Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method.

An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype-specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes I/1a and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype II/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 4d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a.

Hereditary anaemias in Portugal: epidemiology, public health significance, and control.

A countrywide prospective study aimed at establishing the prevalence of the haemoglobinopathy genes in the Portuguese population was carried out by screening 15,208 randomly selected blood samples from young males. This male based survey provided the opportunity of assessing simultaneously the prevalence of the red cell enzyme glucose-6-phosphate dehydrogenase (G6PD) deficiency, thus giving a picture of these important hereditary anaemias in Portugal. The results showed a low average frequency of beta thalassaemia (0.45%) and haemoglobin S (0.32%) carriers as well as G6PD deficiency (0.51%). However, these disorders are unevenly distributed throughout the country with a higher prevalence in some areas, mainly in the south. The relationship of this pattern of haemoglobinopathies to the known haplotypes linked to beta thalassaemia and sickle cell disease, relevant historical events, and local selective pressure was investigated. Hb D and Hb J are the commonest other structural variants. The implemented programme for control of these hereditary anaemias is described.

Unusual molecular basis of Hb H disease in the Azores Islands, Portugal.

An Azorean family with Hb H disease (10% Hb H) was studied in order to elucidate its molecular basis. DNA studies on the patient only revealed a 4.2 kb "leftward" deletion of paternal origin which implies the co-inheritance of a nondeletional alpha-thalassemia determinant. Restriction endonuclease and oligonucleotide analysis allowed the exclusion of five point mutations: initiation codon (at both alpha 1- and alpha 2-globin genes), IVS-I donor splice junction pentanucleotide deletion, codon 125 CTG----CCG substitution, and Saudi Arabian polyadenylation signal mutation. These findings suggest that the molecular basis of this form of Hb H disease is probably different from those described previously.

Red cell membrane alteration involving protein 4.1 and protein 3 in a case of recessively inherited haemolytic anaemia.

We present 2 siblings with a severe congenital haemolytic anaemia. Red cells displayed a variety of abnormal shapes, including leptocytes, schizocytes and elliptocytes. Repeatedly, skeletal protein 4.1 appeared reduced by 30%. The 4.1a/4.1b ratio was normal despite the haemolytic state. No change could be detected in spectrin, nor in sialoglycoproteins. Band 3 was denser, narrower and displaced downward. The parents, who are consanguineous, were devoid of any obvious biochemical abnormality; however, their red cells were not normal. These 2 cases with reduced protein 4.1 clearly depart from 4.1 (-) hereditary elliptocytosis. The possibility of an altered binding of protein 4.1 to some other membrane component is discussed.