Four Novel Partial Deletions of LDL-Receptor Gene in Italian Patients With Familial Hypercholesterolemia
Abstract 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). In both FHRoma and FHChieti/Macerata, the mutant LDL-R mRNA is present in a minute amount, suggesting that the deletion of exons 13 and 14 may increase mRNA degradation. FHPadova-2 is a 2-kb deletion spanning from intron 15 to intron 16 and eliminating the sole exon 16. All deletions except FHPadova-2 produce a shift in the reading frame, leading to either a very short peptide or a truncated protein. In FHPadova-2, elimination of exon 16 does not change the reading frame but is predicted to produce a receptor protein of 813 amino acids, lacking 18 amino acids of the O-linked sugar and 8 amino acids of the transmembrane domain. Ligand blot experiments with rabbit 125I-β VLDL indicate that half the amount of LDL-receptor is present in FHPadova-2 fibroblasts, suggesting that the in-frame deletion of 26 amino acids may disrupt the intracellular transport and/or the insertion of the receptor in the plasma membrane or may increase its degradation.
- Southern analysis
- polymerase chain reaction
- LDL-receptor mRNA
- direct sequencing
- genetic screening
- Received July 27, 1994.
- Accepted October 31, 1994.
Familial hypercholesterolemia (FH) is an autosomal-dominant disorder characterized by an increased level of plasma LDL, which is frequently associated with the development of premature coronary artery disease.1 FH is caused by mutations of the gene encoding the cellular LDL-receptor (LDL-R), which allows the binding, cellular internalization, and degradation of plasma LDL.1 2 A large number of mutations of the LDL-R gene, both gross rearrangements and minute mutations, have been reported.3 4 These mutations disrupt the synthesis, intracellular transport, binding capacity, internalization, and recycling of the LDL-R.3 4 In genetically heterogeneous populations, gross rearrangements (mostly deletions involving more than 25 bp of LDL-R gene)4 account for less than 10% of all mutations.5 6 In a screening of LDL-R gene mutations in 326 Italian FH patients, we discovered four novel partial deletions in FH heterozygous patients. During this survey, we also found that some previously reported deletions were clustered in specific districts of Italy. Haplotype analysis performed in these latter cases indicated that patients bearing these deletions might have had a common ancestor.
Probands (see Table 1⇓ for details) were selected among 326 Italian subjects with the clinical and biochemical features of familial hypercholesterolemia (FH). Patients were selected according to previously reported criteria.7 8 In 130 individuals, the diagnosis of FH was confirmed by assay of LDL-R activity in cultured skin fibroblasts.9
Southern and Northern Blot Analyses
Total cellular RNA was extracted in guanidine-thiocyanate11 from cultured skin fibroblasts that had been maintained in lipoprotein-deficient serum for 15 hours.9 Northern blot analysis was performed as previously reported.8
Reverse Transcription and PCR Amplification
Cellular RNA (1 μg) was reverse-transcribed in a 20-μL reaction mixture containing 8 mmol/L MgCl2, 1 mmol/L of each dNTP, 1 U RNAsin, 100 pmol random hexamers, and 12 U avian myeloblastosis virus (AMV) reverse transcriptase in 1× PCR buffer (30 mmol/L KCl, 50 mmol/L Tris-HCl, pH 9).12 After the sample was heated at 95°C for 5 minutes, 80 μL of 1× polymerase chain reaction (PCR) buffer containing 20 pmol of each primer was added as well as 2.6 U Taq DNA polymerase. MgCl2 concentration in the reaction mixture ranged from 1.5 to 3.5 mmol/L. The following primers were used: (1) 5′ GCT GGA AAT TGC GCT GGA CCG TCG C 3′ (forward primer in exon 1); (2) 5′ CAT GAA CAG GAT CCA CCA C 3′ (reverse primer in exon 10); (3) 5′ ATG ACA CCG TCA TCA GCA GGG A 3′ (forward primer in exon 10); (4) 5′ GTT GTG GAA GAG GAC CAT ATC 3′ (reverse primer in exon 13); (5) 5′ ACC GGA AGA CCA TCT TGG AGG ATG A 3′ (forward primer in exon 12); (6) 5′ CTT GGT GAG ACA TTG TCA CTA TC 3′ (reverse in primer in exon 15); (7) 5′ GAG TGA ACT GGT GTG AGA G 3′ (forward primer in exon 14); and (8) 5′ CAA GGC CGG CGA GGT CTC AGG A 3′ (reverse primer in exon 18). The conditions were 95°C for 5 minutes, 55°C for 2 minutes, and 72°C for 3 minutes for the first time, and subsequently 94°C for 2 minutes, 55°C for 2 minutes, and 72°C for 3 minutes for 28 cycles. PCR products were separated by electrophoresis through a 1.5% agarose gel and sequenced directly with the fmol Sequencing System (Promega Co). Primers used in the sequencing reactions were exon 1 forward primer, exon 10 forward primer, exon 12 forward primer (as specified above), and exon 17 reverse primer (5′ TTC TTC CAT AGA AGG AAG ACC 3′).
PCR Amplification of Genomic DNA
To amplify the region of LDL-R gene encompassing the deletion joint in FHRoma, FHChieti/Macerata, and FHGenova, 1 μg of genomic DNA was amplified in a 100-μL mixture containing 0.2 mmol/L of each dNTP, 50 pmol of each primer, 2.5 U Taq DNA polymerase in 1× PCR buffer (see above), 1.5 to 5 mmol/L MgCl2. Exon 12 forward primer and exon 15 reverse primer (see above) were used for FHRoma and FHChieti/Macerata. Exon 10 forward primer (see above) and intron 13 reverse primer (5′ GTT TCC ACA AGG AGG TTT CAA GGTT 3′) were used for FHGenova. The conditions were 95°C for 5 minutes, 50°C for 90 seconds, 72°C for 120 seconds, and 95°C for 90 seconds for 30 cycles. PCR products were separated by 1% agarose gel electrophoresis after ethidium bromide staining. For FHRoma and FHChieti/Macerata, the appropriate fragments were excised and DNA was extracted from the gel with Quiaex (Diagen, GmbH). Approximately 100 ng of each fragment was digested with 10 UI of XbaI and ApaLI. The fragments obtained after digestion were separated by 1% agarose gel and stained with ethidium bromide.
Ligand blotting experiments were performed in proband P.F. (FHPadova) by using protein extracted from cultured fibroblasts and either human 125I-LDL or rabbit 125I-β VLDL as ligands.13 14 Lipoprotein labeling was carried out according to a standard procedure.9 Membranes were subjected to autoradiography on Hyperfilm-MP (Amersham, UK) for 2 to 4 hours. Densitometric analysis of radiograph films was carried out with an LKB-XL laser densitometer (LKB, Bromma). The radioactive bands corresponding to LDL-R (130 kD) were excised from the membrane and counted directly in a Beckman 5000 gamma counter.
Table 1⇑ gives the clinical and biochemical features of all probands. In all cases, the level of LDL-R activity measured as 125I-LDL degradation by cultured skin fibroblasts was approximately half that found in control cells, indicating that the probands were FH heterozygotes.
Deletion of Exons 3 Through 10 (FHMassa)
Southern blot analysis of proband G.F. (Table 1⇑) DNA digested with several restriction enzymes and hybridized with cDNA probes complementary to exons 1 through 4, 5 through 7, 8 through 10, and 11 through 14 suggested the presence of a large deletion (12 kb) that spanned from the 3′ end of intron 2 to the 5′ end of intron 11 (Fig 1⇓). Northern blot analysis showed a reduced intensity of the normal LDL-R mRNA (5.3 kb) but failed to reveal the presence of mutant mRNA. RNA was reverse-transcribed and amplified by using exon 1 forward primer and exon 15 reverse primer (see “Methods”). Four fragments of approximately 2300, 900, 780, and 640 bp were detected in agarose gel (Fig 2⇓). The nucleotide sequence showed that (1) the 2300-bp fragment corresponded to the normal mRNA (exon 2-exon 3 junction) (Fig 3A⇓); (2) in the 900-bp fragment, exon 2 was followed by exon 11, as expected from the Southern blot results (Fig 3A⇓); (3) in the 640-bp fragment, exon 2 was followed by exon 13 (Fig 3A⇓). We could not obtain a sufficient amount of the 780-bp fragment for sequence analysis. The size of this fragment, however, suggests that it might derive from the joining of exon 2 to exon 12. Thus, the deletion-bearing allele of proband GF generates three mRNA species, the most abundant of which (900-bp fragment) encodes a peptide that contains the 42 amino acids of the NH2 terminal end of the LDL-R and 18 novel amino acids (indicated in italics in Table 2⇓) preceding a stop codon. The other abnormal mRNA species we sequenced (exon 2-exon 13) is expected to encode a protein that contains only 42 amino acids (Table 2⇓). The joining of exon 2 to exon 12 (the 780-bp fragment that was not sequenced) would produce a short receptor devoid of part of the ligand and of EGF precursor homology domains (Table 2⇓). This new deletion found in a single family living in the northwestern part of Tuscany (central Italy) has been designated FHMassa.
Deletion of Exons 11 and 12 (FHGenova)
Southern blot analysis of proband B.M. (Table 1⇑) DNA digested with several restriction enzymes and hybridized with exon 11 through 18 probe suggested the presence of a deletion of ≈4 kb spanning from the 5′ half of intron 10 to the 3′ end of intron 12 and eliminating exons 11 and 12 (Fig 1⇑). PCR amplification of proband genomic DNA obtained by using exon 10 forward primer and intron 13 reverse primer generated a fragment of 2800 bp (data not shown), the size of which was consistent with a 4-kb deletion (Fig 1⇑). Northern blot analysis showed the presence of a single band corresponding to the normal LDL-R mRNA (data not shown). Proband RNA was reverse-transcribed and amplified by using exon 10 forward primer and exon 13 reverse primer. Two fragments of 580 and 320 bp of comparable intensity (data not shown) were obtained and sequenced directly. In the larger fragment, corresponding to the normal mRNA, exon 10 continued with exon 11, whereas in the smaller fragment, exon 10 was followed by exon 13 (Fig 3A⇑). The junction between exon 10 (GG) and exon 13 (GAC) generates a new codon (GGG) that encodes the same amino acid (glycine) as the codon generated by the junction of exon 10 with exon 11 (GGC). However, the junction between exon 10 and exon 13 causes a shift in the reading frame, which produces a sequence of 48 novel amino acids preceding a stop codon (Table 2⇑). This new deletion was named FHGenova after the city of origin of the proband’s family.
Deletions of Exons 13 and 14 (FHRoma and FHChieti/Macerata)
Southern blot analysis of proband M.S. (Table 1⇑) DNA suggested the presence of a deletion of approximately 5 kb downstream of exon 12 (Fig 1⇑). To define the boundaries of the deletion more precisely, we used the following strategies: (1) PCR amplification of genomic DNA using exon 12 forward primer and exon 13 reverse primer, followed by the digestion with ApaLI and Xba I; and (2) PCR amplification of genomic DNA using exon 12 forward primer and exon 15 reverse primer, followed by digestion with ApaLI15 16 (Fig 4⇓). Amplification of the exon 12–exon 15 region, which was possible only in proband DNA (in control DNA, this region is too large to be amenable to PCR amplification), produced a fragment with a size (1800 bp) compatible with a deletion of 4.7 kb (see Fig 4⇓ for details). Northern blot analysis of proband LDL-R mRNA showed the presence of the normal mRNA band with approximately half the intensity of that found in control cells (data not shown). Reverse transcription and amplification of proband RNA with exon 12 forward primer and exon 15 reverse primer produced only a normal fragment of ≈530 bp (data not shown). However, when amplification was performed using exon 10 forward primer/exon 15 reverse primer, we could detect besides the normal fragment (900 bp) a minute amount of an abnormal fragment (650 bp) (Fig 5⇓). The sequence of the latter showed that exon 12 joined exon 15 (Fig 3B⇑), as expected. This new deletion was named FHRoma after the city of origin of proband’s family.
We previously reported this deletion in two members of a family living in the central districts of Italy (FHChieti)8 ; a group from Dallas, Tex, reported this deletion in a homozygous Canadian patient of Italian ancestry.17 The same mutation (see below) has recently been found in three other unrelated subjects (M.L., G.F., M.M.) (Table 1⇑) living in central Italy. Although this deletion eliminates exons 13 and 14, its size and boundaries are different from those defined in FHRoma (Fig 4⇑). More specifically, the PCR amplification of the exon 12–exon 15 region from genomic DNA, followed by digestion with ApaLI, showed that in the FHChieti/Macerata mutation, the deletion (3.7 kb) was smaller than that of FHRoma (Fig 4⇑). As found in FHRoma, reverse transcription of RNA and PCR amplification of the exon 10–exon 15 region produced a trace amount of a 650-bp fragment and the normal 900-bp fragment (Fig 5⇑). The size of the 650-bp fragment is consistent with the idea that the mutant mRNA in FHChieti/Macerata lacks exons 13 and 14. This was confirmed by the direct nucleotide sequence in Fig 3B⇑.
Analysis of the restriction fragment length polymorphisms (RFLPs) of the LDL-R gene in the four patients found to be carriers of this deletion (Table 1⇑) revealed that the haplotype cosegregating with the mutant allele [Sph I (+), Stu I (+), HincII (+), ApaLI 5′ (+), Pvu II (−), Nco I (+), ApaLI 3′ (+)] was the same in all affected subjects. Comparison of this haplotype with that found in the Italian-Canadian patient [Bms I (+), Sph I (+), Stu (+), ApaLI 5′ (+), Pvu II (−), Nco I (+), ApaLI 3′ (+), H.H. Hobbs, personal communication, 1994] strongly suggests that all probands with FHChieti/Macerata deletion shared a common ancestor who had presumably come from the central-eastern districts of Italy, close to Chieti and Macerata.
The joining of exon 12 to exon 15 in mRNA encoded by either the FHRoma or FHChieti/Macerata allele produces a shift of the reading frame, leading to a sequence of 15 novel amino acids before a premature stop codon (Table 2⇑). The amount of this truncated receptor in proband cells is expected to be negligible in view of the fact that in both FHRoma and FHChieti/Macerata, the level of mutant LDL-R mRNA is very low (Fig 5⇑).
Deletion of Exon 16 (FHPadova-2)
Southern blot analysis of proband DNA (P.F.) (Table 1⇑) digested with several restriction enzymes and hybridized with various exon-specific cDNA probes suggested the presence of a 2-kb deletion spanning from the HindIII site in intron 15 to the KpnI site in intron 16 and eliminating the sole exon 16 (Fig 1⇑). Northern blot analysis of proband RNA showed the presence of the 5.3-kb band with an intensity comparable to that found in control cells (data not shown). Reverse transcription of proband mRNA and amplification of the exon 14–exon 18 region produced comparable amounts of two fragments of 600 and 520 bp (data not shown). The nucleotide sequence showed that the 600-bp fragment corresponded to the normal mRNA (exon 15–exon 16 junction), whereas the 520-bp fragment corresponded to the mutant mRNA (exon 15–exon 17 junction) (Fig 3B⇑). The joining of exon 15 with exon 17 does not change the reading frame (Table 2⇑). Thus, the mutant mRNA is translated into a receptor protein of 813 amino acids devoid of the last 18 amino acids downstream of the O-linked sugar domain and the first 8 amino acids of the transmembrane domain. Ligand blotting experiments using rabbit 125I-β VLDL (Fig 6⇓) and human 125I-LDL (data not shown) indicated that proband fibroblasts bound half the amount of both ligands compared with control cells. This new deletion has been designated FHPadova-2 after the city of origin of the proband’s ancestors. A point mutation designated FHPadova (Asp200→Gly) was reported previously by Hobbs et al4 in an Italian-American FH patient. To avoid misunderstanding, we renamed this mutation FHPadova-1.
In the present study, we report four novel partial deletions of the LDL-R gene discovered during the screening of 326 Italian FH patients (living mostly in the northwestern and central districts of Italy). All patients had the clinical and biochemical features of FH; none was found to be a carrier of familial defective apoB-100 (Arg→Gln at codon 3500).18 These deletions, all found in heterozygous patients, were characterized by using a combination of techniques such as Southern blotting, PCR amplification of genomic DNA (whenever possible), Northern blot analysis, reverse transcription, and PCR amplification of LDL-R mRNA (RT-PCR) extracted from skin fibroblasts. In all cases, the use of RT-PCR allowed us to identify the mutant mRNAs even in those situations in which this mRNA had not been detected in Northern blot analysis because of either a small size difference with respect to its normal counterpart or its reduced content. From analysis of the RT-PCR products, three different situations emerged: (1) in FHGenova (deletion of exons 11 and 12) and FHPadova-2 (deletion of exon 16), the amount of the mutant mRNA was apparently identical to that of its normal counterpart; (2) in FHRoma and FHChieti/Macerata (deletion of exons 13 and 14), the mutant mRNA was present in trace amounts; and (3) in FHMassa (deletion of exons 2 through 10), the mutant mRNA consisted of one major and two minor forms (in the major form, exon 1 joined exon 11, as was expected from the genomic DNA analysis, whereas the size of the two minor forms was consistent with the skipping of either exon 11 or exon 12, caused by an alternative splicing). These findings lend further support to the general idea that the intracellular level of mutant LDL-R mRNA varies considerably in relation to both the type of mutation and the occurrence of alternative splicings.8 12
The sequence of the deletion joints shows that the deletion-bearing alleles encode either a very short peptide (FHMassa), a truncated receptor (FHGenova, FHRoma, and FHChieti/Macerata), or a receptor with an in-frame deletion of few amino acids (FHPadova-2) (Table 2⇑). Because it is conceivable that truncated receptors lacking several important domains are rapidly degraded intracellularly, the functional defects caused by these deletions belong to either class I or II.3 4 In FHPadova-2 (deletion of the sole exon 16), the expected mutant receptor protein is devoid of the last 18 amino acids downstream the O-linked region (ie, those amino acids immediately outside the plasma membrane) and the proximal 8 amino acids of the membrane spanning domain. Because the results of the ligand blot with rabbit 125I β-VLDL showed that in the proband fibroblasts there was half the amount of the normal receptor, it is most likely that this mutation reduces the amount of receptor protein available for ligand binding. The membrane spanning domain of the mutant receptor would theoretically contain 8 novel amino acids belonging either to the immediately extracellular region of the O-linked sugar or to the proximal region of the cytoplasmic domain. In this new configuration, the average hydropathy index19 of the novel membrane domain (1.93 to 1.51) would drop below the values usually found in the plasma membrane domain of LDL-R observed in several animal species,20 21 22 23 24 which ranges from 2.123 to 2.54.21 22 It is reasonable to assume that in FHPadova-2 the receptor protein may not be inserted in the plasma membrane, even though it is safely transported through the cytoplasm.
The fact that the deletion of exon 16 is a cause of FH is supported by a previous observation by some Dutch investigators who reported a deletion of this exon in a heterozygous FH patient with a 0.4-kb deletion spanning from intron 15 to the 5′-splice site of exon 16.25
It has not been the aim of this study to analyze the molecular mechanisms responsible for the deletions reported here. It is reasonable to assume, however, that Alu sequence recombinations might be the cause of at least three of the five deletions described: FHRoma, FHChieti/Macerata, and FHPadova-2. Alu sequences are indeed present in introns 12 and 14 (FHChieti/Macerata and FHRoma) and in introns 15 and 16 (FHPadova-2).26 27 28
We have taken advantage of the description of the four new deletions (FHMassa, FHGenova, FHRoma, FHPadova-2) to update the series of deletions found so far in Italian FH patients (Table 3⇓). In our series, large deletions account for approximately 7% of all mutations of LDL-R gene, a value fairly similar to that observed in other populations.5 6 During our survey, we confirmed that some deletions (eg, the previously reported FHChieti/Macerata and FHPavia)8 29 were clustered in specific areas of Italy (northwestern districts for FHPavia; central-eastern districts for FHChieti/Macerata). Thus, despite the genetic heterogeneity of the Italian population, clusters of identical mutations of LDL-R gene are not uncommon.
The small number of patients carrying the same mutation (Table 3⇑) does not allow us to draw firm conclusions on the genotype-phenotype relationship. However, it is of interest that among the probands carrying the FHChieti/Macerata mutation, subject M.M. is free of the typical clinical features of FH (tendon xanthoma, arcus cornealis, and coronary heart disease) despite a fairly high level of plasma LDL cholesterol. A possible explanation for this finding may be the sex and the relatively low Lp(a) level of proband M.M. A survey of the patients of our series confirmed that the clinical features of FH are much less severe and delayed in female FH patients compared with males. On the other hand, the level of Lp(a) in our patients shows a strong correlation with coronary heart disease and carotid atherosclerosis but not with tendon xanthomatosis or arcus cornealis (S.B., M.R., S.C., unpublished observations).
As a corollary to our study, we propose the use of nonradioactive procedures for the identification of some deletions in Table 3⇑. The systematic screening of three deletions (FHGenova, FHRoma, and FHChieti/Macerata) may be successfully performed by PCR amplification of genomic DNA, followed (if necessary) by the digestion of the amplified fragment with appropriate restriction enzymes, as illustrated in Fig 4⇑ for FHChieti/Macerata and FHRoma.
This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR), Progetto Finalizzato Invecchiamento (S.B.), and from CNR, Progetto Finalizzato Ingegneria Genetica (S.C).
Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. New York, NY: McGraw-Hill Publishing Co; 1989:1215-1250.
Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34-47.
Horsthemke B, Dunning A, Humphries S. Identification of deletions in the human low density lipoprotein receptor gene. J Med Genet. 1987;24:144-147.
Sun X-M, Webb JC, Gudnadson V, Humphries S, Seed M, Thompson GR, Knight BL, Soutar AK. Characterization of deletions in LDL receptor gene in patients with familial hypercholesterolemia in the UK. Arterioscler Thromb. 1992;12:762-770.
Daga A, Mattioni T, Balestrieri R, Coviello DA, Bertolini S. Use of three DNA polymorphisms of the LDL receptor gene in the diagnosis of familial hypercholesterolemia. Hum Genet. 1990; 84:412-416.
Lelli N, Ghisellini M, Gualdi R, Tiozzo R, Calandra S, Gaddi A, Ciarrocchi A, Arca M, Fazio S, Coviello DA, Bertolini S. Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia. Arterioscler Thromb. 1991;11:234-243.
Goldstein JL, Basu SK, Brown MS. Receptor mediated endocytosis of low density lipoprotein in cultured cells. In: Colowick SP, Kaplan NO, eds. Methods in Enzymology, Volume 98. Orlando, Fla: Academic Press, Inc; 1983:241-260.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
Lelli N, Garuti R, Zambelli F, Cassanelli S, Tiozzo R, Corsini A, Bertolini S, Riva E, Ortisi MT, Bellù R, Calandra S. Alternative splicing of mutant LDL-receptor mRNA in an Italian patient with familial hypercholesterolemia due to a partial deletion of LDL-receptor gene (FHPotenza). J Lipid Res. 1993;34:1347-1354.
Daniel TO, Schneider WJ, Goldstein JL, Brown MS. Visualization of lipoprotein receptors by ligand blotting. J Biol Chem. 1983;258:4606-4611.
Kowal HH, Herz J, Weisgraber KH, Mahley RW, Brown MS, Goldstein JL. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J Biol Chem. 1990;265:10771-10779.
Leitersdorf E, Hobbs HH. Human LDL receptor gene: two ApaLI RFLPs. Nucleic Acids Res. 1987;15:2782.
Taylor R, Jeenah M, Seed M, Humphries S. Four DNA polymorphisms in the LDL receptor gene: their genetic relationship and use in the study of variation at the LDL receptor locus. J Med Genet. 1988;25:653-659.
Hobbs HH, Leitersdorf E, Goldstein JL, Brown MS, Russell DW. Multiple crm− mutations in familial hypercholesterolemia. J Clin Invest. 1988;81:909-917.
Sudhof TC, Goldstein JL, Brown MS, Russell DW. The LDL-receptor gene: a mosaic of exons shared with different protein. Science. 1985;228:815-822.
Yamamoto T, Bishop RW, Brown MS, Goldstein JL, Russell DW. Deletion in cysteine rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science. 1986;232:1230-1237.
Lee LY, Mohler WA, Schafer BL, Freudenberger JS, Byrne-Connoly N, Eager KB, Mosley ST, Leighton JK, Thrift RN, Davis RA, Tanaka RD. Nucleotide sequence of rat low density lipoprotein receptor cDNA. Nucleic Acids Res. 1989;17:1259-1260.
Mehta KD, Chen W-J, Goldstein JL, Brown MS. The low density lipoprotein receptor in Xenopus laevis. J Biol Chem. 1991; 266:10406-10414.
Lehrman MA, Russell DA, Goldstein JL, Brown MS. Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1986;83:3679-3683.
Bertolini S, Lelli N, Coviello DA, Ghisellini M, Masturzo R, Tiozzo N, Elicio N, Gaddi A, Calandra S. 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 (FHPavia). Am J Hum Genet. 1992:51:123-134.