Mutations in more than 70 genes cause hereditary anemias (HA), a highly heterogeneous group of rare/low frequency disorders in which we included: hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA) and Diamond-Blackfan anemia; hemolytic anemias due to erythrocyte membrane defects, as hereditary spherocytosis and stomatocytosis; hemolytic anemias due to enzymatic defects. The study describes the diagnostic workflow for HA, based on the development of two consecutive versions of a targeted-NGS panel, including 34 and 71 genes, respectively. Seventy-four probands from 62 unrelated families were investigated. Our study includes the most comprehensive gene set for these anemias and the largest cohort of patients described so far. We obtained an overall diagnostic yield of 64.9%. Despite 54.2% of cases showed conclusive diagnosis fitting well to the clinical suspicion, the multi-gene analysis modified the original clinical diagnosis in 45.8% of patients (nonmatched phenotype-genotype). Of note, 81.8% of nonmatched patients were clinically suspected to suffer from CDA. Particularly, 45.5% of the probands originally classified as CDA exhibited a conclusive diagnosis of chronic anemia due to enzymatic defects, mainly due to mutations in PKLR gene. Interestingly, we also identified a syndromic CDA patient with mild anemia and epilepsy, showing a homozygous mutation in CAD gene, recently associated to early infantile epileptic encephalopathy-50 and CDA-like anemia. Finally, we described a patient showing marked iron overload due to the coinheritance of PIEZO1 and SEC23B mutations, demonstrating that the multi-gene approach is valuable not only for achieving a correct and definitive diagnosis, but also for guiding treatment.

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Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca 2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca 2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca 2+ overload, by the Ca 2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca 2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias.

Hereditary erythrocytes disorders include a large group of conditions with heterogeneous molecular bases and phenotypes. We analyzed here a case series of 155 consecutive patients with clinical suspicion of hereditary erythrocyte defects referred to the Medical Genetics Unit from 2018 to 2020. All of the cases followed a diagnostic workflow based on a targeted next-generation sequencing panel of 86 genes causative of hereditary red blood cell defects. We obtained an overall diagnostic yield of 84% of the tested patients. Monogenic inheritance was seen for 69% (107/155), and multi-locus inheritance for 15% (23/155). PIEZO1 and SPTA1 were the most mutated loci. Accordingly, 16/23 patients with multi-locus inheritance showed dual molecular diagnosis of dehydrated hereditary stomatocytosis/xerocytosis and hereditary spherocytosis. These dual inheritance cases were fully characterized and were clinically indistinguishable from patients with hereditary spherocytosis. Additionally, their ektacytometry curves highlighted alterations of dual inheritance patients compared to both dehydrated hereditary stomatocytosis and hereditary spherocytosis. Our findings expand the genotypic spectrum of red blood cell disorders and indicate that multi-locus inheritance should be considered for analysis and counseling of these patients. Of note, the genetic testing was crucial for diagnosis of patients with a complex mode of inheritance.

The erythroferrone (ERFE) is the erythroid regulator of hepatic iron metabolism by suppressing the expression of hepcidin. Congenital dyserythropoietic anemia type II (CDAII) is an inherited hyporegenerative anemia due to biallelic mutations in the SEC23B gene. Patients with CDAII exhibit marked clinical variability, even among individuals sharing the same pathogenic variants. The ERFE expression in CDAII is increased and related to abnormal erythropoiesis. We identified a recurrent low‐frequency variant, A260S, in the ERFE gene in 12.5% of CDAII patients with a severe phenotype. We demonstrated that the ERFE‐A260S variant leads to increased levels of ERFE, with subsequently marked impairment of iron regulation pathways at the hepatic level. Functional characterization of ERFE‐A260S in the hepatic cell system demonstrated its modifier role in iron overload by impairing the BMP/SMAD pathway. We herein described for the first time an ERFE polymorphism as a genetic modifier variant. This was with a mild effect on disease expression, under a multifactorial‐like model, in a condition of iron‐loading anemia due to ineffective erythropoiesis.

The hereditary anemias are a relatively heterogeneous set of disorders that can show wide clinical and genetic heterogeneity, which often hampers correct clinical diagnosis. The classical diagnostic workflow for these conditions generally used to start with analysis of the family and personal histories, followed by biochemical and morphological evaluations, and ending with genetic testing. However, the diagnostic framework has changed more recently, and genetic testing is now a suitable approach for differential diagnosis of these patients. There are several approaches to this genetic testing, the choice of which depends on phenotyping, genetic heterogeneity, and gene size. For patients who show complete phenotyping, single-gene testing remains recommended. However, genetic analysis now includes next-generation sequencing, which is generally based on custom-designed targeting panels and whole-exome sequencing. The use of next-generation sequencing also allows the identification of new causative genes, and of polygenic conditions and genetic factors that modify disease severity of hereditary anemias. In the research field, whole-genome sequencing is useful for the identification of non-coding causative mutations, which might account for the disruption of transcriptional factor occupancy sites and cis-regulatory elements. Moreover, advances in high-throughput sequencing techniques have now resulted in the identification of genome-wide profiling of the chromatin structures known as the topologically associating domains. These represent a recurrent disease mechanism that exposes genes to inappropriate regulatory elements, causing errors in gene expression. This review focuses on the challenges of diagnosis and research into hereditary anemias, with indications of both the advantages and disadvantages. Finally, we consider the future perspectives for the use of next-generation sequencing technologies in this era of precision medicine.

CDA type I is a rare hereditary anemia, characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in one of the two genes: (i) CDAN1 , encoding Codanin-1, which is implicated in nucleosome assembly and disassembly; (ii) C15orf41 , which is predicted to encode a divalent metal ion-dependent restriction endonuclease with a yet unknown function. We described two cases of CDA type I, identifying the novel variant, Y94S, in the DNA binding domain of C15orf41, and the H230P mutation in the nuclease domain of the protein. We first analyzed the gene expression and the localization of C15orf41. We demonstrated that C15orf41 and CDAN1 gene expression is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Moreover, we functionally characterized the two variants, establishing that the H230P leads to reduced gene expression and protein level, while Y94S induces a slight decrease of expression. We demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. However, no altered nuclear-cytosolic compartmentalization of mutated C15orf41 was observed. Both mutants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle in these cells. Our functional characterization demonstrated that the two variants have different effects on the stability of the mutated mRNA, but both resulted in impaired erythroid maturation, suggesting the block of cell cycle dynamics as a putative pathogenic mechanism for C15orf41-related CDA I.

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