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Haplotype analysis and targeted next-generation resequencing allowed us to identify a mutation in the KIF23 gene and to show its association with an autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III). The region at 15q23 where CDA III was mapped in a large Swedish family was targeted by array-based sequence capture in a female diagnosed with CDA III and her healthy sister. Prioritization of all detected sequence changes revealed 10 variants unique for the CDA III patient. Among those variants, a novel mutation c.2747C>G (p.P916R) was found in KIF23, which encodes mitotic kinesin-like protein 1 (MKLP1). This variant segregates with CDA III in the Swedish and American families but was not found in 356 control individuals. RNA expression of the 2 known splice isoforms of KIF23 as well as a novel one lacking the exons 17 and 18 was detected in a broad range of human tissues. RNA interference-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23/MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

Introduction: Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis is also seen in congenital dyserythropoietic anemia type II (CDA II). Reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55 and CD59 on erythrocytes in congenital dyserythropoietic anemia type III (CDA III). 

Methods: Erythrocytes from 16 CDA III positive individuals, 14 CDA III negative relatives and three normal controls per assay were studied with flow cytometry after EMA staining. Flow cytometry after anti-CD55 and anti- CD59 was performed on erythrocytes from 12 CDA III positive and 7 CDA III negative relatives with one normal control per assay. 

Results: CDA III - erythrocytes exhibited marginally stronger fluorescence after EMA-staining than normal controls. Correlation between EMA fluorescence and erythrocyte volume was confirmed. CDA III subjects did not differ from normal controls concerning CD55 and CD59. 

Conclusion: The results of the present study indicate no abnormality of the erythrocyte membrane in CDA III and show that standard flow cytometry cannot be used to discriminate between CDA III and normal controls. 

Background: Congenital dyserythropoietic anemia type III (CDA III) can be caused by mutation in KIF23. CDA III differs from CDA I and II in the sense that secondary hemochromatosis has not been reported. However, we have observed elevated serum ferritin in a CDA III family. Since primary hemochromatosis is common in Northern Europe we decided to screen the family for HFE mutations.

Aim: Study clinical appearance and prevalence of HFE gene mutations, C282Y and H63D, in a CDA III family. 

Methods: DNA from 37 CDA III patients and 21 non-affected siblings was genotyped. Iron status from EDTA plasma was measured in 32 of the CDA III patients and 18 of the non-affected siblings.

Results: Out of 37 CDA III patients, 18 carried heterozygous HFE mutations and six were compound heterozygotes. Out of 21 CDA III negative siblings, nine had heterozygous HFE mutations, two were homozygous (one H63D and one C282Y), and two were compound heterozygous. None of the patients with wt HFE, regardless of CDA III status, suffered from iron overload. Four patients with HFE mutations needed treatment with phlebotomy to normalize ferritin and transferrin iron saturation; one CDA III negative patient with homozygous C282Y, two CDA III patients with heterozygous HFE mutations and one CDA III case with compound heterozygosity.

Conclusion: HFE mutations were found in 65 % of CDA III patients and in 62 % of their CDA III negative siblings. Heterozygous HFE mutation, C282Y and even H63D, can cause iron overload when occurring concomitantly with CDA III.