Dislipidemie aterogene: geni coinvolti nella sindrome da basse HDL

Le dislipidemie aterogene sono alterazioni del metabolismo lipidico associate ad elevato rischio cardiovascolare. Esse sono caratterizzate da bassi valori di HDL ed elevati livelli di trigliceridi, con numerosi geni potenzialmente coinvolti. Ciò rende particolarmente complessa l’analisi mutazionale. In una casistica pediatrica di 55 bambini con valori di colesterolo HDL 150 mg/dl, abbiamo effettuato l’analisi mutazionale di 3 geni candidati, quello per l’apolipoproteina AI (APOA1), quello per la lipoproteinlipasi (LPL) e quello per la lecitina:colesterolo aciltransferasi (LCAT). Come popolazione di controllo è stata utilizzata una popolazione costituita da 174 bambini sani. Questo è stato il primo obiettivo del progetto del dottorato. Nel gene APOA1 sono state identificate le seguenti variazioni di sequenza: 1 nel promotore (-75G>A) e 19 introniche (8 delle quali nuove). Nel gene LPL sono state identificate le seguenti variazioni di sequenza: 1 al 5’UTR (-281T>G), 8 esoniche (N291S, L365V, D9N, S45N (nuova), V108V, E118E, T106T, S447X) e 14 introniche. La N291S, L365V, -281T>G e la D9N sono state precedentemente descritte come varianti associate a fenotipi iperlipidemici e bassi livelli di HDL. Le varianti identificate nel gene LPL sono state ricercate tramite saggio SNaPshot nella popolazione di controllo. La variante N291S è associata al fenotipo di dislipidemia aterogena (p=0.01). L’identificazione delle lesioni molecolari nei geni candidati e la loro caratterizzazione risultano indispensabili al miglioramento dell’abilità diagnostica, prognostica e terapeutica di tale sindrome. Il secondo obiettivo di questo progetto di dottorato, è stato quello di identificare mutazioni nel gene ABCA1 in un probando adulto caratterizzato da livelli molto bassi di colesterolo HDL (HDL-C: 1 mg/dl) al fine di confermare la diagnosi clinica di malattia di Tangier. L’analisi mutazionale e d’espressione del gene ha identificato due varianti introniche (1195-27 G>A; 1510-1 G>A, nuova) che provocano un difetto di splicing e portano alla produzione di una proteina tronca. Il probando è risultato essere eterozigote composto per le due varianti introniche che, quindi, possono essere associate alla malattia di Tangier

A novel splicing mutation in the ABCA1 gene, causing Tangier disease and familial HDL deficiency in a large family.

International audience; Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene. ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86 ± 0.16 mmol/L and 92.2 ± 10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency. Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Analisi mutazionale dei geni ApoA1 e LPL nella dislipidemia aterogena in età pediatrica

Le dislipidemie aterogene sono alterazioni del metabolismo lipidico associate ad elevato rischio cardiovascolare. Esse sono caratterizzate da bassi valori di HDL ed elevati livelli di trigliceridi, con numerosi geni potenzialmente coinvolti e dunque complessa analisi mutazionale. In una casistica pediatrica di 55 bambini con valori di colesterolo HDL 150 mg/dL, abbiamo preliminarmente effettuato l'analisi mutazionale di 2 geni candidati, APOAI e LPL. L'analisi mutazionale è stata condotta per sequenziamento e ottimizzata utilizzando una piattaforma strumentale automatizzata in formato 96-well. Come popolazione di controllo è stata utilizzata una popolazione costituita da 174 bambini senza dislipidemia aterogena. Nel gene APOAI sono state identificate le seguenti variazioni di sequenza: 1 nel promotore (-75G>A) e 19 introniche (8 delle quali nuove). Nel gene LPL sono state identificate le seguenti variazioni di sequenza: 1 al 5'UTR (-281T>G), 8 esoniche tra cui 4 missenso (N291S, L 365V, D9N, S45N( nuova)) e 15 introniche. Le varianti missenso e la -281T >G erano già state precedentemente descritte associate a fenotipi iperlipidemici e bassi livelli di HDL. La frequenza delle varianti identificate nella popolazione target è stata confrontata con quella nella popolazione di controllo mediante un saggio di single nucleotide primer extension. Le varianti più interessanti risultano (già nel gruppo alquanto ridotto da noi esaminato) essere le seguenti: nel gene ApoAl, la -75G>A (frequenza=0.182, "odds ratio"=2.04, p=9.969); nel gene L PL, la N291S, significativamente associata al fenotipo di dislipidemia aterogena (frequenza=0.04, "odds ratio"=8.24, p=0.010) e la S447X (frequenza=0.064, "odds ratio"=0.47, p=0.081), quest'ultima descritta con un aumento di funzione della proteina, cui conseguono bassi livelli sierici di trigliceridi e diminuito rischio cardiovascolare. L'identificazione e la caratterizzazione delle lesioni molecolari, anche estendendo I' analisi ad altri geni candidati ed ampliando la popolazione in studio, risultano indispensabili per una caratterizzazione genetica di questa sindrome

Expression and DNA methylation of ENaC genes

In the airways Cl− ions are secreted by the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel that also limits Na+ absorption by partially repressing the Epithelial Na+ Channel (ENaC). The mechanisms regulating the transcription of the 3 ENaC genes (SCNN1A, SCNN1B and SCNN1G) are not fully elucidated and only sporadic experimental evidence exists on their possible epigenetic regulation. We studied the expression of the 3 ENaC genes and the DNA methylation levels of their 5′-flanking regions in H441, MCF10A, 16HBE, CFBE and HACAT in vitro cell lines, as well as in nasal brushing, granulocytes, lymphocytes and monocytes of subjects with no Cystic Fibrosis (CF)

The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis.

More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies

DNA Methylation Patterns Correlate with the Expression of SCNN1A, SCNN1B, and SCNN1G (Epithelial Sodium Channel, ENaC) Genes

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype–phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, β and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5′-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF

Atherogenic dyslipidemia in children: evaluation of clinical, biochemical and genetic aspects

The precursors of atherogenic dyslipidemia (AD) are not well defined. Therefore, we investigated 62 non-obese, non-diabetic AD and 221 normolipemic children. Anthropometric parameters, blood pressure and biochemical measures were obtained in index children, their parents and all available siblings. The heritability (h(2)) of anthropometric and biochemical traits was estimated by SOLAR. Rare and common variants in APOA1 and LPL genes were screened by re-sequencing. Compared to normolipemic, AD children showed increased body mass index, waist circumference, plasma glucose, insulin, ApoB, HOMA-IR, hs-CRP and lower adiponectin (p<0.001 for all). Metabolic syndrome was present in 40% of AD while absent in controls. All traits (except adiponectin and hs-CRP) showed a strong familial aggregation, with plasma glucose having the highest heritability (89%). Overall, 4 LPL loss-of-function mutations were detected (p.Asp9Asn, p.Ser45Asn, p.Asn291Ser, p.Leu365Val) and their cumulative prevalence was higher in AD than in control children (0.073 vs. 0.026; P=0.038). The LPL p.S447* gain-of-function mutation, resulted to be less frequent in AD than in control children (0.064 vs. 0.126; P=0.082). No variant in the APOA1 gene was found. Our data indicate that AD is a rather common dyslipidemia in childhood; it associates with metabolic abnormalities typical of insulin resistant state and shows a strong familial aggregation. LPL variants may contribute to the development of AD phenotype

Correction: Atherogenic Dyslipidemia in Children: Evaluation of Clinical, Biochemical and Genetic Aspects.

[This corrects the article DOI: 10.1371/journal.pone.0120099.]