Detection of familial defective apolipoprotein B-100 among patients clinically diagnosed with heterozygous familial hypercholesterolemia in maritime Canada

Clinical Biochemistry, Vol. 27, No. 4, pp. 265-272, 1994 Copyright 0 1994 The Canadian Society of Cliiical Chemists Printed in the USA. All rights reserved OCW-912094 $6.00 + .oO

Pergamon ooo9-9120(94)Eoo17-0

Detection of Familial Defective Apolipoprotein B-100 Among Patients Clinically Diagnosed With Heterozygous Familial Hypercholesterolemia in Maritime Canada BARBARA MORASH,’ DUANE L. GUERNSEY,‘72 MENG H. TAN,3*4 GALE DEMPSEY,5 and BASSAM A. NASSAR4*5 ‘Department of Physiolo y and Biophysics, *Division of Molecular Pathology & Molecular Genetics, Departments of s,Medicine and 4Biochemistry, and 5Division of Clinical Chemistry, Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada Familial defective apolipoprotein B-100 (FDB) is a genetic disorder resulting from a mutation in the apolipoprotein B-100 (apo B-l 00) gene, most frequently at position 3500, in which arginine is substituted for glutamine in the mature protein. This mutation drastically decreases the affinity of the mutant apo B-100 particle for the low-density lipoprotein (LDL) receptor, and hence decreases the clearance of cholesterol from the circulation. Familial hypercholesterolemia (FH), also a disorder of lipid metabolism, results from mutations in the gene for the LDL receptor. Both FDB and heterozygous FH occur at approximately the same frequency (1 in 500) among Caucasians and both produce clinical symptoms and signs that can be indistinguishable. Polymerase chain reaction (PCR) amplification and subsequent restriction analysis have been used to detect the substitution at codon 3500 in the apo B-100 gene using mutagenic PCR primers. At least one proband from 10 unrelated families with a history of hypercholesterolemia was screened by mutagenic PCR for FDB. Only one of 10 patients demonstrated the mutation for FDB. The mutant apo B-100 allele was shown to segregate with other clinically affected family members. These results demonstrate that molecular analysis is essential to distinguish between FDB and heterozygous FH in hypercholesterolemic families.

KEY WORDS: familial defective apolipoprotein B-100 (FDB); apolipoprotein B-100 (apt B-100); familial hypercholesterolemia (FH); low-density lipoprotein (LDL) receptor; polymerase chain reaction (PCR); restriction isotyping.

Introduction

A

B-100 (apo B-1001, the only protein in the low-density lipoprotein (LDL) parti-

polipoprotein

Correspondence: Dr. Bassam A. Naasar, Division of Clinical Chemistry, Department of Pathology, Victoria General Hospital, 1278 Tower Road, Halifax, Nova Scotia B3H 2Y9, Canada. Manuscript received December 9,1993; revised March 1 1994; accepted March 3,1994. CLINICAL

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cle, is synthesized in the hepatocytes and secreted into the circulation in very low-density lipoproteins (VLDL) (1). The apo B-100 gene, positioned on the short arm of chromosome 2, is 43 kb in length and consists of 29 exons (2,3). The gene encodes a 4536 amino acid peptide that resides as a single copy in the LDL particle. The apo B-100 protein mediates binding of the LDL particle to the membrane bound LDL receptor (4). Binding of apo B-100 to the LDL receptor results in the cellular internalization and lysozymal degradation of the LDL particle. This process serves in the clearance of cholesterol from the circulation. Although many regions within the apo B-100 molecule contribute to its binding, the critical receptor binding domain is generally accepted to be the region between amino acid residues 3000-4000. Monoclonal antibodies to that region bind poorly to apo B-100 when the LDL particle is bound to its receptor (5-7). Familial defective apo B-100 (FDB) results from a guanine to adenine mutation at nucleic acid 10708 in exon 26 of the apo B-100 gene that leads to a substitution of glutamine for arginine in codon 3500 (arg3500 + gln) of the mature protein (8,9). This substitution involves the putative binding domain of the protein and reduces the affinity of the apo B-100 for the LDL receptor to approximately 3% of normal levels. LDL particles carrying the defective apo B-100 protein accumulate in the blood. Like heterozygous familial hypercholesterolemia (HFH), a disorder that results from mutations in the gene for the LDL receptor (41, FDB occurs at a frequency of approximately l/500 among Caucasians (9,101. Both disorders can produce similar clinical features including: moderate to severe hypercholesterolemia, elevated LDL cholesterol (LDLK!), tendon xanthomas, premature atherosclerosis, and a high inci265

MOFUSHET AL

dence of premature myocardial infarction. Additionally, the levels of triglycerides (TG), VLDL, and high-density lipoprotein (HDL) are normal in these two disorders (9,111. Although the tendon xanthomas have not been uniformly described in FDB patients, in some instances, diagnosis of FDB or HFH is difficult based on clinical evidence alone (9,12,13). Previously, FDB patients were identified by the use of either monoclonal antibodies that bind with higher affinity to the abnormal LDL particle (14) or based on the hybridization of genomic or polymerase chain reaction (PCR) amplified DNA to radiolabeled allele specific oligonucleotide probes complementary to either the normal or mutant apo B-100 gene (12,15,16). These methods are time consuming and inconvenient, requiring the use of radioisotopes. Although the mutation at codon 3500 does not create or abolish a restriction enzyme site, it has been possible to artificially introduce restriction sites at the 3500 FDB locus specific for either the normal or mutant allele using mismatched PCR primers (17). This technique has facilitated the development of two faster and simpler methods for the detection of the substitution at codon 3500 involving PCR amplification of a region of genomic DNA spanning codon 3500 and subsequent restriction analysis of the PCR amplified products (18,191. The first method (18) leads to the creation of an MspI restriction site in the wild type but not the mutant allele. The second (19) involves a two-test approach in which the amplification fragment is first cleaved with S&361 that can cleave the normal allele due to the introduced restriction site by the mutagenic PCR. A false-positive result can be ruled out by the second reaction that introduces a ScaJ restriction site using a second set of primers. The advantage of the latter approach has been attributed to its tolerance to variation in magnesium concentration and annealing temperature. In this study, we utilize both methods to screen 10 unrelated hypercholesterolemic individuals from eastern Canada who were previously clinically diagnosed with HFH and treated accordingly. Materials and methods Su~Ec~s

Ten unrelated patients who presented to our lipid clinic with a family history of hypercholesterolemia, increased total cholesterol, LDLC, tendon xanthomas, and premature coronary heart disease and who had been diagnosed with HFH, were screened for FDB. All patients and some of their participating family members gave informed consent for enrolling in the study. Total cholesterol, TG, and HDLC were assayed by established methods using a Cobas FARA analyzer (Roche Diagnostics, Mississauga, ON). Plasma LDLC was calculated using the Friedewald equation (20) and apo B-100 was measured by electroimmunoassay (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). 266

ISOLATION OF GENOMIC DNA BY THE POLYMERASE MUTAGENIC

PRIMERS

CHAIN AND

AND

AMPLIFICATION

REACTION DIGESTION

USING

MspI

BY

DNA was extracted from peripheral blood leukocytes by standard methods (21). Amplification of a 477 base pair (bp) fragment was performed as recently described by Motti et al. (18) with some modification. The PCR was carried out using a COY Model 60 Tempcycler (Coy Laboratory Products Inc., Ann Arbor, MI, USA) using two 24 base long oligonucleotide primers (Genosys). The sequence of the 5’ PCR primer corresponded to nucleotides 1067510698 and the 3’ primer corresponds to bases 11128-11151. A modification was introduced in the 5’ primer toward its 3’ end where the next to last base (base 10697) was altered from an A to C. The single base substitution in the 5’ primer resulted in the introduction of an MspI restriction site (CCGG) in the PCR product of the wild-type allele at codon 3500 that is not present in the mutant allele product. A second polymorphic MspI restriction site at codon 3611 could be present in either fragment (Figure 1). Absence of the MspZ restriction site at codon 3500 in the PCR amplified product is diagnostic of FDB. PCR was performed in 30-cl.L reaction volumes. The reaction mixture consisted of: 0.2 mmol/L of dATP, dCTP, dGTP, and dl?l?P, 30 pmol of each primer (Genosys), 1 p Thermus aquaticus (Taq) DNA polymerase (Perkin-Elmer), 300 ng genomic DNA, 1.5 mmol/L MgCl,, 10 mmol/L Tris-HCL (pH 7.4), 50 mmol/L KCl, and 0.001% gelatin. Each reaction mixture was overlaid with 30 PL of mineral oil and heated to 95 “C for 5 min for initial denaturation. This was followed by 30 cycles of amplification as follows: denaturation at 95 “C for 1 min, annealing at 60 “C for 1 min, and extension at 70 “C for 1 min. A final extension at 70 “C for 5 min was also performed. Following the PCR amplification, 20 FL of reaction product was digested at 37 “C for 2-3 h (without further purification) in a total volume of 30 FL by adding 20 U of the restriction enzymes Mspl, 3 PL of the respective enzyme buffer supplied by the manufacturer (Boehringer Mannheim Inc., Montreal, PQ, Canada) and made up to a final volume of 30 PL with purified H,O. A fraction of the restriction enzyme digests (15 p,L) was analyzed by polyacrylamide gel electrophoresis (PAGE; 10% w/v) in Tris-borate buffer, stained with ethidium bromide, and photographed in a DNA UV-transilluminator. 3500

l

3611 I

-

23

# ----i 477

bp

121

bp

bp 333

bp

* FDB mutation # benign

polymorphism

Figure 1 - MspI restriction map of polymerase chain reaction amplified apolipoprotein B-100 fragment. CLINICALBIOCHEMISTRY,VOLUME27,AUGUST1994

FDB IN HFH PATIENTS ROUNDS OF DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION USING MUTAGENIC PRIMERS ANII DIGESTION WITH TWO ENZYMES: A ROBUST STRATEGY FOR DETECTION OF FDB

Two

Mammotte and van Bockxmeer (19) described a strategy for the detection of the arg3500 --f gln substitution that eliminates the possibility of false negatives. Their method also involved mutagenic PCR primers that introduce a restriction site into PCR amplified DNA. In this approach, a two-test strategy was applied to prevent the possibility of falsenegative results. In the first test, the downstream primer was deliberately mismatched to the normal apo B-100 gene sequence by substituting a G nucleotide for an A, 4 bp from codon 3500 in the FDB locus that resulted in the formation of a Sau961 restriction site in the amplification product of the normal apo B-100 allele. The mutation at codon 3500 prevented the formation of this restriction site. The 87 bp PCR amplified product of the wild-type allele was cleaved with Sau961 into 67 and 20 bp fragments. For an FDB heterozygote, half of the amplified product representing the mutant allele remained undigested, generating fragments of 87,67, and 20 bp when visualized by PAGE. In doubtful cases, a second PCR reaction was performed using the same upstream primer used in the first reaction with a downstream primer that has two deliberate mismatches (T and G instead of G and A). This resulted in the introduction of a ScaZ restriction site in the mutant apo B-100 allele but not in the normal one. For a normal individual, none of the PCR amplified 87 bp product was digested. However, for an FDB heterozygote, half of the PCR amplified product was digested into 65 and 22 bp fragments resulting in three bands upon PAGE. In this work, the PCR reaction was performed in a volume of 25 FL of reaction mixture composed as follows: 0.25 U of Taq DNA polymerase (Perkin-Elmer; amplitaq), 6.25 pmol of each primer (Calgary DNA laboratory), 10 mmol/L Tris-HCl (pH 7.41, 50 mmol/L KCl, 2.0 mmol/L MgClz, 0.001% (w/v> gelatin, 0.2 mmol/L of each dNTP, and 100 ng DNA. It was subjected to an initial denaturing cycle at 95 “C!for 5 min, followed by 35 cycles as follows: heating to 95 “C for 1 min for denaturation, 46 “C for 1 min for annealing, and 72 “C for 1 min for extension. The products were either digested with ScuZ (24 U) or Sau961 (4 U) (BRL) as described above. Results MUTAGENIC PCR PRIMERS AND SUBSEQUENT MspI DIGESTION FOR DIAGNOSIS OF FDB

We have adapted the procedure of Motti et al. (18) in our laboratory to screen 10 unrelated probands from hypercholesterolemic families; these patients were clinically diagnosed with HFH based on their clinical presentation and lipid profile (22). Figure 2 shows a representative PAGE of the PCR amplified CLINICAL BIOCHEMISTRY, VOLUME 27, AUGUST 1994

DNA following MspI digestion for some of the patients analyzed by this procedure. The DNA from most of the patients (lanes A-D) in this group was digested with MspI at codon 3500 to generate 333 and 23 bp fragments upon electrophoresis, indicating that these individuals are homozygous for the normal apo B-100 allele at codon 3500. The 121 bp fragment in these lanes is due to MspI digestion at the nondiagnostic polymorphic MspI restriction site at codon 3611. In lane E one of the two apo B-100 alleles was digested with MspI at both codons 3500 and 3611 to generate bands of 333,23, and 121 bp, respectively. It was not possible to visualize the 23 bp fragment in any of the analysis due to the small size of the fragment yielding very low fluorescence. The second apo B-100 allele was digested only at codon 3500 to generate fragments of 454 (333 and 121 bp) and 23 bp. This pattern indicates that these individuals have two normal apo B-100 alleles at codon 3500 in phase with the frequent as well as the rare allele at codon 3611. The FDB proband (Figure 2) is a 40-year-old female who was previously clinically diagnosed with HFH. However, the recent development of this procedure allows us to distinguish between HFH and FDB and has identified this patient as FDB instead, as can be seen from the appearance of a band at 356 bp upon PAGE of the MspI digested PCR products. The 356 bp fragment results from the absence of an MspI restriction site at codon 3500 (333 and 23 bpl. The presence of the 333 bp fragment represents the second apo B-100 allele that was normal at codon 3500 and digested with MspI. The 121 bp fragment is due to the polymorphic MspI restriction site at codon 3611. The same procedure was then used to show that the mutation at codon 3500 segregated with the disease in this family (Figure 3). The affected sibling, like the FDB proband, suffered from the substitution at codon 3500 (heterozygous FDB) as indicated by the presence of a 356 bp fragment. The 121 bp fragment resulted from a polymorphic MspI site on the mutant apo B-100 allele. The normal apo B-100 allele differs from the proband in that the MspI site at codon 3611 is absent in the affected sibling generating a band at 454 bp. The unaffected sibling had two normal apo B-100 alleles both of which have polymorphic MspI sites on codons 3611. DIAGNOSIS USING TWO ROUND PCR AMPLIFICATION ANII THE RESTRICTIONENZYMES sCa.z AND Sau96I Diagnosis of heterozygous FDB was confirmed in this family using the method of Mammotte and van Bockxmeer (19) (Figure 41. In the first testing protocol, PCR amplification produced a 87 bp DNA fragment. DNA amplified from the unaffected sibling contained the Sau.961 restriction site and was completely digested into 67 and 20 bp fragments. DNA from the FDB proband and the affected sibling was partially digested @7,67, and 20 bp fragments). The undigested fragment (87 bp) indicated the presence of the mutant allele, because a Saul restriction 267

MORASH ET

AL.

100 bp ladder uncut

A-

unaffected

B -

unaffected

C-

unaffected

D-

unaffected

proband E-

-

FDB

unaffected

Figure 2 - Polyacrylamidegel electrophoresisof the PCR amplifiedapolipoproteinB-100 (ape B-100) fragmentfollowing Msp1 digestion of five individuals with normal apo B-100 alleles at position 3500 (A-E) and one FDB heterozygote (proband FDB). The affected FDB proband was identified by the presenceof a band at 356 bp that was not present in unaffectedindividuals. site could not be introduced. Partial digestion in this case also confirmed heterozygosity for the mutation in these patients. Confirmation of these results by application of the second test strategy, which introduces a ScaI restriction site in amplified DNA of FDB affected members, produced 87,65, and 22 bp fragments in the affected patients but not in the normal sibling, confirming the diagnosis of heterozygous FDB in those patients. Discussion FDB and FH are two related disorders of lipid metabolism. In both abnormalities, there is accumulation of LDL particles in the circulation and deposition of cholesterol in abnormal locations. Although they differ in their etiology, this does not impact on the final outcome where both diseases produce sim-

ilar and in some instances identical, clinical criteria (9,11,23). The similarity in clinical presentation coupled with the fact that both disorders occur at a similar frequency in the population, make the distinction of patients with HFH or FDB difficult when based solely on clinical criteria (13,15). In this study, a group of unrelated patients presenting to our lipid clinic with a family history of hypercholesterolemia and who were previously diagnosed with type IIa hyperlipoproteinemia due to HFH (based on clinical criteria), were screened for FDB by molecular analysis for the G to A substitution at nucleotide 10,708. Our results indicated that only one of the 10 patients carried the mutation leading to the arg3500 --s,gln substitution and precipitating FDB. Corsini et al. (13) have also reported similar findings in a family that was initially diagnosed as HFH based upon clinical presentation. Molecular analysis revealed

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FDB IN HFH PATIENTS

FDB sib

undffected

rib

FDB proband

Figure 3 - Polyacrylamide gel electrophoresis showing the MspZdigestion products of the amplified apo B-100 fragment from an affected and an unaffected sibling of the FDB proband.

that the affected family members suffered from FDB. Others have also reported cases of FDB among a group of 252 patients diagnosed with type IIa hypercholesterolemia (24). The cases of misdiagnosed FDB accounted for approximately 5% of the individuals in the study group. These results demonstrate the necessity for molecular analysis in distinguishing between hypercholesterolemic patients suffering from FDB and those suffering from HFH; diagnosis based on clinical presentation alone may not be sufficient especially in patients with FDB and severe hypercholesterolemia (13,151. Recent reports indicate that the hypercholesterolemia in FDB patients however, may range from moderate to severe (13,241. The clinical features of FDB, such as xanthomas and cornea1 arcus, are at least partially dependent upon the severity of hypercholesterolemia.

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Patients with FDB and moderate hypercholesterolemia may not exhibit the tendinous xanthoma. Application of molecular diagnostic techniques for the detection of FDB has enabled rapid screening of patients suspected with the disease, and demonstration of the segregation of the mutant apo B-100 allele with other clinically affected family members. In this study we compared two recently described PCR-based diagnostic methods to screen for FDB. The first involved amplification of a 477 bp fragment and subsequent restriction isotyping with MspI enzymatic digestion. The second involved a two-test strategy requiring two PCR amplifications, each producing 87 bp PCR products, that were then cleaved with either Sau961 or Scul. Both of these diagnostic methods were equally effective in the diagnosis of FDB; however, when using the former

MORASHET

Sau961

AL.

. . dge.stm

80 bp-, 60 bpe

Figure 4 - Polyacrylamidegel electrophoresisof the PCR amplifiedapo B-100 fragment following digestionwith either Sau96I or Seal of DNA isolated from the FDB proband, a clinically affected sibling and a clinically unaffected sibling confirm diagnosis of heterozygousFDB in this family. approach some considerations might aid in the interpretation of data. It has been observed that the mutant apo B-100 allele regularly segregated in sev-

era1 European populations with specific restriction markers, indicating a common and ancient ancestral origin of the mutation in these populations; and

TABLE 1

Lipid and ApolipoproteinLevels in PatientsDiagnosedWith HeterozygousFamilial HypercholesterolemiaBased on Clinical Phenotype Cholesterol(mmol/L) Age

Sex

Total

HDLC

LDLC

A

50

M

:

5: 37 73 46

E

9.5 11.5 10.8

0.90 0.98 2.10

8.0 9.9 8.3

1.05

:.:

Patient

: E H

~DBP FDBS

40 48 36

40 24

F

F M

:.: 7:6

El

76 8’4 8:4

; F

8.9 6.5

0.90

1.10 1.41 1.20 1.23

1.90 0.65

6:l 5.4 6.7

z*: 5:2

Triglycerides (mmol/L)

Apolipoprotein B-100 (g/L)

1.40 1.46 0.90 2.30 1.84

ND 1.81 1.60 ND 1.58

1.40 1.52 1.06 0.76

1.23 ND 1.65 1.34

0.90 1.45

K

Total, total serum cholesterol; HDLC, high-density lipoproteincholesterol; LDLC, low-density lipoprotein cholesterol; FDBP, familial defective apolipoprotein B-100 (FDB) patient; FDBS, FDB sibling; ND, not determined.Reference ranges for total cholesterol,triglycerides,LDLC, HDLC, and apolipoproteinB-100 were describedelsewhere (22). 270

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FDB IN I-IFHPATIENTS

that newer mutations are rare (12,25,26X Motti et al. (18) have utilized the polymorphic MspI restriction site at codon 3611 in all their FDB patients. The inclusion of this site in the analytical approach they described can create a marker for examining segregation of normal and mutant alleles within the studied families. However, because the presence of this marker is variable, larger size fragments of 477 bp generated from potentially undigested DNA, and the 454 bp generated from the normal allele when cleaved at codon 3500, but not 3611, cannot be easily distinguished. We have observed that an undigested PCR amplified DNA sample should be run on each polyacrylamide gel along with the digested samples for easier interpretation. In addition, PCR amplified and MspI cleaved DNA from an individual with a normal apo B-100 allele (333 bp band), or ideally from a known FDB heterozygote (333 and 356 bp bands), should also be run with any samples to distinguish an FDB homozygote (356 bp fragment) that might be misinterpreted as having two normal apo B-100 alleles (333 bp fragment). Only one FDB homozygote has been described in the literature; this patient reportedly suffered from moderate hypercholesterolemia (8.6 mmol/L) on a normal diet and without any lipid lowering drugs (27). The outcome of this patient differs from those previously reported with homozygous FH (27), thus indicating the importance of differentiation between the diagnoses. The advantage of the second technique, namely the two-test strategy, is the clearer separation of the smaller size fragments and easier interpretation of the data. However, it lacks the power to provide any segregation analysis. It could be suggested, that the most reliable diagnostic test for FDB is perhaps a two-test strategy in which the first amplification involves the Sau961 digest. Any suspected false positives due to uncleaved fragments could be subsequently subjected to a second round of amplification involving either the Sca.l or MspI strategy, any of which could be equally effective as a confirmatory test. In conclusion, comparison of the approach described by Motti et al. (18) to the two-test strategy described by Mamotte and Bockxmeer (19) shows that the first approach can be useful in segregation analysis because it includes a polymorphic marker in addition to its ability to identify the FDB mutation. This MspI site was described earlier as a marker for hypercholesterolemia (28). The latter approach however has the advantage of easy interpretation that makes its application as a diagnostic tool highly practical. Acknowledgements We thank K. Buth for her technical assistance. This research was supported by a research award grant from the University Avenue Laboratory Medicine Associates. BA Morash was the recipient of a Medical Research Council of Canada Studentship end an Izaak Walton Killam Memorial Studentship. CLINICALBIOCHEMISTRY,VOLUME 27, AUGUST 1994

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