Two Italian kindreds carrying the Arg136-->Ser mutation of the Apo E gene: development of premature and severe atherosclerosis in the presence of epsilon 2 as second allele.

BACKGROUND AND AIMS: Type III hyperlipoproteinemia, or dysbetalipoproteinemia, is commonly associated with apolipoprotein E2 homozygosity (Cys112, Cys158). Apo E2-Christchurch (Arg136-->Ser), a rare mutation of the Apo E gene, located in the receptor-binding domain of the protein, has been found to be associated in the vast majority of cases of dysbetalipoproteinemia. METHODS AND RESULTS: This is the first report of two Italian kindreds carrying the Arg136-->Ser mutation. One family is a four-generation kindred from Genoa (Liguria, Italy) with a high rate of mortality due to coronary artery disease: the proband was a 51-year-old woman with previous myocardial infarction and residual angina, severe carotid atherosclerosis, peripheral arterial vascular disease and arterial hypertension. The other family was identified in Palermo (Sicily, Italy): the proband was an overweight 62-year-old man with a mixed form of hyperlipidemia. The mutation, which was identified by means of Apo E genotyping followed by direct sequencing, co-segregated with the same haplotype in the two families. CONCLUSIONS: The family histories and clinical examinations of these subjects clearly show that the Apo E Arg136-->Ser variant fully expresses a type III phenotype in association with a second allele coding for Apo E2, and only partially in association with a second allele coding for Apo E4.

Hypocomplementemic type II membranoproliferative glomerulonephritis in a male patient with familial lecithin-cholesterol acyltransferase deficiency due to two different allelic mutations.

Patients with familial lecithin-cholesterol acyltransferase (LCAT) deficiency very often show progressive glomerulosclerosis with evolution to end-stage disease. High levels of an abnormal lipoprotein (lipoprotein X) cause glomerular capillary endothelial damage. The ultrastructural study of renal biopsy specimens shows characteristic glomerular deposits of membrane-like, cross-striated structures and vacuole structures. The gene encoding for LCAT has been mapped to chromosome 16q22.1, and several mutations of this gene cause LCAT deficiency which is inherited as an autosomal recessive trait and which is characterized by corneal opacities, normochromic normocytic anemia, and renal dysfunction. Herein we report clinical features and renal histological findings concerning a 24-year-old male patient with classical familial LCAT deficiency due to two different allelic mutations: a nonsense mutation inherited from the father and a missense mutation inherited from the mother. Moreover, the patient showed glomerular histological lesions and an immunofluorescent glomerular pattern typical of hypocomplementemic membranoproliferative type II glomerulonephritis (dense-deposit disease). The nature of electron-dense material that characterizes dense-deposit disease is still unknown, but there are suggestions that some chemical modifications might occur in the renal basement membranes. Therefore, this clinical case might induce to consider possible relations between disorders of the lipoprotein metabolism and renal dense-deposit disease.

Italian familial defective apolipoprotein B patients share a unique haplotype with other Caucasian patients.

Familial defective apolipoprotein (apo) B-100 together with familial hypercholesterolemia are the two common genetic conditions that cause hypercholesterolemia. Familial defective apolipoprotein B-100 is due to mutations around codon 3500 of the apo B gene. The most-characterized mutation is a G>A transition at nucleotide 10,708 that results in the substitution of arginine by glutamine at codon 3500 (Apo B Arg3500Gln). Two other mutations are caused by a C>T transition, one at nucleotide 10,800 (Apo B Arg3531Cys) and the other at nucleotide 10,707 (apo B Arg3500Trp). In the present study we describe three new Italian cases of familial defective apolipoprotein B-100 (Apo B Arg3500Gln), one from the Liguria region and two from Sicily, and the haplotype of the apo B gene co-segregating with the mutation. By screening two groups of probands, clinically diagnosed as having Familial Hypercholesterolemia (700 from mainland Italy and 305 from Sicily), the prevalence of familial defective apolipoprotein B-100 due to Arg3500Gln was found to be very low (0.28% and 0.65%, respectively). The Arg3531Cys mutation was not detected in any proband. In the three new families with Arg3500Gln mutation in the present study and in one previously described in Italy, the mutation was associated with a unique apo B haplotype, which is consistent with data previously reported for Caucasian patients [XbaI-, MspI+, EcoRI-, presence of the 5' signal peptide insertion (Ins) allele, and the 49-repeat allele of the 3'-VNTR].

Root resorption in elderly patients.

Root resorption in permanent teeth is a frequently observed pathology that may originate in various causes. Life expectancy is progressively rising, odontological preventive care is becoming more widespread and professionals are educating their patients in the importance of preventive practices. Because senior citizens are thus losing fewer teeth prematurely they will be conversely more at risk for dental problems later in life. The knowledge of the alterations that may appear in the roots of geriatric patients is particularly relevant to devising therapy and establishing prognosis. The aim of the present study was to evaluate the nature and magnitude of the histologic and histomorphometric features of root resorption and the eventual possibility of repair in elderly people. Seventy-seven uniradicular teeth of patients aged between 65 and 90 years and 18 premolars of patients aged between 14 and 20 years, were removed, fixed in 90% formalin, decalcified in EDTA and embedded in paraffin. Vestibulo-lingual sections were stained with hematoxylin-eosin and employed to perform histological and histomorphometric studies. The results showed that 30% of the teeth of younger patients and 94% of the teeth of elderly patients exhibited areas of root resorption. From the 416 resorptive areas found in elderly patients, 173 exhibited signs of repair being the volume/surface ratio of these areas 0.69 +/- 0.06. These data show that root resorption is a frequent finding in the older population under study. Resorptions are characterized by scarce depth, large areas and a high incidence of repair despite the old age of the patients.

Root resorption in elderly patients

Root resorption in permanent teeth is a frequently observed pathology that may originate in various causes. Life expectancy is progressively rising, odontological preventive care is becoming more widespread and professionals are educating their patients in the importance of preventive practices. Because senior citizens are thus losing fewer teeth prematurely they will be conversely more at risk for dental problems later in life. The knowledge of the alterations that may appear in the roots of geriatric patients is particularly relevant to devising therapy and establishing prognosis. The aim of the present study was to evaluate the nature and magnitude of the histologic and histomorphometric features of root resorption and the eventual possibility of repair in elderly people. Seventy-seven uniradicular teeth of patients aged between 65 and 90 years and 18 premolars of patients aged between 14 and 20 years, were removed, fixed in 90

formalin, decalcified in EDTA and embedded in paraffin. Vestibulo-lingual sections were stained with hematoxylin-eosin and employed to perform histological and histomorphometric studies. The results showed that 30

of the teeth of younger patients and 94

of the teeth of elderly patients exhibited areas of root resorption. From the 416 resorptive areas found in elderly patients, 173 exhibited signs of repair being the volume/surface ratio of these areas 0.69 +/- 0.06. These data show that root resorption is a frequent finding in the older population under study. Resorptions are characterized by scarce depth, large areas and a high incidence of repair despite the old age of the patients.

Root resorption in elderly patients.

Root resorption in permanent teeth is a frequently observed pathology that may originate in various causes. Life expectancy is progressively rising, odontological preventive care is becoming more widespread and professionals are educating their patients in the importance of preventive practices. Because senior citizens are thus losing fewer teeth prematurely they will be conversely more at risk for dental problems later in life. The knowledge of the alterations that may appear in the roots of geriatric patients is particularly relevant to devising therapy and establishing prognosis. The aim of the present study was to evaluate the nature and magnitude of the histologic and histomorphometric features of root resorption and the eventual possibility of repair in elderly people. Seventy-seven uniradicular teeth of patients aged between 65 and 90 years and 18 premolars of patients aged between 14 and 20 years, were removed, fixed in 90

formalin, decalcified in EDTA and embedded in paraffin. Vestibulo-lingual sections were stained with hematoxylin-eosin and employed to perform histological and histomorphometric studies. The results showed that 30

of the teeth of younger patients and 94

of the teeth of elderly patients exhibited areas of root resorption. From the 416 resorptive areas found in elderly patients, 173 exhibited signs of repair being the volume/surface ratio of these areas 0.69 +/- 0.06. These data show that root resorption is a frequent finding in the older population under study. Resorptions are characterized by scarce depth, large areas and a high incidence of repair despite the old age of the patients.

Clinical expression of familial hypercholesterolemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype.

Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients' age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (+18%) and high density lipoprotein cholesterol lower (-5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients >30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the epsilon2 allele and a raising effect of the epsilon4 allele on the LDL cholesterol level in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol. Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregating with the mutation, thus suggesting the presence of common ancestors.

Pseudodominance of lipoprotein lipase (LPL) deficiency due to a nonsense mutation (Tyr302>Term) in exon 6 of LPL gene in an Italian family from Sardinia (LPL(Olbia)).

We analyzed the molecular defect in the lipoprotein lipase (LPL) gene of a young boy from Sardinia who had primary hyperchylomicronemia, pancreatitis, and a complete LPL deficiency in post-heparin plasma. Analysis of LPL gene was performed by using single strand conformation polymorphism (SSCP) and direct sequencing of SSCP-positive region. The proband was homozygous for a C > A transversion in exon 6, which converts the codon for tyrosine at position 302 into a termination codon and eliminates an RsaI restriction site; this allowed the rapid screening of the proband's family members, among whom nine heterozygotes and one additional homozygote were identified. The homozygote was the proband's paternal grandmother who had shown the first clinical manifestation (recurrent pancreatitis) of LPL deficiency at the age of 54 years. LPL mutation carriers showed a mild dyslipidemic phenotype characterized by a reduction of high density lipoprotein-cholesterol (HDL-C) levels, HDL-C/total cholesterol ratio, and low density lipoprotein (LDL) size, associated with a variable increase of triglyceride levels. Five of these carriers were also heterozygotes for beta-thalassemia (Q39X mutation). In these double mutation carriers, plasma HDL-C levels were higher and plasma triglycerides tended to be lower than in carriers of LPL mutation alone. The Tyr302 > Term mutation encodes a truncated protein of 301 amino acids that is probably not secreted by the LPL producing cells. This is the first mutation of LPL gene found in Sardinians.

Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia.

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.

Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia.

The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996. The age of the patients ranged from 1 to 64 years; one third of them were older than 30. Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L. The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r=-0.665; P<0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (

A 'de novo' point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia.

Severe hypercholesterolemia was found in an 11-year-old boy with no family history of familial hypercholesterolemia. The reduced LDL-receptor activity in cultured skin fibroblasts (40% 125I-LDL degradation as compared with a control cell line) indicated the presence of an LDL-receptor defect. The analysis of the promoter region and the exons of LDL-receptor gene by single strand conformation polymorphism revealed an abnormal migration pattern in exon 1, which was due to a T --> A transversion at nucleotide 28 of the cDNA. This novel mutation causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) and introduces an AviII restriction site in exon 1. Screening of the mutation by polymerase chain reaction (PCR) amplification of exon 1 and AviII digestion revealed that none of the proband's family members carried the mutation. Non-paternity was excluded after the analysis of a battery of 14 short tandem repeats located in 13 different chromosomes. These results are consistent with the hypothesis that the proband is heterozygous for a 'de novo' mutation of the LDL-receptor gene producing a non-conservative amino acid substitution. We suggest that the change in the net charge of the signal peptide, caused by the addition of a positively charged amino acid, impairs the co-translational translocation of the nascent receptor protein across the endoplasmic reticulum membrane.

Apolipoprotein E epsilon 4 allele frequency is not increased in progressive supranuclear palsy.

We examined apolipoprotein E (ApoE) immunoreactivity and allele frequency in 12 autopsied cases of progressive supranuclear palsy (PSP), a neurodegenerative disease characterized by diffuse neurofibrillary tangle (NFT) formation without beta-amyloid deposits. In spite of the ApoE immunoreactivity associated with NFTs, in PSP the ApoE allele frequency was comparable with that of age-matched normal controls. This suggests that in Alzheimer's disease the increased frequency of ApoE epsilon 4 does not influence neurofibrillary degeneration, but is probably linked to beta-amyloid deposition.

Occurrence of multiple aberrantly spliced mRNAs of the LDL-receptor gene upon a donor splice site mutation that causes familial hypercholesterolemia (FHBenevento).

A novel point mutation of the LDL-receptor gene was found in an Italian patient with homozygous familial hypercholesterolemia. The SSCP analysis of the promoter and of 16 out of the 18 exons of the LDL-receptor gene was negative, suggesting that the mutation might be located in the region of the gene encompassing exons 14 and 15, a region that had not been amenable to polymerase chain reaction (PCR) amplification from genomic DNA. This region was amplified from cDNA by reverse transcription PCR (RT-PCR). RT-PCR of proband cDNA generated three fragments of 800, 600, and 550 bp, respectively, as opposed to a single 720 bp fragment obtained from control cDNA. The sequence of these fragments showed that: i) in the 800-bp fragment exon 14 continued with the 5' end of intron 15 (90 nucleotides), which in turn was followed by exon 16; ii) in the 600-bp fragment exon 14 was followed by the 5' end of exon 15 (50 nucleotides), which continued with exon 16; iii) in the 550-bp fragment exon 14 joined directly to exon 16. These abnormally spliced mRNAs resulted from a G-->A transition at the +1 nucleotide of intron 15, which changed the invariant GT dinucleotide of the 5' donor splice site. That was associated with the activation of two cryptic donor splice sites in intron 15 and exon 15, respectively, and the use of an alternative splicing leading to the skipping of exon 15. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was one-fourth that found in control cells. These abnormally spliced mRNAs are predicted to encode three abnormal receptor proteins: the first would contain an insertion of 30 novel amino acids; the second would be a truncated protein of 709 amino acids; the third would be devoid of the 57 amino acids of the O-linked sugar domain. Ligand blot experiments indicated that the amount of LDL-receptor present in proband's fibroblasts was approximately one-tenth that found in control cells.

Four novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia.

In this study, we report four new partial deletions of the LDL-receptor (LDL-R) gene discovered during a survey of 326 Italian patients with familial hypercholesterolemia (FH). All deletions were found in FH heterozygotes whose LDL-R activity in skin fibroblasts ranged from 52% to 43% of the values found in control cells. The size and boundaries of the deletions were defined by Southern blotting and, in some cases, by polymerase chain reaction (PCR) amplification of genomic DNA. The sequence of the deletion joint was performed after the reverse transcription and PCR amplification of the appropriate regions of LDL-R mRNA. FHMassa is a 12-kilobase deletion spanning from intron 2 to intron 10. RT-PCR showed that the mutant allele is transcribed into one major and two minor mRNAs. In the most abundant mRNA species, exon 2 joins exon 11, as expected from DNA analysis. In one minor mRNA, which was sequenced, exon 2 joins exon 13, with exons 11 and 12 skipped as a result of an alternative splicing. FHGenova is a 4-kb deletion spanning from intron 10 to intron 12 and eliminating exons 11 and 12. FHRoma is a 4.7-kb deletion spanning from the 5' end of intron 12 to the middle of intron 14 and eliminating exons 13 and 14. This deletion differs in size from the previously described deletion (FHChieti/Macerata), which is located in the same region of the LDL-R gene but is smaller (3.7 kb).(ABSTRACT TRUNCATED AT 250 WORDS)

A new missense mutation (Cys297-->Phe) of the low density lipoprotein receptor in Italian patients with familial hypercholesterolemia (FHTrieste).

During a survey of the mutations of the low density lipoprotein receptor (LDL-R) gene in Italian patients with familial hypercholesterolemia (FH), we identified a novel point mutation, that creates a new EcoRI site at the 5' end of exon 7, in a heterozygous FH subject (FH-100). The sequence of a cDNA fragment encompassing exon 7 showed the presence of a G-->T transversion in codon 297; this created a new EcoRI site and produced a missense mutation, leading to a Cys297-->Phe substitution in repeat A of the epidermal growth factor (EGF) precursor homology domain of LDL-R. Since the substitution of Cys297 disrupts the intracellular transport of the LDL-R protein, as previously demonstrated by site-directed mutagenesis, we suggest that this mutation is the cause of FH in the FH-100 proband. We screened the DNA of 303 Italian FH patients by amplification of exon 7 from genomic DNA followed by digestion with EcoRI or by Southern blotting. Two individuals (FH-64 and FH-127) were found to be carriers of the Cys297-->Phe mutation. Restriction fragment length polymorphism analysis demonstrated that, in two kindreds (FH-64 and FH-100), the haplotype in linkage with the Cys297-->Phe mutation was the same, suggesting the presence of a common ancestor. The Cys297-->Phe mutation has been designated FHTrieste after the name of the city in Northern Italy from which probands FH-100 and FH-127 originate.