Noël Peretti

Lessons from chylomicron retention disease: a potential new approach for the treatment of hypercholesterolemia?

During the last decades, the identification of different rare diseases inducing fat intestinal malabsorption, namely the genetic hypocholesterolemias, has led to the characterization of the physiological steps involved in the intestinal absorption of lipids. Chylomicrons are essential for intestinal absorption of lipids since they are the main carriers of dietary lipids. These triglyceride-rich lipoproteins are secreted exclusively from the enterocyte. The SAR1B gene was identified as responsible for chylomicron retention disease (CMRD), previously called Anderson’s disease (OMIM #246700) [1]. Historically, the first clinical description was in 1961 of a 7-month-old child with a persistent neonatal diarrhea [2]. Finally, in 2003, genotyping demonstrated that Anderson’s disease and CMRD were in fact the same disease [1]. The identification of the gene SAR1B in the pathophysiology of this disease improved our understanding of the physiological secretion of chylomicron. The SAR1B gene is abundant in the intestine, the liver, and muscles. This gene encodes the SAR1b protein, which is involved in chylomicron transport from the endoplasmic reticulum (ER) to the Golgi apparatus [3]. SAR1b is also involved in the hepatic secretion of very low-density lipoprotein (VLDL). Genetic experiments highlight the pivotal role of SAR1B in lipid metabolism. First, SAR1B surexpression in rodents increased the risk of metabolic syndrome, as revealed by incremental weight gain, fat deposition, dyslipidemia, hepatic steatosis, insulin insensitivity, and intestinal fat absorption [4]. In contrast, deletion of the 2 SAR1 isoforms (A and B) suppressed the secretion of chylomicron in vitro by Caco2 cells [5]. However, this genetic approach also demonstrates the limits of experimental models. The SAR1 protein has 2 isoforms, namely SAR1B the most abundant in the human intestine (3-higher) which is depleted in CMRD, and SAR1A. There is incongruity between patients with CMRD who have only a mutation of SAR1B and the cellular or animal models who need a double deletion of the 2 SAR1 isoforms to induce the severe phenotype of the disease [5,6]. Indeed, the deletion of SAR1B alone did not nullify the secretion of chylomicron in Caco2 cells [5]. Interestingly, in duodenal biopsies of patients with CMRD, the compensatory increase of SAR1A (1.5 times) seems insufficient to compensate for the staggering decrease in SAR1B expression [7]. Therefore, it seems reasonable to suppose that surexpression of SAR1A in patients could not compensate the SAR1B suppression. The genotype-phenotype correlation is not obvious in CMRD [8], since less deleterious mutations such as missense mutations are not necessarily associated with less severe phenotypes [9]. Interestingly, the mutation of SAR1B may be involved in different tissue-specific failures: hepatocyte secretion of nascent VLDL in a SAR1B-dependent mechanism may be involved in the hepatic steatosis described in CMRD. Furthermore, the skeletal muscle and the myocardium have been shown to express the SAR1B gene; therefore, increased creatinine kinase and cardiomyopathy described in some patients may be also related to this mutation [10]. Diagnosing this extremely rare disease (probably fewer than 1 in 1,000,000 people worldwide have this autosomal recessive disease) will be the first challenge for the physician. Until now, around 60 cases have been described; but only 40 with their genotype, including about 20 different mutations. Furthermore, the nonspecificity of the initial symptoms (diarrhea and steatorrhea, abdominal distension, vomiting and a rapid failure to thrive) explain the delayed diagnosis. Only one-third of children described in the literature were diagnosed with CMRD during their first year of life [10]. Therefore, a newborn with a chronic diarrhea without any confirmed classical diagnosis should have a lipid profile performed. A severe decrease in total and low-density lipoprotein (LDL) cholesterol (50% normal value) but a moderate decrease in high-density lipoprotein (HDL) and normal triglycerides (TG). This is very different in patients with other intestinal primary hypocholesterolemias associated with intestinal malabsorption, namely abetalipoproteinemia (ABL) and homozygous familial hypobetalipoproteinemia type I (FHBL-1), who have almost undetectable LDL, a normal HDL, and very low TG levels. Others biological abnormalities may point to the diagnosis of CMRD: Liposoluble vitamins are decreased with a severe and permanent vitamin E deficiency even with supplementation. In contrast, vitamins D and K are normalized easily with oral supplementation. Acanthocytosis on blood film is rare in CMRD, in contrast to ABL and FHBL-1. A transaminitis is frequent and early but not specific, with an elevated transaminases (1.5–3 times normal), associated with normal gamma-glutamyltransferase

Single, short in-del, and copy number variations detection in monogenic dyslipidemia using a next-generation sequencing strategy

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next‐generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia‐causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype‐phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia.