Novel mutations in Darier disease and association to self-reported disease severity

<div><p>Darier disease is a rare and severe autosomal dominant skin disease characterised by malodorous keratotic papules in seborrheic areas of the skin. Darier disease affects up to 1 in 30 000 people and is caused by mutations in the <i>ATP2A2</i> gene, which encodes to the sarco/endoplasmic reticulum calcium-ATPase isoform 2 that pumps calcium into the endoplasmic reticulum. Although many <i>ATP2A2</i> variants have been described, it is not known if genotype correlates with phenotype, which could be important for prognosis and treatment. This is the first study to use whole exome sequencing to screen the <i>ATP2A2</i> gene in a cohort of 28 clinically diagnosed Darier disease patients. Twenty-one different disease causing variants were identified and 15 of these were novel. Sixteen of the 21 variants were predicted to be pathogenic using <i>in silico</i> prediction programs. There were seven missense, four intronic/splice-sites, three frameshifts, two in-frame deletions, four nonsense and one synonymous mutations. This study also found ten patients who harbour more than one <i>ATP2A2</i> variant. The phenotype of the patient cohort was assessed by photography and by patient questionnaires. The genotype-phenotype association was examined for all variants in relation to the patient’s disease severity score, and no correlation could be established.</p></div

Patient clinical characteristics.

<p>Patient clinical characteristics.</p

The effect of AICAR on pAMPK and mitotracker red stain in control and patient fibroblasts.

<p>Control and patient fibroblasts were grown on coverslips in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Cells were than incubated with mitotracker red (MTR), fixed, stained with anti pAMPK antibodies and visualized by fluorescent (pAMPK) secondary antibodies. The coverslips were examined by confocal fluorescent microscopy (10×40). A: depicts a representative migrograph of pAMPK stain in green and MTR stain in red. The graphs represent green intensity per cell (B) and red intensity per cell (C) +/− standard deviation (*p<0.05). All micrographs were taken under the same conditions.</p

The effect of AICAR on mitochondrial content and Δψ.

<p>Control 1 and an NDUFS2 patient's fibroblasts were grown in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Cells were then incubated with TMRE (red) and Mitrotracker green (green) and examined by confocal fluorescent microscopy. A: depicts a representative micrograph TMRE stain in red and MTG stain in green. The graphs represent green intensity per cell (B) and red:green ratio (C), +/− standard deviation (*p<0.05). All micrographs were taken under the same conditions.</p

Medication effect (1–5), disease severity (mild–severe) and disease severity (1–5) scores sorted by mutation type.

<p>Medication effect and disease severity score (Top and bottom), a score of 1 = bad, 2 = acceptable, 3 = good, 4 = very good or 5 = excellent. Disease severity rate (Middle), a score of 1 = severe, 2 = moderate and 3 = mild. A score of 0 corresponds to patient’s lack of answer to a particular category. The type of <i>ATP2A2</i> mutation is shown on the x-axis, and they are sorted into seven categories: No mutations, benign, missense, in-frame deletion, splice-site, frameshift and nonsense mutations. ● represent patients receiving systemic treatment; ▲represents patients receiving topical treatment; ◆ represent patients receiving no treatment and represent patients who have received laser treatment. The different coloured points represent the patients with two <i>ATP2A2</i> variants. Points with the same colours are variants in the same patient.</p

Patient data.

<p>Patient data.</p

The effect of various compounds on growth, ROS and ATP in control and patient fibroblasts.

<p>Fibroblast from three separate controls and seven patients (C20ORF7,NDUFS2,NDUFS4,C6ORF66, FOXRED1 and B17.2L) were grown in microtiter wells in GLU or GAL medium or in GAL medium, supplemented with one of eight various compounds (BEZA,AICAR,OLTI,SBP,RZV,GENI,EGCG,GSI) in GAL for 72hrs. Growth was assessed by MB (A). ROS was measured by DCF and normalized to MB (B). ATP was measured by luciferin-luciferase and normalized to MB (C). Values (A-C) are presented as relative values graphically presented as heatmaps representing the mean of triplicates performed on at least 2 separate occasions. Growth in GAL without additives was set as the value of 1(black). Values < 1 are presented in increasingly green and values >1 in increasingly red. All parameters are summarized in (D) and evaluated as a significantly positive (+), negative (−) or nonsignificant (ns) effect. Increased values for growth and ATP were regarded as positive while negative for increased ROS. Positive only is shaded in red, negative only in green, nonsignificant in dark grey and mixed responses in gray.</p

Oxygen consumption.

<p>Control 1 (A) and NDUFS2 patient's (B) fibroblasts were grown in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Media were changed to unbuffered media and oxygen consumption rates (OCR) were measured by an XF24 instrument. Basal OCR was measured (basal) before the addition of uncoupler and maximal rate (CCCP) after. The results are presented as rates subtracted by non mitochondrial oxygen consumption (in the presence of rotenone and antimycin) and normalized to cell amount measured by methylene blue (MB)at A₆₂₀, +/− standard deviation. *p<0.05.</p

Measurement of growth, ROS and ATP in control and patient fibroblasts.

<p>Fibroblast from control and patient (NDUFS2) were grown in microtiter wells in GLU, GAL or GAL supplemented with AICAR. The amount of control cells, measured by methylene blue (MB) at A₆₂₀ was compared to viable count by trypan blue at 72h (A). Growth of control and patient cells was measured at 24,48,72h and 144h by MB at A₆₂₀ (B,C). Growth with AICAR was assessed at 72h (C). ROS measured by DCF at 72h is expressed as relative fluorescent units (RFU) divided by the amount of cells measured by MB at A₆₂₀ (D). ATP was measured at 72h by luciferin-luciferase is expressed as relative luminescence units (RLU) divided by the amount of cells measured by MB at A₆₂₀ (E). Values are presented as mean of triplicates +/- standard deviation. *p<0.05.</p

Islets are highly uncoupled; mice islets more than human.

<p>A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.</p