the study of familial hypercholesterolemia in italy a narrative review

Abstract In this review we outline our experience in the clinical and molecular diagnosis of familial hypercholesterolemia (FH), built up over more than three decades. We started our work by selecting FH patients on the basis of stringent clinical criteria, including extensive family studies. In most patients we confirmed the clinical diagnosis by showing a reduced LDLR activity in skin fibroblasts. After the isolation of LDLR cDNA and the characterization of the corresponding gene by the Dallas group, we started a systematic molecular investigation of our patients first using Southern blotting, and, subsequently Sanger sequencing. Up to now we have been able to identify 260 mutations of LDLR gene in more than 1000 genotyped FH patients, including 68 homozygotes. During this survey we identified 13 mutation clusters located in different geographical districts, which gave us the chance to compare the phenotype of patients carrying the most common mutations. We also found that mutations in APOB and PCSK9 genes were a rare cause of FH in our cohort. Despite our efforts, we failed to identify mutations in candidate genes in ∼20% of cases of definite FH. An exome-wide study, conducted within the context of an international collaboration, excluded the presence of other major genes in our unexplained FH cases. Recently, we have adopted sequencing technology of the next generation (NGS) with the parallel sequencing of a panel of FH targeted genes as a way of obtaining a more comprehensive picture of the gene variants potentially involved in the disease

Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of specific alleles allowed the rapid detection of a point mutation (missense Gly528 → Asp) in exon 11 of the low density lipoprotein receptor gene which was otherwise not detectable by exon amplification and enzymatic digestion as it does not modify the normal restriction pattern. The mutant allele, designated as FH-Palermo-1 from the origin of the first carrier family identified, gave a specific PCR product of 109 bp clearly distinct from the product of 168 bp obtained from other alleles with a nonspecific couple of primers. This method allowed us to distinguish one positive sample mixed with up to 11 parts of normal DNA. Furthermore, the specific amplification product was characterized by a Bsm I restriction site not present in nonspecific products.—Cantafora, A., I. Blotta, E. Mercuri, S. Calandra, and S. Bertolini. Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction. J. Lipid Res. 1998. 39: 1101–1105

A novel mutation in the sterol 27-hydroxylase gene of a woman with autosomal recessive cerebrotendinous xanthomatosis

<p>Article abstract</p> <p>Mutations of the gene encoding the mitochondrial enzyme sterol 27-hydroxylase (<it>CYP27A1 </it>gene) cause defects in the cholesterol pathway to bile acids that lead to the storage of cholestanol and cholesterol in tendons, lenses and the central nervous system. This disorder is the cause of a clinical syndrome known as cerebrotendinous xanthomatosis (CTX). Since 1991 several mutations of the <it>CYP27A1 </it>gene have been reported. We diagnosed the clinical features of CTX in a caucasian woman. Serum levels of cholestanol and 7α-hydroxycholesterol were elevated and the concentration of 27-hydroxycholesterol was reduced. Bile alcohols in the urine and faeces were increased. The analysis of the <it>CYP27A1 </it>gene showed that the patient was a compound heterozygote carrying two mutations both located in exon 8. One mutation is a novel four nucleotide deletion (c.1330-1333delTTCC) that results in a frameshift and the occurrence of a premature stop codon leading to the formation of a truncated protein of 448 amino acids. The other mutation, previously reported, is a C - > T transition (c. c.1381C > T) that converts the glutamine codon at position 461 into a termination codon (p.Q461X). These truncated proteins are expected to have no biological function being devoid of the cysteine residue at position 476 of the normal enzyme that is crucial for heme binding and enzyme activity.</p

Hypertriglyceridaemia and low plasma HDL in a patient with apolipoprotein A-V deficiency due to a novel mutation in the APOA5 gene

APOA5 encodes a novel apolipoprotein (apo A-V) which appears to be a modulator of plasma triglyceride (TG). In apoA5 knock out mice plasma TG level increases almost fourfold, whereas in human APOA5 transgenic mice it decreases by 70%. Some SNPs in the APOA5 gene have been associated with variations in plasma TG in humans. In addition, hypertriglyceridaemic (HTG) patients have been identified who carried rare nonsense mutations in the APOA5 gene (Q139X and Q148X), predicted to result in apo A-V deficiency. In this study we report a 17-year-old male with high TG and low high density lipoprotein cholesterol (HDL-C), who at the age of two had been found to have severe HTG and eruptive xanthomas suggesting a chylomicronaemia syndrome. Plasma postheparin LPL activity, however, was normal and no mutations were found in LPL and APOC2 genes. The sequence of APOA5 gene revealed that the patient was homozygous for a point mutation (c.289 C&gt;T) in exon 4, converting glutamine codon at position 97 into a termination codon (Q97X). Apo A-V was not detected in patient's plasma, indicating that he had complete apo A-V deficiency. The administration of a low-fat and low-oligosaccharide diet, either alone or supplemented with ω-3 fatty acids, started early in life, reduced plasma TG to a great extent but had a negligible effect on plasma HDL-C. Loss of function mutations of APOA5 gene may be the cause of severe HTG in patients without mutations in LPL and APOC2 genes

Increased Circulating Soluble Cd14 Is Associated With High Mortality In Gram-Negative Septic Shock

The soluble glycoprotein sCD14 binds lipopolysaccharide, a complex that activates endothelial cells and that may be crucial in gram-negative sepsis, Therefore, serum sCD14 was analyzed in 54 patients with gram-negative septic shock and in 26 healthy controls, sCD14 was tested by ELISA and Western blotting, Patients had higher sCD14 concentrations than controls (median, 3.23 vs. 2.48 µg/mL, P = .002). Increased levels were associated with high mortality (median, 4.2 µg/mL in nonsurvivors vs. 2.8 µg/mL in survivors, P = .001). sCD14 was found in two isoforms (49 and 55 kDa) in monocyte cultures. In sera only one of either form was detectable. Controls had the 49-kDa form, and patients had either the 49- or 55-kDa form, but patients with high levels of sCD14 had only the 55-kDa form. Twenty-one (53%) of 39 with the 55-kDa form and 8 (57%) of 14 with the 49-kDa form died. Thus, the level of sCD14 but not its biochemical form had a prognostic value in patients with gram-negative septic shoc

APOE MODENA : A NOVEL APOE MUTANT CAUSING LIPOPROTEIN GLOMERULOPATHY

Lipoprotein glomerulopathy (LPG) is a rare disease characterized by laminated lipid thrombi in the lumina of dilated glomerular capillaries. Apolipoproteins E and B can be demonstrated in these lipid deposits. The plasma lipid profile of LPG patients is similar to that of dysbetalipoproteinemia with elevated IDL and ApoE. Patients often present nephrotic proteinuria but their lipid profile differs from that of nephrotic syndrome secondary to other kidney diseases. LPG has been mainly reported in Japanese and Chinese subjects, associated with novel mutations in APOE gene encoding ApoE. We have identified LPG in an Italian women who at the age of 47 was found to have a combined hyperlipidemia (TC 376 and TG 306 mg/dl) and subsequently developed proteinuria in the nephrotic range. Renal biopsy demonstrated lipoprotein thrombi in glomerular capillaries suggesting LPG. The sequence of APOE gene showed that the patient was: i) homozygous for \uf0652 allele [Cys112 and Cys158 in the mature protein] and ii) heterozygous for a novel mutation in exon 4: c.502 C>T [Arg 150>Cys in the mature protein]. Since the mutant ApoE has a new cysteine residue it is likely that it forms a disulfide bridge with the other Cys residues of the E2 isoforms, resulting in ApoE polymerization (dominant negative effect). This is probably the cause of both dyslipidemia and lipid thrombi in the glomerular capillaries. In view of the poor response of plasma lipids and renal histology and function to statin treatment, the patient started lipid-apheresis with reduction of both dyslipidemia and proteinuria

Niemann-Pick type C disease mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NP44406) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (−1026T/G and −1186T/C or −238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T→G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene

Lipoprotein glomerulopathy associated with a mutation in apolipoprotein e

Lipoprotein glomerulopathy is a pathological condition characterized by lipid accumulation in the glomerular capillaries that has been associated with the presence of rare mutants of apolipoprotein E (ApoE). We describe a 51-year-old Italian patient presenting Type III hyperlipidemia and proteinuria in whom renal biopsy showed capillary ectasia and intraluminal lipid deposits, suggesting the diagnosis of lipoprotein glomerulopathy. The patient, who had elevated plasma ApoE level, was found to be heterozygous for a mutation in ApoE (Arg150Cys), designated apoEMODENA. This mutation induces the formation of ApoE dimers that are detectable under non-reducing conditions. Treatment with hypolipidemic drugs did not result in a complete remission of the proteinuria and was accompanied by a slow but progressive worsening of renal function with the persistence of intracapillary lipid thrombi. The introduction of low-density lipoprotein aphaeresis combined with a more aggressive lipid lowering and antihypertensive therapy resulted in the remission of proteinuria and a substantial improvement of renal function. Switching from low-density lipoprotein aphaeresis to plasma filtration did not result in an equivalent control of renal damage. The patient died of intracranial hemorrhage during an acute episode of malignant hypertension