Lipodystrophies are a heterogeneous group of rare disorders characterized by lack or dysfunction of white adipose tissue (WAT) with perturbation in its mass or distribution (1, 2) and consequent alterations in adipokines levels, mainly leptin and adiponectin (3–5). Lipodystrophies can be classified according to their etiology in genetic or acquired syndromes or based on the extension of the lack of subcutaneous adipose tissue as generalized, partial, or localized (6–10). Manifestations of congenital generalized lipodystrophies (CGLs) appear early, typically at birth or immediately after, and are associated with the development of metabolic complications (11, 12). CGL has four main subtypes, all with an autosomal recessive transmission pattern (13, 14). Their estimated prevalence is approximately 0.23 cases/million and 0.96 cases/million, worldwide and in Europe, respectively (15, 16). Several patients with CGL have been diagnosed among consanguineous individuals, particularly in families from Brazil, Lebanon, Scandinavia, and of African ancestry, probably due to the high rate of founder mutations and endogamy in these ethnic groups (17). To date, about 30 patients with congenital generalized lipodystrophy type 4 (CGL4) have been described, most of them in families of Omani, Japanese, Hispanic, Moroccan, and Turkish origin. In Europe, this phenotype is extremely rare, and only two cases have been reported so far in United Kingdom and Russia (18, 19), with patients reporting consanguinity between their parents or being immigrant families from certain regions where consanguineous relationships are common.

CGL4, first described in 2009 (20), is due to mutations in the polymerase I and transcript release factor (PTRF) gene located on chromosome 17q21.2 (21). PTRF gene encodes a cytoplasmatic protein called caveolae-associated protein 1 (Cavin-1), which, together with caveolin 1, is responsible for the biogenesis of caveolae and co-localizes in the adipocytes (22, 23), being a master regulator of cell differentiation and adipose tissue expandability (24). Beside adipocytes, Cavin-1 is expressed in several tissues, including muscles (7). Common findings in CGL4 subjects are the following: a progressive loss of adipose tissue during infancy, emaciated face due to the absence of Bichat fat pads, acromegalic features (mainly face, hands, and feet), prominent superficial subcutaneous veins (pseudo-flebomegaly), accelerated growth, umbilical hernia, hirsutism, clitoromegaly and amenorrhea, osteopenia and distal metaphyseal deformation with joint stiffness, pyloric stenosis, atlantoaxial instability, and percussion-induced muscle mounding (2, 8, 25). Patients display lack of the subcutaneous adipose tissue, while the mechanical adipose tissue (primary in the retro-orbital region and in small amounts in the buccal, masticator, para-pharyngeal, infrapatellar, and popliteal regions) and bone marrow fat are preserved (21, 26, 27). Regarding their metabolic profile, subjects may present with hypertriglyceridemia, hepatomegaly and hepatic steatosis, hyperinsulinemia, and insulin resistance, although clear evidence of type 2 diabetes has not been described (1, 11, 19, 28). A peculiar feature of this CGL subtype is the involvement of the skeletal and cardiac muscles. Patients display congenital myopathy with high creatine kinase serum levels, local protracted muscle contractions (also known as “mounding”), and severe arrhythmias usually exercise induced (such as long QT syndrome, catecholaminergic polymorphic ventricular tachycardia) causing an increased risk for sudden cardiac death (18, 29). Hypertrophic cardiomyopathy, with or without hypertension, associated with an ectopic cardiac fat deposition and/or lipotoxicity has been described (30). Some case reports have described nephrosis and transient immunoglobulin A deficiency (20, 31, 32). Patients affected by this disease are expected to be hyperphagic due to the impairment of the feedback between adipocytes and central feeding mechanisms (10). One of the master regulators of this signaling is leptin, an adipokine produced in the WAT, whose levels are directly proportional to body fat mass and are, therefore, extremely low in subjects with CGL (3). Leptin modulates food intake, satiety, and energy balance through its action on hypothalamic neurons (33); therefore, low levels of leptin in lipodystrophy can trigger hyperphagia (34).

We herein describe the clinical phenotypes of two pediatric siblings with CGL4, carrying a novel mutation in exon 1 of the PTRF gene in the homozygous state.

This study describes the phenotypic and clinical characteristics of two pediatric siblings affected by CGL type 4 due to a novel homozygous mutation in the PTRF/CAVIN 1 gene.

CGL4 is a recently identified subtype of CGL (CGL4, MIM 613327) characterized by distinctive clinical features such as myopathy, gastrointestinal abnormalities, cardiac arrythmias, and cervical spine instability (36). Initially, CGL4 was phenotypically described in two Mexican American siblings as a novel subtype of CGL (37). Afterwards, mutations in the polymerase I and transcript release factor (PTRF) gene, encoding a cytoplasmatic protein called caveolae-associated protein 1 (Cavin-1), essential for caveolae formation, were described as the etiology (20). To date, 15 mutations (involving 30 patients) of this gene have been described in HGMD (http://www.hgmd.cf.ac.uk/ac/index.php) and diagnosed in several countries (Japan, Saudi Arabia, Oman, Egyptian, Turkey, the United Kingdom, Germany, Morocco, and Mexico). Previously, five missense/nonsense mutations, two splice sites, five small deletions, and three small insertions were discovered in the CAVIN1/PTRF gene (18–20, 28, 30, 36–46). We herein describe a novel homozygous mutation (c.21T>A; p.Tyr7Ter) that causes a change in the reading frame to stop codon, resulting in a premature protein truncation with a likely loss of function effect. Interestingly, the majority of patients with CGL4 reported so far had null mutations in the CAVIN1/PTRF gene, exhibiting classical characteristics of the disease similar to our cases. Although limited, milder phenotypes have been described in rare cases, suggesting phenotypic variability in CGL4. A single base deletion (c.947delA) resulting in a frameshift, depletion of the normal stop codon, and extension of the 3′ untranslated region (UTR) reading frame of the CAVIN1/PTRF gene, producing a predicted protein of higher molecular weight, was described in a Moroccan child who was reported to have a subtle phenotype of CGL4 (41). This prompted the question of whether less severe mutations, such as missense mutations, could be associated with CGL4 of less severe phenotypes. Mild metabolic disorders in heterozygous carriers were reported, suggesting that these individuals may be at risk for mild metabolic disturbances even though they do not exhibit overt lipodystrophy or myopathy (18, 25).

Both of our patients showed early metabolic complications such as hypertriglyceridemia, low HDL cholesterol levels, hepatomegaly, and hepatic steatosis with elevated levels of liver enzymes. A slight increase in HOMA-IR was observed only in one patient. The metabolic phenotype was similar to other previously described CGL4 cases (19, 38, 39, 45, 47). Dyslipidemia is a common finding among these patients, even in the first years of life, whereas diabetes tends to develop during puberty, but subjects can express elevated fasting insulin levels and mild insulin resistance at a younger age. In line with previous reports, none of our siblings had proteinuria or impaired renal function (31, 34). This could be explained by the younger age of our cases compared to the expected age of the onset of this comorbidity (31, 34). The relationship between generalized lipodystrophy and metabolic derangements is well known. Looking specifically at CGL4, PTRF-CAVIN protein is a master regulator in the process of caveolae stabilization (22, 23). The lack of caveolae can affect the regulation of lipolysis, fatty acid flux, triglyceride synthesis, and alteration of insulin signaling in adipocytes, as caveolae are known to concentrate insulin receptors along their margins (14, 39). Caveolae function is extremely complex, including the compartmentalization of multiple signaling pathways not related to the metabolism. A recent case report has shown how the immune system of a subject with CGL4 did not mount a normal response to a single dose of lipid nanoparticle encapsulated mRNA anti-SARS-CoV-2 vaccine but responded to a single dose of the Adenovirus-based vaccine (48). The authors hypothesized that the poor patient’s immune response could be related to the reduced import of the vaccine due to the lack of caveolin, since the lipid nanoparticle encapsulated mRNA is internalized by caveolae-mediated endocytosis.

Patients with CGL4 have been described to present a milder metabolic profile compared to the other subtypes of CGL (20, 28, 36, 38), but several biases may intervene to confound the clinical picture, including the age at diagnosis, the dietary regimen, and lifestyle of various affected subjects. In the past, authors hypothesized that the missense mutations frequently observed in patients with CGL4 could cause a milder metabolic phenotype (28). Another possible explanation could be related to a relatively later loss of secretory function of adipocytes, mainly leptin and adiponectin than other CGLs (10). However, both our patients showed very low/undetectable levels of leptin and adiponectin at very young ages, similarly to most children with CGL4 described by others (31–33, 40). In addition, patients with CGL1 or CGL2, the subtype associated with the most severe metabolic phenotypes, had not only low leptin and adiponectin levels but also very low circulating free leptin and high-soluble leptin receptor levels (49). Since the circulating free leptin isoform is the biologically active subtype, this form could determine the clinically divergent metabolic phenotype. We could therefore speculate that patients with CGL4 have higher circulating free leptin levels and lower protein-bound forms than other CGLs, causing the milder phenotype. Further studies on these patients are necessary to confirm this hypothesis. Other possible explanations for the milder phenotype could be related to the younger age of our patients, preventing the natural disease progression, which typically occurs after early childhood, or related to the gastrointestinal disease potentially limiting food intake or the hypertrophic muscles potentially consuming a portion of surplus energy.

Body composition studies highlighted a reduction in whole subcutaneous adipose tissue, especially over the extremities. MRI scans revealed the characteristic pattern of fat distribution, with generalized loss of SAT but preserved mechanical fat in the retroorbital and periarticular regions, over the palms and soles and in the bone marrow fat. These results were similar to those demonstrated in several studies regarding CGL4 (19, 29, 37, 38), except for the presence of adipose tissue in the palms and soles that was not described in older patients (38). We could hypothesize that the adipose tissue loss in the hands and feet could manifest later in life, typically when the children are in puberty, contrary to the metabolic adipose tissue loss that is already evident from the first months of life.

However, despite the decreased body fat, children had normal height and growth rate was accelerated, likely a result of the relatively high IGF-1 values detected in the patients despite normal GH pulsatility. Since caveolae constitute one pathway for the internalization of GH in vitro (18, 46), the elevated growth velocity and IGF-1 levels, observed in our children with CGL4, could be associated with the reducing caveolae formations, typical of this subtype of lipodystrophy, since GH pulsatility was normal.

The role of leptin as a regulator of bone mass is controversial (50, 51). However, children and adults affected by CGL had no alterations in bone metabolism markers, despite leptin deficiency (52, 53). In our patients, hand and wrist X-ray of our siblings showed a reduction in the calcium content with trabecular bone mass alteration, while baseline serum markers of bone metabolism were within the normal range, except for hypovitaminosis D, and no past fractures were reported. The low calcium content could be related to hypovitaminosis D and the increased growth rate at the time of diagnosis. The follow-up will help clarify bone metabolism during growth.

Apart from the bone, CGL4 importantly affects skeletal, cardiac, and gastrointestinal muscles (21, 23). Bioptic, electron microscopy, and US studies have shown chronic dystrophic changes, including marked variation in muscle fiber size, internal nucleation, a few necrotic and regenerating fibers, increased interstitial fibrosis with reduced caveolae formation, and accumulation of lipid droplets (20, 29, 39–41). Although the exact pathogenesis of myopathy remains unclear, it is common thought that the lack of caveolae has a crucial role in the pathogenesis of the muscle phenotype (30). In our patients, muscle abnormality was denounced during the first years of life, as demonstrated by rapidly deteriorating walking ability and extremely elevated serum CK levels. The muscle biopsy performed on the boy showed variation in fiber size, necrotic fibers, and atypical regenerating forms. At ultrasound, muscles appeared hyperechogenic due to replacement of the hypoechogenic muscle bundles with adipocytes. Tight Achilles tendon is another hallmark of CGL4 caused by hypertrophic calves. Patients are not able to walk on their feet but are forced to stay on their toes, which dramatically increases forefoot pressures (29). In agreement with the literature, both of our patients suffered from Achilles tendon retraction from the first years of life, with the boy presenting a more severe phenotype. Other interesting features in both children were scoliosis and atlantoaxial dislocation, whereas we did not observe percussion-induced muscle mounding, a peculiar feature of CGL4 and of muscular dystrophy due to mutations in the CAV3 gene (19, 29, 40). In the latter disease, preservation of sarcolemmal caveolin-3 staining pattern was observed in young patients compared to older subjects who showed a complete deficiency of caveolin-3, associated with percussion-induced muscle mounding (20, 40, 41). We may hypothesize that in patients with CGL4, secondary reduction in caveolin-3 might arise with age and/or disease progression, and only when deficiency of both proteins (caveolin-3 and cavin-1) develops that muscle mounding can be observed.

Gastrointestinal tract disorders have been described in СGL4 to underline the role of cavin-1 in muscle functionality (18, 20, 28, 38, 42, 45). Massive smooth muscle hypertrophy and thick muscularis mucosa have been shown in CGL4 patients (18, 30). Our male patient had a history of pyloric stenosis, while his sister did not show signs of gastrointestinal involvement, suggesting a phenotypic variability in the presence of the same mutation. The ultrasound study of the gut was normal, but tight dysmotility could be hypothesized since both children, mainly the male one, frequently suffer from abdominal pain not responsive to classic gastrointestinal care advice.

Cardiac arrhythmia have been reported in many CGL4 patients, with an increased risk of sudden death, likely secondary to ventricular arrhythmia also during adolescence (18, 20, 28, 30, 38, 41, 45, 54). The underlying mechanism of paroxysmal ventricular tachycardia (PVT) in patients with CGL4 is unclear. Autoptic studies showed adipocytes interdigitating muscle fibers and mild fibro-fatty infiltration close to the coronary sinus area and atrioventricular node (30). Therefore, the novel cardiac pathology could promote the development of progressive cardiomyopathy, with left ventricular hypertrophy and interstitial/perivascular fibrosis around cardiomyocytes with an alteration of membrane polarization. Mutations in CAV3 gene, also involved in the caveolae formation, induce an increase in late sodium current by affecting the sodium channels located in the caveolae (28); we could hypothesize that PTRF/CAVIN1 mutations in patients affect the sodium channel or the other cardiac channels. In resting conditions, no arrhythmia episodes are described in patients with CGL4. However, during exercise when the heart is subjected to a more significant physical effort, due to the possible mechanism previously described, arrhythmias could develop. Since cardiac arrhythmia is a severe and potentially life-threatening condition, occurring frequently after emotions or exercise, patients with CGL4 should be closely monitored by ECG and, if necessary, fitted with an implanted pacemaker and cardioverter defibrillator (ICD) device. The girl, but not the boy, suffered from paroxysmal supraventricular tachycardia (PSVT) at 3 months of age, causing a cardiac arrest, suggesting again a significant degree of phenotypic variability. After cardiopulmonary resuscitation, the child started therapy with beta-blocker and digoxin until she was 1 year old. No other episodes have been described since then. Echocardiography did not show signs of ventricular hypertrophy in both children. However, they are under tight control, and physical activity has been scheduled after cardiologist and physiatrist indications and monitoring.

We have herein described the phenotype of two pediatric siblings affected by CGL4 due to a novel homozygous mutation of the CAVIN1/PTRF gene. The coexistence of generalized lack of fat, myopathy with elevated CK levels, arrhythmias, gastrointestinal dysmotility, and skeletal abnormalities should prompt a diagnosis of CGL4, although phenotypic variability could be present.

CGL4 in Europe is extremely rare, and only two other cases have been described so far, while the majority of patients come from families of Omani, Japanese, Hispanic, Moroccan, and Turkish origin.

Most patients from these countries, described so far, have identical point mutations, suggesting that they have all the same origin, while in Europe, the genetic etiology of CGL4 previously reported is more complex due to a variability in patients with different ethnic backgrounds, and the presence of immigrant families form different parts of the world. The diagnosis of patients with CGL4 with a heterogeneous molecular etiology is fundamental to detail variable phenotypic expressions, as was observed in our patients.

The datasets presented in this article are not readily available because of ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by the Ethics Committee of Maggiore Hospital Novara (protocol code 69/19 and date of approval 2019). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the participants/patients' legal guardian/next of keen for the publication of this case report.

VM, TD, MR, and FP collected the anamnestic and the biochemical data for the patients. VM, AB, and TD performed the literature search, reviewed, and extracted data from the papers. MG, SM, and CP performed the genetic and hormones analysis. VM and SB performed the figures and table designing. VM, FP, SM, and MG wrote the manuscript. FP, BA, GA, GC, FS, and IR verified the analytical methods and supervised the manuscript drafting. All authors discussed the results and contributed to the final manuscript. All authors contributed to the article and approved the submitted version.