scholarly journals Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2196-2201 ◽  
Author(s):  
AF Roux ◽  
F Morle ◽  
D Guetarni ◽  
P Colonna ◽  
K Sahr ◽  
...  
Keyword(s):  
North Africa ◽  
Molecular Basis ◽  
Blot Hybridization ◽  
Molecular Defect ◽  
Dot Blot ◽  
Hereditary Elliptocytosis ◽  
Black Patients ◽  
Allele Specific ◽  
Alpha Spectrin ◽  
Pcr Method

Abstract Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

scholarly journals Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2196-2201
Author(s):  
AF Roux ◽  
F Morle ◽  
D Guetarni ◽  
P Colonna ◽  
K Sahr ◽  
...  
Keyword(s):  
North Africa ◽  
Molecular Basis ◽  
Blot Hybridization ◽  
Molecular Defect ◽  
Dot Blot ◽  
Hereditary Elliptocytosis ◽  
Black Patients ◽  
Allele Specific ◽  
Alpha Spectrin ◽  
Pcr Method

Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

scholarly journals Alpha Thalassemia Genotypes in Kuwait

2020 ◽  
Author(s):  
Adekunle Adekile ◽  
Jalaja Sukumaran ◽  
Diana Thomas ◽  
Thomas D'Souza ◽  
Mohammad Haider
Keyword(s):  
Blot Hybridization ◽  
Thalassemia Major ◽  
Dot Blot ◽  
Low Frequencies ◽  
Kuwait University ◽  
Alpha Thalassemia ◽  
Reverse Dot Blot Hybridization ◽  
Oligonucleotide Hybridization ◽  
Allele Specific ◽  
Kuwaiti Population

Abstract Background: The frequency of the alpha thalassemia trait is approximately 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of patients who were referred for molecular diagnosis over a 20-year period.Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was performed by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot blot hybridization (Vienna Lab Strip Assay).Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families. Most (90.8%) were Kuwaiti nationals. The commonest genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3% of the samples, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. PA-1 was therefore the most frequent allele (0.59). The frequency of the α0 (--MED) allele was 0.017. Rare alleles that were found in very low frequencies included α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but nondeletional PA-1 is by far the most common cause of the moderate to severe HbH (β4 tetramer) disease phenotype. The α0 (–MED) allele is also encountered, which has implications for premarital counseling, especially for the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

scholarly journals Spectrum of Alpha Thalassemia Alleles in Kuwait

2020 ◽  
Author(s):  
Adekunle Adekile ◽  
Jalaja Sukumaran ◽  
Diana Thomas ◽  
Thomas De Souza ◽  
Mohammad Haider
Keyword(s):  
Blot Hybridization ◽  
Thalassemia Major ◽  
Dot Blot ◽  
Low Frequencies ◽  
Kuwait University ◽  
Alpha Thalassemia ◽  
Oligonucleotide Hybridization ◽  
Alpha Globin ◽  
Allele Specific ◽  
Kuwaiti Population

Abstract Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period. Methods: Blood samples from suspected cases were sent to the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University. A retrospective study of the molecular characterization of samples from 1994 to 2015 was carried out. The alpha globin genotypes were determined by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay). Results: 400 samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years (~60% children and adolescents). Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3%, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.733). The frequency of the α0, –MED was 0.19. Rare alleles that were found in very low frequencies included the α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana,and Hb Icaria. Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH disease phenotype. The α0 (–MED) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

scholarly journals First Report of Grapevine Virus A in Spain

Plant Disease ◽  
1997 ◽  
Vol 81 (7) ◽  
pp. 830-830 ◽  
Author(s):  
I. Zabalgogeazcoa ◽  
C. de Blas ◽  
C. Cabaleiro ◽  
A. Segura ◽  
F. Ponz
Keyword(s):  
Blot Hybridization ◽  
Stem Bark ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
First Report ◽  
Grapevine Virus A ◽  
Mediterranean Countries ◽  
Pcr Product ◽  
Pcr Method ◽  
Rna Blots

Grapevine trichovirus A (GVA), a flexuous, filamentous, phloem-limited virus with an approximately 7.3-kbp RNA genome, is widespread in grapevines showing symptoms of leafroll and/or rugose wood. The virus can be mechanically inoculated to Nicotiana benthamiana and N. clevelandii. A field survey of diseased Vitis vinifera white and red cultivars was carried out in Pontevedra (northwest Spain) during the autumn of 1993. We detected the presence of GVA in vines showing leafroll symptoms by an immunocapture-reverse transcription-polymerase chain reaction (PCR) method (2) with GVA-specific primers (1). Bands of the expected size of 430 bp were obtained with extracts from petioles and stem bark as reaction substrates. To verify these results, Northern (RNA) blots with double-stranded (ds) RNAs isolated from grapevines were prepared. Hybridization was positive in two out of 10 samples analyzed. The probe was a 32P-labeled 430-bp PCR product amplified from extracts of N. benthamiana plants infected with GVA strain Is151 (gift of A. Mina-fra). The specificity of this probe was confirmed in dot blot hybridization, as a positive signal was obtained with extracts from GVA-inoculated N. benthamiana, but not with extracts of phosphate buffer-inoculated N. benthamiana, turnip mosaic potyvirus-inoculated Arabidopsis thaliana, or potato potyvirus Y-inoculated N. xanthi plants. The probe did not hybridize to dsRNAs extracted from enzyme-linked immunosorbent assay-positive GLRaV-III-infected grapevines. GVA has been identified in other Mediterranean countries, but to our knowledge this is the first report of the detection of GVA in Spain. References: (1) A. Minafra and A. Hadidi. J. Virol. Methods 47:175, 1994. (2) G. Nolasco et al. J. Virol. Methods 45:201, 1993.

scholarly journals Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2343-2348 ◽  
Author(s):  
S Lyman ◽  
RH Aster ◽  
GP Visentin ◽  
PJ Newman
Keyword(s):  
Heavy Chain ◽  
Blot Hybridization ◽  
Membrane Glycoprotein ◽  
Dot Blot ◽  
Amino Acid Polymorphism ◽  
Single Nucleotide ◽  
Nucleotide Base ◽  
Allele Specific ◽  
Diagnostic Approaches ◽  
Homozygous Individual

Abstract The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3′ end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.

scholarly journals Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French Northern Alps

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2113-2119 ◽  
Author(s):  
S Feddal ◽  
G Brunet ◽  
L Roda ◽  
S Chabanis ◽  
N Alloisio ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Blot Hybridization ◽  
Limited Area ◽  
Dot Blot ◽  
Western Blots ◽  
Genetic Transmission ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Heterozygous Form ◽  
Northern Alps

4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.

scholarly journals Alpha Thalassemia Genotypes in Kuwait

2020 ◽  
Author(s):  
Adekunle Adekile ◽  
Jalaja Sukumaran ◽  
Diana Thomas ◽  
Thomas De Souza ◽  
Mohammad Haider
Keyword(s):  
Blot Hybridization ◽  
Thalassemia Major ◽  
Dot Blot ◽  
Low Frequencies ◽  
Kuwait University ◽  
Alpha Thalassemia ◽  
Reverse Dot Blot Hybridization ◽  
Oligonucleotide Hybridization ◽  
Allele Specific ◽  
Kuwaiti Population

Abstract Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period.Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay).Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families.. Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3%, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.59). The frequency of the α0, –MED was 0.017. Rare alleles that were found in very low frequencies included the α0 (--FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.Conclusion: There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH (β4 tetramer), disease phenotype. The α0 (–MED) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

scholarly journals Molecular analysis of hereditary elliptocytosis with reduced protein 4.1 in the French Northern Alps

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2113-2119 ◽  
Author(s):  
S Feddal ◽  
G Brunet ◽  
L Roda ◽  
S Chabanis ◽  
N Alloisio ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Blot Hybridization ◽  
Limited Area ◽  
Dot Blot ◽  
Western Blots ◽  
Genetic Transmission ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Heterozygous Form ◽  
Northern Alps

Abstract 4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.

scholarly journals Alpha Thalassemia Genotypes in Kuwait

2020 ◽  
Author(s):  
Adekunle Adekile ◽  
Jalaja Sukumaran ◽  
Diana Thomas ◽  
Thomas De Souza ◽  
Mohammad Haider
Keyword(s):  
Blot Hybridization ◽  
Thalassemia Major ◽  
Dot Blot ◽  
Low Frequencies ◽  
Kuwait University ◽  
Alpha Thalassemia ◽  
Reverse Dot Blot Hybridization ◽  
Oligonucleotide Hybridization ◽  
Allele Specific ◽  
Kuwaiti Population

Abstract Background: The frequency of alpha thalassemia trait is about 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of the patients who were referred for molecular diagnosis over a 20-year period. Methods: This is a retrospective study of the a-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot-blot hybridization (Vienna Lab Strip Assay). Results: Four hundred samples were characterized and analyzed from individuals aged <1 month to 80 years, with a median of 6 years from 283 unrelated families.. Most (90.8%) were Kuwaiti nationals. The common genotype was homozygosity for the polyadenylation-1 mutation (α PA-1 α/α PA-1 α) in 33.3%, followed by heterozygosity (αα/α PA-1 α) for the same mutation in 32.3%. The PA-1 was therefore the most frequent allele (0.59). The frequency of the α 0 , – MED was 0.017. Rare alleles that were found in very low frequencies included the α 0 (-- FIL ) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria. Conclusion : There is a wide variety of alpha thalassemia alleles among Kuwaitis, but the nondeletional PA-1 is by far the most common cause of moderate to severe HbH (β4 tetramer), disease phenotype. The α 0 (– MED ) allele is also encountered, which has implications for pre-marital counselling especially the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

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