Detection of mutations in the apolipoprotein CII gene by denaturing gradient gel electrophoresis. Identification of the splice site variant apolipoprotein CII-Hamburg in a patient with severe hypertriglyceridemia

¹ Division of Clinical Chemistry, Department of Medicine, Albert Ludwigs-University, 79106 Freiburg, Germany.

² Department of Clinical Chemistry, University Hospital, 5000 Odense, Denmark.

³ Department of Pediatrics, Johann Wolfgang Goethe-University, 60590 Frankfurt, Germany.

⁴ Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0131.

⁵ Lipid Research and Atherosclerosis Center, Institute of Internal Medicine and Geriatry, University of Palermo, 90127 Palermo, Italy.

⁶ Klinisch-Chemisches Zentrallabor der Universitätskliniken des Saarlandes, 66421 Homburg/Saar, Germany.
^a Address correspondence to this author at: Department of Medicine, Division of Clinical Chemistry, Hugstetter Strasse 55, 79106 Freiburg i. Br., Germany. Fax 49-761-270 3444; e-mail msnauck{at}mzl200.ukl.uni-freiburg.de.

	Abstract

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Familial apolipoprotein (apo) CII deficiency is a rare autosomal recessive inborn error of metabolism clinically resembling lipoprotein lipase deficiency. A number of mutations of the apo CII gene are known to date; they are located in the promoter region, the coding exons, or in the splice junctions. We present a simple assay based on PCR and denaturing gradient gel electrophoresis, which allows scanning of the promoter, the entire coding sequence, and the splice junctions of the apo CII gene for sequence variants. All gene fragments are amplified using a common PCR protocol and are examined for mutations on a single gradient gel. Using this method and direct sequencing, we identified homozygosity for a donor splice-site mutation in the second intron, previously designated apo CII-Hamburg, as the genetic cause of apo CII deficiency in a 9-year-old boy presenting with chylomicronemia, eruptive xanthoma, and pancreatitis. In addition, the method allowed us to detect all of six different other known mutations of the apo CII gene. We conclude, therefore, that our assay is highly sensitive; in addition, it is easy to perform and may facilitate the differential diagnosis of disorders of lipoprotein metabolism at the genetic level.

	Introduction

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Apolipoprotein CII is a 79-amino acid apolipoprotein (apo).¹ It serves as a cofactor for lipoprotein lipase (LPL) (1). LPL hydrolyzes triglycerides present in chylomicrons and VLDLs to monoglycerides, diglycerides, and free fatty acids. Apo CII is primarily synthesized in the liver (2) and is usually present in human plasma at concentrations ranging from 22 to 55 mg/L (3).

The importance of apo CII as a physiological activator of LPL has been established in vitro (4) and by the identification of patients with excessive hypertriglyceridemia due to a deficiency of apo CII (5)(6). Affected patients present with chylomicronemia, eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, and recurrent attacks of pancreatitis, which may ultimately lead to chronic pancreatic insufficiency. Apo CII deficiency is transmitted in an autosomal recessive fashion. The biochemical findings in homozygous individuals include severe fasting hypertriglyceridemia and chylomicronemia; heterozygous carriers are typically normolipidemic (7). A transient normalization of plasma triglyceride concentrations and a marked improvement in the clinical course is achieved by infusion of apo CII in these patients (8). Interestingly, however, transgenic mice overexpressing the human apo CII gene are hypertriglyceridemic, suggesting that apo CII has functions in the metabolism of plasma triglycerides beyond activating LPL (9), for example, in the regulation of the receptor-mediated catabolism of remnant lipoproteins. The human apo CII gene is located on chromosome 19q13.2; it is ~3.4 kb in length and consists of three introns and four exons. The first intron is located within the 5' untranslated region of the gene; the second intron interrupts the codon for amino acid 11 of the signal peptide, whereas the last intron interrupts the codon for amino acid 44 of the mature protein (10). Molecular defects leading to deficiency of apo CII have been observed in a number of unrelated kindred with familial hyperchylomicronemia (8)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). To date, only point mutations of apo CII rather than major gene rearrangements have been identified. These produce either amino acid exchanges, abnormal RNA splicing, frameshifts, or premature termination codons. Recently, a single nucleotide substitution in the promoter of the apo CII gene has been identified, which diminishes binding of a transcription factor to a positive cis-acting element. Ultimately, this mutation results in complete deficiency of apo CII in the plasma (22).

Apo CII deficiency can be diagnosed biochemically by measuring apo CII (23), by isoelectric focusing (IEF) to detect charge variants (24)(25), or by functional assays in which the capacity of apo CII to restore LPL activity to normal is determined (6). Each of these approaches has major limitations. Reference values for plasma concentrations of apo CII determined by immunoassay do not exclude the presence of dysfunctional variants. Mutant apo CII lacking LPL cofactor activity may have an unaffected isoelectric point, as shown for apo CII _{St. Michael} (12), and both IEF and functional assays are cumbersome. In addition, heterozygous carriers of apo CII mutations are hardly detectable by these methods. One strategy to overcome these problems is to analyze the apo CII gene directly.

In recent years, several reports have shown that electrophoresis in thermally and chemically denaturing gradients is a powerful method to screen for disease-related mutations and polymorphisms (26)(27)(28)(29). In this study, we report the development of a simple and highly sensitive denaturing gradient gel electrophoresis (DGGE)-based assay to detect mutations in the promoter and coding regions of the apo CII gene including the flanking splice sites. Using this method, we identified a donor splice-site mutation in the second intron of the apo CII gene in a 9-year-old boy with severe chylomicronemia. The sensitivity of our assay was further established by its ability to detect all of six other previously characterized apo CII gene mutations.

	Materials and Methods

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human subjects
The male index patient (E.D.) suffered from recurrent vomiting during the perinatal phase. As early as after 2 months of breast feeding, he was switched to medium chain triglycerides. Hypertriglyceridemia was diagnosed at early childhood, but the underlying genetic abnormality was not identified. At that time, the parents repeatedly received dietary advice. Nevertheless, the patient continued to experience recurrent episodes of gastrointestinal discomfort, vomiting, and diarrhea. In December 1991, when the patient was 9 years old, he presented with eruptive xanthoma of the skin and was referred to the Department of Pediatrics of the Frankfurt University Hospital. Plasma triglycerides and cholesterol were 33.0 mmol/L and 6.63 mmol/L, respectively, and the biochemical diagnosis of apo CII deficiency was established (see below). There was no evidence of hepatospleno-megaly. There was no pathological finding on neurological examination. Blood glucose, thyroid-stimulating hormone, thyroid hormones, urea, and creatinine were all within the appropriate reference intervals. The xanthomas gradually disappeared during the weeks to follow, while the patient received a fat-restricted diet. At the age of 11 years, the boy developed two bouts of acute pancreatitis, which resolved within a few days of standard therapeutic regimen including discontinuation of oral fat intake and hypocaloric parenteral nutrition. Since then, additional severe gastrointestinal symptoms have not occurred, and amylase and lipase activities remained within the appropriate reference ranges despite marked fluctuations of the plasma triglycerides.

The patient is the son of clinically healthy parents of Turkish descent living in Germany. It is not clear whether there is consanguinity. Their history was insignificant for hyperlipoproteinemia, atherosclerotic vessel disease, or other metabolic disorders. The sister of the patient was normolipidemic. There was no family history of premature atherosclerosis. The brother of the mother, however, was reported to have high plasma triglycerides as well, but he and other family members were not available for genetic and biochemical characterization.

lipids, lipoproteins, and apolipoproteins
Blood from fasting individuals was drawn into tubes containing EDTA·K₂ (final concentration, 1.5–2 g/L). Plasma was recovered by centrifugation. Cholesterol and triglycerides were measured using enzymatic reagents (Boehringer Mannheim and Wako). VLDL (density <1.0063 kg/L) were isolated by preparative ultracentrifugation (30)(31). HDL-cholesterol was determined by precipitating apo B-containing lipoproteins in the density >1.0063 kg/L infranate (32). The presence of chylomicrons was assessed by inspection of the serum. Analysis of the apolipoproteins of triglyceride-rich lipoproteins by IEF in immobilized pH gradients (Immobiline Dry Plates 4–7, Pharmacia) and immunoblotting of apo CII was carried out as described (24)(25). The dried gel was scanned densitometrically using the OneDScan(TM) software from MWG-Biotech, and the relative concentration of apo CII was expressed as a percentage of the entire apo CII/CIII complex (apo CII/apo CII apo CIII₀ apo CIII₁ apo CIII₂).

dna isolation
Genomic DNA was isolated from peripheral leukocytes with a commercial blood extraction kit (Diagen GmbH, Düsseldorf, Germany). DNA was suspended in Tris-EDTA buffer (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA·K₂) at concentrations between 50 and 200 mg/L and stored at -20 °C.

pcr primer design and melting map predictions
PCR primers corresponding to intronic sequences flanking the four exons of the apo CII gene were designed on the basis of the sequence published by Das et al. (33). Each PCR primer was designed to include at least the first four intron bases flanking each exon in the respective amplification product, thus allowing detection of splice site mutations. The fragment of the short and untranslated first exon was extended to cover also a substantial part of the 5' regulatory region of the gene. To allow for amplification of all fragments under identical conditions, primers with melting temperatures (T_m) as similar as possible were chosen. To achieve the highest possible sensitivity, we introduced a GC clamp into one primer of each pair such that the amplicon enclosed a single domain melting at low temperature. Melting maps of each genomic fragment with a GC clamp attached to either the 5' or the 3' primer were generated with the MELT 87 computer algorithm devised by Lerman and Silverstein (34). MELT 87 calculates the T_m of a given sequence as a function of its nucleotide sequence.

amplification of the promoter and the exons of apo cii gene by pcr
All genomic regions examined were amplified using identical reaction mixtures and thermal cycling conditions. Amplifications were performed in volumes of 50 µL, containing 100 ng of genomic DNA, 0.8 µmol/L of each primer, 200 µmol/L of each deoxyribonucleotide, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl₂, 50 mmol/L KCl, 0.1 g/L gelatin, 100 mL/L dimethyl sulfoxide, and 1 U Thermus aquaticus DNA polymerase (Boehringer Mannheim). The protocol included an initial denaturation step at 95 °C for 3 min, followed by touch-down cycling in a programmable thermocycler (UNO II, Biometra). The first 15 cycles consisted of a denaturation step of 1 min at 94 °C, an annealing step of 1 min at 57 °C, and an elongation step of 1 min at 72 °C. In the next 15 cycles, the annealing temperature was reduced to 55 °C for 1 min; the last 15 cycles were run with an annealing step of 53 °C for 1 min, followed by a final elongation step of 72 °C for 10 min. Subsequently, a denaturation/renaturation program was performed that included 99 °C for 7 min, 65 °C for 60 min, 37 °C for 60 min, and final cooling to 4 °C. The abundance and quality of the DNA fragments were analyzed by electrophoresis on 20 g/L agarose gels, followed by ethidium bromide staining and inspection under ultraviolet light.

dgge
To assess the sensitivity of the DGGE method, we examined DNA harboring previously identified mutations in the apo CII gene. DGGE was carried out according to Fischer and Lerman (26), as modified by Nissen et al. (35). GC-clamped amplified DNA fragments were run on 60 g/L polyacrylamide gels (acrylamide:bisacrylamide, 19:1, by weight; gel size,16 x 20 x 0.1 cm) containing a linear gradient of 30–70% denaturing agent (100% corresponding to 7 mol/L urea and 400 mL/L deionized formamide). Electrophoresis was performed at 150 V for 4 h at 60 °C in TAE buffer (40 mmol/L Tris-acetate, pH 7.5, 1 mmol/L EDTA·Na₂), using the thermostated DGENE(TM) electrophoresis chamber from Bio-Rad. After electrophoresis, the gels were stained with ethidium bromide and examined under ultraviolet illumination.

dna sequencing and restriction typing
A 857-bp fragment of the apo CII gene was amplified using the primer pair 5'-AGT CAG CCT GCC ACA TGA CAC CCC-3' (forward primer, located in the first intron) and 5'-GGG ACT CTC CCC TTG TCC ACT GAT-3' (reverse primer, located in the fourth exon), the amplification mixture described above, and the following cycler protocol: initial denaturation step at 95 °C for 3 min, followed by 30 cycles of denaturation (1 min at 95 °C), annealing (1 min at 65 °C), and elongation (1 min at 72 °C). Amplification was completed by an elongation reaction at 72 °C for 10 min. Direct sequencing of the double-stranded PCR product was performed using the dideoxynucleotide chain termination method and a fluorescently labeled forward primer. Reaction products were developed on an A.L.F. DNA sequencer (Pharmacia). To analyze the apo CII gene by restriction enzyme typing, the 857-bp fragment of the apo CII gene was digested with HphI and DdeI (37 °C for 3 h), analyzed on a 2% agarose gel, and visualized by ethidium bromide staining.

	Results

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We studied lipids and lipoproteins in a boy with a longstanding history of hypertriglyceridemia. He was hospitalized when he developed eruptive xanthoma at the age of 9 years. Triglycerides and cholesterol were 33.0 mmol/L and 6.63 mmol/L, respectively. LDL and HDL were markedly reduced. Inspection of the fasting plasma kept in the refrigerator overnight revealed ample amounts of chylomicrons and a clear infranate. The patient was thus diagnosed as having type I hypertriglyceridemia. A fat-free diet produced decreases of his triglycerides and cholesterol to 4.75 and 2.95 mmol/L, respectively, within 3 days of hospital admission. Lipid and lipoprotein concentrations of the first degree relatives of the patient are shown in Table 1 . We prepared triglyceride-rich lipoproteins by ultracentrifugation at density <1.0063 kg/L and analyzed their apolipoprotein composition by IEF in an immobilized pH gradient. Only trace amounts of apo CII were detectable in two independent samples from the index patient (Fig. 1 ). The densitometric analysis of two independent samples from the index patient revealed that apo CII contributed 1.1% and 2.5%, respectively, to the total of apo CII and apo CIII. Compared with the abundance of apo CIII, apo CII was reduced in the mother and in the father of the index patient (16.0% and 16.2%, respectively). In contrast, the relative concentration of apo CII was 23.7% in the patient's sister.

View larger version (64K): [in this window] [in a new window]   Figure 1. IEF and immunoblotting of urea-soluble apolipoproteins of triglyceride-rich lipoproteins in a 9-year-old boy with chylomicronemia and in his first degree relatives. Triglyceride-rich lipoproteins were prepared by preparative ultracentrifugation at a density of 1.0063 kg/L; delipidated; solubilized in 20 mmol/L TRIS-HCl, pH 10.0, 6 mol/L urea, 10 g/L sodium dodecyl sulfate, and 10 mmol/L dithiothreitol; and subjected to IEF in an immobilized pH gradient (pH 4–7) (24)(25). Gels were stained for protein, using Coomassie Brilliant Blue (24) (left panel) or blotted to a polyvinyldifluoride membrane (25) and probed with a polyclonal antiserum specific for apo CII (23) (right panel). After drying, the stained gel was scanned densitometrically, and the relative concentration of apo CII was expressed as a percentage of the entire apo CII/CIII complex (apo CII/apo CII + apo CIII0 + apo CIII1 + apo CIII2). The percentages of the scanned apo CII were 1.1% and 2.5% for two independent samples from the index patient, 23.7% for the patient's sister, and 16.2% and 16.0% for the mother and father of the index patient, respectively.

To identify the molecular defect underlying the apo CII deficiency in our patient and to establish an effective procedure to rule out apo CII deficiency in patients with excessive hypertriglyceridemia, we developed a DGGE method to screen for sequence abnormalities of the apo CII gene. Oligonucleotide primers were designed to amplify the promoter region and exon 1, the translated exon sequences, and the flanking intron sequences of the apo CII gene (33). The fragment covering the 5' regulatory region of the apo CII gene extended into the promoter up to position 235 upstream from the transcription initiation site. To increase the sensitivity of our assay, an artificially high melting domain was incorporated into the amplification products, using GC-clamped oligonucleotide primers. For each fragment, melting maps containing GC clamps of variable lengths at either the 5' or the 3' end were evaluated (34). Ultimately, the lengths and sites of attachment of the GC clamp producing the most uniform low-melting domain were chosen. Sequences of the primers are listed in Table 2 . Melting profiles of the GC-clamped PCR products were generated. As examples, the maps encompassing exon 3 and exon 4 are shown in Fig. 2 . With the exception of the exon 4 fragment, all melting maps indicated that the respective fragments contain a uniformly low-melting domain, thus predicting high sensitivity to DNA sequence variations (Fig. 2 ). In the case of the fourth exon, the ideal profile was not achieved because two domains largely differing by their T_m were present (Fig. 2 ). The domain with the lower T_m, however, was very small. For this reason, we assumed that it had no effect on the sensitivity to detect sequence variations in the large domain melting at higher temperatures.

View larger version (11K): [in this window] [in a new window]   Figure 2. Theoretical melting profiles for the GC-clamped PCR products containing exons 3 and 4 of the apo CII gene, respectively.

When we examined the second exon of the apo CII gene, we found a single band halted at an aberrant position and a four-band pattern in our index patient and in his mother, suggesting homozygosity and heterozygosity, respectively, for a sequence abnormality in this part of the apo CII gene (Fig. 3 ). To identify the underlying mutation, we directly sequenced this DNA fragment. As shown in Fig. 4 A, the index patient was homozygous for a point mutation of the donor splice site of the second intron of the apo CII gene (1 G C). This mutation, also known as apo CII-Hamburg, was previously described in another Turkish family living in Germany (14) and in one Japanese family (17). Using digestion with HphI and DdeI of a 857-bp fragment of the apo CII gene generated by PCR, we confirmed that our index patient was homozygous for this mutation; both parents were heterozygous carriers, whereas the sister was not affected (Fig. 4B ). The (1 G C) substitution of apo CII-Hamburg gives rise to abnormally spliced apo CII messenger RNA, which contains a termination codon next to the site of the mutation. Ultimately, a truncated form of apo CII is produced, which is not secreted into the plasma. As described by Fojo et al. (14), a small proportion of the apo CII mRNA undergoes splicing, thus explaining the presence of trace amounts of apo CII also found in the plasma of our homozygous patient.

View larger version (47K): [in this window] [in a new window]   Figure 3. DGGE of apo CII gene fragments. (A) Promoter and first exon. Lanes 1 and 4, controls; lane 2, - 86 A G (homozygous); lane 3, - 86 A G (heterozygous). (B) Second exon and flanking regions. Lanes 1 and 4, controls; lane 2, second intron + 1 G C (apo CII-Hamburg, homozygous); lane 3, second intron + 1 G C (apo CII-Hamburg, heterozygous). (C) Third exon and flanking regions. Lanes 1 and 7, controls; lane 2, Lys19 Thr (heterozygous); lanes 3 and 4, Glu38 Lys (heterozygous); lane 5, Tyr37 TER (homozygous); lane 6, Tyr37 TER (heterozygous). (D) Fourth exon and flanking regions. Lanes 1 and 4, controls; lane 2, Lys55 Gln (heterozygous); lane 3, frameshift 70 + G (heterozygous).

View larger version (26K): [in this window] [in a new window]   Figure 4. Identification of a G C nucleotide substitution (apo CII-Hamburg) as the molecular defect in a patient with chylomicronemia and apo CII deficiency. (Top) Direct sequencing of an apo CII gene fragment. The nucleotide sequence of the antisense strand is shown. The sequence indicates homozygosity for a G-to-C transition, changing the consensus donor splice site sequence of the second intron. This produces an abnormally spliced apo CII messenger RNA containing a termination codon next to the site of the nucleotide substitution. (Bottom) Detection of the C-for-G substitution characteristic of apo CII-Hamburg in the index patient and his first degree relatives by restriction typing of in vitro amplified DNA with HphI and DdeI. A 857-bp fragment of the apo CII gene was amplified as described in Materials and Methods and digested with HphI and DdeI. HphI digestion of the "wild-type" allele produced four fragments, 514, 202, 114, and 27 in length, respectively, whereas the mutant allele gave rise to three fragments of 514, 316, and 27 bp, respectively. DdeI digestion of the wild-type and mutant allele produced eight (429,193, 92, 37, 37, 37, 18, and 14 bp) and nine fragments (323, 193, 92, 106, 37, 37, 37, 18, and 14 bp), respectively. The 551-bp bands in lanes 3 and 4 and the 74-bp bands in lanes 9 and 10, respectively, are due to a 37-bp insertion polymorphism within the third intron of the apo CII gene described previously by Hegele et al. (55). Smaller restriction fragments are not labeled.

To further evaluate the sensitivity of our assay, we sought documentation of its ability to detect other sequence variations. We analyzed samples from previously identified homozygous or heterozygous carriers of mutations in the apo CII gene (Table 3 ). We obtained altered migration patterns when we examined the regions in which the mutations were reported to occur. Samples heterozygous for a particular mutation produced a characteristic four-band pattern, the faster migrating pair of bands representing the homoduplexes of the mutant and wild-type strands, respectively, and the slower migrating pair of bands corresponding to heteroduplexes of the two alleles. When we studied DNA containing the same mutation on both alleles, we observed single homoduplex bands that were halted at aberrant positions in the gel. In the cases of the homozygous mutation in the promoter (-86 A G, fragment 1, lane 2, Fig. 3 ) and in the third exon (Tyr TER, lane 5, Fig. 3 ), respectively, we artificially generated heterozygous states by mixing the amplification products of the mutant and the wild-type before heat denaturation and renaturation. As expected, this produced four-band patterns typical for heterozygosity (fragment 1, lane 3, and exon 3, lane 6, Fig. 3 ).

	Discussion

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Established biochemical methods for diagnosing a deficiency of apo CII have been based on the immunochemical quantification of apo CII (23), IEF (24)(25), or on functional assays in which the capacity of apo CII to restore LPL activity to normal is measured (6). The latter method is laborious and not suited for screening purposes. Apo CII immunoassays and IEF may fail to unravel a functional deficiency of apo CII (36), and mutant apo CII may have an unaffected isoelectric point (11). Examination of the apo CII gene, on the other hand, permits definite determination of the defect in individuals with suspected apo CII deficiency. There is no evidence of founder gene effects in apo CII deficiency, and most defects in the apo CII gene identified thus far were unique. Any newly identified case or family is, therefore, expected to have another mutation. Although substantial improvements of direct DNA sequencing methods have been achieved, they are still expensive and labor-intensive, and heterozygosity of mutations might produce equivocal results. We, therefore, wished to develop a simple technique capable of detecting point mutations anywhere in the apo CII gene.

A number of different mutation screening strategies are available today, including chemical cleavage (37), RNase cleavage (38), and heteroduplex analysis (39)(40). These techniques have different levels of sensitivity, simplicity of use, speed of mutation detection, environmental safety, and cost-effectiveness (41)(42). One of the most widely used techniques is single-strand conformation polymorphism (SSCP) analysis; it is rather sensitive and, most important, is easy to establish. To ensure optimal sensitivity of SSCP methods, however, the PCR products from each DNA region must be run under several different conditions. This may include the use of nondenaturing polyacrylamide gels, both with and without addition of glycerol, and running the gels at different power and temperature (43). DGGE, which has been used in the examination of many genes associated with dyslipidemia, for example, in studies of the low-density lipoprotein receptor (35)(44)(45), the codon 3500 region of apo B (46), apo E (47)(48), and lipoprotein lipase (49) compares favorably to the SSCP technique. Most important, DGGE is generally accepted as being more sensitive than SSCP (50)(51). No previously undetected mutations were found when samples in which no mutation was detected by DGGE were completely sequenced, thus giving this technique ~100% sensitivity (52)(53). Another advantage of DGGE is that identical electrophoresis conditions can be used for a number of DNA regions (35)(54). Thus, once the DGGE conditions are established for a particular sequence of interest, DGGE may be markedly simpler than SSCP.

Pivotal for the convenience and sensitivity of DGGE is the design of the primers, which should afford common cycling and electrophoresis conditions for all fragments and the highest sensitivity. Both demands have been accomplished in the method presented here. All fragments are amplified using the same reaction and cycling conditions, and all fragments are scanned for mutations on a single denaturing gradient gel. Together, these features allow us to study individual patients one at a time rather than to collect larger series of patients and analyze them exon by exon.

Our assay scans not only the entire coding sequence and splice junctions of the apo CII gene, but also a major part of the promoter region of the apo CII gene, encompassing those sites of sequence variations of the 5' regulatory region known to date (21)(22). Because there are no known frequent polymorphisms in the apo CII gene that are covered by the amplified fragments, each deviation from the usual electrophoretic pattern is likely to represent a mutation. Thus, after the suspected mutation-carrying exon has been identified, sequencing can be confined to this particular exon. Once a specific mutation is detected in a family with apo CII deficiency, the DGGE method can also be used to trace the mutation in the respective family.

Clinically and biochemically, apo CII deficiency closely simulates LPL deficiency (hyperlipoproteinemia type Ia). It is, therefore, referred to as hyperlipoproteinemia type Ib. Although mutations in the LPL gene more frequently appear to be the cause of type I hyperlipoproteinemia, our DGGE assay will be a valuable complement to the differential diagnosis of type I hyperlipoproteinemia. It will not only provide a tool for the definite genetic diagnosis of apo CII deficiency but also open the opportunity to identify new mutations having only mild impacts on the clinical and/or biochemical phenotype. This, in turn, will extend the understanding of the relationship between genotype and phenotype of apo CII mutations.

Identification of variants exhibiting unaffected LPL cofactor activity in patients with hyperlipoproteinemia may help to unravel thus far unknown functions of apo CII. Evidence for one such function comes from the finding that transgenic mice overexpressing human apo CII exhibit hypertriglyceridemia (9). In these mice, the clearance of VLDL was delayed, whereas the production of VLDL was unaffected. VLDL prepared from the transgenic animals showed markedly decreased binding to heparin, raising the possibility that apo CII modulates the interaction of lipoproteins with cell surface glycosaminoglycans.

In conclusion, we have shown here that DGGE is a reliable and rapid screening method for the detection of sequence variations in the apo CII gene. Considering its high capacity and sensitivity, this method affords rapid diagnosis of apo CII deficiency and will contribute to the elucidation of the possible role of apo CII variants in mild hypertriglyceridemia.

	Acknowledgments

 
We thank L. S. Lerman for providing the MELT 87 program. We gratefully acknowledge the excellent technical assistance of Sabine von Karger and Ulrike Stein.

	Footnotes

 
¹ Nonstandard abbreviations: apo, apolipoprotein; IEF, isoelectric focusing; LPL, lipoprotein lipase; DGGE, denaturing gradient gel electrophoresis; T_m, melting temperature; and SSCP, single-strand conformation polymorphism.

	References

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