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Fatal cardiac arrhythmia and long-QT syndrome in a new form of congenital generalized lipodystrophy with muscle rippling (CGL4) due to PTRF-CAVIN mutations.

Rajab A, Straub V, McCann LJ, Seelow D, Varon R, Barresi R, Schulze A, Lucke B, Lützkendorf S, Karbasiyan M, Bachmann S, Spuler S, Schuelke M - PLoS Genet. (2010)

Bottom Line: Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability.Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3.In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%.

View Article: PubMed Central - PubMed

Affiliation: Genetics Unit, Ministry of Health, Directorate General of Health Affairs, Royal Hospital, Muscat, Oman.

ABSTRACT
We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae.

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Cell-biological consequences of PTRF-CAVIN depletion.(A) Confocal microscopic image of the punctate distribution of caveolin-1 which labels the caveolae on the surface of a fibroblast. (B) Severe reduction of the punctate caveolin-1 distribution in the absence of PTRF-CAVIN and its unstructured distribution within the cytoplasm. (C) Normal punctate distribution of PTRF-CAVIN on the fibroblast surface. (D) Absence of all PTRF-CAVIN immunoreactivity on a patient fibroblast. (E–G) Each panel depicts two patient fibroblasts, one untransfected (right) and one transfected with PTRF-FLAG construct (left). In the untransfected cell caveolin-1 is only found in the Golgi-apparatus. Reexpression of PTRF-CAVIN in the left cell redirects the caveolin-1 staining to the caveolae in the cell periphery where the two proteins co-localize (yellow dots in panel G).
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pgen-1000874-g006: Cell-biological consequences of PTRF-CAVIN depletion.(A) Confocal microscopic image of the punctate distribution of caveolin-1 which labels the caveolae on the surface of a fibroblast. (B) Severe reduction of the punctate caveolin-1 distribution in the absence of PTRF-CAVIN and its unstructured distribution within the cytoplasm. (C) Normal punctate distribution of PTRF-CAVIN on the fibroblast surface. (D) Absence of all PTRF-CAVIN immunoreactivity on a patient fibroblast. (E–G) Each panel depicts two patient fibroblasts, one untransfected (right) and one transfected with PTRF-FLAG construct (left). In the untransfected cell caveolin-1 is only found in the Golgi-apparatus. Reexpression of PTRF-CAVIN in the left cell redirects the caveolin-1 staining to the caveolae in the cell periphery where the two proteins co-localize (yellow dots in panel G).

Mentions: We used caveolin-1 immunoreactivity as a marker for caveolae on the fibroblast surfaces. Control fibroblasts showed a punctate staining pattern at the cell periphery that dissolved in the absence of PTRF-CAVIN (Figure 6A and 6B, Figure 7E and 7F) while the perinuclear caveolin-1 staining within the Golgi-apparatus remained unchanged. As caveolin-1 was present in patient fibroblasts in normal quantities (Figure 4D) lack of PTRF-CAVIN seems to disable recruitment of caveolin-1 into the caveolar microdomains of the outer cell membrane. However, transfection of a construct into patient fibroblasts, which contained the full-length, wild-type PTRF-CAVIN gene cloned downstream of the eukaryotic CMV-promoter, reconstituted the punctate staining pattern at the cell periphery that corresponds to the caveolae (Figure 6E–6G). Morphometric analysis by transmission electron microscopy of the quantity of caveolae on the cell surface revealed a severe reduction by >97% in the patient's fibroblasts (Figure 7A and 7B). Complementary to this technique we employed medium and high resolution Atomic Force Microscopy (AFM) to study the cell surface of native and mildly fixated cells from patient and control. This revealed a comparatively smooth cell surface in the patient with only occasional indentations of 50–100 nm corresponding to caveolae (Figure 7C and 7D). In the patient, overlay between AFM and confocal images revealed the absence of the punctate caveolin-1 staining at the cell surface especially within the major indentations of the fibroblast cell membrane. Instead, the caveolin-3 immunoreactivity was diffusely distributed within the cytoplasm. As expected, PTRF-CAVIN immunoreactivity was entirely absent from the cell surface of patient fibroblasts (Figure 7H).


Fatal cardiac arrhythmia and long-QT syndrome in a new form of congenital generalized lipodystrophy with muscle rippling (CGL4) due to PTRF-CAVIN mutations.

Rajab A, Straub V, McCann LJ, Seelow D, Varon R, Barresi R, Schulze A, Lucke B, Lützkendorf S, Karbasiyan M, Bachmann S, Spuler S, Schuelke M - PLoS Genet. (2010)

Cell-biological consequences of PTRF-CAVIN depletion.(A) Confocal microscopic image of the punctate distribution of caveolin-1 which labels the caveolae on the surface of a fibroblast. (B) Severe reduction of the punctate caveolin-1 distribution in the absence of PTRF-CAVIN and its unstructured distribution within the cytoplasm. (C) Normal punctate distribution of PTRF-CAVIN on the fibroblast surface. (D) Absence of all PTRF-CAVIN immunoreactivity on a patient fibroblast. (E–G) Each panel depicts two patient fibroblasts, one untransfected (right) and one transfected with PTRF-FLAG construct (left). In the untransfected cell caveolin-1 is only found in the Golgi-apparatus. Reexpression of PTRF-CAVIN in the left cell redirects the caveolin-1 staining to the caveolae in the cell periphery where the two proteins co-localize (yellow dots in panel G).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837386&req=5

pgen-1000874-g006: Cell-biological consequences of PTRF-CAVIN depletion.(A) Confocal microscopic image of the punctate distribution of caveolin-1 which labels the caveolae on the surface of a fibroblast. (B) Severe reduction of the punctate caveolin-1 distribution in the absence of PTRF-CAVIN and its unstructured distribution within the cytoplasm. (C) Normal punctate distribution of PTRF-CAVIN on the fibroblast surface. (D) Absence of all PTRF-CAVIN immunoreactivity on a patient fibroblast. (E–G) Each panel depicts two patient fibroblasts, one untransfected (right) and one transfected with PTRF-FLAG construct (left). In the untransfected cell caveolin-1 is only found in the Golgi-apparatus. Reexpression of PTRF-CAVIN in the left cell redirects the caveolin-1 staining to the caveolae in the cell periphery where the two proteins co-localize (yellow dots in panel G).
Mentions: We used caveolin-1 immunoreactivity as a marker for caveolae on the fibroblast surfaces. Control fibroblasts showed a punctate staining pattern at the cell periphery that dissolved in the absence of PTRF-CAVIN (Figure 6A and 6B, Figure 7E and 7F) while the perinuclear caveolin-1 staining within the Golgi-apparatus remained unchanged. As caveolin-1 was present in patient fibroblasts in normal quantities (Figure 4D) lack of PTRF-CAVIN seems to disable recruitment of caveolin-1 into the caveolar microdomains of the outer cell membrane. However, transfection of a construct into patient fibroblasts, which contained the full-length, wild-type PTRF-CAVIN gene cloned downstream of the eukaryotic CMV-promoter, reconstituted the punctate staining pattern at the cell periphery that corresponds to the caveolae (Figure 6E–6G). Morphometric analysis by transmission electron microscopy of the quantity of caveolae on the cell surface revealed a severe reduction by >97% in the patient's fibroblasts (Figure 7A and 7B). Complementary to this technique we employed medium and high resolution Atomic Force Microscopy (AFM) to study the cell surface of native and mildly fixated cells from patient and control. This revealed a comparatively smooth cell surface in the patient with only occasional indentations of 50–100 nm corresponding to caveolae (Figure 7C and 7D). In the patient, overlay between AFM and confocal images revealed the absence of the punctate caveolin-1 staining at the cell surface especially within the major indentations of the fibroblast cell membrane. Instead, the caveolin-3 immunoreactivity was diffusely distributed within the cytoplasm. As expected, PTRF-CAVIN immunoreactivity was entirely absent from the cell surface of patient fibroblasts (Figure 7H).

Bottom Line: Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability.Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3.In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%.

View Article: PubMed Central - PubMed

Affiliation: Genetics Unit, Ministry of Health, Directorate General of Health Affairs, Royal Hospital, Muscat, Oman.

ABSTRACT
We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae.

Show MeSH
Related in: MedlinePlus