RESUMO
Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life-threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next-generation sequencing to provide a high-throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion-dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next-generation sequencing panel could be a cost-effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.
Assuntos
Anemia Hemolítica Congênita/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Anemia Hemolítica Congênita/genética , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Enzimas/genética , Componentes do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Hiperbilirrubinemia Hereditária/diagnóstico , Lactente , Recém-Nascido , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Mutação , Adulto JovemRESUMO
The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD(202A) and G6PD(376G) alleles and alpha-thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD(202A,376G) (G6PD A-) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide. The prevalence of G6PD(202A,376G) was 13.6% in males and 3.3% in females with an overall prevalence of 8.7%. G6PD(202A,376G) was associated with a 10 g/l decrease in haemoglobin concentration (P = 0.008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate-aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P > 0.09). Similar results were found within a sub-group of children who were not receiving hydroxycarbamide. By comparison, single and double alpha-globin deletions were associated with progressively higher haemoglobin concentrations (P = 0.005 for trend), progressively lower values for haemolytic component (P = 0.007), and increased severe pain episodes (P < 0.001). In conclusion, G6PD(202A,376G) may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.