高木 敦子

Triglyceride deposit cardiomyovasculopathy (TGCV) is a phenotype primarily reported in patients carrying genetic mutations in PNPLA2 encoding adipose triglyceride lipase (ATGL) which releases long chain fatty acid (LCFA) as a major energy source by the intracellular TG hydrolysis. These patients suffered from intractable heart failure requiring cardiac transplantation. Moreover, we identified TGCV patients without PNPLA2 mutations based on pathological and clinical studies. We provided the diagnostic criteria, in which TGCV with and without PNPLA2 mutations were designated as primary TGCV (P-TGCV) and idiopathic TGCV (I-TGCV), respectively. We hereby report clinical profiles of TGCV patients. Between 2014 and 2018, 7 P-TGCV and 18 I-TGCV Japanese patients have been registered in the International Registry. Patients with I-TGCV, of which etiologies and causes are not known yet, suffered from adult-onset severe heart disease, including heart failure and coronary artery disease, associated with a marked reduction in ATGL activity and myocardial washout rate of LCFA tracer, as similar to those with P-TGCV. The present first registry-based study showed that TGCV is an intractable, at least at the moment, and heterogeneous cardiovascular disorder.

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.

Human LPL is a glycoprotein enzyme with a molecular mass of 61 kDa, and it plays a key role in regulating the triglyceride (TG) levels in circulation by hydrolyzing TGs in TG-rich lipoproteins at the first step in their metabolism. Homozygous or compound heterozygous LPL deficiency causes severe fasting hypertriglyceridemia. Heterozygous LPL deficiency usually results in a normolipidemic state, but this may cause mild hypertriglyceridemia if heterozygotes are exposed to factors, such as high alcohol intake and/or a hyperinsulinemic state. Severe fasting hypertriglyceridemia is mainly caused by abnormalities of the LPL gene, whereas there are some cases caused by gene defects relating to synthesis and transport of LPL such as LMF and GPIHBP1, and by an autoantibody to LPL acting as inhibitor of LPL.

Elevations in plasma triglyceride (TG) and free fatty acid (FFA) concentrations are generally thought to play a role in the pathogenesis of insulin-resistant diabetes. The objective of this study was to investigate the relationship between hypertriglyceridemia and glucose-stimulated insulin responsiveness in non-diabetic patients. Forty subjects were divided into three BMI-matched groups as follows: one group consisted of 8 patients with a lipoprotein lipase (LPL) deficiency, another consisted of 12 patients with hypertriglyceridemia and a third consisted of 20 subjects with normal TG levels. In response to a 75 g oral glucose tolerance test, plasma insulin levels in the LPL-deficient subjects were higher (106 +/- 11 mu U/ml) than those in the hypertriglyceridemic (69 +/- 16 mu U/ml) and normolipidemic (29 3 mu U/ml) subjects, at 30 min. On the other hand, their plasma glucose levels (127 +/- 6 mg/dl) were less than those seen in the normolipidemic group (165 +/- 9 mg/dl) after 90 min. Thus, LPL-deficient subjects with hypertriglyceridemia displayed an enhanced glucose-stimulated insulin response as well as lower blood glucose levels, the latter of which is not generally seen in those with hypertriglyceridemia and normolipidemia. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Temperature-controlled on-chip capillary electrophoresis (CE) was applied to multi-temperature single-strand conformation polymorphism (SSCP) analysis for the detection of three lipoprotein lipase (LPL) gene mutations. In the SSCP analysis, five different temperature conditions (10, 15, 20, 25, and 30 degrees C) were used to obtain an SSCP pattern. Differences between the electropherograms of Cy5-labeled PCR products of wild/wild homozygote and wild/mutant heterozygote were clearly detected for all four LPL mutations in at least one appropriate temperature condition. These results indicate that our SSCP system is applicable to high-throughput and accurate SSCP screening for locating unknown as well as known single nucleotide polymorphisms (SNPs).

Objectives of this work are to study changes in the immunoreactive HTGL mass during storage under various conditions. In addition, the shelf-life of the HTGL ELISA kit was confirmed. The immunological reactivity of HTGL in PHP stored in the liquid, frozen, or lyophilized state was monitored using purified human PHP-HTGL as the standard material. Furthermore, the long-term stability of the HTGL ELISA kit was ascertained. The immunoreactive HTGL mass in the lyophilized PHP maintained its initial immunological reactivity for at least 26 months at 4 degrees C or lower. The other reagents included in the HTGL ELISA kit also have a long shelf-life when they are stored at 4 degrees C or less. HTGL in PHP was stabilized by lyophilization and can be used as the standard material for HTGL ELISA; the HTGL ELISA kit has a long shelf-life, i.e., more than two years.

A ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) was applied to the detection of two mutations in human lipoprotein lipase (LPL) gene, G188E (one base transition) and Arita (one base deletion). A probe oligodeoxyribonucleotide of 13 bases representing the wild type (WT) sequence of LPL was immobilized on a gold electrode, followed by hybridization with a sample PCR product of 350 base pairs under conditions in which both WT and mutated (MT) sequences could form a duplex with the probe. The hybridized electrodes were soaked in an electrolyte containing FND under conditions in which only the mismatched duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex remaining on the electrode to,give rise to a current signal. Blind tests were run to judge the genotype (WT/WT, WT/MT, or MT/MT) of 10 samples each for the G188E and Arita mutations and then, 8 and 10 of them were judged correctly, respectively.

We previously characterized the patients with autosomal recessive hypercholesterolemia (ARH) as having severe hypercholesterolemia and retarded plasma low-density lipoprotein (LDL) clearance despite normal LDL receptor ( LDLR) function in their cultured fibroblasts, and we identified a mutation in the ARH locus in these patients. ARH protein is an adaptor protein of the LDL and reportedly modulates its internalization. We developed ARH knockout mice (ARH(-/-)) to study the function of this protein. Plasma total cholesterol level was higher in ARH(-/-) mice than that in wild-type mice (ARH(+/+)), being attributed to a 6-fold increase of LDL, whereas plasma lipoprotein was normal in the heterozygotes (ARH(+/-)). Clearance of I-125-LDL from plasma was retarded in ARH(-/-) mice, as much as that found in LDLR-/- mice. Fluorescence activity of the intravenously injected 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-LDL was recovered in the cytosol of the hepatocytes of ARH(+/+) mice, but not in those of ARH(-/-) or LDLR-/- mice. Also, less radioactivity was recovered in the liver of ARH(-/-) or LDLR-/- mice when [H-3]cholesteryl oleyl ether (CE)-labeled LDL was injected. In contrast, uptakes of [H-3]CE-labeled LDL, I-125-LDL, and DiI-LDL were all normal or slightly subnormal when the ARH(-/-) hepatocytes were cultured. We thus concluded that the function of the hepatic LDLR is impaired in the ARH(-/-) mice in vivo, despite its normal function in vitro. These findings were consistent with the observations with the ARH homozygous patients and suggested that certain cellular environmental factors modulate the requirement of ARH for the LDLR function.

We describe the development of a new type of DNA array chip that utilizes electrochemical reactions and a novel method of simultaneously identifying multiple genetic mutations on an array chip. The electrochemical array (ECA) uses a threading intercalator specific to double-stranded nucleotides, ferrocenylnaphthalene diimide (FND), as the indicator. ECA does not require target labeling, and the equipment is simple, durable and less expensive. The simultaneous multiple mutation detection (SMMD) system using an ECA chip and FND utilizes an enzyme to simultaneously distinguish several genetic mutations such as single nucleotide polymorphism (SNP), insertion, deletion, translocation and short tandem repeat. We examined this SMMD system using an ECA chip, by detecting seven different mutations on the lipoprotein lipase (LPL) gene for 50 patients in a blind test. It turned out that all the results obtained were concordant with the sequencing results, demonstrating that this system is a powerful tool for clinical applications.

The present study describes how to process human postheparin plasma (PHP) containing hepatic triglyceride lipase (HTGL) that is utilized as a standard material of HTGL for the quantification of HTGL mass in human plasma. The optimal storage conditions for PHP were established by monitoring the stability of HTGL molecules in PHP as an antigen, which was stored in the liquid, frozen, or lyophilized state, using purified human PHP-HTGL as the standard material and a commercial HTGL ELISA MARUPI kit, which is a direct sandwich enzyme-linked immunosorbent assay (ELISA). The HTGL ELISA MARUPI kit, for which the validity was confirmed by precision and dilution tests, showed that the immunoreactive mass of HTGL in lyophilized PHP remained stable for at least 12 months at a storage temperature of 4degreesC or lower. These results indicate that lyophilized PHP stored at a temperature of less than 4degreesC can be utilized as the standard material for the quantification of HTGL in human plasma using the HTGL ELISA MARUPI kit.

Previously we have reported on siblings with severe hypercholesterolemia, xanthomas, and premature atherosclerosis without any impairment of low-density lipoprotein receptor in their fibroblasts as a first characterization of autosomal recessive hypercholesterolemia (ARH). Recently, mutations were identified for this disease in a gene encoding a putative adaptor protein. The purpose of this study was to examine the molecular pathogenesis of ARH in Japanese siblings. A novel insertion mutation was discovered in the ARH gene of the siblings. An insertion of an extra cytosine residue was identified in a locus comprising eight consecutive cytosines at positions 599 through 606 in exon 6, resulting in a sequence of nine cytosines and generating an early stop codon at 657 - 659. The mother was heterozygous for this mutation. Neither transcription product nor protein of ARH was detected in the fibroblasts of the homozygous patients. A single nucleotide polymorphism was discovered among the normal control subjects at position 604 ( cytosine to thymine: ARH-604C to ARH-604T), which changes the proline residue at 202 to serine. Interestingly, ARH is caused by a mutation of cytosine to adenine at this same position. Both siblings exhibited fatty liver, which may also be related to this mutation.

A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, 5447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degreesC], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for: WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.

An electrochemical hybridization assay has been devised that enables the rapid analysis of a heterozygous deficiency of the human lipoprotein lipase (LPL) gene. PCR products of 350 base pairs (bp) containing the wild-type sequence, a mutated G(818) --> A transition or a G(916) deletion of the LPL gene were subjected to hybridization with a probe DNA of 13 or 15 bases that represented either the wild-type or the mutated sequence immobilized on a gold electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in the presence of ferrocenylnaphthalene diimide (FND) at 460 mV. The measured change in peak current, Deltai, was defined by (i - i(o))i(o) x 100%, where i. and i represent the current before and after hybridization, respectively. Matched combinations of sample and probe gave Deltai values of 40-90%, whereas mismatched combinations gave values of 20-35%, enabling the discrimination of matched hybrids from mismatched ones across a slim margin. Because the heterozygote contains both the wild-type and mutated sequences, however, it alone gives large Deltai values with both the wild- and mutant-type probes. This system was validated on 10 unknown samples of each of the two types of LPL mutation, which were correctly identified in every case.

48歳男.腹痛と嘔吐を主訴とし,重症急性膵炎Stage2と判断した.習慣性飲酒歴があり44歳時に高脂血症を指摘されたが放置し,46歳時に膵炎の既往がある.入院時検査でトリグリセリド(TG)が高値であったが,入院直後は絶食とし,その後の脂肪制限食により第9病日に正常化した.血中リポ蛋白-TG水解に関与しているヘパリン静注後血漿リポプロテインリパーゼ(LPL)は一過性に減少したが,LPLは遺伝子解析で異常を認めず他に由来すると判断した.本症例の病態と成因は,習慣性過量飲酒により高TG血症と膵炎をきたし,炎症性サイトカインによりLPLが減少したため強度の高TG血症となり,さらに膵炎を増悪したと考察した

Case report: A case is presented of predisposing a patient's father with obligate heterozygous lipoprotein lipase (LPL) deficiency to mild hypertriglyceridemia in Japanese I-family members (n = 8) with patient DI, who was a compound heterozygote for a novel missense mutation of G154V (GG(716)C-->GTC/Gly(154)Val) in exon 5 and a novel splice mutation (Int8/5'-dss/t( + 2)c; a T-to-C transition in the invariant GT at position + 2 of the 5' donor splice site (dss)) in intron 8 of the LPL gene. Results: The patient's father and paternal grandmother were heterozygotes for the Int8/5'-dss/t( + 2)c allele, while the patient's mother and maternal grandmother were heterozygotes for the G154V allele. These four heterozygous carriers with one defective LPL allele showed 45-57% of the mean LPL activity and mass in the post-heparin plasma (PHP) observed in normal individuals. Among the four heterozygous carriers, the patient's father, who was <40 years old, nonobese and hyperinsulinemia, manifested mild hypertriglyceridemia (type IV hyperlipoproteinemia). The remaining three healthy heterozygous carriers (two were >40 years old and the other was < 40 years old) were all normolipidemic state. Conclusion: In this family, hyperinsulinemia as a marker of insulin resistance may be a strong determinant of hypertriglyceridemia in the carrier with heterozygous LPL deficiency. (0 2002 Elsevier Science B.V. All rights reserved.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents, Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma, The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3 ' acceptor splice site (3 ' -ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5 ' donor splice site (5 ' -dss) of intron 8 (Int8/5 ' -dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3 ' -consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; G (G) under bar (716)C(-->)G (T) under barC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin, The 1nt8/5 ' -dss/t(+2)c mutation inactivated the authentic 5 ' splice site of intron 8 and led to the utilization of a cryptic 5 ' -dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8.jlr These additional mutations in the consensus sequences of the 3 ' and 5 ' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.

We systematically investigated the molecular defects resulting in primary lipoprotein lipase (LPL) deficiency in a Japanese male infant thereafter called 'the patient') with severe fasting hypertriglyceridaemia (type I hyperlipoproteinaemia). The primary LPL deficiency was diagnosed on the basis of the findings that no LPL activity was detected in post-heparin plasma (PHP) and that the immunoreactive LPL mass in PHP was less than 2% of the control level. The patient was a compound heterozygote for a novel missense mutation (G(568)GA --> AGA/Gly(105) --> Arg; G105R) in exon 3 and a missense mutation (GAC(867) --> GAG/Asp(204) --> Glu; D204E) in exon 5 of the LPL gene. The biological significance of both missense mutations was examined by an in vitro study of the expression of the mutant G105R LPL cDNA and D204E LPL cDNA in COS-I cells. Both mutant LPLs were catalytically inactive and were barely released by heparin from the expressing COS-I cells. These findings explain the failure to detect LPL activity and immunoreactive LPL mass in the patient's PHP. The G105R allele could be detected by digestion with the BsmAI restriction enzyme, and the D204E allele by digestion with Hindi. The patient inherited the G105R allele from his mother and the D204E allele from his father. His parents were heterozygotes for the corresponding mutant allele, but normolipidaemic. The novel G105R missense mutation could not be detected by conventional analysis of single-strand conformation polymorphism, but it was identified by extensive sequencing of the entire exons and their flanking regions in the LPL gene.

We have developed a direct sandwich-enzyme-linked immunosorbent assay (ELISA) for quantification of the hepatic triglyceride lipase (HTGL) immunoreactive mass in human plasma. This direct sandwich-ELISA uses a combination of two distinct monoclonal antibodies (MAbs), which recognize different epitopes on the HTGL molecule: a horseradish peroxidase (HRP)-labeled anti-human HTGL MAb (2(4)F12C12) as an enzyme-linked MAb, and an anti-human HTGL MAb (1(11)A3H3) coated on a microtiter plate as a solid-phase MAb, Purified human post-heparin plasma (PHP)-HTGL was used as the standard material. The detection range of the sandwich-ELISA was 40-800 ng of HTGL protein per mi of plasma. The intra- and inter-assay coefficients of variation were less than 2.0% and 2.3%, respectively. The recovery tests resulted in variation only between 97.7% and 103.5%. No significant assay interference was caused by a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. The reliability of the HTGL mass values obtained with the direct sandwich-ELISA was assessed by comparison with the HTGL mass values determined by our earlier one-step sandwich-enzyme immunoassay (EIA), The two sets of values showed a highly significant correlation (r = +0.952, n = 64). Strong correlation (r = +0.959, n = 50) was also found between the HTGL masses with the direct sandwich-ELISA and the HTGL activities determined with a selective immunoinactivation assay. The HTGL mass concentrations in PHP from 64 healthy subjects were 1916 +/- 841 ng/ml by the direct sandwich-ELISA and 1925 +/- 785 ng/ml (mean +/- standard deviation (SD)) by the one-step sandwich-EIA. The present direct sandwich-ELISA permits rapid identification of certain HTGL abnormalities in PHP samples from patients with hypertriglyceridemia or diseases such as hypothyroidism or renal failure, which affect HTGL. (C) 2000 Elsevier Science B.V. All rights reserved.

A reliable method for detection of small-sized low-density lipoprotein (LDL) produced during transient hypertriglyceridaemia induced by ingestion of fat is described. Electrophoresis using a commercial non-denaturing 2.0-16% polyacrylamide gradient gel is commonly utilized to determine the size of LDL particles, but it failed to detect a minor change in LDL size induced during postprandial hypertriglyceridaemia. Detection of small-sized LDL was achieved by adjusting the polyacrylamide concentration. Electrophoresis using a nondenaturing 1.5-10% polyacrylamide gradient gel gave the best resolution for detecting small-sized LDL induced during postprandial hypertriglyceridaemia. This improved method may be useful for elucidating the underlying mechanism of production of small-sized LDL (small dense LDL) in subjects with chronic hypertriglyceridaemia.

We herein report a case of a 5-month-old Japanese female (patient AN) with fasting hyperchylomicronemia due to a primary lipoprotein lipase (LPL) deficiency. Patient AN was compound heterozygous for a missense mutation (GG(818)G-->GAG/Gly(188) -->Glu; G188E) in exon 5 and a nonsense mutation (TGG(1401)-->TGA/Trp(382) --> Stop; W382X) in exon 8 of the LPL gene. This resulted in less than 10% of the control levels for both the LPL activity and immunoreactive LPL mass in the postheparin plasma. A G188E mutation was thus identified for the first time in a Japanese, and the haplotype of this G188E allele was different from that of the G188E alleles identified in other ethnic groups. This additional mutation might be useful for early diagnosis of LPL gene aberrations in Japanese patients with fasting hyperchylomicronemia. (C) 1999 Elsevier Science B.V. All rights reserved.

Objective: The purpose of this study was to develop and evaluate a direct sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the lipoprotein lipase (LPL) immunoreactive mass in human plasma using monoclonal antibodies (MAbs) directed against LPL purified from human postheparin plasma (PHP). Design: The DNA fragment containing exon 1 through 7 of the LD-H gene were amplified by PCR and directly sequenced. Total RNA was prepared from venous blood and the proportion of LD-H cDNA to total LD cDNA was semiquantified. Methods and results: The direct sandwich-ELISA was performed using a combination of two distinct types of MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of human LPL was specifically measured using a horseradish peroxidase-labeled anti-human LPL MAb [1(1)D2B2] as an enzyme-linked MAb, and an anti-human LPL MAb [2(10)F8F9] coated on a polystyrene microtiter plate as a solid-phase MAb. Purified human PHP-LPL was used as a standard material. The detection range of the sandwich-ELISA was 3.6-460 ng of LPL protein per mt of plasma. The intra- and interassay coefficients of variation were less than 5.9% and 3.3%, respectively. The validity of this method was additionally assured by the recovery test, which resulted in the Variation only between 97.5% and 105.1%, and also by the interference test, which resulted in noninterference of LPL assay with a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. To assess the reliability of the LPL mass values obtained with the direct sandwich-ELISA, they were compared with LPL mass values determined by the one-step sandwich-EIA (MARKIT-F LPL EIA kit) previously established by us. This comparison showed a highly significant correlation (r = +0.990) between the two sets of values. The LPL mass concentrations in PHP from 33 healthy subjects were 267 +/- 53 and 257 +/- 59 ng/mL (mean +/- SD), respectively. Conclusion: The present direct sandwich-ELISA is useful for rapidly identifying certain abnormalities of LPL in PHP samples from patients with hypertriglyceridemia. Copyright (C) 1999 The Canadian society of clinical Chemists.

WE herein report a case of a 40-year-old Japanese woman (patient IT) with a history of recurrent aggravation of hypertriglyceridaemia, pancreatitis and miscarriages in three previous pregnancies. However, strict dietary intervention was applied during a fourth pregnancy. As a result, acute pancreatitis was avoided, and the patient gave birth to a healthy infant. In patient IT, the underlying etiology of the recurrent aggravation of hypertriglyceridaemia during pregnancy was a lipoprotein lipase (LPL) gene aberration, She was homozygous for LPL deficiency due to a nonsense mutation (TGG(1401) --> TGA/Trp(382) --> Stop) in exon 8 of the LPL gene, which resulted in the absence of LPL activity and immunoreactive LPL mass, Our findings indicate that, in LPL deficiency pregnancy seriously exacerbates hypertriglyceridaemia and increases the risk of acute pancreatitis, which endangers both the mother and fetus. Early diagnosis of LPL deficiency and appropriate management thereof are essential for normal childbirth.

Objective: The purpose of this study was to develop an improved method of direct DNA sequencing, which makes it possible to identify heterozygous mutations of the lipoprotein lipase (LPL) gene in order to understand the underlying genetic disorder of type IV hyperlipoproteinemia. Methods and Results: The direct sequencing method was improved by devising primers for amplifying the LPL gene and for sequencing DNA amplified by the polymerase chain reaction (PCR), since the reported base sequences of the introns flanking exons of the LPL gene were limited to 40 bases. Improvement was achieved by attaching nine additional bases to both the PCR amplification primer and sequencing primer, and by optimizing the Tm Value of the sequencing primers by adjusting the sequence of the nine extra bases. Use of the sequencing primers having suitable Tm values (48 degrees C similar to 58 degrees C) made it possible to reduce nonspecific bands on the sequence ladder pattern and to identify heterozygous mutation sites in LPL gene exons 5 and 6 as model cases. Conclusion: Our improved direct sequencing method is useful for identifying heterozygous mutation sites in human LPL gene exons and splicing consensus regions.

We have systematically investigated a genetic defect resulting in a primary lipoprotein lipase (LPL) deficiency in a proband TN and his affected brother SN, both manifesting familial hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in postheparin plasma from the two patients. Immunocytochemical and biosynthetic studies on the proband's monocyte-derived macrophages with rabbit antihuman LPL antiserum revealed that no immunochemically detectable LPL protein was found in either the cells or culture medium, whereas LPL having a molecular mass of 61 kD was detected in normal cells. No detectable LPL mRNA was identified from poly(A)+RNA of the proband's macrophages by Northern blot analysis, and grossly visible LPL gene rearrangement was not observed by Southern blot analysis. Sequence analysis of polymerase chain reaction-amplified LPL gene exons detected one base deletion of G (first position of Ala221) at base 916 in exon 5 which leads to a premature termination by a frameshift. This mutation, designated as LPL(Arita) and resulting in the loss of an AluI restriction enzyme site, was newly identified. We further analyzed the LPL gene from the two patients and their family members by digestion with AluI. Both patients were homozygous for LPL(Arita) allele, while their spouses did not have this mutation. As genetically expected, their children were all heterozygous for LPL(Arita). We conclude that primary LPL deficiency in the proband was caused by a lack of enzyme synthesis due to the absence of LPL mRNA resulting from one base deletion of G in exon 5, and that heterozygous LPL(Arita) deficient subjects show almost half value of control LPL mass.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67 200 and 65 500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60 600 and 64 600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apoliproprotein C-II were 1.0 mM and 0.6 μM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum. © 1989.