Biallelic CAV1 null variants induce congenital generalized lipodystrophy with achalasia

in European Journal of Endocrinology
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  • 1 Department of Pediatric Gastroenterology, Hepatology and Nutrition, Mersin University Faculty of Medicine, Mersin, Turkey
  • | 2 Sorbonne University, Inserm UMR_S938, Saint-Antoine Research Centre, Institute of Cardiometabolism and Nutrition, Paris, France
  • | 3 Department of Radiology, Mersin University Faculty of Medicine, Mersin, Turkey
  • | 4 Department of Gastroenterology, Mersin University Faculty of Medicine, Mersin, Turkey
  • | 5 Sorbonne University, Inserm UMR_S1155, LUMIC, Tenon Imagery and Cytometry Platform, Paris, France
  • | 6 Nantes University, CNRS, Inserm UMR_S1087, Institut du Thorax, Nantes, France
  • | 7 Department of Endocrinology, Diabetology and Reproductive Endocrinology, Assistance Publique-Hôpitaux de Paris, Saint-Antoine University Hospital, National Reference Center for Rare Diseases of Insulin Secretion and Insulin Sensitivity (PRISIS), Paris, France
  • | 8 Department of Molecular Biology and Genetics, Assistance Publique-Hôpitaux de Paris, Saint-Antoine University Hospital, Paris, France

Correspondence should be addressed to A N Karhan or C Vigouroux; Email: asunurkar83@gmail.com or corinne.vigouroux@inserm.fr
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Objective

CAV1 encodes caveolin-1, a major protein of plasma membrane microdomains called caveolae, involved in several signaling pathways. Caveolin-1 is also located at the adipocyte lipid droplet. Heterozygous pathogenic variants of CAV1 induce rare heterogeneous disorders including pulmonary arterial hypertension and neonatal progeroid syndrome. Only one patient was previously reported with a CAV1 homozygous pathogenic variant, associated with congenital generalized lipodystrophy (CGL3). We aimed to further delineate genetic transmission, clinical, metabolic, and cellular characteristics of CGL3.

Design/Methods

In a large consanguineous kindred referred for CGL, we performed next-generation sequencing, as well as clinical, imagery, and metabolic investigations. We studied skin fibroblasts from the index case and the previously reported patient with CGL3.

Results

Four patients, aged 8 months to 18 years, carried a new homozygous p.(His79Glnfs*3) CAV1 variant. They all displayed generalized lipodystrophy since infancy, insulin resistance, low HDL-cholesterol, and/or high triglycerides, but no pulmonary hypertension. Two patients also presented at the age of 15 and 18 years with dysphagia due to achalasia, and one patient had retinitis pigmentosa. Heterozygous parents and relatives (n = 9) were asymptomatic, without any metabolic abnormality. Patients’ fibroblasts showed a complete loss of caveolae and no protein expression of caveolin-1 and its caveolin-2 and cavin-1 partners. Patients’ fibroblasts also displayed insulin resistance, increased oxidative stress, and premature senescence.

Conclusions

The CAV1 null variant investigated herein leads to an autosomal recessive congenital lipodystrophy syndrome. Loss of caveolin-1 and/or caveolae induces specific manifestations including achalasia which requires specific management. Overlapping phenotypic traits between the different CAV1-related diseases require further studies.

Abstract

Objective

CAV1 encodes caveolin-1, a major protein of plasma membrane microdomains called caveolae, involved in several signaling pathways. Caveolin-1 is also located at the adipocyte lipid droplet. Heterozygous pathogenic variants of CAV1 induce rare heterogeneous disorders including pulmonary arterial hypertension and neonatal progeroid syndrome. Only one patient was previously reported with a CAV1 homozygous pathogenic variant, associated with congenital generalized lipodystrophy (CGL3). We aimed to further delineate genetic transmission, clinical, metabolic, and cellular characteristics of CGL3.

Design/Methods

In a large consanguineous kindred referred for CGL, we performed next-generation sequencing, as well as clinical, imagery, and metabolic investigations. We studied skin fibroblasts from the index case and the previously reported patient with CGL3.

Results

Four patients, aged 8 months to 18 years, carried a new homozygous p.(His79Glnfs*3) CAV1 variant. They all displayed generalized lipodystrophy since infancy, insulin resistance, low HDL-cholesterol, and/or high triglycerides, but no pulmonary hypertension. Two patients also presented at the age of 15 and 18 years with dysphagia due to achalasia, and one patient had retinitis pigmentosa. Heterozygous parents and relatives (n = 9) were asymptomatic, without any metabolic abnormality. Patients’ fibroblasts showed a complete loss of caveolae and no protein expression of caveolin-1 and its caveolin-2 and cavin-1 partners. Patients’ fibroblasts also displayed insulin resistance, increased oxidative stress, and premature senescence.

Conclusions

The CAV1 null variant investigated herein leads to an autosomal recessive congenital lipodystrophy syndrome. Loss of caveolin-1 and/or caveolae induces specific manifestations including achalasia which requires specific management. Overlapping phenotypic traits between the different CAV1-related diseases require further studies.

Introduction

Caveolae are plasma membrane microdomains that act as signaling platforms in several cell types, including adipocytes, smooth muscle cells, endothelial cells, and fibroblasts. The integral membrane protein caveolin-1, encoded by the CAV1 gene, is required for caveolae formation and is the main protein component of caveolae. Caveolin-1 interacts, among others, with the insulin receptor and contributes to the compartmentalization of insulin signaling pathways (1). Caveolin-1 is also a fatty acid-binding protein that able to translocate from the plasma membrane to the adipocyte lipid droplets, therefore contributing to the regulation of lipid storage (2, 3). In addition, it plays a role in tumor suppression and oxidative stress-induced cellular senescence (4). Loss of caveolin-1 expression in mice induces several defects including progressive lipodystrophy with insulin resistance and hypertriglyceridemia (5, 6), cardiomyopathy, and pulmonary hypertension (7, 8).

The phenotypic spectrum of the rare CAV1 genetic defects in humans remains difficult to delineate. A single patient has been previously reported with a homozygous CAV1 pathogenic variant. This patient was a young woman described with congenital generalized lipodystrophy (CGL), severe insulin-resistant diabetes, and hypertriglyceridemia, referred to as CGL3 (9). Heterozygous CAV1 null variants, including nonsense and frameshift mutations, have been reported so far in ten patients with different phenotypes, that is partial lipodystrophy with neurological involvement (10), precocious and severe pulmonary arterial hypertension (11, 12, 13), and/or neonatal progeroid syndromes (14, 15, 16).

In this study, we investigated a large consanguineous kindred referred for CGL and identified a novel homozygous CAV1 frameshift variant in four affected young patients. Besides variable insulin resistance-associated metabolic abnormalities, esophageal achalasia, leading to severe dysphagia, emerges as specific comorbidity. One affected patient also displayed atypical retinitis pigmentosa. By studying cultured skin fibroblasts from the index case and from the previously reported patient with a homozygous CAV1 nonsense variant (9), we show that CGL3 is associated with a complete loss of protein expression of caveolin-1 and its partners caveolin-2 and cavin-1 and with the absence of caveolae at the plasma membrane. Fibroblasts from patients also displayed insulin resistance, increased oxidative stress, and premature senescence.

Patients and methods

Patients

This study includes 19 individuals from a large Turkish consanguineous family investigated at Mersin University, Department of Pediatric Gastroenterology, Hepatology and Nutrition, Turkey. Genetic studies were performed in the Department of Molecular Biology and Genetics, and the disease features were reviewed in the French Reference Center for Rare Diseases of Insulin Secretion and Insulin Sensitivity (PRISIS), both at Assistance-Publique Hôpitaux de Paris, Saint-Antoine Hospital, Paris, France. Clinical and molecular studies and skin biopsy were performed after full written informed consent, according to the Ethics Committee of Mersin University. The study was approved by a French institutional research ethics board (CPP Ile de France 5). Written informed consent for publication of their clinical details and/or clinical images was obtained from the patients’ parents, from patients aged above 12, and from their relatives.

Genetic analyses

Exons and flanking intronic sequences of a panel of 23 genes involved in lipodystrophic and/or insulin resistance syndromes, including CAV1, were sequenced from genomic DNA in patient 1, as described (17) (Fig. 1). Sanger sequencing was performed with the Big Dye Terminator v3.1 sequencing kit (Thermo Fisher Scientific) after PCR. Data were analyzed on a 3500xL Dx device with the SeqScape v2.7 software (Thermo Fisher Scientific). CAV1 variants were described based on the longest isoform (NM_001753.4) using Alamut 2.11 (Sophia Genetics, Switzerland) and Human Genome Variation Society guidelines.

Figure 1
Figure 1

Genealogical tree of the studied family. Patients 1 to 4 diagnosed with congenital generalized lipodystrophy and homozygous CAV1 p.(His79Glnfs*3) variant (M/M) are depicted by filled symbols. Half-filled symbols (heterozygous subjects, +/M) and patients with normal genotype (+/+) were not affected by the disease. Patient 1 (P1, arrow) is the index case.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Fibroblast cultures

Primary fibroblast cultures from patient 1 were established after skin biopsy. Cultured fibroblasts from the previously reported patient with CGL3, carrying the homozygous CAV1 p.Glu38* pathogenic variant (9), and from two non-obese, non-diabetic, normotensive women who underwent plastic surgery (18) were studied at similar passages. Cells were grown in DMEM low glucose with pyruvate (#31885049; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (D. Dutscher, Bernolsheim, France), 1% penicillin/streptomycin, and 2 mM glutamine (Invitrogen).

Transmission electron microscopy

Cultured fibroblasts were fixed in 2.5% glutaraldehyde, 0.1 M cacodylate buffer at 4°C, rinsed in 0.2 M cacodylate, post-fixed in 1.5% potassium ferrocyanide 1% osmium tetroxide, dehydrated using graded alcohol series, and then embedded in epoxy resin. Semi-fine sections (0.5 µm) were stained with toluidine blue. Ultrathin sections (70 nm) were contrasted with UranyLess and lead citrate (Delta Microscopies, France) and examined using an electron microscope (JEOL 2110 HC, Croissy, France) with a 2Kx2K Veleta CCD camera (Olympus).

Western blot

Protein expression studies were performed on whole-cell extracts using antibodies described in Supplementary Table 1 (see section on supplementary materials given at the end of this article).

Cellular response to insulin

To measure fibroblasts' ability to bind insulin, they were maintained for 16 h in serum-free medium supplemented with 0.1% albumin (Sigma–Aldrich), then incubated with 125I-insulin (0.3 ng/mL, PerkinElmer) with or without unlabeled insulin (5 × 10−8 M) in HEPES buffer at pH 7.65, 15°C, for 5 h. Radioactivity was measured in a gamma counter (PerkinElmer 2470 Wizard2) and results were normalized to protein content. Insulin effect on glycogen synthesis was evaluated by the incorporation of 14C-glucose as previously described (19). Results were normalized to the protein content and expressed as a percentage of the basal value.

Oxidative stress and cellular senescence

The production of reactive oxygen species (ROS) was assayed by quantifying the oxidation of 5-6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). The blue staining produced by hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-β-dgalactopyranoside) by senescence-activated ββ-galactosidase at pH 6.0 was used as a biomarker of cellular senescence, as previously described (20). The ratio of pH 6/pH 4 staining of blue X-gal was quantified at 630 nm.

Statistical analyses

GraphPad Prism software (GraphPad Software Inc.) was used to calculate statistical significance with a threshold at P < 0.05. Gaussian distribution was tested with Kolmogorov–Smirnov test. Differences between two groups were assessed by unpaired two-sample t-tests or Mann–Whitney tests, and multiple comparisons between more than two groups were conducted by ANOVA with Bonferroni test or Kruskal–Wallis test for post hoc analysis. All data are means ± s.e.m. of at least three independent experiments.

Results

Identification of a novel CAV1 pathogenic variant responsible for an autosomal recessive congenital generalized lipodystrophy syndrome

Patient 1, a 15-year-old girl from a large consanguineous Turkish family, was referred for genetic investigation of CGL (Fig. 1). We identified a novel homozygous CAV1 variant (NM_001753.4): c.237_238del, p.(His79Glnfs*3) through sequencing of a panel of 23 genes involved in lipodystrophy and/or insulin resistance syndromes. This variant, confirmed by Sanger sequencing, was absent from the gnomAD and ExAC databases that list genetic variants from the general population and is classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria (21). The deletion of two nucleotides in exon 3 leads to a shift in the reading frame. If the transcript is expressed, it is predicted to result in the synthesis of an abnormal form of caveolin-1, truncated in its oligomerization domain and deprived of its scaffolding and intra-membrane domains (Fig. 2). Sequencing of the gene panel did not reveal any other pathogenic variant. Patient 1 was thus diagnosed with CGL3.

Figure 2
Figure 2

Schematic representation of CAV1 gene and caveolin-1 protein. CAV1 pathogenic variants (NM_001753.4) identified in this study or previously reported (9, 10, 11, 14, 15) are indicated, with the corresponding phenotypes. OD, oligomerization domain, CSD, caveolin-scaffolding domain. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

The same CAV1 variant was observed in the homozygous state in patient 2, who was subsequently referred with similar symptoms, and in patients 3 and 4, diagnosed with CGL after familial investigations. The parents of patients 1, 2, 3 and4 were asymptomatic. Genetic investigations, performed in seven of them, revealed that they were heterozygous carriers of the variant. Unaffected siblings of the patients (n = 8) were heterozygous for the variant or had a normal genotype.

This family thus showed an autosomal recessive transmission of CGL3 due to a novel p.(His79Glnfs*3) CAV1 pathogenic variant (Figs 1 and 2).

Phenotype of the disease

Table 1 summarizes the patients’ main phenotypic features.

Table 1

Main phenotypic features of patients 1–4. Height, weight, and BMI centiles are determined according to CDC (Centers for Disease Control and Prevention, USA); Mid-parental target height is calculated according to Tanner et al. (44). Values given in bold are outside the normal range.

Patient 1Patient 2Patient 3Patient 4Normal range
Age at evaluation15 years18 years10 years8 months
GenderFemaleMaleFemaleFemale
Height (cm)15315513462
 Centile<10th<3rd<25th<3rd
 Mid-parental target height in postpubertal patients (cm)160170
Weight (kg)424930.58.6
 Centile10th3rd40th75th
BMI (kg/m2)17.920.41722.4
 Centile25th25th50thND
Lipodystrophy onsetEarly infancyEarly infancyEarly infancyEarly infancy
Generalized lipoatrophy sparing palms and soles++++
Failure to thrive in infancy++++
Increased appetite+---
Triangular face++++
Muscular hypertrophy+++-
Acanthosis nigricans++--
Hirsutism+NA--
Polycystic ovaries+NA--
Liver steatosis (ultrasonography)+---
Blood pressure, cardiac examination, and ECGNormalNormalNormalNormal
EchocardiographyNormalNormalNDND
Estimated right ventricular systolic pressure (mmHg)2527NDND12–57
Megaesophagus++--
Muscular strength and neurological examinationNormalNormalNormalNormal
Ophthalmological examinationNormalAtypical retinitis pigmentosaNDND
Fasting glucose (mmol/L)5.04.85.34.73.5–5.6
Fasting insulin (mIU/L)3008.19.69.42–9
HbA1c (%)5.85.35.45.14.8–6
Triglycerides (mmol/L)5.40.93.42.30.3–1.5
Total cholesterol (mmol/L)4.03.83.14.23–5.1
HDL-cholestrol (mmol/L)0.730.680.750.650.9–1.8
Serum and urinary calciumNormalNormalNormalNormal
Urinary calcium/creatinine ratio0.0550.0530.0440.048<0.14
PTH (pg/mL)39.941.3NDND15–65
25-OH-Vitamin D (nmol/L)28.22334.452.450–200
Bone mineral density (lumbar spine Z-score)−2.78−3.85NDND

The listed signs are indicated as present (+) or absent (−) in each patient.

NA, not applicable; ND, not determined.

Patient 1

Patient 1 was referred at the age of 15 to Mersin University with difficulty in swallowing liquids and solids. She was born from first-cousin parents of Turkish origin, at term after an uneventful pregnancy, with birth weight and height of 2800 g and 48 cm, respectively. At examination, her height was 153 cm, BMI 17.9, and Tanner pubertal stage 5. She showed a triangular and acromegaloid face, with a generalized loss of fat sparing palms and soles, a taut and thin mottled skin with visible dermal vessels, extensive acanthosis nigricans in the armpits and groins, a prominent musculature, and enlarged hands and feet (Fig. 3A, B and C). She also complained about hirsutism, with secondary amenorrhea since the age of 14. The family noticed her dysmorphic facial and body appearance during early infancy. Although her linear growth was delayed with poor weight gain during infancy, she was described with a voracious appetite. She did not present with any skeletal deformity, joint contractures, or muscular functional defect. No cognitive delay was observed and she had normal developmental milestones. Neurological examination and blood pressure were normal.

Figure 3
Figure 3

Photographs from patients with congenital generalized lipodystrophy due to the novel CAV1 p.(His79Glnfs*3) homozygous pathogenic variant. Photographs from patient 1 (A, B and C), patient 2 (D), patient 3 (E), and patient 4 (F) are shown. Triangular face with empty cheeks and generalized lipoatrophy are observed in all patients. Muscular hypertrophy is visible in patients 1–3. Photographs from patient 1 also show mottled skin (A, B and C), acromegaloid features (A), and acanthosis nigricans (B). A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Laboratory parameters showed hypertriglyceridemia (5.4 mmol/L), low HDL-cholesterol (0.73 mmol/L), major fasting hyperinsulinemia (300 mIU/L) with normal glycemia (5 mmol/L), and HbA1c (5.8%). Liver tests and creatine kinase as well as kidney and thyroid function were normal. Androgen levels were in the normal range. Serum luteinizing hormone (LH,: 13.9 IU/L) was higher than follicle-stimulating hormone (FSH: 5.8 IU/L). Estradiol levels were in the normal range for the follicular phase. Calcemia and parathormone serum and urinary levels were normal, with decreased vitamin D level and bone mineral density (Z score: −2.78 at lumbar spine).

Generalized lipoatrophy was confirmed by MRI (Fig. 4) and by very low leptin levels (0.3 ng/mL), also consistent with hyperphagia. MRI also revealed enlarged polycystic ovaries (Fig. 5). ECG and echocardiogram, including estimated right ventricular systolic pressure, were normal. Liver steatosis was diagnosed with ultrasonography. Medical history, physical examination, and results of metabolic investigations allowed the diagnosis of CGL. A treatment with metformin and medium-chain triglycerides was initiated.

Figure 4
Figure 4

MRI of patient 1 showing generalized lipoatrophy. Magnetic resonance T1-weighted images without fat suppression of patient 1 and of age, sex, and ethnically matched control subject are shown. Brain/cranial (A and B) and abdomen (C) axial images, and pelvis coronal images (D and E) of patient 1 show that lipoatrophy is generalized, sparing only periorbital and bone marrow regions. Hepatic steatosis and muscular hypertrophy are also visible.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Figure 5
Figure 5

MRI of patient 1 showing enlarged polycystic ovaries. Magnetic resonance T1-weighted coronal images of patient 1, showing polycystic right (A) and left (B) ovaries of enlarged size (20 × 15 × 39 mm and 37 × 35 × 30 mm, respectively) (arrows).

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Dysphagia was investigated with several procedures. Barium esophagogram series showed a dilated esophagus with a distal bird’s beak deformity (Fig. 6A), and severe stenosis of the distal esophagus was confirmed by endoscopy. High-resolution esophageal manometry showed the absence of lower esophageal sphincter relaxation with increased integrated relaxation pressure (51 mmHg) and decreased peristalsis, consistent with esophagogastric junction outflow obstruction (22) (Fig. 6B and C). Based on clinical, endoscopic, radiological, and manometric findings, an early stage of achalasia was diagnosed. Peroral endoscopic myotomy (POEM) was performed leading to a significant relief in dysphagia-related symptoms.

Figure 6
Figure 6

Barium esophagogram series and high-resolution manometry showing achalasia in patients 1 and 2. Barium esophagogram series in patient 1 (A) and patient 2 (D and E). Arrows show the bird’s beak deformity at the distal part of esophagus (A and D) with a dilated esophageal body (E). High-resolution manometry, showing a pressure topography during a swallow, in a control subject (B), patient 1 (C), and patient 2 (F). The horizontal axis refers to time and the vertical axis to length along the esophagus, with the lower esophageal sphincter (LES) depicted below. The pressure magnitude, encoded in color (blue, green, yellow, and red from low to high pressure), is shown along the horizontal line. In a control subject, the pressure topography graph during a swallow shows a normal distal propagation of esophageal peristalsis, with LES relaxation over time (B). Weak peristalsis in patient 1 defines esophagogastric junction outflow obstruction (C). A continuous high pressure without LES relaxation allows the diagnosis of type 2 achalasia in patient 2 (F). A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Patient 2

Patient 2 was an 18-year-old cousin of patient 1 (Fig. 1) referred for difficulty in swallowing. Generalized lipodystrophy and mild dysmorphic features were present from early infancy. His symptoms and physical examination were very similar to those of patient 1 (Fig. 3D and Table 1). However, his metabolic abnormalities were milder, with low HDL-cholesterol but normal triglyceridemia. Although glucose tolerance was normal, normal-to-high 2 h-postprandial insulin (45.8 mIU/L, Normal: 4–52.5 at T120 min after oral glucose tolerance test) (23) and C-peptide levels (8.32 µg/L, Normal: 1.1–4.4) suggested insulin resistance. Blood pressure and cardiac examination including echocardiography were normal, as well as liver, renal, and thyroid function and testosterone and FSH levels. Type 2 achalasia diagnosed upon radiological, endoscopic, and manometric findings (Fig. 6D, E and F), was successfully treated by POEM. Patient 2 also complained of trouble seeing at night for 2 years. Although neurological examination and ophthalmological fundus analysis were normal, optical coherence tomography (OCT) revealed bilateral loss of ellipsoid line and atrophy of the retinal pigment epithelium, and defective outer limiting membrane in the left eye, suggesting atypical retinitis pigmentosa.

Patient 3 and patient 4

Patient 3, a 10-year-old girl, and patient 4, an 8-month-old girl, were investigated during the systematic familial screening (Fig. 1). As patients 1 and 2 were born from consanguineous parents, at term after normal pregnancies, with normal birth weight and height, their linear growth was delayed and they presented with a dysmorphic appearance and generalized lipoatrophy, which were noticed by the family during early infancy (Fig. 3E and F). Their physical examination was otherwise normal. They both achieved normal developmental milestones. Laboratory investigations revealed increased levels of triglycerides and insulin and decreased HDL-cholesterol (Table 1).

Patients’ relatives

Relatives of patients 1, 2, 3 and 4 (n = 15) (Fig. 1) had no complaint and did not show any sign of lipodystrophy. Their anthropometric measurements and physical examination were normal, except for the older brother of patient 1 who presented with mild mental retardation of unknown origin. Their laboratory evaluation including full blood count, fasting glucose and insulin, HbA1c, liver function tests, and serum lipids was normal.

Homozygous CAV1 p.(His79Glnfs*3) and p.(Glu38*) pathogenic variants induce a loss of protein expression of caveolin-1, caveolin-2 ,and cavin-1 and a loss of caveolae formation in patients’ fibroblasts

We compared cultured fibroblasts from patient 1 and from the previously reported patient with CGL due to the homozygous p.(Glu38*) CAV1 pathogenic variant (9) to those from controls. The p.(His79Glnfs*3) and the previously described p.(Glu38*) CAV1 pathogenic variants predict to interrupt the caveolin-1 amino acid sequence at the N-terminal part of the protein, proximally to its scaffolding and intra-membrane domains (24) (Fig. 2). As expected, we show that, similarly to the fibroblasts carrying the homozygous CAV1 p.(Glu38*) variant (9), cells from patient 1 with the novel CAV1 p.(His79Glnfs*3) variant do not exhibit detectable expression of caveolin-1 (Fig. 7A). We evaluated the protein expression of caveolin-2 and cavin-1, which are binding partners of caveolin-1. We show that caveolin-2 and cavin-1 protein levels are strongly decreased in patients’ cells (Fig. 7A). Notably, pathogenic variants in CAVIN1 (previously referred to as PTRF), encoding cavin-1, lead to a form of CGL previously associated with achalasia (25). As expected from the major role of caveolin-1 and cavin-1 for caveolae formation, electron microscopy shows that caveolae, abundant in control cells, are completely absent in fibroblasts from patients with CAV1 null variants (Fig. 7B).

Figure 7
Figure 7

Homozygous CAV1 p.(His79Glnfs*3) and p.(Glu38*) pathogenic variants induce a loss of protein expression of caveolin-1, caveolin-2, and cavin-1 and a loss of caveolae formation in patients’ fibroblasts. (A) Protein expression of caveolin-1, caveolin-2, and cavin-1 in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (B) Representative images of electron microscopy showing caveolae at the plasma membrane of control fibroblasts (arrows), which were completely absent in patients’ cells.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Homozygous CAV1 null variants impair insulin signaling

We then investigated the insulin-mediated activation of proximal signaling intermediates from the metabolic and mitogenic insulin pathways (i.e. IRß, IRS1, AKT, and ERK1/2) and showed that it is strongly impaired in fibroblasts from patients with CAV1 null variants (Fig. 8A). In addition, the effect of insulin on its distal signaling, as evaluated by glycogen synthesis, is also severely inhibited in patients’ cells (Fig. 8B). Although the total amount of insulin receptors is not significantly decreased in whole protein extracts from patients’ cells (Fig. 8A), the absence of caveolae, which are normally enriched at the plasma membrane with insulin receptors (1), could impair the initiation of the insulin signal. To test this hypothesis, we evaluated the capacity of fibroblasts to bind insulin and showed that it was decreased by ~20% in cells bearing CAV1 null variants as compared to control cells (Fig. 8C). These results suggest that the loss of caveolae could contribute, in part, to the insulin resistance observed in cells from patients carrying CAV1 null pathogenic variants.

Figure 8
Figure 8

Homozygous CAV1 null variants impair insulin signaling in patients’ fibroblasts. (A) Insulin-mediated activation of insulin receptor β-subunit (IRβ), insulin receptor substrate-1 (IRS1), protein kinase B (AKT/PKB), and extracellular-regulated kinase (ERK)1/2 was evaluated by Western blot in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Cells were maintained for 24 h in a serum-free medium, then incubated or not with 50 nmol/L human insulin (#I9278, Sigma–Aldrich) for 8 min, and the total protein expression of signaling intermediates as well as their phosphorylated activated forms was evaluated. Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (B) The effect of insulin on glycogen synthesis and (C) the ability of cells to bind insulin to its receptor were evaluated as described in Methods section. Results are expressed as the percentage of the first control without insulin. **P < 0.01, ns, not significant.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Homozygous CAV1 null variants increase oxidative stress and senescence in fibroblasts

Caveolin-1 has been shown to modulate oxidative stress-induced cellular senescence (4), a pathway that has been involved in lipodystrophic diseases (26). We thus aimed to evaluate the effects of CAV1 pathogenic variants on the production of reactive oxygen species (ROS) and on cellular senescence. Patients’ fibroblasts, as compared to control cells, display a major increase in oxidative stress (Fig. 9A), as well as elevated levels of senescence markers (Fig. 9B) and increased senescence-activated β-galactosidase activity (Fig. 9C and D).

Figure 9
Figure 9

Homozygous CAV1 null variants promote oxidative stress and senescence in patients’ fibroblasts. (A) Reactive oxygen species (ROS) production was assessed by oxidation of 5-6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Results are normalized to DNA content measured by DAPI and are expressed as the percentage of the first control. (B) Cellular senescence was evaluated by the protein expression levels of the cell cycle arrest and senescence protein markers p16, p21, and phospho-p53 as compared to total p53. Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (C and D) Senescence-associated β-galactosidase activity (SA-β-gal) was assessed by X-gal staining at pH6 compared to non-specific staining at pH 4. Representative immunofluorescence images from triplicate experiments are shown. Scale bar is 100 µm. The ratio of pH 6.0/pH 4.0 staining, which represents SA-β-galactosidase activity, is expressed as the percentage of the first control. ****P < 0.0001, ns, not significant. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

Citation: European Journal of Endocrinology 185, 6; 10.1530/EJE-21-0915

Discussion

We report here a novel homozygous null pathogenic variant in CAV1 in four patients with CGL3 and show the autosomal recessive transmission of the disease in a large consanguineous family. This study adds important phenotypic data, since CGL3 was previously described in only one patient (9). By revealing the consequences of CAV1 loss-of-function in patients’ cells, this study also highlights the involvement of caveolin-1 and caveolae in cellular insulin response, oxidative stress, and cellular senescence, which could contribute to specific clinical manifestations.

Insulin resistance and related signs, including acanthosis nigricans, prominent veins, muscular hypertrophy, hypertriglyceridemia, low HDL-cholesterol, hirsutism and/or polycystic ovary syndrome, and hepatic steatosis, are consistently associated with lipodystrophy syndromes (27).

These manifestations, together with generalized lipoatrophy, were present in the previously described patient with CGL3 (9) as well as, with variable severity, in the affected patients from this study. As previously described in other CGL subtypes, with the exception of CGL2 (9, 28), lipoatrophy only spared mechanical adipose tissue (from palms, plantar, and retro-orbital regions) and bone marrow fat. It can be hypothesized that different adipose tissue depots, which have specific developmental origin and gene expression, could be differently impacted by CGL-associated pathogenic variants (29).

Lipodystrophy-associated insulin resistance was shown to result, at least in part, from defective adipocyte lipid storage and leptin deficiency leading to cellular lipotoxicity (30). Our studies in patients’ fibroblasts suggest that deficient caveolin-1 and cavin-1 protein expression and/or the complete absence of caveolae could also directly contribute to insulin resistance.

The CGL3 phenotype was previously associated with short stature, hypocalcemia, osteopenia, and megaesophagus (9), but whether these signs were due to the homozygous CAV1 null variant remained elusive. We show that achalasia, diagnosed at ages 15 and 18 in two affected patients, should be considered as main complication of CGL3 requiring careful investigations and specific management. Endoscopic myotomy was successful in the two affected patients and avoided invasive surgical procedures. Three affected patients from this study had short stature, and one patient (patient 3) had a height percentile under 25th, contrasting with the accelerated growth frequently described in patients with CGL1 and CGL2, due to AGPAT2 or BSCL2 pathogenic variants, respectively (27). Whether this could be due to the severe impairment of insulin-activated mitogenic signaling pathways linked to the loss of caveolae requires further studies. The four patients did not present with any clinical bone or joint abnormality. However, we did observe osteopenia in two of the investigated patients, with a Z-score below −2.5 at the lumbar spine level, contrasting with the increased bone density usually observed in patients with CGL1 or CGL2 (27, 31). Interestingly, osteopenia was confirmed by DEXA at the age of 21 in the previously described patient with CGL3, with a lumbar spine Z-score of −3 (Dr Chong Kim, personal communication). The decreased bone mass observed in patients 1 and 2 was not explained by vitamin D nor sex steroid hormone levels. In addition, in contrast to hypocalcemia with hypercalciuria observed in the previously reported patient (9), and in caveolin-1 knockout mice (32), serum and urinary calcium were normal in patients from this study. Detailed investigations of calcium homeostasis remain to be performed in CGL3.

The biallelic pathogenic variants responsible for CGL3, located in exon 2 and proximal exon 3 of CAV1, lead to similar phenotypes associated with a loss of protein expression of caveolin-1 and of its partner caveolin-2 (9). Our results show that these variants also strongly inhibit cavin-1 protein expression and lead to a complete loss of caveolae at the plasma membrane of patients’ fibroblasts. Heterozygous subjects were asymptomatic, suggesting that half amount of functional caveolin-1 is sufficient to avoid any specific pathological consequences. In contrast, CAV1 variants affecting the C-terminal domain of the protein were reported as pathogenic in the heterozygous state and lead to rare but heterogeneous diseases (Fig. 2) that may result from different pathophysiological mechanisms. Functional studies of the CAV1 p.(Leu159Serfs*22) variant, responsible for autosomal dominant pulmonary arterial hypertension, showed that the mRNA and protein expression of the mutated allele is preserved and that the resulting abnormal protein could act via a dominant-negative effect on the trafficking of caveolin-1 to the plasma membrane (11, 12, 13). The CAV1 p.(Phe160*) variant, reported in one patient with a neonatal complex progeroid syndrome associated with pulmonary artery hypertension and lipodystrophy, was also shown to be transcribed, without increased RNA degradation (14, 15). In accordance, caveolae were present at the cell surface of the patient’s fibroblasts, but caveolin-1 oligomers displayed decreased stability and weakened interactions with cavin-1 (14, 15, 16).

Cavin-1, encoded by the CAVIN1 gene, is a caveolin-1-interacting protein, which, like caveolin-1, stabilizes caveolae structures. Cavin-1 is located at adipocyte lipid droplets and contributes to the adipocyte differentiation process (2, 3, 18, 33). Biallelic CAVIN1 pathogenic variants are responsible for CGL4, a form of CGL also characterized by skeletal and/or cardiac muscular dystrophy (30, 31), with achalasia in some patients (25, 34, 35). Achalasia is due to the dysfunction of nitrergic inhibitory motor neurons that innervate the circular smooth muscle of the distal esophagus, resulting in impaired relaxation of lower esophageal sphincter (36). We and others have previously shown that CGL4-related CAVIN1 pathogenic variants severely impair cavin-1 and caveolin expression, as well as caveolae formation (18, 35, 37). Since caveolae have been shown to regulate calcium homeostasis and excitation-contraction coupling in smooth muscle (38, 39), their deficiency could promote the development of uncontrolled esophageal contractions, which may evolve to achalasia. Oxidative stress could also contribute to achalasia, as reported in the triple A (alacrima – achalasia – adrenal insufficiency) syndrome (OMIM 231550) (40). In addition, oxidative stress and/or premature senescence could participate in the pathophysiology of lipodystrophy (26, 27, 30, 41).

Patient 2 from this study was diagnosed with atypical retinitis pigmentosa. Interestingly, caveolin-1 has been shown to modulate retinal neuroprotective signaling (42, 43), and retinitis pigmentosa was reported in two patients with a syndrome of partial lipodystrophy, congenital cataracts, and neurological abnormalities, due to the heterozygous CAV1 p.(Lys135Argfs*4) variant (10). However, ophthalmological fundus examination did not show any sign of retinitis pigmentosa in the previously described patient with CGL3, investigated at the age of 26 (9, and Dr Chong Kim, personal communication). Whether this sign could be due to CGL3-associated CAV1 variants remains hypothetical. Dysmorphic features and/or mottled skin were observed in the affected patients from this study and in the two patients reported with neonatal premature aging syndrome due to de novo CAV1 variants (14, 15) (Fig. 2), but no other progeroid sign was observed in patients with CGL3. Although right heart catheterization was not performed, clinical examination and echocardiography did not reveal any sign of pulmonary arterial hypertension in patients with CGL3.

Other phenotypic studies are required to better delineate the clinical relationships between the different CAV1-related diseases, due to loss-of-function or dominant-negative mechanisms. However, in addition to careful metabolic monitoring, we suggest that gastrointestinal, cardiac, ophthalmological, and neurological evaluation should also be included in the follow-up of patients with CGL3.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/EJE-21-0915

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by institutional fundings from the Institut National de la Santé et de la Recherche Médicale (Inserm), Sorbonne Université, the Centre National de la Recherche Scientifique, the French Ministry of Solidarity and Health, Assistance-Publique Hôpitaux de Paris, and by Fondation pour la Recherche Médicale (grant number EQU201903007868).

Author contribution statement

A N Karhan, J Zammouri, Y Usta, I Jéru, and C Vigouroux are equally contributing authors.

Acknowledgements

The authors thank the patients and their families for their participation in this study. They thank Audrey Geeverding and Michael Trichet from Sorbonne University, Department of Electron Microscopy, Paris-Seine Biology Institute, Paris, France for expert advice and help with electron microscopy, Dr Chong Kim, Department of Pediatrics, da Criança Institute, University of Sao Paulo, Brazil for updated clinical data of the previously reported patient with CGL3, and Dr Sonja Janmaat, from the National Reference Center for Rare Diseases of Insulin Secretion and Insulin Sensitivity (PRISIS), Assistance Publique-Hôpitaux de Paris, France, for editorial assistance. Corinne Vigouroux is a member of the European Reference Network on Rare Endocrine Conditions – Project ID No 739527.

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    Genealogical tree of the studied family. Patients 1 to 4 diagnosed with congenital generalized lipodystrophy and homozygous CAV1 p.(His79Glnfs*3) variant (M/M) are depicted by filled symbols. Half-filled symbols (heterozygous subjects, +/M) and patients with normal genotype (+/+) were not affected by the disease. Patient 1 (P1, arrow) is the index case.

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    Schematic representation of CAV1 gene and caveolin-1 protein. CAV1 pathogenic variants (NM_001753.4) identified in this study or previously reported (9, 10, 11, 14, 15) are indicated, with the corresponding phenotypes. OD, oligomerization domain, CSD, caveolin-scaffolding domain. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

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    Photographs from patients with congenital generalized lipodystrophy due to the novel CAV1 p.(His79Glnfs*3) homozygous pathogenic variant. Photographs from patient 1 (A, B and C), patient 2 (D), patient 3 (E), and patient 4 (F) are shown. Triangular face with empty cheeks and generalized lipoatrophy are observed in all patients. Muscular hypertrophy is visible in patients 1–3. Photographs from patient 1 also show mottled skin (A, B and C), acromegaloid features (A), and acanthosis nigricans (B). A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

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    MRI of patient 1 showing generalized lipoatrophy. Magnetic resonance T1-weighted images without fat suppression of patient 1 and of age, sex, and ethnically matched control subject are shown. Brain/cranial (A and B) and abdomen (C) axial images, and pelvis coronal images (D and E) of patient 1 show that lipoatrophy is generalized, sparing only periorbital and bone marrow regions. Hepatic steatosis and muscular hypertrophy are also visible.

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    MRI of patient 1 showing enlarged polycystic ovaries. Magnetic resonance T1-weighted coronal images of patient 1, showing polycystic right (A) and left (B) ovaries of enlarged size (20 × 15 × 39 mm and 37 × 35 × 30 mm, respectively) (arrows).

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    Barium esophagogram series and high-resolution manometry showing achalasia in patients 1 and 2. Barium esophagogram series in patient 1 (A) and patient 2 (D and E). Arrows show the bird’s beak deformity at the distal part of esophagus (A and D) with a dilated esophageal body (E). High-resolution manometry, showing a pressure topography during a swallow, in a control subject (B), patient 1 (C), and patient 2 (F). The horizontal axis refers to time and the vertical axis to length along the esophagus, with the lower esophageal sphincter (LES) depicted below. The pressure magnitude, encoded in color (blue, green, yellow, and red from low to high pressure), is shown along the horizontal line. In a control subject, the pressure topography graph during a swallow shows a normal distal propagation of esophageal peristalsis, with LES relaxation over time (B). Weak peristalsis in patient 1 defines esophagogastric junction outflow obstruction (C). A continuous high pressure without LES relaxation allows the diagnosis of type 2 achalasia in patient 2 (F). A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

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    Homozygous CAV1 p.(His79Glnfs*3) and p.(Glu38*) pathogenic variants induce a loss of protein expression of caveolin-1, caveolin-2, and cavin-1 and a loss of caveolae formation in patients’ fibroblasts. (A) Protein expression of caveolin-1, caveolin-2, and cavin-1 in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (B) Representative images of electron microscopy showing caveolae at the plasma membrane of control fibroblasts (arrows), which were completely absent in patients’ cells.

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    Homozygous CAV1 null variants impair insulin signaling in patients’ fibroblasts. (A) Insulin-mediated activation of insulin receptor β-subunit (IRβ), insulin receptor substrate-1 (IRS1), protein kinase B (AKT/PKB), and extracellular-regulated kinase (ERK)1/2 was evaluated by Western blot in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Cells were maintained for 24 h in a serum-free medium, then incubated or not with 50 nmol/L human insulin (#I9278, Sigma–Aldrich) for 8 min, and the total protein expression of signaling intermediates as well as their phosphorylated activated forms was evaluated. Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (B) The effect of insulin on glycogen synthesis and (C) the ability of cells to bind insulin to its receptor were evaluated as described in Methods section. Results are expressed as the percentage of the first control without insulin. **P < 0.01, ns, not significant.

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    Homozygous CAV1 null variants promote oxidative stress and senescence in patients’ fibroblasts. (A) Reactive oxygen species (ROS) production was assessed by oxidation of 5-6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) in fibroblasts from controls, from patient 1, carrying the CAV1 p.(His79Glnfs*3) homozygous pathogenic variant, and from the patient previously described with a homozygous CAV1 p.(Glu38*) variant responsible for CGL3 (9). Results are normalized to DNA content measured by DAPI and are expressed as the percentage of the first control. (B) Cellular senescence was evaluated by the protein expression levels of the cell cycle arrest and senescence protein markers p16, p21, and phospho-p53 as compared to total p53. Representative images of Western blots performed in triplicate are shown. Tubulin is used as an index of the cellular protein level. Numbers on the left correspond to the expected protein molecular weight. (C and D) Senescence-associated β-galactosidase activity (SA-β-gal) was assessed by X-gal staining at pH6 compared to non-specific staining at pH 4. Representative immunofluorescence images from triplicate experiments are shown. Scale bar is 100 µm. The ratio of pH 6.0/pH 4.0 staining, which represents SA-β-galactosidase activity, is expressed as the percentage of the first control. ****P < 0.0001, ns, not significant. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0915.

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