Introduction Multiple symmetric lipomatosis (MSL) (syn.: Launois-Bensaude Syndrome, benign symmetric lipomatosis) is a rare disease of fatty tissue. The pathophysiology of MSL still remains unclear, although several approaches have been described in order to understand it. Beside morphological characteristics and some molecular cell biological approaches, little is known about the histological and immunohistochemical characterization of adipose tissue from patients with MSL. Methods From the 45 patients with MSL in our database, 10 were included in the study. Fat tissue samples were collected from affected and unaffected areas. The forearm served as a control area as this area is not affected in MSL. The specimens were analyzed after selected stainings were taken (hematoxylin-eosin = HE, Elastica van Gieson,
Background:Multiple symmetric lipomatosis (MSL) is defined as a disorder of nonencapsulated adipose tissue growth. Its prevalence is indicated as 1:25,000 and affects, as stated in the literature, mainly Mediterranean males (male:female ratio of 15:1). Phenotypes are still classified as defined by Donhauser in 1991. We report clinical and phenotypic data of the largest patient cohort investigated in Germany so far.Methods:Forty-five patients diagnosed with MSL at the University Hospital Regensburg between 2007 and 2017 were photographed, clinically examined, and blood samples were taken. Based on the photographs (n = 33), 11 independent observers assessed patients using the Donhauser classification. Furthermore, the bodies of all patients were subdivided into 12 body areas, and the viewers had to indicate all MSL-affected areas per patient. Prevalence was calculated, comorbidities were assessed, and blood samples were analyzed.Results:According to the established Donhauser classification, less than 50% of the patients could be classified. Therefore, based on the constellations of MSL-affected body areas, a new classification that divides phenotypes of MSL into 5 types (Ia, Ib, Ic, II, and III) was set up and was able to cover 100% of our patients. The male to female ratio was found to be 1:2.5 (male:female). Prevalence of MSL in the catchment area was found to be 1:25,000. Hypercholesterinemia and hypothyroidism were frequent comorbidities, and blood analyses were normal besides a hypercholesterinemia.Discussion:The new proposed classification system describes 5 subtypes and allowed to classify all assessed patients. Male to female ratio (1:2.5) contradicted most previous publications.
Little is known on the causes and pathogenesis of the adipose tissue disorder (familial) Multiple Symmetric Lipomatosis (MSL). In a four-generation MSL-family, we performed whole exome sequencing (WES) in 3 affected individuals and 1 obligate carrier and identified Calcyphosine-like ( CAPSL ) as the most promising candidate gene for this family. Screening of 21 independent patients excluded CAPSL coding sequence variants as a common monogenic cause, but using immunohistochemistry we found that CAPSL was down-regulated in adipose tissue not only from the index patient but also in 10 independent sporadic MSL-patients. This suggests that CAPSL is regulated in sporadic MSL irrespective of the underlying genetic/multifactorial cause. Furthermore, we cultivated pre-adipocytes from MSL-patients and generated 3T3-L1-based Capsl knockout and overexpressing cell models showing altered autophagy, adipogenesis, lipogenesis and Sirtuin-1 (SIRT1) expression. CAPSL seems to be involved in adipocyte biology and perturbation of autophagy is a potential mechanism in the pathogenesis of MSL. Downregulation of CAPSL and upregulation of UCP1 were common features in MSL fat while the known MSL genes MFN2 and LIPE did not show consistent alterations. CAPSL immunostainings could serve as first diagnostic tools in MSL clinical care with a potential to improve time to diagnosis and healthcare options.
Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cD-NAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG(r) tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The a-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.
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