Vol. 178, No. 2, 1991 July 31, 1991

BIOCHEMICAL

LECITHIN-CHOLESTEROL WITH A MISSENSE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 460-466

ACYLTRANSFERASE (LCAT) DEFICIENCY MUTATION IN EXON 6 OF THE LCAT GENE

Eiichi Maeda*,Yoshiko Naka, TakashiMatozaki, Maki Sakuma YasuoAkanuma$Gen Yoshino, and Masato Kasuga SecondDepartmentof Internal Medicine, Kobe University School of Medicine 7-5, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650 Japan nSakumaHospital, Kita- Nishi-2, Kucchan-cho,Abuta, Hokkaido 044Japan §TheInstitute for DiabetesCare andResearch,Asahi Life Foundation l-6-1 Marunouchi, Chiyoda-ku, Tokyo 100Japan Received

June

5,

1991

The plasma enzyme, human lecithin-cholesterol acyltransferase(LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation. e 1991Academic Press,Inc.

Familial lecithin-cholesterol acyltransferase(LCAT, E.C.2.3.1.43) deficiency was first reported by Norum and Gjone in 1967 (1,2). It is a rare autosomal recessive condition clinically characterized by cornea1 opacity, anemia and frequently proteinuria; and by decreasedlevels of plasmacholesterol esters and HDL-cholesterol. LCAT, a glycoprotein of apparent Mr-63,000(3), catalyzes the transfer of acyl groups from the C-2 position of lecithin to the 3-hydroxy group of cholesterol to form the majority of cholesterol ester in plasma(4). LCAT is synthesized by the liver and is secreted into plasma where it mediatesa key step in the reverse cholesterol transport system which is the pathway

* To whom correspondenceshouldbe addressed. 0006-291X/91 Copyright Ail rights

$1.50

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

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to transfer excess cholesterol in peripheral tissues to the liver (5). Free cholesterol the cell membrane is uptaken by high density lipoprotein (HDL) esterified by LCAT Once cholesterol

is esterified,

on

particle and whereby

it moves to the core of the HDL

particle or is transfered to other lipoprotein fractions, thereby maintaining a normal form of HDL-particle

and a concentration gradient between cell membrane and plasma.

Both the cDNA

sequence (6) and the genomic structure (7) of human LCAT

have

been elucidated. The gene contains 6 exons spanning 4.2Kb and encodes a protein of 440 amino acids which becomes a mature protein of 416 residues after cleavage of a signal peptide. A mutation in a LCAT deficient gene was first reported by Taramelli et al. (8). They sequenced one of the clones from a LCAT deficient patient. According allele-specific oligonucleotide hybridization,

that was considered as a heterozygous

for the position 1447 (C to G; Arg t47->Trp) work

to case

and another undetected mutation.

In this

enzymatic amplification and direct sequencing of all exons encoding LCAT

led to

the identification of homozygous

missense mutation at amino acid residue 293 of the

mature enzyme.

MATERIALS

AND

METHODS

Patient The Japanese 56-year-old male patient investigated in this report was diagnosed as a familial LCAT deficiency homozygote on the basis of the clinical and biochemical features characteristic of this disease (9). He had suffered from cornea1 opacity since childhood and from normocytic normochromic anemia. His HDL-cholesterol level was extremely reduced to 8mg/dl and only 34% of the plasma total cholesterol was esterified. The plasma LCAT activity was 12% of normal controls by Glomset-Wright’s method (10) and was 9% by NagasakiAkanuma’s method (11). His parents were first cousins, and his sister was also lacked plasma LCAT activity. He had a two children who were in good health and had no cornea1 opacity or anemia. His sister and children were available for DNA analysis.

Enzvmatic amvlification of aenomic DNA Genomic DNA was extracted from white blood cells as described (12). To amplify genomic DNA, a polymerase chain reaction (PCR) (13) was performed in a total volume of 1OOyl containing 1Pg of genomic DNA digested with HindIII, lOOpmo1 of each primer (sense and antisense), 50mM KCl, 1OmM TrisaHCl pH8.3*25”C, 1SmM MgC12, O.Ol%(w/v) gelatin, 0.2mM each deoxyribonucleotide triphosphate and 2.5 units of Taq DNA polymerase (Cetus/Perkin-Elmer). PCR reaction was carried out for 30 cycles of denaturation(93’C,dOsec), anealing(55-60°C,60sec) and polymerization (73”C,9Osec) using a thermal cycler (Asteck). The location of each segment amplified by first PCR is indicated in Fig 1. In the subsequent asymmetrical PCR (second PCR) (14) to generate single-stranded DNA for direct sequencing, 0.5t.t.l of first PCR reaction mixture (containing S-long of synthesized DNA) was used as the template and 100 pmol of only one of the primers (either the sense and the antisense primer) was included in a total volume of 100~1. The second PCR was carried out for 25 cycles (conditions for each cycle were identical to that for first PCR).

SeauencinP of amolified sir&e-stranded DNA

The product of the second PCR was extracted with an equal volume of choloroform and diluted to 2ml with distilled water. Single-stranded DNA was separated from the oligonucleotides and deoxyribonucleotide triphosphates by filtration through a Centricon 100

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(Amicon). The retentate was dried in a Speed Vat concentrator (Savant) and stored at -20°C. DNA sequencing primers (Fig.1) that are different from the oligonucletides used as primer in the PCR was synthesized and radiolabeled at the 5’ terminus with [y-s*P]ATP as described (15). Single-stranded DNA was resuspended in 5~1 of distilled water immediately before use in the sequencing and mixed with 3~1 of s*P-labeled primer(=25pmol) and 21.~1of 5x polymerase buffer (250mM TlisHCl pH8.3, 40mM MgC12, 150mM KCl, 50mM dithiothreitol)(l6). After anealing at 95°C for lOmin, the mixture was dispensed into each of four tubes to which 21.~1of the appropriate DNA sequencing/termination mix [containing all four deoxyribonucleotide triphosphates (each at a concentration of SOpM), the appropriate dideoxyribonucleotide triphosphate (8pM) and 1.3units of modified T7 DNA polymerase (Sequenase, United States Biochemical)] was added. After a 40-min incubation at 50°C the reaction was terminated by adding 3~1 of stop solution [95%(v/v) f ormamide, 20mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyan01 FF]. After heating to 95°C for 3min, the reaction mixtures were analyzed by electrophoresis through a 6% polyacrylamide/7M urea gel and autoradiography. Ndel digestion studv DNAs from family members were amplified by PCR using two primers which were synthesized to encompass the transition in exon 6. PCR products were partially purified by phenol/chloroform extraction and concentrated by ammonium acetate/ethanol precipitation. The fragments (1 pg) were digested with 24units of restriction enzyme NdeI (Takara Syuzo) at 37°C for 2 hour in a total volume of 50~1 containing 50mM Tris*HCl pH7.5, 1OOmM NaCl, 1OmM MgCl2, ImM dithiothreitol. Digested DNAs were analyzed by electrophoresis through a 1.8% agarose gel. RESULTS Southern blotting analysis, using genomic DNA was digested with LCAT

cDNA,

three restriction

showed

obtained from the patient which

enzymes (TaqI, PstI and BamHI)

neither major insertions

therefore amplified five segments (shown

and the normal

nor deletions (data not shown).

in Fig 1) of the genomic DNA

We

of the patient

by the polymerase chain reaction (PCR). Subsequently to the first PCR, single-stranded DNA was generated by the asymmetric PCR (second PCR) (10) using only one of the primers that are used in the first sequencing. Analysis

PCR

and was

of all six exons and exon-intron

used as a template junctions

revealed only a single

G to A transition at position 4660 of the nucleotide sequence (numbering McLean et al. (7)) of the gene. This transition 293 in exon 6: a methionine codon (ATG) 1000

II

I Exon

II

1 1 t

5

De.

I

I

I

6 1 t1

It1 t

direct

I

II II

1 t

codon

5000

mm

1

into an isoleucine

I

34

m 1

was converted

IIll

2

according to

causes a missense mutation at codon

2000

IIll

for direct

t

t

1 t

1

1. Schematic diagram of the LCAT gene and strategy for amplification and sequencing of essential regions of the gene. Five segments indicatedby black

barwereamplifiedby the first andsecondPCR.Arrows indicatethe locationfor thesequncing primers:upword;for the antisense-strand template, downword; for the sense-strand template.

Upper scale indicates nucleotide number according to McLean et al. (7). 462

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A. Normal G

A

T

RESEARCH

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B. Mutant C

GATC

E~P. 2, Partial nucleotide sequence of the sense strand of exon 6 of the LCAT gene from normal subject (A) and from the patient (B). Direct sequencing of singlestranded DNA generated by the PCR was performed according to the dideoxyribonucleotide chain termination method. Genomic DNA from normal subject had the same sequence as described(7). 4660 Genomic DNA from the patient affected by LCAT deficiencyhada transitionof nucleotide (G to A), leading to the substitution of methionine for isoleucine at residue 293 of the mature enzyme.

(ATA). The patient was homozygous for this mutation as judged by the fact that only single band corresponding to A could be detected in the sequence ladder (Fig 2). In addition, as this mutation creates a new hexanucleotide recognition site for NdeI located at nucleotide 4657-4662 of the gene (from CATGTG

to CATATG),

NdeI digestion

study of genomic DNAs from the patient and relatives was performed. As shown in Fig 3, this patient and his sister were homozygous and his children were heterozygous for this mutation. The plasma LCAT activities assayed by Nagasaki-Akanuma’smethod (11) were extremely reduced(less than 10% of nor&l, controls) in homozygous subjects and were was

about

found

50-60%

of normal

in HDL-cholesterol

raises the possibility

controls

level

that this mutation

in heterozygous

in heterozygous accounts

subjects.

and homozygous

for the reduced

LCAT

Similar

change

subjects. activity

This

found

in

this family. DISCUSSION Many reports have been available on the physiological role of LCAT through detailed research on the patients affected by familial LCAT deficiency (1,2,4).

Also,

several number of knowledge about functional regions and structural model of LCAT were derived from amino acid sequenceof this enzyme(7,17). However, the molecular basis for LCAT deficiency has not been fully investigated. In this study, an enzymatic 463

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Fir. 3. Pedigree chart of the proband and four relatives studied (A) and the fhgment length analysis in restriction enzyme NdeI digestion study (B). Filled, half-filled, and open symbols indicate, respectively, LCAT deficiency homozygote, LCAT deficiency heterozygote, and normal subject determined according to the clinical findings. LCAT activity was assayed by Nagasaki-Akanuma’s method(l1) ( normal limits: male 87.WO.2, female 74.4+10.6 nmol/ml/hr/37”C). 437bp length DNA fragments were generated from the patient and four relatives by the PCR using two primers [sense primer: nucleotide 4377-4396, antisense primer: nucleotide 4793-48131. These fragments were digested with NdeI and were analyzed by electrophoresis. Each lane number corresponds to that indicated at pedigree chart. M: 4x174 DNA digested with HincIl as a size marker. The mutation, G4660 to A, creates a new recognition site for NdeI so that amplified DNA derived from mutated allele is cleaved into two fragments (283 and 154bp) upon digestion with NdeI. Thus subject 1 and 5 were identified molecular genetically to be homozygote, subject 2 and 3 to be he&zygote for this mutation and subject 4 to be wild type according to the formation of fragment length.

amplification

and direct sequencing

of the genomic

DNA from a LCAT deficient patient

was performed in order to elucidate the molecular basis for LCAT deficiency. Analysis of the entire coding regions and exon-intron boundaries revealed that all nucleotide sequencewas identical to published sequence(7) except one nucleotide at position 4660 in exon 6. This G to A transition causes a substitution of methionine residue for isoleucine residue at 293 of mature enzyme. In addition, this mutation creates a new recognition site for NdeI. NdeI digestion study demonstrated the relationship between the number of the mutated allele and the reduction of plasma LCAT activity. Thus, this mutation can account for the reduceedLCAT activity found in this patient. This

single

point

mutation

we

found

LCAT gene. The active site of LCAT

in this

study

was within

exon

6 of

the

is identified as Ser181(17) or Serzr6 (18)

encoded within exon 5 according to selective chemical modification of amino acid residuesor to its homology with other serine-type esterases.Cys3l (exon 2) and Cysla (exon 5) are reported to be adjacent to the active site serine and also to have important role in this enzyme activity since they are likely to act an acyl acceptor from active site serine(19,20). Although exon 6 encodes nearly half of the protein, any information concerning functional characteristicsof exon 6 is not available. Therefore the mechanism 464

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of the enzymatic defect caused by this mutation is unclear. But this mutation warrants further study on unidentified functional regions and a structural The patients affected by LCAT deficiency cholesterol

level and by abnormal structure

are characterized

and composition

reduced fractional LCAT rate and decreased HDL-cholesterol were

found in patients with

coronary

responsible for LCAT deficiency

model of this enzyme.

of HDL

HDL-

particles (1) A

per plasma cholesterol ratio

heart disease (21). Thus,

may provide new DNA

by suppressed

search

for

mutations

diagnostic probes that could

be employed for patients at high risk of coronary heart disease. We reported here a mutation in the LCAT

gene that is different

by Taramelli et al.@) who found one point mutation Their patient was clinically

diagnosed

as a LCAT

mutation was identified to be heterozygous

at the fourth exon of this gene. deficiency

for position

another undetected mutation also responsible for patient described here is, to our knowledge,

from the report

homozygote,

but the

1447. Thus, there should be

this enzyme defect. Therefore,

the first

case with

LCAT

the

deficiency

molecular genetically defined as homozygous for a mutated allele. Our present findings can be a new information contributing specificity

to better understanding of molecular basis of the

and mode of action of LCAT. ACKNOWLEDGMENTS

We are indebted to

Drs. T. Kadowaki,

Department

of Internal Medicine

of University

performing

DNA

work

analysis. This

was

Scientific Research from the Japanese Ministry 63570395 and 03671153).

M. Odawara of Tokyo supported

and

H. Sakura,

for their valuable in part

by

Third help in

Grant-in-Aid

for

of Education, Science and Culture (No.

REFFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11)

Norum, K.R., and Gjone, E. (1967) Stand. J. Clin. Lab. Invest. 20, 231-243. Gjone, E., and Norum, K.R. (1968) Acta. Med. Stand. 183, 107-112. Doi, Y., and Nishida, T. (1983) J. Biol. Chem. 258, 5840-5846. Glomset, J.A. (1968) J. Lipid. Res. 9,155-170. Fielding, L.J., and Fielding, P.E. (1982) Med. Clin. North. Am. 66,363-373. McLean, J., Fielding, C., Drayna, D., Dieplinger, H., Baer, B., Kohr, W., Henzel, W., and Lawn, R. (1986) Proc. Natl. Acad. Sci. USA 83,2335-2339. McLean, J., Wion, K., Drayna, D., Fielding, C., and Lawn, R. (1986) Nucleic Acids Res. 14,9397-9406. Taramelli, R., Pontoglio, M., Candiani, G., Ottolenghi, S., Dieplinger, H, Catapano, A., Albers, J., Vergani, C., and McLean, J. (1990) Hum. Genet. 85,195-199. Sakuma, M., Akanuma, Y., Kodama, N., Yamada, N., Murata, S.,Murase, T, Itakura, H., and Kosaka, K. (1982) Acta. Med. Stand. 212,225-232. Glomset, J.A., and Wright, J.L. (1964) Biochim. Biophys. Acta,89,266-276. Nagasaki, T., and Akanuma, Y., (1977) Clin. Chim. Acta 75,371-375

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12) Maniatis, T, Fritsch E-F., and Sambrook, J. (1989) Molecular Cloning: a laboratory manual 2nd edition. pp. 9.17-9.23. Cold Spring Harbor Labratory Press, NY. 13) Saiki, R.K., Gelfand, D.H., Stoffel, S., Sharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239,487-491. 14) Gyllensten, U.B., and Erlich, H.A. (1988) Proc. Natl. Acad. Sci. USA,85,7652-7656. 15) Kadowaki, T, Kadowaki, H., and Taylor, S.I. (1990) Proc. Natl. Acad. Sci. USA 87,658-662. 16) Gibbs, R.A., Nguyen, P-N., McBride, L.J., Koepf, SM., and Caskey, CT. (1989) Proc. Natl. Acad. Sci. USA 86,1919-1923. 17) Yang, C.-Y., Manoogian, D., Pao, Q., Lee, F-S., Knapp, R.D., Gotto, Jr., A.M. and Pownall, H.J. (1987) J...Biol. Chem. 262,3086-3091. 18) Park, Y.B., Yiiksel, K.U., Gracy, R.W., and Lacko, A.G. (1987) Biochem. Biophys. Res. Comm. 143,360-363. 19) Jauhiainen, M., and Dolphin, P (1986) J. Biol. Chem. 261,7032-7043. 20) Jauhiainen, M., Ridgway, N-D., and Dolphin, F! (1987) Biochim. Biophys. Acta 918,175-188. 211 Wallentin, L., and Moberg, B. (1982) Atherosclerosis 41,155-165.

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