Hum Genet (1990) 84:439-445
9 Springer-Vertag1990
Apolipoprotein A1 Baltimore (Argl0 >Leu), a new ApoA1 variant John A. A . Ladias 1, Peter O. Kwiterovich, Jr. 2, Hazel H. Smith 2, Sotirios K. Karathanasis 3, and Stylianos E. Antonarakis 1 i Genetics Unit, Department of Pediatrics, The Johns Hopkins University, School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205, USA 2Lipid Research Unit, Department of Pediatrics, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA 3Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA Received April 24, 1989 / Revised August 28, 1989
Summary. A new apolipoprotein A1 (APOA1) gene variant has been identified in a family ascertained through a proband undergoing coronary angiography. The variant, ApoA1 Baltimore, was due to a mutation at codon 34 of the third exon of the A P O A 1 gene ( C G A to CTA) that resulted in an arginine-to-leucine substitution at the tenth amino acid of the mature ApoA1 and a change in charge of - 1 . The mutation abolishes a TaqI restriction site and it is easily detectable after polymerase chain reaction amplification of genomic DNA. The proband was heterozygous for the mutation. Eight other members of the pedigree had the same ApoA1 variant. Cosegregation of the variant with hypoalphalipoproteinemia could not be demonstrated and the association of this mutation with hypoalphalipoproteinemia was confined to three affected members of the nuclear family. No effect of the mutant on any lipoprotein phenotype could be established.
Introduction H u m a n apolipoprotein A-1 (ApoA1), a major protein constituent of H D L (high density lipoprotein), is synthesized from a single gene in the liver and small intestine as a 267-residue preproapolipoprotein (preproapoA-I; Zannis and Breslow 1985; Breslow 1985). The gene for A P O A 1 has been cloned and characterized (Karathanasis et al. 1983; Shoulders and Baralle 1982). The presegment, 18 amino acid residues long, is cleaved co-translationally by a signal peptidase (Zannis and Breslow 1985; Breslow 1985). The resulting proapoA-I contains a hexapepfide prosegment covalently linked to the NH2 terminus of mature ApoA1; it is secreted into plasma and lymph (Shoulders and Baralle 1982) and undergoes extracellular post-translational cleavage to the mature 243-residue ApoA1 (Zannis and Breslow 1985; Breslow 1985; Bojanovski 1985). A p o A 1 serves as a cofactor for the plasma enzyme lecithin: cholesterol acyltransferase, which is responsible for the formation of
Offprint requests to: S. E. Antonarakis
most cholesteryl esters in plasma (Saltar et al. 1975). Decreased concentration of A p o A 1 in plasma (hypoalphalipoproteinemia) have been correlated with increased risk of premature coronary artery disease (Maciejko et al. 1983). Polymorphic forms of A p o A 1 occur in the human population (Breslow 1985). A number of such A p o A 1 variants have now been identified. For example, two of these variants, ApoA1 Milano (Arglv3---~Cys) and A p o A 1 Giessen (Pro143-->Arg), are associated with mildly reduced levels of H D L (Francheschini et at. 1985; U t e r m a n n et al. 1984). Heterozygous subjects with the A p o A 1 variant (Pro143--~Arg) all had a decreased level of the mutant protein in plasma and a functional abnormality of the mutant A p o A 1 (Utermann et al. 1984). Several other variants are not associated with any detectable dyslipoproteinemia (Breslow 1985). For example, the three probands with A p o A 1 Marburg (Lysl07--->0) all had low H D L levels but cosegregation of the variant with hypoalphalipoproteinemia could not be demonstrated (Utermann et al. 1982). We describe here a new A p o A 1 variant, ApoA1 Baltimore (Argl0-~Leu), in a family that was large enough to allow a study of the possible cosegregation of the variant with lipoprotein phenotypes and premature coronary artery disease.
Materials and methods Subjects The proband (IV-25) and his family (Fig. 1) were ascertained through The Johns Hopkins University Coronary Artery Disease (JHU-CAD) Study. This study consisted of 203 index cases (99 males 50 years of age or younger and 104 females 60 years of age or younger) undergoing elective coronary arteriography, their spouses, and first-degree relatives. Thirty-eight relatives (including spouses) of the proband were examined and studied (see pedigree in Fig. 1).
Blood samples Plasma was obtained from blood drawn after a 12-h overnight fast into tubes containing solid EDTA (ethylenediamine tetraacetic
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11
12
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[ 15
15
29
30
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Fig. 1. Pedigree with A P O A 1 Baltimore. A, B, C, D in the nuclear family of the proband (individual shown by arrow) refer to the D N A polymorphism haplotype as explained in the results. [~O Not examined; mO examined, non-carriers; []1~ examined, cartiers; []| not examined, carriers by pedigree
2
3
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5
6
Restriction endonuclease analysis of genomic D N A
The plasma levels of total cholesterol and triglycerides and the concentrations of V L D L (very low density lipoprotein) cholesterol, LDL (low density lipoprotein) cholesterol, and H D L cholesterol were determined using methods of the Lipid Research Clinics Program (Manual of Laboratory Operations 1982), as modified previously (Kwiterovich et al. 1987).
Nuclear D N A was isolated from the leukocytes of Na2EDTAtreated peripheral blood as described (Kunkel et al. 1977), and 5 10pg D N A was digested with one of various restriction endonucleases using conditions recommended by the commercial suppliers. The resulting D N A fragments were separated according to size by electrophoresis in 1% agarose gels, transferred to nitrocellulose membranes, fixed, and hybridized with 32p-labeled probes. Washing of filters and autoradiography were carried out as described (Scott et al. 1979). The probes used were: (1) a 2.2-kb PstI genomic D N A fragment that contains the APOA1 gene and detects TaqI, MspI, XmnI and SstI polymorphic sites (Ordovas et al. 1986; Antonarakis et al. 1988); (2) a 1.2-kb PstI-PvuII genomic D N A fragment 3' to the A P O A 4 gene (Antonarakis et al. 1988; Oettgen et al. 1986). The first probe was used for the mapping of the abnormal TaqI site within the APOA1 gene, and both probes were used for the detection of several D N A polymorphic sites in the A P O A 1 - A P O C 3 - A P O A 4 gene cluster.
Plasma L D L B protein assay
Enzymatic D N A amplification and sequencing
The content of LDL B protein in plasma was measured by radial immunodiffusion (RID) using the M-Partigen Apolipoprotein B Kit (Calbiochem-Behring Corp., LaJolla, Calif.), as described previously (Kwiterovich et al. 1987).
Enzymatic amplification was performed using the procedure of Saiki et al. (1986). One microgram of genomic D N A was incubated in a 100-~tl reaction mixture with 1.5 ~tM each of the two oligonucleotide primers synthesized on an Applied Biosystems 780A D N A synthesizer (primer 1 and 2); 20011M each dGTP, dATP, TTP and dCTP; 10~tM Tris-C1 (pH 8.3); 1.51aM MgCI2; 50 pM NaC1; 0.01% gelatin and 2.5 units of D N A polymerase from Thermophilus Aquaticus (Saiki et al. 1988). The reaction mixture was incubated initially at 94~ for 5 rain, then at 37~ for 3min, and at 70~ for 3 min. Subsequent cycles were consisted of incubations at 94~ for 2 min, 37~ for 2 rain and 70~ for 2 min. After 45 cycles, 10% of the reaction mixture (10 pl) was electrophoresed in a 4% Nusieve agarose gel, stained with ethidium bromide and the amplified D N A fragment was visualized under ultraviolet irradiation. The remaining 90% of the reaction mixture was extracted with phenol, phenol/chloroform (1:1), and precipitated with ethanol. The D N A was digested with PstI and HindIII and ligated to PstI/HindIII linearized M13mp19 vector. The ligation mix was used to transform JM103 host cells. Individual clones were sequences using the dideoxytermination method of Sanger et al. (1977).
acid). The subjects were following a regular diet and none was taking lipid-lowering medication.
Lipid and lipoprotein cholesterol quantitation
Plasma ApoA1 assay ApoA1 was measured by RID in commercially prepared agarose plates containing monospecific goat antibody to ApoA1 as specified by the manufacturer (DiffuGen ApoA1 plates, catalogue no. 1901, Tago, Burlingame, Calif.) as described previously (Kwiterovich et al. 1987). The combined intra- and interassay coefficients of variation for the LDLB and ApoA1 assays were 4 % - 5 % for each.
Normal plasma lipid, lipoprotein cholesterol, and apolipoprotein levels' An elevated or depressed level for a plasma lipid or lipoprotein cholesterol was defined as a value above the 95th percentile, or below the 5th percentile, respectively, age and sex specific, using cutpoints from the Lipid Research Clinics Program (Lipid Research Clinics Population Studies Data Book 1980). A low plasma ApoA1 level was a value in our control population below the 5th percentile (105 mg/dl for 299 normal males, aged 19 to 36 years, and 119 mg/dl for 168 normal females, aged 19 to 35 years). A high plasma LDLB level was a value above 134mg/dl, which was the 95th percentile for both the 299 males and 169 females. To assess those with a higher LDLB but lower ApoA1 levels, the ratio of LDLB/ApoA1 was computed: a high ratio was a value above the 95th percentile from the same control groups, namely, > 1.04 for the men and >0.88 for the women.
Apolipoprotein A1 electrophoresis H D L was isolated from fasting subjects by sequential ultracentrifugation in a Beckman 60Ti rotor (Beckman Instrument, Palo Alto, Calif.) between densities 1.063g/ml and 1.210g/ml (Havel et al. 1955). The isolated H D L was recentrifuged at 1.210 g/ml, dialyzed extensively against 0.01 M NHgHCO3 (pH 8.2), lyophilized, and delipidated with chloroform-methanol 3 : 1as previously described (Sprecher et al. 1984). Delipidated HDLs were analyzed by twodimensional gel electrophoresis utilizing a pH 4 to 6 gradient for isoelectrofocusing (Sprecher et al. 1984).
441 I probe
I
3'
5'
t I I B
Fig. 2. A Autoradiogram from restriction endonuclease analysis after digestion with TaqI and hybridization with the APOA1 probe. This is family 2 in the Johns Hopkins University Coronary Artery Disease Study. Each lane contains DNA from an individual numbered as in Fig. 1. C Control DNA; DNA fragment sizes are measured in kilobases (kb). B Restriction TaqI map of the APOA1 gene area. The gene is shown as a box. Blackareasrepresent exons and whiteareasrepresent intervening sequences. Fragment sizes are shown in kilobases. TaqI cleavage sites are shown with arrows. The polymorphic TaqI site is shown with an asterisk. The TaqI site that is absent in one APOA1 gene of the proband is shown as striped arrow
Results
Abnormal APOA1 gene fragment after TaqI digestion After digestion of h u m a n genomic D N A with TaqI restriction endonuclease and hybridization with the A P O A 1 probe, the following fragments are detected: 1.2 and 2.0kb, which are invariant in all individuals, and the variable 5.4 kb or 11.2 kb in the presence or absence, respectively, of the polymorphic TaqI site 5' of the A p o A 1 gene (Fig. 2 A , B ) . A 34-year-old male index case (IV-25 of Fig. 1) f r o m the J H U - C A D study was found to be heterozygous for an abnormal A P O A 1 gene fragment after digestion of his D N A with TaqI and hybridization with the A P O A 1 probe. The proband exhibited the 11.2-kb polymorphic TaqI fragment, a novel 6.6-kb fragment, and one copy of the 1.2-kb fragment (Fig. 2). The same abnormal 6.6-kb TaqI D N A fragment was present in the p r o b a n d ' s sister (IV-26), his father (III-14), two of his father's sisters (III-10, III-12), his first cousin (IV-22), and his father's first cousin (III-4) and her two daughters ( I V - l , IV-2). Further restriction endonuclease analysis revealed no other D N A abnormality and was conclusive that the abnormal fragment was due to a loss of the TaqI site in the
s.~ kb
11.2
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6.6
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third exon of A P O A 1 gene around codon 10 (data not shown). H a p l o t y p e analysis was p e r f o r m e d on different m e m bers of the p r o b a n d ' s nuclear family using several reported D N A polymorphisms on the A P O A 1 - A P O C 3 A P O A 4 gene cluster. The polymorphic sites used were TaqI and XmnI 5' to the A P O A 1 gene, MspI in the third intron of A P O A 1 , PstI 3' to A P O A 1 , SstI on exon 4 of the A P O C 3 gene, two PstI sites 3' to the A P O A 4 gene, and BqIII also 3' to the A P O A 4 gene (Antonarakis et al. 1988). The haplotype associated with the absent TaqI site in exon 3 of the A P O A 1 gene is: + 3 + + - - + + for the eight polymorphic sites scored ( + refers to the presence of a polymorphic site, - refers to the absence of a polymorphic site and 3 refers to allele 3 for the XmnI polymorphism). For simplicity this haplotype is n a m e d B. The p r o b a n d IV-25 has haplotype B and C (C is -2-+ .... ). His father III-14 has haplotypes A and B (A is + 3 + + - + - - ) and his m o t h e r III-15 has haplotypes C and D (D is + 3 + + - - - + ) . Individual IV-23, the p r o b a n d ' s brother, who does not have the TaqI mutation, inherited haplotypes A and D and his sister IV-26 inherited haplotypes B and D (see pedigree in Fig. 1).
Polymerase chain reaction amplification and nucleotide sequencing To identify the molecular defect that resulted in the loss of the TaqI site in exon 3 of the A P O A 1 gene, 164 nucleotides around the m u t a n t TaqI site were amplified from the p r o b a n d ' s D N A , using the polymerase chain reaction. Figure 3 shows the nucleotide sequences of the oligonucleotide primers used in the reaction. Artificial restriction sites (PstI and HindIII) were incorporated into the oligonucleotide primer sequences and used for cloning of the amplification product into M13mp19 vector. Seven independent clones were subjected to nucleotide sequence analysis. Six had the mutant sequence and one had the normal sequence. The mutation shown in Fig. 4 was a G-to-T transversion at codon 34 of the A P O A 1 gene which codes for amino acid residue 10 of the mature A P O A 1 . This nucleotide substitution changes the C G A codon for arginine to C T A , which is the codon for leucine. The presence of the mutation in other m e m bers of the family was ascertained by Southern blot analysis, or by TaqI digestion after polymerase chain reaction amplification of the 164 nucleotides that include the TaqI site of exon 3 of the A P O A 1 gene.
Two-dimensional gel electrophoresis of the variant ApoA1 protein The protein encoded from the m u t a n t A P O A 1 gene was studied using two-dimensional gel electrophoresis. As
442 Primer 1 Pst,
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GTG CTC AhA GAC AGC GGC AGA GAC TAT GTG TCC CAG TIT GAA C~C TCC GCC ...3' Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Set Gln Phe Glu Gly Set Ala GAG 'l~I~fCTG TCG CCG TCT CTTHindCGAIII AAC A
Fig. 3. DNA sequence of the amplified APOA1 fragment. The two primers for amplification are shown in boxes as primer 1 and primer 2. Primer 1 contained a PstI site and primer 2 contained an artificial HindIII site. The TaqI site that has mutated in APOA1 Baltimore is also boxed. The junction between IVS 2 and exon 3 is shown
Primer 2
Fig. 4. Nucleotide sequence of the TaqI site mutation in the APOA1 Baltimore gene. Sequences from three independent mutant M13 clones are shown shown in Fig. 5 the migration of the mutant A P O A 1 Baltimore (Argl0-+Leu) is compatible with a - 1 charge change. T h e r e is approximately equal quantity of the normal A p o A 1 and A p o A 1 Baltimore in the patient's plasma.
Plasma lipids, lipoproteins, and apolipoproteins The plasma levels of lipids, lipoprotein cholesterol, and A p o A 1 and L D L B were determined in the proband (IV-25) and in thirty-one blood relatives and seven spouses of the relatives (Fig. 1, Table 1). The data in the affected relatives in the nuclear family differed from those in the other relatives with A p o A 1 Baltimore. The proband (IV-25) and both his carrier brother (III-14) and sister (IV-26) had low H D L cholesterol levels, or low A p o A 1 levels, on one or m o r e occasions. This was not true for the other six relatives with the mutation who had normal levels of H D L cholesterol and A p o A 1 (Table 1). The three affected persons in the nuclear family also had H D L cholesterol and A p o A 1 levels that fluctuated, an observation that was confirmed when a 3-year follow-up sample was obtained. Such fluctuation was not true of the other relatives with the mutation or of the unaffected brother (IV-23) of the proband.
Fig. 5. Two-dimensional gel electrophoretograms of HDL ApoA1 isoforms from a normal subject (a) and a patient with ApoA1 Baltimore (b). Only the portion of the gel containing the ApoA1 isoforms is presented. The ApoA-I+2 isoform is proapoA1, and ApoA1 isoforms 0 to - 2 are mature ApoA1 isoforms. The ApoA1 Baltimore patient has approximately equal quantities of the normal (ApoAlo) and mutant (ApoA1) isoforms
The plasma cholesterol, triglyceride, and other lipoprotein values in the three affected persons in the nuclear family were normal, suggesting a hypoalphalipoproteinemia lipoprotein pattern. While only three of the blood relatives of the proband (including the mother) had a high H D L cholesterol value ( > the 95th percentile, age and sex specific; Lipid Research Clinics Population Studies D a t a Book 1980), seven other relatives of the index case had borderline high H D L cholesterol values, between the 75th and 95th percentiles. While hyperalphalipoproteinemia was not a dominant phenotype in this family, the H D L cholesterol values were noticeably shifted toward higher values. The expression of the A P O A 1 variant in the heterozygous state may have been affected by the expression of another A P O A 1 allele or other genes. Neither the proband nor any of his relatives had hypertriglyceridemia with one exception (III-6, type I V lipoprotein pattern), and only one had a high L D L cholesterol level (V-6, type IIa lipoprotein pattern). In regard to L D L B, eight of the relatives of the proband had a plasma L D L B level above 134 mg/dl, the currently used upper limit of normal in our laboratory. All but one of these relatives also had a low ratio of L D L cholesterol to L D L B (_< 1.2) suggesting that the phenotype of hyper-
443 Table 1. Plasma levels of lipids, lipoprotein cholesterol, and apolipoproteins in proband and relatives. ND, Not determined Position in pedigree
Sex
Age
Total cholesterol
Total triglyceride
VLDL cholesterol
LDL cholesterol
LDLB
HDL cholesterol
HDL ApoA1
LDLB/ApoA1 ratio
Proband IV-25
M
35 36
167 196 204
67 107 102
20 43 6
109 113 149
104 105 149
46 (22) 49
(90) a 124 115
1.16 b 0.85 1.30
22 22 23 23 23
137 131 124 c 131 c 147 c
25
142
63 82 94 98 88 68
ND ND 15 ND 24 20
82 66 80 ND 91 83
82 71 ND ND ND 84
42 49 (29) (29) (32) 39
131 (99) ND ND ND (117)
0.63 0.72 ND ND ND 0.72
38
Rela~ves with ApoA-1 Baltimore IV-26
F
III-14
M
65 66 68
208 185 211
107 58 100
34 ND 19
150 146 138
149 100 160
(24) (27) 39
(101) ND 117
1.48 ND 1.37
III-10
F
68 69
213 287
54 76
29 ND
137 220
123 139
50 52
128 142
0.96 0.98
III-12
F
69 70
220 232
80 113
5 40
180 140
139 137
55 55
155 132
0.90 1.04
Ill-4
F
62 63
221 197
(54) 76
20 ND
139 104
132 123
62 78
154 139
0.86 0.88
IV-1
F
44
190
91
3
137
125
50
165
0.76
IV-2
F
39
142
41
1
86
87
55
137
0.64
IV-22
F
46
151
(39)
4
98
87
49
132
0.66
208
ND
115
128
49
113
1.13
Rela~ves without ApoA-1 Bal~more III-1
M
60
206
11I-5
F
66
228
181
ND
131
14__99
61
(94)
1.59
III-6
M
64
187
297
ND
85
118
43
108
1.09
III-8
M
62
223
69
ND
139
118
70
156
0.76
III-9
M
73
229
182
ND
142
147
51
120
1.23
III-13
M
67
214
(50)
22
130
103
62
189
0.54
II1-15
F
61
237
50
ND
107
80
104
273
0.29
III-16
F
48
236
125
ND
137
153
74
228
0.67
III-17
F
84
232
107
ND
133
112
78
152
0.74
IV-9
F
32
171
56
ND
104
101
56
120
0.84
IV-10
F
24
196
109
ND
120
106
54
139
0.76
IV-11
F
30
168
48
ND
78
76
80
181
0.42
50
ND
114
103
50
113
0.91
IV-14
M
29
174
IV-15
F
31
167
70
ND
95
94
58
134
0.70
ND
136
168
42
120
1.40
116 103
90 106
59 57
123 167
0.73 0.63
IV-20
M
41
201
133
IV-23
M
40 43
192 180
86 123
ND 20
IV-27
F
25
188
63
ND
99
90
76
139
0.65
ND
137
133
81
174
0.76
IV-28
F
21
235
73
IV-29
F
64
201
85
ND
102
127
82
171
0.74
ND
129
129
82
185
0.70
IV-30
F
54
220
57
V-1
M
21
197
86
ND
131
103
49
108
0.95
V-5
M
12
155
98
ND
97
82
38
ND
ND
214
51
ND
156
110
48
136
0.81
V-6
F
8
444 Table 1 (continued) Position in pedigree
Sex
Age
Total cholesterol
Total triglyceride
VLDL cholesterol
LDL cholesterol
LDLB
F F M M M F F
62 60 62 62 37 37 39
306 229 206 220 204 217 217
136 143 115 120 91 108 (33)
ND ND 29 ND ND ND ND
220 116 141 137 141 139 133
201 133 127 149 123 149 91
HDL cholesterol
HDL LDLB/ApoA1 ApoA1 ratio
SpotIscs III-2 III-7 III-11 IV-8 IV-12 IV-19 IV-24
59 84 36 59 45 56 77
186 186 109 136 111 161 ND
1.08 0.72 1.17 1.10 1.11 0.93 ND
a Lipid and lipoprotein values in brackets are < 5th percentile, age and sex specific (Lipid Research Clinics Population Studies Data Book 1980). Plasma ApoA1 values < 5th percentile (< 105 mg/dl for males and < 119 mg/dl for females) are also in brackets b Lipid and lipoprotein values that are underlined are > 95th percentile, age and sex specific (Lipid Research Clinics Population Studies Data Book 1980). Plasma LDLB values that are > 134 mg/dl, the 95th percentile for both control men and women, are also underlined. Ratios of LDLB/ApoA1 >95th percentile (1.04 for males, and 0.88 for females, respectfully) are also underlined c Determined at the Molecular Disease Branch, National Heart, Lung and Blood Institute
apo B (Kwiterovich 1988) was prevalent in this family. We therefore examined the ratio of L D L B / A p o A 1 in this kindred to see if a high ratio might provide a better chemical marker. O f the nine persons with the A p o A 1 variant, four had an elevated L D L B / A p o A 1 ratio. Five of the twenty-three relatives of the p r o b a n d without the variant had an elevated ratio. Thus, we were unable to detect any lipoprotein pattern that was consistently associated with the A p o A 1 Baltimore variant. The finding of the mutant A p o A 1 Baltimore in the first cousin of the p r o b a n d ' s father indicated that this mutant was being transmitted through the proband's paternal grandfather's (individual II-10) side of the family. This finding further indicates that the paternal grandfather of the p r o b a n d carried the mutant allele. This is of interest since the paternal grandfather (II-10) developed angina pectoris early in his forties, and died at the age of 49 years f r o m myocardial infarction. The father of the p r o b a n d (III-14) had a myocardial infarction at the age of 35 years. The p r o b a n d himself underwent elective diagnostic coronary arteriography because of atypical angina pectoris, but did not have significant coronary atherosclerosis ( > 50% blockage). The other four family m e m b e r s with the A p o A 1 variant did not have any clinical evidence of coronary artery disease.
Discussion A new apolipoprotein A1 variant, n a m e d A p o A 1 Baltim o r e , has been detected in a family in which the proband had low, or borderline low, H D L cholesterol and A p o A 1 levels, indicative of an hypoalphalipoproteinemia lipoprotein pattern. The mutation in codon 34 of the third exon of the A P O A 1 gene was a G-to-T-substitution that resulted in an Arg-to-Leu amino acid substitution at the tenth residue of mature A p o A 1 . Interestingly, the mutation changed a C G dinucleotide to CT and therefore was an exception to the CG--+TG mutation rule, in which methylation-deamination of the C in
the C p G dinucleotide results in a C- to T-substitution (Youssoufian et al. 1986). Classical linkage analysis between the phenotype of hypoalphalipoproteinemia or coronary artery disease and A P O A 1 Baltimore does not result in a tight linkage between the phenotype and the genetic marker. Explanations for the lack of linkage include: (1) the A P O A 1 Baltimore gene has no relationship with the phenotype of hypoalphalipoproteinemia. (2) The A P O A 1 Baltim o r e gene is related to hypoalphalipoproteinemia but there are other gene loci that modify the phenotype. It is of note that in the nuclear family of the proband there is linkage between the presence of the A P O A 1 Baltimore gene and hypoalphalipoproteinemia. Studies on the metabolic fate of A p o A 1 Baltimore polypeptide are currently being performed. Preliminary results from such metabolic studies in the sister of the proband indicated increased catabolism of A p o A 1 (Schaefer et al. 1988). However, both the A p o A 1 Baltim o r e and the wild-type A p o A 1 were catabolized at an increased rate in the proband's affected sister, suggesting that the A p o A 1 variant is associated with an abnormality that facilitates the catabolism of apolipoprotein A1. One candidate is hyperapo B, a condition in which the elevated small, dense, L D L particles are often accompanied by low A p o A 1 and H D L cholesterol levels (Kwiterovich 1988; Kwiterovich and Sniderman 1983). Further studies are underway to determine what factors may influence the metabolism of A p o A 1 and H D L in affected family members. A p o A 1 Baltimore is a rare variant. F r o m more than 400 unrelated chromosomes studied in the Johns Hopkins University Coronary Artery Disease Study, and the 196 chromosomes from unrelated Mediterranean and American black individuals studied for haplotype analyses (Antonarakis et al. 1988), only one chromosome (that of the proband) carried the mutant chromosome A p o A 1 Baltimore gene. While low levels of A p o A 1 and H D L cholesterol are prevalent in patients with coronary artery disease, they are often accompanied by elevated
445 levels o f t r i g l y c e r i d e , o r L D L c h o l e s t e r o l , o r L D L B p r o tein ( K w i t e r o v i c h a n d S n i d e r m a n 1983). It is of i n t e r e s t t h a t t h e p r o b a n d (IV-25, Fig. 1) is o n e o f t h e few i n d e x cases w h o h a d h y p o a l p h a l i p o p r o t e i n e m i a at b a s e l i n e exa m i n a t i o n in t h e J o h n s H o p k i n s U n i v e r s i t y C o r o n a r y Artery Disease Study. Cosegregation between APOA1 Baltimore and hypoalphalipoproteinemia could not be demonstrated. Nevertheless, t h e v a r i a n t p r o v i d e s an o p p o r t u n i t y to s t u d y t h e in vivo a n d in vitro m e t a b o l i s m o f an A p o A 1 m o l e c u l e t h a t has lost a p o s i t i v e l y c h a r g e d r e s i d u e , a r g i n i n e , at p o s i t i o n 10 in t h e m a t u r e p o l y p e p t i d e . T h e m u t a t i o n m a y also p e r m i t t h e i d e n t i f i c a t i o n o f p a t i e n t s w h o h a v e an a s s o c i a t e d a b n o r m a l i t y t h a t a d v e r s e l y affects l i p o p r o tein m e t a b o l i s m a n d i n c r e a s e s risk for p r e m a t u r e c o r o nary artery disease.
Acknowledgements. We thank the members of the family described in this paper for their cooperation during the study. We also thank Dr. B. Brewer and Richard Gregg for the two-dimensional electrophoresis of HDL-ApoA1, J. G. Lewis for expert technical assistance, J. Strayer for the art work, and P. Divel for typing this manuscript. The study was performed by grants from the National Institutes of Health HL 31497 and HD 19591.
References Antonarakis SE, Oettgen P, Chalaravarti A, Halloran SL, Hudson RR, Feisse L, Karathanasis SK (1988) DNA polymorphism haplotypes of the human apolipoprotein ApoA1-ApoC3ApoA4 gene cluster. Hum Genet 80: 265-273 Bojanovski D, Gregg RE, Chiselli G, Schaefer EJ, Light JA, Brewer HB Jr (1985) Human apolipoprotein AoI isoprotein metabolism: proapoA-I conversion to mature ApoA1. J Lipid Res 26:185-193 Breslow JL (1985) Human apolipoprotein molecular biology and genetic variation. Annu Rev Biochem 54 : 699-727 Francheschini G, Sirzori CR, Bosisio E, Gualandri V, Orsini GB, Mogavero AM, Gapurso A (1985) Relationship of the phenotype expression of the ApoA1 Milano apoprotein with plasma lipid and lipoprotein patterns. Atherosclerosis 58 : 159-174 Havel RJ, Eder HA, Bragdon JH (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345-1353 Karathanasis SK, Zannis VI, Breslow JL (1983) Isolation and characterization of the human apolipoprotein A1 gene. Proc Natl Acad Sci USA 80: 6147-6151 Kunkel LM, Smith KD, Boyer SH, Borgaonkar SD, Wachtel SS, Miller OJ, Breg WR, Jones HW, Rary JM (1977) Analysis of human Y chromosome specific reiterated DNA in chromosome variants. Proc Natl Acad Sci USA 74 : 1245-1249 Kwiterovich PO Jr (1988) HyperapoB: a pleiotropic phenotype characterized by dense low density lipoproteins and associated with coronary artery disease. Clin Chem 34 [Suppl B-1 to B-135] B71-B83 Kwiterovich PO Jr, Sniderman AD (1983) Atherosclerosis and apoproteins B and A1. Prev Med 12 : 815-834 Kwiterovich PO Jr, White S, Forte T, Bachorik PS, Smith H, Sniderman D (1987) Hyperapobetalipoproteinemia in a family with familial combined hyperlipidemia and familial hypercholesterolemia. Arteriosclerosis 7:211-225 Lipid Research Clinics Population Studies Data Book (1980) The prevalence study, vol 1. (NIH publication no 80-1527) US Department of Health and Human Services, Washington, DC
Maciejko J J, Holmes DR, Kottke BA, Zinsmeister AR, Dinh DM, Mao SJT (1983) Apolipoprotein A1 as a marker of angiographically assessed coronary artery disease. N Engl J Med 309 : 385-389 Manual of Laboratory Operations (1982) Lipid and lipoprotein analysis. (LRC Program, vol 1) DHEW publication no. (NIH) 75-628, May 1974 (revised October 1982) Oettgen P, Antonarakis SE, Karathanasis SK (1986) BglII polymorphism site downstream to the human ApoA4 gene. Nucleic Acids Res 14: 7138 Ordovas JM, Schaefer EJ, Salem D, Ward RH, Glueck CJ, Vergani C, Wilson PWF, Karathanasis SK (1986) Apolipoprotein A1 gene polymorphism associated with premature coronary artery disease and familial hypoalphalipoproteinemia. N Engl J Med 314 : 671-677 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1986) Enzymatic amplification of beta globin genomic sequenced and restriction site analysis for diagnosis of sickle cell anemia. Science 120:1350-1354 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich H A (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 : 487-491 Saltar AK, Garnev CW, Baker HN, Sparrow JT, Jackson RL, Gotto AM, Smith LC (1975) Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase. Biochemistry 14: 30573062 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 Schaefer JR, Gregg RE, Zech LA, Meng MS, Ronan R, Antonarakis SE, Bachorik PS, Kwiterovich PO, Brewer HB (1988) Comparison of the kinetics of metabolism of normal apolipoprotein A1 and a mutant apolipoprotein A1, ApoA1 Baltimore (abstract). Clin Res 36 : 545A Scott AF, Phillips JA, Migeon BR (1979) Restriction endonuclease analysis for localization of human B and S globin genes on chromosome eleven. Proc Natl Acad Sci USA 76:45634565 Shoulders CC, Baralle FE (1982) Isolation of the human HDL apoprotein A1 gene. Nucleic Acids Res 10: 4873-4885 Sniderman A, Shapiro S, Marpole D, Malcolm I, Skinner B, Teng B, Kwiterovich PO Jr (1980) The association of coronary atherosclerosis and hyperapobetalipoproteinemia (increased protein but normal cholesterol content in human plasma low density lipoprotein). Proc Natl Acad Sci USA 77 : 604-608 Sprecher DL, Taam L, Brewer HB Jr (1984) Human plasma apolipoproteins: analysis by two-dimensional gel electrophoresis. Clin Chem 30: 2084-2092 Utermann G, Steinmetz A, Paetzold R, Wilk J, Feussner G, Kaffarnik H, Mueller-Eckhardt C, Seidel D, Vogelberg KH, Zimmer F (1982) Apolipoprotein A1 Marburg. Studies on two kindreds with a mutant human apolipoprotein A1. Hum Genet 61 : 329-337 Utermann G, Haas J, Steinmetz A, Paetzold R, Rail CA, Weisgraber KH Jr, Mahley RW (1984) Apolipoprotein A-l~i..... (Pro143----~Arg): a mutant that is defective in activating lecithin : cholesterol acyltransferase. Eur J Biochem 144 : 325331 Youssoufian H, Kazazian HH Jr, Phillips DG, Aronis S, Tsiftis G, Brown VA, Antonarakis SE (1986) Recurrent mutations in hemophilia A give evidence for CpG mutation hotspots. Nature 324 : 380-382 Zannis VI, Breslow JL (1985) Genetic mutation affecting human apolipoprotein metabolism. Adv Hum Genet 14: 125-215
9 Springer-Vertag1990
Apolipoprotein A1 Baltimore (Argl0 >Leu), a new ApoA1 variant John A. A . Ladias 1, Peter O. Kwiterovich, Jr. 2, Hazel H. Smith 2, Sotirios K. Karathanasis 3, and Stylianos E. Antonarakis 1 i Genetics Unit, Department of Pediatrics, The Johns Hopkins University, School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205, USA 2Lipid Research Unit, Department of Pediatrics, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA 3Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA Received April 24, 1989 / Revised August 28, 1989
Summary. A new apolipoprotein A1 (APOA1) gene variant has been identified in a family ascertained through a proband undergoing coronary angiography. The variant, ApoA1 Baltimore, was due to a mutation at codon 34 of the third exon of the A P O A 1 gene ( C G A to CTA) that resulted in an arginine-to-leucine substitution at the tenth amino acid of the mature ApoA1 and a change in charge of - 1 . The mutation abolishes a TaqI restriction site and it is easily detectable after polymerase chain reaction amplification of genomic DNA. The proband was heterozygous for the mutation. Eight other members of the pedigree had the same ApoA1 variant. Cosegregation of the variant with hypoalphalipoproteinemia could not be demonstrated and the association of this mutation with hypoalphalipoproteinemia was confined to three affected members of the nuclear family. No effect of the mutant on any lipoprotein phenotype could be established.
Introduction H u m a n apolipoprotein A-1 (ApoA1), a major protein constituent of H D L (high density lipoprotein), is synthesized from a single gene in the liver and small intestine as a 267-residue preproapolipoprotein (preproapoA-I; Zannis and Breslow 1985; Breslow 1985). The gene for A P O A 1 has been cloned and characterized (Karathanasis et al. 1983; Shoulders and Baralle 1982). The presegment, 18 amino acid residues long, is cleaved co-translationally by a signal peptidase (Zannis and Breslow 1985; Breslow 1985). The resulting proapoA-I contains a hexapepfide prosegment covalently linked to the NH2 terminus of mature ApoA1; it is secreted into plasma and lymph (Shoulders and Baralle 1982) and undergoes extracellular post-translational cleavage to the mature 243-residue ApoA1 (Zannis and Breslow 1985; Breslow 1985; Bojanovski 1985). A p o A 1 serves as a cofactor for the plasma enzyme lecithin: cholesterol acyltransferase, which is responsible for the formation of
Offprint requests to: S. E. Antonarakis
most cholesteryl esters in plasma (Saltar et al. 1975). Decreased concentration of A p o A 1 in plasma (hypoalphalipoproteinemia) have been correlated with increased risk of premature coronary artery disease (Maciejko et al. 1983). Polymorphic forms of A p o A 1 occur in the human population (Breslow 1985). A number of such A p o A 1 variants have now been identified. For example, two of these variants, ApoA1 Milano (Arglv3---~Cys) and A p o A 1 Giessen (Pro143-->Arg), are associated with mildly reduced levels of H D L (Francheschini et at. 1985; U t e r m a n n et al. 1984). Heterozygous subjects with the A p o A 1 variant (Pro143--~Arg) all had a decreased level of the mutant protein in plasma and a functional abnormality of the mutant A p o A 1 (Utermann et al. 1984). Several other variants are not associated with any detectable dyslipoproteinemia (Breslow 1985). For example, the three probands with A p o A 1 Marburg (Lysl07--->0) all had low H D L levels but cosegregation of the variant with hypoalphalipoproteinemia could not be demonstrated (Utermann et al. 1982). We describe here a new A p o A 1 variant, ApoA1 Baltimore (Argl0-~Leu), in a family that was large enough to allow a study of the possible cosegregation of the variant with lipoprotein phenotypes and premature coronary artery disease.
Materials and methods Subjects The proband (IV-25) and his family (Fig. 1) were ascertained through The Johns Hopkins University Coronary Artery Disease (JHU-CAD) Study. This study consisted of 203 index cases (99 males 50 years of age or younger and 104 females 60 years of age or younger) undergoing elective coronary arteriography, their spouses, and first-degree relatives. Thirty-eight relatives (including spouses) of the proband were examined and studied (see pedigree in Fig. 1).
Blood samples Plasma was obtained from blood drawn after a 12-h overnight fast into tubes containing solid EDTA (ethylenediamine tetraacetic
44O
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III
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2
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9
(• 10
11
12
13
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13
14.
[ 15
15
29
30
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Fig. 1. Pedigree with A P O A 1 Baltimore. A, B, C, D in the nuclear family of the proband (individual shown by arrow) refer to the D N A polymorphism haplotype as explained in the results. [~O Not examined; mO examined, non-carriers; []1~ examined, cartiers; []| not examined, carriers by pedigree
2
3
z,,
5
6
Restriction endonuclease analysis of genomic D N A
The plasma levels of total cholesterol and triglycerides and the concentrations of V L D L (very low density lipoprotein) cholesterol, LDL (low density lipoprotein) cholesterol, and H D L cholesterol were determined using methods of the Lipid Research Clinics Program (Manual of Laboratory Operations 1982), as modified previously (Kwiterovich et al. 1987).
Nuclear D N A was isolated from the leukocytes of Na2EDTAtreated peripheral blood as described (Kunkel et al. 1977), and 5 10pg D N A was digested with one of various restriction endonucleases using conditions recommended by the commercial suppliers. The resulting D N A fragments were separated according to size by electrophoresis in 1% agarose gels, transferred to nitrocellulose membranes, fixed, and hybridized with 32p-labeled probes. Washing of filters and autoradiography were carried out as described (Scott et al. 1979). The probes used were: (1) a 2.2-kb PstI genomic D N A fragment that contains the APOA1 gene and detects TaqI, MspI, XmnI and SstI polymorphic sites (Ordovas et al. 1986; Antonarakis et al. 1988); (2) a 1.2-kb PstI-PvuII genomic D N A fragment 3' to the A P O A 4 gene (Antonarakis et al. 1988; Oettgen et al. 1986). The first probe was used for the mapping of the abnormal TaqI site within the APOA1 gene, and both probes were used for the detection of several D N A polymorphic sites in the A P O A 1 - A P O C 3 - A P O A 4 gene cluster.
Plasma L D L B protein assay
Enzymatic D N A amplification and sequencing
The content of LDL B protein in plasma was measured by radial immunodiffusion (RID) using the M-Partigen Apolipoprotein B Kit (Calbiochem-Behring Corp., LaJolla, Calif.), as described previously (Kwiterovich et al. 1987).
Enzymatic amplification was performed using the procedure of Saiki et al. (1986). One microgram of genomic D N A was incubated in a 100-~tl reaction mixture with 1.5 ~tM each of the two oligonucleotide primers synthesized on an Applied Biosystems 780A D N A synthesizer (primer 1 and 2); 20011M each dGTP, dATP, TTP and dCTP; 10~tM Tris-C1 (pH 8.3); 1.51aM MgCI2; 50 pM NaC1; 0.01% gelatin and 2.5 units of D N A polymerase from Thermophilus Aquaticus (Saiki et al. 1988). The reaction mixture was incubated initially at 94~ for 5 rain, then at 37~ for 3min, and at 70~ for 3 min. Subsequent cycles were consisted of incubations at 94~ for 2 min, 37~ for 2 rain and 70~ for 2 min. After 45 cycles, 10% of the reaction mixture (10 pl) was electrophoresed in a 4% Nusieve agarose gel, stained with ethidium bromide and the amplified D N A fragment was visualized under ultraviolet irradiation. The remaining 90% of the reaction mixture was extracted with phenol, phenol/chloroform (1:1), and precipitated with ethanol. The D N A was digested with PstI and HindIII and ligated to PstI/HindIII linearized M13mp19 vector. The ligation mix was used to transform JM103 host cells. Individual clones were sequences using the dideoxytermination method of Sanger et al. (1977).
acid). The subjects were following a regular diet and none was taking lipid-lowering medication.
Lipid and lipoprotein cholesterol quantitation
Plasma ApoA1 assay ApoA1 was measured by RID in commercially prepared agarose plates containing monospecific goat antibody to ApoA1 as specified by the manufacturer (DiffuGen ApoA1 plates, catalogue no. 1901, Tago, Burlingame, Calif.) as described previously (Kwiterovich et al. 1987). The combined intra- and interassay coefficients of variation for the LDLB and ApoA1 assays were 4 % - 5 % for each.
Normal plasma lipid, lipoprotein cholesterol, and apolipoprotein levels' An elevated or depressed level for a plasma lipid or lipoprotein cholesterol was defined as a value above the 95th percentile, or below the 5th percentile, respectively, age and sex specific, using cutpoints from the Lipid Research Clinics Program (Lipid Research Clinics Population Studies Data Book 1980). A low plasma ApoA1 level was a value in our control population below the 5th percentile (105 mg/dl for 299 normal males, aged 19 to 36 years, and 119 mg/dl for 168 normal females, aged 19 to 35 years). A high plasma LDLB level was a value above 134mg/dl, which was the 95th percentile for both the 299 males and 169 females. To assess those with a higher LDLB but lower ApoA1 levels, the ratio of LDLB/ApoA1 was computed: a high ratio was a value above the 95th percentile from the same control groups, namely, > 1.04 for the men and >0.88 for the women.
Apolipoprotein A1 electrophoresis H D L was isolated from fasting subjects by sequential ultracentrifugation in a Beckman 60Ti rotor (Beckman Instrument, Palo Alto, Calif.) between densities 1.063g/ml and 1.210g/ml (Havel et al. 1955). The isolated H D L was recentrifuged at 1.210 g/ml, dialyzed extensively against 0.01 M NHgHCO3 (pH 8.2), lyophilized, and delipidated with chloroform-methanol 3 : 1as previously described (Sprecher et al. 1984). Delipidated HDLs were analyzed by twodimensional gel electrophoresis utilizing a pH 4 to 6 gradient for isoelectrofocusing (Sprecher et al. 1984).
441 I probe
I
3'
5'
t I I B
Fig. 2. A Autoradiogram from restriction endonuclease analysis after digestion with TaqI and hybridization with the APOA1 probe. This is family 2 in the Johns Hopkins University Coronary Artery Disease Study. Each lane contains DNA from an individual numbered as in Fig. 1. C Control DNA; DNA fragment sizes are measured in kilobases (kb). B Restriction TaqI map of the APOA1 gene area. The gene is shown as a box. Blackareasrepresent exons and whiteareasrepresent intervening sequences. Fragment sizes are shown in kilobases. TaqI cleavage sites are shown with arrows. The polymorphic TaqI site is shown with an asterisk. The TaqI site that is absent in one APOA1 gene of the proband is shown as striped arrow
Results
Abnormal APOA1 gene fragment after TaqI digestion After digestion of h u m a n genomic D N A with TaqI restriction endonuclease and hybridization with the A P O A 1 probe, the following fragments are detected: 1.2 and 2.0kb, which are invariant in all individuals, and the variable 5.4 kb or 11.2 kb in the presence or absence, respectively, of the polymorphic TaqI site 5' of the A p o A 1 gene (Fig. 2 A , B ) . A 34-year-old male index case (IV-25 of Fig. 1) f r o m the J H U - C A D study was found to be heterozygous for an abnormal A P O A 1 gene fragment after digestion of his D N A with TaqI and hybridization with the A P O A 1 probe. The proband exhibited the 11.2-kb polymorphic TaqI fragment, a novel 6.6-kb fragment, and one copy of the 1.2-kb fragment (Fig. 2). The same abnormal 6.6-kb TaqI D N A fragment was present in the p r o b a n d ' s sister (IV-26), his father (III-14), two of his father's sisters (III-10, III-12), his first cousin (IV-22), and his father's first cousin (III-4) and her two daughters ( I V - l , IV-2). Further restriction endonuclease analysis revealed no other D N A abnormality and was conclusive that the abnormal fragment was due to a loss of the TaqI site in the
s.~ kb
11.2
I
II ~2 I I 1
6.6
2.o
I
]
third exon of A P O A 1 gene around codon 10 (data not shown). H a p l o t y p e analysis was p e r f o r m e d on different m e m bers of the p r o b a n d ' s nuclear family using several reported D N A polymorphisms on the A P O A 1 - A P O C 3 A P O A 4 gene cluster. The polymorphic sites used were TaqI and XmnI 5' to the A P O A 1 gene, MspI in the third intron of A P O A 1 , PstI 3' to A P O A 1 , SstI on exon 4 of the A P O C 3 gene, two PstI sites 3' to the A P O A 4 gene, and BqIII also 3' to the A P O A 4 gene (Antonarakis et al. 1988). The haplotype associated with the absent TaqI site in exon 3 of the A P O A 1 gene is: + 3 + + - - + + for the eight polymorphic sites scored ( + refers to the presence of a polymorphic site, - refers to the absence of a polymorphic site and 3 refers to allele 3 for the XmnI polymorphism). For simplicity this haplotype is n a m e d B. The p r o b a n d IV-25 has haplotype B and C (C is -2-+ .... ). His father III-14 has haplotypes A and B (A is + 3 + + - + - - ) and his m o t h e r III-15 has haplotypes C and D (D is + 3 + + - - - + ) . Individual IV-23, the p r o b a n d ' s brother, who does not have the TaqI mutation, inherited haplotypes A and D and his sister IV-26 inherited haplotypes B and D (see pedigree in Fig. 1).
Polymerase chain reaction amplification and nucleotide sequencing To identify the molecular defect that resulted in the loss of the TaqI site in exon 3 of the A P O A 1 gene, 164 nucleotides around the m u t a n t TaqI site were amplified from the p r o b a n d ' s D N A , using the polymerase chain reaction. Figure 3 shows the nucleotide sequences of the oligonucleotide primers used in the reaction. Artificial restriction sites (PstI and HindIII) were incorporated into the oligonucleotide primer sequences and used for cloning of the amplification product into M13mp19 vector. Seven independent clones were subjected to nucleotide sequence analysis. Six had the mutant sequence and one had the normal sequence. The mutation shown in Fig. 4 was a G-to-T transversion at codon 34 of the A P O A 1 gene which codes for amino acid residue 10 of the mature A P O A 1 . This nucleotide substitution changes the C G A codon for arginine to C T A , which is the codon for leucine. The presence of the mutation in other m e m bers of the family was ascertained by Southern blot analysis, or by TaqI digestion after polymerase chain reaction amplification of the 164 nucleotides that include the TaqI site of exon 3 of the A P O A 1 gene.
Two-dimensional gel electrophoresis of the variant ApoA1 protein The protein encoded from the m u t a n t A P O A 1 gene was studied using two-dimensional gel electrophoresis. As
442 Primer 1 Pst,
]
IVS2
IGAGCCTC~AaCTC~CCTC~ATCT~WCTC I 5'...tcagatctcagcccacagctggcctgatctgggtctcccctcccaccctcag
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Taq I CAG CAA GAT GAA CCC CCC GAG AGC CCC TGG C~T CCh] GTG AAG GAS CTG GCC ACT GTG TAC GTG GAT Gln Gln Asp Glu Pro Pro Gin Set Pro Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp
GTG CTC AhA GAC AGC GGC AGA GAC TAT GTG TCC CAG TIT GAA C~C TCC GCC ...3' Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Set Gln Phe Glu Gly Set Ala GAG 'l~I~fCTG TCG CCG TCT CTTHindCGAIII AAC A
Fig. 3. DNA sequence of the amplified APOA1 fragment. The two primers for amplification are shown in boxes as primer 1 and primer 2. Primer 1 contained a PstI site and primer 2 contained an artificial HindIII site. The TaqI site that has mutated in APOA1 Baltimore is also boxed. The junction between IVS 2 and exon 3 is shown
Primer 2
Fig. 4. Nucleotide sequence of the TaqI site mutation in the APOA1 Baltimore gene. Sequences from three independent mutant M13 clones are shown shown in Fig. 5 the migration of the mutant A P O A 1 Baltimore (Argl0-+Leu) is compatible with a - 1 charge change. T h e r e is approximately equal quantity of the normal A p o A 1 and A p o A 1 Baltimore in the patient's plasma.
Plasma lipids, lipoproteins, and apolipoproteins The plasma levels of lipids, lipoprotein cholesterol, and A p o A 1 and L D L B were determined in the proband (IV-25) and in thirty-one blood relatives and seven spouses of the relatives (Fig. 1, Table 1). The data in the affected relatives in the nuclear family differed from those in the other relatives with A p o A 1 Baltimore. The proband (IV-25) and both his carrier brother (III-14) and sister (IV-26) had low H D L cholesterol levels, or low A p o A 1 levels, on one or m o r e occasions. This was not true for the other six relatives with the mutation who had normal levels of H D L cholesterol and A p o A 1 (Table 1). The three affected persons in the nuclear family also had H D L cholesterol and A p o A 1 levels that fluctuated, an observation that was confirmed when a 3-year follow-up sample was obtained. Such fluctuation was not true of the other relatives with the mutation or of the unaffected brother (IV-23) of the proband.
Fig. 5. Two-dimensional gel electrophoretograms of HDL ApoA1 isoforms from a normal subject (a) and a patient with ApoA1 Baltimore (b). Only the portion of the gel containing the ApoA1 isoforms is presented. The ApoA-I+2 isoform is proapoA1, and ApoA1 isoforms 0 to - 2 are mature ApoA1 isoforms. The ApoA1 Baltimore patient has approximately equal quantities of the normal (ApoAlo) and mutant (ApoA1) isoforms
The plasma cholesterol, triglyceride, and other lipoprotein values in the three affected persons in the nuclear family were normal, suggesting a hypoalphalipoproteinemia lipoprotein pattern. While only three of the blood relatives of the proband (including the mother) had a high H D L cholesterol value ( > the 95th percentile, age and sex specific; Lipid Research Clinics Population Studies D a t a Book 1980), seven other relatives of the index case had borderline high H D L cholesterol values, between the 75th and 95th percentiles. While hyperalphalipoproteinemia was not a dominant phenotype in this family, the H D L cholesterol values were noticeably shifted toward higher values. The expression of the A P O A 1 variant in the heterozygous state may have been affected by the expression of another A P O A 1 allele or other genes. Neither the proband nor any of his relatives had hypertriglyceridemia with one exception (III-6, type I V lipoprotein pattern), and only one had a high L D L cholesterol level (V-6, type IIa lipoprotein pattern). In regard to L D L B, eight of the relatives of the proband had a plasma L D L B level above 134 mg/dl, the currently used upper limit of normal in our laboratory. All but one of these relatives also had a low ratio of L D L cholesterol to L D L B (_< 1.2) suggesting that the phenotype of hyper-
443 Table 1. Plasma levels of lipids, lipoprotein cholesterol, and apolipoproteins in proband and relatives. ND, Not determined Position in pedigree
Sex
Age
Total cholesterol
Total triglyceride
VLDL cholesterol
LDL cholesterol
LDLB
HDL cholesterol
HDL ApoA1
LDLB/ApoA1 ratio
Proband IV-25
M
35 36
167 196 204
67 107 102
20 43 6
109 113 149
104 105 149
46 (22) 49
(90) a 124 115
1.16 b 0.85 1.30
22 22 23 23 23
137 131 124 c 131 c 147 c
25
142
63 82 94 98 88 68
ND ND 15 ND 24 20
82 66 80 ND 91 83
82 71 ND ND ND 84
42 49 (29) (29) (32) 39
131 (99) ND ND ND (117)
0.63 0.72 ND ND ND 0.72
38
Rela~ves with ApoA-1 Baltimore IV-26
F
III-14
M
65 66 68
208 185 211
107 58 100
34 ND 19
150 146 138
149 100 160
(24) (27) 39
(101) ND 117
1.48 ND 1.37
III-10
F
68 69
213 287
54 76
29 ND
137 220
123 139
50 52
128 142
0.96 0.98
III-12
F
69 70
220 232
80 113
5 40
180 140
139 137
55 55
155 132
0.90 1.04
Ill-4
F
62 63
221 197
(54) 76
20 ND
139 104
132 123
62 78
154 139
0.86 0.88
IV-1
F
44
190
91
3
137
125
50
165
0.76
IV-2
F
39
142
41
1
86
87
55
137
0.64
IV-22
F
46
151
(39)
4
98
87
49
132
0.66
208
ND
115
128
49
113
1.13
Rela~ves without ApoA-1 Bal~more III-1
M
60
206
11I-5
F
66
228
181
ND
131
14__99
61
(94)
1.59
III-6
M
64
187
297
ND
85
118
43
108
1.09
III-8
M
62
223
69
ND
139
118
70
156
0.76
III-9
M
73
229
182
ND
142
147
51
120
1.23
III-13
M
67
214
(50)
22
130
103
62
189
0.54
II1-15
F
61
237
50
ND
107
80
104
273
0.29
III-16
F
48
236
125
ND
137
153
74
228
0.67
III-17
F
84
232
107
ND
133
112
78
152
0.74
IV-9
F
32
171
56
ND
104
101
56
120
0.84
IV-10
F
24
196
109
ND
120
106
54
139
0.76
IV-11
F
30
168
48
ND
78
76
80
181
0.42
50
ND
114
103
50
113
0.91
IV-14
M
29
174
IV-15
F
31
167
70
ND
95
94
58
134
0.70
ND
136
168
42
120
1.40
116 103
90 106
59 57
123 167
0.73 0.63
IV-20
M
41
201
133
IV-23
M
40 43
192 180
86 123
ND 20
IV-27
F
25
188
63
ND
99
90
76
139
0.65
ND
137
133
81
174
0.76
IV-28
F
21
235
73
IV-29
F
64
201
85
ND
102
127
82
171
0.74
ND
129
129
82
185
0.70
IV-30
F
54
220
57
V-1
M
21
197
86
ND
131
103
49
108
0.95
V-5
M
12
155
98
ND
97
82
38
ND
ND
214
51
ND
156
110
48
136
0.81
V-6
F
8
444 Table 1 (continued) Position in pedigree
Sex
Age
Total cholesterol
Total triglyceride
VLDL cholesterol
LDL cholesterol
LDLB
F F M M M F F
62 60 62 62 37 37 39
306 229 206 220 204 217 217
136 143 115 120 91 108 (33)
ND ND 29 ND ND ND ND
220 116 141 137 141 139 133
201 133 127 149 123 149 91
HDL cholesterol
HDL LDLB/ApoA1 ApoA1 ratio
SpotIscs III-2 III-7 III-11 IV-8 IV-12 IV-19 IV-24
59 84 36 59 45 56 77
186 186 109 136 111 161 ND
1.08 0.72 1.17 1.10 1.11 0.93 ND
a Lipid and lipoprotein values in brackets are < 5th percentile, age and sex specific (Lipid Research Clinics Population Studies Data Book 1980). Plasma ApoA1 values < 5th percentile (< 105 mg/dl for males and < 119 mg/dl for females) are also in brackets b Lipid and lipoprotein values that are underlined are > 95th percentile, age and sex specific (Lipid Research Clinics Population Studies Data Book 1980). Plasma LDLB values that are > 134 mg/dl, the 95th percentile for both control men and women, are also underlined. Ratios of LDLB/ApoA1 >95th percentile (1.04 for males, and 0.88 for females, respectfully) are also underlined c Determined at the Molecular Disease Branch, National Heart, Lung and Blood Institute
apo B (Kwiterovich 1988) was prevalent in this family. We therefore examined the ratio of L D L B / A p o A 1 in this kindred to see if a high ratio might provide a better chemical marker. O f the nine persons with the A p o A 1 variant, four had an elevated L D L B / A p o A 1 ratio. Five of the twenty-three relatives of the p r o b a n d without the variant had an elevated ratio. Thus, we were unable to detect any lipoprotein pattern that was consistently associated with the A p o A 1 Baltimore variant. The finding of the mutant A p o A 1 Baltimore in the first cousin of the p r o b a n d ' s father indicated that this mutant was being transmitted through the proband's paternal grandfather's (individual II-10) side of the family. This finding further indicates that the paternal grandfather of the p r o b a n d carried the mutant allele. This is of interest since the paternal grandfather (II-10) developed angina pectoris early in his forties, and died at the age of 49 years f r o m myocardial infarction. The father of the p r o b a n d (III-14) had a myocardial infarction at the age of 35 years. The p r o b a n d himself underwent elective diagnostic coronary arteriography because of atypical angina pectoris, but did not have significant coronary atherosclerosis ( > 50% blockage). The other four family m e m b e r s with the A p o A 1 variant did not have any clinical evidence of coronary artery disease.
Discussion A new apolipoprotein A1 variant, n a m e d A p o A 1 Baltim o r e , has been detected in a family in which the proband had low, or borderline low, H D L cholesterol and A p o A 1 levels, indicative of an hypoalphalipoproteinemia lipoprotein pattern. The mutation in codon 34 of the third exon of the A P O A 1 gene was a G-to-T-substitution that resulted in an Arg-to-Leu amino acid substitution at the tenth residue of mature A p o A 1 . Interestingly, the mutation changed a C G dinucleotide to CT and therefore was an exception to the CG--+TG mutation rule, in which methylation-deamination of the C in
the C p G dinucleotide results in a C- to T-substitution (Youssoufian et al. 1986). Classical linkage analysis between the phenotype of hypoalphalipoproteinemia or coronary artery disease and A P O A 1 Baltimore does not result in a tight linkage between the phenotype and the genetic marker. Explanations for the lack of linkage include: (1) the A P O A 1 Baltimore gene has no relationship with the phenotype of hypoalphalipoproteinemia. (2) The A P O A 1 Baltim o r e gene is related to hypoalphalipoproteinemia but there are other gene loci that modify the phenotype. It is of note that in the nuclear family of the proband there is linkage between the presence of the A P O A 1 Baltimore gene and hypoalphalipoproteinemia. Studies on the metabolic fate of A p o A 1 Baltimore polypeptide are currently being performed. Preliminary results from such metabolic studies in the sister of the proband indicated increased catabolism of A p o A 1 (Schaefer et al. 1988). However, both the A p o A 1 Baltim o r e and the wild-type A p o A 1 were catabolized at an increased rate in the proband's affected sister, suggesting that the A p o A 1 variant is associated with an abnormality that facilitates the catabolism of apolipoprotein A1. One candidate is hyperapo B, a condition in which the elevated small, dense, L D L particles are often accompanied by low A p o A 1 and H D L cholesterol levels (Kwiterovich 1988; Kwiterovich and Sniderman 1983). Further studies are underway to determine what factors may influence the metabolism of A p o A 1 and H D L in affected family members. A p o A 1 Baltimore is a rare variant. F r o m more than 400 unrelated chromosomes studied in the Johns Hopkins University Coronary Artery Disease Study, and the 196 chromosomes from unrelated Mediterranean and American black individuals studied for haplotype analyses (Antonarakis et al. 1988), only one chromosome (that of the proband) carried the mutant chromosome A p o A 1 Baltimore gene. While low levels of A p o A 1 and H D L cholesterol are prevalent in patients with coronary artery disease, they are often accompanied by elevated
445 levels o f t r i g l y c e r i d e , o r L D L c h o l e s t e r o l , o r L D L B p r o tein ( K w i t e r o v i c h a n d S n i d e r m a n 1983). It is of i n t e r e s t t h a t t h e p r o b a n d (IV-25, Fig. 1) is o n e o f t h e few i n d e x cases w h o h a d h y p o a l p h a l i p o p r o t e i n e m i a at b a s e l i n e exa m i n a t i o n in t h e J o h n s H o p k i n s U n i v e r s i t y C o r o n a r y Artery Disease Study. Cosegregation between APOA1 Baltimore and hypoalphalipoproteinemia could not be demonstrated. Nevertheless, t h e v a r i a n t p r o v i d e s an o p p o r t u n i t y to s t u d y t h e in vivo a n d in vitro m e t a b o l i s m o f an A p o A 1 m o l e c u l e t h a t has lost a p o s i t i v e l y c h a r g e d r e s i d u e , a r g i n i n e , at p o s i t i o n 10 in t h e m a t u r e p o l y p e p t i d e . T h e m u t a t i o n m a y also p e r m i t t h e i d e n t i f i c a t i o n o f p a t i e n t s w h o h a v e an a s s o c i a t e d a b n o r m a l i t y t h a t a d v e r s e l y affects l i p o p r o tein m e t a b o l i s m a n d i n c r e a s e s risk for p r e m a t u r e c o r o nary artery disease.
Acknowledgements. We thank the members of the family described in this paper for their cooperation during the study. We also thank Dr. B. Brewer and Richard Gregg for the two-dimensional electrophoresis of HDL-ApoA1, J. G. Lewis for expert technical assistance, J. Strayer for the art work, and P. Divel for typing this manuscript. The study was performed by grants from the National Institutes of Health HL 31497 and HD 19591.
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