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Congenital dyserythropoietic anemia type III (CDA III) is caused by a mutation in kinesin family member, KIF23
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, no 23, p. 4791-4799Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Washington: American Society of Hematology , 2013. Vol. 121, no 23, p. 4791-4799
National Category
Hematology
Identifiers
URN: urn:nbn:se:umu:diva-70201DOI: 10.1182/blood-2012-10-461392ISI: 000321895700025PubMedID: 23570799Scopus ID: 2-s2.0-84881035005OAI: oai:DiVA.org:umu-70201DiVA, id: diva2:620098
Available from: 2013-05-07 Created: 2013-05-07 Last updated: 2023-03-23Bibliographically approved
In thesis
1. Congenital Dyserythropoietic Anemia type III (CDA III): diagnostics, genetics and morbidity
Open this publication in new window or tab >>Congenital Dyserythropoietic Anemia type III (CDA III): diagnostics, genetics and morbidity
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Congenital Dyserythropoietic Anemias (CDA) are rare hereditary hemolytic disorders with large bi- to multi-nucleated erythroblasts in the bone marrow. Hemolysis is negative in a direct antiglobulin test (DAT). Based on morphology and clinical picture, three major forms of CDAs, type I, II, and III have been defined. CDA III, dominantly inherited, constitutes the rarest type with a majority of cases belonging to a family in Västerbotten, Sweden. The genetic background of CDA I and CDA II has been linked to mutations in CDAN1 and SEC23B respectively. The mutation of CDA III has been linked to 15q22 in earlier studies.

In this project we have defined the causative genetic lesion in two families with CDA III. The novel mutation KIF23 c.2747C>G (p.P916R) was shown to segregate with CDA III in the Swedish and American CDA III families and was absent in 356 healthy controls. KIF23 encodes mitotic kinesin-like protein 1 (MKLP1), which plays a central role in the last step of cytokinesis. RNAi-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, resulting in increasing number of bi-nuclear cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23, encoding MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

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 CDA II, while reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55, and CD59 on erythrocytes in CDA III. We found no abnormality of the erythrocyte membrane in CDA III and concluded that standard flow cytometry cannot be used to discriminate between CDA III and normal controls.

In CDA I and CDA II a majority of patients, including those who are not transfusion dependent, suffer from iron overload, which, according to earlier studies, is not the case in CDA III. We found that individuals of the Västerbotten CDA III family carry mutations in the hemochromatosis (HFE) gene. Three CDA III patients with heterozygous or compound HFE mutations need treatment with phlebotomy due to iron overload. One of them carries heterozygous H63D mutation, which is not reported to lead to iron overload by itself in otherwise healthy individuals. We propose that molecular genetic testing of the HFE gene is indicated in all patients with CDA, including CDA III.

Place, publisher, year, edition, pages
Umeå: print och media, 2016. p. 54
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1784
Keywords
congenital dyserythropoietic anemia, KIF23, hereditary hemochromatosis, iron overload, flow cytometry
National Category
Hematology
Identifiers
urn:nbn:se:umu:diva-117454 (URN)978-91-7601-424-0 (ISBN)
Public defence
2016-04-22, E04, By 6E, 901 85, Norrlands Universitets sjukhus, Umeå, 19:35 (Swedish)
Opponent
Supervisors
Available from: 2016-04-15 Created: 2016-02-29 Last updated: 2018-06-07Bibliographically approved
2. Mitotic Kinesin-Like Protein 1 (MKLP1/KIF23) in hereditary congenital dyserythropoietic anemia type III and in cancer
Open this publication in new window or tab >>Mitotic Kinesin-Like Protein 1 (MKLP1/KIF23) in hereditary congenital dyserythropoietic anemia type III and in cancer
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A hereditary form of autosomal dominant congenital dyserythropoietic anaemiatype III (CDA III) has been reported in four families from Sweden, Argentina, Cuba and USA. CDA III patients might experience signs of mild anaemia and some of them need occasional blood transfusions. Other clinical features seen in CDA III patients are retinal angioid streaks, monoclonal gammopathy of undetermined significance and multiple myeloma. Their bone marrow is characterised by presence of giant erythroblasts with up to 12 nuclei. Previously, CDA III was mapped to a region on chromosome 15q21-q25.

In this study we aimed to identify the genetic cause of CDA III, investigate the reasons why erythroid lineage in the patients’ bone marrow is mostly affected, and seek the explanation of increased rate of cancer in the Swedish CDA III family.

We identified the genetic cause of CDA III using targeted next generation sequencing. A novel missense mutation c.2747C>G, p.P916R in kinesin familymember 23 gene (KIF23) segregated with the disease in both the American and Swedish family, and was absent in databases of sequence variants from healthy individuals. Knock-down and rescue experiments in HeLa Kyoto cells showed that the P916R mutation caused cytokinesis failure which resulted in large cells with several nuclei. This was consistent with the CDA III phenotype.

To reveal interaction partners of wild-type and mutant KIF23 proteins, pull-down experiments followed by mass spectrometry and Western blot analysis were performed. This identified Coatomer Protein Complex I (COPI), a vesicle forming complex responsible for intracellular transport, as a KIF23 interactor. By using immunofluorescence and fluorescence microscopy, we showed that COPI subunits COPα and COPβ localize to the midbody during cytokinesis. These findings indicate involvement of vesicle transport proteins in mitosis and cytokinesis, though the significance of COPI-KIF23 interaction in cell division remains to be uncovered.

To address the question if other cells are affected by the KIF23 P916R mutation, we created a knock-in mouse model with Kif23 c.2726C>G, p.P909R, which corresponds to the human KIF23 c.2747C>G. However, the mice did not developany phenotype indicating CDA III. This result was consistent with the studies ofother CDA subtypes where mouse models failed, suggesting that CDA occur only in humans. Our study of human and mouse KIF23/Kif23 expression revealed novel, previously not annotated transcripts, one in human and two in mice. Expression analysis of total mRNA using droplet digital PCR demonstrated an extensive variation of KIF23 and Kif23 expression levels in all tissues. The shortest Kif23 transcript lacking exon 17 and 18 was prevalent in mice, while corresponding transcript in human was the least expressed.

Considering the importance of KIF23 in cytokinesis and KIF23 association with cancer, we hypothesized that somatic KIF23 mutations might be overrepresented in cancer. For this purpose, we screened KIF23 and its promoter in non-small cell lung cancer samples that previously demonstrated KIF23 overexpression. No pathogenic driving KIF23 variants were detected by Sangersequencing; however, subsequent genome-wide genotyping (SNP-array) detected gain of chromosome 15 in most cases that could possibly explain KIF23 overexpression.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2021. p. 59
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2117
Keywords
Alternative splicing, Cell division, COPI, Congenital dyserythropoietic anemia, Cytokinesis, Gene expression, KIF23 protein human, Cancer, MKLP1 protein mouse, Mutation
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-179788 (URN)978-91-7855-470-6 (ISBN)978-91-7855-471-3 (ISBN)
Public defence
2021-03-05, Hörsal Betula, byggnad 6M, målpunkt L, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
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Note

P.g.a. covid 19 uppmanas åhörare att delta digitalt via Zoom. Länk: https://umu.zoom.us/j/65108113265

Available from: 2021-02-12 Created: 2021-02-10 Last updated: 2021-02-10Bibliographically approved

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Liljeholm, MariaNorberg, AnnaSandström, HerbertWahlin, AndersGolovleva, Irina

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