PS1198 MOLECULAR DIAGNOSIS OF RARE CONGENITAL ANEMIAS USING TARGETED NEXT-GENERATION SEQUENCING IN INDIAN PATIENTS

Abstract

Listen

Translate article

Background: Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin-5’-maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next-generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear. Aims: To improve accuracy, robustness, and cost-effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS. Methods: We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA-binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α-spectrin, β-spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations. Results: A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations. The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry-derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry-derived parameter EImax (<0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin >166 mOsm/kg and EImax <0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty-seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values <40 had mutations only in the SPTA1 gene.Summary/Conclusion: Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and <0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.