Molecular study of red cell pyruvate-kinase deficiency in 15 patients withchronic haemolytic anaemia

BACKGROUND: Identification of RBC pyruvate-kinase (PK) gene mutations by po lymerase chain reaction (PCR) and single strand conformation polymorphism ( SSCP) followed by PK gene sequencing in positive cases has been assessed an d the results obtained with a preliminary study of 15 unrelated patients of Spanish origin are presented, PATIENTS AND METHODS: Patients have been classified into two different grou ps: group 1, propositus (15 cases), and group 2, relatives of the patients included in group 1 (10 males and 5 females). In group 1, a PCR was followe d by SSCP and sequencing, and in group 2, the PCR was followed by digestion with specific restriction endonucleases [PCR-ER). RESULTS: Group 1: from 15 patients included in the study 2 were identified as homozygous, 4 as heterozygous and 9 as compound heterozygous. In this gr oup, were identify 26 affected alleles with 11 different mutations: T-1456 10 alleles (38.6%), T-721 3 alleles (11.6%), A(1010), C-514, C-1015 and T-1 223 2 alleles (7.7%), and C-1070, A(1291) T-1508, A(1595) y T-1675 one alle le, Relatives from 8 out of 15 patients from group 1 showed the following p attern: homozygous tone case), heterozygous (10 cases), compound heterozygo us (2 cases) and normal (2 cases), CONCLUSIONS: SSCP procedure followed by direct gene sequencing in positive cases is fast and simple enough to allow the identification of PK deficient variants, avoiding the need of biochemical characterisation of semipurifie d deficient enzyme, which is more cumbersome and time consuming. In additio n, the PCR-ER method is a very useful tool for screening of the most freque nt molecular variants, as well as, for the detection of the carrier conditi on of this enzymopathy (family studies).