PS1196 INNOVATIVE DIAGNOSTIC APPROACH FOR RARE ANEMIA
Authors: Rizzuto V. , Álvarez A. Blanco , Vega B. Tazón , Pastor D. Beneitez , Pereira M.D. M. Mañú 
Published: June 2019

Background:

Rare anemia (RA) embrace a highly heterogeneous group of ultra‐rare disorders which severe forms are life‐threatening with complex genotype‐phenotype correlations. Both conventional and NGS present bottlenecks.

Aims:

To establish an integrative diagnostic algorithm encompassing conventional and new methodologies for reducing the number of misdiagnosed/undiagnosed cases.

Methods:

53 patients with chronic hemolytic/hyporegenerative anemia included and categorized based on a) clinical criteria b) EMA‐Binding test (EBT) and osmotic gradient ektacytometry (LoRRca) and c) t‐NGS (69 disease causing and candidate genes).

EBT conventional cut‐off for Hereditary Spherocytosis (HS) was used (decreased fluorescence >21%). LoRRca cut‐offs already established by the group. Genetic variants (coverage >100x) were annotated on variant effect and MAF and IGV assessed. Disease causing variants on inheritance pattern and VarSome plus Human Splicing Finder. Interpretation and nomenclature according to ACMG. t‐NGS was considered as gold standard for conclusive diagnosis and tests sensitivity.

Results:

Clinical orientation/conclusive diagnosis correlation is shown in the figure.

HS oriented patients were stratified according to CPG: 1 to 4 HS diagnosis criteria (family history, peripheral spherocytes, raised MCHC, % hyperdense). Only 5/43 oriented HS patients fulfill all criteria, in which EBT, LoRRca and t‐NGS confirmed HS diagnosis. EBT was HS positive in 31 of 38 remaining cases (81.6%) while LoRRca showed HS pattern in the 38. After t‐NGS, conclusive HS diagnosis was reached in 36 of the 38 cases. 5 of them also showed a disease causing mutation on RBC enzymes. 2 cases remained undiagnosed, both with EBT and LoRRca HS compatible profiles. In addition, correlation among EBT and HS causative genes showed lower range of values (x = −19%) for SPTA1 variants in contrast with SLC4A1 variants (x = −35%). Five from 6 clinically oriented as Hereditary Elliptocytosis (HE) were confirmed by t‐NGS, resulting in 3 HE and 2 sever forms, Hereditary Piropoikilocytosis (HPP). EMA BT was HS positive in one HPP case showing a double population. LoRRca showed HE profile in 5 cases and HS profile in 1 case corresponding with the third undiagnosed case which showed a disease causing CDAN1 variant and a SPTB VUS in heterozygous status. One case oriented ad Hereditary Xerocytosis (HX) was diagnosed by both, LoRRca and t‐NGS, with a mild form of HX. From the two cases oriented as chronic non‐spherocytic hemolytic anemia, 1 was diagnosed by t‐NGS as HX (LoRRca and EBT negative) and 1 correspond with the fourth undiagnosed case which presented EBT in the “gray zone” and non‐conclusive LoRRca profile. In 1 patient with compensated hemolysis, conclusive diagnosis was reached based on a HBA1 variant coding for an unstable Hb (Evans) causing mild hemolytic anemia. Genetic diagnosis of 92% (49/53) of the included cases could be determined. The 4 undiagnosed patients will be further analyzed by 33 candidate genes and WES (ERN‐EuroBloodNet –SOLVE). LoRRca showed a sensitivity of 100% for HS (83% for HE). EBT showed a total sensitivity of 85% for HS (cut‐off >21%) while increasing the cut‐off to >16% would increase sensitivity to 93% without increasing false positive, confirming results already reported (Llaudet, 2017).

Summary/Conclusion:

Establishing an integrative harmonized diagnostic algorithm, including LoRRca and t‐NGS has been revealed essential for high diagnosis performance and reduce misdiagnosis, thus, directly impacting on better personalized diagnosis for RA.

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