An apparent inconsistency in parent to offspring transmission of point mutations of LDLR gene in familial hypercholesterolemia.

Background: Familial Hypercholesterolemia (FH), the most common form of autosomal co-dominant hypercholesterolemia, is due to mutations in the LDLR gene, mostly minute or point mutations in the coding sequence.

Methods: Analysis of LDLR gene was performed by direct resequencing and multiplex ligation-dependent probe amplification (MLPA).

Results: LDLR gene resequencing showed that proband I.

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.

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.

Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia.

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated.

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.

Two novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia (FH Siracusa and FH Reggio Emilia).

In the present study we report two novel partial deletions of the LDL-R gene. The first (FH Siracusa), found in an FH-heterozygote, consists of a 20 kb deletion spanning from the 5' flanking region to the intron 2 of the LDL-receptor gene. The elimination of the promoter and the first two exons prevents the transcription of the deleted allele, as shown by Northern blot analysis of LDL-R mRNA isolated from the proband's fibroblasts.

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).

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.

Partial duplication of the EGF precursor homology domain of the LDL-receptor protein causing familial hypercholesterolemia (FH-Salerno).

A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian family hypercholesterolemia (FH) patient during a screening of 300 FH patients. The proband as well as her daughter were found to be heterozygotes for the mutation. Binding, internalization, and degradation of 125I-labeled LDL by the proband's fibroblasts were reduced to approximately 50% compared to values found in control cells.

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.

Chorionic DNA analysis for the prenatal diagnosis of familial hypercholesterolaemia.

Prenatal diagnosis for familial hypercholesterolaemia (FH) was performed by using restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene on chorionic villi DNA taken during the 10th week of pregnancy. Both parents were FH heterozygotes and had previously had a healthy son and an FH homozygous son. Two RFLPs were informative in this family and revealed that the fetus was unaffected by FH.

A large deletion in the LDL receptor gene--the cause of familial hypercholesterolemia in three Italian families: a study that dates back to the 17th century (FH-Pavia).

In the LDL-receptor gene, a large rearrangement causing hypercholesterolemia was detected in three apparently unrelated families living in northern Italy. In all probands, binding, internalization, and degradation of 125I-LDL measured in skin fibroblasts were found to be 40%-50% of control values, indicative of heterozygous familial hypercholesterolemia (FH). Southern blot analysis revealed that the probands were heterozygous for a large (25-kb) deletion of the LDL-receptor gene eliminating exons 2-12.

Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia.

Three gross rearrangements of the low density lipoprotein receptor (LDL-R) gene were recognized during a survey of 23 unrelated Italian subjects with familial hypercholesterolemia (FH). Restriction endonuclease data were obtained by Southern blotting and hybridization with exon-specific probes. Proband FH-29 is heterozygous for a 4-kb deletion, which eliminates exons 13 and 14.

Duplication of exons 13, 14 and 15 of the LDL-receptor gene in a patient with heterozygous familial hypercholesterolemia.

During a survey of Italian patients with familial hypercholesterolemia (FH), we identified an FH heterozygous patient with a gross rearrangement of the low density lipoprotein (LDL) receptor gene. Southern blot analysis of the proband's DNA digested with restriction enzymes PvuII, BamHI, BglII and XbaI and hybridization with cDNA probes complementary to the 3' end of the gene revealed the presence of abnormal fragments that were approximately 7 kb larger than their normal counterparts. DNA digestion with other enzymes (EcoRV, NcoI, KpnI and StuI) and hybridization with probes complementary to exons 13-17 generated normal fragments and an abnormal fragment of 6.

Audiological findings in elderly patients with chronic renal failure.

The audiological results of 46 patients (m/f 27/19, mean age; 57.4 +/- 11.1) with chronic renal failure (CRF) undergoing dialysis were compared with those of an age- and gender-matched control group (n = 25).

Modulation of the synthesis of apolipoproteins in rat hepatoma cells.

The present study was designed to investigate whether plasma lipoproteins and albumin can affect the basal synthetic rate of apolipoproteins in differentiated rat hepatoma cells (Fao) incubated in serum-free medium. The synthesis of apolipoproteins was measured by the incorporation of [35S]methionine into medium lipoproteins isolated by density gradient ultracentrifugation. Under all the experimental conditions used, Fao cells synthesized almost exclusively apolipoprotein E.

Changes of the main isoform of human apolipoprotein A-I following incubation of plasma.

This study was aimed to ascertain whether the more acidic isoforms of plasma apo A-I (A-I-1 and A-I-2) could originate in vitro from the main plasma isoform (A-I0). Apo A-I isoforms were separated by two-dimensional gel electrophoresis before and after a prolonged incubation of serum or EDTA-plasma at 37 degrees C. Incubated plasma there was a marked decrease of apo A-I0 and a concomitant linear increase of apo A-I-1 + and A-I-2.

Isoforms of rat apolipoprotein A-I isolated from the lipoproteins of hepatic Golgi apparatus and plasma.

We compared apo A-I isolated from the lipoproteins of the Golgi apparatus of rat liver with apo A-I found in plasma lipoproteins. Golgi apo A-I consists of 3 main isoforms with a molecular weight of approximately 28000 and isoelectric points (pI) of 5.97, 5.

Separation of the isoprotein forms of apoprotein A-I of rat, rabbit and human HDL by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis.

The distribution and the relative content of the isoprotein forms (isoforms) of apoprotein A-I (apo A-I) of HDL isolated from rat, rabbit and human plasma were studied by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Rat apo A-I consists of seven isoforms having the same molecular weight (27,000) and moving in the 6.44-5.

Plasma and urine lipoproteins during the development of nephrotic syndrome induced in the rat by adriamycin.

The changes of plasma lipoproteins which occur during the development of nephrotic syndrome induced in the rat were investigated by the administration of the antineoplastic drug adriamycin. Rats received a single intravenous injection of the drug (7.5 mg/Kg) and were sacrificed 5, 10, 15, 20, 25, and 30 days after treatment.