Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2793-2797, April 1991 Medical Sciences

Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene (high density lipoprotein cholesterol/coronary heart disease/skin xanthomas/apolipoprotein A-I polymorphism)

TOMOYUKI MATSUNAGA*, YOSHIKAZU HIASAt, HISAKO YANAGI*, TOSHIHIRO MAEDAt, NAOKO HATrORI*, KIMIKO YAMAKAWA*, YASUKO YAMANOUCHI*, ISAO TANAKAf, TAKASHI OBARAt, AND HIDEO HAMAGUCHI*§ *Department of Human Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305, Japan; tDepartment of Cardiology, Komatsushima Red Cross Hospital, Komatsushima 773, Japan; and tTsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba 305, Japan

Communicated by Alexander G. Bearn, December 13, 1990

The molecular genetic defect of a female ABSTRACT patient with apolipoprotein A-I (apoA-I) deficiency and premature atherosclerosis was examied. Her parents were first cousins. Her plasma density fraction from 1.063 to 1.21 g/ml contained no apoA-I on SDS/PAGE and no measurable high density lipoprotein cholesterol. Southern blot hybridization showed no gross abnormality to be present in the patient's apoA-I gene and homozygosity for a haplotype of restriction fragment length polymorphisms in the apoA-I gene region. Sequencing after amplification by PCR revealed a codon 84 nonsense mutation (CAG -* TAG, Gin -- stop) of exon 4 and a codon 37 missense mutation (GCC -- ACC, Ala -- Thr) of exon 3 in the patient's apoA-I gene. The data from dot-blot hybridization with allele-specific oligonucleotide probes indicated that she was homozygous for the apoA-I gene with regard to the two mutations. The codon 37 missense mutation was also detected in the apoA-I gene of 6 out of 60 controls, who all had normal levels of apoA-I and high density lipoprotein cholesterol, suggesting that the missense mutation is polymorphic and not associated with apoA-I deficiency. These findings indicate that homozygosity for the apoA-I gene with codon 84 nonsense mutation causes the deficiency of apoA-I and of high density lipoprotein cholesterol in the patient.

tions in the apoA-I gene have been described in review articles, although no concrete data were supplied (12, 13). One of the patients with apoA-I deficiency described above is a Japanese female, an offspring from a first-cousin marriage. She was initially reported as having apoA-I and apoCIII deficiency (9). A later reexamination of the patient's plasma apolipoproteins, however, revealed that only apoA-I was undetectable, apoC-III being present, as shown in this paper. To define the molecular genetic defect of the Japanese patient, the apoA-I gene has been analyzed by Southern blot hybridization, sequencing after amplification using PCR, and dot-blotting using allele-specific oligonucleotide probes. In this report we present an apoA-I deficiency due to a nonsense mutation of the apoA-I gene.

MATERIALS AND METHODS

Human apolipoprotein A-I (apoA-I) is a single polypeptide chain composed of 243 amino acids (1) that plays a central role in high density lipoprotein (HDL) metabolism as the major protein of HDL and as a cofactor for lecithincholesterol acyltransferase (2). The apoA-I gene is adjacent to the genes encoding apolipoprotein C-III (apoC-Il) and apolipoprotein A-IV on the long arm of chromosome 11 (3). The apoA-I gene has been localized to the chromosome region 11q23 (4). Decreased plasma apoA-I has been found a risk factor for coronary heart disease by epidemiologic studies (5). Four pedigrees of patients with apoA-I deficiency and very low HDL levels have been described (6-9). The patients all suffered from premature coronary heart disease, supporting the concept of a role for apoA-I in the pathogenesis of atherosclerosis. Gross abnormalities extending over the apoA-I gene and the adjacent gene(s) have been detected as the molecular genetic defect in two of the pedigrees: an inversion of "'.5-kilobase (kb) pairs of DNA containing portions of the apoA-I and apoC-III genes resulted in familial apoA-I and C-III deficiency in one family (10); and the complete deletion of the apolipoprotein A-I, C-Ill, and A-IV genes caused familial apolipoprotein A-I, C-III, and A-IV deficiency in the other family (11). In addition, pedigrees of two patients with apoA-I deficiency due to frameshift muta-

Patient. A detailed clinical finding of the patient, a 60-yrold Japanese woman, has been described (9). Briefly, she was noted to have planar xanthomas at age 18 years; her symptom of angina pectoris started at age 52 years, and she was admitted to the hospital at age 55 years. Cardiac catheterization demonstrated 90%o stenosis of the proximal left descending artery, 75-90%o stenosis of the circumflex branch, and complete occlusion of the proximal right artery (Fig. 1). She had symmetric, yellow-orange, planar xanthomas on her neck, elbow, and the posterior of her thigh. HDL cholesterol level was markedly reduced to 3 mg/dl, as measured by the heparin-Ca2+ precipitation procedure, and apoA-I was not detected by a single radial immunodiffusion analysis. She received coronary-bypass surgery, and improvement of her symptoms was noted. Recently, however, she has suffered a relapse of coronary heart disease. Her parents were first cousins (Fig. 2). Her father died of a cerebrovascular disease at age 70, and her mother died of an unknown disease at age 64. One of her six uncles is reported to have died of coronary heart disease. She has a 68-year-old and a 64-year-old sister, a 33-year-old and a 29-year-old daughter, and a 24-year-old son, all of whom are apparently healthy. All siblings and children of the patient had decreased levels of HDL cholesterol (21 to =34 mg/dl) (9). Our request for her family blood samples for the present study was declined by the family. Plasma Apolipoprotein and Lipid Analysis. Blood samples were obtained the morning after an overnight fast. Lipoproteins were separated essentially by standard sequential preparative ultracentrifugal techniques (14). All separations were done by using the Beckman TL-100 ultracentrifuge with a 100.3 rotor at 100,000 rpm at 40C. The very low density lipoprotein [density (p) < 1.006 g/ml] was removed after 2.5hr centrifugation. The intermediate density lipoprotein (p =

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Abbreviations: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; apoC-11, apolipoprotein C-III. gTo whom reprint requests should be addressed.

2793

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et

al.

Proc. Natl. Acad. Sci. USA 88

(1991)

B

FIG. 1. Coronary angiograms of the patient. (A) Left coronary artery. The arrow indicates 90%o stenosis of the proximal left descending artery, and the arrowhead shows 75-90% stenosis of the circumflex branch. (B) Right coronary artery. Complete occlusion of the proximal right artery is indicated by an arrow.

1.006-1.019 g/ml) and low density lipoprotein (p = 1.0191.063 g/ml) were each removed after 4-hr centrifugation. HDL (p = 1.063-1.21 g/ml) was obtained after 4-hr centrifugation. To analyze the apoA-I in the HDL samples, 0.1% SDS/12.5% PAGE was performed by the method of Laemmli (15). apoC-Ill was analyzed by isoelectric focusing, as described (16), after delipidation of the very low density lipoprotein samples. SDS/PAGE and isoelectric focusing gels were stained with Coomassie brilliant blue R. The apoA-I in the HDL samples and the apoC-III in the very low density lipoprotein samples were also analyzed by two-dimensional gel electrophoresis as described (16). Two-dimensional electrophoresis gels were stained by the silver stain technique (17). The bands and spots of apoA-I, apoC-III, and apolipoprotein E were identified on SDS/PAGE gels or twodimensional electrophoresis gels by the method of immunoblotting with goat anti-human apolipoprotein A-I, C-III, and E, respectively (Dai-ichi Pure Chemicals, Tokyo) with fluorescein-conjugated rabbit anti-goat immunoglobulin G (Cappel Laboratories). Apolipoprotein bands in isoelectric focusing gels were identified by comparisons with the results of two-dimensional gel electrophoresis and the published data (18). Concentrations of plasma apolipoproteins A-I, A-Il, B, C-Il, C-Ill, and E were measured by the single radial immunodiffusion technique with 1% agarose gel plates each containing an antibody against the respective apolipoprotein (Dai-ichi Pure Chemicals). Concentrations of cholesterol and triacylglycerol (triglyceride) were determined enzymatically (19, 20). As a control, concentrations of plasma apolipopro-

11 III IV v

(a)

Dead

FIG. 2. Pedigree of a kindred with apoA-I deficiency. The patient with apoA-I deficiency is indicated by the arrow.

teins, cholesterol, and triacylglycerol were also measured in healthy Japanese women with a mean age of 60 years. HDL cholesterol levels in controls were measured by the heparin-Ca2+-Ni2+ precipitation procedure (21). Southern Blot Hybridization. Genomic DNA was isolated from the leukocytes in 10 ml of peripheral blood of the patient and of 60 healthy Japanese subjects of ages between 22 and 55 years, essentially according to the method of Kunkel et al. (22). Five-microgram samples of patient and control DNA were digested with the restriction enzymes Taq I, Xmn I, Msp I, Pst I, and Sac I (Boehringer Mannheim). Southern blot hybridization was done as described (23) with a 2.2-kb Pst I fragment containing the entire coding region plus introns of the human apoA-I gene (24) as probe. Oligonucleotide Synthesis. Synthetic oligonucleotides were synthesized by the phosphoramidite method of oligonucleotide synthesis in a DNA synthesizer (Applied Biosystems). PCR, Direct Cloning, and DNA Sequencing. Two genomic DNA fragments of the apoA-I gene were amplified from 1-,ug samples of DNA from the patient and each of the controls by the automated PCR technique (25) for 30 cycles using Taq DNA polymerase (Promega). The sequence and position of the primers are given in Table 1. The PCR reactions were performed with 3-min extension at 72°C, 90-s denaturation at 93°C, and 2-min primer hybridizing at 55°C for primers P1 and P2 and at 45°C for primers P3 and P4. The amplified regions, 723-base-pair (bp) fragment I (bases 467-1189) and 802-bp fragment II (bases 1555-2356), include all exons and splice junctions of the apoA-I gene (26). For cloning and sequencing, an aliquot of each amplified product from the patient and one of the controls was purified from an agarose gel, ligated to pUC 18, and cloned using Escherichia coli DH5a as host (27). Double-stranded DNA sequencing of the cloned apoA-I gene fragments was done by the dideoxynucleotide chaintermination method of Sanger et al. (28). Dot-Blot Hybridization with Allele-Specific Oligonucleotide Probes. Five microliters of amplified DNA was dot-blotted onto nylon membrane. The membrane was prehybridized at 59°C to test for nonsense mutation and at 50°C to test for missense mutation for 1 hr in 5x SSPE (1x SSPE is 0.15 M 55

Table 1. Sequences of oligonucleotides used as primers Position Number Sequence 467-486 P1 5'-TTAGAGACTGCGAGAAGGAG-3' P2 5'-CTCATCAGATATTAGGTGAGGACT-3' 1166-1189 P3 5'-TTGAGAGTGTACTGGAAATGCTA-3' 1555-1577 P4 2335-2356 5'-CCCAAAAGAAAGAAGCTGCTTC-3' Positions are numbered according to Shoulders et al. (26).

Medical Sciences: Matsunaga et aL NaCI/10 mM phosphate, pH 7.4/1 mM EDTA)/5x Denhardt's solution/0.5% SDS/salmon sperm DNA at 0.1 mg/ ml. Each allele-specific oligonucleotide probe, 5'-end labeled with [y-32P]ATP, was then added to the same buffer, and hybridization occurred for 1 hr. The membrane was rinsed twice with 2x SSPE/0.1% SDS and washed once with 5x SSPE/0.1% SDS for 10 min before exposure to x-ray film at -80'C for 4 hr. The oligonucleotide probe specific for the nonsense mutation has the sequence 5'-GGCCTGAGGTAGGAGATGA-3', whereas the normal counterpart has the sequence 5'-GGCCTGAGGCAGGAGATGA-3'. These probes were hybridized at 590C. The temperature for washing was 61'C for the nonsense mutation probe and 630C for the normal counterpart. The probe specific for the missense mutation has the sequence 5'-GAAGGCTCCACCTTGGGAA-3', whereas the normal counterpart has the sequence 5'-GAAGGCTCCGCCTTGGGAA-3'. These probes were hybridized at 50'C. The temperature for washing was 580C for the missense mutation probe and 550C for the normal counterpart.

RESULTS Plasma Apolipoprotein and Lipid Analysis. Table 2 shows the lipid and apolipoprotein concentrations of the plasma in the patient and female controls with a mean age of 60 years. Lipid concentrations oflipoprotein subfractions in the patient are also shown in Table 2. In the patient's plasma, cholesterol, triacylglycerol (triglyceride), apolipoprotein B, and apolipoprotein E concentrations were within the normal range, but levels of apolipoprotein A-II and C-II were decreased. The patient had no HDL cholesterol measurable by ultracentrifugation. apoA-I was not detected in the plasma by a single radial immunodiffusion analysis or in the HDL fraction by SDS/PAGE (Fig. 3A). Contrary to the previous report (9), apoC-III was detected in the patient's serum by a single radial immunodiffusion analysis, although its level was reduced. The presence of apoC-III in the patient was confirmed by isoelectric focusing of the very low density lipoprotein fractions (Fig. 3B). Two-dimensional gel electrophoresis also showed the absence of apoA-I in the HDL fraction Table 2. Lipid and apolipoprotein concentrations in patient and controls Patient Controls Cholesterol 214 ± 33 154 Plasnna 4 VLD; IDL ND LDL 147 ND 51 ± 12 HDL

Triacylglycerol 84 136 ± 67 Plasmia 16 VLD]L IDL ND LDL 58 ND HDL Plasma apolipoprotein ND A-I 138 ± 20 34 ± 4.6 4.8 A-II 89 91 ± 22 B 3.7 ± 1.6 1.6 C-II 2.8 8.4 ± 3.4 C-III 5.1 5.3 ± 1.2 E Values given are means ± SD in mg/dl. Controls were 55 healthy Japanese women with a mean age of 60 years (mean ± SD, 60 ± 5.8). VLDL, very low density lipoprotein fractions; IDL, intermediate density lipoprotein fractions; LDL, low density lipoprotein fractions; and ND, nondetectable.

2795

Proc. Natl. Acad. Sci. USA 88 (1991) Patient

A Control

-_

B C

p

Aib

-E3 -E2

E _o

A

IC111-0

_*-

w

- -C 11 C 111-1

~C1l1-2

FIG. 3. Electrophoretic analysis of lipoprotein fractions of the patient and a control. (A) SDS/PAGE of HDL fractions. Alb, albumin; E, apolipoprotein E; Al, apoA-I (Coomassie staining). (B) Isoelectric focusing of very low density lipoprotein fractions. ApoCIII is shown by CIII-0, CIII-1, and CIII-2. C, control; P, patient; E3 and E2, major bands of apolipoproteins E3 and E2, respectively; CII, apolipoprotein C-II (Coomassie staining).

and the presence of apoC-III in the very low density lipoprotein fraction in the patient (data not shown). Southern Blot Hybridization. The apoA-I gene specific bands of the patient were analyzed with five different enzymes, Taq I, Xmn I, Msp I, Pst I, and Sac I. The apoA-I gene-specific bands were all normal in the patient, indicating no gross abnormality in her apoA-I gene (data not shown). As to restriction fragment length polymorphisms detectable in the apoA-I and apoC-III gene region with the five enzymes (24, 29-31), she was homozygous for the restriction fragment length polymorphism haplotype T2X1M1PlS1 (data not shown). Direct Cloning and DNA Sequencing of the Amplified DNA. Fig. 4 illustrates the genomic organization of the apoA-I gene, the cloned regions, and the positions of the base substitutions detected in the patient's gene. Clones carrying the apoA-I gene fragment I contain exons 1-3 and their splice junctions. Clones carrying fragment II include exon 4 and its splice junctions. The entire insert offour clones ofeach apoA-I gene fragment was selected at random and sequence-analyzed. For the controls, no nucleoside change was evident in fragment I and II when the sequences were compared with the published sequence for human apoA-I gene (26, 32). For the patient, one notable nucleoside substitution was discerned in fragment II of all four clones (Fig. 5): a C -- T transition within the codon (CAG) for amino acid 84 (Gln) that produced a nonsense codon (TAG). In addition, another nucleoside change was detected in fragment I of all four clones: a G -* A transition within the codon (GCC) for amino acid 37 (Ala) that produced a missense codon (ACC) for threonine.

P1

P2

P4

P3

3'

5' 2

Exon 1

3

4

GlySerAla e

LeuArg Glne CTGAG6IGGAGATG

Control sequence

GGCTCMCTTGGGA

Patient sequence

GGCTCCNCCTTGGGA

35 36 37 38 39

Gly Ser Thr Leu Gly

82 83 84 85 86

CTGAGG[lkGGAGATG LeuArg Stop

FIG. 4. Patient and normal sequences in the apoA-I gene. The G A and C T substitutions are boxed. Exons of the apoA-I gene are illustrated by open bars. Positions of primers P1, P2, P3, and P4 are shown. Fragment I was amplified by using primers P1 and P2, and fragment II was amplified by using primers P3 and P4. --

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Medical Sciences: Matsunaga et al. Normal A C G T

Patient A C G T

89 Asp _

Lys

Ser Met Glu 84

Qw-

G AG

Om--

to

do-

GAG

G

m 40w

84

Arg Leu

Gly

-s

GAG G

I,,

_W

FIG. 5.

-

'lwW

mmo

78 Glu

patient and

foAGG

.-

AG Gly GAG Glu Thr GAG

Cholesterol,

ATG

he

AG

Leu

T

A

-8

GAG

Gn

Arg

Table 3. Lipid and apoA-I levels in controls heterozygous for codon 37 missense mutation

~ ~~GC

~

AGC A

Proc. Natl. Acad. Sci. USA 88 (1991)

_m

Glu

AW Thr TAG Gn Glu

78

Autoradiography of sequencing gels of DNA from the a normal subject near the region of the nonsense

mutation. The C

--

T substitution that results in the introduction of

premature termination codon is indicated by the arrowhead and box.

a

Dot-Blot Hybridization with Allele-Specific Oligonucleotide Probes. The amplified DNA of the patient and 60 healthy Japanese was dot-blot-hybridized with allele-specific oligonucleotide probes for the nonsense mutation, missense mutation, and their normal counterparts. Fig. 6 shows the results of the patient and a control. The data confirm the presence of the codon 84 nonsense and codon 37 missense mutations in the patient's apoA-I gene and indicate homozygosity of the gene with regard to the two mutations. The apoA-I gene with the codon 37 missense mutation was observed in six of the 60 controls in a heterozygous state with the normal allele. Levels of plasma HDL cholesterol and apoA-I were normal

(Table 3). DISCUSSION In the present study, we have analyzed the genetic defect that leads to apoA-I deficiency in a Japanese female patient. Because the absence of apoA-I and the presence of apoC-III in the patient were demonstrated by single radial immunodiffusion analyses and electrophoretic methods, the patient was regarded as having apoA-I deficiency. In the previous study (9), the reduced level of apoC-III in the patient was mistaken for absence of apoC-III in the analysis with single radial immunodiffusion. No gross abnormality was seen in her apoA-I gene by Southern blot hybridization. Therefore, the genomic DNA of the patient was analyzed for possible alterations in the nucleotide sequence of the apoA-I gene. PCR amplification of the apoA-I gene of the patient, followed by cloning into a bacterial plasmid and nucleotide sequencing, revealed a nucleoside substitution of deoxyguanosine for deoxyadenosine in exon 3 and another nucleoside substitution of deoxycytidine for thymidine in exon 4. The presence of the two nucleoside substitutions in the patient was confirmed by dot-blot hybridization with allele-specific oligonucleotide probes. The nucleoside substitution of deoxyguanosine for deoxyadenosine in exon 3 results in the substitu-

B

A Patient Normal

probe

Mutant

probe

Normal

Normal probe 0

Variant probe

Patient Normal 0 0

FIG. 6. Dot-blot hybridization of amplified DNA of the patient and a normal subject. (A) Hybridization with allele-specific oligonucleotide probes for the normal and the C T substitution at codon 84 (mutant probe). (B) Hybridization with allele-specific oligonucleotide

probes for the normal and the G

-*

A substitution at codon 37

(variant probe). The patient is homozygous for both mutations.

Subject ST

Sex M M M M

Age 54 HH 51 IT 38 TM 24 KK M 23 iT F 22 M, male; F, female.

mg/dl Serum HDL 208 45.4 261 51.7 188 81.1 153 46.5 153 46.3 147

67.4

Triacyl-

glycerol,

apoA-I,

mg/dl 223

mg/dl

96 65 113 77 54

141 118 156 118 121 143

tion Ala -* Thr at residue 37 of apoA-I. A population study suggests that this amino acid change is polymorphic and not associated with apoA-I deficiency (Table 3). On the other hand, the nucleoside substitution of deoxycytidine for thymidine leads to the introduction of a premature termination codon at a position corresponding to amino acid 84 of the normal 243-amino acid mature apoA-I and thus to the synthesis of a truncated protein. Considering the role of nonsense mutations in single-gene disorders, the codon 84 nonsense mutation of the apoA-I gene is very likely responsible for the apoA-I deficiency in the patient. Moreover, since the data from the dot-blot hybridization with allele-specific oligonucleotide probes demonstrate the presence of the apoA-I gene with the nonsense mutation and the absence of the normal allele in the patient, homozygosity for the apoA-I gene with the codon 84 nonsense mutation can be considered confirmed. This homozygosity was supported by the data on polymorphisms. Dot-blot hybridization with allele-specific oligonucleotide probes indicated that the patient is homozygous for the apoA-I gene with regard to the codon 37 missense mutation. Because 6 of the 60 controls were heterozygous for the missense mutation, the frequency of the gene with the missense mutation is 0.05, and the probability of occurrence of the homozygote for the missense mutation by chance is estimated to be -0.006 in an offspring from a first-cousin marriage. In addition, the patient is homozygous for the restriction fragment length polymorphism haplotype T2X1M1P1S1 in the apoA-I gene region. The homozygous codon 84 nonsense mutation should be considered the cause of the apoA-I deficiency in the present case, the identical mutant apoA-I gene most likely being transmitted to the patient from both parents, who were first cousins. Considering the role of apoA-I in HDL metabolism (2), the unmeasurably low level of HDL cholesterol, the planar xanthomas, and the premature atherosclerosis in the patient are likely to be the consequence of the abnormal apoA-I due to the nonsense mutation of the apoA-I gene. As described in the Introduction, markedly reduced levels of HDL cholesterol and premature atherosclerosis were noted in two sisters with familial apoA-I and C-III deficiency due to an inversion of DNA containing portions of the apoA-I and apoC-III genes and in another patient with familial apolipoprotein A-I, C-IIl, and A-IV deficiency due to complete deletion of the apolipoprotein A-I, C-III, and A-IV genes (7, 8, 10, 11). Planar xanthomas were seen in the two sisters with familial apoA-I and C-Ill deficiency but not in the patient with familial apolipoprotein A-I, C-III, and A-IV deficiency (7, 8). Corneal clouding was noted in all three cases with the rearrangement extending over the apoA-I gene and adjacent gene(s) (7, 8) but was not detected in the present case (9). Hepatosplenomegaly and abnormal tonsils were not observed in any case (7-9). These findings suggest that the clinical feature caused by the single-gene defect of the apoA-I gene includes a marked

Medical Sciences: Matsunaga et A reduction of HDL cholesterol levels and premature atherosclerosis. We thank Dr. S. E. Humphries (The Charing Cross Sunley Research Centre) for providing us with a human apoA-I gene clone and Mr. N. Shirato for preparation of the photographs. This study was supported by Scientific Research Grant 63571084 from the Ministry of Education, Science and Culture of Japan and research grants from the Ministry of Health and Welfare of Japan.

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