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• W4385752199 abstract "The diagnostic definition of mild haemolysis is often difficult and complex; this may be due to the lack of univocal laboratory features, and, in some cases, to the impossibility of identifying genetic variants that can fully explain the clinical picture of the patients. In fact, despite the availability of sensitive and specific laboratory investigation and sophisticated molecular approaches it is still not possible to clarify all cases of congenital haemolytic anaemia, even in the face of a clear hereditary aetiology.1-3 We describe five patients with a lifelong history of haemolysis that remained undefined after classical diagnostic workflow, representing an example of a diagnostic challenge often encountered in cases, mostly adults, with faint clinical features. Patients displayed mild or compensated haemolysis with reticulocytosis and decreased aptoglobin (Table 1). Red blood cell (RBC) morphology was slightly altered, showing moderate anysocytosis, the presence of spherocytes (2%–12%), ovalocytes (4%–8%) and very rare stomatocytes. Osmotic fragility tests were mostly normal, acidified glycerol lysis test (AGLT50) and Pink test only were positive in three patients, and NaCl osmotic fragility was slightly increased in one. Eosin-5' maleimide (EMA)-binding, considered the gold standard test for diagnosis of hereditary spherocytosis (HS),4 was borderline in one patient (case 2, 11% decrease in fluorescence) and normal in the others. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis did not show any quantitative abnormality in RBC membrane proteins. Osmoscan curve performed by LoRRca MaxSis (Mechatronics)5 was slightly altered and suggestive of an RBC membrane defect, even if not typical for HS (Figure 1). In particular, the area under the curve (AUC) was decreased in all patients, Omin was normal or at high reference limits, and EImax was normal or only slightly decreased; Ohyper was decreased in three out of five patients. A new parameter was analysed, defined as ‘Ohypo’ and corresponding to the osmolality at 50% of EImax in the hypo-osmolar part of the curve; interestingly, cases I.1 and II.1, belonging to the same family, had overlapping curves with decreased Ohyper, whereas patients 2, 3 and 4 showed similar curves characterized by increased ‘Ohypo’. 4% ovalocytes 4% stomatocytes 2% spherocytes 8% ovalocytes 4% spherocytes 7% ovalocytes 6% spherocytes rare schistocytes 12% spherocytes 6% ovalocytes rare stomatocytes 8% ovalocytes 3% spherocytes 2% schistocytes rare stomatocytes SPTA1 αLELY HET SPTA1 αLEPRA HET Diagnostic uncertainty after haematological and biochemical investigations prompted us to investigate the genetic defect using a targeted-next-generation sequencing (NGS) panel containing 49 genes associated with congenital haemolytic anaemias (Table S1) on a MiSeq platform (Illumina). Molecular analysis showed in all patients the presence of a heterozygous null allele (non-sense, frameshift, splicing) in SPTA1 gene (per se not considered sufficient to cause hemolysis), together with a low-expression polymorphic SPTA1 allele (αLELY: c.6531-12C>T, c.5572C>G; αLEPRA: c.4339-99C>T). No other pathogenic variants were identified in genes associated with congenital haemolytic anaemias. Family studies performed in families 1, 3 and 4 confirmed the in trans segregation of αLELY allele (Figure S1), whereas it was present at a homozygous state in patient 2. Alpha-spectrin is overproduced in normal RBCs at least fourfold, thus explaining why pathogenic SPTA1 variants resulting in quantitative defect cause HS transmitted in a recessive way. The two low-expression SPTA1 polymorphisms detected in this series are well known in the literature. In particular, αLELY (Low Expression LYon) with a minor allele frequency (MAF) of 25.6% (ExAC, gnomAD), causes partial skipping of exon 46 leading to about 50% reduced amount of alpha-spectrin6; αLEPRA (Low Expression PRAgue) (MAF 0.426%), located in intron 30, partially activate an alternative acceptor splice site at position −70, resulting in frameshift and premature termination of translation with decreased alpha-spectrin production of about 16%.7 αLELY allele was considered not affecting the phenotype when transmitted in trans with a SPTA1 variant associated with HS,8, 9 whereas it enhances the severity of the disease when is found in compound heterozygosity with SPTA1 pathogenic variants causing elliptocytosis.10 Due to the αLEPRA more drastic reduction in spectrin content, its in trans transmission with a null SPTA1 allele has been shown to cause severe autosomal recessive HS that resolves with splenectomy.7, 9 We herein hypothesize that a null SPTA1 allele in compound heterozygosity with a low-expression polymorphism may result in a spectrin quantitative reduction per se not sufficient to be detected by SDS–PAGE analysis or to alter specific laboratory tests such as EMA-binding, but enough to destabilize the cytoskeleton thus resulting in a mild form of HS. Actually, our patients are characterized by mild or compensated anaemia, some laboratory markers comparable to what was reported in a large series of HS cases11 (median absolute reticulocytes number 220 vs. 240 × 109/L; median unconjugated bilirubin 1.46 vs. 1.8 g/dL, undetectable aptoglobin), and lower median spherocyte number (5% [range 2–12] vs. 8% [1–56]). However, negative EMA-binding test and SDS-PAGE not allowed a diagnosis of HS. In addition, the LoRRca Osmoscan curve was slightly altered and all the canonical parameters diagnostic for HS (Omin, EI, and Ohyper), were normal or at borderline values; despite this, the patients displayed a constantly reduced AUC indicating a decreased RBC deformability (77% reduction of normal area vs. 73% of normal in typical HS patients5) the right shift of the new point of the curve, defined as ‘Ohypo’ parameter, contributes to the reduction of the AUC value. Similar results were reported by Svidnicki et al.,12 who also found a very mild clinical phenotype in relatives carrying only one null SPTA1 allele, suggesting a dominant negative effect of these variants; in our series, the only informative relative with heterozygous null allele was clinically and haematologically normal (Figure S1C), as already reported in other families.1 In conclusion, the data reported in this study are suggestive of a phenotypic effect of compound heterozygosity of αLELY and a null SPTA1 allele, resulting in mild haemolytic anaemia that may be missed by the diagnostic tests specific for HS. The frequency of such cases may be underestimated due to the slight clinical manifestation and in the absence of molecular characterization; in fact, recent studies have demonstrated that recessive HS due to SPTA1 biallelic variants may be more common than expected.2, 8, 13 A careful analysis of Osmoscan curve may provide an indication, and molecular investigation by NGS may clarify the underlying genotype14 leading to an accurate diagnosis; this may be also of importance for genetic counselling since it is known that null SPTA1 variants are associated with a severe phenotype when inherited at homozygous or compound heterozygous level. This research was funded by the Italian Ministry of Health - Current research IRCCS, Fondazione IRCCS Ca' Granda Policlinico Milano, project n. RC2022 175/05. This work is generated within the European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet, FPA 739541). WB, PB, and EF are EuroBloodNet members. Elisa Fermo and Paola Bianchi designed the study, analysed the results and prepared the manuscript; Anna Zaninoni performed ektacytometric analysis; Cristina Vercellati and Anna Paola Marcello performed laboratory diagnosis of patients; Paola Castronovo and Ilaria Bestetti performed NGS sequencing; Wilma Barcellini and Bruno Fattizzo cared for the patients. All the authors provided a critical review of the manuscript. All data generated or analysed during this study are included in this published article. All the authors declare no conflicts of interest related to this study. The study received the approval from the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Ethic Commitee (Study Cytopan: approval Milano Area 2, 201_2021bis). Peripheral blood was collected during diagnostic procedures after obtaining informed consent and approval from the Institutional Human Research Committee, in accordance with the Helsinki International ethical standards on human experimentation. Figure S1. Table S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W4385752199 date "2023-08-11" @default.
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• W4385752199 title "When alpha spectrin null alleles meet low expression alpha spectrin polymorphisms" @default.
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• W4385752199 doi "https://doi.org/10.1111/bjh.19038" @default.
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