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Mutations in C16orf57 and normal-length telomeres unify a subset of patients with dyskeratosis congenita, poikiloderma with neutropenia and Rothmund-Thomson syndrome.

Walne AJ, Vulliamy T, Beswick R, Kirwan M, Dokal I - Hum. Mol. Genet. (2010)

Bottom Line: Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families.Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls.These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Paediatrics, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Barts and The London Children’s Hospital, 4 Newark Street, London, UK. a.walne@qmul.ac.uk

ABSTRACT
Dyskeratosis congenita (DC) is an inherited poikiloderma which in addition to the skin abnormalities is typically associated with nail dystrophy, leucoplakia, bone marrow failure, cancer predisposition and other features. Approximately 50% of DC patients remain genetically uncharacterized. All the DC genes identified to date are important in telomere maintenance. To determine the genetic basis of the remaining cases of DC, we undertook linkage analysis in 20 families and identified a common candidate gene region on chromosome 16 in a subset of these. This region included the C16orf57 gene recently identified to be mutated in poikiloderma with neutropenia (PN), an inherited poikiloderma displaying significant clinical overlap with DC. Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families. In addition, three of six families previously classified as Rothmund-Thomson syndrome (RTS-a poikiloderma that is sometimes confused with PN) were also found to have homozygous C16orf57 mutations. Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls. These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS. This study also highlights the multi-system nature (wider than just poikiloderma and neutropenia) of the clinical features of affected individuals (and therefore house-keeping function of C16orf57), a possible role for C16orf57 in apoptosis, as well as a distinct difference from previously characterized DC patients because telomere length was normal.

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Effects of knockdown of C16orf57 on apoptosis and cell cycle. (A) Levels of knockdown achieved in 7 days after treatment with lentiviral-shRNA. (B) Corresponding apoptosis levels in HT1080 cells. (C) Corresponding cell cycle analysis. Vertical stripes, unmanipulated cells; horizontal stripes, cells transduced with a negative shRNA lentiviral construct; solid black bars, cells transduced with a C16orf57 shRNA lentiviral construct. Numbers on graphs relate to the data point for each experiment.
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DDQ371F5: Effects of knockdown of C16orf57 on apoptosis and cell cycle. (A) Levels of knockdown achieved in 7 days after treatment with lentiviral-shRNA. (B) Corresponding apoptosis levels in HT1080 cells. (C) Corresponding cell cycle analysis. Vertical stripes, unmanipulated cells; horizontal stripes, cells transduced with a negative shRNA lentiviral construct; solid black bars, cells transduced with a C16orf57 shRNA lentiviral construct. Numbers on graphs relate to the data point for each experiment.

Mentions: In light of the lack of functional information relating to the role of C16orf57 in the cell, we undertook a series of experiments to determine whether it had any role in cell biology either in terms of apoptosis or in the cell cycle. All experiments were performed on an unsorted population of HT1080 cells either 7 or 14 days after transduction with viral constructs encoding either a C16orf57 shRNA or a negative shRNA to observe the effects of knockdown of the C16orf57 transcript (Fig. 5A). Unmanipulated HT1080 cells were assayed at the same time. Apoptosis levels after knockdown with the C16orf57 shRNA showed a small increase in the proportion of cells that were either apoptotic or post-apoptotic relative to either the unmanipulated cells or the cells treated with the negative shRNA viral construct, suggesting the effect is specific to the action of reducing the C16orf57 levels in the cell (Fig. 5B). Repetition of these experiments show that this increase in the percentage of cells that are apoptotic following C16orf57 knockdown, although subtle, is statistically significant (Student's t-test P = 0.016, Supplementary Material, Table S3). Concomitant with this, the proportion of cells observed in the G2/M phase of the cell cycle was increased in the C16orf57 knockdown group (Fig. 5C) relative to the unmanipulated cells, but this failed to reach significance.Figure 5.


Mutations in C16orf57 and normal-length telomeres unify a subset of patients with dyskeratosis congenita, poikiloderma with neutropenia and Rothmund-Thomson syndrome.

Walne AJ, Vulliamy T, Beswick R, Kirwan M, Dokal I - Hum. Mol. Genet. (2010)

Effects of knockdown of C16orf57 on apoptosis and cell cycle. (A) Levels of knockdown achieved in 7 days after treatment with lentiviral-shRNA. (B) Corresponding apoptosis levels in HT1080 cells. (C) Corresponding cell cycle analysis. Vertical stripes, unmanipulated cells; horizontal stripes, cells transduced with a negative shRNA lentiviral construct; solid black bars, cells transduced with a C16orf57 shRNA lentiviral construct. Numbers on graphs relate to the data point for each experiment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2957322&req=5

DDQ371F5: Effects of knockdown of C16orf57 on apoptosis and cell cycle. (A) Levels of knockdown achieved in 7 days after treatment with lentiviral-shRNA. (B) Corresponding apoptosis levels in HT1080 cells. (C) Corresponding cell cycle analysis. Vertical stripes, unmanipulated cells; horizontal stripes, cells transduced with a negative shRNA lentiviral construct; solid black bars, cells transduced with a C16orf57 shRNA lentiviral construct. Numbers on graphs relate to the data point for each experiment.
Mentions: In light of the lack of functional information relating to the role of C16orf57 in the cell, we undertook a series of experiments to determine whether it had any role in cell biology either in terms of apoptosis or in the cell cycle. All experiments were performed on an unsorted population of HT1080 cells either 7 or 14 days after transduction with viral constructs encoding either a C16orf57 shRNA or a negative shRNA to observe the effects of knockdown of the C16orf57 transcript (Fig. 5A). Unmanipulated HT1080 cells were assayed at the same time. Apoptosis levels after knockdown with the C16orf57 shRNA showed a small increase in the proportion of cells that were either apoptotic or post-apoptotic relative to either the unmanipulated cells or the cells treated with the negative shRNA viral construct, suggesting the effect is specific to the action of reducing the C16orf57 levels in the cell (Fig. 5B). Repetition of these experiments show that this increase in the percentage of cells that are apoptotic following C16orf57 knockdown, although subtle, is statistically significant (Student's t-test P = 0.016, Supplementary Material, Table S3). Concomitant with this, the proportion of cells observed in the G2/M phase of the cell cycle was increased in the C16orf57 knockdown group (Fig. 5C) relative to the unmanipulated cells, but this failed to reach significance.Figure 5.

Bottom Line: Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families.Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls.These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Paediatrics, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Barts and The London Children’s Hospital, 4 Newark Street, London, UK. a.walne@qmul.ac.uk

ABSTRACT
Dyskeratosis congenita (DC) is an inherited poikiloderma which in addition to the skin abnormalities is typically associated with nail dystrophy, leucoplakia, bone marrow failure, cancer predisposition and other features. Approximately 50% of DC patients remain genetically uncharacterized. All the DC genes identified to date are important in telomere maintenance. To determine the genetic basis of the remaining cases of DC, we undertook linkage analysis in 20 families and identified a common candidate gene region on chromosome 16 in a subset of these. This region included the C16orf57 gene recently identified to be mutated in poikiloderma with neutropenia (PN), an inherited poikiloderma displaying significant clinical overlap with DC. Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families. In addition, three of six families previously classified as Rothmund-Thomson syndrome (RTS-a poikiloderma that is sometimes confused with PN) were also found to have homozygous C16orf57 mutations. Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls. These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS. This study also highlights the multi-system nature (wider than just poikiloderma and neutropenia) of the clinical features of affected individuals (and therefore house-keeping function of C16orf57), a possible role for C16orf57 in apoptosis, as well as a distinct difference from previously characterized DC patients because telomere length was normal.

Show MeSH
Related in: MedlinePlus