Abstract

Abstract 4028

Poster Board III-964

CDAII, the most frequent type of congenital dyserythropoietic anemia family, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblasts' morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Recent studies indicated that CDAII is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. Linkage analysis located the CDAII gene in a 5 cM region on chromosome 20 and several candidate genes have been excluded. We recently investigated the cytoplasmic proteome of human red blood cells (RBCs) using a combinatorial peptide ligand library as a capturing agent to amplify the signal of low- and very-low abundance proteins: 1578 proteins, most of them unexpected, were identified allowing a deep exploration of the RBC pathways (Roux-Dalvai, Mol Cell Proteomics, 2008). In this study we used a proteomic-genomic approach to identify the candidate gene for CDAII by matching the data on the cytoplasmic proteome of human RBC with the chromosomic localization of CDAN2 locus. The analysis of RBCs cytoplasmic proteome allowed us to identify 17 proteins codified by genes located in the chromosomic region between 20p11.23 and 20q11.23: SNX5, SEC23B, DTD1, NAT5, GINS1, BCL2L1, MAPRE1, CHMP4B, EIF2S2, AHCY, ACSS2, GSS, EIF6, CPNE1, EPB41L1, RPRD1B(C20orf77), TGM2. Most of them were excluded because found to be associated to other diseases, already excluded in CDAII by previous works, or because not related by function to CDAII. Among the remaining proteins we focus on SEC23B for its possible role in the endoplasmic reticulum-to Golgi trafficking and its localisation on 20p11, the region with the highest LOD-score in CDAII after the recent mapping of the markers on the current contigs (Denecke et al, Biochim Biophys Acta, 2009).

The 20 exons and intronic flanking regions of SEC23B gene were analysed by direct sequencing in 16 CDA II patients from 13 families; 13 different mutations were detected among the 28 mutated alleles identified: 6 of them were missense, 2 frameshift, 1 splicing and 4 stop codon. All the missense mutations affected highly conserved aminoacids, and were not found in 100 normal alleles examined. Two of them (c.40C>T and c.325G>A) were detected in various unrelated patients.

Despite all exons and flanking intronic regions were sequenced, one patient failed to show mutations and two cases displayed only one mutation, suggesting the possibility that a second gene could be involved in CDAII. Patients' data are summarised on the table (*,° = members of the same family).

SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles. Very recently it has been shown that knockdown of Zebrafish SEC23B leads to aberrant erythrocyte development (Schwarz et al, Nat Genet, 2009). Even if the exact function of human SEC23B is not completely clarified, abnormalities in this gene are likely to disturb ER-to Golgi trafficking affecting different glycosylation pathways, and ultimately accounting for the cellular phenotype observed in CDA II.

Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 
N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 
C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 
Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 
Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 
S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 
C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 
8° N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
9° N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
10° N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
11 N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 
12* S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 
13* S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 
14 N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
15 N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 
16 S Italy 10.7 87 Yes ?/? ?/? ?/? 
Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 
N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 
C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 
Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 
Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 
S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 
C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 
8° N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
9° N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
10° N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
11 N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 
12* S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 
13* S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 
14 N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 
15 N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 
16 S Italy 10.7 87 Yes ?/? ?/? ?/? 

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.