Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1

Abstract

Congenital dyserythropoietic anemias (CDAs) constitute a rare group of inherited red-blood-cell disorders associated with dysplastic changes in late erythroid precursors. CDA type I (CDAI [MIM 224120], gene symbol CDAN1) is characterized by erythroid pathological features such as internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane, carrying cytoplasmic organelles into the nucleus. A cluster of 45 highly inbred Israeli Bedouin with CDAI enabled the mapping of the CDAN1 disease gene to a 2-Mb interval, now refined to 1.2 Mb, containing 15 candidate genes on human chromosome 15q15 (Tamary et al. 1998). After the characterization and exclusion of 13 of these genes, we identified the CDAN1 gene through 12 different mutations in 9 families with CDAI. This 28-exon gene, which is transcribed ubiquitously into 4738 nt mRNA, was reconstructed on the basis of gene prediction and homology searches. It encodes codanin-1, a putative o-glycosylated protein of 1,226 amino acids, with no obvious transmembrane domains. Codanin-1 has a 150-residue amino-terminal domain with sequence similarity to collagens and two shorter segments that show weak similarities to the microtubule-associated proteins, MAP1B (neuraxin) and synapsin. These findings, and the cellular phenotype, suggest that codanin-1 may be involved in nuclear envelope integrity, conceivably related to microtubule attachments. The specific mechanisms by which codanin-1 underlies normal erythropoiesis remain to be elucidated.

Figure 1

Physical map and genomic organization of the CDAN1 locus. a, Relative positions of landmark microsatellite markers are indicated in the CDAI candidate interval defined by markers D15S779 and D15S778. The CDA1 interval is indicated by green and brown arrows. An informative SNP (pink) within the newly defined transcript LOC146050 enabled us to restrict the linkage interval. The genes and transcripts identified are boxed: known genes in green, unknown transcripts in blue, inferred genes in purple, CDAN1 in red. The expression patterns of these genes were tested by RT-PCR on RNA from erythroid precursor cells (Pope et al. 2000) and are marked by a plus sign (+) or a minus sign (−) in the Express lane. Resequenced genes are marked by a plus sign (+) or a minus sign (−) in the Reseq lane. b, Expanded view of the 15-kb segment showing the genomic organization of CDAN1 and of two alternative transcripts isolated and sequenced from erythroid and fibroblast cells. Coding and noncoding exons are depicted as filled and open boxes, respectively. Red and blue exons are based on two partial transcripts, DKFZp434G2127 and BI855138, respectively. Green and pink exons are based on gene prediction and RT-PCR, respectively. The lengths of the corresponding transcripts and inferred proteins are indicated to the right.

Figure 2

The mutation in a Bedouin family. A, Pedigree of a Bedouin family, showing the segregation of the founder chromosome with the disease. B, Sequence chromatogram from a wild-type (WT) and diseased (M) genotype showing a C→T substitution in exon 25, converting arginine to tryptophan at codon 863. C, Segregation of the Bedouin mutation, as assayed by NcoI restriction. A 412-bp PCR product is generated between the F and R primers. The mutation results in a gain of restriction site and, therefore, the restriction digest produced fragments of 175 and 237 bp, instead of the 412-bp band observed in healthy individuals.

Figure 3

Northern blot analysis. Panels A and B show CDAN1 and β-actin mRNA exposed for 1 week and overnight, respectively. The membrane was purchased from Clontech and was hybridized according to the manufacturer’s instructions. The membrane was first probed with codanin-1 (exons 26–28 [fig. 1]) and subsequently reprobed with β-actin cDNA as an internal control.

Figure 4

Multiple alignment of human codanin-1. Multiple alignment with its putative mouse and fugu orthologs. Identical and similar residues in the multiple-alignment chart are represented by asterisks (*) and colons (:), respectively; weak conservation is represented by periods (.). Conserved BLOCKS are confined within blue boxes. Shaded boxes are used for known block similarity: green, fibrillar collagens (IPB00885) (A, G); pink box, MAP1B (IPB000102) (C, E). Arrows indicate positions of mutated residues; green and blue arrows specify missense mutations within families I–VI and VII–IX, respectively, and red arrows specify null mutations. Note the cluster of mutations around aa 1034–41 and 866–867. The thick black arrow indicates the region with shared similarity to fibrillar collagens. The thin black line represents a split between exons.