PS1204 SEVERE HEREDITARY HAEMOLYTIC ANAEMIA DUE TO THREE NOVEL SPTA1 MUTATIONS IN TWO COMPOUND HETEROZYGOUS UNRELATED PATIENTS
Authors: Krishnevskaya E. , Rodriguez I. Hernandez , Serra A. Ancochea , Payán‐Pernía S. , Rizzuto V. , Magariño M. Molero , Vives‐Corrons J.‐L. 
Published: June 2019

Background:Red blood cell (RBC) membrane defects are a cause of hereditary haemolytic anaemia (HHA) due to mutations in the genes that encode cytoskeletal proteins leading to a reduced cell deformity. Using next generation (NGS) and whole exome sequencing (WES), in two unrelated patients with HHA due to hereditary pyropoikilocytosis (HPP) and hereditary spherocytosis (HS), we have identified a complex set of mutations that affect SPTA1. Three are new: two due to an abnormal splicing and one to a micro election of chromosome 1Aims:The main objective of our task is to provide a fast and efficient diagnosis of HHA using NGS and WESMethods:NGS that includes a panel of 35 genes responsible for membranophaty (ANK1, EPB41, EPB42, SLC4A1, SPTB, SPTA1), haemoglobinopathy (HBA1, HBA2, HBB), enzymopathy (ADA, AK1, ALDOA, BPBM, CYB55, G6PD, GCLC, GPI, GSR, GSS, HK1, NT5C3A, PFKM, PGK1,) and CDA (CDAN1, C15orf41, SEC23B, KLF1, GATA1, KIF23). NGS and WES have been performed using an Illumina HiSeq2000 deviceResults:Patient 1. A 40 year‐old woman with HPP. CBC: Hb: 102 g/l, MCV: 101fl and Retics: 260 x109/l. The study was performed after splenectomy and the OGE curve was typical of severe HS. NGS revealed two canonical splice‐site mutations in SPTA1 gene: c.6842+2T>C and c.1599+2T>C. The c.6842+2T>C mutation is found in the lateral nucleation zone of a‐spectrin with b‐spectrin and produces an aberrant splicing eventually in 100% of the cases with a production of α‐chains unsuitable for spectrin dimerization, and a decrease of binding to skeleton during the heterodimers assembly. The c.1599+2T>C mutation is in the zone of association of spectrin dimers leading to an aberrant splicing eventually in 100% of the cases and probably a change in the protein conformation that produces a breakage of tetramerisation and a loss of skeleton stabilityPatient 2. A 2 year‐old boy with HS. CBC: 72 g/l, MCV 95 fl and Retics:182 x109/l. Splenomegaly. The OGE curve was typical of moderate HS. The NGS analysis demonstrated a coinheritance for two different mutations in SPTA1 gene: n.158537345_158849273del and c.4339–99C>T. The n.158537345_158849273del, confirmed by WES, leads to a micro deletion of chromosome 1 that removes the entire SPTA1, leading to the absence of α‐spectrin. The c.4339–99C>T, also known as spectrin α‐LEPRA, is an autosomic recessive mutation that causes a decrease of a‐chain of 84%. Both SPTA1 gene mutations are autosomal recessive and their co‐inheritance explains the severe HS.Summary/Conclusion:HPP and H are heterogeneous disorders of RBC membrane that can result from mutations in the genes encoding α‐spectrin (SPTA1). The resulting defects an abnormal horizontal and/or vertical cytoskeletal associations affecting the stability and deformability of RBC membrane. The clinical diagnosis of HE and HS is based on the identification of the characteristic RBC morphology and specific membrane rheological abnormalities using the OGE. Here, two unrelated patients with severe elliptocytosis (HPP) and HS are described with the joint inheritance of a complex set of mutations (four in total) in the SPTA1 gene; three are new and one is the known as α‐LEPRA. The correlation between genetic mutations NGS and EOG is discussed. Our study demonstrates the existence of complex interactions between SPTA1 gene mutations leading to severe haemolytic anaemia highlighting the usefulness of molecular diagnostics in patients with no identifiable family history of disorders of the erythrocyte cytoskeleton