ADDITIVE EFFECT OF MULTIPLE GENETIC VARIANTS IN SEC23B-PIEZO1 ON IRON METABOLISM DYSHOMEOSTASIS

Antonella NOSTROSO1,2, Teresa CEGLIE6, Achille IOLASCON1,2, Immacolata ANDOLFO1,2, Roberta RUSSO1,2, Barbara Eleni ROSATO1,2, Roberta MARRA1,2, Federica Maria ESPOSITO1,2, Vanessa D'ONOFRIO1,2, Michela RIBERSANI3, Anna BULLA4, Giovanni Carlo DEL VECCHIO5, Giorgia MANDRILE6

1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli ‘Federico II’, Napoli, Italy
2CEINGE Biotecnologie Avanzate Franco Salvatore, Napoli, Italy
3Università Sapienza di Roma Sezione Ematologia, Dipartimento di Medicina Traslazionale e di Precisione, Roma, Italy
4Azienda Ospedaliera Universitaria Policlinico “G. Rodolico – San Marco” , Catania, Italy
5U.O.C. Pediatria Generale e Specialistica " Bruno Trambusti", Bari, Italy
6SSD Microcitemie, AOU San Luigi Gonzaga, Regione Gonzole 10, Orbassano, Italy

Hereditary anemias (HA) are heterogeneous conditions characterized by a complex genotype-phenotype correlation. The two main groups include hypo-productive anemias and hemolytic anemias due to altered deformability of erythrocytes. Within these groups, the most frequent conditions are, respectively, congenital dyserythropoietic anemia type II (CDAII) (caused by biallelic mutations in the SEC23B) and dehydrated stomatocytosis (DHS) (mostly caused by gain-of-function mutations in the PIEZO1). Although the different pathogenesis, iron overload, and reduced hepcidin levels represent their main complications.

Hereditary anemias can be caused by multi-locus inheritance. Indeed, in our cohort of patients (n=583), 34% exhibit multi-locus inheritance. Among them, 8 patients exhibit pathogenic variants in both SEC23B and PIEZO1 genes. They show intermediate levels of anemia (RBC 3.7x106/µL, Hb 10.1 g/dL, MCV 92.3 fL) but a more pronounced iron overload (ferritin 646.3 ng/mL, IS 83.4%) compared to patients with isolated defects. In vitro functional assays demonstrated that silencing of SEC23B in a cellular hepatic model (Hep3B) engineered with R2456H-PIEZO1-GoF variant (Hep3B-KI) induced a stronger downregulation of HAMP gene expression compared to Hep3B-WT, Hep3B-shSEC23B, and Hep3B-KI. Additionally, shSEC23B/PIEZO1-KI showed increased ferritin expression at both mRNA and protein levels. To further characterize the alteration of iron status in shSEC23B/PIEZO1-KI, we assessed the inhibition of the BMP6/SMADs pathway. Analysis of target genes revealed a more pronounced downregulation of SMAD6, ID1, and ID3 in shSEC23B/PIEZO1-KI, compared to Hep3B-shSEC23B and Hep3B-KI. These data confirm the additive effect of the two causative genes on iron dyshomeostasis and the importance of accurate diagnostic classification for personalized medicine.