[Molecular analysis of alpha globin gene deletions among patients with microcytic hypochromic anemia in Kermanshah-Iran]

The majority of alpha-thalassemi mutations are deletions of one or both alpha- globin genes. Since the Iranian populaion is a mixture of different ethnic groups, frequency and distribution of globin mutations in various regions of the country need to be clarified. The aim of this study was to determine the common alpha globin gene deletions among individuals with hypochromic microcytic anemia in Kermanshah province. Following the initial evaluation, 92 patients [47 women and 45 men] were found as microcytic hypochromic [MCV < 80 fl and MCH< 27 pg] anemia and selected for this study. All samples were analyzed for detection of four alpha-gene deletions [-alpha3.7,-alpha4.2,- [alpha] 20.5 and --MED] by GAP-PCR technique. After amplification, 10microl of PCR product was electrophoresed through 1.2% agarose gel and bands were visualized by staining gel in ethidium bromide solution and photographed under a UV transilluminater. 45 patients had -alpha 3.7 single gene deletion. In patients with -alpha3.7 deletion, in both homozygous and heterozygous states, MCH was lower than normal ranges. However, the percent of HbA2 was in normal range. In this study, other common deletional mutations, including - [alpha]20.5, -alpha4.2 and --MED were not found. The results of persent study showed that the frequency of -alpha3.7 single gene deletion among patients with microcytic hypochromic anemia in Kermanshah province was 48.9%

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Identificação e caracterização de variantes novas e raras da hemoglobina humana: [revisão] / Identification of characterization of novel and rare variants of human hemoglobin: [review]

As anormalidades estruturais da hemoglobina estão entre as doenças genéticas mais comumente encontradas nas populações humanas. O Laboratório de Hemoglobinopatias do Departamento de Patologia Clínica da Faculdade de Ciências Médicas da Universidade Estadual de Campinas - Unicamp, localizado em Campinas, no estado de São Paulo, região Sudeste do Brasil, realizou, em seus 27 anos de existência, cerca de 130.000 diagnósticos. Entre as variantes estruturais detectadas, as hemoglobinas S, C e D-Punjab foram, como esperado, as mais freqüentes, porém um número expressivo de outras hemoglobinas anômalas, novas e raras, também foi encontrado. Esses achados estão sumarizados no presente artigo.

Hemoglobin structural abnormalities are among the most commonly found human genetic diseases. The Laboratory of Hemoglobinopathies in the Clinical Pathology Department of the Medical Sciences School of the State University in Campinas - Unicamp, São Paulo, Southeastern Brazil, carried out, in its 27 years of activity, about 130,000 diagnoses. As expected, hemoglobins S, C and D were the most frequently observed variants, but an expressive number of other abnormal, novel and rare hemoglobins, was also detected. These findings are summarized in the present article.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2 percent) patients had CAR/Ben genotype; 36 (28.8 percent) Ben/Ben; 18 (14.4 percent) CAR/CAR; 2 (1.6 percent) CAR/Atypical; 2 (1.6 percent) Ben/Cam; 1 (0.8 percent) CAR/Cam; 1 (0.8 percent) CAR/Arab-Indian, and 1 (0.8 percent) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

Human erythroid progenitors from adult bone marrow and cord blood in optimized liquid culture systems respectively maintained adult and neonatal characteristics of globin gene expression

In vitro suspension culture procedures for erythroid progenitor cells make it possible for us to obtain large cultures of erythrocyte populations for the investigation of globin gene switching. In this study we aimed to establish optimized culture systems for neonatal and adult erythroblasts and to explore the globin expression patterns in these culture systems. To culture CD34+ cells purified from human umbilical cord blood (CB) and adult bone marrow (BM), we respectively replaced the fetal bovine serum (FBS) with human cord serum and human adult serum. These CD34+ cells were then induced to erythroid differentiation. All the globin mRNA (including alfa-, xi-, vita-, gama-and epsilón-globin), the hemoglobin (Hb)-producing erythroid cells and the cellular distribution of fetal hemoglobin (Hb F) were identified during the culture process. The results showed that the globin expression pattern during erythroid differentiation in our culture systems closely recapitulated neonatal and adult patterns of globin expression in vivo, suggesting that our specially optimized culture systems not only overcame the higher Hb F levels in the BM-derived CD34+ culture in FBS-containing medium but also eliminated the disadvantages of low cell proliferation rate and low globin mRNA levels in serum-free medium.

Molecular characterization of alpha-thalassemia in the Mexican population / Caracterización molecular de talasemia alfa en una población mexicana

Background. α-Thalassemia (α-Thal) has been poorly characterized at the molecular level in Mexico. Methods. 106 consecutive individuals identified in Laboratorios Clínicos de Puebla, with either hypochromia (MCH < 24 pg) and/or microcytosis (MCV < 75 fl in women or < 80 fl in man), without iron deficiency, with or without anemia were investigated in this study, along a 16 month-period, α and β-Thal were looked for, the former were characterized at the molecular level. Results. Out of the 106 consecutive cases with hypochromia and/or microcytosis and normal levels of protoporphyrin zinc complex, 48 cases (45.3%) had thalassemia (37 cases of β-Thal and 11 cases of α-Thal), whereas in 58 cases (54.7%) a definite diagnosis could not be established. Of the α-Thal cases, 8 were heterozygous and two were homozygous for the -α3.7 deletion, whereas one case was heterozygous for the α2Hph allele. Conclusions. Only few of the α-Thal alleles tested were found, thus the α-thalassemic mutations, present in the studied population, seem to be rather heterogeneous.

Antecedentes. En México, la α-talasemia (α-Thal) ha sido pobremente caracterizada a nivel molecular. Mátodos. Se estudiaron 106 individuos consecutivos identificados en los Laboratorios Clínicos de Puebla, con hipocromia (CMH < 24 pg) y lo microcitosis (VCM < 75 fl en mujeres o 80 fl en hombres), sin deficiencia de hierro, con o sin anemia, durante un periodo de 16 meses. Se investigaron α y β-Thal; las primeras fueron caracterizadas a nivel molecular. Resultados. De los 106 casos consecutivos estudiados con hipocromia y/o microcitosis, y niveles normales del complejo de protoporfirina-cinc, 48 casos (45.3%) tenían talasemias (37 de ellos β-Thal y 11 α-Thal), mientras que en 58 casos (54.7%) no pudo establecerse un diagnóstico definitivo. De las talasemias α, ocho casos eran heterocigotos y dos homocigotos para la deleción -α3.7, mientras que sólo un caso resultó heterocigoto para el alelo α2Hph. Conclusiones. De los alelos α-Thal estudiados sólo se encontraron algunos, de lo que se infiere que en la población estudiada esas mutaciones parecen ser bastante heterogáneas.

Gene amplification using DNA from human spot urine samples.

The purpose of this study was to test the amplification of DNA from human urinary sediment for molecular epidemiological studies. Twenty-six urine samples were obtained from healthy volunteers. Polymerase chain reactions (PCR) for methylenetetrahydrofolate reductase (MTHFR), beta-globin, and N-acetyltransferase 2 (NAT2) was conducted using genomic DNA isolated from the urine. The MTHFR and beta-globin genes were amplified successfully from all the urine DNA samples while the NAT2 gene was amplified in 88.5% of cases. The median yield of DNA was 0.28 microg from the 10 ml urine samples, sufficient amounts of DNA being contained in urinary sediments for amplification of all three genes. This result indicates that urine can be used as a DNA source for PCR-based molecular epidemiological studies.

Análise dos haplótipos do gene da betaS-globina no Ceará / Analysis of betaS-globin gene haplotypes in Ceará, Brazil

No presente trabalho abordam-se vários aspectos relacionados à natureza molecular da anemia falciforme (AF), desordem hematológica de caráter hereditário. A descoberta do polimorfismo do DNA no grupamento do gene betaS, originando diferentes haplótipos da doença, permitiu ampliar o conhecimento em torno da heterogeneidade clínica observada nos pacientes falcêmicos nas mais diversas regiões do mundo. Analisaram-se os diferentes haplótipos e seus parâmetros hematológicos, presentes em um grupo de 22 pacientes naturais e procedentes do estado do Ceará. A distribuição das freqüências dos haplótipos encontrados foi de 55,9 por cento para Benin; 41,2 por cento para República Centro-Africana (CAR); e de 2,9 por cento para o haplótipo Senegal. Esses dados, em comparação com os demais estudos realizados no Brasil, mostram associação entre os seus valores para um alfa de 5 por cento (p < 0,05). Os resultados obtidos possibilitam um entendimento mais completo da fisiopatologia desta doença e da sua complexidade clínica em nosso meio, bem como permitem um conhecimento mais amplo da AF em nosso país.

Estudio comparativo de detección del virus papiloma humano (VPH) en muestras citológicas y biopsias de cuello uterino / Detection of human papilloma virus in cytologic samples or biopsies of the cervix

Background: The DNA quality for the detection and typification of Human Papilloma Virus (HPV) varies according to the type of sample in which it is studied. This may affect the sensitivity and specificity of the method employed. Aim: To study the yield and specificity of HPV detection and typification in uterine cervical samples obtained by cervical brushing fresh frozen and formalin fixed tissue. Material and methods: Cytological, fresh frozen and fixed tissue samples from 44 patients (nine with low grade lesions and 35 with high grade lesions) were studied. Nested polymerase chain reaction for genes E6/E7 was used to typify HPV groups as low risk or high risk. Results: Of all the cytological samples obtained by brushing 84% of fixed samples and 43% of fresh frozen samples were positive for HPV. The yields were significantly different when comparing brushing with fixed tissue or fresh frozen tissue and fixed tissue with fresh frozen tissue (p <0.05). The frequency of high risk HPV fluctuated from 41% in fresh frozen tissue to 98% in cytological samples. Low risk HPV was detected in 16% of fresh frozen tissue and 68% of cytological samples. A mixed infection was detected in 66%, 41% and 14% of cytological, fresh frozen and fixed tissue samples respectively. Conclusions: Cytological samples obtained by brushing had the highest yield for the detection of cervical infection with HPV.

Hemoglobin E-beta thalassemia: factors affecting phenotype.

The phenotype of E-beta-thalassemia is affected by several genetic factors. The aim of this study was to analyze severity of E-beta-thalassemia and correlate with HbE, HbF, E/F ratios, beta-mutation and Xmn I polymorphism. Thirty cases of E-beta-thalassemia (23 with childhood onset) were studied. HbE levels were quantitated by HPLC. Xmn1 polymorphism and beta-mutations were studied by PCR-RFLP and ARMS respectively. Commonest features were pallor (100%), splenomegaly (74%), and hepatomegaly (65%), 43% (10/23) were on regular transfusions at diagnosis. One case presented with paraplegia. Patients heterozygous for Xmn I polymorphism (+/-) had later onset (>3 yrs) compared to homozygous (-/-) absence (0.5-2.8 yrs). Most (69.6%) showed beta-mutation IVS 1-5 (G-->C). Negative correlation was found between age of onset and HbE. Thus, presentation is similar to previously reported Thai cases. Heterozygosity of Xmn I polymorphism also delays disease onset. Early diagnosis facilitates appropriate management and prenatal diagnosis.

Beta-globin gene cluster haplotypes in Iranian patients with beta-thalassemia

Beta-globin gene cluster haplotypes are useful in diagnosis of particular molecular defects in Beta-thalassemia, prenatal diagnosis of Beta-thalassemia, and elucidating population affinities. Beta-globin gene cluster haplotypes were studied in 150 Beta-thalassemia minor and 52 healthy in-dividuals from the Fars province of Iran. DNA was extracted from leukocytes of whole blood by phe-nol-chloroform. Haplotype was determined by PCR-RFLP technique. There were 26 out of 150 with homozygous haplotypes. Haplotype I was found as the most prevalent haplotype among both patients and normal individuals. Out of 26 patients bearing homozy-gous haplotypes, 12 [46.2%] had typical haplotype I and 3 [11.5%] had atypical haplotype I. The prevalence of haplotype I in normal control subjects was around 43% [45 out of 104 Beta A chromo-somes]. The second prevalent haplotype was haplotypes V [15.4%] and III [15.4%] for homozygous patients and controls, respectively. The most frequent mutation in patients was IVS II.1 [G-->A] that was not linked to a single haplotype. IVS I.110 [G-->A] mutation was linked to haplotype I. Mutation in codon 30 [G -->A] was associated with haplotype V. Being Haplotype I the most prevalent haplotype in Beta-thal and BetaA chromosomes, implies that Beta-thalassemia mutations might have arisen in the chromosomal background common in the popula-tion, rather than due to selection pressure or gene flow [migration]. Patients with haplotype IX had the highest HbF levels compared to other haplotypes

Rapid detection of alpha + thalassaemia deletion & alpha-globin gene triplication by Gap-PCR in Indian subjects.

BACKGROUND & OBJECTIVES: Southern blot hybridization is the commonly used method to delineate alpha globin gene defects. This technique is time consuming, requires a large amount of genomic DNA and radioactive probes for detecting the mutations, which limits its use in diagnosis. The present paper emphasizes the efficacy of a well-established Gap-PCR technique in the Indian set up to detect defects of the alpha globin gene in clinics and laboratories engaged in the diagnosis of thalassaemias. METHODS: A total of 190 normal subjects (voluntary blood donors), 183 individuals with heterozygous beta-thalassaemia and 19 with homozygous beta-thalassaemia were screened for -alpha 3.7, -alpha 4.2 deletions and for triplication of alpha gene (alpha alpha alpha anti 3.7). RESULTS: The use of normal and mutant primers in Gap-PCR revealed eight (4.2%) normal individuals, 22 (12%) individuals with heterozygous beta-thalassaemia and 1 (5.2%) with homozygous beta-thalassaemia as carriers of single- -alpha 3.7 deletion. Amplified fragment of 1.8 kb indicated the presence of alpha gene triplication (alpha alpha alpha anti 3.7) in 4 subjects with heterozygous beta-thalassaemia. Normal alpha-genotype (alpha alpha/alpha alpha) was found in 250 samples. However, none of the studied samples revealed the presence of -alpha 4.2 deletion. INTERPRETATION & CONCLUSION: Gap-PCR is a robust, simple, rapid and non-radioactive technique thus useful in diagnostic laboratories for the detection of common alpha-thalassaemia mutations.

Hb G Makassar (beta 6:Glu-Ala) in a Thai family.

Hemoglobin G Makassar was identified in a family of Thai origin, which was found to share identical properties with hemoglobin S in routine hemoglobin separation both by cellulose acetate electrophoresis and cation-exchange HPLC. It is, therefore, subjects with hemoglobin G Makassar and hemoglobin S who may sometimes be mistakenly identified for each other. In this study, identification of hemoglobin G Makassar was achieved by DNA sequence analysis, which revealed a single nucleotide substitution GAG-->GCG at codon 6 of the beta-globin gene. The hemoglobin variant was hemoglobin G Makassar [beta 6:Glu-->Ala]. This is the first report of hemoglobin G Makassar in Thailand.

RFLP Haplotypes of beta-Globin Gene Complex of beta-Thalassemic Chromosomes in Koreans

Korea is in the low-prevalence area of beta-thalassemia and the Korean population has relatively homogenous racial characteristics. Recently, we identified some causative mutations of the Korean beta-thalassemia patients. In order to elucidate the genetic background of beta-thalassemia alleles in Koreans, we determined the restriction fragment length polymorphism (RFLP)-haplotype and framework (FW) in nine beta-thalassemia chromosomes of five different causative mutations by PCR-based method and family linkage study. The result that the haplotype and the framework linked to the initiation codon ATG-->AGG mutation were -+-++-+ and FW3A, respectively, in all of three families in this study suggests a common origin of this mutation at least in Koreans. A novel beta-thalassemia mutation, codons 89/90 -TG, showed discrepancy between -++--++- and FW1, which could be explained by gene conversion. A case of codons 8/9 +G frameshift mutation had +----++ and FW1. The linkage of the two beta-thalassemia mutations, codon 17 AAG-->TAG and codons 41/42 -TTCT, with specific haplotypes and frameworks common to the Koreans and the neighboring countries suggests that those mutations are influenced by the genetic flow from the south China.

The Agamma-195 (C->G) mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression

Alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population

A beta-thalassaemia allele with 3 base substitution in codons 4/5 & 6 (ACT CCT GAG-> ACA TCT TAG) detected by denaturing gradient gel electrophoresis & sequencing.

We report the analysis of a beta-thalassaemia gene involving three bases in codons 4/5 and 6 (ACT CCT GAG-> ACA TCT TAG) in a confirmed carrier whose child had beta-thalassaemia major. The fragment of the gene carrying the mutation was detected by denaturing gradient gel electrophoresis (DGGE) using GC clamped primers, followed by direct sequencing. DGGE analysis indicated that one gene was the wild type (normal) while the sequence changes observed were all in the other gene causing beta-thalassaemia major in the child. This confirms a single case report from Lucknow (UP) and adds to the beta-thalassaemia mutations identified in the beta-globin gene in India and will help in the thalassaemia control programme.

Effect of single mismatches at 3'-end of primers on polymerase chain reaction

Objective and Method - To investigate the effect of three different mismatches [G/T, G/A or G/G] at the 3'-end of a primer to amplify a 268 bp [base pair] region of the human beta-globin gene using different annealing temperatures [45 to 65°C]. Results - The primer with the G/T mismatch was as efficient as the normal primer [G/C match] in the amplification of a 268 bp product at all temperatures tested. However, the primers having G/A or G/G mismatches at the 3'-end did not produce any specific polymerase chain reaction [PCR] fragment at all the annealing temperatures used, except a barely detectable 268 bp product for the G/G mismatch at 45 and 50°C. Conclusion - We conclude that our PCR system was refractory to amplification when one of the primers contained a G/A or G/G mismatch at the 3'-end with template DNA

Variable clinical severity of Hb E beta-thalassemia among Indians.

OBJECTIVE: The aim of this preliminary report was to look at the effect of genetic variations in the alpha, beta and gamma globin genes in 7 cases of hemoglobin E/beta-thalassemia with diverse clinical expression of the disease. METHODS: beta-thalassemia mutations were characterized by PCR and dot blot hybridization. G gamma gene polymorphism (Xmnl) was determined by PCR followed by restriction enzyme digestion and polyacrylamide gel electrophoresis. alpha genotyping was done by Southern blot hybridization. RESULTS: Six cases had a severe beta+ mutation [IVS 1 nt 5 (G-->C)] and one case had a beta zero mutation [F/S 41/42 (-CTTT)]. Hence, the beta-thalassemia mutation does not seem to contribute towards the clinical diversity. alpha-genotyping showed a single alpha-gene deletion of the rightward type in three of the five milder cases. The -158 G gamma (C-->T) substitution was present at least in heterozygous state (+/-) in all the milder cases. CONCLUSIONS: Deletional alpha thalassemia and presence of the -158 G gamma (C->T) substitution are the two factors which appear to be more important in decreasing the severity of the disease rather than the nature of the beta thalassemia mutation.