Apolipoprotein B-52 mutation associated with hypobetalipoproteinemia is compatible with a misaligned pairing deletion mechanism
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Apolipoprotein B-52 mutation associated with
hypobetalipoproteinemia is compatible with a
misaligned pairing deletion mechanism
W. Antoinette Groenewegen, Elaine S. Krul, and Gustav Schonfeld'
Department of Medicine, Division of Atherosclerosis and Lipid Research, Washington University School
of Medicine, 4566 Scott Avenue, Box 8046, St. Louis, MO 63110
Abstract We have identified a nt 4 truncation of apoB in a of both have been implicated in individual cases (2, 3).
large kindred with hypobetdipoprt cteinemia that arose by an At the gene level, truncated forms of apoB have arisen
ambiguous deletion of one of four dilferent groups of base-pairs. in a number of ways: I) by a nonsense mutation convert-
Eleven affected members of the kindred had total cholesterols
* * * ing a codon for a n amino acid into a termination codon;
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(C) of 114 28, LDL-Cs of 46 21, and apoBs of 47 25 (all
*
in mg/dl, mean SD). These levels were lower (P < 0.0001) 2) by deletion of a large part of the gene; 3) by a point mu-
than in 15 unaffected relatives. On Western blotting, apoB-100 tation or a deletion involving a n introdexon splicing site;
and a second major band corresponding to apoB-52 were seen and 4) by small single or multi-base deletions leading to
in the affected individuals. The majority of the plasma apoB-52 a frameshift and a premature termination codon.
was associated with a smaller than normal low density lipopro-
We previously identified four truncation mutants of
tein (LDL) particle. The molecular basis for this apoB-52 trun-
cation is a 5-bp deletion, converting the sequence between apolipoprotein B-100, namely apoB-89 (4, 5), apoB-40 (4,
cDNA nucleotide 7276 and 7283 from 5'-AAGTTAAG-3' into 5), apoB-54.8 (6), and apoB-75 (3). Here we report the
the mutant sequence 5'-AAG-3'. This results in a frameshift identification and characterization of a new apoB trunca-
starting at amino acid residue 2357 and a termination codon at tion, apoB-52, which could be caused by the deletion of
amino acid residue 2362. Deletion of one of four different groups
several different five base pair cassettes. Because of the
of five consecutive bases, Le., AAGTT, AGTTA, GTTAA, and
TTAAG, all result in the same mutant sequence. Thus, the pre- ambiguous nature of this mutation, we analyzed other
cise deletion is ambiguous. We propose that a misaligned mutations described for the apoB gene and surprisingly
pairing mechanism involving repeat sequences is compatible found several others to be ambiguous.
with this deletion mutation. We have noted similar ambiguous
deletions associated with apoB-37, apoB-40, and a number of
single base deletions and some may also be explained by a mis-
aligned pairing mechanism. Small ambiguous deletions appear METHODS
to constitute a major proportion of the apoB gene mutation
spectrum suggesting that it may be a suitable model for studying Blood collection and plasma analyses
the mechanisms of such mutations-Gmenewegen, W. A., T h e proband was identified through screens of blood
E. S. Krul, and G. Schonfeld. Apolipoprotein B-52 mutation donors obtained from the St. Louis Red Cross Blood
associated with hypobetalipoproteinemia is compatible with a
misaligned pairing deletion mechanism. J Lipid Res. 1993. 34: Bank and volunteers at community screening as previ-
971-981. ously described (6). Blood from fasted donors was drawn
into tubes containing EDTA (1.0 mg/ml). Small aliquots
Supplementary key words low density lipoprotein mutagene- of whole blood were removed and stored at -7OOC for
sis genetic disease haplotype frameshift
isolation of genomic DNA from the leucocytes (see below).
T h e remaining sample was centrifuged and small aliquots
of plasma were stored at - 7OoC for immunoblotting (see
A number of truncated forms of apolipoprotein B-100
have been described (1). These have all been associated
with hypobetalipoproteinemia. Although the gene defects
have been identified, the cell biologic and physiologic Abbreviations: bp, base pair; apoB, apolipoprotein B; PCR, polymer-
mechanisms by which these truncated apoB proteins con- ase chain reaction, LDL, low density lipoprotein; VLDL, ve'y low den-
sity lipoprotein; IDL, intermediate density lipoprotein; FPLC, fast pro-
tribute to hypobetalipoproteinemia are not uniform. De- tein liquid chromatography.
creased production, rapid clearance, a n d a combination 'To whom correspondence should be addressed.
Journal of Lipid Research Volume 34, 1993 971below). For some experiments, blood cells were separated 7346 according to the sequence of Knott et al. (10). The
by low speed centrifugation and the leucocytes were im- 425 bp PCR product contained an internal PstI site near
mediately isolated from packed blood cells using Histo- the 5'-end and an internal Hind11 site near the 3'-end.
paque-1077 (Sigma, St. Louis, MO) and DNA was iso- Genomic DNA (2.5 pg) and 50 pmol of each forward and
lated as described below. Total plasma lipids and reverse primer were amplified in 5 x 100 pl reactions with
lipoprotein-lipids were determined in the Washington recombinant Taq polymerase as r(,portcd previously (11).
University Lipid Research Center Core Laboratory using The DNA was denatured at 95OC for 5 min and 40 cycles
protocols of the Lipid Research Clinics (7). ApoB and of PCR at 95OC for 30 sec, 55OC for 30 sec, and 72OC for
apoA-I plasma concentrations were determined by im- 3 min were performed in an automated temperature
munonephelometry (Behring, Somerville, NJ). cycler (CoyTempCycler, Ann Arbor, MI). Annealing at
55°C for 30 sec followed by a final extension step at 72OC
Electrophoresis and immunoblotting for 7 min ended the amplification procedure. After check-
Electrophoresis of plasma or isolated lipoproteins on ing a small aliquot (5 ~ 1of) each reaction for the forma-
3-676 gradient SDS-PAGE gels was performed as de- tion of the correct PCR product, the five reactions were
scribed previously (4). In some cases, gels were silver- combined, extracted with phenol-chloroform 1:l (vh)
stained using Silver Stain Plus (Bio-Rad, Richmond, and precipitated with ethanol. The recovered DNA was
CA). Electrotransfer of proteins to Immobilon-P mem- digested with PstI and Hind11 (both from Boehringer
branes (Millipore Corporation, Bedford, MA) was per- Mannheim, Indianapoli IN) according to the manufac-
formed and immunoblotting with anti-apoB monoclonal turer's instructions, gel purified, and recovered using
antibodies was carried out as described previously (3). Geneclean (BiolOl, La Jolla, CA). The purified DNA was
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ligated into pGEM3Zfl') that had been digested with the
FPLC separation of plasma same restriction enzymes as above and gel-purified using
Plasma (1.5 ml) was chromatographed on two 25-ml Geneclean. E. coli J M 109 were transformed with the
Superose 6 columns at room temperature as described recombinant plasmid. Clones containing the correct in-
previously (4). The column eluent was analyzed enzymat- sert were identified by hybridization with an oligonucleo-
ically for cholesterol (Wako Pure Chemicals, Richmond, tide internal to the amplified sequence (cDNA position
VA). Equal aliquots (35 pl) were removed from each 7241 to 7260) as described (12). Briefly, clones were
column fraction and applied to 3-6% gradient SDS- streaked in identical grid patterns onto duplicate agar
PAGE gels for electrophoresis and immunoblotting as de- plates. One of the plates contained a nitrocellulose filter
scribed above. Bands corresponding to apoB-100 and and both plates were incubated overnight at 37OC. The
apoB-52 on the resulting autoradiograph were scanned colonies grown on the nitrocellulose disk were lysed in situ
using a laser densitometer. Areas under the peaks were while the other plate was kept as a master plate and stored
determined using Sigmascan Uandel Scientific, Corte at 4OC. DNA bound to the nitrocellulose filter was hybri-
Madera, CA). dized to the internal 32P-end-labeled oligonucleotide and
positive clones were identified on the resulting autoradio-
DNA preparation graph. Positive clones were recovered from the master
Genomic DNA was isolated from whole blood as de- plate and expanded by standard methods and plasmid
scribed by Kawasaki (8). Briefly, whole blood was cen- DNA was prepared by the Magic Miniprep procedure
trifuged and the white cells were washed several times in (Promega, Madison, WI). One pg of plasmid DNA was
T E (10 mM Tris-HC1, 1 mM EMlA pH 7.4) until no sequenced by the Sequenase dideoxy sequencing proce-
more red cells remained. The cells were treated with a dure (United States Biochemical Corp., Cleveland, OH)
buffer containing proteinase K at 56OC for 45 min, fol- (13) using the universal primers T7 and SP6 supplied with
lowed by 95OC for 10 min to inactivate the protease. The the kit.
DNA was stored at -2OOC. When larger amounts of
genomic DNA were required, DNA was isolated from a MseI restriction enzyme digests
white cell pellet (obtained by Histopaque-1077 separa- The region of the apoB gene from cDNA position 7163
tion) (9). to 7346 (10) was amplified from genomic DNA using
primer B52-3 (cDNA nucleotide position 7163 to 7186)
Polymerase chain reaction and DNA sequencing and B52-2 (see above) to yield a 184 bp fragment.
Two PCR primers, B52-1 and B52-2, encompassing the Genomic DNA was denatured at 94OC for 4 min and 30
region of the apoB gene predicted to contain the mutation cycles of PCR were carried out at 92OC for 1 min and at
were synthesized by the Protein Chemistry Core Facility 58OC for 5 min. An aliquot (15 p1) of each PCR reaction
at Washington University Medical School. The forward was digested with MseI (New England Biolabs, Beverly,
primer, B52-1 (21 bp), started at cDNA position 6922 and MA) according to the manufacturer's conditions. The
the reverse primer B52-2 (23 bp) started at cDNA position resultant DNA fragments were separated by electrophore-
972 Journal of Lipid Research Volume 34, 1993TABLE 1. Clinical characteristics of the apoB-52 kindred
Subject Sex Age BMI TChol TG LDL-C HDL-C ApoB ApoA-I
Y' kg/mz mg/dl
Affected apoB-100/apoB-52
11-5 M 74 24.5 128* 101 68 * 38 33 110
11-9 F 62 23.8 173* 155 96' 43 94 128
111-5 F 56 21.5 130. 53' 37. 82 36 188
111-9 M 42 24.7 87' 30' 37' 42 33 105
111-12 M 44 22.6 141 72 62 * 62 51 140
111-20 F 38 21.1 127' 50 47' 67 44 167
IV-9" M 27 22.1 97' 40 34. 55 30 124
IV-11 M 26 21.5 86 * 38' 31' 47 30 115
IV-13 M 10 16.1 86 * 29' 25' 53 24 139
IV- 14 M 6 18.3 96' 36' 41 * 46 37 131
IV-25 M 9 16.6 103' 40 32* 61 30 133
Mean * SD 35.8 f 22.6 21.1 + 3.0 114 + 28.0* 58.5 f 38.3' 46.4 f 21.0d 54.2 f 13.0 40.2 f 19.3* 134.5 f 24.5
Unaffected apoB-1001apoB-100
11-1 M 82 24.9 234 106 169 41 142 115
11-2 F 80 24.1 267 81 178 69 145 162
11-6 F 68 18.1 151' 68 70 65 68 149
11-7 F 70 24.4 235 107 158 54 127 131
11-10 M 64 25.1 212 195 116 55 117 153
111-14 M 39 24.9 203 295 115 29. 130 107
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111-16 M 36 22.0 207 325'' 106 36 129 131
111-17 F 37 20.1 199 51 119 66 97 175
111-22 M 35 28.1 173 150 103 38 101 112
111-25 F 33 22.2 199 141 113 55 109 166
IV-12 F 15 17.9 138 50 79 46 78 118
IV-18 F 9 16.7 129 105.' 62* 43 69 135
IV- 19 F 7 15.0 132 166** 63' 33 * 86 114
IV-20 F 5 15.4 144 142'. 73 40 86 133
IV-24 M 12 21.5 183 210 100 38 104 118
Mean * SD 39.4 f 27.2 21.4 f 4.0 187.1 f 41.9 146.1 f 82.2 108.3 f 37.0 47.2 f 12.7 105.9 f 25.4 134.6 f 21.7
Plasma lipid profiles were determined using protocols of the Lipid Research Clinics, and plasma apoB and apoA-I concentrations were determined
by immunonephelometry. apoB phenotypes were assigned on the basis of Western blot analysis. BMI, body mass index; TChol, total plasma cholesterol;
TG, total plasma triglyceride; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; ApoB, apolipoprotein B; ApoA-I, apolipoprotein A-I; *, 5 5th
percentile; * * 295th percentile.
"Proband.
bP < 0,0001(unpaired t-test) compared to B-100/B-100 phenotype.
'P = 0.0012 (Mann-Whitney two-sample test).
dP < 0.0001 (Mann-Whitney two-sample test).
sis on a 12% polyacrylamide gel and stained with total and LDL-cholesterol and total apoB concentrations
ethidium bromide (14). < 5th percentile for his age, race, and sex. On immuno-
blotting of his plasma, apoB-100 was present as the pre-
3' VNTR analysis dominant form of apoB (Fig. 1).In addition, an unusual
Genomic DNA was isolated from all the members of apoB, apoB-52 was identified. This protein was also
the kindred as described above. The polymorphic region identified in the plasma of his mother, brother, and eight
in the 3' untranslated region of the apoB gene was am- additional relatives and it is associated with a -40%
plified by PCR using primers and conditions as described decrease in total plasma cholesterol concentration and a
by Boerwinkle et al. (15). The various alleles were as- - 55% decrease in plasma LDL-cholesterol and apoB
signed by comparison with known alleles, kindly provided concentrations (Fig. 2 and Table 1). As shown in Fig. 2,
by Lawrence Chan. we predict that 11-3, the maternal grandmother of the
proband, was an obligate heterozygote for the apoB-52
mutation. She died at the age of 71 of lymphoma. In-
RESULTS dividual 11-6 (Table l), in whom no truncated protein was
demonstrated, also had a total cholesterol concentration
Proband and his kindred below the 5th percentile, while her other lipid levels were
The proband was an asymptomatic 27-year-old man, in the normal range. Also in the non-affected group
who was taking no medication at the time of blood sam- (Table l), individuals IV-18 and IV-19 had LDL-choles-
pling, (subject IV-9 in Table 1 and Figs. 1 and 2) with terol concentrations below the 5th percentile while their
Groenewegen, Kml, and Schonfeld Misaligned pairing deletion mechanism in apoB-52 973total cholesterol levels were in the normal range. The
reasons for their low cholesterol concentrations are not
clear.
Lipoprotein distribution of apoB-52
The proband's plasma was fractionated by gel exclusion
- 874 chromatography as described in the Methods section.
ApoB-100 was present in the fractions corresponding to
B 52-
- 854.8 VLDL and IDL and exhibited a major peak correspond-
B48- ing to the LDL-cholesterol fractions (Fig. 3A). ApoB-52
LDL overlapped with apoB-100 LDL fraction, but the
majority of the apoB-52 LDL was present in a retarded
(smaller) LDL fraction, suggesting that two types of LDL
-826 particles were present in the proband's plasma, one con-
taining apoB-100 and one containing apoB-52. O n the
FPLC profile no apoB-52 was detected in fractions cor-
responding to the VLDL or HDL size ranges. However,
when a VLDL fraction was separated on a 3-6% acryla-
Fig. 1. Identification of apoB-52 protein. Immunoprecipitated apoB mide gel and silver-stained, a band of the apoB-52 size
from the plasma of the proband (IV-9), his brother (IV-11) and mother was visible (see inset in Fig. 3A). Plasma from an in-
(111-5) were run on a 3-6% SDS-PAGE gel. A control sample applied
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on the left identified the positions of apoB-100 and apoB-48. Thrombin- dividual previously identified to contain the apoB-54.8
digested LDL apoB-100 (t-LDL) was applied on the right side of the gel truncation (individual 111-17 from ref. 6) was also frac-
to identify the positions of apoB-74 and apoB-26. For comparison a tionated and an FPLC profile was constructed as above
sample containing previously identified apoB-54.8 truncation was ap-
plied in the 2nd lane from the right. Immunoblotting with an anti-apoB (Fig. 3B). The lipoprotein distribution of the truncated
monoclonal antibody (C1.4) was performed as described in the Methods apoB-54.8 was very similar to apoB-52 in that the
Section. The resultant autoradiograms are shown with the apoB species majority of apoB-54.8 appeared associated with a smaller
as determined by relative molecular weights indicated.
LDL particle. A small proportion of the apoB-54.8 trun-
cation was also found in the VLDL to IDL density range.
T
I
II -"r T
1
37/37
2
35/37 37/69 35/29
@-El
7
35/37
a
6 5 7 9 10
3%9 3%9
+ I
11 12 13 14 18 19 20 21 24 25
29/37 29/37 29/37 37/37 37/69
Fig. 2. Pedigree of the apoB-52 kindred and 3'VNTR analysis. ApoB phenotypes were determined by Western blot analysis. ApoB-52/apoB-100
phenotype (@a);apoB-lOO/apoB-100 phenotype (m, 0);and non-tested relatives (0,O); deceased relatives (7). The proband is identified by an arrow.
The clinical data are given in Table 1. The results from the 3'VNTR analysis carried out as described in the Methods section are given below each
appropriate member of the pedigree. The 3'VNTR results are abbreviated as follows: 3'&37/3'@-49 corresponds to 37/49 and so on. The results for
each member of the kindred tested are shown.
974 Journal of Lipid Research Volume 34, 1993I I I I I I I ,20
1 40 - CHOL
100 4
-8-54.8 w
02
20 - <
- 15
00 - b
w
3:
0
2
L
80 - - 10
P
60 - 1 : 4
4
,-,. " b
' .', ;-'! 0
40 -
,
I ) .
-.
I , L'
$8
i
,
L
''
'
:
- 5rz.
b
20 - I
I : ,
I
,. I
. .,'1. ,
I
0 '. , -"I I ' 1 I
0 10 20 30 40 50 60
B
FRACTION (0.5rnl)
Fig. 3. Separation of plasma apoB-100 from apoB-52 (A) or apoB-54.8 (B). Plasma (1.5 ml) was subjected to gel permeation chromatography on
two Superose 6 columns as described in the Methods. The dotted line represents Cglfraction total cholesterol determined in each fraction. The dashed
and solid lines represent percentages of the total summed densitometric areas for apoB-100 and truncated apoB, respectively, determined in each
Downloaded from www.jlr.org by guest, on October 11, 2015
column fraction. Normal plasma VLDL, LDL, and HDL elute between fractions 5-9, 25-27, and 44-47, respectively. The inset in (A) is a silver-
stained SDS-PAGE gel of VLDL isolated from the B-100/B-52 subject's plasma by ultracentrifugation at a density of 1.006 g/ml. Despite the apparent
absence ofapoB-52 in this subject's VLDL fraction as separated by FPLC (as a percent, the amount of apoB-52 is barely detectable), the silver-stained
gel confirms that apoB-52 is present in the VLDL fraction.
ApoB-52 mutation
An appropriate region of theapoB gene, estimated
from the size of the apoB truncation, was amplified by
PCR from genomic DNA using primers and procedures
described in the Methods section. The PCR product was
cloned into pGEM3Zfi+). Ninety-six clones were
screened for the apoB insert and 28 clones showed a posi-
tive reaction with the internal oligonucleotide B52-3. Ten
clones were sequenced at both the 5'- and 3'-ends of the
cloned apoB gene fragment using the universal primers
T7 and SP6. Five clones contained a deletion offive
nucleotides converting the sequence between cDNA
nucleotide 7276 and 7283 from 5'-AAGTTAAG-3' into a
mutant sequence of 5'-AAG-3' (Fig. 4 and Fig. 5). This
deletion causes a frameshift resulting in fivenew amino
acid residues, starting at amino acid residue 2357, and a
termination codon ( E A ) at amino acid residue 2362.
The predicted truncated apoB proteinwould contain 2361
amino acids which represent 52% of the 4536 amino acids
contained in apoB-100. We thus name this mutant protein
apoB-52. Comparison of the normal and mutantapoB se-
quence revealed that the deletion of any of four overlap-
ping 5 bpcassettes could result in the sequence associated Fig. 4. Identification of the apoB-52 mutation. The region of the apoB
with the apoB-52 genotype (Fig. 5). It is therefore not pos- gene predicted to contain the mutation, as determined by the size of the
sible tostate precisely which cDNA nucleotides were protein on the Western blot, was amplified by PCR using primers and
conditions described in the Methods Section. The fragment was cloned
deleted. into pGEM3ZfC) and clones containing the correct insert were identified
The sequencing results from ten individual clones re- as described in the Methods Section. Clones were sequenced by standard
vealed a sequence variation at cDNA nucleotide 7064 dideoxysequencing procedure. Ten clones were sequenced and five
-
( 219 bp upstream of the apoB-52 mutation), which con-
yielded the mutant sequence shown in B. The five bases indicated by *
is one possible deletion shown in Fig. 4 as mt-1; term, termination
verts the consensus apoB gene sequence of GAC (10) to codon.
Gmmewegen, Kml, and Schonfld Misaligned pairing deletion mechanism in apoB-52 9752355
u i n o acid 0111 Gln Val Lys Lys Asp Tyr Phe Glu
wildtype
/
sequence
7212
\ cDNA a c A A GTT M0 ATA AM GAT TAC "IT! GAQ
Fig. 5 . Possible origins of the mutation giving rise to
& ATA
the apoB-52 truncation. The top part of the figure
shows the wildtype amino acid and cDNA sequences.
The middle part shows the possible groups of 5 bp that
ATA may have been deleted indicated by 5 ''---n in mt-1 to
ATA mt-4. The bottom part shows the resulting mutant
cDNA and amino acid sequences. The underlined
ATA nucleotides in the wildtype and mutant cDNA se-
quences indicate the areas before and after the muta-
& cDNA
7272
CAA -AT IUA AGA TTA CTT M A
tion, respectively.
mutant
/
saquance
\ amino acid
2355
Gln Gln Asp Ly4 L.u m u term
GAT (amino acid codon 2285). This polymorphism was with the mutant allele in all the affected and none of the
Downloaded from www.jlr.org by guest, on October 11, 2015
previously described by a number of investigators (16, 17 non-affected members of the kindred.
and references therein). Five clones contained the GAC
codon and the 5 bp deletion and the other five clones con- Characteristics of other deletion mutations in the
tained the GAT codon and the wildtype sequence. This apoB gene
finding provides evidence that the GAC codon and the The ambiguous nature of the 5 bp deletion associated
5 bp deletion are on the same chromosome and argues with the apoB-52 truncation led us to examine previously
against artificial sequences caused during cloning or identified deletion mutations in the apoB gene. The muta-
during the PCR amplification. tion associated with apoB-37 is a 4 bp deletion. Examina-
tion of the DNA sequence surrounding this mutation also
M s e I digestion and S'VNTR analysis of the revealed four alternative groups of 4 bp that, when
apoB-52 mutation deleted, would result in the same mutant sequence
The apoB-52 mutation disrupts an MseI restriction site (Fig. 7). Further, a 2 bp deletion associated with the
(TTAA) and MseI digests of PCR amplified DNA were apoB-40 truncation can arise by two different deletions
used to confirm the presence of the deletion mutation. A (Fig. 8). A larger deletion resulting in apoB-61 (deletion
PCR primer B52-3 upstream of the mutation together of 37 bp) is unambiguous and the 694 bp deletion that
with primer B52-2 were used to amplify a 184 bp frag- would give rise to apoB-25 was suggested to have arisen
ment containing the mutation site as described in the by recombination involving Alu repeated sequences (18).
Methods section. This fragment contains two MseI sites in The remainder of the identified deletion mutations in
addition to the potentially mutated site. If the mutation apoB are single base deletions. Table 2 shows their se-
is not present, i.e., the wildtype allele, the major bands quence context. Four out of the nine single base deletions
formed by digestion with MseI are 82 and 60 bp. If one are ambiguous.
allele contains the mutation, an additional band of 142 bp
should be seen when the digestion products are separated
by electrophoresis as described in the Methods section. DISCUSSION
When genomic DNA from the proband and his kindred
were analyzed by this method, the presence of a 142 bp In this report we describe a novel truncation of apoB,
band indicating a mutant allele cosegregated with the apoB-52. This truncated form of apoB was associated
presence of the apoB-52 protein by immunoblotting. with a hypobetalipoproteinemic phenotype (Fig. 2 and
Fig. 6 shows representative examples of an apoB-100/ Table 1). The majority of plasma apoB-52 was found asso-
apoB-52 heterozygote (lane 2) and an apoB-lOO/apoB-lOO ciated with a slightly smaller than normal LDL particle
homozygote (lane 3). Thus, this is further confirmation (Fig. 3A). We previously described a different apoB trun-
that the five nucleotide deletion is the cause of the forma- cation, apoB-54.8 (6), which shares a very similar profile
tion of the apoB-52 truncation found in these individuals. to apoB-52 upon gel filtration of plasma (Fig. 3B). Only
3'VNTR analysis of the apoB gene (Fig. 2) demonstrated trace amounts of apoB-52 or apoB-54.8 were found as-
that the allele carrying the 3' fl 49 repeat co-segregates sociated with VLDL-IDL-sized particles. The lipopro-
976 Journal of Lipid Research Volume 34, 19931726
u i n o acid EiK Thr A8n Ser Leu A m
\ CDKA 5385
CAC ACA AAC AOT CTO AAC
rt-1 CAC ACA --- -OT CTO AAC
po..ible It-2 CAC AC- --- AOT CTO AAC
deletion. rt-3 CAC A-- --C AQT CTO AAC
It4 CAC --- -AC AOT CTO AAC
5385
CDKA CAC ACA On: =A
mutant /
66- .OqUence
\ 1726
u i n o acid Eis Thr Val tam
Fig. 7. Possible origins of the apoB-37 mutation, a 4 bp deletion. For
detailed description, see legend to Fig. 5.
1 2 3
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Fig. 6. Detection of apoB-52 mutation by MseI digestion. The figure cluded that the 5 bp deletion mutation is the cause of the
shows separation of the digested fragments by 12% polyacrylamide gel apoB-52 truncated protein seen in the plasma of the
electrophoresis stained by ethidium bromide. The DNA marker used affected members of this kindred.
was an @X714HinfI digest (Promega, Madison, WI) and the indicated
fragment sizes are in bps. Lane 1: uncut PCR product of 111-5; lane 2: The ambiguities associated with the apoB-52 mutation
individual 111-5, apoB-52 protein present by immunoblot; lane 3: reported here and with some of the other apoB mutations
individual 11-7, no apoB-52 present by immunoblotting. The 82 and have not been commented on previously. Figs. 5, 7, and
60 bp bands are digestion products of the wildtype allele. The 142 bp
band is indicative of the mutant allele. The lower band in lanes 2 and 8 illustrate the ambiguities associated with the apoB-52,
3 is an additional digestion product (36 bp) which is present irrespective apoB-37, and apoB-40 mutations, respectively. In addi-
of the mutation. The higher molecular weight bands in lanes 1 and 2 tion, approximately 50% of the single base deletions were
may be heterodupleces.
also ambiguous (Table 2). We have compared the charac-
teristics of these small ambiguous deletion mutations with
tein distribution profiles for both the truncated and mechanisms proposed in the literature. We shall consider
apoB-100 proteins were obtained by expressing the amount the role of DNA sequences as well as the role of DNA
of truncated apoB in each fraction as a percentage of the polymerases in the mutagenesis process.
total amount of truncation present. This resulted in the
apparent absence of truncations in the VLDL-IDL size
range. However, in fact, as shown in the inset of Fig. 3A, 1827
amino,acid Asp Thr Val Ala Lys
apoB-52 protein was present in the VLDL fraction, and
/
the presence of apoB-54.8 in VLDL has been reported wildtype
previously (6). Thus, both .apoB-52 and apoB-54.8 were
distributed similarly to apoB-100, i.e., the vast majority of
apoB-100 was in LDL and 5 10% was in VLDL and IDL.
sequence
' cDNA QAC
5688
ACP QTT QCJ! AAQ
This suggests that the VLDL+IDL+ LDL cascade prob-
ably is intact in these subjects and operates effectively for
\1
It-1 QAC ACT --T GCT AAQ
both their apoB-100- and apoB-truncation-containing possible
deletions
lipoproteins. mt-2 QAC AC- -TT QCJ! AAQ
The molecular basis for the apoB-52 truncation was
identified as a 5 bp deletion between nucleotides 7276 and
7283 which predicts a protein of 2361 amino acids, or 5688
52% of the total apoB protein according to the centile sys- cDNA GAC Am TQC TAA
tem. Confirmation of the apoB-52 mutation in the re- mutant /
mainder of the kindred was obtained using the absence or sequence
1827
presence of an MseI site on the apoB gene as a marker \ u i n o acid Asp Thr Cys term
(Fig. 6) and also by noting the segregation of the S'VNTR Fig. 8. Possible origins of the apB-40 mutation, a 2 bp deletion. For
allele 3'6 49 with the apoB-52 truncation. Thus, it is con- detailed description, see legend to Fig. 5.
Gmenewegen, Kml, and Schonfld Misaligned pairing deletion mechanism in apoB-52 977TABLE 2. Characteristics of l b p deletion mutations in apoB with the deletion mutations found in the lacl gene of E. coli
No Ambiguities Ambiguous
(see above). Fig. 9 illustrates the intermediates that are
proposed to form during a misaligned pairing mechanism
ApoB-3 1 AAA S T T T A involving the AAG repeat. The configurations during
ApoB-39 AAG C T G C A T either continuous or discontinuous strand synthesis are il-
ApoB-52.8 G A T T A G TTT
lustrated in Figs. 9A and 9B, respectively. In each case the
ApoB-52.8 TTT AAA ACA“
boxed areas indicate the repeated trinucleotide sequence.
ApoB-67 GGG &G ATA
ApoB-75 A C T ACT G T G
ApoB-86 AAA AAC AAA L
5’CAAC&AG:3’
ApoB-87
ApoB-89 GAA GAG GCA
CAG E A ATG
A 1 -J G T T G L T T A [ T C I T A T 5‘
The sequence context of apoB truncations that result from a l b p dele- 0 0
tion is shown (28). In the column “no ambiguities” the underlined base
indicates the deleted base in each case. In the “ambiguous” column one
of the underlined bases is deleted in each case.
f7
‘Linton, M . F., R. Farese, and S. G . Young, personal communi- 5‘C A A C S A G 3’
cation.
3aGT T C I T ~ T A T S
Small deletions or insertions often seem to occur in
@@@ \ /
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three DNA sequence contexts (19): a) in runs of reiterated C
bases (e.g., ...AAAAAA...), b) in runs of tandem repeats
(e.g., ...ACACACAC...) and c) in direct repeats separated
by interposed DNA sequences (e.g., ...AGC .....AGC ...).
.a
The misaligned pairing model (20) is applicable to these
contexts. For single nucleotide deletions, the model 3 5 ’ C A A Cl-----J
:xiGATA3’
predicts that a bulge of a single nucleotide occurs within ’’1 1 A A G 3 ’ deleted = mt -1
a run of reiterated bases on the template strand. If the
primer-strand is extended as if it were properly aligned,
the “looped out” base on the template strand is not repli-
cated, resulting in the deletion of the base. The same
mechanism could also account for insertions if the bulge B 0 0
” C A A C W A G ] T T I X I A T A 3‘
occurs on the primer-strand. The model can also be ap- 1
plied to tandem repeats of two or more bases and provides 3’ [jIfICi
T A T 5’
a mechanism for deletion or duplication of such repeat se- c--
quences. Alternatively, a nonrelated sequence may be in-
terposed between two direct repeats. In that case, the
primer-strand temporarily dissociates and ‘‘slips’’ until it
hybridizes to the next repeat sequence, creating a bulge
on the template strand. If the primer strand is extended
in the misaligned configuration, one of the repeat se-
quences and all of the intervening sequence will be
OT
{y
’ ‘ C A A C G I A T A 3’
2 r----3
deleted. Many examples of deletions that end in repeat se- 3’ LlJ-C! TAT 5
quences have been described for the lacl gene of E. coli (21)
and thus these mutations are ambiguous. Direct repeat f7
sequences of between 2 and 8 bp are also frequently found r - - ---I
near human gene deletions (22). 3 3‘GT TGIT-T__C_;TAT’
The sequence around the apoB-52 deletion contains 5‘AAGTT 3’deleted=mt-4
several repeat sequences. Two dinucleotide repeats, AA Fig. 9. Diagrammatic representation of the proposed misaligned pair-
and AG, (Fig. 5 ) would account for all four possible ing mechanism for the apoB-52 mutation. Continuous and discontinu-
ous DNA strand synthesis are shown in A and B, respectively. Solid
groups mt-1 to mt-4 that may have been deleted. In addi- boxed bases are the repeat sequences on the template strand and the
tion, however, a AAG repeat is located at the ends of the dashed boxed bases are the newly synthesized base repeats on the primer
deleted sequence. The underlined sequence in Fig. 5 strand. Solid arrow indicates direction of synthesis; 1) synthesis of the
primer strand including repeat sequence 1; 2) primer misalignment with
clearly shows that only one of the AAG repeat sequences repeat 2; 3) final sequence of the primer strand after the mutation. The
remains as a result of the mutation and this is consistent group of bases deleted is shown separately (see also Fig. 5).
978 Journal of Lipid Research Volume 34, 1993Studies on this type of mechanism (23) have shown that Thus far we have considered the sequence context of
repeat 2 is deleted together with the intervening sequence. the apoB gene mutation sites in the production of trunca-
Thus, the misaligned pairing mechanism is compatible tion mutations. However, different DNA polymerases ex-
with the apoB-52 mutation and either mt-1 or mt-4 (see hibit differing degrees of fidelity of replication, indicating
Fig. 5) represent the more probable deletions. that the enzymes also may play significant roles in gener-
Similar to the apoB-52 mutation reported here, the se- ating mutations (27). The mutation spectrum of the apoB
quence context of the 4 bp deletion for apoB-37 (Fig. 7) truncations, consisting of 24/25 (ref. 28, Linton, M. F.,
contains two dinucleotide repeats (AC and CA) as well as R. Farese, and S. G. Young, personal communication)
a 3 bp repeat sequence (ACA). A misaligned pairing deletions plus base substitutions, bears a remarkable
mechanism may also account for this deletion and a resemblance to the spectra described for some mam-
scheme similar to that shown in Fig. 9 for the apoB-52 de- malian DNA polymerases. The spectrum for DNA poly-
letion may be drawn for this mutation (not shown). It also merase /3, a putative repair and gap-filling enzyme, con-
would propose mt-1 and mt-4 (Fig. 7) as the most proba- sists of 90% frameshift plus base substitution mutations
ble deletions. (29). Also for the apoB gene, single base deletions (ref. 28,
The ambiguous 2 bp deletion associated with the Linton, M. F., R. Farese, and S. G . Young, personal com-
apoB-40 truncation involves a T-residue on either side of munication) accounted for 64% of all deletions (9 out of
the G-residue that is deleted (Fig. 8). It is not clear 14) and no insertions have yet been reported. This again
whether this would constitute a repeat sequence. Very few is similar for DNA polymerase p , where the mutation
2 bp deletions have been identified and most do not ap- spectrum showed that the loss of a single base was much
pear in repeat sequences (19). Studies on the bacterio- more frequent than the addition of a base and much more
phage T 4 rIIB gene did find a number of ambiguous 2 bp frequent than the loss of two or more bases (29). The rela-
Downloaded from www.jlr.org by guest, on October 11, 2015
deletions but no consensus sequence was identified (24). tive importance of DNA sequence, DNA polymerase
It has been suggested that mechanisms other than mis- fidelity, and the interactions between sequences and fidel-
aligned pairing contribute to 2 bp deletions (19). ity in mutagenesis remains to be determined.
The single base deletions described for the apoB gene In addition to the 14 deletions in the apoB gene (see
are approximately equally divided between both unam- results), there are also 11 mutations of the apoB gene
biguous and ambiguous ones (Table 2). The ambiguous truncations associated with base substitutions (ref. 28,
single base deletions may also have involved a misaligned Linton, M. F., R. Farese, and S. G. Young, personal com-
pairing mechanism. They occur in mononucleotide re- munication). The mechanisms for some of these nonsense
peats, the longest being a repeat of four A-residues in mutations have been discussed elsewhere (30). Further-
apoB-52.8 (Linton, M. F., R. Farese, and S. G. Young, more, at least 85 base changes have been reported (17,
personal communication). The unambiguous deletion of 31-34) although a number may be sequencing Errors.
the C-residue in apoB-86 rimy also have arisen by a Thus, 14 of 110 (12%) mutations/variations of the apoB
misalignment-type mechanism as this occurs within a run gene result from deletions and 7 of the deletions are am-
of eight A-residues. The minimum number of reiterated biguous. It therefore appears that besides base substitu-
bases necessary for the misaligned pairing mechanism is tion mutations, small (ambiguous) deletions constitute a
not clear (for review, see ref. 25). However, a single base significant proportion of the mutations in the apoB' gene.
deletion hotspot sequence 5'-TTTT-3' (26) was identified This is in contrast to the defects described for some other
in the lacZa gene and when this sequence was mutated, genes involved in lipid metabolism, where most do not in-
first to 5 ' - T E T - 3 ' and then to 5'-CXT-3', the mutation volve small (ambiguous) deletions. At least 12 gede defects
frequency decreased dramatically with each alteration in (35) have been described for the apoE gene and, except for
the hotspot sequence. It was concluded that 97% of the a duplication event in the variant E3-Leiden, all are base
single base deletions at this 4 bp repeat hotspot involve a substitutions. Ten of the 18 mutations (36-39) described
misalignment mechanism although other mechanisms for the LDL receptor gene are large deletions '( >1 kb)
cannot be ruled out. Single base deletions that do not which appear to have arisen by homologous recombina-
occur in mononucleotide repeats, and that are therefore tion involving Alu repeat sequences. However, three small
not ambiguous, probably involve distinctly different deletions (37-39) in the LDL receptor gene have been
mechanisms which are presently not understood. Valida- reported, all of which are ambiguous. The majority of the
tion of the proposed mechanism for the apoB mutations 15 mutations (40-42) in the lipoprotein lipase gene are
could be achieved in an in vitro system similar to Kunkel's missense mutations except for two frameshift mutations
(26) by cloning the target sequence into a suitable vector (41, 42), neither of which are ambiguous. Eighteen muta-
and observing the frequency of mutations in the wildtype tions have been described for the apoC-I1 gene, including
sequence and after site-directed mutagenesis of the puta- five small deletions, four (43, 44) of which are ambiguous.
tive repeat sequences. Of the 24 mutations described for the apoA-I gene, three
Gmenewegm, KNI, and Schonjeld Misaligned pairing deletion mechanism in apoB-52 979(45-47) are small deletions and two of these are ambigu- D. H. Gelfond, J. J. Sninsky and T. G. White, editors. Aca-
ous. In addition, 8/16 small /3-hemoglobin gene deletions demic Press, Inc. San Diego, CA. 146-152.
(48) are ambiguous but the majority of the 288 mutations 9. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986.
Methods in Molecular Biology. Elsevier Publishing Co.,
described for this gene are base substitutions. New York, NY. 44-46.
In conclusion, we have attempted to reconcile the mu- io. Knott, T. J., L. M. Powell, R. J. Pease, A. J. Lusis, S. C.
tations .found in the apoB gene with published data and Wallis, B. J. McCarthy, R. W. Mahley, B. Levy-Wilson, J.
mechanisms. A number of deletions identified in the apoB Scott, and B. Blackhart. 1986. Complete cDNA and derived
gene, including the apoB-52 and apoB-37 mutations, are protein sequence of human apolipoprotein B-100. Nucleic
A c i h &s. 14: 7501-7503.
compatible with a misaligned pairing mechanism and 11. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf,
DNA polymerase enzymes may also play a significant role R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich.
in the production of mutations. Small ambiguous muta- 1988. Primer-directed enzymatic amplification of DNA
tions appear frequently in the apoB gene mutation spec- with a thermostable DNA polymerase. Science. 239:
trum and this gene may thus provide a suitable target for 487-491.
12. Sambrook, J., E. E Fritsch, and T. Maniatis. 1989.
further studies of small DNA deletion mutations. I Molecular Cloning. A Laboratory Manual, 2nd Edition.
Cold Spring Harbor Laboratory Press, Cold Spring Har-
We thank Lawrence Chan for providing the 3'VNTR standards, bor, NY. 1.98-1.99.
Clay.Semenkovich for his critique of the manuscript, and Tom 13. Sanger, E, A. R. Coulson, and S. Nicklen. 1977. DNA se-
Kitchens and Tish Kettler for their expert technical assistance. quencing with chain-terminating inhibitors. Proc. Natl.
We are grateful to ST, the proband, and his family for their help- Acad. Sci. USA. 74: 5463-5467.
ful cooperation and to Diana Tessereau for obtaining the blood 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989.
Molecular Cloning. A Laboratory Manual, 2nd Edition.
samples, and Cheryl Doyon for preparing the manuscript. This
Cold Spring Harbor Laboratory Press, Cold Spring Har-
Downloaded from www.jlr.org by guest, on October 11, 2015
work was supported by NIH Grant R01 HL42460-04. bor, NY. 6.39-6.44.
Manuscript received 12 November 1992 and in revisedjorm 15 January 1993 15. Boerwinkle, E., E. Fourest, L. Chan, and W. Xiong. 1989.
Rapid typing of tandemly repeated hypervariable loci by
the polymerase chain reaction applications to the apolipo-
protein B S'hypervariable region. Pmc. Natl. Acad. Sci. USA.
86: 212-216.
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Gmenewegen, Krul, and Schonfcld Misaligned pairing deletion mechanism in apoB-52 981You can also read
