Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I

Abstract

The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.

Figure 1.

Pedigrees of three families with novel non-synonymous variants of C15ORF41. Black filled symbols indicate individuals with CDA-I. Chromatograms from a control individual (upper) and the proband (lower) show sequence changes identified, together with the amino acids encoded by the change and by adjacent codons. Positions of nucleotide changes are shown above the chromatogram and are given according to numbering of the base within the open reading frame in the C15ORF41 cDNA (ENST00000566621). The lower two panels also show electron micrographs of intermediate erythroblasts from each proband clearly showing the spongy heterochromatin (arrowed) indicative of CDA-I. Probands are indicated by arrows. Below each symbol the status of that individual for the change identified in the proband is shown.

Figure 2.

C15ORF41 gene and protein structure. (A) Schematic representation of the C15ORF41 gene, exons are shown to scale with coding sequence shown in white and UTRs in black. Red numbers above lines indicate intron sizes (not to scale); numbers above exons indicate exon number; asterisks indicate the two exons in which mutations have been identified. Black arrow heads indicate locations of primers used to amplify the C15ORF41 transcript. The lower section shows the C15ORF41 protein with annotated domains shown to scale. (B,C) Predicted tertiary structure of the conserved domains identified in C15ORF41. Missense changes found in CDA-I patients are shown using sticks (Y94C and L178Q) and labeled in black. (B) Two helix-turn-helix domains predicted for the N-terminal of C15ORF41 (amino acids 4–129). Helices are numbered and putative DNA interaction helices are shown in blue (H3 and H6). The displayed DNA molecule was extracted from Rhee et al. (C) The PD-(D/E)XK nuclease domain predicted for the C-terminal region of C15ORF41 (amino acids 161–259). Highly conserved residues in the PD-(D/E)XK nuclease superfamily that form part of its active centre are labeled in red and side chains are shown using sticks. (D) Purified recombinant C15ORF41 protein was treated with varying concentrations of trypsin (lanes 2–4) for 1 hour at 37°C and digests were analyzed by SDS-PAGE. As a control, another recombinant protein (human RECQ1) was similarly digested (lanes 5–7). Lane 1: size markers. Lanes 2, 5: no trypsin. Lanes 3, 6: 4 μg/mL trypsin. Lanes 4, 7: 100 μg/mL trypsin. The arrow indicates the location of trypsin, * is C15ORF41, ** is RECQ1.