Trend of human papillomavirus genotypes in cervical neoplasia observed in a newly developing township in Yangon, Myanmar.

Persistent infection with oncogenic types of human papillomavirus (HPV) is the most important risk factor associated with cervical cancer. This study detected the oncogenic HPV genotypes in cervical neoplasia in relation to clinicopathological findings using a cross-sectional descriptive method in 2011 and 2012. Cervical swabs and colposcopy-directed cervical biopsy tissues were collected from 108 women (median age 45 years;range 20-78) showing cervical cytological changes at Sanpya General Hospital, Yangon, Myanmar. HPV DNA testing and genotyping were performed by polymerase chain reaction and restriction fragment length polymorphism. HPV was identified in women with cervical intraepithelial neoplasia (CIN) 1 (44.4%), CIN2 (63.2%), CIN3 (70.6%), and squamous cell carcinoma (SCC) (74.1%). The association between cervical neoplasia and HPV positivity was highly significant (p＝0.008). Most patients infected with HPV were between 40-49 years of age, and the youngest were in the 20- to 29-year-old age group. The most common genotype was HPV 16 (65.6%) with the following distribution:70% in CIN1, 41.7% in CIN2, 91.7% in CIN3, and 60% in SCC. HPV-31 was the second-most frequent (21.9%):30% in CIN1, 33.3% in CIN2, 8.3% in CIN3, and 15% in SCC. The third-most frequent-genotype was HPV-18 (7.8%):8.3% in CIN1, and 20% in SCC. Another genotype was HPV-58 (4.7%):16.7% in CIN1 and 5% in SCC. The majority of CIN/SCC cases were associated with HPV genotypes 16, 31, 18, and 58. If oncogenic HPV genotypes are positive, the possibility of cervical neoplasia can be predicted. Knowledge of the HPV genotypes distribution can predict the effectiveness of the currently used HPV vaccine.

Prevalence of high-risk human papillomavirus (HR-HPV) infection among women with normal and abnormal cervical cytology in Myanmar.

This study aimed to determine the prevalence of normal and abnormal cervical cytology in women who attended the cervical cancer screening clinic of the Department of Medical Research in Lower Myanmar, and to determine the proportion of high-risk (HR) human papillomavirus (HPV) infection and HPV genotypes in women with normal and abnormal cervical cytology. A total of 1,771 women were screened from 2010 to 2011. Among them, 762 women (43.0%) had a normal smear, and 866(48.9%) and 87 (4.9%) were diagnosed with inflammatory smears and atypical squamous cells of undetermined significance (ASCUS), respectively. Diagnoses of low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) numbered 42 (2.3%) and 11 (0.6%) respectively. Three cases of squamous cell carcinoma (SCC) (0.2%) were detected. Cervical swabs were collected from 96 women with abnormal cervical cytology and 20 with normal cytology. HR-HPV DNA testing was performed by polymerase chain reaction (PCR) with pU1M/pU2R primers. HR-HPV were identified in 35.5% (22/62) of inflammatory smears, 60% (6/10) of ASCUS, 86.7% (13/15) of LSIL, 50% (3/6) of HSIL, 100% (3/3) of SCC and 5% (1/20) of normal cytology. In PCR-positive cases, HPV genotyping was analyzed by the cleaved amplification polymorphism method. The most prevalent HPV genotypes were HPV-16 (60.4%) followed by HPV-31 (14.6%), HPV-18 (12.5%) and HPV-58 (12.5%). Women with abnormal cervical cytology were 10 times more likely to be HR-HPV positive than those with normal cytology (p=0.0001). This study suggests that the implementation of a cervical cytology screening program and routine vaccination against HPV in preadolescent and adolescent groups are needed to reduce the burden of HPV-associated cervical cancer.

[Simple and rapid analysis of beta-thalassemia mutation by sequence-specific amplification].

This study was done to detect and diagnose beta-thalassemia (beta-Thal) gene quickly. We applied sequence specific Amplification (SSA) method to the analysis. 13 kinds of beta-Thal and two kinds of hemoglobin variants were able to detect under the same PCR condition. These mutations were found frequently in ten countries of Asian region (the southern part of China, Vietnam, Cambodia, Thailand, Myanmar, Malaysia, Singapore, Indonesia, Pakistan, India), and 15 kinds in total (-28CapA-->G, CD5-CT, CD8/9+-G, CD15G-->A, CD17A-->T, IVSI-1G-->T, CD41/42-4del, CD16-C, CD26G-->A(betaE), IVSI-5G-->C, CD35C-->A, CD71/72 +A, CD6A-->T (betaS), -619del, IVSII-654C-->T). More than 80% of patients are included in these mutations. To make the reagents a kit, the procedure became simple and rapid. DNA was extracted by salting out method. The PCR product was detected by polyacrylamide gel electrophoresis and silver staining. The confirmation of the variant was done by the PCR-direct sequencing method. It took approximately six hours for PCR reaction, electrophoresis and staining. This method made us to detect and diagnose beta-Thal in one day.

[Case of deletion type beta(0)-thalassemia found in an Asian (Malaysian) family living in Japan].

Hb and gene analyses of a Malaysian mother and her two daughters with microcytic anemia living in Japan were performed. Hb analyses of their hemolysates by IEF and DEAE-HPLC revealed high values of Hb A2 and HbF, but abnormal Hbs such as Hb E and Hb Constant Spring, which cause beta- and alpha-thalassemia traits, were not detected. From these data, they were suspected to be beta-thalassemia carriers. The thalassemic mutations commonly found in the Asian area by ARMS and nucleotide sequencing methods were not detected, and the frameworks of the beta-globin gene and the haplotypes of the beta-like globin gene cluster between the mother and daughters were not identical. These results led us to conclude that there was a beta(0)-thalassemia mutation with a large deletion from the beta-globin gene beyond the 3'beta/BamHI polymorphic site 3' downstream to the beta-globin gene. However, the range of the deletion from the beta-like globin gene cluster has not yet been completed in detail. Recently, there have been many foreigners mainly from Asian countries in Japan. We may encounter people with the rare type thalassemic mutation described in the text besides the mutations frequently found in Asian countries.

Hb Constant Spring [alpha 142, Term-->Gln (TAA>CAA in alpha2)] in the alpha-thalassemia of anemic patients in Myanmar.

Hb Constant Spring (Hb CS), the gene (alpha(CS)) of which arises from a point mutation in the termination codon of the alpha2-globin gene, is the most prevalent variety of nondeletional alpha-thalassemia (alpha-thal) in Asian populations. It is a major cause of Hb H disease in compound heterozygotes who have Hb CS combined with a duplicated alpha gene deletion (--/alpha(CS)alpha), and it tends to be more severe than Hb H disease which is caused by a triple alpha gene deletion (--/-alpha). Hb CS is often missed by routine electrophoresis but not by polymerase chain reaction (PCR) methods. During alpha-thal screening and genotyping of 235 patients diagnosed by laboratory tests hemoglobin (Hb), MCV, MCH and Hb H inclusion bodies] using the gap-PCR method, 175 patients were diagnosed to be carriers of an alpha-thal gene, genotypes of which were 133 alpha-thal-2, 34 alpha-thal-1 (including one only by laboratory test) and eight with Hb H disease. Detection of the alpha(CS) gene for the carriers of alpha-thal-1 and Hb H disease was done by the mismatched PCR-RFLP (restriction fragment length polymorphism) method and the alpha(CS) gene was found in the homozygous state in an alpha-thal-1 patient and a single gene form in two Hb H disease patients. These genotypes were characterized by the PCR-sequencing method. These patients clinically presented the aspects of Hb H disease and of a homozygote form of alpha-thal-1. The description of the alpha(CS) gene in Myanmar is of great value in the development of an effective procedure for prenatal diagnosis of Hb Bart's hydrops fetalis syndrome.

Multiple cytokines are involved in the early events leading to the Alzheimer's disease pathology.

It is likely that neuroinflammation begins well before detectable cognitive impairment in Alzheimer's disease (AD) occurs. Clarifying the alterations occurring prior to the clinical manifestation of overt AD dementia may provide valuable insight into the early diagnosis and management of AD. Herein, to address the issue that neuroinflammation precedes development of AD pathology, we analyzed cytokine expression profiles of the brain, with focus on non-demented control patients with increasing AD pathology, referred to as high pathology control (HPC) cases, who provide an intermediate subset between AD and normal control cases referred to as low pathology control (LPC) cases. With a semi-quantitative analysis of cytokine mRNA, among 15 cytokines and their related molecules tested, we found the involvement of eight: interleukin-1(IL-1) receptor antagonist (IL-1ra), IL-1 converting enzyme (ICE), IL-2, IL-6, IL-8, tumor necrosis factor (TNF) α, macrophage-colony stimulating factor (M-CSF) and transforming growth factor (TGF) ß1 during the development from LPC to HPC, while decreases in IL-1ra, IL-8, MCP-1 and TNFα, and an increase in TACE were implicated in the later development from HPC to AD. These findings indicate that neuroinflammation precedes the clinical manifestation of overt dementia, rather than being involved at the later stages of AD.

A case of alpha-thalassemia-2 associated with pulmonary infarction.

Pulmonary infarction is an entity of medical significance that develops concurrently in beta-thalassemia but not in alpha-thalassemia. The reason for this difference is yet to be elucidated. We have evaluated a 21-year-old male alpha-thalassemia-2 patient who had profound microcytic anemia and pulmonary infarction. Analysis of the alpha-globin gene revealed -alpha3.7/alpha alpha genotype. His mother also had the same heterozygous gene deletion, though she had neither anemia nor pulmonary infarction. Since the patient had no other predisposition to pulmonary infarction, it is suggested that there is a close etiologic relationship between morphologic abnormality of the erythrocytes caused by alpha-thalassemia-2 and development of pulmonary infarction.

Common alpha-thalassemia deletions in transfusion-dependent thalassemia patients in the Southeast Asia region of Myanmar.

Screening of 3 common alpha-thalassemia (thal) deletions (-alpha3.7, -alpha4.2 and --SEA) in Southeast Asia was done by polymerase chain reaction in 170 unrelated Myanmar thal patients receiving transfusions. Thal deletions were detected in 27 patients (15.9%) as: (1) alpha-thal-2 (-alpha3.7/alphaalpha) in 12 heterozygous or hemoglobin (Hb) E-beta-thal cases; (2) alpha-thal-1 in 7 patients (2-alpha3.7/-alpha3.7 and 5 --SEA/alphaalpha); and (3) Hb H (-alpha3.7/--SEA) in 8 patients. The latter 15 alpha-thal-1 and Hb H patients had no beta-thal mutations and represented 8.8% of the overall patients seeking transfusion for refractory anemia in Myanmar. This is the first description of alpha-thal in Myanmar from the molecular aspect, and its clinical and racial heterogeneity are described and discussed.

High incidence of 3-thalassemia, hemoglobin E, and glucose-6-phosphate dehydrogenase deficiency in populations of malaria-endemic southern Shan State, Myanmar.

Samples from 916 members of various ethnic groups from malaria-endemic southern Shan State, Myanmar, were analyzed for 3-thalassemia (3-thal), 3-thalassemia (3-thal), abnormal hemoglobin variants, and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of these subjects, 530 (57.9%) were found to have at least one of these red cell genetic disorders. The overall frequencies for the various red cell genetic disorders were as follows: 3-thal, 37.5% (343/916); hemoglobin E (Hb-E), 20.3% (186/916); G6PD-Mahidol, 17.5% (160/916); and 3-thal, 0.3% (3/916). The frequencies of combined disorders were 6.9% (63/ 916) for 3-thal/Hb-E, 5.7% (52/916) for 3-thal/G6PD-Mahidol, 2.8% (26/916) for Hb-E/G6PD-Mahidol, 1.1% (10/916) for 3-thal/Hb-E/G6PD-Mahidol, and 0.1% (1/916) for 3-thal/3-thal/G6PD-Mahidol. Of the various ethnic and non-ethnic groups, the Bamar population showed the highest frequencies of 3-thal (56.9%, 177/311), Hb-E (28.3%, 88/311), and G6PD-Mahidol (21.2%, 66/311) (all duplicated and triplicated cases were included). In addition, 2 new mutations, an 3 gene triplication (/333(anti3.7); 0.2%, 2/916) and Hb-Neapolis (0.1%, 1/916), were detected. Our results showed that race was the dominant factor affecting the frequencies of red cell genetic disorders in malaria-endemic areas of Myanmar.

Two children with thalassemia identified during screening for anemia in junior high school.

We present two Japanese students with thalassemia identified during screening for anemia in their junior high school. Blood test results revealed marked hypochromic and microcytic erythrocytosis in one patient and microcytic anemia in the other. Both cases showed a mean corpuscular volume/red blood cell (MCV/RBC) ratio less than 13. Their beta/alpha synthesis ratio was elevated. Deletion of psialpha2, psialpha1, alpha2, alpha1 and theta1 genes in the alpha-globin gene clusters were noted in the first case. This pattern of gene deletion was consistent with heterozygous alpha-thalassemia 1 of the Southeast Asian type. On the other hand, an increased hemoglobin A2 level and reduced beta/alpha synthesis ratio were found in the second case. Direct cloning and DNA sequencing identified a point mutation (guanine to adenine) at position 1 of intervening sequence II in the beta-globin gene (IVS II-1 G-->A). These results suggest that this patient had heterozygous beta0-thalassemia. Diagnosis of thalassemia should be confirmed by molecular analysis in cases with microcytic anemia or hypochromic microcytosis with a MCV/RBC ratio of 13 or less.

Pseudoreticulocytosis in a patient with hemoglobin Köln due to autofluorescent erythrocytes enumerated as reticulocytes by the Cell-Dyn 4000.

Pseudoreticulocytosis in a 25-year-old female patient with hemoglobin Köln is reported. The abnormal hemoglobin, hemoglobin Köln (beta chain, Val98-->Met), had previously been confirmed in the patient at the age of 21 years, as well as in her mother, by polymerase chain reaction-based direct sequence analysis of the beta globin gene. The patient underwent splenectomy at the age of 22 years. On her admission to our hospital for treatment of an immunoglobulin A nephropathy, an analysis by an automated hematology analyzer, the Abbott Cell-Dyn 4000 (CD4000), reported a marked reticulocytosis. Staining by the Brecher method with new methylene blue indicated a moderate reticulocytosis (5.7%) of a lesser extent than that indicated by the CD4000 (51.1%). The frequencies of red blood cells (RBC) with Pappenheimer bodies (13.8%), Heinz bodies (32.7%), and Howell-Jolly bodies (0.3%) were increased. The CD4000 detects RBC with RNA fluorescently stained with CD4K530 as reticulocytes. Autofluorescence of RBC with hemoglobin Köln, as we demonstrated by flow cytometry and fluorescent microscopy, was considered to have caused the pseudoreticulocytosis on the fully automated reticulocyte enumeration by the CD4000.

Evaluation of automated glycohemoglobin analyzer HLC-723G7 beta-thalassemia analysis mode.

We evaluated the new automated glycohemoglobin analyzer HLC-723G7 (G7) intended for beta-thalassemia diagnosis to determine correlation of hemoglobin (Hb) F and Hb A2 quantitation with the Variant beta-Thalassemia Short Program and to evaluate the performance of the G7 analyzer. Two hundred fifty EDTA blood samples with Hb A, A2, and F by Variant were analyzed for Hb A2 and Hb F by G7 device. Recovery, precision, and interference of the G7 analyzer were studied. The R2 values for Hb A2 and Hb F were 0.963 and 0.640, respectively. Recovery of Hb A2 and Hb F of low and high control samples ranged from 96% to 100% and 91% to 101%, respectively. The coefficients of variation (CVs) of intraassay precision ranged from 1.3% to 3.9% for Hb F and 0.0 to 1.9% for Hb A2. The CVs of interassay precision ranged from 1.9% to 5.6% for Hb F and 2.5% to 3.0% for Hb A2. The CVs for Hb F in both conjugated bilirubin and chyle substance groups were higher than the CVs for Hb A2. This evaluation revealed very good correlation of Hb A2 measurement between the G7 and the Variant devices. The performance evaluation demonstrated good recovery, precision, and low interference of both Hb A2 and Hb F measurements.

Hb Bristol-Alesha presenting thalassemia-type hyperunstable hemoglobinopathy.

Hemoglobin (Hb) Bristol-Alesha is caused by a GTG --> ATG mutation at codon 67 in the Hb beta chain, resulting in abnormal beta globin chains with mutated molecules from normal beta67 valine (Val) to beta67 methionine (Met) or beta67 aspartate (Asp). We describe a Japanese child with this rare hemoglobinopathy and a very unstable Hb molecule phenotype. The diagnosis of hemolytic anemia was made when the patient was 6 months of age. Development of marked splenomegaly necessitated red blood cell transfusions twice a month. After splenectomy when the patient was 4 years of age, laboratory findings of hemolytic anemia became more prominent. Specific abnormal Hb molecules initially were not detected, and the alpha/beta globin synthesis ratio was abnormal at 2.22. After splenectomy, we identified the presence of abnormal beta-globin chains with a beta67Val:beta67Met:beta67Asp molecule ratio of 74:11:15. We speculate that the high fraction of the beta67Met molecule in this patient, compared with that in previously reported cases, caused extreme Hb instability, which resulted in thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.

Neonatal hyperbilirubinemia in Japanese neonates: analysis of the heme oxygenase-1 gene and fetal hemoglobin composition in cord blood.

Neonatal hyperbilirubinemia is frequent and severe in Japanese infants. Although the G71R mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene is associated with severe neonatal hyperbilirubinemia in this population, it accounts for only half of the neonates with severe hyperbilirubinemia. It was suggested that increased bilirubin production would also be associated with severe neonatal hyperbilirubinemia in Japanese infants. To elucidate the genetic factors causing severe hyperbilirubinemia in these patients, we studied two notable factors associated with bilirubin production: heme oxygenase-1, a key enzyme of heme metabolism, and fetal Hb composition, a factor possibly associated with heme load in neonates. We first determined the sequences of promoter and all coding regions of the heme oxygenase-1 gene in Japanese neonates who had undergone phototherapy, but found no mutation except for the polymorphic (GT)n repeats in the promoter region. These repeats modulate the transcription of the heme oxygenase-1 gene, and the longer repeat sequences are known to reduce the transcription. We detected a significant difference in the allele frequencies of each number of (GT)n repeats between Japanese and German populations. However, we could not find a relation between those polymorphisms and neonatal hyperbilirubinemia. We next analyzed the state of Hb switching of the gamma- to beta-globin chain and the phenotype of gamma-globin chain isoforms in cord blood. We found no relation between fetal Hb composition and neonatal hyperbilirubinemia. Further studies are required to elucidate genetic or environmental factors in neonatal hyperbilirubinemia in Japanese infants.

Hb Nishinomiya [Leu-Gly-inserted between codons 69(E13) and 70(E14) of beta]: a novel unstable hemoglobin with reduced oxygen affinity found in a patient with spherocytic hemolysis.

Substantially decreased oxygen saturation levels were incidentally detected by pulseoxymetry in a patient with spherocytic hemolysis who was undergoing laparoscopic splenectomy. Molecular analysis revealed that he was carrying hemoglobin (Hb) Nishinomiya, a novel Hb variant [Leu-Gly-inserted between codons 69(E13) and 70(E14) of beta]. Amino acid substitutions around positions 70-73(E13-17) of the beta chain are likely to change stability and oxygen affinity, as has been demonstrated in several Hb variants including Hb Seattle. The apparent substitution of the amino acid residues in the heme pocket of the beta chain explains the decreased stability and oxygen affinity of Hb Nishinomiya.

A wider molecular spectrum of beta-thalassaemia in Myanmar.

Two hundred and nine beta-thalassaemia (beta-Thal) alleles of 158 unrelated Myanmar patients (107 HbE-beta-Thal; 51 beta-Thal major) were analysed for beta-globin gene mutations. Amplification refractory mutation system (ARMS) characterized six beta-thal mutations known to Myanmar [betaIVSI-1(G-->T), codon 41/42(-TCTT), betaIVSI-5(G-->C), codon 17(A-->T), betaIVS II-654(C-->T), and -28 Cap (A-->G)] in 166/209 (79.4%) alleles. DNA sequencing of 24 alleles from 43 ARMS-negative samples (20.6%) identified an additional 12 new mutations, to produce a total of 18 different mutations. Nineteen alleles (9.1%) remained for further characterization. The molecular spectrum of Myanmar beta-Thal is wider and more heterogeneous than previously reported.