The diagnosis of CDA I was based on history, clinical findings, laboratory data, morphological analysis of both peripheral blood and marrow smears, and genetic testing.

Local university ethical committees approved both the DNA sampling and the collection of patients’ data from Medical Genetics Ambulatory in Naples (University Federico II, DAIMedLab).

Written informed consent was obtained from the patients for the participation in the study and the publication of the case report.

Genomic DNA preparation and mutational screening for CDAN1, SEC23B, and C15orf41 genes by direct sequencing were performed as previously described (Russo et al., 2013). High-throughput sequencing by the custom multi-gene panel for hereditary anemias was performed as described (Russo et al., 2018).

The pathogenicity of the novel exonic variants has been evaluated by InterVar, a bioinformatics software tool for clinical interpretation of genetic variants based on the ACMG/AMP 2015 guideline¹. Mainly, the pathogenicity of each variant was assessed by gathering evidence from various sources: population data, computational and predictive data, functional data, localization of the variant in a mutational hotspot and critical and well-established functional domain, and segregation data (Richards et al., 2015; Russo et al., 2018).

cDNA encoding full-length wild-type (WT) C15orf41 sequence was cloned in the pCMV-Tag1 vector for mammalian cell expression (Invitrogen) in the BglII and XhoI sites, to obtain an N-terminal tagged protein with FLAG. The point mutations c.281A > C, p.Tyr94Ser (Y94S) and c.689A > C, p.His230Pro (H230P) were introduced into the pCMV-Tag1 vector by using a QuikChange site-directed mutagenesis kit (Stratagene) (Russo et al., 2017). The coding sequence was sequenced after mutagenesis.

Hek-293, HepG2, HuH7, MG-63, HEL, and K562 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) or RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin (Invitrogen), and 100 mg/mL streptomycin (Invitrogen) in a humidified 5% CO₂ atmosphere at 37°C, according to the manufacturer’s instructions. Hek-293 cells (400 × 10³) were transfected with pCMV-Tag1-C15orf41 plasmids (2.5 μg/well) using the DNA Transfection Reagent (TransFectin Lipid Reagent, Bio-Rad) according to the manufacturer’s procedures. Cells were collected 16, 24, and 48 h after the transfection to perform RNA and protein extractions. For generating K562 stably over-expressing C15ORF41 gene, 10⁶ cells were transfected with pCMV-Tag1-C15orf41 plasmids using Hily Max DNA Transfection Reagent (Dojindo Laboratories). After 48 h, G418 (0.6 mg/mL) was added as a selection marker. Clones were generated according to the limiting dilution method (see Supplementary Material for further details).

Erythroid differentiation of K562-C15orf41 stable clones (2 × 10⁵/mL) was performed adding 50 μM hemin (Sigma) to the culture medium, after 24 h of starvation (Andolfo et al., 2010). Cells were collected before hemin addition (0 days) and two days after hemin addition (2 days). For cell cycle analysis, K562 stable clones were harvested by centrifugation, resuspended in PBS containing 3.75% Nonidet P-40, 100 μg/ml RNase A and 40 μg/ml propidium iodide, and incubated at room temperature for 3 h in the dark. The cell antigen profile was analyzed by flow cytometry through evaluation of CD71 (proerythroblasts) and CD235a (proerythroblasts and orthochromatic erythroblasts). Samples were analyzed on a FACS flow cytometer (Becton Dickinson Immunocytometry Systems, BDIS).

Total RNA was extracted either from peripheral blood leukocytes (PBLs), reticulocytes and from cell lines using TRIzol reagent (Life Technologies). Synthesis of cDNA from total RNA (2 μg) was performed using SensiFAST^TM cDNA Synthesis Kit (Bioline). Quantitative RT-PCR (qRT-PCR) using Power SYBR Green PCR Master Mix (Applied Biosystems) was performed on Applied Biosystems 7900HT Sequence Detection System using standard cycling conditions. β-actin was used as internal control, while the Neomycin resistance gene was used as a control of transfection efficiency for K562 stable clones. Relative gene expression was calculated by using the 2^−ΔCt method, as described (Russo et al., 2013).

Proteins were extracted from cell lines using RIPA lysis buffer containing protease inhibitor cocktail (1×). Subcellular fractionation in nuclear and cytoplasmic proteins was performed using NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific^TM). Equal amounts of protein from each lysate, as determined by a Bradford assay, were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto polyvinylidene difluoride membranes (Biorad). Detection was performed with mouse anti-FLAG antibody (1:1000) (Sigma-Aldrich) and rabbit anti-C15orf41 (1:500) (Atlas Antibodies HPA061023). Since this antibody was recommended for immunofluorescence (IF) we tested its specificity for western blotting (WB) by using C15orf41 over-expression cells as a positive control (Bordeaux et al., 2010) (Supplementary Figure S1).

Mouse anti-TBP (TATA Binding Protein) (1:1000) (Sigma-Aldrich) and mouse anti-α-TUBULIN (1:5000) (Abcam) were used as a control for equal loading for cytosolic and nuclear proteins’ extracts, respectively. Mouse anti-β-actin (1:12000) (Sigma-Aldrich) was used as a loading control for total proteins’ extracts. Labeled bands were visualized and densitometric analysis performed with the BioRad Chemidoc using Quantity One software (BioRad) to obtain an integrated optical density (OD) value.

For IF analysis 3 × 10⁵ cells were fixed for 10 min in 4% Paraformaldehyde (PFA, Sigma) and washed in 50 mM PBS/NH4Cl (Sigma-Aldrich, Milan, Italy). After washing in PBS 1×, cells were allowed on 35 mm IBIDI μ-Dishes (Ibidi GmbH, Martinsried, Germany) coated with 0.05% poly-L-lysine (Sigma-Aldrich, Milan, Italy) to adhere. Permeabilization was performed with 0.2% Triton/PBS, followed by blocking with 1% BSA/PBS. The seeded cells were immunologically stained with rabbit anti-C15orf41 antibody (1:25) (Atlas Antibodies HPA061023), mouse anti-NUCLEOPHOSMIN (1:200), and secondary antibodies (1:200) (Alexa Fluor 546 anti-rabbit, Life Technologies and Alexa Fluor 488 anti-mouse). Nuclei were stained with 1 μg/ml DRAQ5 in PBS for 15 min at room temperature. Cells were preserved in PBS 1× and imaged using a LEICA TCS SP8 meta confocal microscope, equipped with an oil immersion plan Apochromat 63× objective 1.4 NA. The following settings were used: Green channel excitation of Alexa488 by the argon laser 488 nm line was detected with the 505–550 nm emission bandpass filter. Red channel excitation of Alexa546 by the Helium/Neon laser 543 nm line was detected with the 560–700 nm emission bandpass filter (using the Meta monochromator). Blue channel excitation of DRAQ5 by the blue diode laser 647 nm and emission bandpass filter.

Statistical significance of differences in protein and gene expression was determined using the Mann–Whitney test or Student’s t-test. Correlation analysis of C15orf41 with CDAN1 gene expression was performed by Pearson correlation test. A two-sided p-value < 0.05 was considered statistically significant.

For the in silico correlation analysis between C15orf41 and CDAN1 gene expression in normal hematopoietic cell subpopulations we used the dataset “Normal Hematopoietic Subgroups – (GEO ID: gse19599),” stored in the R2: Genomics Analysis and Visualization Platform², a biologist-friendly, web-based genomics analysis, and visualization application.

Clinical features and genetic data of the two probands are summarized in Table 1. Case 1 (A-II.2) was a 7-years-old female, second child from healthy non-consanguineous parents of Italian origin (Sardinia). At birth, cholestatic hepatopathy, dysmorphic features (bilateral syndactyly of the IV–V toes), and severe anemia (Hb 5.5 gr/dl) were observed. Family history was not indicative of anemia. At diagnosis, the proband presented transfusion-dependent normocytic anemia with a blood transfusion frequency every 15–20 days, and low reticulocyte count (Table 1). BM analysis showed: erythroid hyperplasia with 6% of cells showing megaloblastic features, nuclear abnormalities, and nuclear/cytoplasmic maturation asynchrony; 4% of erythroblasts were bi- and tri-nucleated; the granulopoietic/erythropoietic ratio (G:E) = 0.53. A substantial percentage of erythroblasts showed inter-nuclear bridges (5%), a typical feature of CDA I. Accordingly, genetic testing for CDAN1 was performed, but no causative variants were identified. Conversely, when we analyzed C15orf41 gene, we observed the presence of the transversion c.281A > C in the homozygous state, resulting in a novel aminoacidic substitution p.Tyr94Ser (Y94S). It is an ultra-rare variant (rs587777101) with a minor allele frequency (MAF) C = 0.00001 in the ExAC database. In agreement with the recessive inheritance pattern, both parents were heterozygous (Figure 1A).

Case 2 was a 2.4-years-old male, born from 3rd degree consanguineous parents of Turkish origin. At birth, recurrent pneumonia, thoracic dysplasia, and short limbs were observed. Family history was negative for anemia or jaundice. The proband presented transfusion-dependent normocytic anemia (12 transfusions/year), low reticulocyte count, growth retardation, and increased ferritin level, suggesting an iron loading condition (Table 1). No splenomegaly was observed at physical examination and abdominal echography. BM analysis showed severe megaloblastic changes and normoblasts with double or multiple nuclei, a morphological feature suggestive of CDA II. Accordingly, we firstly performed Sanger sequencing analysis for CDA II-disease gene SEC23B, finding no causative variants. Then, as a second-step analysis, we enrolled the patient in our multi-gene panel for hereditary anemias, identifying the transversion c.689A > C in C15orf41 in the homozygous state, resulting in the amino acid substitution p.His230Pro (H230P), as reported (Russo et al., 2018). In agreement with the recessive inheritance pattern, both parents were heterozygous (Figure 1B).

To evaluate the effect of the two identified mutations on C15orf41 gene expression, we initially analyzed C15orf41 expression in PBLs isolated from the two probands and healthy controls (HCs). To note, C15orf41 is a ubiquitous gene, showing a comparable level of expression in both PBLs and reticulocytes (Supplementary Figure S2). No difference in gene expression levels of the proband A-II.2 compared to those detected in HCs was observed, suggesting that Y94S variant does not affect gene expression. Conversely, we found a marked down-regulation of C15orf41-H230P in the second proband B-II.1 (Figure 2A). Likewise, we saw a similar trend of CDAN1 expression in the two patients. Notably, the proband A-II.2 did not show any alterations of CDAN1 expression compared to those seen in HCs, while the B-II.1 proband revealed a decrease of CDAN1 expression level, although not statistically significant (Figure 2B). Of note, a direct correlation between C15orf41 and CDAN1 expression genes in healthy subjects was observed (r = 0.62, p = 0.0006) (Figure 2C). We confirmed the ex vivo data on C15orf41-CDAN1 correlation by in silico analysis of the expression dataset for normal hematopoietic cell subpopulations, obtained by R2 database (Figure 2D). Additionally, we achieved comparable results by gene expression profiling of different human cell lines (Hek-293, HepG2, HuH7, MG-63, HEL, and K562 cells), where a significant direct correlation between C15orf41 and CDAN1 expression was observed (Figure 2E).

We first assessed the turnover and localization of the C15orf41 protein in Hek293 cells transiently transfected with pCMV-tag1-C15orf41. Time-course analysis showed a gradual increase of C15orf41 gene expression in cells transfected with WT clone at 16, 24, and 48 h compared to those transfected with empty vector (EV) (Figure 3A). Conversely, WB analysis on the same harvested cells revealed a marked increase of C15orf41 protein level at 16 h after transfection, with a progressive decrease of the C15orf41-FLAG signal, which resulted highly down-regulated at 48 h after the transfection (Figure 3B).

To investigate C15orf41 localization, we assessed the endogenous protein levels and localization of the protein by both WB and IF on a nuclear and a cytosolic fraction of Hek-293 cells (Figure 3C,D). Both analyses confirmed that the protein was mainly expressed in the nucleus, but also in the cytosol compartment, even if in a smaller amount, suggesting a role of the protein in these two cellular compartments (Figure 3D and Supplementary Figure S3). No co-localization of C15orf41 with nucleoli was observed (Supplementary Figure S3).

To study in vitro the pathogenetic effect of the two variants, we evaluated gene expression and protein level of both C15orf41-H230P and C15orf41-Y94S mutants at 16 h after transfection in Hek-293. In agreement with the ex vivo data on both patients, we observed a sharp decrease of both gene expression and protein levels in cells over-expressing C15orf41-H230P mutant compared to C15orf41-WT ones (Figure 4A,B and Supplementary Figure S1). Conversely, only a slight reduction in gene expression and protein level in cells over-expressing C15orf41-Y94S was observed (Figure 4A,B and Supplementary Figure S1).

To obtain a reliable cellular model, able to be induced to erythroid differentiation, we developed K562 cells stably over-expressing C15orf41-WT and both mutants. Over-expressing clones were selected by measuring Neomycin relative gene expression in each K562 clone and comparing the digestion pattern of mutant vs. WT clones (Supplementary Figure S4). K562 selected clones WT#3 and Y94S#5 showed strong over-expression of C15orf41 compared to those observed in K562 EV#3 clone. Instead, H230P#10 cells showed a marked gene down-regulation respect to WT#3 cells (Figure 5A). WB analysis confirmed the same trend for all the clones (Figure 5B).

To investigate if both mutants could affect erythroid differentiation, we treated K562 cells with hemin. Evaluation of CD71 and CD235 differentiation markers showed a statistically significant decreased percentage of CD71⁺/CD235⁺ cells in both Y94S and H230P clones compared to the WT one (Figure 5C). Moreover, we observed a slight increase of the rate of S-phase at cell cycle analysis in K562 cells over-expressing Y94S and H230P mutants compared to WT, although not statistically significant (Figure 5D and Supplementary Figure S5). To note, immunolocalization analysis of C15orf41 protein in K562 stable clones highlighted a preferential localization of the mutated proteins within nuclear compartment compared to WT one, similarly to those observed in Hek-293 cells transiently over-expressing C15orf41 mutants (Figure 5E).