Summary. Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A3G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the 272A3G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the 272A3G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C3T had a mild condition.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans. © 1998 by The American Society of Hematology.

Hb Lepore is one of the most common abnormal haemoglobins in Caucasians in Central Portugal and in the Spanish Alta Extremadura (0.28% in a survey of school children). A group of 19 Portuguese and 14 Spanish Hb Lepore carriers (all unrelated) was characterised at the molecular level by the polymerase chain reaction, sequencing and restriction enzyme analysis. The Portuguese and one Spanish carrier were heterozygous for Hb Lepore-Baltimore, whereas all other Spanish subjects were Hb Lepore-Washington-Boston carriers. Sequencing of the Hb Lepore-Baltimore gene further established the crossover at delta 68-beta 84, a region two codons (CDs) shorter than that previously described and easily confirmed by digestion with MaeI and BanI. Data from haplotype analysis suggest that this crossover occurred as an independent event on the Iberian Peninsula. The haematological data were similar in both groups except for the levels of Hb F and the G gamma chain, which were significantly higher in the Hb Lepore-Baltimore heterozygotes. Quantification of the globin chains and the mRNA transcripts showed that the delta beta gene is transcribed at a higher level than the delta gene with levels of translation giving rise to 10%-15% of Hb Lepore. The different levels of Hb F observed in the two groups are the results of the higher transcription rate of the gamma genes in Hb Lepore-Baltimore heterozygotes and an apparently less efficient translation of G gamma genes in Hb Lepore-Washington-Boston heterozygotes.

Allele alpha LELY is a low-expression allele of the erythroid spectrin alpha-gene. It carries mutations in exon 40 (alpha V/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of alpha-chains by beta-chains, and would eventually account for the low-expression character. When it occurs in trans to an alpha-allele responsible for hereditary elliptocytosis (alpha HE allele; alpha HE/alpha LELY diplotype), allele alpha LELY enhances the severity of elliptocytosis. Because allele alpha LELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele alpha LELY will be designated alpha HE-LELY allele. The HE component was the known alpha 28 Arg-->His mutation. This alpha HE-LELY allele was investigated within the alpha HE-LELY/alpha LELY diplotype, a diplotype not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the alpha LELY component in cis of the alpha HE mutation counteracted the like component in trans.

Summary.In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G → C on exon 12 and two 5 0 splice donor site (GT) mutations on intron 8 [IVS8(þ2)T → G] and intron 10 [IVS10(þ1)G → C]. Two previously described missense mutations, 1456C → T and 993C → A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(þ1)G → C or IVS8(þ2)T → G.Keywords: pyruvate kinase deficiency, human PK-LR gene mutations, haemolytic anaemia, splice site, Portugal.Pyruvate kinase (PK EC 2.7.1.40) deficiency is the most common enzyme abnormality in the erythrocyte glycolytic pathway causing hereditary nonspherocytic haemolytic anaemia (HNSHA). The PK-deficient anaemia is transmitted as an autosomal recessive disorder with a heterogenous clinical phenotype, ranging from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring exchange transfusion. Splenectomy is effective in decreasing haemolysis in most patients. Heterozygotes are usually asymptomatic, with a 30-50% decrease in the PK activity levels (Miwa et al, 1979). PK deficiency was first described by Valentine et al (1961) and since then more than 400 cases have been reported, with the biochemical characteristics of the residual enzyme differing markedly between patients. The International Committee for Standardization in Haematology (ICSH) established the standard methods for enzymatic studies; however, the low activity, different stability and the hybrid tetramers formed in the compound heterozygous causes difficulty in characterization of the PK variants (Miwa et al, 1979). A molecular approach, identifying the gene mutations, enables a better characterization of the PK-deficient patients with a more precise genotype/phenotype correlation.It is also useful for diagnosis among transfusion-dependent patients with chronic haemolytic anaemia and prenatal diagnosis.Four PK isoenzymes have been described in mammals, M1, M2, L and R, which are tissue specific and differ in their enzymatic properties (Imamura & Tanaka, 1972). In mammals there are only two different PK genes: the PK-M gene that codes for the isoenzymes M1 (muscle and brain) and M2 (fetal and most adult tissues) using alternative RNA splicing (Noguchi et al, 1986) and the PK-LR gene, that codes for the L-type (liver) and R-type (erythrocyte) isoenzymes using two different tissue-specific promoters (Noguchi et al, 1987). More than 70 mutations have been identified in the coding and flank...

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.

We have characterized 14 patients in 10 families with a mild form of homozygous beta thalassemia which has not been previously well defined. As these patients originate from a small area of northern Portugal we propose to call this beta + thalassaemia--Portuguese type. Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and they are characterized by low levels of HbF, less than 20%, indicating only a mild deficit in beta globin production. Heterozygotes are indistinguishable from those with the more common types of beta thalassaemia as regards red cell morphology, haemoglobin analysis and globin chain synthesis studies. Globin gene mapping excluded the presence of alpha thalassaemia in these patients and demonstrated no abnormalities in the beta-like globin gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are identical on both chromosomes in all of the homozygotes, confirming their homogeneity.

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