Novel mutations in the microsomal triglyceride transfer
protein gene causing abetalipoproteinemia
Ken Ohashi,* Shun Ishibashi,1,* Jun-ichi Osuga,* Ryu-ichi Tozawa,* Kenji Harada,*
Naoya Yahagi,* Futoshi Shionoiri,* Yoko Iizuka,* Yoshiaki Tamura,* Ryozo Nagai,†
D. Roger Illingworth,§ Takanari Gotoda,* and Nobuhiro Yamada**
Department of Metabolic Diseases,* and Department of Cardiology,† Faculty of Medicine, Universityof Tokyo, Tokyo, 113-8655, Japan; Department of Medicine,§ Division of Endocrinology, Diabetes,
and Clinical Nutrition, Oregon Health Sciences University, Portland, OR 97201; and Departmentof Endocrinology, Metabolism, and Atherosclerosis,** Institute of Clinical Medicine,
University of Tsukuba, Tsukuba, Japan 305-8575

Abstract Abetalipoproteinemia (ABL) is an inherited disease characterized by the virtual absence of apolipoprotein
B (apoB)-containing lipoproteins from plasma. Only limited
numbers of families have been screened for mutations in
the microsomal triglyceride transfer protein (MTP) gene.
To clarify the genetic basis of clinical diversity of ABL, mutations of the MTP gene have been screened in 4 unrelated
patients with ABL. Three novel mutations have been identified: a frameshift mutation caused by a single adenine deletion at position 1389 of the cDNA, and a missense mutation,
Asn780Tyr, each in homozygous forms; and a splice site mutation, 2218-2A➝G, in a compound heterozygous form. The
frameshift and splice site mutations are predicted to encode truncated forms of MTP. When transiently expressedin Cos-1 cells, the Asn780Tyr mutant MTP bound protein
disulfide isomerase (PDI) but displayed negligible MTP activity. It is of interest that the patient having the Asn780Tyr
mutation, a 27-year-old male, has none of the manifestations characteristic of classic ABL even though his plasma
apoB and vitamin E were virtually undetectable. These
results indicated that defects of the MTP gene are the proximal cause of ABL.—Ohashi, K., S. Ishibashi, J-i. Osuga, R-i.
Tozawa, K. Harada, N. Yahagi, F. Shionoiri, Y. Iizuka, Y.
Tamura, R. Nagai, D. R. Illingworth, T. Gotoda, and N. Yamada. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia. J. Lipid Res.
2000. 41: 1199–1204.
Supplementary key words microsomal triglyceride transfer protein •abetalipoproteinemia • apolipoprotein B • triglycerides • cholesterol •lipoproteins

Abetalipoproteinemia (ABL) is an autosomal recessive
disorder characterized by the virtual absence of apolipoprotein B (apoB)-containing lipoproteins from plasma(1). Affected individuals develop a wide spectrum ofsymptoms including retinitis pigmentosa, spinocerebellar
degeneration, and a bleeding tendency due to malabsorption of vitamin K. Symptoms observed in some patientswith homozygous ABL are indistinguishable from those withhomozygous hypobetalipoproteinemia (HBL), an autosomal codominant disorder arising from molecular defects in
apoB (2, 3).
Wetterau, Sharp, Shoulders et al. (4 –6) have delineatedthat ABL is caused by molecular abnormalities in microsomal triglyceride transfer protein (MTP). MTP forms a heterodimer with protein disulfide isomerase (PDI), an ubiquitous endoplasmic reticulum (ER) resident protein involved
in disulfide bond formation (7) and functions in the assembly of apoB-containing lipoproteins. Without MTP, asin the case of ABL, apoB is unable to be properly lipidated
and undergoes rapid presecretory degradation (8–11).
On the basis of its homology with vitellogenin (VTG), an
ancient transport and storage lipoprotein found in egglaying vertebrates, the MTP molecule is predicted to consist of three major structural domains: N-terminal b-barrel(residues 22–297), middle a-helical (residues 298–603),
and C-terminal domains (residues 604 –894) (12, 13). The
N-terminal b-barrel domain mediates the interaction with
the N terminus of apoB (13); the middle a-helical domain
mediates the interaction with both PDI and apoB (14, 15);
and the C terminal mediates the lipid-binding and transfer catalytic activity of MTP. The first two domains areglobular in shape and closely related in structure to VTG(13). Amino acids 666–736 of MTP are homologous toamino acids 389–458 of cholesteryl ester transfer protein(CETP) (16).
To date, 14 separate mutations have been described in
the MTP gene and/or cDNA from patients with ABL (5, 6,

Abbreviations: ABL, abetalipoproteinemia; apo, apolipoprotein; CETP,
cholesteryl ester transfer protein; ER, endoplasmic reticulum; HBL, hypobetalipoproteinemia; MTP, microsomal triglyceride transfer protein; PCR,
polymerase chain reaction; PDI, protein disulfide isomerase; RFLP, restriction fragment length polymorphism; VTG, vitellogenin.
1 To whom correspondence should be addressed.

Journal of Lipid Research Volume 41, 2000

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12, 17, 18). Most of these are either major truncations orlarge deletions. Only one encodes a missense mutation,
Arg540His, in a compound heterozygous form (14). At
the present time it remains uncertain whether MTP deficiency is the sole cause of ABL and it is possible that
other defects in the assembly and secretion of apoBcontaining lipoproteins cause expression of the clinicalphenotype of ABL.
In the present study we have investigated a cohort of 2
Japanese and 2 American patients with ABL for defects in
MTP. Mutations of the MTP gene, including 1 missense mutation, were identified in both alleles of all individuals. The
missense mutation is the second to be reported and was located in the middle of the C-terminal domain, the region
that contains a putative lipid-binding and transfer domain.

MATERIALS AND METHODSClinical data
Patient 1: A 32-year-old Japanese female (150 cm, 42 kg) was
identified during routine cholesterol screening as having extremely low cholesterol levels ( Table 1). Her parents were consanguineous; their laboratory data were not available. Her sister
was normolipemic with low plasma apoB levels (Table 1). Except
for notable intolerance for fat-rich meals, her development was
uneventful. Neurological examination revealed absent ankle
and knee jerks and positive Romberg’s sign. But she had normal
nerve conduction velocities. Fine mottling was noted in the retina .
Marked acanthocytosis with a hemoglobin level of 12.8 g/dL wasfound. The duodenal mucosa appeared white endoscopicallyand the cytoplasm of the enterocytes was vacuolated.
Patient 2: A 27-year-old Japanese male (173 cm, 66 kg) withno history of steatorrhea or other medical problems was foundto be extremely hypolipemic during a routine medical examination (Table 1). His parents were consanguineous; their laboratory data were not available. His general health had been excellent. Neither neurological nor ophthalmologic abnormalities
were found. Acanthocytosis and mild fatty liver were detected;
otherwise, his laboratory data were normal.
Patients 3 and 4: Clinical characteristics of patients 3 and 4
were described previously (19); patients 3 and 4 were cases 1 and

2 in the original article, respectively. In brief, patient 3 hadshown intermittent diarrhea and steatorrhea since birth and the
diagnosis of ABL was made at age 15, at which time he presented
with loss of deep tendon reflexes, an ataxic gait, and a positive
Romberg’s sign. Patient 4 was diagnosed at 7 months of age,when he was evaluated for failure to thrive. Both patients havebeen managed by restricting dietary intake of long-chain fats
and supplementation of fat-soluble vitamins A, E, and K. There
was no evidence of consanguinity in either family. Plasma lipid
profiles of these patients and their parents are shown in Table 1.
Informed consent was obtained from the subjects, and theprocedures described in this study were approved by ethics committees of the University of Tokyo (Tokyo, Japan) and the Oregon Health Sciences University (Portland, OR).

PCR amplification and sequencing of the MTP gene
Genomic DNA was extracted from either blood leukocytes(patients 1 and 2) or skin fibroblasts in culture (patients 3 and
4). The promoter, 189 bp of 59 flanking sequences, and all 18
exons and their associated exon –intron junctions of the MTP gene
were amplified by polymerase chain reaction (PCR) according
to Narcisi et al. (12). PCR products were directly sequenced by
the dideoxy chain-termination method on an ABI 373 DNA sequencer (Perkin-Elmer Cetus, Applied Biosystems, Foster City,
CA). Each mutation was verified by sequencing the PCR products from 6 independent amplifications. Compound heterozygosity of patient 4 was established by sequencing the subcloned
PCR fragments.

PCR-restriction fragment length polymorphism
For easy screening of the Asp780Tyr missense mutation, a pairof mismatched primers was designed so that the PCR productfrom the mutant allele specifically contains an SspI restriction
site. The primer sequences were as follows: for ward primer, 59ACACAACTCAAATGGAATTATCTACAGCAG-39; reverse primer,
59-TAAATGTATAATGCATAAACACTTACCAAT-39.

Construction of the mutant MTP cDNA
Site-directed mutagenesis was per formed by recombinant
PCR (20). The template was pTARGET/MTP, which contain acDNA sequence encompassing the entire coding region of MTP
from the plasmid pSV7D/MTP (a gift from C. C. Shoulders and
J. Scott, MRC Molecular Medicine Group of Hammersmith Hospital, London, UK) (10) in the mammalian expression vectorpTARGET (Promega, Madison, WI). The fragment spanning nu-

TABLE 1. Clinical characteristics of patients with abetalipoproteinemia
Family(Country)

Age/Sex

Cholesterol

Triglyceride

HDLcholesterol

ApoB

mg/dL
Family 1, Japan
Proband
SisterFamily 2, Japan
Proband
Family 3, America
ProbandFatherMotherFamily 4, America
ProbandFatherMother

mg/L

32/F
34/F

42
140

0.2
43

36
65

0.9
46

,1
12

27/M

34

2.6

23

0.6

,1

15/M

25
208
315

5
98
96

—
—
—

0
114
154

1–2
—
—

0.6/M

30
227
138

5
210
83

—
—
—

0
107
61

1.1
—
—

Ages of the probands are at the diagnosis. Clinical data of families 3 and 4 are adopted from ref. 19.

1200

Vitamin E

Journal of Lipid Research Volume 41, 2000

cleotides 1818 –2342 was amplified by primer set 1: forward
primer, MPCR1, 59-CCGTTTCTCCAGGAGTGGATC-39; reverse
primer, 16M39, 59-CTATATTCACTCGGGTTTTAGACT-39. The
fragment spanning nucleotides 2318 –2751 was amplified byprimer set 2: forward primer, 16M59, 59-AGTCTAAAACCCGAGTGAAATATAG-39; reverse primer, cDNA11, 59-ACTTAGTCATGCCACATTGT-39. The two PCR products were mixed to form aheteroduplex and a second PCR was performed with the primers
MPCR1 and cDNA11, yielding a fragment spanning nucleotides
1818–2751. The fragment was restricted with PflMI and wascloned back into the partially digested pTARGET/MTP.

Transient expression of recombinant MTPs in Cos-1 cells
Plasmids encoding wild-type or mutant MTP were transfected into Cos-1 cells by electroporation as previously described (12). Approximately 48 h after transfection, the cells
were washed three times in ice-cold phosphate-buffered saline(PBS) and harvested in homogenization buffer (5) (50 mm Tris
[pH 7.4], 50 mm KCl, 5 mm EDTA, leupeptin [5 mg/mL], 2 mm
phenylmethylsulfonyl fluoride). Soluble microsomal content
was isolated as described (4). HepG2 cells were used as a positive control.

Assay of MTP activity
MTP activity was measured according to Wetterau et al. (4)and Leiper et al. (10). Lipid transfer activity is expressed as thepercentage of the total [ 14C]triglyceride transferred from donor
small unilamellar vesicles (SUVs) to acceptor SUVs in a 1-h assay
as a function of the amount of protein.

Immunoblot analyses
Samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel (3 –15%) electrophoresis in a reducing condition. After transfer to a polyvinylidene difluoride (PVDF) membrane, immunoblot analyses were per formed with a kit (ECL;
Amersham, Arlington Heights, IL), using a rabbit anti-human
MTP antibody (a gift from C. C. Shoulders and J. Scott, MRC
Molecular Medicine Group of Hammersmith Hospital) (12).

Immunoprecipitation with anti-PDI antibody
After transfection, the cells were harvested and homogenizedin 15/40 buffer (15 mm Tris [pH 7.4], 40 mm NaCl, 1 mm EDTA,
0.02% NaN3) (4). The homogenates were solubilized with sodium
deoxycholate as described (4), and ultracentrifuged at 105,000 g
for 1 h. The supernatants ( ,1 mL) were mixed with 5 ml of protein G–Sepharose 4 Fast Flow beads (Amersham Pharmacia Biotech, Piscataway, NJ)) to which 2 mg of the anti-bovine PDI antibody (rabbit polyclonal IgG; Upstate Biotechnology, Lake Placid,NY) or 5 ml of nonimmune rabbit serum had been absorbed. The
mixture was incubated at 4 8C for 4 h on a rotary shaker and thebeads were extensively washed with 15/40 buffer. The resulting
immunoprecipitates were further subjected to immunoblot analyses with the anti-MTP antibody as described above.

RESULTS
Four unrelated ABL individuals have been screened formutations in the MTP gene. Three mutations in a homozygous form (patients 1–3) and two mutations in acompound heterozygous form (patient 4) were identified(Fig. 1). Of these, three are novel mutations.
Frameshift mutations
Sequence analysis of the PCR products amplified fromDNA of patient 1 revealed a single adenine deletion inexon 11, within a run of five adenines between nucleotides 1385 and 1389 of the MTP cDNA. This deletioncauses a frameshift after the codon for amino acid 462
and generates a premature termination codon (UGA)
after coding 14 amino acids in the new reading frame. Patient 1 was homozygous for the deletion.
Patient 3 was found to be homozygous for an insertionof a single adenine in exon 4, between positions 419 andFig. 1. Identification of the mutations in the MTP gene from abetalipoproteinemic subjects. All sequences were determined on bothstrands by direct sequencing of the amplified DNA and the regions across a mutation were sequenced on the separately amplified DNAs.
Note that the chromatogram of the 1783C➝T mutation in patient 4 shows the sequence of the antisense strand. del, Deletion of a specified
nucleotide; ins, insertion of a specified base. Mutations are indicated by arrows.

Ohashi et al. Mutations in MTP gene causing abetalipoproteinemia

1201

420 of MTP cDNA. This insertion is located within a runof four adenines. The mutation results in the generationof a new reading frame after amino acid 140 and causes apremature stop codon (UGA) one amino acid into the
new reading frame.
Nonsense mutation
Patient 4 was found to have a C-to-T transition in exon
13, at nucleotide 1783 in one allele of the MTP gene. Thischanges an arginine codon (CGA) to a premature stopcodon (TGA). A truncated translation product of 594
amino acids is predicted.
Splice site mutation
The second mutation in patient 4 was shown to be a single A-to-G substitution at the second to last position of the
39 acceptor splice site of intron 15. This substitution alters
the invariant AG dinucleotide consensus sequence at asplice acceptor site, from 59-AG:GAACTT-39 to 59-GG:
GAACTT-39. Alteration of this invariant dinucleotide sequence is associated with aberrant splicing that could
cause either exon skipping, activation of a nearby cryptic
splice site, or the production of an incompletely spliced
mRNA (21). The first would give rise to an mRNA thatlacks exon 16, nucleotide 2218–2342, resulting in a newreading frame after the codon for amino acid 739 and apremature stop codon. Failure to splice intron 15 would
give rise to an mRNA encoding 751 amino acids, 12 arising from the unspliced intron.
Missense mutation
Patient 2 was homozygous for each of the reported polymorphic sites (12, 14) and the only sequence abnormality
present involved a single A-to-T mismatch at nucleotide
2338 of the cDNA, in exon 16. This substitution results inthe conversion of an asparagine to a tyrosine at position
780 of the protein, which is in the middle of its carboxylterminal domain (amino acids 603 –894). None of 50 alleles from unrelated normal Japanese subjects carried themutation, ruling out the possibility that it is a common
polymorphism.
Functional characterization of the mutant MTP
To examine whether the Asn780Tyr missense mutation
leads to a nonfunctional protein, we expressed the mutant
cDNA in Cos-1 cells and measured MTP activity (Fig. 2).
Even though comparable amounts of MTP protein were
present in the cell lysates, the cells expressing the
Asn780Tyr mutant had negligible levels of MTP activity
compared with Cos-1 cells expressing the wild-type MTP,
or HepG2 cells. After subtracting the background levels ofactivity present in the mock-transfected Cos-1 cells, it wascalculated that the mutant MTP displayed negligible MTP
activities compared with wild-type MTP. These results indicate that the Asn780Tyr mutant MTP is functionally defective. It is important to note that the mutant MTP was recovered as soluble protein. Because binding with PDI isbelieved to be essential for solubility of the protein, theinteraction between the mutant and PDI seemed to be in1202

Journal of Lipid Research Volume 41, 2000

Fig. 2. Transient expression of normal and mutant MTP in Cos-1
cells. Expression constructs containing either normal or mutant(Asn780Tyr) MTP cDNA were transfected into Cos-1 cells and expressed for 48 h. Cell homogenates were used for immunoblot
analyses (A) and for measurement of triglyceride transfer activities(B). Transfer activities are expressed as the percentage of the total[14C]triglyceride transferred from donor to acceptor membranes in
1 h. Columns and bars represent means 6 SD (n 5 3). * P , 0.0001
by ANOVA. N.S., Not significant.

tact. To confirm the proper association between the
Asn780Tyr mutant and PDI, immunoprecipitation experiments were carried out with anti-PDI antibody. As shownin Fig. 3, both the wild-type and the mutant MTP were
coimmunoprecipitated by anti-PDI antibody, supporting
the notion that the binding with PDI was intact in the mutant protein.

DISCUSSION
The results presented here support the view that mutations of the MTP gene are the primary cause of ABL in
both Japanese and American patients. Two mutations
identified herein, the exon 4 insertion and the exon 13
nonsense mutation, have been described in other families(12). The exon 4 insertion mutation was previously foundin a white American family of British origin, an English
and an Anglo/Irish family. Because a similar family background was observed in patient 3 (19), the families withthe exon 4 insertion may have common ancestors. The
other reported patient who shares the exon 13 mutation

Fig. 3. The mutant MTP is associated with PDI. Expression constructs containing either normal or mutant (Asn780Tyr) MTP cDNA
were transfected into Cos-1 cells and expressed for 36 h. Cell lysates
were immunoprecipitated with either anti-PDI antibody or nonimmune serum. The immunoprecipitates were subjected to immunoblot analyses for MTP. Both the wild-type and the mutant protein
were detected in anti-PDI immunoprecipitates, but not in the nonimmune precipitates. This result indicated that the Asn780Tyr mutant
was coimmunoprecipitated by anti-PDI antibody, supporting the notion that the binding with PDI was intact in the mutant protein.

was homozygous for the change, whereas our patient was
a compound heterozygote. It is plausible that the patientswith the mutation come from common ancestors (12).
Three mutations have been newly identified: one frameshift, one splice site, and one missense. Because both
frameshift and splice site mutations theoretically produce
severely truncated proteins, it is reasonable to consider thatthese mutant MTP proteins are functionally defective. Clinically, it is noteworthy that the sister of patient 1 and themother of patient 4, who were supposed to be obligatory
heterozygotes having one allele of the nonfunctional MTP
protein, were relatively hypocholesterolemic and had low
plasma apoB levels (Table 1). These observations are apparently against the notion that ABL is an autosomal recessive
disease (1). Raabe et al. (22) reported that heterozygotes of
MTP knockout mice had reduced plasma apoB levels, supporting the possibility that human MTP deficiency is inherited in a codominant manner at least in some families.
On the other hand, it is possible that the MTP with amissense mutation, Asn780Tyr, is functional. To examine
this possibility, we performed expression experiments; theresults showed that triglyceride transfer activity was markedly reduced in the cells expressing the Asn780Tyr mutant MTP compared with those expressing the wild-type
MTP, even though equivalent amounts of protein were
produced from the two expression vectors. In view of thefact that this mutation was not found in 50 alleles from unrelated normal Japanese subjects, we conclude that
Asn780Tyr is a functionally defective missense mutation. It
is noteworthy that this is the first report of a patient who ishomozygous for a missense mutation that is also the second missense mutation reported to date.
The other missense mutation identified so far changes
Arg-540 to histidine (14). Ricci et al. (17) reported another subtle mutation, a 30-amino acid truncation of the
carboxyl terminus of the 894-amino acid protein. In bothcases, the mutations disrupted the proper association with
PDI, which is thought to be essential for solubility of theprotein, and the mutant proteins became insoluble. In
contrast, the Asn780Tyr mutant MTP was recovered as soluble protein in our expression experiments and was coimmunoprecipitated with PDI by anti-PDI antibody. Thesefindings indicate that the association with PDI was maintained in the Asn780Tyr mutant.
Residue 780 is in the middle of the carboxyl terminus of
the protein, the region that contains a putative lipid-binding
domain. Asparagine and tyrosine are both neutral aminoacids, but the latter contains a hydrophobic aromatic ring.
The change in hydrophobicity in this region might affectthe tertiary structure of the carboxyl terminus of MTP,
thus disrupting its lipid-binding capabilities. To date, nomissense mutation affecting lipid binding/transfer has
been reported. We tried to demonstrate whether the mutant
protein bound lipids by employing the method describedby Jamil et al. (23). However, we failed to detect significant triglyceride binding that was specific for MTP, probably because the amounts of the expressed MTP proteins
were not large enough in our experimental system inwhich Cos-1 cells were used. Further studies are needed todetermine the lipid-binding capacity of the mutant MTP.
From a clinical standpoint, patient 2, who was homozygous for the Asn780Tyr mutation, is of particular interest
because he was free from any of the accepted clinicalmanifestations characteristic of ABL despite having undetectable levels of plasma vitamin E (Table 1). Usually ABL
patients develop neurological symptoms within the first or
second decade of life (1). It seems reasonable to assume
that the missense mutations, like the one found in patient
2, may encode a protein with residual lipid transfer activity in vivo and may therefore account for his milder oratypical phenotype. The patient’s plasma concentrationsof vitamin E were as low as those seen in other ABL patients, yet the vitamin E content in organs, particularly inthe retina and nervous system, may be higher, masking
the development of neurological symptoms. Alternatively,
his vitamin E consumption might be relatively high, again
delaying the development of the neurological symptoms.
In conclusion, we present three novel mutations in the
MTP gene leading to ABL. These results provide further
insight into the genetic basis of the phenotypic diversityof ABL.

NOTE ADDED IN PROOF
While this manuscript was under revision, six novel mutations including two missense mutations, Ser590Ile andGly746Glu, were reported in Canadian subjects with ABL(24). No expression studies were provided concerning themissense mutations.
We are indebted to Dr. Carol C. Shoulders for MTP antibody
and pSV7D/MTP plasmid. We thank Dr. Y. Q. Ling and Dr. Tamio
Teramoto for instruction on the MTP assay, and Alyssa H.
Hasty for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, the Promotion of Fundamental Studies in Health Science of the Organization for

Ohashi et al. Mutations in MTP gene causing abetalipoproteinemia

1203

Pharmaceutical Safety and Research (OPSR), and by Health
Sciences Research Grants (Research on Human Genome andGene Therapy) from the Ministry of Health and Welfare.
Manuscript received 9 November 1999 and in revised form 13 April 2000.

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