Apolipoprotein B-52 mutation associated with hypobetalipoproteinemia is compatible with a misaligned pairing deletion mechanism

Page created by Julio Cunningham
 
CONTINUE READING
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;

                                                                                                                                               Downloaded from www.jlr.org by guest, on October 11, 2015
(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                   971
below). 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

                                                                                                                           Downloaded from www.jlr.org by guest, on October 11, 2015
                                                            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, 1993
TABLE 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

                                                                                                                                                         Downloaded from www.jlr.org by guest, on October 11, 2015
  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                         973
total 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

                                                                                                                                                    Downloaded from www.jlr.org by guest, on October 11, 2015
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, 1993
I             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                                                                975
2355
               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, 1993
1726
                                                                                               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

                                                                                                                                                       Downloaded from www.jlr.org by guest, on October 11, 2015
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                    977
TABLE 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
                                                                                                   @@@    \       /

                                                                                                                                                             Downloaded from www.jlr.org by guest, on October 11, 2015
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, 1993
Studies 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.
                          REFERENCES                                      16.   Huang, L. S., H. Kayden, R. J. Sokol, and J. L. Breslow.
                                                                                1991. ApoB gene nonsense and splicing mutations in a com-
 1. Young, S. G. 1990. Recent progress in understanding                         pound heterozygote for familial hypobetalipoproteinemia.
    apolipoprotein-B. Circulation. 82: 1574-1594.                               J. Lipid Res. 32: 1341-1348.
 2. Parhofer, K. G., P. H. R. Barrett, D. M. Bier, and G.                 17.   Yang, C., Z-W. Gu, S. Weng, T. W. Kim, S-H. Chen, H.
    Schonfeld. 1992. Lipoproteins containing the truncated                      J. Pownall, P. M. Sharp, S-W. Liu, W-H. Li, A. M. Gotto,
      apolipoprotein, apoB-89, are cleared from human plasma                    Jr,, and L. Chan. 1989. Structure of apolipoprotein B-100
      more rapidly than apoB-100-containing lipoproteins in                     of human low density lipoproteins. Arteriosclerosis. 9: 96-108.
      vivo. J. Clik Invest. 89: 1931-1937.                                18.   Huang, L. S., M. E. Ripps, S. H. Korman, R. J. Deckel-
 3.   Krul, E. S., K. G. Parhofer, P. H. R. Barrett, R. D.                      baum, and J. L. Breslow. 1989. Hypobetalipoproteinemia
      Wagner, and G. Schonfeld. 1992. ApoB-75, a truncation of                  due to an apolipoprotein B gene exon 21 deletion derived
      apolipoprotein-B associated with familial hypobetalipo-                   by alu-alu recombination. J. Biol. Chem. 264: 1394-1400.
      proteinemia: genetic and kinetic studies. J. Lipid Res. 33:         19.   Ripley, L. S. 1990. Frameshift mutation: determinants of
      1037-1050.                                                                specificity. Annu. Rev. Genet. 24: 189-213.
 4.   Krul, E. S., M. Kinoshita, P. Talmud, S. E. Humphries,              20.   Streisinger, G., Y. Okada, J. Emrich, J. Newton, A.
      S. Turner, A. C. Goldberg, K. Cook, E. Boerwinkle, and                    Tsugita, E. Terzaghi, and M. Inouye. 1966. Frameshift mu-
      G. Schonfeld. 1989. Two distinct truncated apolipoprotein                 tations and the genetic code. Cold Spring Harbor Symp. @ant.
      B species in a kindred with hypobetalipoproteinemia.                      Biol. 31: 77-84.
      Arteriosclerosis. 9: 856-868.                                       21.   Farabaugh, P.J., U. Schmeissner, M. Hofer, and J. H.
 5.   Talmud, P., L. Kingunderwood, E. Krul, G. Schonfeld,                      Miller. 1978. Genetic studies of the lac repressor. VII. On
      and S. Humphries. 1989. The molecular basis of truncated                  the molecular nature of spontaneous hotspots in the lac1
      forms of apolipoprotein B in a kindred with compound                      gene of Escherichia coli, J. Mol. Biol. 126: 847-863.
      heterozygous hypobetalipoproteinemia. J Lipid Res. 30:              22.   Krawczak, M.,and D. N. Cooper. 1991. Gene deletions
      1773-1779.                                                                causing human genetic disease: mechanisms of mutagenesis
 6.   Wagner, R. D., E. S. Krul, J. J. Tang, K. G. Parhofer,                    and the role of the local DNA sequence environment. Hum.
      K. Garlock, P. Talmud, and G. Schonfeld. 1991. ApoB-                      Genet. 86: 425-441.
      54.8, a truncated apolipoprotein found primarily in VLDL,           23.   Papanicolaou, C., and L. S. Ripley. 1989. Polymerase-
      is associated with a nonsense mutation in the apoB gene                   specific differences in the DNA intermediates of frameshift
      and hypobetalipoproteinemia.J. Lipid Res. 32: 1001-1011.                  mutagenesis. J. Mol. Biol. 207: 335-353.
 7.   Lipid Research Clinics Program. 1974. Manual of Labora-             24.   de Boer, J. G., and L. S. Ripley. 1988. An in vitro assay for
      tory Operations. Vol. 1. Lipid and Lipoprotein Analysis.                  frameshift mutations: hotspots for deletions of 1 bp by
      DHEW Publication No. (NIH) 75-628. Washington, DC:                        Klenow-fragment polymerase share a consensus DNA se-
      US Government Printing Office.                                            quence. Genetics. 118: 181-191.
 8.   Kawasaki, E. S. 1990. Sample preparation from blood,                25.   Kunkel, T. A. 1990. Misalignment-mediated DNA synthe-
      cells, and other fluids. In PCR Protocols. M. A. Innis,                   sis errors. Biochemistry. 29: 8003-8011.

980      Journal of Lipid Research          Volume 34, 1993
26. Kunkel, T. A. 1986. Frameshift mutagenesis by eucaryotic                 terolemia in Ashkenazi Jews. Am. J Hum. Genet. 49:
     DNA polymerase in vitro. J Biol. Chem. 261: 13581-13587.                443-449.
27. Kunkel, T. A. 1985. The mutational specificity of DNA             39.    Koivisto, U-M., H. Turtola, K. Aalto-Setiila, B. Top, R. R.
     polymerases-cr and -y during in vitro DNA synthesis.                    Frants, P. T. Kovanen, A-C. Syvhen, and K. Kontula.
    J. Biol. Chem. 2 6 0 12866-12874.                                        1992. The familial hypercholesterolemia (FH)-North
28. Farese, R. V., Jr., M. F. Linton, and S. G. Young. 1992.                 Karelia mutation of the low density lipoprotein receptor
     Apolipoprotein-B gene mutations affecting cholesterol                   gene deletes seven nucleotides of exon 6 and is a common
    levels. J. Intern. Mcd. 231: 643-652.                                    cause of FH in Finland. J. Clin. Invest. 90: 219-228.
29. Kunkel, T. A. 1985. The mutational specificity of DNA             40.    Santamarina-Fojo, S., and H. B. Brewer, Jr. 1991. The
     polymerase-@during in vitro DNA synthesis.J. Biol. Chem.                familial hyperchylomicronemia syndrome. New insights
     2 6 0 5787-5796.                                                        into underlying genetic defects. J. Am. Med. Assoc. 265:
30. Collins, D. R., T. J. Knott, R. J. Pease, L. M. Powell, S. C.            904-908.
     Wallis, S. Robertson, C. R. Pullinger, R. W. Milne, Y. L.        41.    Henderson, H. E., R. Devlin, J. Peterson, J. D. Brunzell,
     Marcel, S. E. Humphries, P. J. Talmud, J. K. Lloyd, N. E.               and M. R. Hayden. 1990. Frameshift mutation in exon 3
     Miller, D. Muller, and J. Scott. 1988. Truncated variants of            of the lipoprotein lipase gene causes a premature stop
     apolipoprotein B cause hypobetalipoproteinemia. Nucleic                 codon and lipoprotein lipase deficiency. Mol. Biol. Med. 7 :
    Acidr Res. 16: 8361-8375.                                                511-517.
31. Soria, L. F., E. H. Ludwig, H. R. G. Clarke, G. L. Vega,          42.    Takagi, A,, Y. Ikeda, Z. Tsutsumi, T. Shoji, and A.
     S. M. Grundy, and B. J. McCarthy. 1989. Association be-                 Yamamoto. 1992. Molecular studies on primary lipoprotein
     tween a specific apolipoprotein B mutation and familial                 lipase (LPL) deficiency. One base deletion (G916) in exon 5
     defective apolipoprotein B-100. Pmc. Natl. Acad. Sci. USA.              of LPL gene causes no detectable LPL protein due to the
     86: 587-591.                                                            absence of LPL mRNA transcript. J. Clin. Invest. 89:
32. Huang, L. S., M. E. Ripps, and J. L. Breslow. 1990.                      581-591.
     Molecular basis of five apolipoprotein B gene poly-              43.    Fojo, S. S., A. F. H. Stalenhoef, K. Marr, R. E. Gregg,

                                                                                                                                              Downloaded from www.jlr.org by guest, on October 11, 2015
     morphisms in noncoding regions. J. Lipid Res. 31: 71-77.                R. S. Ross, and H. B. Brewer,Jr. 1988. A deletion mutation
33. Levy-Wilson, B., L. Soria, E. H. Ludwig, M. Argyres,                     in the apoC-I1 gene (apoC-IINi,,,Fn) of a patient with a
    A. R. Brooks, B. D. Blackhart, W. Friedl, and B. J.                      deficiency of apolipoprotein C-11. J. Clzn. Inuest. 82:
     McCarthy. 1991. A polymorphism in a region with en-                     1489-1494.
     hancer activity in the second intron of the human apolipo-       44.    Xiong, W., W-H. Li, I. Posner, T. Yamamura, A.
     protein B gene. J. Lipid Res. 32: 137-145.                              Yamamoto, A. M. Gotto, Jr., and L. Chan. 1991. No severe
34. Ladias, J. A. A., P. 0. Kwiterovich, Jr., H. H. Smith,                   bottleneck during human evolution: evidence from two
     M. Miller, P. S. Bachorik, T. Forte, A. J. Lusis, and S. E.             apolipoprotein C-I1 deficiency alleles. Am. J. Hum. Genet.
    Antonarakis. 1989. Apolipoprotein B-100 Hopkins (argi-                   48: 383-389.
     nine,al9+ tryptophan): a new apolipoprotein B-100 variant       45.    Rall, S. C., Jr., K. H. Weisgraber, R. W. Mahley, Y.
     in a family with premature atherosclerosis and hyperapo-               Ogawa, C. J. Fielding, G. Utermann, J. Haas, A. Stein-
    betalipoproteinemia. J. Am. Med. Assoc. 262: 1980-1988.                 metz, H-J. Menzel, and G. Assmann. 1984. Abnormal leci-
35. Schonfeld, G. 1990. Inherited disorders of lipid transport.             thin:cholesterol acyltransferase activation by a human
    Endocrinol. Metab. Clin. North Am. 19: 229-257.                         apolipoprotein A-I variant in which a single lysine residue
36. Russell, D. W., V. Esser, and H. H. Hobbs. 1989. Molecu-                is deleted. J. Biol. Chem. 259: 10063-10070.
    lar basis of familial hypercholesterolemia. Arteriosclerosis     46.    Funke, H., A. von Eckardstein, P. H. Pritchard, M. Karas,
    S~fipl.9: 1-8-1-13.                                                     J. J. Albers, and G. Assmann. 1991. A frameshift mutation
37. Leitersdorf, E., H. H. Hobbs, A. M. Fourie, M. Jacobs,                  in the human apolipoprotein A-I gene causes high density
     D. R. van der Westhuyzen, and G. A. Coetzee. 1988. Dele-               lipoprotein deficiency, partial lecithin:cholesterol acyltrans-
    tion in the first cysteine-rich repeat of low density lipo-             ferase deficiency, and corneal opacities. J. Clin. Inuest. 87:
    protein receptor impairs its transport but not lipoprotein              371-376.
    binding in fibroblasts from a subject with familial hyper-       47.    Deeb, S. S., M. C. Cheung, R. Peng, A. C. Wolf, R. Stern,
    cholesterolemia. Pmc. Natl. Acad. Sci. USA. 85: 7912-7916.              J. J. Albers, and R. H. Knopp. 1991. A mutation in the
38. Meiner, V., D. Landsberger, N. Berkman, A. Reshef, P.                   human apolipoprotein A-I gene. J. Biol. Chem. 266:
    Segal, H. C. Seftel, D. R. van der Westhuyzen, M. S.                    13654-13660.
    Jeenah, G. A. Coetzee, and E. Leitersdorf. 1991. A com-          48.    Honig, G. R., and J. G. Adams 111. 1986. Human Hemo-
    mon Lithuanian mutation causing familial hypercholes-                   globin Genetics. Springer-Vedag, Wien. 308-341.

                                   Gmenewegen, Krul, and Schonfcld   Misaligned pairing deletion mechanism in apoB-52                 981
You can also read