Hb Köln [<FONT FACE=Symbol>a</font>2<FONT FACE=Symbol>b</font>298(FG5) val-met] identified by DNA analysis in a Brazilian family

Abstract

Hb Köln was identified by DNA analysis in a Brazilian patient. A four-year old Brazilian female, with jaundice since birth, presented an abnormal band, between A2 and S, in hemoglobin electrophoresis on a cellulose acetate membrane, and a band with electrophoretic migration similar to Hb C on agar gel. Thermic instability and isopropanol precipitation tests were positive. Heinz bodies were observed in the patient’s peripheral blood. Sequencing of the three exons of the <FONT FACE="Symbol">b</font> globin gene detected a transition from G to A in the first position of codon 98. This alteration does not create or abolish any known restriction site. In this case, confirmation of the mutation was accomplished by allele-specific oligonucleotide hybridization, which is a simple and fast identification method when the clinical data and hematological and electrophoretic patterns are suggestive of Hb Köln.<br>Hb Köln resulta da substituição do aminoácido valina por metionina na posição 98 da cadeia da hemoglobina. Embora seja a mais freqüente das hemoglobinas instáveis, possui poucas características que permitem distinguíla sem a realização de análise estrutural da molécula. Com o objetivo de desenvolver um procedimento alternativo à análise protéica, este trabalho descreve a identificação da Hb Köln através de análise de DNA. Uma paciente branca, sexo feminino, 4 anos de idade, com icterícia desde o nascimento, foi analisada, apresentando uma banda anômala entre A2 e S em eletroforese em acetato de celulose, e migração eletroforética semelhante a Hb C em gel de agar. Os testes de instabilidade térmica e de precipitação pelo isopropanol foram positivos e foram observados corpúsculos de Heinz no sangue periférico. Os três exons do gene da globina foram seqüenciados e uma transição de G <FONT FACE="Symbol">®</font> A foi identificada na primeira posição do codon 98. Essa alteração não cria ou abole nenhum sítio conhecido de enzima de restrição. Desta forma, a confirmação da mutação foi levada a efeito através de hibridização com oligonucleotídeos aleloespecíficos, o que permitiu o estabelecimento de um método simples e rápido de identificação de novos casos quando os dados clínicos e o padrão hematológico e eletroforético sugerem Hb Köln

Frequency and spectrum of hemoglobinopathy mutations in a Uruguayan pediatric population

“…The most common α-globin deletions (−α 3.7 , −α 4.2 , - - SEA , - - FIL , - - MED , -(α) 20.5 and - - THAI ) were identified by multiplex PCR while non-deletional forms were identified by restriction fragment length polymorphism (RFLP) from products amplified by PCR (α Hph α, α NcoI α and αα NcoI ) (Hall et al , 1993; Tan et al , 2001). The HBB gene was amplified and sequenced to confirm and identify β-globin mutations (Miranda et al , 1997). In samples with the HbS mutation, five polymorphic sites in the β-globin gene cluster were analyzed: 1) HincII 5′ε, 2) HindIII G γ, 3) HindIII A γ, 4) HincII 5′ ψβ and 5) HincII 3′ ψβ.…”

alpha-thalassemia, HbS, and beta-globin gene cluster haplotypes in two Afro-Uruguayan sub-populations from northern and southern Uruguay

“…In order to ascertain the presence of structural hemoglobinopathies and b-thalassemias, a b-globin gene DNA segment of 770 bp, located between positions -161 in the promoter and +565 in intron 2, was amplified and automatically sequenced with a Big Dye Terminator Version II Sequencing Kit (Applied Biosystems), using an internal primer located in the +2 position relative to the CAP site (Miranda et al, 1997).We also analyzed eight polymorphic sites in the b-globin gene cluster: 1) HincII 5'e; 2) HindIII-G g; 3) HindIII -A g; 4) HincII-yb; 5) HincII-3'yb; 6) HinfI-5'b; 7) AvaII-b; and 8) HinfI-3b. Fragments containing each of these sites were amplified by PCR, using primers and conditions previously described (Saiki et al, 1988;Sutton et al, 1989;Guerreiro et al, 1992).…”

Hb Florida: A novel elongated C‐terminal β‐globin variant causing dominant β‐thalassemia phenotype

“…The patient presented with moderate chronic hemolytic anemia (RBC ¼ 3.8 Â 10 6 /mm 3 , Hb ¼ 8.6 g/dl, Hct ¼ 28%, MCV ¼ 73.0 fl, MCH ¼ 22.6 pg, RDW ¼ 28.0%), pallor, jaundice, and liver and spleen enlargement, having required blood transfusion five times during viral and bacterial infections. Peripheral blood analysis revealed a remarkable degree of anisocytosis with microcytosis, poikilocytosis (with ovalocytes and schistocytes), hypochromia, basophilic stippling, and 13% reticulocytes and 2% erythroblasts (Figure 1a).…”

“…b-Globin gene sequencing (3.77 ABI, Applied Biosystems, CA), with primers described elsewhere [3], revealed a deletion of one nucleotide (-C) in between codons 140 and 141 (GCC/CTG?GCC/TG) (Figure 1d), which modified the C-terminal sequence and added 10 more residues to the b-chain [(141)TrpPro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-Leu-Leu-SerAsn-Phe-(156)Tyr-COOH]. No other alteration was found in the b-alleles.…”