Atherosclerosis 144 (1999) 443 – 447

Letter to the Editor

Detection of a new compound heterozygote (del G916/G1401A) for
lipoprotein lipase deficiency and a comparative haplotype analysis ofthe mutant lipoprotein lipase gene from Japanese patients
Minoru Okubo a,*, Shigeaki Inoue b, Asako Horinishi a, Takeshi Ogihara b,1,Ken-ichiro Kaneko b, Takanari Gotoda c, Nobuhiro Yamada c, Toshio Murase aa

Department of Endocrinology and Metabolism, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, 2 -2 -2 Toranomon,Minato-ku, Tokyo 105, Japanb
Department of Pediatrics, Urayasu Hospital, Juntendo Uni6ersity, Chiba 279, Japanc
The Third Department of Internal Medicine, Faculty of Medicine, Uni6ersity of Tokyo, Tokyo 113, Japan
Received 30 April 1998; received in revised form 2 November 1998; accepted 7 January 1999

Keywords: Lipoprotein lipase deficiency; Chylomicronemia; Hypertriglyceridemia; Point mutation; Compound heterozygote;
Haplotype; Founder effectDear Editors
A patient with early-onset chylomicronemia from a
non-consanguineous family to be a compound heterozygote for two different mutations in the lipoprotein
lipase (LPL) gene has been demonstrated. Reports oncompound heterozygotes for LPL deficiency have beenlimited.
The patient, a 2-month-old Japanese boy, was admitted to Urayasu Hospital because of hyperlipidemia. He
showed eruptive exanthomas on his face and buttocks,
but had no signs of pancreatitis or hepatosplenomegaly.
The patient’s plasma triglyceride (TG) and total cholesterol (TC) concentrations were 14 183 and 763 mg/dl,
respectively (Table 1). The apolipoprotein (apo) CII
concentration was a predictable value for plasma TG
levels from a previous study [1]. LPL activity, assayed
as described elsewhere [2], and mass, determined with asandwich enzyme-linked immunosorbent assay (ELISA)* Corresponding author. Tel.: +81-3-3588-1111; fax: + 81-3-35827068.
E-mail address: QFG00550@nifty.ne.jp (M. Okubo)
1
Present address: Department of Medicine, Urayasu Hospital,
Juntendo University, Chiba 279, Japan.

kit (Dai-nippon Pharmaceutical, Japan) [3], in the patient’s postheparin plasma were virtually undetectable.
The father’s plasma lipid levels were within the normal
range, whereas the mother exhibited moderate hypertriglyceridemia shortly after the delivery. The LPL activities of the parents were nearly normal, but their
LPL masses were approximately half the normal value.
Informed consent for molecular analysis was obtained
from the parents.
Nucleotide sequence analysis of the patient’s LPL
gene showed two separate point mutations: a single
guanosine deletion at nucleotide 916 (del G916) in exon
5, leading to premature termination at codon 224 by aframeshift; and a G-to-A transition at nucleotide 1401
(G1401A) in exon 8, resulting in a substitution oftryptophan (TGG) at codon 382 by a stop codon(TGA). Southern blot hybridization was performed
first, as described previously [4], but showed no major
rearrangements (data not shown). The polymerase
chain reaction (PCR) was then carried out as previously
described [4]. PCR products were directly inserted intoa plasmid vector pCRII (Invitrogen, USA), and thenucleotide sequence of subcloned plasmids were determined in both directions by dideoxy termination withan AmpliTaq cycle sequencing kit (Perkin-Elmer, USA)0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 ( 9 9 ) 0 0 0 0 8 - 8

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444

using a DNA genetic analyzer PRISM 310 (PerkinElmer, USA). Analysis of subclones comprising exon 5in the patient’s LPL gene indicated that half of thoseexamined had the del G916, while the other half had anormal sequence (Fig. 1). Nucleotide analysis of subclones inserted in the patient’s exon 8 showed that halfof the subclones had a G1401A transition, while theother half had a normal sequence (Fig. 1). No mutations were found in other exons or exon-intron junc-tions. In order to confirm these mutations, analysis byrestriction fragment length polymorphism (RFLP) wasperformed as described previously [4,5]. Mutational
study of the patient’s family showed that the del G916
was inherited from his father, and G1401A from hismother (data not shown). These results established thatthe patient was a compound heterozygote for two
different LPL gene mutations. Combination of these
two mutations in a single patient had never been re-Table 1
Biochemical data in the lipoprotein lipase (LPL)-deficient kindred

Age
Triglyceride (mg/dl)
Total cholesterol (mg/dl)
HDL-cholesterol (mg/dl)
Apo CII (mg/dl)
LPL activity (mmol FFA/ml per h)
LPL mass (ng/ml)
H-TGL activity (mmol FFA/ml per h)
Apo E genotypeaFather

PatientMother24 years
144
216
42
3.6
4.3
139
4.3
3/3

2 months
14 183
763
61
21.0
B0.6
B20
9.9
3/324 years
465
226
34
11.3
5.2
131
3.6
3/3

Normala3.7 91.3
6.4 9 2.1
223 966
8.892.9

Normal values are expressed as mean 9 S.D.

Fig. 1. Nucleotide sequence of the polymerase chain reaction (PCR)-amplified lipoprotein lipase (LPL) gene from the patient. The allele ofpaternal origin (upper panel) and the allele of maternal origin (lower panel) are identified by mutational analysis of the kindred. The sequence
data in exon 5 in the antisense strand shows a deletion of G (C in antisense) in the allele of paternal origin, which results in a stop codon by aframeshift, and a normal sequence in the allele of maternal origin. The sequence data in exon 8 in the sense strand shows a normal sequence inthe allele of paternal origin and a G-to-A transition in the allele of maternal origin, which causes the nonsense mutation. The patient was acompound heterozygote for the two mutations.

M. Okubo et al. / Atherosclerosis 144 (1999) 443–447

445Fig. 2. Analysis of GT microsatellites flanking the lipoprotein lipase (LPL) gene analysis in the patient’s kindred. The left panel shows theelectrophoretic patterns from the LPL 5%-GT microsatellite, and the right panel shows the patterns from the 3%-GT microsatellite. The patient’s
GT repeats are indicated by arrows, and the exact numbers determined by sequence analysis are shown. The patient’s 21 5%-GT repeats and 233%-GT repeats were inherited from the father, and the 16 5%-GT repeats and 26 3%-GT repeats from the mother. Arrows whose shafts are brokenlines point to the peak of the size standard.

ported, although each of them had been reported independently previously [4 – 6].
The LPL gene mutations in this patient were thought
to be null alleles, because virtually no LPL mass oractivity was detected in the patient’s postheparin
plasma. This is consistent with previous results of invitro expression studies. G1401A transition has been
indicated to significantly reduce both LPL mass andactivity [4,7]. Although the stop codon at codon 382[W382X] in the patient was caused by the G1401A
transition, the same G-to-A transition at nucleotide
1400, which also resulted in W382X, has been reported
and shown to be a null allele as well [8]. Previous
reports showed that G916 deletion caused a null allele[5,6]. These results were compatible with the findingsthat the patient’s LPL mass and activity wereundetectable.
The patient’s LPL gene haplotype was investigated inorder to compare it with those of apparently unrelated
LPL-deficient patients in Japan with the same LPL
mutation [4,5]. The del G916 mutation was previously
reported in two families with LPL deficiency [5,6], andthe family adds one more case of the del G916 mutation.
The G1401A transition was also found in a homozygous patient with LPL deficiency [4]. These findings raised the question of whether the mutated alleles
originated from a common ancestor or had occurred
independently. RFLP haplotyping, constructed using
P6uII and HindIII RFLP by PCR and BamHI RFLP
by Southern blot hybridization as described previously[4,9], showed that the patient was HindIII ( +/+),
BamHI (− /− ), P6uII (+ /− ). Analysis of the patient’s family indicated that the del G916 allele had thehaplotype A [HindIII ( + ), BamHI ( −), P6uII ( −)],
while the G1401A allele had the haplotype C [HindIII(+ ), BamHI (−), P6uII (+ )], according to a previous
report [4]. These findings suggested that the patient’sdel G916 and G1401A allele had RFLP haplotypes
identical to those of unrelated patients with the samemutation.
GT microsatellite haplotypes were further analyzed
in the LPL gene. Polymorphic GT microsatellites havebeen reported to be more informative markers thanRFLPs at the LPL locus [10,11]. The numbers of 5%and 3%-GT microsatellite repeats in the patient’s LPLgene alleles, determined by sequencing analysis, were 16
and 21 5%-GT and 23 and 26 3%-GT repeats. The size ofthe GT microsatellite alleles of the patient’s parents andof families was compared with LPL deficiency previously reported [4,5], primarily based on the method ofWood et al. [10], which was modified by using fluorescence end-labeled primers, GS-500 as a size standard,and Performance Optimizing Polymer 4 on DNA genetic analyzer PRISM 310, according to the manufacturer’s instructions. Analysis of microsatellites with theDNA genetic analyzer showed the relative sizes of theGT repeats, and comparison of the patient’s and parents’ microsatellites established that the del G916 allele
had 21 repeats of the 5%-GT microsatellite and 23
repeats of the 3%-GT microsatellite, while the G1401A

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446

Table 2Distribution of 44 unrelated Japanese lipoprotein lipase (LPL) chromosome haplotypes and the LPL-deficient kindred’s haplotypes
LPL 3%-GT

LPL 5%-GTTotal

11161720212221230013000000142611102629323538001123(G1401A)
82
1001(del G916)
001000000020001008621Total

338213148allele had 16 repeats of the 5%-GT microsatellite and 26
repeats of the 3%-GT microsatellite (Fig. 2). These GT
microsatellites were compared in the del G916 mutationin our patient and an LPL-deficient patient heterozygous for the del G916 mutation [5], and found thatthey were the same. The patient’s G1401A allele had aGT microsatellite haplotype identical to that of a patientwith the same mutation [4]. In addition, the GT microsatellite haplotypes were examined in 11 unrelated
Japanese families. The distribution of 44 Japanese LPL
chromosome haplotypes plus four haplotypes from thepatient’s kindred are shown in Table 2. These haplotype
analyses disclosed two points: (1) the G1401A allele wason the commonest GT microsatellite haplotype among
Japanese, whereas the del G916 allele was on a uniquemicrosatellite haplotype; and (2) each patient’s mutatedallele had a haplotype identical to that of the patientwith the same mutation, who is unrelated to the one
described here. These findings suggest that the del G916
mutation had a common Japanese ancestor, and that theG1401A transition may also have originated from acommon ancestor. However, the GT microsatellite haplotype on the del G916 mutation was not found among
44 chromosomes, indicating that the del G916 mutation
is not very prevalent in the Japanese population, eventhough this mutation has occasionally been found inpatients with LPL deficiency.
Just recently, a systemic survey of nucleotide-sequencevariation in 71 individuals from three populations reported 88 variable sites in a 9.7-kb region of the LPL
gene, and a total of 88 distinct haplotypes were identified[12]. Because more polymorphic markers and more
independent chromosomes should be investigated tocharacterize all the possible haplotypes which occur in
the Japanese population, further studies will be needed
to define the identity of the haplotypes of the LPL-deficient patients mentioned above.
Heterozygous states of LPL mutations have attracted
attention as one of the causes of postprandial hyperlipi-demia [13]. Hyperlipidemia during pregnancy has beenfound to occur in women with heterozygous LPL mutations [8], and the patient’s mother may be an example,because moderate hypertriglyceridemia was observed
shortly after delivery. As the apo E isoform has beenshown to influence expression of hyperlipidemia[8,14,15], the apo E genotype of the patient’s family wasdetermined by digestion of PCR-amplified DNA with
HhaI according to Hixon and Vernier [16], and foundthe mother’s genotype to be E3/E3, a wild-type isoform.
This may explain why her hyperlipidemia was notsevere.
In summary, a new case of familial LPL deficiency
was described and a founder effect by performing haplotype analysis of the patient was investigated.

Acknowledgements
This work was supported in part by the Japan Motorcycle Racing Organization.

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