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Unité Mixte de Recherche 585 Institut National de la Santé et de la Recherche Médicale/Institut National des Sciences Appliquées (V.P.-D., C.M., M.L., P.M.), Physiopathologie des Lipides et Membranes, 69621 Villeurbanne cedex, France; Laboratoire de biochimie (C.M., M.D.), Centre hospitalier Lyon-Sud, Pierre Bénite, France; Unité 11 (L.P., P.M.), Hôpital Cardiovasculaire Louis Pradel, Lyon-Bron, France; Laboratoire de biochimie (A.S.), Hôpital neurologique, Lyon, France; and Service d’Endocrinologie (B.E.), Hôpital Bellevue, Saint-Etienne, France
Address all correspondence and requests for reprints to: Valérie Pruneta-Deloche, Unité Mixte de Recherche 585 Institut National de la Santé et de la Recherche Médicale/Institut National des Sciences Appliquées, Bâtiment Louis Pasteur, 20 avenue Albert Einstein, 69621 Villeurbanne cedex, France. E-mail: valerie.pruneta-deloche{at}insa-lyon.fr.
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Abstract |
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Design: Upon investigation of a patient suffering from recurrent hypertriglyceridemic pancreatitis without family history or conventional secondary cause of dyslipidemia, we identified a previously unreported nonsense heterozygous lipoprotein lipase (LPL) gene mutation S172fsX179 associated with an antihuman LPL IgG.
Results: This autoantibody partially inhibited wild-type LPL activity in vitro. Furthermore, the patient’s plasma triglyceride concentrations were efficiently decreased under immunosuppressive treatment, and this was confirmed by sequential withdrawal/reintroduction tests.
Conclusions: We consider that this unique combination of a genetic defect and an autoimmune disease results in chronic major hypertriglyceridemia. Because immunosuppressive treatment can improve this dyslipidemia, assessment of anti-LPL autoantibody is worthwhile in unmanageable chronic major hypertriglyceridemia, even in the presence of a heterozygous LPL deficiency.
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Introduction |
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Subjects and Methods |
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The LPL gene nucleotide sequence was first analyzed through single-strand conformation polymorphism screening and sequencing of variants as previously reported (12). The absence of a deleterious mutation on the second LPL allele was further confirmed through direct sequencing. The entire coding sequence (exon 1 through exon 9) and the intron-exon junctions and the proximal promoter were studied.
Lipids, lipoproteins, and apo
The concentrations of total plasma cholesterol and TG were measured enzymatically using commercial kits (BioMérieux, Marcy l’Etoile, France). LDL-C and HDL-C were measured after lipoprotein fractions were separated by sequential ultracentrifugation. We determined apo AI, apo B, and apo E by automated rate nephelometry, and apo CII and apo CIII by immunoturbidimetry.
Determination of LPL activity
Fasting plasma samples were obtained at 0 min and 15 min after an iv injection of heparin (50 IU/kg body weight). LPL activity was quantified in duplicate as previously described (13). Briefly, the activity of LPL was measured using triolein emulsion labeled with glycerol-14C-oleate (Amersham Pharmacia Biotech) as a substrate, and after selective inhibition of hepatic lipase by preincubation with a specific goat antibody (kindly provided by Dr. S. Griglio, France). To investigate the presence of a circulating inhibitor of LPL in the proband’s plasma, inhibition assay of LPL activity was performed by preincubating control postheparin plasma (2 h; 4 C) in the presence of increasing volumes of the patient’s lipoprotein-free plasma before the assay. LPL activity of the human source of LPL was 10.1 ± 0.4 µmol nonesterified fatty acids (NEFA) released per hour and per milliliter (mean ± SD). For the patient, the mean of two independent experiments is presented.
Detection of a circulating LPL autoantibody in patient serum
LPL partially purified from postheparin plasma was submitted to 10% sodium dodecyl sulfate-polyacrylamide gel and blotted to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA) as previously described (13). The blots were incubated with the patient’s plasma or with control plasma. Immunodetection was accomplished using a goat antihuman IgGs-horseradish peroxidase conjugate as secondary antibody (1:1000 dilution; Sigma, St. Louis, MO) and the enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).
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Results |
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A 50-yr-old man was referred to our lipid clinic for evaluation of a severe unmanageable type V hyperlipoproteinemia with a fasting chylomicronemia. His lipid profile showed an accumulation of both chylomicrons and very low-density lipoprotein with low LDL-C and HDL-C and increased plasma apo CII and CIII concentrations (Table 1
). He was found hypertriglyceridemic for 30 yr and suffered from four recurrent episodes of acute pancreatitis between the ages of 46 and 49 yr. He remained for at least 8 yr with a documented major type V hyperlipoproteinemia refractory to both strict dietary restriction and fibrate treatment (Fig. 1). He was not treated with any drug known to induce hypertriglyceridemia and had neither disease leading to secondary dyslipidemia nor familial history of dyslipidemia. He had no personal history of autoimmune disease, but his father had a vitiligo.
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The patient’s LPL activity was measured on four occasions and found consistently decreased. The activity varied and was, in some instances, mildly reduced as in heterozygous LPL deficiency, or in other instances severely reduced as in homozygous LPL deficiency (Table 1
). The patient was found heterozygote for a previously unreported nonsense S172fsX179 LPL mutation due to the deletion of the second nucleotide of codon 172. The second LPL allele was shown not to harbor additional nucleotide sequence variation, and therefore the LPL gene alteration alone accounted neither for the severity of dyslipidemia nor for the drastic LPL deficiency observed in vitro (Table 1). Additional study of common polymorphisms did not uncover TG-raising variants of potential modifier genes: Apo E genotype was E3/E3; similarly in the chromosome 11 AV-AI-CIII-AIV gene cluster, only the common alleles were present for 2 apo AV polymorphisms (c-3A>G and S19W) as for 3 apo CIII polymorphisms (g-482C>T, g-455T>C, and the Sst-I RFLP).
Autoimune inhibitor of LPL
We searched for a direct autoimmune inhibition of LPL activity and introduced an immunosuppressive treatment as a diagnostic test, taking into account the variable LPL activity that was occasionally lower than expected for heterozygote deficiency, the family history of autoimmune disease, and the unusual resistance to hypotriglyceridemic treatment. The patient had only a very mild increase in antinuclear autoantibody (titer 1/80, sensitivity limit 
1/80) and remained negative for antithyroid autoantibody and rheumatoid factor. As shown in Fig. 1, the introduction of azathioprine treatment upon the withdrawal of fibrates resulted in a 40% decrease in the plasma TG concentrations. To confirm the hypotriglyceridemic effect of immunosuppressive treatment, three sequential tests of azathioprine withdrawal/reintroduction established that, under treatment, plasma TG concentration was consistently decreased by 38% from an average without treatment of 27.0 ± 8.9 mmol/liter to an average under treatment of 17.7 ± 7.3 mmol/liter (P < 0.0001). No recurrent acute pancreatitis was observed during the following 18 months, whereas four episodes had occurred within the 4 yr before introduction of immunosuppression.
A specific IgG directed against human LPL was detected in the patient’s plasma by Western blotting (Fig. 2). The ability of the anti-LPL IgG to inhibit LPL activity was studied ex vivo by the preincubation of the patient’s lipoprotein free plasma with a postheparin human control plasma as a source of active LPL. In two independent experiments, the lipoprotein free plasma induced a dose-dependent, but partial, inhibition of the control LPL activity (Fig. 3).
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Discussion |
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S172fsX179 mutation is predicted to encode an inactive truncated peptide. However, heterozygote LPL deficiency alone could not account for an unmanageable type V hypertriglyceridemia as in our patient. It is likely that the heterozygote LPL S172sfX179 mutation alone would account for an heterozygote LPL deficiency phenotype with only a mild decrease in postheparin plasma LPL activity (around 50%), leading at most to a late-onset moderate and easily manageable type IV hyperlipoproteinemia (5, 6, 7). In our patient, postheparin LPL activity was found severely reduced on some occasions (Table 1), suggesting an additional LPL impairment. Subjects with heterozygote LPL deficiency are prone to severe type-V hyperlipoproteinemia when combined with secondary causes of dyslipidemia such as type 2 diabetes, hypothyroidism, pregnancy, and alcoholism (9, 10, 11, 12). However, these were excluded in our patient. Risk haplotypes in the apo AV-AI-CIII-AIV gene cluster on chromosome 11 are highly overrepresented in type V hyperlipidemia, compared with population studies, and may enhance this dyslipidemia (12), but these haplotypes were absent in our patient.
Autoimmunization toward LPL may exceptionally generate circulating LPL inhibitors, leading to hyperchylomicronemia in autoimmune disease (14). We had previously reported a complete normalization of plasma TG under immunosuppressive treatment in a case of nonfamilial hyperchylomicronemia with wild-type LPL. Strict normalization of hypertriglyceridemia was obtained subsequent to the disappearance of an anti-LPL autoantibody (13). We now report the first case of severe LPL deficiency caused by the combination of an inherited heterozygote nonsense LPL mutation and an acquired circulating LPL inhibitor. This anti-LPL IgG provided only partial LPL inhibition, unlike our previous report where the LPL inhibitor fully impaired wild-type LPL activity in vitro (13). Therefore the combination with heterozygote LPL deficiency is likely to account for the severity of LPL impairment leading to type V hyperlipoproteinemia in our patient. Moreover, the underlying heterozygote LPL deficiency may also explain why decrease in plasma TG concentration was only partial upon immunosuppressive therapy, which might have only partially suppressed the circulating LPL inhibitor. In our previous report (13), the circulating inhibitor accounted alone for hyperchylomicronemia. Even partial suppression of the autoantibody upon azathioprine had rescued sufficient LPL activity to normalize plasma TG concentration (13). On the other hand, in the present case, LPL was expressed from a single allele, likely reducing enzyme availability. This may explain why azathioprine treatment failed to rescue enough LPL activity to normalize plasma TG concentration, although recurrent pancreatitis was efficiently prevented.
S172fsX179 mutation is predicted to encode a truncated peptide likely to be poorly secreted, as reported in expression models (15, 16, 17). It is unknown whether peptide truncation triggered the anti-LPL autoimmunization. However, due to family history, we speculate that the patient is prone to present autoimmune diseases. Kihara et al. (18) first advanced the concept of autoimmune hyperchylomicronemia in a patient with idiopathic thrombocytopenic purpura and Graves’ disease. Interestingly, two recent reports highlighted a high frequency of autoantibodies directed against LPL in patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (19, 20, 21, 22). Up to 40% prevalence was reported, but it is very likely that these autoantibodies had a very low LPL inhibitory capacity, as evidenced by a mild 10% increase in plasma TG concentration in these patients. Our results suggest that these mild circulating inhibitors, when combined with low prevalence LPL heterozygote deficiency, may trigger more severe hypertriglyceridemia.
In summary, our case report strongly suggests that autoimmune hyperchylomicronemia should be investigated in refractory severe hypertriglyceridemia when genetic alterations alone cannot account for the intensity of the phenotype and when the patient has either a personal or a family history of autoimmune disease. Indeed, the identification of an anti-LPL autoantibody (circulating LPL inhibitor), leading to the adjunction of an immunosuppressive treatment, provides a unique opportunity to handle an otherwise unmanageable dyslipidemia and prevent acute pancreatitis.
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Acknowledgments |
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Footnotes |
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First Published Online April 19, 2005
Abbreviations: apo, Apolipoprotein(s); HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LPL, lipoprotein lipase; NEFA, nonesterified fatty acids; TG, triglyceride.
Received January 31, 2005.
Accepted April 12, 2005.
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References |
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