases. In direct sequencing alone, EGFR mutation was detected in 50 cases. In ARMS assay alone, EGFR mutation was detected in 83 cases. The mutation rates of direct sequencing and ARMS assay were 40.4% and 47.7%, respectively. Therefore, the mutation detection rate of ARMS assay was significantly higher than that of direct sequencing (P

Bovine parainfluenza 3 virus (PI3) causes respiratory infections in cattle and sheep with great economic losses in livestock. The aim of this investigation was to determine the significance of molecular methods in the identification of isolated strains of PI3 virus. Twenty cattle nasal swabs were analyzed for the presence of PI3 using the standard virology method of virus isolation in MBDK cell line and virus neutralization test. The identification of isolated strains was confirmed by RT-PCR and method of direct sequencing with primers for PI3 fusion (F) protein gene. PI3 virus was isolated and identified in four nasal swabs using the standard virology method and RT-PCR. The analysis of nucleotide sequences of isolated PI3 strains showed high similarity with sequences isolated from cattle in Asia. Our results showed that molecular methods are very useful in the diagnosis of PI3 infections as well as for the identification and characterization of PI3 strains in Serbia. [Projekat Ministarstva nauke Republike Srbije, br. 31008 i br. 175073]

An epidemic caused by influenza A (H7N9) virus was recently reported in China. Deep sequencing revealed the full genome of the virus obtained directly from a patient’s sputum without virus culture. The full genome showed substantial sequence heterogeneity and large differences compared with that from embryonated chicken eggs.

This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB , 315 in katG , 18 in rpsL , and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA , and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb. [ABSTRACT FROM AUTHOR]