Atherosclerosis 210 (2010) 173–176
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Pseudoxanthoma elasticum and familial hypercholesterolemia: A deleterious combination of cardiovascular risk factors Livia Pisciotta a , Patrizia Tarugi b , Claudia Borrini a , Antonella Bellocchio a , Raffaele Fresa a , Deanna Guerra b , Daniela Quaglino b , Ivonne Ronchetti b,∗∗ , Sebastiano Calandra b , Stefano Bertolini a,∗ a b
Department of Internal Medicine, University of Genoa, Italy Department of Biomedical Sciences, University of Modena and Reggio Emilia, Italy
a r t i c l e
i n f o
Article history: Received 8 November 2009 Accepted 17 November 2009 Available online 24 November 2009 Keywords: Pxeudoxanthoma elasticum (PXE) Familial Hypercholesterolemia (FH) ABCC6 gene LDLR gene Coronary Artery Disease (CAD)
a b s t r a c t Background and objective: Pseudoxanthoma Elasticum (PXE), an autosomal recessive disease due to mutations in ABCC6 gene, is characterised by fragmentation of elastic fibres with involvement of the cardiovascular system. We investigated a 60-year-old female with angina pectoris found to have PXE, associated with elevated plasma LDL-C suspected to be due to autosomal-co-dominant hypercholesterolemia. Methods: ABCC6, LDLR, PCSK9 and exon 26 of APOB genes were re-sequenced. Cardiovascular involvement was assessed by coronary angiography, single-photon emission computed tomography (SPECT) and ultrasound examination. Results and conclusions: The patient was a compound heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and heterozygous for a novel LDLR mutation (p.R574H). She had severe coronary stenosis and calcification of the arteries of the lower limbs. Treatment with ezetimibe/simvastatin 10/60 mg/day, maintained over a 4.5-year period, reduced of LDL-C and the myocardial ischemic area. In PXE patients LDL-lowering treatment might contribute to delay macrovascular complications. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder characterised by progressive calcification and fragmentation of elastic fibres [1]. The most severe clinical manifestations appear in the dermis, in the retina, and in the vessel wall, leading to ruptures, aneurisms and arterial occlusion [1,2]. PXE is caused by mutations in ABCC6 gene, which encodes for multidrug resistanceassociated protein 6 (MRP6 or ABCC6), whose physiological role is poorly understood [3]. Autosomal co-dominant hypercholesterolemias represent a genetically heterogeneous group of disorders which include: (i) Familial Hypercholesterolemia (FH) due to a large variety of mutations in LDLR gene; (ii) Familial Defective apo B-100 (FDB) caused by some mutations in APOB gene, resulting in defective binding of LDL-apo B100 to the LDL-receptor;
∗ Corresponding author at: Department of Internal Medicine, University of Genoa, Viale Benedetto XV 6, I-16132 Genoa, Italy. Tel.: +39 010 3537992; fax: +39 010 3537797. ∗∗ Corresponding author at: Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41100 Modena, Italy. Tel.: +39 059 2055418; fax: +39 059 2055426. E-mail addresses:
[email protected] (I. Ronchetti),
[email protected] (S. Bertolini). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.11.028
(iii) Autosomal Dominant Hypercholesterolemia 3 (ADH3) caused by gain of function mutations in the PCSK9 gene, which encodes PCSK9 protein involved in the post-translational degradation of LDL-receptor [4–6]. Here we describe the clinical features and the molecular characterization of a patient with PXE associated with severe hypercholesterolemia and coronary artery disease, who was found to be compound heterozygous for two known mutations in the ABCC6 gene, as well as simple heterozygous for a novel mutation in LDLR gene.
2. Methods 2.1. Kindred GO The proband (I.1 in Fig. 1) was a 60-year-old female from Sicily, with clinical diagnosis of PXE, autoimmune hypothyroidism (treated with levothyroxine replacement) and severe hypercholesterolemia. She was found to have arcus cornealis, variable-threshold angina pectoris, but no tendon xanthomatosis. She had intermittent claudication since the age of 35. The diagnosis of PXE was made at the age of 44 when she had a sudden central visual field loss due to a retinal haemorrhage. Detailed physical examination is reported in Supplementary Material. The clinical,
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Fig. 1. Pedigree of kindred GO. The proband, indicated by an arrow, was a compound heterozygous for the following ABCC6 gene mutations: (i) c.951 C > A in exon 8 (p.S317R); (ii) c.3421 C > T in exon 24 (p.R1141X). She was also heterozygous for a mutation in LDLR gene: c.1721 G > A in exon 12 (p.R574H), indicated by an asterisk.
biochemical and genetic investigation was extended to proband’s sisters, offspring and grandchildren (Fig. 1). 2.2. Laboratory investigations The methods for the morphological analysis of the skin, the assessment of plasma lipid profile and the analysis of ABCC6 gene and the candidate genes for autosomal co-dominant hypercholesterolemia are illustrated in Supplementary Methods. 3. Results 3.1. Biochemical analyses Routine laboratory tests performed in the proband (subject I.1 in Fig. 1) did not reveal significant alterations except for increased levels of fibrinogen (540 ± 78 mg/dL), C-reactive protein (11.4 ± 2.6 mg/L) and total and LDL-cholesterol (TC, LDL-C) (Table 1). One proband’s sister and offspring (Fig. 1) had moderate hypercholesterolemia. Two of the grandchildren had plasma
cholesterol level between the 90th and 95th percentile of the distribution in subjects from the general population of similar age and gender (Table 1). Histopathology of the skin showed areas where elastic fibers were polymorphic, fragmented and mineralised. The resting electrocardiogram and two-dimensional echocardiography did not reveal significant alterations. Pharmacological stress echocardiography with dipyridamole showed hypokinesis of the mean portion of the lateral and posterior myocardial wall. Coronary angiography revealed the presence of severe stenosis (>70%) in the proximal left circumflex and in one diagonal branch of mean diameter and 40% stenosis of the right coronary artery. After exercise stress, single-photon emission computed tomography (SPECT with Tc-99m) showed myocardial ischemia in the distal lateral wall and apex (Supplementary Fig. 1a). B-mode ultrasound examination revealed fibrous-calcific plaques with 20% stenosis in the carotid arteries and increased intima–media thickness (IMT) with calcifications in the arteries of the lower limbs.
Table 1 Plasma lipids and clinical data of Family GO. Subject (gender)
I.1 (F)
I.2 (F)
I.3 (F)
II.1 (F)
II.2 (F)
II.3 (M)
III.1 (F)
III.2 (F)
III.3 (M)
ABCC6 genotype LDLR genotype Age (years) BMI (kg/m2 ) TC (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L) ApoA-I (mg/dL) ApoB (mg/dL) APOE genotype
M1/M2 W/M3 60 24.9 11.50 ± 0.59 9.08 ± 0.54 1.70 ± 0.16 1.93 ± 0.30 173 ± 10 246 ± 13 33
W/M2 W/W 56 20.5 7.88 5.44 2.01 0.92 209 144 33
W/W W/W 34 24.1 5.53 3.29 1.83 0.87 202 97 33
W/M1 W/M3 40 20.8 8.22 4.96 2.66 1.32 226 132 23
W/M1 W/M3 39 23.4 7.03 4.78 1.89 0.77 182 122 34
W/M1 W/M3 36 28.3 7.19 5.22 0.90 2.31 112 135 23
W/M1 W/W 7 17.7 4.55 2.75 1.55 0.53 192 68 23
W/W W/M3 15 19.9 5.74 3.75 1.52 1.03 183 92 33
W/M1 W/M3 8 13.6 5.71 3.77 1.68 0.57 194 95 34
Values are mean ± SD; all values are before pharmacological treatment; ABCC6 genotype: W = wild type, M1 = c.951 C > A (p.S317R), M2 = c.3421 C > T (p.R1141X); LDLR genotype: W = wild type, M3 = c.1721 G > A (p.R574H).
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Fig. 2. Plasma LDL cholesterol concentrations in the proband, before and after lipid-lowering treatment (Ezetimibe 10 mg/day associated with Simvastatin at progressively increased doses of 5, 10, 15, 20, 30, 40 and 60 mg/day), over 4.5-year follow-up.
Carotid ultrasound evaluation in proband’s offspring showed increased max-IMT in subject II.1 (1.08 mm) and in subject II.3 (1.00 mm). 3.2. Sequence of ABCC6 gene The proband was compound heterozygous for mutations in ABCC6 gene: (i) a C > A transversion in exon 8 (c.951 C > A), which results in arginine for serine conversion at position 317 (p.S317R); (ii) a C > T transition in exon 24 (c.3421 C > T), which converts arginine codon at position 1141 into a termination codon (p.R1141X). One of the proband’s sisters (subject I.2) was a carrier of the p.R1141X mutation. The proband’s three offspring and two of the grandchildren were carriers of p.S317R mutation (Fig. 1). 3.3. Sequence analysis of candidate genes for autosomal co-dominant hypercholesterolemia The sequence of LDLR gene showed that the proband was heterozygous for a G > A transition in exon 12 (c.1721 G > A), which causes a histidine for arginine substitution at position 574 of the LDL-receptor (p.R574H). No carrier of this mutation was found in 106 controls and in 60 Sicilian FH patients (Supplementary Table 1). The presence of a major rearrangement in LDLR gene was excluded by MLPA analysis. In view of the very high plasma cholesterol level found in the patient, we also sequenced exon 26 of the APOB gene and the whole PCSK9 gene searching for mutations associated with high plasma LDL. Neither mutations nor functional polymorphisms were detected in these genes. The proband’s offspring and two grandchildren (subjects III.2 and III.3) were found to be heterozygous for the p.R574H mutation. 3.4. In silico analysis of the ABCC6 and LDLR missense mutations The ABCC6 p.S317R mutation resulted to be “possibly damaging” according to PolyPhen and “pathogenic” according to PANTHER, but was predicted to be “tolerated” according to SIFT. The LDLR p.R574H mutation was predicted to be pathogenic by all three programmes (Supplementary Table 2). 3.5. Screening for the ABCC6 mutations Both mutations, p.S317R and p.R1141X, were screened in 106 healthy controls. No carriers of these mutations were found. The p.R1141X was also screened in 173 patients with premature CAD (before 55 years in males and 65 years in females) (Supplementary Table 1) Three carriers (two males and one female) were found among subjects of this group (1.7%).
3.6. LDL lowering therapy and clinical follow-up of the proband Treatment with ezetimibe/simvastatin 10/60 mg/day (started when the patient was 60) has been carried out for more than 4 years with substantial reduction of plasma cholesterol levels (Fig. 2). During follow-up the CRP rapidly decreased and remained within the normal range and no episodes of angina were recorded. SPECT showed a significant reduction of the myocardial ischemic area (in the distal lateral wall and apex) after exercise test (Supplementary Fig. 1b). Carotid ultrasound examination, performed after 2 and 4 years of lipid-lowering therapy, revealed a mild increase of arterial stenosis (+8%). 4. Discussion Our proband is the second patient reported to have PXE and FH due to mutations in LDLR gene. The first case reported in literature was a Japanese patient with hypercholesterolemia (TC 7.88 mmol/L) and tendon xanthomatosis, who was homozygous for a frameshift mutation in ABCC6 gene (resulting in a premature stop codon) and heterozygous for a 6 kb deletion in LDLR gene. This patient had no ocular manifestations but had a history of cerebral bleeding resulting in right hemiparesis at 55. She also had a remarkable arterial wall calcification from the ascending aorta to femoral arteries as well in the coronary arteries [7]. Our proband suffered from variable-threshold angina and coronary angiography documented severe stenosis in left coronary arteries. These vascular alterations were probably the result of multiple factors related to both PXE and FH. With regard to cardiovascular manifestations of PXE, a recent study of 42 molecularly defined patients, showed that the prevalence of angina pectoris and myocardial infarction was 12% and 5%, respectively, while the prevalence of intermittent claudication was 22% [2]. It is well established that hypercholesterolemia due to LDLR gene mutations predisposes to premature CAD. In our series of 644 molecularly characterised heterozygous FH patients over 30 years of age (mean age 49.0 ± 12.0 years) the prevalence of myocardial infarction, angina and silent myocardial ischemia was 20.6%, 7.8% and 2.0%, respectively (Supplementary Table 3). Symptomatic arterial disease of lower limbs was not found in our series of FH patients. It is most likely that the combination of the arterial lesions related to PXE and FH has accelerated the arterial stenosis responsible for both angina and ischemia in the lower limbs. In this context it is noteworthy the observation that the frequency of the p.R1141X mutation of ABCC6 gene (the most common mutation in PXE) was found to be increased (3.3% vs. 0.8%) in
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a large group of patients with premature coronary heart disease [8]. This observation was confirmed in the present study suggesting that even haplo-insufficiency of ABCC6 (as expected in heterozygotes for a null allele) might result in subtle alterations of the structure of arterial wall (e.g. in coronary artery), or in a mild chronic oxidative stress (recently demonstrated in PXE) [9–11] which induces oxidation of LDL and possibly intimal injury [12]. Since in our patient cardiovascular revascularization was considered unfeasible because of the risk of severe haemorrhages in the retina and other districts (due to the antithrombotic therapy required to prevent restenosis after percutaneous transluminal coronary angioplasty), the most effective therapeutic option to reduce the risk of CAD was the use of LDL-lowering drugs. The patient was treated with a combination of ezetimibe and simvastatin, which resulted in a stable decrease of LDL cholesterol level (−70%), resolution of angina and a significant reduction of the myocardial ischemic area. This study suggests that LDL-lowering drugs might be indicated in PXE patients (even in the absence of elevated plasma LDL-C (Supplementary Table 4) to delay plaque formation and reduce macrovascular complications. Conflict of interest statement The authors have no conflict of interest. Acknowledgements This work was supported by grants from the University of Genoa to S.B. and from European Union - GENESKIN n.512117 and PXEInternational to I.R.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2009.11.028. References [1] Finger RP, Issa PC, Ladewing MS, et al. Pseudoxanthoma elasticum: genetics, clinical manifestations and therapeutic approaches. Surv Ophthalmol 2009;54:272–85. [2] Vanakker OM, Leroy BP, Coucke P, et al. Novel clinico-molecular insights in pseudoxanthoma elasticum provide an efficient molecular screening method and a comprehensive diagnostic flowchart. Hum Mutat 2008;29:205. [3] Li Q, Jiang Q, Pfendner E, Varadi A, Uitto J. Pseudoxanthoma elasticum: clinical phenotypes, molecular genetics and putative pathomechanisms. Exp Dermatol 2009;18:1–11. [4] Soutar AK, Naumova RP. Mechanisms of disease: genetic causes of familial hypercholesterolemia. Nat Clin Pract Cardiovasc 2007;4:214–25. [5] Varret M, Abifadel M, Rabès J-P, Boileau C. Genetic heterogeneity of autosomal dominant hypercholesterolemia. Clin Genet 2008;73:1–13. [6] Abifadel M, Rabes J-P, Devillers M, et al. Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease. Hum Mutat 2009;30:520–9. [7] Noji Y, Inazu A, Higashikata T, et al. Identification of two novel missense mutations (p.R1221C and p.R1357W) in the ABCC6 (MRP6) gene in a Japanese patient with pseudoxanthoma elasticum (PXE). Intern Med 2004;43:1171–6. [8] Trip MD, Smulders YM, Wegman JJ, et al. Frequent mutation in the ABCC6 gene (R1141X) is associated with a strong increase in the prevalence of coronary artery disease. Circulation 2002;106:773–5. [9] Garcia Fernandez MI, Gheduzzi D, Boraldi F, et al. Parameters of oxidative stress are present in the circulation of PXE patients. Biochim Biophys Acta 2008;1782:474–81. [10] Pasquali-Ronchetti I, Garcia-Fernandez MI, Boraldi F, et al. Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations. J Pathol 2006;208:54–61. [11] Li Q, Jiang Q, Uitto J. Pseudoxanthoma elasticum: oxidative stress and antioxidant diet in a mouse model (Abcc6−/−). J Invest Dermatol 2008, 128-1160-4. [12] Fraley AE, Tsimikas S. Clinical applications of circulating oxidized lowdensity lipoprotein biomarkers in cardiovascular disease. Curr Opin Lipidol 2006;17:502–9.
