Hum Genet (199(I) 86:91-93

�9 Springer-Verlag 1990

Detection by denaturing gradient gel electrophoresis of a new polymorphism in the apolipoprotein B gene

Maria Navajas t, Anne-Marie Laurenr Jean-Franqois Moreel=, Ashraf Ragab 3, Jean-Pierre Cambou 3, G6rard Cuny l, Franqois Cambien 2, and G6rard Roiz/~s I ICNRS UPR 841)2 and INSERM U.249, Institut de Biologie, Boulevard Henri IV, F-34060 Montpellier Cedex. France 2INSERM U.258, INSERM SC 7, H6pital Broussais, 96. Rue Didot. F-75675 Paris Cedex 14, France )INSERM U. 101, CHU Purpan. Place Baylac, F-31059 Toulouse Cedex, France

Received March 16, 1990 / Revised July 12. 1990

Summary. The apol ipoprote in B gene is subject to muta- tions that may be impor tant in co ronary heart diseases. We have used polymerase chain reaction and denatur ing gradient gel e lectrophoresis to characterize a single nuc- leotide substitution in the apol ipoprote in B gene, This mutat ion affects amino acid 4311 of the protein and con- verts asparagine to serine. It was found in 24% of the 81 unrelated individuals analyzed. Moreover , another mu- tation was detected by sequencing in a single individual.

Introduction

The apol ipoprote in componen t of low density l ipopro- tein ( L D L ) is apol ipoprote in B (apo B). It both main- tains the integrity of the particle (Yan et al, 1986) and serves as ligand to the L D L receptor in the cellular up- take of cholesterol (Brown and Goldstein 1983).

More and more point mutat ions are recognized in the apo B gene since it has been cloned and sequenced (Blackhart et al. 1986: Knot t et al. 1986: Law et al. 1986a). Methods for finding new mutat ions have been rapidly developing since the polymerase chain reaction (PCR) has appeared (Saiki et al. 1985). One of them, called denatur ing gradient gel e lectrophoresis ( D G G E ) has been shown to be efficient provided the muta t ion to be detected is present in the first fusion domain of the D N A molecule (Fischer and Le rman 1983: Myers et al. 1985: Noll and Collins 1987). It has been improved more recently by adding a GC clamp at the end of oligonuc- leotides used for amplification (Sheffield et al. 1989).

We have used this technique in a port ion of exon 29 of the apo B gene and detected a new mutat ion, which is present in 24% of the 162 ch romosomes analyzed. It segregates in a Mendel ian fashion. It has been charac- terized by sequencing as affecting amino acid 4311, which is conver ted from Asn to Ser in the varying allele.

Qffprint requests to: G. Roiz6s

Materials and methods

Genomic DNA isolated from blood samples was studied in 8[ un- related individuals.

A 924-base pair (bp) fragment from exon 29 of apo B-gene was amplified by PCR according the following conditions: I pg of geno- mic DNA was mixed with 75 pmol of each oligonucleotide and 75 nmol of each deoxyribonueleoside triphosphate in 50~1 of the buffer recommended by the supplier: 2.5 units of Taq polymerase (Genofit. Geneva) were added. Except for an incubation time of 5 rain at 94~ during the first cycle, the conditions were as follows for 29 cycles: 5 s at 90~ 30 s at 63~ 2 min at 70~ plus one cycle of extension at 7(1~ for 10 rain. A 5-p,l sample of the reaction pro- ducts was analyzed on a 1% agarose minigel. The sequences of the oligonucleotides used were as follows:

primer 1 (20 mer) 5' CTGGCTI'GCTAACCTCTCTG 3'.

primer 2 (2(3 mer) 5' GAGAAGCTI'CCTGAAGCTGC 3'.

The gel apparatus, buffers, and conditions for DGGE were the same as described in Myers et al. (1987). A fraction (3 gl) of each amplified fragment was subjected to electrophoresis in a 6.5% acrylamide gel with a linearly increasino gradient from 0% to 100% or 20% to 70% denaturant (100% denaturant = 7 M urea and 40%, vol/vol, formamide) at 150 V for 4 h.

Amplified double-stranded DNA was selectively precipitated in ethanol to remove the primers and ssDNA synthesized by PCR using one of the original primers (20 pmole). After 20 rounds of amplification, the ssDNA was phenol extracted and ethanol pre- cipitated. A fraction of the resuspended sample was annealed to an internal primer and sequenced using sequenase protocol (Tabor and Richardson 1987). Single-stranded templates were prepared according to Sanger et al. (t977), The sequence reactions were performed using the dideoxy chain termination procedure. Two in- ternal sequencing primers were used to sequence both strands: primer 3 (17 met) 5' CACTATGTTCATAAGGG 3' and primer 4 ( 17 mer) 5' ATACCTCTTGGGCTTCT 3',

Results and discussion

The analysis of the PCR products of 81 individuals by the D G G E method demons t ra ted a new polymorphism in the region of exon 29 of the h u m a n apo B gene. Two alleles were detectable. The slow migrat ing allele with

92

Fig.l. Denaturing gradient etectrophoresis polymorphism of am- plified exon 29 of apolipoprotein B of human DNA. Two alleles (A and B) are found. Bands corresponding to A and B alleles are indicated by arrows. The gel shows the effect of mutation on frag- ment migration detected by a 20%-70% denaturing gradient

E 5' { ,;, 3,

Exon 29 bp 350 92/+

m,oo <i0 I I position ~037 43~5

a Primer I Primer 2

To determine the position and the nature of the mu- tation, the 574-bp E c o R I fragment was sequenced (Fig. 2a). Figure 2b shows the mutation at nucleotide 823 of the PCR product. This result was also confirmed by sequencing the PCR products of one heterozygote after cloning in M13 mpl l according to Messing (1983). The position of the mutation agrees with the calculated melting map of the exon 29 region, in which the early- melting domain is located between nucleotides 714 and 924 (data not shown). This mutation is affecting amino acid 4311 in the protein by changing Asn to Set (AAT- -+ACT) .

Analysis is in progress to ascertain whether this new polymorphism has a different frequency in patients with hyperlipidemia or coronary heart disease.

In the course of this study, sequencing also revealed in one homozygote for the wild type another yet unde- scribed mutation, which was not detectable by D G G E . It affects amino acid 4243 and changes an arginine to a threonine (AGG--*ACG) (data not shown). We looked for its presence by the ASO method, in a collection of 96 unrelated individuals, but we were not able to find it in other samples.

Wild type Mutated type ( homozygous ) ( heferozy.qous )

T E O A T C G A

. . . . ~ ~ - -= , , . m ~'~ " G T

A f ~ . w ~ " " -

G T J~A

~ A C

Fig. 2. a Partial restriction map of amplified of cxon 29 of apolipo- protein B (924 bp). The 574-bp EcoRl fragment was sequenced. b Sequence gel analvsis of the mutated region

the milder denaturing conditions was designated as A, and the other as B (Fig. 1). The three expected geno- types were detected. The A A and BB samples gave a single band in the gel (Fig. i). In addition to the two ex- pected bands, the heterozygotes (AB) exhibited two supplementary ones corresponding to the heteroduplex DNA fragments formed upon annealing of the D N A strands of both A and B alleles. As already observed by Sheffield et al. (1989) and by Traystman et al. (1990), above the bands corresponding to the detected alleles, a second minor band, sometimes a third one, plus a smear are visible. In spite of this, the two alleles were in all cases unambiguously detectable by this method.

Inheritance of the mutation was tested in a 29-member family. Both alleles segregated in a Mendelian fashion (data not shown). The distribution of the three genotypes is: 45 AA: 33 AB, and 3 BB. This genotype distribution is close to that expected from the Hardy-Weinberg pre- diction. The frequency of the mutant allele B is 24%.

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