- Email: [email protected]
Cholesteryl Ester Storage Disease (CESD) due to novel mutations in the LIPA gene
Molecular Genetics and Metabolism 97 (2009) 143–148
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Cholesteryl Ester Storage Disease (CESD) due to novel mutations in the LIPA gene Livia Pisciotta a, Raffaele Fresa a, Antonella Bellocchio a, Elisabetta Pino a, Virgilia Guido a, Alfredo Cantafora b, Maja Di Rocco c, Sebastiano Calandra d,*, Stefano Bertolini a,* a
Department of Internal Medicine, University of Genoa, Viale Benedetto XV 6, I-16132 Genoa, Italy Deparment of Clinical Medicine, University of Rome ‘‘La Sapienza”, Rome, Italy c Institute G. Gaslini, Paediatrics II, University of Genoa, Italy d Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41100 Modena, Italy b
a r t i c l e
i n f o
Article history: Received 5 January 2009 Received in revised form 17 February 2009 Accepted 17 February 2009 Available online 26 February 2009 Keywords: Cholesteryl Ester Storage Disease (CESD) Lysosomal acid lipase (LAL) deficiency LIPA gene mutations Hepatosplenomegaly Mixed hyperlipidemia Hypoalphalipoproteinemia
a b s t r a c t Cholesteryl Ester Storage Disease (CESD) is a rare recessive disorder due to mutations in LIPA gene encoding the lysosomal acidic lipase (LAL). CESD patients have liver disease associated with mixed hyperlipidemia and low plasma levels of high-density lipoproteins (HDL). The aim of this study was the molecular characterization of three patients with CESD. LAL activity was measured in blood leukocytes. In two patients (twin sisters) the clinical diagnosis of CESD was made at 9 years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (5% of control) and heteroalleic mutations in LIPA gene coding sequence: (i) the common c.894 G>A mutation and (ii) a novel nonsense mutation c.652 C>T (p.R218X). The other patient was an 80 year-old female who for several years had been treated with simvastatin because of severe hyperlipidemia associated with low plasma HDL. In this patient the sequence of major candidate genes for monogenic hypercholesterolemia and hypoalphalipoproteinemia was negative. She was found to be a compound heterozygote for two LIPA gene mutations resulting in 5% LAL activity: (i) c.894 G>A and (ii) a novel complex insertion/deletion leading to a premature termination codon at position 82. These findings suggest that, in view of the variable severity of its phenotypic expression, CESD may sometimes be difficult to diagnose, but it should be considered in patients with severe type IIb hyperlipidemia associated with low HDL, mildly elevated serum liver enzymes and hepatomegaly. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Lysosomal acid lipase (LAL)1 (GenBank a.n. CAA83495) is a lysosomal enzyme which hydrolyzes cholesteryl esters (CE) and triglycerides (TG) internalized via receptor-mediated endocytosis of plasma lipoprotein particles [1–6]. The LAL-mediated release of free cholesterol (FC) within the cell causes down-regulation of HMG-CoA
* Corresponding authors. Fax: +39 059 2055426 (S. Calandra), +39 010 3537797 (S. Bertolini). E-mail addresses: [email protected] (S. Calandra), [email protected] (S. Bertolini). 1 Abbreviations used: LAL, lysosomal acid lipase; CESD, Cholesteryl Ester Storage Disease; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; CE, cholesteryl esters; FC, free cholesterol; TG, triglycerides; apoA-I, apolipoprotein A-I; apoB, apolipoprotein B; LDL, low-density lipoproteins; VLDL, very low-density lipoproteins; ACAT, acyl-CoA:cholesterol acyltrasferase; LIPA, HMGR, LDLR, APOB, PCSK9, APOE, ABCA1, APOA1 and LCAT genes: genes encoding lysosomal acid lipase, hydroxyl-methylglutaryl coenzyme A reductase, low-density lipoprotein receptor, apoB, proprotein covertase subtilisin/kexin type 9, apoE, ATP-binding cassette transporter A1, apoA-I and lecithin–cholesterol acetyltransferase, respectively; FCH, familial combined hyperlipidemia; NASH, non-alcoholic steato-hepatitis. 1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2009.02.007
reductase (HMGR) and LDL receptor (LDLR) genes and up-regulation of cholesterol esterification by the activation of ACAT enzyme [1,5]. In humans LAL is encoded by the LIPA gene (GenBank a.n. NG008194) located on chromosome 10 (10q23.2-q23.3) [7,8]. Homozygous and compound heterozygous mutations of this gene resulting in complete LAL deficiency are the cause of Wolman’s disease (OMIM +278000), a rare recessive disorder [9], characterized by massive storage of CE and TG in most tissues, failure to thrive and death, usually before one year of age [5,10]. Subjects carrying mutations in the LIPA gene which result in residual LAL activity (i.e. 2–8% of controls in blood leukocytes) develop the less severe disorder known as Cholesteryl Ester Storage Disease (CESD) [5,11–13]. CESD may be diagnosed in childhood or late in life, as its phenotypic expression shows a broad spectrum of severity of clinical manifestations [5,12,13]. The frequency of CESD in the population is presently unknown. A population survey of the heterozygous carriers of the most frequent LIPA gene mutation (c.894 G>A in exon 8) found in CESD patients suggests that the prevalence of CESD may be around 2.5/100,000 [14]. In the present work we report the molecular characterization of three patients with CESD, who were found to be compound
144
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
heterozygous, carrying the common LIPA gene mutation (c.894 G>A, del p.S275_Q298), in combination with two novel mutations resulting in null alleles. Methods
available for the study (subjects II.2 and III.1) were apparently healthy. Informed consent was obtained from probands and relatives involved in this study. The study protocol was approved by the institutional human investigation committee of each participating institution.
Family 1 Biochemical analyses The probands (subjects III.2 and III.3) (Fig. 1) were biovular twin sisters, in whom the clinical diagnosis of CESD was made at 9 years of age, when one of them was admitted to a paediatric hospital for mononucleosis infection. The first clue to the diagnosis was the finding of a persistent elevation of plasma liver enzymes and the presence of an otherwise unexplained hepatomegaly which required liver biopsy. The results of liver biopsy (see below) and of the assay of LAL activity in peripheral blood leukocytes led to the clinical diagnosis of CESD. At the age of 18 the sisters were referred to our Lipid Clinic for molecular diagnosis and treatment of a primary mixed hyperlipidemia. The probands’ parents (II.1 and II.2) and the probands’ sister (III.1) (Fig. 1) were apparently healthy, apart from moderate hypercholesterolemia. Family 2 The proband (subject I.2) (Fig. 1) was referred to the Lipid Clinic at the age of 68 because of a severe mixed hyperlipidemia, associated with low levels of HDL-C and apoA-I. After the exclusion of secondary hyperlipidemia, she started treatment with high doses of HMGR inhibitor (simvastatin 40 mg/day). She had been under treatment up to the age of 78. At the age of 68 she was found to have moderate hepatosplenomegaly and a mild elevation of serum liver enzymes. The monitoring of serum liver enzymes during statin treatment did not reveal significant changes with respect to pre-treatment values. At the age of 78 she was found to have myelodysplasia, mainly involving the erythroid lineage. For this reason statin therapy was discontinued. The molecular and clinical diagnosis of CESD was made at the age of 80 (see Results). The patient died from acute heart failure at 82. The other family members
Family 1
Genetic analyses Genomic DNA was extracted from peripheral blood leukocytes by a standard procedure [17]. All coding exons (exons 2–10) and flanking regions of the LIPA gene were amplified using the primers reported by Tadiboyina et al. [18] and sequenced directly by automatic sequencer CEQ2000 DNA Analysis System (Beckman Coulter, Fullerton, CA). The amplification conditions are specified in the Supplementary methods. The LIPA gene mutations found by sequencing were confirmed using independent methods, as described in Supplementary methods. In view of the high level of plasma cholesterol in the proband of Family 2, we analyzed the major candidate genes for monogenic hypercholesterolemia (LDLR, APOB and PCSK9) in this subject, using previously described methods [15]. Furthermore, since the proband of Family 2 had very low levels of HDL-C, we sequenced APOA1, ABCA1 and LCAT genes on the assumption that she had a monogenic form of HDL deficiency [19]. All subjects investigated were also genotyped for APOE polymorphism [20]. The LIPA gene mutations found in our patients were designated according to the Human Genome Variation Society (the numerical
Family 2
2
1
I
1
II
III
Plasma TC, TG, HDL-C, apoA-I and apoB concentrations were determined as described [15]. The assay of LAL activity was performed in probands’ peripheral blood leukocytes using p-nitrophenyl palmitate as substrate [16]. The results were expressed as nmol substrate hydrolyzed per mg soluble protein per min (the control values were 56.1 ± 7.6 nmol/mg protein/min).
1
2
2
II
3
1
I
III
1
2
2
1
c.894 G>A, del c.823_894 (del p.S275_Q298) c.652 C>T (p.R218X) c.230 ins 35 nt, del c.232_245 (p.G77fsX82) Fig. 1. Pedigrees of the three patients with CESD; the probands are indicated by an arrow. The probands of Family 1 were compound heterozygotes for the following LIPA gene mutations: (i) c.652 C>T in exon 6 (p.R218X), inherited from the mother and maternal grandfather; (ii) c.894 G>A in exon 8 (del p.S275_Q298), inherited from the father. The proband of Family 2 was a compound heterozygote for the following LIPA gene mutations: (i) c.230 ins 35 nt, del c.232_245 in exon 4 (p.G77fsX82); (ii) c.894 G>A in exon 8 (del p.S275_Q298). This latter mutation was transmitted to her daughter.
145
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148 Table 1 Genotypes, plasma lipids, liver function tests and clinical data of Family 1. Subject
I.1
I.2
II.1
II.2
III.1
III.2
III.3
LIPA genotype Age (years) BMI (kg/m2) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) ApoA-I (mg/dL) ApoB (mg/dL) AST (U/L) () ALT (U/L) () GGT (U/L) () Bilirubin (mg/dL) LAL activity () APOE genotype
W/M1 86 27.7 4.78 1.83 1.06 110 86
W/W 81 22.4 8.12 1.84 1.91 222 148
W/M2 52 26.2 6.08 1.05 1.75 140 103
W/M1 49 23.3 6.46 1.50 1.65 210 119
W/W 23 19.0 6.59 1.58 1.75 212 118
e3e3
e3e4
e3e3
e3e3
e3e3
M1/M2 18 20.7 8.26 ± 0.50 2.60 ± 0.25 0.91 ± 0.05 90.4 ± 4.5 159.6 ± 11.2 83.9 ± 19.1 186.7 ± 39.1 52.1 ± 6.5 1.42 ± 0.34 1.6 e3e3
M1/M2 18 17.3 7.20 ± 0.44 1.65 ± 0.34 0.97 ± 0.06 96.2 ± 4.2 132.6 ± 7.2 72.7 ± 16.8 114.5 ± 16.3 47.6 ± 7.2 1.44 ± 0.24 2.5 e3e3
Values are mean ± SD; () reference values: AST (10–30 U/L), ALT (10–36 U/L), GGT (7–39 U/L), LAL activity (56.1 ± 7.6 nmol/mg protein/min); LIPA genotype: W = wild type, M1 = c.652 C>T (p.R218X), M2 = c.894 G>A (del p.S275_Q298).
series of codons includes the sequence of the signal peptide, www.hgvs.org/mutnomen). Results Family 1 Plasma lipids Table 1 shows the pre-treatment plasma lipid levels in the two probands (III.2 and III.3) and their relatives. The affected sisters were found to have a marked elevation of TC, TG and ApoB, associated with a moderate reduction of HDL-C and apoA-I. Liver biopsy The liver biopsy was performed only in proband III.3 when she was 9 years old. Light-microscopy and immunohistochemistry showed massive vesicular steatosis, portal and septal fibrosis, portal infiltration of macrophages (CD68-positive) and lymphoid cells with abnormal lipid deposits and lipid droplets in hepatocytes and Kupffer cells. Electron microscopy demonstrated lysosomal lipid storage in hepatocytes, macrophages and Kupffer cells, as well as the presence of birefringent cholesteryl ester crystals in hepatocytes. LAL activity LAL activity measured in peripheral blood leukocytes was slightly below 5% of the control values (Table 1). DNA sequence analysis The sequence of the LIPA gene (Fig. 2) showed that both probands were compound heterozygous for the following mutations: 1) c.652 C>T transition in exon 6, which converts the CGA codon (Arginine) into a termination codon (TGA) (p.R218X) – this mutation was present in probands’ mother (II.2) and in the maternal grandfather (I.1); 2) c.894 G>A transition in the last nucleotide of exon 8, which does not change the amino acid (p.Q298Q) but causes the skipping of exon 8 (in-frame deletion of 24 amino acids: del p.S275_Q298), as previously demonstrated by Klima et al. [21] – this mutation was present in probands’ father (II.1) (Figs. 1 and 2). No mutations were found in the probands’ sister (III.1). Family 2 Plasma lipids Table 2 shows the pre-treatment plasma lipid levels of the proband (I.2) and her relatives (II.2 and III.1). The striking elevation of
TC and apoB found in the proband suggested that she might have heterozygous familial hypercholesterolemia, possibly associated with some defect of HDL metabolism, causing plasma HDL deficiency [19]. DNA sequence analysis On the assumption that the proband had familial hypercholesterolemia, we sequenced first the three major candidate genes underlying this disorder (LDLR, APOB and PCSK9), but no pathogenic mutations were detected. In view of the very low level of HDL-C, we sequenced APOA1, ABCA1 and LCAT genes, but no pathogenic mutations were found in any of these genes. Next, taking into account that the proband had type IIb hyperlipidemia, associated with an otherwise unexplained hepatosplenomegaly, and that many cases of CESD reported in literature were found to have low HDL levels, we decided to sequence the LIPA gene. We found that the proband was compound heterozygous for the following mutations: (1) c.894 G>A in exon 8 [21]; (2) a complex rearrangement involving exon 4, consisting of: (a) the insertion of the last 33 nucleotides of intron 3 and of the first two nucleotides of exon 4 (c.229 and c.230) downstream from nucleotide c.230; (b) the deletion of 14 nucleotides at the 50 of exon 4 (del c.232_245) (Fig. 3). This mutation disrupts the reading frame, causing the insertion of a premature termination codon at position 82 (Fig. 4). The predicted product of this mutant allele is a short peptide of 81 amino acids, as compared to the 378 amino acids of the mature wild type LAL. The proband’s daughter (II.2) was found to carry the c.894 G>A mutation. LAL activity In view of this unexpected finding, we measured LAL activity in proband’s peripheral blood leukocytes and found that the residual LAL activity was close to 5% of the control values (Table 2). Screening of the LIPA gene mutations in control subjects The three LIPA gene mutations found in the two families were screened in 150 randomly selected control subjects. No carriers of these mutations were found. Screening of the common LIPA gene mutation (c.894 G>A) in patients with type IIb hyperlipidemia Since the lipoprotein pattern observed in CESD is similar to that found in subjects with type IIb hyperlipidemia, we screened 150 unrelated type IIb patients, including 100 patients with Familial Combined Hyperlipidemia (FCH) (102 males and 48 females, 50.0 ± 9.8 years of age, TC 8.32 ± 0.85, HDL-C 1.08 ± 0.19 and TG 3.69 ± 0.98 mmol/L, apoB 145 ± 13 mg/dL), for the most common
146
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
Fig. 2. LIPA gene mutations in the probands of Family 1. (A) Left panel: partial sequence of exon 6 of the LIPA gene in one of the probands of Family 1. The nucleotide substitution c.652 C>T is highlighted at the top of the panel. (B) Right panel: agarose gel electrophoresis of digestion fragments of exon 6 generated by the restriction enzyme BclI. Mw = molecular weight markers; M = carriers of c.652 C>T mutation; W = control subject (see Supplementary methods). (C) Left panel: partial sequence of exon 8-intron eight junction of the LIPA gene in one of the probands of Family 1. The nucleotide substitution c.894 G>A is highlighted at the top of the panel. (D) Right panel: agarose gel electrophoresis of digestion fragments of exon 8 generated by the restriction enzyme Sty I. Mw = molecular weight marker; M = carriers of c.894 G>A mutation; W = control subject (see Supplementary methods).
Discussion
Table 2 Genotypes, plasma lipids, liver function tests and clinical data of Family 2. Subject
I.2
II.2
III.1
LIPA genotype Age (years) BMI (kg/m2) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) ApoA-I (mg/dL) ApoB (mg/dL) AST (U/L) () ALT (U/L) () GGT (U/L) () Bilirubin (mg/dL) LAL activity () APOE genotype
M2/M3 80 17.6 10.17 ± 0.68 3.36 ± 0.46 0.56 ± 0.10 87.2 ± 6.6 197.8 ± 15.4 46.2 ± 2.3 45.0 ± 3.0 29 ± 2.0 0.80 ± 0.21 3.0 e3e3
W/M2 52 24.9 6.69 0.63 2.02 198 118
W/W 18 20.2 4.24 0.69 1.19 150 57
e3e3
e3e3
Values are mean ± SD; () reference values: AST (10–30 U/L), ALT (10–36 U/L), GGT (7–39 U/L), LAL activity (56.1 ± 7.6 nmol/mg protein/min); LIPA genotype: W = wild type, M2 = c.894 G>A (del p.S275_Q298), M3 = c.230 ins 35 nt, del c.232_245 (p.G77>frameshift > 82).
LIPA gene mutation (c.894 G>A). One subject was found to be heterozygous for this mutation. The sequence of the LIPA gene in this subject failed to reveal other mutations. This observation suggests that the frequency of this mutant allele in our patients with type IIb hyperlipidemia (1:150) is close to that expected in the general population (1:200) [14].
In this paper we describe three patients, from two families, affected by CESD. In the twin sisters of Family 1 the clinical diagnosis was made in the first decade of life, following the fortuitous observation of a persistent elevation of serum liver enzymes in apparently healthy children, which prompted the paediatrician to perform liver biopsy, followed by LAL activity assay in peripheral blood leukocytes. In striking contrast, the diagnosis of CESD in the proband of Family 2 was made late in life (80 years), during the investigation of the genetic bases of her complex dyslipidemia. The proband of Family 2 is the oldest CESD patient reported so far (see Supplementary Table and references on line), suggesting that some patients may be asymptomatic for many years and accidentally discovered during routine clinical investigations. The three subjects we investigated were found to be compound heterozygous for LIPA gene mutations. They shared the c.894 G>A substitution in exon 8, which is the most frequent mutant allele in CESD patients (see Supplementary Table and references on line). This mutation causes an abnormal splicing with the formation of an abnormal mRNA devoid of exon 8, whose translation product is a peptide of 375 amino acids (354 in the mature protein) due to the in-frame deletion of 24 amino acids (del p.S275_Q298) [21]. This abnormal protein has no activity [22]. However, despite the presence of the mutation, 3–5% of LIPA pre-mRNA is spliced correctly, accounting for a residual LAL activity in peripheral blood leukocytes corresponding to about 5% of control values [12,23,24].
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
147
Fig. 3. LIPA gene mutations in the proband of Family 2. (A) Left panel: partial sequence of intron 3-exon four junction of the LIPA gene in the probands of Family 2. The initial portion of nucleotide sequence inserted (GCTACT) is superimposed on the wild type sequence (CCCAAA); both sequences are highlighted at the top of the panel. (B) Right panel: polyacrylamide gel electrophoresis of the amplified fragments of intron 3-exon four junction. mw = molecular weight markers; M = carrier of the insertion of 35 nucleotides after position c.230; W = control subjects (see Supplementary methods). (C) Left panel: partial sequence of exon 8-intron eight junction of the LIPA gene in the proband of Family 2. The nucleotide substitution c.894 G>A is highlighted at the top of the panel. (D) Right panel: agarose gel electrophoresis of digestion fragments of exon 8 generated by the restriction enzyme Sty I. mw = molecular weight marker; M = carrier of c.894 G>A mutation; W = control subjects (see Supplementary methods).
WILD type Exon 4
3’ Intron 3
…ggt gct act gcc tcc taa aca atg aat gtt ttt cag GT Exon 3
Exon 4
… AAA G GT CCC AAA CCA GTT GTC TTC CTG CAA CAT GGCTTC … Lys Gly77 Pro Lys Pro Val Val Phe Leu Gln His Gly Leu88 MUTANT (ins 35 nt, del 14 nt) Exon 3
Exon 4
… AAA G GT gct act gcc tcc taa aca atg aat gtt ttt cag GTC TTC CTG CAA … Lys Gly77 Ala ThrAla Ser Term 82 Fig. 4. Top figure shows the wild type nucleotide sequence of exon 3-exon four junction and its translation product. The inserted sequence, after the nucleotide c.230 T of exon 4, which includes the last 33 nucleotides of intron 3 and the first two nucleotides of exon 4, is boxed. The nucleotide sequence (c.232_245) which is deleted in the mutant allele is underlined. Bottom figure shows the sequence of the mutant allele in proband of Family 2 and the predicted translation product (frameshift after Gly77>Term82).
The other LIPA gene mutations present in our patients (p.R218X and p.G77>frameshift78>X82) are expected to abolish LAL activity. In this context the residual capacity to hydrolyze CE and TG in hepatocytes and Kupffer cells of our patients depends entirely on the amount of normally spliced LIPA mRNA available in these cells. The percentage of correctly spliced mRNA (and the corresponding residual LAL activity) in the liver might be different among CESD patients carrying c.894 G>A mutation. Indeed, the residual LAL activity measured in fibroblasts of patients homozygous for c.894 G>A mutation was found to vary from 2% to 12% [24–26].
One of the key features of our patients was mixed hyperlipidemia (type IIb hyperlipidemia). The pre-treatment levels of TC, TG and apoB in our patients (Tables 1 and 2) were comparable to those found in 57 CESD patients reported in literature (TC 8.47 ± 1.60, TG 2.48 ± 1.03 mmol/L and apoB 196.0 ± 41.9 mg/dL). The mechanism underlying this hyperlipidemia is related to an increased production rate of apoB in VLDL and LDL with a normal fractional catabolic rate [27,28]. In our probands we found low levels of HDL-C and apoA-I, comparable with those previously reported in other CESD patients (HDL-C 0.72 ± 0.22 mmol/L and apoA-I 86.4 ± 20.9 mg/dL) (see Supplementary references on line). These levels are below the 5th percentile of the distribution in the population and similar to those observed in subjects heterozygous for mutations in ABCA1, APOA1 and LCAT genes [19,29,30], but lower that those found in patients with familial combined hyperlipidemia [29]. The mechanism responsible for the low plasma HDL is presently unknown. In CESD cells the FC pool is reduced and, most likely, less FC is transported from the intracellular compartments to the plasma membrane. This defect could affect ABCA1-mediated cholesterol efflux from the cell membrane to extracellular acceptors, such as lipid-poor apoA-I particles, a process which leads to the formation of pre-b-HDL in the liver and ultimately to the formation of mature plasma HDL [31]. In view of these findings the criteria for the clinical diagnosis of probable CESD might be the following: (i) a recessive form of severe mixed hyperlidemia with markedly reduced plasma HDL levels; (ii) the presence of enlarged liver of unknown etiology associated with increased serum liver enzymes; (iii) the exclusion of some clinical conditions such as metabolic syndrome, type 2 diabetes, NASH and FCH.
148
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
Acknowledgments This work was supported by grants from the University of Genova (S.B.) and University of Modena and Reggio Emilia (S.C.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymgme.2009.02.007. References [1] J.L. Goldstein, S.E. Dana, J.R. Faust, A.L. Beaudet, M.S. Brown, Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease, J. Biol. Chem. 250 (1975) 8487–8495. [2] G.N. Sando, V.L. Henke, Recognition and receptor-mediated endocytosis of the lysosomal acid lipase secreted by cultured human fibroblasts, J. Lipid Res. 23 (1982) 114–123. [3] D. Ameis, M. Merkel, C. Eckerskorn, H. Greten, Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase, Eur. J. Biochem. 219 (1994) 905–914. [4] S. Sheriff, H. Du, G.A. Grabowski, Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression, J. Biol. Chem. 270 (1995) 27766–27772. [5] G. Assmann, U. Seedorf, Acid Lipase Deficiency: Wolman Disease and Cholesteryl Ester Storage Disease, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic & Molecular Bases of Inherited Disease, McGrawHill, New York, 2001, pp. 3551–3572. [6] R.A. Anderson, G.N. Sando, Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases, J. Biol. Chem. 266 (1991) 22479–22484. [7] R.A. Anderson, N. Rao, R.S. Byrum, C.B. Rothschild, D.W. Bowden, R. Hayworth, M. Pettenati, In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in Wolman disease to chromosome 10q23.2-q23.3, Genomics 15 (1993) 245–247. [8] C. Aslanidis, H. Klima, K.J. Lackner, G. Schmitz, Genomic organization of the human lysosomal acid lipase gene (LIPA), Genomics 20 (1994) 329–331. [9] P.J. Meikle, J.J. Hopwood, A.E. Clague, W.F. Carey, Prevalence of lysosomal storage disorders, JAMA 281 (1999) 249–254. [10] B.K. Burton, S. Reed, Acid lipase cross-reacting material in Wolman Disease and Cholesterol Ester Storage Disease, Am. J. Hum. Genet. 33 (1981) 203–208. [11] J.M. Hoeg, S.J. Demosky, O.H. Pescovitz, H.B. Brewer Jr, Cholesteryl ester storage disease and Wolman disease: phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency, Am. J. Hum. Genet. 36 (1984) 1190– 1203. [12] C. Aslanidis, S. Ries, P. Fehringer, C. Büchler, H. Klima, G. Schmitz, Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity, Genomics 33 (1996) 85–93. [13] P. Lohse, S. Maas, P. Lohse, M. Elleder, J.M. Kirk, G.T.N. Besley, D. Seidelet, Compound heterozygosity for a Wolman mutation is frequent among patients with cholesteryl ester storage disease, J. Lipid Res. 41 (2000) 23–31. [14] S. Muntoni, H. Wiebusch, M. Jansen-Rust, S. Rust, U. Seedorf, H. Schulte, K. Berger, H. Funke, G. Assmann, Prevalence of cholesteryl ester storage disease, Artrioscler. Thromb. Vasc. Biol. 27 (2007) 1866–1868.
[15] L. Pisciotta, C.P. Oliva, A.B. Cefalu, D. Noto, A. Bellocchio, R. Fresa, A. Cantafora, D. Patel, M. Averna, P. Tarugi, S. Calandra, S. Bertolini, Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia, Atherosclerosis 186 (2006) 433–440. [16] A.D. Patrick, P. Willcox, R. Stephens, V.G. Kenyon, Prenatal diagnosis of Wolman’s disease, J. Med. Genet. 13 (1976) 49–51. [17] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. [18] V. Tadiboyina, D.M. Liu, B.A. Miskie, J. Wang, R.A. Hegele, Treatment of dyslipidemia with lovastatin and ezetimibe in an adolescent with cholesterol ester storage disease, Lipids Health Dis. 4 (2005) 26 (On line). [19] L. Pisciotta, I. Hamilton-Craig, P. Tarugi, A. Bellocchio, T. Fasano, P. Alessandrini, G. Bittolo Bon, D. Siepi, E. Mannarino, L. Cattin, M. Averna, B. Cefalù, A. Cantafora, S. Calandra, S. Bertolini, Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders, Atherosclerosis 172 (2004) 329–340. [20] J.E. Hixson, D.T. Vernier, Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI, J. Lipid Res. 31 (1990) 545–548. [21] H. Klima, K. Ullrich, C. Aslanidis, P. Fehringer, K.J. Lackner, G. Schmitz, A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease, J. Clin. Invest. 92 (1993) 2713–2718. [22] F. Pagani, R. Pariyarath, R. Garcia, C. Stuani, A.B. Burlina, G. Ruotolo, M. Rabusin, F.E. Baralle, New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease, J. Lipid Res. 39 (1998) 1382–1388. [23] D. Ameis, G. Brokmann, R. Knoblich, M. Merkel, R.E. Ostlund Jr., J.W. Yang, P.M. Coates, J.A. Cortner, S.V. Feinman, H. Greten, A 5’ splice-site mutation and a dinucleotide deletion in the lysosomal acid lipase gene in two patients with cholesteryl ester storage disease, J. Lipid Res. 36 (1995) 241–250. [24] H. Du, S. Sheriff, J. Bezerra, T. Leonova, G.A. Grabowski, Molecular and enzymatic analyses of lysosomal acid lipase in cholesteryl ester storage disease, Mol. Genet. Metab. 64 (1998) 126–134. [25] R.A. Anderson, G.M. Bryson, J.S. Parks, Lysosomal acid lipase mutations that determine phenotype in Wolman and Cholesteryl Ester Storage Disease, Mol. Genet. Metab. 68 (1999) 333–345. [26] S. vom Dahl, K. Hazer, A. Rolfs, B. Albrecht, C. Niedereau, C. Vogt, S. van Weely, J. Aerts, G. Müller, D. Häussinger, Hepatosplenomegalic lipidosis: what unless Gaucher? Adult cholesteryl ester storage disease (CESD) with anemia, mesenteric lipodystrophy, increased plasma chitotriosidase activity and a homozygous lysosomal acid lipase-1 exon 8 splice junction mutation, J. Hepatol. 31 (1999) 741–746. [27] H.N. Ginsberg, N.-A. Le, M.P. Short, R. Ramakrishnan, R.J. Desnick, Suppression of apolipoprotein B production during treatment of Cholesteryl Ester Storage Disease with Lovastatin. Implications for regulation of apolipoprotein B synthesis, J. Clin. Invest. 80 (1987) 1692–1697. [28] M. Cummings, G.F. Watts, Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in cholesteryl ester storage disease, Clin. Chem. 41 (1995) 111–114. [29] L. Pisciotta, T. Fasano, L. Calabresi, A. Bellocchio, R. Fresa, C. Borrini, S. Calandra, S. Bertolini, A novel mutation of apolipoprotein A-I gene in a family with familial combined hyperlipidemia, Atherosclerosis 198 (2008) 145–151. [30] L. Pisciotta, L. Calabresi, G. Lupattelli, G. Siepi, M.R. Mannarino, E. Moleri, A. Bellocchio, A. Cantafora, P. Tarugi, S. Calandra, S. Bertolini, Combined monogenic hypercholesterolemia and hypoalphalipoproteinemia caused by mutations in LDL-Receptor and LCAT genes, Atherosclerosis 182 (2005) 153– 159. [31] D.J. Rader, Molecular regulation of HDL metabolism and function: implication for novel therapies, J. Clin. Invest. 116 (2006) 3090–3100.
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Cholesteryl Ester Storage Disease (CESD) due to novel mutations in the LIPA gene Livia Pisciotta a, Raffaele Fresa a, Antonella Bellocchio a, Elisabetta Pino a, Virgilia Guido a, Alfredo Cantafora b, Maja Di Rocco c, Sebastiano Calandra d,*, Stefano Bertolini a,* a
Department of Internal Medicine, University of Genoa, Viale Benedetto XV 6, I-16132 Genoa, Italy Deparment of Clinical Medicine, University of Rome ‘‘La Sapienza”, Rome, Italy c Institute G. Gaslini, Paediatrics II, University of Genoa, Italy d Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41100 Modena, Italy b
a r t i c l e
i n f o
Article history: Received 5 January 2009 Received in revised form 17 February 2009 Accepted 17 February 2009 Available online 26 February 2009 Keywords: Cholesteryl Ester Storage Disease (CESD) Lysosomal acid lipase (LAL) deficiency LIPA gene mutations Hepatosplenomegaly Mixed hyperlipidemia Hypoalphalipoproteinemia
a b s t r a c t Cholesteryl Ester Storage Disease (CESD) is a rare recessive disorder due to mutations in LIPA gene encoding the lysosomal acidic lipase (LAL). CESD patients have liver disease associated with mixed hyperlipidemia and low plasma levels of high-density lipoproteins (HDL). The aim of this study was the molecular characterization of three patients with CESD. LAL activity was measured in blood leukocytes. In two patients (twin sisters) the clinical diagnosis of CESD was made at 9 years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (5% of control) and heteroalleic mutations in LIPA gene coding sequence: (i) the common c.894 G>A mutation and (ii) a novel nonsense mutation c.652 C>T (p.R218X). The other patient was an 80 year-old female who for several years had been treated with simvastatin because of severe hyperlipidemia associated with low plasma HDL. In this patient the sequence of major candidate genes for monogenic hypercholesterolemia and hypoalphalipoproteinemia was negative. She was found to be a compound heterozygote for two LIPA gene mutations resulting in 5% LAL activity: (i) c.894 G>A and (ii) a novel complex insertion/deletion leading to a premature termination codon at position 82. These findings suggest that, in view of the variable severity of its phenotypic expression, CESD may sometimes be difficult to diagnose, but it should be considered in patients with severe type IIb hyperlipidemia associated with low HDL, mildly elevated serum liver enzymes and hepatomegaly. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Lysosomal acid lipase (LAL)1 (GenBank a.n. CAA83495) is a lysosomal enzyme which hydrolyzes cholesteryl esters (CE) and triglycerides (TG) internalized via receptor-mediated endocytosis of plasma lipoprotein particles [1–6]. The LAL-mediated release of free cholesterol (FC) within the cell causes down-regulation of HMG-CoA
* Corresponding authors. Fax: +39 059 2055426 (S. Calandra), +39 010 3537797 (S. Bertolini). E-mail addresses: [email protected] (S. Calandra), [email protected] (S. Bertolini). 1 Abbreviations used: LAL, lysosomal acid lipase; CESD, Cholesteryl Ester Storage Disease; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; CE, cholesteryl esters; FC, free cholesterol; TG, triglycerides; apoA-I, apolipoprotein A-I; apoB, apolipoprotein B; LDL, low-density lipoproteins; VLDL, very low-density lipoproteins; ACAT, acyl-CoA:cholesterol acyltrasferase; LIPA, HMGR, LDLR, APOB, PCSK9, APOE, ABCA1, APOA1 and LCAT genes: genes encoding lysosomal acid lipase, hydroxyl-methylglutaryl coenzyme A reductase, low-density lipoprotein receptor, apoB, proprotein covertase subtilisin/kexin type 9, apoE, ATP-binding cassette transporter A1, apoA-I and lecithin–cholesterol acetyltransferase, respectively; FCH, familial combined hyperlipidemia; NASH, non-alcoholic steato-hepatitis. 1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2009.02.007
reductase (HMGR) and LDL receptor (LDLR) genes and up-regulation of cholesterol esterification by the activation of ACAT enzyme [1,5]. In humans LAL is encoded by the LIPA gene (GenBank a.n. NG008194) located on chromosome 10 (10q23.2-q23.3) [7,8]. Homozygous and compound heterozygous mutations of this gene resulting in complete LAL deficiency are the cause of Wolman’s disease (OMIM +278000), a rare recessive disorder [9], characterized by massive storage of CE and TG in most tissues, failure to thrive and death, usually before one year of age [5,10]. Subjects carrying mutations in the LIPA gene which result in residual LAL activity (i.e. 2–8% of controls in blood leukocytes) develop the less severe disorder known as Cholesteryl Ester Storage Disease (CESD) [5,11–13]. CESD may be diagnosed in childhood or late in life, as its phenotypic expression shows a broad spectrum of severity of clinical manifestations [5,12,13]. The frequency of CESD in the population is presently unknown. A population survey of the heterozygous carriers of the most frequent LIPA gene mutation (c.894 G>A in exon 8) found in CESD patients suggests that the prevalence of CESD may be around 2.5/100,000 [14]. In the present work we report the molecular characterization of three patients with CESD, who were found to be compound
144
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
heterozygous, carrying the common LIPA gene mutation (c.894 G>A, del p.S275_Q298), in combination with two novel mutations resulting in null alleles. Methods
available for the study (subjects II.2 and III.1) were apparently healthy. Informed consent was obtained from probands and relatives involved in this study. The study protocol was approved by the institutional human investigation committee of each participating institution.
Family 1 Biochemical analyses The probands (subjects III.2 and III.3) (Fig. 1) were biovular twin sisters, in whom the clinical diagnosis of CESD was made at 9 years of age, when one of them was admitted to a paediatric hospital for mononucleosis infection. The first clue to the diagnosis was the finding of a persistent elevation of plasma liver enzymes and the presence of an otherwise unexplained hepatomegaly which required liver biopsy. The results of liver biopsy (see below) and of the assay of LAL activity in peripheral blood leukocytes led to the clinical diagnosis of CESD. At the age of 18 the sisters were referred to our Lipid Clinic for molecular diagnosis and treatment of a primary mixed hyperlipidemia. The probands’ parents (II.1 and II.2) and the probands’ sister (III.1) (Fig. 1) were apparently healthy, apart from moderate hypercholesterolemia. Family 2 The proband (subject I.2) (Fig. 1) was referred to the Lipid Clinic at the age of 68 because of a severe mixed hyperlipidemia, associated with low levels of HDL-C and apoA-I. After the exclusion of secondary hyperlipidemia, she started treatment with high doses of HMGR inhibitor (simvastatin 40 mg/day). She had been under treatment up to the age of 78. At the age of 68 she was found to have moderate hepatosplenomegaly and a mild elevation of serum liver enzymes. The monitoring of serum liver enzymes during statin treatment did not reveal significant changes with respect to pre-treatment values. At the age of 78 she was found to have myelodysplasia, mainly involving the erythroid lineage. For this reason statin therapy was discontinued. The molecular and clinical diagnosis of CESD was made at the age of 80 (see Results). The patient died from acute heart failure at 82. The other family members
Family 1
Genetic analyses Genomic DNA was extracted from peripheral blood leukocytes by a standard procedure [17]. All coding exons (exons 2–10) and flanking regions of the LIPA gene were amplified using the primers reported by Tadiboyina et al. [18] and sequenced directly by automatic sequencer CEQ2000 DNA Analysis System (Beckman Coulter, Fullerton, CA). The amplification conditions are specified in the Supplementary methods. The LIPA gene mutations found by sequencing were confirmed using independent methods, as described in Supplementary methods. In view of the high level of plasma cholesterol in the proband of Family 2, we analyzed the major candidate genes for monogenic hypercholesterolemia (LDLR, APOB and PCSK9) in this subject, using previously described methods [15]. Furthermore, since the proband of Family 2 had very low levels of HDL-C, we sequenced APOA1, ABCA1 and LCAT genes on the assumption that she had a monogenic form of HDL deficiency [19]. All subjects investigated were also genotyped for APOE polymorphism [20]. The LIPA gene mutations found in our patients were designated according to the Human Genome Variation Society (the numerical
Family 2
2
1
I
1
II
III
Plasma TC, TG, HDL-C, apoA-I and apoB concentrations were determined as described [15]. The assay of LAL activity was performed in probands’ peripheral blood leukocytes using p-nitrophenyl palmitate as substrate [16]. The results were expressed as nmol substrate hydrolyzed per mg soluble protein per min (the control values were 56.1 ± 7.6 nmol/mg protein/min).
1
2
2
II
3
1
I
III
1
2
2
1
c.894 G>A, del c.823_894 (del p.S275_Q298) c.652 C>T (p.R218X) c.230 ins 35 nt, del c.232_245 (p.G77fsX82) Fig. 1. Pedigrees of the three patients with CESD; the probands are indicated by an arrow. The probands of Family 1 were compound heterozygotes for the following LIPA gene mutations: (i) c.652 C>T in exon 6 (p.R218X), inherited from the mother and maternal grandfather; (ii) c.894 G>A in exon 8 (del p.S275_Q298), inherited from the father. The proband of Family 2 was a compound heterozygote for the following LIPA gene mutations: (i) c.230 ins 35 nt, del c.232_245 in exon 4 (p.G77fsX82); (ii) c.894 G>A in exon 8 (del p.S275_Q298). This latter mutation was transmitted to her daughter.
145
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148 Table 1 Genotypes, plasma lipids, liver function tests and clinical data of Family 1. Subject
I.1
I.2
II.1
II.2
III.1
III.2
III.3
LIPA genotype Age (years) BMI (kg/m2) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) ApoA-I (mg/dL) ApoB (mg/dL) AST (U/L) () ALT (U/L) () GGT (U/L) () Bilirubin (mg/dL) LAL activity () APOE genotype
W/M1 86 27.7 4.78 1.83 1.06 110 86
W/W 81 22.4 8.12 1.84 1.91 222 148
W/M2 52 26.2 6.08 1.05 1.75 140 103
W/M1 49 23.3 6.46 1.50 1.65 210 119
W/W 23 19.0 6.59 1.58 1.75 212 118
e3e3
e3e4
e3e3
e3e3
e3e3
M1/M2 18 20.7 8.26 ± 0.50 2.60 ± 0.25 0.91 ± 0.05 90.4 ± 4.5 159.6 ± 11.2 83.9 ± 19.1 186.7 ± 39.1 52.1 ± 6.5 1.42 ± 0.34 1.6 e3e3
M1/M2 18 17.3 7.20 ± 0.44 1.65 ± 0.34 0.97 ± 0.06 96.2 ± 4.2 132.6 ± 7.2 72.7 ± 16.8 114.5 ± 16.3 47.6 ± 7.2 1.44 ± 0.24 2.5 e3e3
Values are mean ± SD; () reference values: AST (10–30 U/L), ALT (10–36 U/L), GGT (7–39 U/L), LAL activity (56.1 ± 7.6 nmol/mg protein/min); LIPA genotype: W = wild type, M1 = c.652 C>T (p.R218X), M2 = c.894 G>A (del p.S275_Q298).
series of codons includes the sequence of the signal peptide, www.hgvs.org/mutnomen). Results Family 1 Plasma lipids Table 1 shows the pre-treatment plasma lipid levels in the two probands (III.2 and III.3) and their relatives. The affected sisters were found to have a marked elevation of TC, TG and ApoB, associated with a moderate reduction of HDL-C and apoA-I. Liver biopsy The liver biopsy was performed only in proband III.3 when she was 9 years old. Light-microscopy and immunohistochemistry showed massive vesicular steatosis, portal and septal fibrosis, portal infiltration of macrophages (CD68-positive) and lymphoid cells with abnormal lipid deposits and lipid droplets in hepatocytes and Kupffer cells. Electron microscopy demonstrated lysosomal lipid storage in hepatocytes, macrophages and Kupffer cells, as well as the presence of birefringent cholesteryl ester crystals in hepatocytes. LAL activity LAL activity measured in peripheral blood leukocytes was slightly below 5% of the control values (Table 1). DNA sequence analysis The sequence of the LIPA gene (Fig. 2) showed that both probands were compound heterozygous for the following mutations: 1) c.652 C>T transition in exon 6, which converts the CGA codon (Arginine) into a termination codon (TGA) (p.R218X) – this mutation was present in probands’ mother (II.2) and in the maternal grandfather (I.1); 2) c.894 G>A transition in the last nucleotide of exon 8, which does not change the amino acid (p.Q298Q) but causes the skipping of exon 8 (in-frame deletion of 24 amino acids: del p.S275_Q298), as previously demonstrated by Klima et al. [21] – this mutation was present in probands’ father (II.1) (Figs. 1 and 2). No mutations were found in the probands’ sister (III.1). Family 2 Plasma lipids Table 2 shows the pre-treatment plasma lipid levels of the proband (I.2) and her relatives (II.2 and III.1). The striking elevation of
TC and apoB found in the proband suggested that she might have heterozygous familial hypercholesterolemia, possibly associated with some defect of HDL metabolism, causing plasma HDL deficiency [19]. DNA sequence analysis On the assumption that the proband had familial hypercholesterolemia, we sequenced first the three major candidate genes underlying this disorder (LDLR, APOB and PCSK9), but no pathogenic mutations were detected. In view of the very low level of HDL-C, we sequenced APOA1, ABCA1 and LCAT genes, but no pathogenic mutations were found in any of these genes. Next, taking into account that the proband had type IIb hyperlipidemia, associated with an otherwise unexplained hepatosplenomegaly, and that many cases of CESD reported in literature were found to have low HDL levels, we decided to sequence the LIPA gene. We found that the proband was compound heterozygous for the following mutations: (1) c.894 G>A in exon 8 [21]; (2) a complex rearrangement involving exon 4, consisting of: (a) the insertion of the last 33 nucleotides of intron 3 and of the first two nucleotides of exon 4 (c.229 and c.230) downstream from nucleotide c.230; (b) the deletion of 14 nucleotides at the 50 of exon 4 (del c.232_245) (Fig. 3). This mutation disrupts the reading frame, causing the insertion of a premature termination codon at position 82 (Fig. 4). The predicted product of this mutant allele is a short peptide of 81 amino acids, as compared to the 378 amino acids of the mature wild type LAL. The proband’s daughter (II.2) was found to carry the c.894 G>A mutation. LAL activity In view of this unexpected finding, we measured LAL activity in proband’s peripheral blood leukocytes and found that the residual LAL activity was close to 5% of the control values (Table 2). Screening of the LIPA gene mutations in control subjects The three LIPA gene mutations found in the two families were screened in 150 randomly selected control subjects. No carriers of these mutations were found. Screening of the common LIPA gene mutation (c.894 G>A) in patients with type IIb hyperlipidemia Since the lipoprotein pattern observed in CESD is similar to that found in subjects with type IIb hyperlipidemia, we screened 150 unrelated type IIb patients, including 100 patients with Familial Combined Hyperlipidemia (FCH) (102 males and 48 females, 50.0 ± 9.8 years of age, TC 8.32 ± 0.85, HDL-C 1.08 ± 0.19 and TG 3.69 ± 0.98 mmol/L, apoB 145 ± 13 mg/dL), for the most common
146
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
Fig. 2. LIPA gene mutations in the probands of Family 1. (A) Left panel: partial sequence of exon 6 of the LIPA gene in one of the probands of Family 1. The nucleotide substitution c.652 C>T is highlighted at the top of the panel. (B) Right panel: agarose gel electrophoresis of digestion fragments of exon 6 generated by the restriction enzyme BclI. Mw = molecular weight markers; M = carriers of c.652 C>T mutation; W = control subject (see Supplementary methods). (C) Left panel: partial sequence of exon 8-intron eight junction of the LIPA gene in one of the probands of Family 1. The nucleotide substitution c.894 G>A is highlighted at the top of the panel. (D) Right panel: agarose gel electrophoresis of digestion fragments of exon 8 generated by the restriction enzyme Sty I. Mw = molecular weight marker; M = carriers of c.894 G>A mutation; W = control subject (see Supplementary methods).
Discussion
Table 2 Genotypes, plasma lipids, liver function tests and clinical data of Family 2. Subject
I.2
II.2
III.1
LIPA genotype Age (years) BMI (kg/m2) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) ApoA-I (mg/dL) ApoB (mg/dL) AST (U/L) () ALT (U/L) () GGT (U/L) () Bilirubin (mg/dL) LAL activity () APOE genotype
M2/M3 80 17.6 10.17 ± 0.68 3.36 ± 0.46 0.56 ± 0.10 87.2 ± 6.6 197.8 ± 15.4 46.2 ± 2.3 45.0 ± 3.0 29 ± 2.0 0.80 ± 0.21 3.0 e3e3
W/M2 52 24.9 6.69 0.63 2.02 198 118
W/W 18 20.2 4.24 0.69 1.19 150 57
e3e3
e3e3
Values are mean ± SD; () reference values: AST (10–30 U/L), ALT (10–36 U/L), GGT (7–39 U/L), LAL activity (56.1 ± 7.6 nmol/mg protein/min); LIPA genotype: W = wild type, M2 = c.894 G>A (del p.S275_Q298), M3 = c.230 ins 35 nt, del c.232_245 (p.G77>frameshift > 82).
LIPA gene mutation (c.894 G>A). One subject was found to be heterozygous for this mutation. The sequence of the LIPA gene in this subject failed to reveal other mutations. This observation suggests that the frequency of this mutant allele in our patients with type IIb hyperlipidemia (1:150) is close to that expected in the general population (1:200) [14].
In this paper we describe three patients, from two families, affected by CESD. In the twin sisters of Family 1 the clinical diagnosis was made in the first decade of life, following the fortuitous observation of a persistent elevation of serum liver enzymes in apparently healthy children, which prompted the paediatrician to perform liver biopsy, followed by LAL activity assay in peripheral blood leukocytes. In striking contrast, the diagnosis of CESD in the proband of Family 2 was made late in life (80 years), during the investigation of the genetic bases of her complex dyslipidemia. The proband of Family 2 is the oldest CESD patient reported so far (see Supplementary Table and references on line), suggesting that some patients may be asymptomatic for many years and accidentally discovered during routine clinical investigations. The three subjects we investigated were found to be compound heterozygous for LIPA gene mutations. They shared the c.894 G>A substitution in exon 8, which is the most frequent mutant allele in CESD patients (see Supplementary Table and references on line). This mutation causes an abnormal splicing with the formation of an abnormal mRNA devoid of exon 8, whose translation product is a peptide of 375 amino acids (354 in the mature protein) due to the in-frame deletion of 24 amino acids (del p.S275_Q298) [21]. This abnormal protein has no activity [22]. However, despite the presence of the mutation, 3–5% of LIPA pre-mRNA is spliced correctly, accounting for a residual LAL activity in peripheral blood leukocytes corresponding to about 5% of control values [12,23,24].
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
147
Fig. 3. LIPA gene mutations in the proband of Family 2. (A) Left panel: partial sequence of intron 3-exon four junction of the LIPA gene in the probands of Family 2. The initial portion of nucleotide sequence inserted (GCTACT) is superimposed on the wild type sequence (CCCAAA); both sequences are highlighted at the top of the panel. (B) Right panel: polyacrylamide gel electrophoresis of the amplified fragments of intron 3-exon four junction. mw = molecular weight markers; M = carrier of the insertion of 35 nucleotides after position c.230; W = control subjects (see Supplementary methods). (C) Left panel: partial sequence of exon 8-intron eight junction of the LIPA gene in the proband of Family 2. The nucleotide substitution c.894 G>A is highlighted at the top of the panel. (D) Right panel: agarose gel electrophoresis of digestion fragments of exon 8 generated by the restriction enzyme Sty I. mw = molecular weight marker; M = carrier of c.894 G>A mutation; W = control subjects (see Supplementary methods).
WILD type Exon 4
3’ Intron 3
…ggt gct act gcc tcc taa aca atg aat gtt ttt cag GT Exon 3
Exon 4
… AAA G GT CCC AAA CCA GTT GTC TTC CTG CAA CAT GGCTTC … Lys Gly77 Pro Lys Pro Val Val Phe Leu Gln His Gly Leu88 MUTANT (ins 35 nt, del 14 nt) Exon 3
Exon 4
… AAA G GT gct act gcc tcc taa aca atg aat gtt ttt cag GTC TTC CTG CAA … Lys Gly77 Ala ThrAla Ser Term 82 Fig. 4. Top figure shows the wild type nucleotide sequence of exon 3-exon four junction and its translation product. The inserted sequence, after the nucleotide c.230 T of exon 4, which includes the last 33 nucleotides of intron 3 and the first two nucleotides of exon 4, is boxed. The nucleotide sequence (c.232_245) which is deleted in the mutant allele is underlined. Bottom figure shows the sequence of the mutant allele in proband of Family 2 and the predicted translation product (frameshift after Gly77>Term82).
The other LIPA gene mutations present in our patients (p.R218X and p.G77>frameshift78>X82) are expected to abolish LAL activity. In this context the residual capacity to hydrolyze CE and TG in hepatocytes and Kupffer cells of our patients depends entirely on the amount of normally spliced LIPA mRNA available in these cells. The percentage of correctly spliced mRNA (and the corresponding residual LAL activity) in the liver might be different among CESD patients carrying c.894 G>A mutation. Indeed, the residual LAL activity measured in fibroblasts of patients homozygous for c.894 G>A mutation was found to vary from 2% to 12% [24–26].
One of the key features of our patients was mixed hyperlipidemia (type IIb hyperlipidemia). The pre-treatment levels of TC, TG and apoB in our patients (Tables 1 and 2) were comparable to those found in 57 CESD patients reported in literature (TC 8.47 ± 1.60, TG 2.48 ± 1.03 mmol/L and apoB 196.0 ± 41.9 mg/dL). The mechanism underlying this hyperlipidemia is related to an increased production rate of apoB in VLDL and LDL with a normal fractional catabolic rate [27,28]. In our probands we found low levels of HDL-C and apoA-I, comparable with those previously reported in other CESD patients (HDL-C 0.72 ± 0.22 mmol/L and apoA-I 86.4 ± 20.9 mg/dL) (see Supplementary references on line). These levels are below the 5th percentile of the distribution in the population and similar to those observed in subjects heterozygous for mutations in ABCA1, APOA1 and LCAT genes [19,29,30], but lower that those found in patients with familial combined hyperlipidemia [29]. The mechanism responsible for the low plasma HDL is presently unknown. In CESD cells the FC pool is reduced and, most likely, less FC is transported from the intracellular compartments to the plasma membrane. This defect could affect ABCA1-mediated cholesterol efflux from the cell membrane to extracellular acceptors, such as lipid-poor apoA-I particles, a process which leads to the formation of pre-b-HDL in the liver and ultimately to the formation of mature plasma HDL [31]. In view of these findings the criteria for the clinical diagnosis of probable CESD might be the following: (i) a recessive form of severe mixed hyperlidemia with markedly reduced plasma HDL levels; (ii) the presence of enlarged liver of unknown etiology associated with increased serum liver enzymes; (iii) the exclusion of some clinical conditions such as metabolic syndrome, type 2 diabetes, NASH and FCH.
148
L. Pisciotta et al. / Molecular Genetics and Metabolism 97 (2009) 143–148
Acknowledgments This work was supported by grants from the University of Genova (S.B.) and University of Modena and Reggio Emilia (S.C.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymgme.2009.02.007. References [1] J.L. Goldstein, S.E. Dana, J.R. Faust, A.L. Beaudet, M.S. Brown, Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease, J. Biol. Chem. 250 (1975) 8487–8495. [2] G.N. Sando, V.L. Henke, Recognition and receptor-mediated endocytosis of the lysosomal acid lipase secreted by cultured human fibroblasts, J. Lipid Res. 23 (1982) 114–123. [3] D. Ameis, M. Merkel, C. Eckerskorn, H. Greten, Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase, Eur. J. Biochem. 219 (1994) 905–914. [4] S. Sheriff, H. Du, G.A. Grabowski, Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression, J. Biol. Chem. 270 (1995) 27766–27772. [5] G. Assmann, U. Seedorf, Acid Lipase Deficiency: Wolman Disease and Cholesteryl Ester Storage Disease, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic & Molecular Bases of Inherited Disease, McGrawHill, New York, 2001, pp. 3551–3572. [6] R.A. Anderson, G.N. Sando, Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases, J. Biol. Chem. 266 (1991) 22479–22484. [7] R.A. Anderson, N. Rao, R.S. Byrum, C.B. Rothschild, D.W. Bowden, R. Hayworth, M. Pettenati, In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in Wolman disease to chromosome 10q23.2-q23.3, Genomics 15 (1993) 245–247. [8] C. Aslanidis, H. Klima, K.J. Lackner, G. Schmitz, Genomic organization of the human lysosomal acid lipase gene (LIPA), Genomics 20 (1994) 329–331. [9] P.J. Meikle, J.J. Hopwood, A.E. Clague, W.F. Carey, Prevalence of lysosomal storage disorders, JAMA 281 (1999) 249–254. [10] B.K. Burton, S. Reed, Acid lipase cross-reacting material in Wolman Disease and Cholesterol Ester Storage Disease, Am. J. Hum. Genet. 33 (1981) 203–208. [11] J.M. Hoeg, S.J. Demosky, O.H. Pescovitz, H.B. Brewer Jr, Cholesteryl ester storage disease and Wolman disease: phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency, Am. J. Hum. Genet. 36 (1984) 1190– 1203. [12] C. Aslanidis, S. Ries, P. Fehringer, C. Büchler, H. Klima, G. Schmitz, Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity, Genomics 33 (1996) 85–93. [13] P. Lohse, S. Maas, P. Lohse, M. Elleder, J.M. Kirk, G.T.N. Besley, D. Seidelet, Compound heterozygosity for a Wolman mutation is frequent among patients with cholesteryl ester storage disease, J. Lipid Res. 41 (2000) 23–31. [14] S. Muntoni, H. Wiebusch, M. Jansen-Rust, S. Rust, U. Seedorf, H. Schulte, K. Berger, H. Funke, G. Assmann, Prevalence of cholesteryl ester storage disease, Artrioscler. Thromb. Vasc. Biol. 27 (2007) 1866–1868.
[15] L. Pisciotta, C.P. Oliva, A.B. Cefalu, D. Noto, A. Bellocchio, R. Fresa, A. Cantafora, D. Patel, M. Averna, P. Tarugi, S. Calandra, S. Bertolini, Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia, Atherosclerosis 186 (2006) 433–440. [16] A.D. Patrick, P. Willcox, R. Stephens, V.G. Kenyon, Prenatal diagnosis of Wolman’s disease, J. Med. Genet. 13 (1976) 49–51. [17] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. [18] V. Tadiboyina, D.M. Liu, B.A. Miskie, J. Wang, R.A. Hegele, Treatment of dyslipidemia with lovastatin and ezetimibe in an adolescent with cholesterol ester storage disease, Lipids Health Dis. 4 (2005) 26 (On line). [19] L. Pisciotta, I. Hamilton-Craig, P. Tarugi, A. Bellocchio, T. Fasano, P. Alessandrini, G. Bittolo Bon, D. Siepi, E. Mannarino, L. Cattin, M. Averna, B. Cefalù, A. Cantafora, S. Calandra, S. Bertolini, Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders, Atherosclerosis 172 (2004) 329–340. [20] J.E. Hixson, D.T. Vernier, Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI, J. Lipid Res. 31 (1990) 545–548. [21] H. Klima, K. Ullrich, C. Aslanidis, P. Fehringer, K.J. Lackner, G. Schmitz, A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease, J. Clin. Invest. 92 (1993) 2713–2718. [22] F. Pagani, R. Pariyarath, R. Garcia, C. Stuani, A.B. Burlina, G. Ruotolo, M. Rabusin, F.E. Baralle, New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease, J. Lipid Res. 39 (1998) 1382–1388. [23] D. Ameis, G. Brokmann, R. Knoblich, M. Merkel, R.E. Ostlund Jr., J.W. Yang, P.M. Coates, J.A. Cortner, S.V. Feinman, H. Greten, A 5’ splice-site mutation and a dinucleotide deletion in the lysosomal acid lipase gene in two patients with cholesteryl ester storage disease, J. Lipid Res. 36 (1995) 241–250. [24] H. Du, S. Sheriff, J. Bezerra, T. Leonova, G.A. Grabowski, Molecular and enzymatic analyses of lysosomal acid lipase in cholesteryl ester storage disease, Mol. Genet. Metab. 64 (1998) 126–134. [25] R.A. Anderson, G.M. Bryson, J.S. Parks, Lysosomal acid lipase mutations that determine phenotype in Wolman and Cholesteryl Ester Storage Disease, Mol. Genet. Metab. 68 (1999) 333–345. [26] S. vom Dahl, K. Hazer, A. Rolfs, B. Albrecht, C. Niedereau, C. Vogt, S. van Weely, J. Aerts, G. Müller, D. Häussinger, Hepatosplenomegalic lipidosis: what unless Gaucher? Adult cholesteryl ester storage disease (CESD) with anemia, mesenteric lipodystrophy, increased plasma chitotriosidase activity and a homozygous lysosomal acid lipase-1 exon 8 splice junction mutation, J. Hepatol. 31 (1999) 741–746. [27] H.N. Ginsberg, N.-A. Le, M.P. Short, R. Ramakrishnan, R.J. Desnick, Suppression of apolipoprotein B production during treatment of Cholesteryl Ester Storage Disease with Lovastatin. Implications for regulation of apolipoprotein B synthesis, J. Clin. Invest. 80 (1987) 1692–1697. [28] M. Cummings, G.F. Watts, Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in cholesteryl ester storage disease, Clin. Chem. 41 (1995) 111–114. [29] L. Pisciotta, T. Fasano, L. Calabresi, A. Bellocchio, R. Fresa, C. Borrini, S. Calandra, S. Bertolini, A novel mutation of apolipoprotein A-I gene in a family with familial combined hyperlipidemia, Atherosclerosis 198 (2008) 145–151. [30] L. Pisciotta, L. Calabresi, G. Lupattelli, G. Siepi, M.R. Mannarino, E. Moleri, A. Bellocchio, A. Cantafora, P. Tarugi, S. Calandra, S. Bertolini, Combined monogenic hypercholesterolemia and hypoalphalipoproteinemia caused by mutations in LDL-Receptor and LCAT genes, Atherosclerosis 182 (2005) 153– 159. [31] D.J. Rader, Molecular regulation of HDL metabolism and function: implication for novel therapies, J. Clin. Invest. 116 (2006) 3090–3100.