S902 THE COMPLEXITY OF GENOTYPE‐PHENOTYPE CORRELATIONS IN HEREDITARY SPHEROCYTOSIS: A COHORT OF 95 PATIENTS

Abstract

Background:Hereditary spherocytosis (HS) presents as a highly heterogeneous disease, both phenotypically and genetically. Disease severity is directly related to the extent of loss of surface area of the red cell. The diagnosis is multifaceted and includes functional testing of red cell deformability. Until recently, establishing or confirming the diagnosis at the DNA level was only rarely done. With increased use of Next‐Generation‐Sequencing (NGS) techniques in diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. However, insight into the impact of genetic defects on the function of membrane proteins, and consequently on phenotypic presentation, is still limited.Aims:Explore genotype‐phenotype correlation in a large cohort of patients with HS.Methods:Retrospective analysis on a cohort of 95 HS patients genotyped by targeted‐NGS (SPTA1, SPTB, ANK1, SLC4A1, EPB41, EPB42, RHAG) as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands).Results:Our cohort included 95 patients suspected to have HS. In 85/95 (89%) of the patients a pathogenic mutation was identified, including 56 novel mutations. Mutations in SPTA1 were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 mutations showed autosomal dominant inheritance. ANK1 mutations accounted for 27% (23/85 patients) and SPTB mutations for 20% (17/85 patients) of the cases, all showing autosomal dominant inheritance. Moderate or severe HS was more frequently observed in patients with SPTB or ANK1 mutations, reflected by significantly lower hemoglobin concentrations (SPTB 10.5 g/dL, ANK1 10.8 g/dL compared to SLC4A1 13.2 g/dL, both p < 0.05) and higher absolute reticulocyte counts (SPTB 580x10^9/L, ANK1 506x10^9/L compared to SPTA1 298x10^9/L and SLC4A1 259x10^9/L, all p < 0.01) in non‐splenectomized patients.We investigated the correlation between phenotype and genetic mutations using protein structure information (Figure). Mutations positioned in the spectrin‐association domains of ANK1, SPTA1 and SPTB resulted in a more severe phenotype. Interestingly, deletion of the whole SPTB gene resulted in a mild phenotype, while the majority of other pathogenic mutations in SPTB were associated with a more severe disease phenotype, suggesting that incorporation of truncated or dysfunctional protein occurs, which might be more harmful than an absolute decrease of otherwise normal protein.Our cohort included nine families and broad phenotypic heterogeneity was observed in four of them. Phenotypic heterogeneity could not be directly explained by the underlying genotype, suggesting that other (genetic) yet‐unknown modifiers contribute to disrupted interactions in the red cell cytoskeleton. To identify alternative predictors of clinical severity we analyzed red cell Osmoscan profiles from the Laser‐assisted Optical Rotational Cell Analyzer. Data were available in a subset of our cohort (44 not splenectomized patients). We observed a clear association between clinical disease severity and the maximal deformability, and the area under the curve (respectively r = −0.48, p < 0.01, and r = −0.38, p = 0.01).Summary/Conclusion:Genotype‐phenotype prediction in HS requires additional insight in the consequences of pathogenic mutations for assembly of and dynamic interactions in the red blood cell cytoskeleton. Currently, in conjunction to the molecular deficit, functional analysis of red cell deformability adds to the prediction of phenotypic variability.image