Abstract
Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.
Highlights
Congenital hemolytic anemias (CHAs) comprise a group of very heterogeneous and rare disorders caused by alterations in structure, transport functions, metabolism, or defective production of red blood cells (RBCs)
Enzyme Defects Eight cases from six families had a biochemical diagnosis of enzyme defect; two were confirmed to have pyruvate kinase (PK) deficiency, three from two families glucose-6-phosphate dehydrogenase (G6PD) deficiency and one was found to have one mutation in GPI gene, in compound heterozygosity with the polymorphism c.489A > G (p.Gly163 = rs1801015), possibly associated to splicing alterations (Fermo et al, 2019); in two related cases no mutations in PKLR gene were found in spite of a slightly decreased PK activity detected by enzymatic assay
Among these cases with enzyme defects, in four patients from three families a coinheritance of hereditary stomatocytosis was suspected due to the presence of stomatocytes at peripheral blood smear and left-shifted Osmoscan curve; only in two of them (5-1 and 5-2) in addition to G6PD deficiency we found a mutation in PIEZO1 gene causing hereditary stomatocytosis
Summary
Congenital hemolytic anemias (CHAs) comprise a group of very heterogeneous and rare disorders caused by alterations in structure, transport functions, metabolism, or defective production of red blood cells (RBCs). Since the pathophysiology of some rare forms is poorly understood, these disorders represent a group of diseases that still lack easy-to-apply tools for diagnosis, clinical management, and patient stratification. The laboratory diagnostic pathway of CHAs was historically based on sequential steps using a panel of functional analyses that investigate the RBC membrane and metabolism. Even after extensive and complete investigation, the differential diagnosis of these disorders may be difficult, and some patients remain undiagnosed with a consequent negative impact on clinical follow up, risk of inappropriate therapeutic decisions and lack of access to new specific treatments
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