A Frame-Shift Mutation in CAV1 Is Associated with a Severe Neonatal Progeroid and Lipodystrophy Syndrome

Abstract

A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.

Fig 1. Expression heatmaps of RNA analyzed from whole blood.

All genes are displayed that reach significance after correction for multiple testing FDR and have an FPKM above 1. Interesting dysregulated pathways and subpathways are: Regulation of apoptosis (p = 3.0 x 10–8), DNA replication, with affected sub-pathways regulation of DNA replication (p = 1.4 x 10–5), M/G1 transition (p = 5.2 x 10–5) and the synthesis of DNA (p = 2.1 x 10–4)). DNA repair is also dysregulated, especially the fanconi anemia pathway (Translocation of ub-FANCD2 and ub-FANCI to chromatin; p = 4.0 x 10–4 and regulation of the Fanconi anemia pathway 4.4 x 10–3). Also membrane trafficking is affected, especially subpathway Endosomal Sorting Complex Required For Transport (ESCRT) (p = 1.8 x10-5). The citric acid (TCA) cycle and respiratory electron transport pathway is significantly dysregulated (p = 9.3x10-6) and cellular responses to stress as well (p = 8.6 x 10–3). For signal transduction, especially signaling by Wnt is affected (p = 3.2 x 10–4).

Fig 2

A. Expression bar plots of RNA analyzed from whole blood involved in other progeroid syndromes. B. AGPAT2, lipodystrophy. C. Known Cav1 pathways and D. Direct interaction partners. * Reach significance after correction for multiple testing (FDR). LPIN1 is not significantly different between case and controls. 95% FPKM cufflinks confidence intervals for estimates were obtained using a Bayesian inference method based on importance sampling from the posterior distribution [21].

Fig 3

A. western blot of lysates from case and control fibroblasts. Two antibodies were used, one that recognized an internal epitope in CAV1 (LS-B192), and one that recognized a C-terminal epitope (ORB47981; would only recognize WT CAV1). Beta-actin was used as a loading control. Calculation of intensity of the CAV1 isoform bands compared to the loading control, illustrated that WT expression is decreased in the case compared to the control. B: Immunocytochemistry of case and control fibroblast cells with anti-PRTF and anti-CAV1 antibodies. In the control, co-localization of CAV1 and PTRF (Caveolae marker) is very clear. In the proband’s cells, this co-localization is less consistant. Green bar = 28um.