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A novel mutation in the ABCA1 gene causing an atypical phenotype of Tangier disease
Journal of Clinical Lipidology (2013) 7, 82–87
Case Study
A novel mutation in the ABCA1 gene causing an atypical phenotype of Tangier disease Smita I. Negi, MD†, Ariel Brautbar, MD†, Salim S. Virani, MD, Aashish Anand, MD, Eliana Polisecki, PhD, Bela F. Asztalos, PhD, Christie M. Ballantyne, MD, Ernst J. Schaefer, MD, Peter H. Jones, MD* Center for Atherosclerosis and Lipoprotein Research, Baylor College of Medicine, 6565 Fannin St, Suite B157, Houston, TX 77030, USA (Drs. Negi, Brautbar, Virani, Ballantyne, and Jones); Boston Heart Diagnostics, Framingham, MA, USA (Drs. Polisecki, Asztalos, and Schaefer); Lipid Metabolism Laboratory, Human Nutrition Research Center on Aging at Tufts University and Tufts University School of Medicine, Boston, MA, USA (Drs. Asztalos and Schaefer); Department of Neurosciences, Baylor College of Medicine, Houston, TX, USA (Dr. Anand); and Michael E. DeBakey VA Medical Center Health Services Research and Development Center of Excellence, Houston, TX, USA (Dr. Virani) KEYWORDS: ABCA1 gene; Central nervous system; High-density lipoprotein; Peripheral neuropathy; Tangier disease
Abstract: Tangier disease is a rare autosomal-recessive disorder caused by mutation in the ATP binding cassette transporter 1 (ABCA1) gene. Typically, Tangier disease manifests with symptoms and signs resulting from the deposition of cholesteryl esters in nonadipose tissues; chiefly, in peripheral nerves leading to neuropathy and in reticulo-endothelial organs, such as liver, spleen, lymph nodes, and tonsils, causing their enlargement and discoloration. An association with early cardiovascular disease can be variable. We describe a patient with a unique phenotype of Tangier disease from a novel splice site mutation in the ABCA1 gene that is associated with a central nervous system presentation resembling multiple sclerosis, and the presence of premature atherosclerosis. Ó 2013 National Lipid Association. All rights reserved.
Low levels of high-density lipoprotein cholesterol (HDL-C) are known to be a strong independent risk factor for cardiovascular disease (CVD).1 Tangier disease (TD), caused by mutations in the gene encoding ATP-binding cassette A1 (ABCA1) protein, is a rare familial condition that leads to a severe deficiency in HDL-C and apolipoprotein A-1 (ApoA-1) levels.2 HDL particles play a key role in the reverse transport of cholesterol from peripheral cells to the liver for biliary excretion.3 ABCA1, a membrane transporter, is abundantly found in macrophages and
† First authors. * Corresponding author. E-mail address: [email protected] Submitted May 21, 2012. Accepted for publication September 16, 2012.
mediates cholesterol and phospholipid efflux to lipid-poor ApoA-1, the precursor of mature HDL.4 ABCA1 is expressed in the small intestine, liver, brain, and cells of the reticulo-endothelial system.4 It is synthesized in the endoplasmic reticulum and transported to the plasma membrane via vesicles.4 Functionally defective ABCA1 variants fail to mediate lipid efflux to ApoA-1 and, as a consequence, the nonlipidated ApoA-1 is unable to undergo maturation into a larger HDL particle and, ultimately, undergoes rapid renal clearance.5 There are several phenotypic presentations of TD and several mutational defects in the ABCA1 gene have been described.6,7 The clinical presentations are related to the accumulation of cholesterol in the peripheral tissues, which include peripheral neuropathies, hepatosplenomegaly, and corneal opacification.8 The risk of CVD in patients with
1933-2874/$ - see front matter Ó 2013 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2012.09.004
Negi et al
Novel mutation in Tangier disease
83
TD varies, and although the carriers of mutations in the ABCA1 gene are reported to have an increased risk of atherosclerosis, not all patients with TD experience premature CVD.9 So far, there are more than 90 identified ABCA1 mutations described in the literature.10,11 Due to the variability of the ABCA1 mutations associated with TD, varying degrees of impairment in lipid transfer activity may occur,12 which results in different phenotypic presentations. In this article, we describe a unique TD homozygous splice-site mutation in the ABCA1 gene resulting in multiple sclerosis-like neurologic presentation that was also associated with premature CVD on screening.
Methods and results Index patient The proband was a 38-year old white woman who presented to the lipid specialty clinic at the Baylor College of Medicine for evaluation of very low HDL-C levels on a routine lipid profile. Her medical history was significant for relapsing-remitting neurologic symptoms since age 8 that included recurrent episodes of disabling ataxia, nystagmus, strabismus, bilateral internuclear ophthalmoplegia, oscillopsia, optic neuritis, and dysarthria. These episodes typically lasted from a few weeks to several months and resolved with no lasting clinical sequelae. The resolution of symptoms was not affected by the use of steroids or other immunomodulating agents. She also reported isolated pressure palsies of ulnar and median nerve leading to surgical decompression for presumed nerve entrapment.
Family history A three-generation pedigree demonstrated consanguinity in the family. The patient’s paternal grandfather was the uncle of the patient’s maternal grandfather (Fig. 1). The inbreeding coefficient was calculated at 1/32 (ie, 1/32 of the patient’s genome is predicted to be homozygous and of the same origin). There was no family history of premature CVD or chronic neurological condition. The patient’s first degree relatives, parents, sibling and children, had low normal HDL-C level of routine lipid testing (Fig. 1).
Physical evaluation Findings of cardiovascular, abdominal, and central and peripheral nervous system examinations were normal at the time of presentation to the lipid clinic. There were no corneal opacities visible to the naked eye. However, on slitlamp examination, there were focal whitish deposits in the corneal stroma (Fig. 2). There were no arcus, xanthelasmas, or tendon xanthomas on examination. Both tonsils were normal in size and color. Carotid intima media thickness was at the 66th percentile for age and gender with an average thickness of 0.6
Figure 1 Pedigree chart depicting HDL-C distribution among index case and family.
mm. No carotid plaque was seen. The total coronary artery calcium score was 12 (above the 98th percentile for age and gender matched controls). Both these findings of an elevated carotid intima media thickness and an elevated coronary calcium score in this young female represent presence of subclinical atherosclerosis.13
Routine and specialized laboratory analysis Standard fasting lipid panel showed a total cholesterol of 124 mg/dL, triglycerides of 138 mg/dL, low-density lipoprotein cholesterol (LDL-C) of 106 mg/dL, and an HDL-C of ,5 mg/dL. On nuclear magnetic resonance (NMR) spectroscopy testing, HDL-C was ,5 mg/dL with triglycerides of 98 mg/dL. The LDL particle number (LDL-P) was 2100 nmol/L (reference value: ,1000 nmol/L) with an LDL-C of 115 mg/dL. Statin therapy was prescribed given the presence of subclinical atherosclerosis, and after 4 months led to an improvement in the LDL-C to 50 mg/dL but the LDL-P remained at 1900 nmol/L. Additional studies showed ApoA-1 0.7 mg/dL (markedly decreased), ApoB 71 mg/dL (normal), and lipoprotein (a) 3.6 mg/dL (normal). The between-run coefficients of variation were all ,5%. HDL particle analysis by two-dimensional gel electrophoresis followed by ApoA-1 antibody immunoblotting in mg/ dl of ApoA-1 measured as previously described revealed: precursor very small pre-beta-1 HDL 0.2 (very low), very small alpha-4 HDL 0.1 (very low), small alpha-3 HDL 0.1 (very low), medium alpha-2 HDL 0.1 (very low), and large HDL alpha-1 0.0 (undetectable, normal .20; Fig. 3). Sizeexclusion chromatography of the patients plasma total lipoproteins after ultracentrifugal flotation at d 5 1.21 g/mL was performed. The profiles were analyzed by absorbance and cholesterol mass (Fig. 4).
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Figure 2 Slit-lamp examination of the cornea under high (A) and low (B) magnification showing small dots representing lipid deposition within the corneal stroma.
Serum markers of inflammation were measured. Highsensitivity C-reactive protein was 0.7 mg/L (reference value: ,3.0 mg/L), Lp-PLA2 (lipoprotein-associated phopholipase A2) was ,100 ng/mL (reference value: 1202342 ng/mL), plasma homocysteine levels were 9.6 mmol/L (reference value: 3.0-15 mmol/L), and the ApoE genotype was 2/3.
Diagnostic imaging Repeated extensive work-up for chronic neurologic disorders, especially multiple sclerosis, including serial magnetic resonance imaging of brain and spinal cord, spinal fluid analysis, and visual-evoked response studies were negative during acute episodes and in remission over 20 years. Four years before our visit, the patient had repeated bouts of lower abdominal colicky pain with change in bowel habits. A computed tomography scan of the abdomen revealed small low-density oval hypoechoic lesions measuring up to 7 mm in the liver parenchyma, possibly related to fatty accumulation, with no enlarged lymph nodes or hepatosplenomegaly. Colonoscopy showed heavy pigmentation of the colonic mucosa that extended through the entire length of the colon (Fig. 5). Multiple pieces of yellow-tan tissue were biopsied which, on microscopic examination, revealed foamy xanthoma cells both within the lamina propria and muscularis mucosa.
Muscle biopsies Muscle biopsy revealed scattered atrophic fibers with a striking absence of lipid droplets surrounding the muscle fibers (Fig. 6). Glycogen content, carnitine content, and glycogen spectrum wavelength of muscle fibers were all normal. Autoimmune screen was negative. Peripheral Myelin Protein 22 gene analyses showed no deletions, duplications, or sequence alterations.
Mutation analysis The combination of a very low HDL-C and a family history of consanguinity were suggestive of a hypoalphalipoproteinemia with an autosomal-recessive inheritance pattern. Thus, we sequenced ABCA1, ApoA-1, and lecithin-cholesterol acyltransferase. Sequencing of the ABCA1 gene revealed a novel homozygous G-C splice site mutation adjacent to exon 33 at the 21 position.
Discussion In this report, we describe a case of TD associated with a novel homozygous splice-site mutation in the ABCA1 gene that resulted in a long history of atypical neurologic symptoms and signs. The patient presented with a multiple
Figure 3 HDL particle analysis by two-dimensional gel electrophoresis followed by apoA-I antibody immunoblotting in mg/dL of apoA-I measured (A) compared with those of a normal subject (left) and of a patient with premature heart disease (center) in the and with a schematic of the individual HDL particles on the right (B).
Negi et al
Novel mutation in Tangier disease
Figure 4 Size-exclusion chromatography of the patient’s plasma total lipoproteins after ultracentrifugal flotation at d 5 1.21 g/mL center. VLDL, very-low-density lipoprotein.
sclerosis-like phenotype, which has so far not been described in TD. In addition, she also had evidence of mild pressure palsies of the peripheral nerves of the hand, which has been described in TD.14 She also had atherosclerosis on noninvasive screening tests, which occurred prematurely considering her age and gender. The occurrence of premature CVD in patients with TD varies, and our
85 decision to start a statin was in response to the subclinical measurement of early atherosclerosis, not the LDL-P level.15 The first case of TD was described by Fredrickson in two siblings from Tangier Island, near the Chesapeake Bay.16 These children had enlarged, orange-colored tonsils filled with CE-laden macrophages, with similar deposition in the liver and spleen, resulting in hepatosplenomegaly, and in the lymph nodes. They also were noted to have marked HDL-C deficiency, decreased LDL-C, and moderate hypertriglyceridemia. Homozygotes are severely deficient in apoA-I and apoA-II, but other apolipoprotein levels were relatively normal, whereas heterozygotes have HDL-C levels that are 50% of normal.17 Clinically, both homozygotes and heterozygotes can develop peripheral neuropathy, corneal opacification, hepatosplenomegaly, omental masses, and premature CHD.9 Variants of ABCA1 in the general population also are linked to premature CVD.6 Two-dimensional gel electrophoresis of apoA-I containing HDL particles from homozygotes generally show very small discoidal pre-beta 1 migrating HDL, whereas heterozygotes have a marked reduction in large spherical alpha 1 and alpha 2 migrating HDL.9 Metabolic studies performed in homozygotes documented that the LDL particles in cases of TD are very small and rich in triglyceride and beta carotene.18 Because of the lack of ABCA1 function in these
Figure 5 Distal colon on colonoscopic examination showing numerous areas of yellowish orange pigmentation. These were found to be collection of foamy xanthoma cells on histopathological examination.
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Figure 6
Journal of Clinical Lipidology, Vol 7, No 1, February 2013
Trichome stain (A) and Oil Red O stain (B) of the muscle biopsy specimen showing atrophic fibers and scanty lipid granules.
individuals, no cellular cholesterol is taken up by pre-beta 1 HDL, which results in rapid catabolism of nascent HDL, probably by the kidney. This lack of CE formation and hence, lack of transfer of CE from HDL to apoB containing lipoproteins19 results in the production of small, cholesterol-poor LDL particles that are prone to greater uptake by non-adipose tissues.19 Our case is atypical and novel on several accounts. First, the clinical presentation is unique for the predominantly central nervous system (CNS) findings, including optic neuritis, internuclear ophthalmoplegia, ocular palsies, dysarthria, and gait problems. These occurred simultaneously with peripheral nervous system involvement, which was less prominent. The clinical heterogeneity in presentation of TD raises the possibility of different metabolic errors or a common metabolic error subject to genetic influences. Second, the patient lacked the orange-colored tonsils and hepatosplenomegaly classically described in TD. However, she did have the corneal stromal opacities and gastrointestinal lipid deposits. Another interesting finding was the total absence of lipid deposits in the muscle fibers on muscle biopsy. Finally, the case is unique with respect to the plasma lipoprotein distribution. Despite both HDL cholesterol and ApoA-1 levels being ,1 mg/dL, a very small amount of ApoA-1 was found in pre-beta 1 HDL, as well as in alpha 4, alpha 3, and alpha 2 HDL (Fig. 3). Her LDL-C was low on a standard lipid panel; however, her LDL particle number was very elevated on NMR analysis. One possible explanation could be that the triglyceride-enriched and cholesterol-depleted LDL particles emanate a strong LDL NMR signal despite the low LDL-C content, leading to an overestimation of the particle number. Our patient did have significant asymptomatic, subclinical atherosclerosis for her age and gender on the basis of both the carotid intima media measurement and coronary calcium score,13 and this was our rationale for use of a statin, not in response to the high LDL-P. The diagnosis of TD was reached on genetic analysis. She was found to have a homozygous splice-site mutation at the 21 position 50 to exon 33 in the ABCA1 gene. The ABCA1 gene contains 49 exons with exons 3 to 49 contributing to the amino acid sequence of ABCA1. There are 2052 base pairs contained within exons 33249, and
therefore an abnormal splice site would result in the loss of 684 amino acids from the terminal end of ABCA1.20 Other splice-site mutations have been reported, but the mutation at 21 to exon 33 is novel.20 Plasma lipoproteins are believed to regulate ganglioside biosynthesis in the CNS. ABCA1 is widely expressed throughout the body, including cells within the CNS, but its role in brain lipid metabolism is not yet fully understood. In the brain, glia synthesize the apolipoproteins involved in CNS lipid metabolism.21 It has been demonstrated that glial ABCA1 is required for cholesterol efflux to apoA-I and also plays a key role in facilitating cholesterol efflux to apoE, which is the major apolipoprotein in the brain. In both astrocytes and microglia, ABCA1 deficiency reduces lipid efflux to exogenous apoE. The impaired ability to efflux lipids in ABCA12/2 glia results in lipid accumulation in both astrocytes and microglia.21 In addition, apoE secretion is compromised in ABCA12/2 astrocytes and microglia.21 In vivo, deficiency of ABCA1 results in a 65% decrease in apoE levels in whole brain, and a 75% to 80% decrease in apoE levels in hippocampus and striatum. More evidence is emerging that glial ABCA1 is a key influence on apoE metabolism in the CNS. Further research on the functions of ABCA1 in the CNS may lead to improved understanding of the plethora of clinical presentations of TD, including the reason for the atherogenicity associated with very low HDL. The transient neurologic symptoms in TD, similar to the relapsing-remitting CNS symptoms in this case, are seen in nearly 50% of TD cases.22 Histopathology and electron microscopy of biopsied peripheral nerves have revealed preferential lipid deposition in the paranodal regions of myelinating Schwann cells. It has been speculated that this accumulation of lipid may physically separate the myelin sheath from the axon at this site, and in turn, leads to paranodal malfunction, slowing of nerve conduction or conduction block, and the resulting clinical symptoms. Paranodal myelin remodelling eventually leads to recovery of paranodal function and to clinical recovery. The reconstituted paranodal region may be vulnerable to further accumulation of lipid deposits, leading to a cycle of breakdown and repair, consistent with the remitting-relapsing clinical course.22
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Novel mutation in Tangier disease
Conclusion We present a unique phenotype of TD, related to a novel splice site mutation in ABCA1, that highlights an unusual constellation of neurologic symptoms mimicked multiple sclerosis, as well as evidence of premature atherosclerosis.
Acknowledgments The authors acknowledge Boston Heart Laboratories, Houston Eye Associates, and Yadollah Harati, MD, Department of Neuromuscular Sciences, Baylor College of Medicine, for their contribution in the preparation of the manuscript.
References 1. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am J Med. 1977;62:707–714. 2. Zannis VI, Chroni A, Krieger M. Role of ApoA–I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL. J Mol Med. 2006;84:276–294. 3. Clee SM, Kastelein JJ, van Dam M, et al. Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes. J Clin Invest. 2000;106:1263–1270. 4. Tang C, Oram JF. The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes. Biochim Biophys Acta. 2009;1791:563–572. 5. Batal R, Tremblay M, Krimbou L, et al. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I. Arterioscler Thromb Vasc Biol. 1998;18:655–664. 6. Brunham LR, Singaraja RR, Hayden MR. Variations on a gene: rare and common variants in ABCA1 and their impact on HDL cholesterol levels and atherosclerosis. Ann Rev Nutr. 2006;26:105–129. 7. Cohen JC, Kiss RS, Pertsemlidis A, Marcel YL, McPherson R, Hobbs HH. Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Science. 2004;305:869–872. 8. Schaefer EJ. Clinical, biochemical, and genetic features in familial disorders of high density lipoprotein deficiency. Arteriosclerosis. 1984;4:303–322.
87 9. Asztalos BF, Brousseau ME, McNamara JR, Horvath KV, Roheim PS, Schaefer EJ. Subpopulations of high density lipoproteins in homozygous and heterozygous Tangier disease. Atherosclerosis. 2001;156: 217–225. 10. Rhyne J, Mantaring MM, Gardner DF, Miller M. Multiple splice defects in ABCA1 cause low HDL-C in a family with hypoalphalipoproteinemia and premature coronary disease. BMC Med Genet. 2009 8;10:1. 11. Bocchi L, Pisciotta L, Fasano T, et al. Multiple abnormally spliced ABCA1 mRNAs caused by a novel splice site mutation of ABCA1 gene in a patient with Tangier disease. Clin Chim Acta. 2010;411: 524–530. 12. Repa JJ, Turley SD, Lobaccaro JA, et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science. 2000;289:1524–1529. 13. Folsom AR, Kronmal RA, Detrano RC, et al. Coronary artery calcification compared with carotid intima-media thickness in the prediction of cardiovascular disease incidence: the Multi-Ethnic Study of Atherosclerosis (MESA). Arch Intern Med. 2008;168:1333–1339. 14. Pareyson D. Diagnosis of hereditary neuropathies in adult patients. J Neurol. 2003;250:148–160. 15. Asztalos BF, Cupples LA, Demissie S, et al. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 2004;24:2181–2187. 16. Fredrickson DS. The inheritance of high density lipoprotein deficiency (Tangier disease). J Clin Invest. 1964;43:228–236. 17. Alaupovic P, Schaefer EJ, McConathy WJ, Fesmire JD, Brewer HB Jr. Plasma apolipoprotein concentrations in familial apolipoprotein A-I and A-II deficiency (Tangier disease). Metabolism. 1981;30:805–809. 18. Schaefer EJ, Brousseau ME, Diffenderfer MR, et al. Cholesterol and apolipoprotein B metabolism in Tangier disease. Atherosclerosis. 2001;159:231–236. 19. Schaefer EJ, Santos RD, Asztalos BF. Marked HDL deficiency and premature coronary heart disease. Curr Opin Lipidol. 2010;21: 289–297. 20. Brooks-Wilson A, Marcil M, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet. 1999; 22:336–345. 21. Hirsch-Reinshagen V, Zhou S, Burgess BL, et al. Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain. J Biol Chem. 2004;279:41197–41207. 22. Cai Z, Blumbergs PC, Cash K, et al. Paranodal pathology in Tangier disease with remitting-relapsing multifocal neuropathy. J Clin Neurosci. 2006;13:492–497.
Case Study
A novel mutation in the ABCA1 gene causing an atypical phenotype of Tangier disease Smita I. Negi, MD†, Ariel Brautbar, MD†, Salim S. Virani, MD, Aashish Anand, MD, Eliana Polisecki, PhD, Bela F. Asztalos, PhD, Christie M. Ballantyne, MD, Ernst J. Schaefer, MD, Peter H. Jones, MD* Center for Atherosclerosis and Lipoprotein Research, Baylor College of Medicine, 6565 Fannin St, Suite B157, Houston, TX 77030, USA (Drs. Negi, Brautbar, Virani, Ballantyne, and Jones); Boston Heart Diagnostics, Framingham, MA, USA (Drs. Polisecki, Asztalos, and Schaefer); Lipid Metabolism Laboratory, Human Nutrition Research Center on Aging at Tufts University and Tufts University School of Medicine, Boston, MA, USA (Drs. Asztalos and Schaefer); Department of Neurosciences, Baylor College of Medicine, Houston, TX, USA (Dr. Anand); and Michael E. DeBakey VA Medical Center Health Services Research and Development Center of Excellence, Houston, TX, USA (Dr. Virani) KEYWORDS: ABCA1 gene; Central nervous system; High-density lipoprotein; Peripheral neuropathy; Tangier disease
Abstract: Tangier disease is a rare autosomal-recessive disorder caused by mutation in the ATP binding cassette transporter 1 (ABCA1) gene. Typically, Tangier disease manifests with symptoms and signs resulting from the deposition of cholesteryl esters in nonadipose tissues; chiefly, in peripheral nerves leading to neuropathy and in reticulo-endothelial organs, such as liver, spleen, lymph nodes, and tonsils, causing their enlargement and discoloration. An association with early cardiovascular disease can be variable. We describe a patient with a unique phenotype of Tangier disease from a novel splice site mutation in the ABCA1 gene that is associated with a central nervous system presentation resembling multiple sclerosis, and the presence of premature atherosclerosis. Ó 2013 National Lipid Association. All rights reserved.
Low levels of high-density lipoprotein cholesterol (HDL-C) are known to be a strong independent risk factor for cardiovascular disease (CVD).1 Tangier disease (TD), caused by mutations in the gene encoding ATP-binding cassette A1 (ABCA1) protein, is a rare familial condition that leads to a severe deficiency in HDL-C and apolipoprotein A-1 (ApoA-1) levels.2 HDL particles play a key role in the reverse transport of cholesterol from peripheral cells to the liver for biliary excretion.3 ABCA1, a membrane transporter, is abundantly found in macrophages and
† First authors. * Corresponding author. E-mail address: [email protected] Submitted May 21, 2012. Accepted for publication September 16, 2012.
mediates cholesterol and phospholipid efflux to lipid-poor ApoA-1, the precursor of mature HDL.4 ABCA1 is expressed in the small intestine, liver, brain, and cells of the reticulo-endothelial system.4 It is synthesized in the endoplasmic reticulum and transported to the plasma membrane via vesicles.4 Functionally defective ABCA1 variants fail to mediate lipid efflux to ApoA-1 and, as a consequence, the nonlipidated ApoA-1 is unable to undergo maturation into a larger HDL particle and, ultimately, undergoes rapid renal clearance.5 There are several phenotypic presentations of TD and several mutational defects in the ABCA1 gene have been described.6,7 The clinical presentations are related to the accumulation of cholesterol in the peripheral tissues, which include peripheral neuropathies, hepatosplenomegaly, and corneal opacification.8 The risk of CVD in patients with
1933-2874/$ - see front matter Ó 2013 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2012.09.004
Negi et al
Novel mutation in Tangier disease
83
TD varies, and although the carriers of mutations in the ABCA1 gene are reported to have an increased risk of atherosclerosis, not all patients with TD experience premature CVD.9 So far, there are more than 90 identified ABCA1 mutations described in the literature.10,11 Due to the variability of the ABCA1 mutations associated with TD, varying degrees of impairment in lipid transfer activity may occur,12 which results in different phenotypic presentations. In this article, we describe a unique TD homozygous splice-site mutation in the ABCA1 gene resulting in multiple sclerosis-like neurologic presentation that was also associated with premature CVD on screening.
Methods and results Index patient The proband was a 38-year old white woman who presented to the lipid specialty clinic at the Baylor College of Medicine for evaluation of very low HDL-C levels on a routine lipid profile. Her medical history was significant for relapsing-remitting neurologic symptoms since age 8 that included recurrent episodes of disabling ataxia, nystagmus, strabismus, bilateral internuclear ophthalmoplegia, oscillopsia, optic neuritis, and dysarthria. These episodes typically lasted from a few weeks to several months and resolved with no lasting clinical sequelae. The resolution of symptoms was not affected by the use of steroids or other immunomodulating agents. She also reported isolated pressure palsies of ulnar and median nerve leading to surgical decompression for presumed nerve entrapment.
Family history A three-generation pedigree demonstrated consanguinity in the family. The patient’s paternal grandfather was the uncle of the patient’s maternal grandfather (Fig. 1). The inbreeding coefficient was calculated at 1/32 (ie, 1/32 of the patient’s genome is predicted to be homozygous and of the same origin). There was no family history of premature CVD or chronic neurological condition. The patient’s first degree relatives, parents, sibling and children, had low normal HDL-C level of routine lipid testing (Fig. 1).
Physical evaluation Findings of cardiovascular, abdominal, and central and peripheral nervous system examinations were normal at the time of presentation to the lipid clinic. There were no corneal opacities visible to the naked eye. However, on slitlamp examination, there were focal whitish deposits in the corneal stroma (Fig. 2). There were no arcus, xanthelasmas, or tendon xanthomas on examination. Both tonsils were normal in size and color. Carotid intima media thickness was at the 66th percentile for age and gender with an average thickness of 0.6
Figure 1 Pedigree chart depicting HDL-C distribution among index case and family.
mm. No carotid plaque was seen. The total coronary artery calcium score was 12 (above the 98th percentile for age and gender matched controls). Both these findings of an elevated carotid intima media thickness and an elevated coronary calcium score in this young female represent presence of subclinical atherosclerosis.13
Routine and specialized laboratory analysis Standard fasting lipid panel showed a total cholesterol of 124 mg/dL, triglycerides of 138 mg/dL, low-density lipoprotein cholesterol (LDL-C) of 106 mg/dL, and an HDL-C of ,5 mg/dL. On nuclear magnetic resonance (NMR) spectroscopy testing, HDL-C was ,5 mg/dL with triglycerides of 98 mg/dL. The LDL particle number (LDL-P) was 2100 nmol/L (reference value: ,1000 nmol/L) with an LDL-C of 115 mg/dL. Statin therapy was prescribed given the presence of subclinical atherosclerosis, and after 4 months led to an improvement in the LDL-C to 50 mg/dL but the LDL-P remained at 1900 nmol/L. Additional studies showed ApoA-1 0.7 mg/dL (markedly decreased), ApoB 71 mg/dL (normal), and lipoprotein (a) 3.6 mg/dL (normal). The between-run coefficients of variation were all ,5%. HDL particle analysis by two-dimensional gel electrophoresis followed by ApoA-1 antibody immunoblotting in mg/ dl of ApoA-1 measured as previously described revealed: precursor very small pre-beta-1 HDL 0.2 (very low), very small alpha-4 HDL 0.1 (very low), small alpha-3 HDL 0.1 (very low), medium alpha-2 HDL 0.1 (very low), and large HDL alpha-1 0.0 (undetectable, normal .20; Fig. 3). Sizeexclusion chromatography of the patients plasma total lipoproteins after ultracentrifugal flotation at d 5 1.21 g/mL was performed. The profiles were analyzed by absorbance and cholesterol mass (Fig. 4).
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Figure 2 Slit-lamp examination of the cornea under high (A) and low (B) magnification showing small dots representing lipid deposition within the corneal stroma.
Serum markers of inflammation were measured. Highsensitivity C-reactive protein was 0.7 mg/L (reference value: ,3.0 mg/L), Lp-PLA2 (lipoprotein-associated phopholipase A2) was ,100 ng/mL (reference value: 1202342 ng/mL), plasma homocysteine levels were 9.6 mmol/L (reference value: 3.0-15 mmol/L), and the ApoE genotype was 2/3.
Diagnostic imaging Repeated extensive work-up for chronic neurologic disorders, especially multiple sclerosis, including serial magnetic resonance imaging of brain and spinal cord, spinal fluid analysis, and visual-evoked response studies were negative during acute episodes and in remission over 20 years. Four years before our visit, the patient had repeated bouts of lower abdominal colicky pain with change in bowel habits. A computed tomography scan of the abdomen revealed small low-density oval hypoechoic lesions measuring up to 7 mm in the liver parenchyma, possibly related to fatty accumulation, with no enlarged lymph nodes or hepatosplenomegaly. Colonoscopy showed heavy pigmentation of the colonic mucosa that extended through the entire length of the colon (Fig. 5). Multiple pieces of yellow-tan tissue were biopsied which, on microscopic examination, revealed foamy xanthoma cells both within the lamina propria and muscularis mucosa.
Muscle biopsies Muscle biopsy revealed scattered atrophic fibers with a striking absence of lipid droplets surrounding the muscle fibers (Fig. 6). Glycogen content, carnitine content, and glycogen spectrum wavelength of muscle fibers were all normal. Autoimmune screen was negative. Peripheral Myelin Protein 22 gene analyses showed no deletions, duplications, or sequence alterations.
Mutation analysis The combination of a very low HDL-C and a family history of consanguinity were suggestive of a hypoalphalipoproteinemia with an autosomal-recessive inheritance pattern. Thus, we sequenced ABCA1, ApoA-1, and lecithin-cholesterol acyltransferase. Sequencing of the ABCA1 gene revealed a novel homozygous G-C splice site mutation adjacent to exon 33 at the 21 position.
Discussion In this report, we describe a case of TD associated with a novel homozygous splice-site mutation in the ABCA1 gene that resulted in a long history of atypical neurologic symptoms and signs. The patient presented with a multiple
Figure 3 HDL particle analysis by two-dimensional gel electrophoresis followed by apoA-I antibody immunoblotting in mg/dL of apoA-I measured (A) compared with those of a normal subject (left) and of a patient with premature heart disease (center) in the and with a schematic of the individual HDL particles on the right (B).
Negi et al
Novel mutation in Tangier disease
Figure 4 Size-exclusion chromatography of the patient’s plasma total lipoproteins after ultracentrifugal flotation at d 5 1.21 g/mL center. VLDL, very-low-density lipoprotein.
sclerosis-like phenotype, which has so far not been described in TD. In addition, she also had evidence of mild pressure palsies of the peripheral nerves of the hand, which has been described in TD.14 She also had atherosclerosis on noninvasive screening tests, which occurred prematurely considering her age and gender. The occurrence of premature CVD in patients with TD varies, and our
85 decision to start a statin was in response to the subclinical measurement of early atherosclerosis, not the LDL-P level.15 The first case of TD was described by Fredrickson in two siblings from Tangier Island, near the Chesapeake Bay.16 These children had enlarged, orange-colored tonsils filled with CE-laden macrophages, with similar deposition in the liver and spleen, resulting in hepatosplenomegaly, and in the lymph nodes. They also were noted to have marked HDL-C deficiency, decreased LDL-C, and moderate hypertriglyceridemia. Homozygotes are severely deficient in apoA-I and apoA-II, but other apolipoprotein levels were relatively normal, whereas heterozygotes have HDL-C levels that are 50% of normal.17 Clinically, both homozygotes and heterozygotes can develop peripheral neuropathy, corneal opacification, hepatosplenomegaly, omental masses, and premature CHD.9 Variants of ABCA1 in the general population also are linked to premature CVD.6 Two-dimensional gel electrophoresis of apoA-I containing HDL particles from homozygotes generally show very small discoidal pre-beta 1 migrating HDL, whereas heterozygotes have a marked reduction in large spherical alpha 1 and alpha 2 migrating HDL.9 Metabolic studies performed in homozygotes documented that the LDL particles in cases of TD are very small and rich in triglyceride and beta carotene.18 Because of the lack of ABCA1 function in these
Figure 5 Distal colon on colonoscopic examination showing numerous areas of yellowish orange pigmentation. These were found to be collection of foamy xanthoma cells on histopathological examination.
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Figure 6
Journal of Clinical Lipidology, Vol 7, No 1, February 2013
Trichome stain (A) and Oil Red O stain (B) of the muscle biopsy specimen showing atrophic fibers and scanty lipid granules.
individuals, no cellular cholesterol is taken up by pre-beta 1 HDL, which results in rapid catabolism of nascent HDL, probably by the kidney. This lack of CE formation and hence, lack of transfer of CE from HDL to apoB containing lipoproteins19 results in the production of small, cholesterol-poor LDL particles that are prone to greater uptake by non-adipose tissues.19 Our case is atypical and novel on several accounts. First, the clinical presentation is unique for the predominantly central nervous system (CNS) findings, including optic neuritis, internuclear ophthalmoplegia, ocular palsies, dysarthria, and gait problems. These occurred simultaneously with peripheral nervous system involvement, which was less prominent. The clinical heterogeneity in presentation of TD raises the possibility of different metabolic errors or a common metabolic error subject to genetic influences. Second, the patient lacked the orange-colored tonsils and hepatosplenomegaly classically described in TD. However, she did have the corneal stromal opacities and gastrointestinal lipid deposits. Another interesting finding was the total absence of lipid deposits in the muscle fibers on muscle biopsy. Finally, the case is unique with respect to the plasma lipoprotein distribution. Despite both HDL cholesterol and ApoA-1 levels being ,1 mg/dL, a very small amount of ApoA-1 was found in pre-beta 1 HDL, as well as in alpha 4, alpha 3, and alpha 2 HDL (Fig. 3). Her LDL-C was low on a standard lipid panel; however, her LDL particle number was very elevated on NMR analysis. One possible explanation could be that the triglyceride-enriched and cholesterol-depleted LDL particles emanate a strong LDL NMR signal despite the low LDL-C content, leading to an overestimation of the particle number. Our patient did have significant asymptomatic, subclinical atherosclerosis for her age and gender on the basis of both the carotid intima media measurement and coronary calcium score,13 and this was our rationale for use of a statin, not in response to the high LDL-P. The diagnosis of TD was reached on genetic analysis. She was found to have a homozygous splice-site mutation at the 21 position 50 to exon 33 in the ABCA1 gene. The ABCA1 gene contains 49 exons with exons 3 to 49 contributing to the amino acid sequence of ABCA1. There are 2052 base pairs contained within exons 33249, and
therefore an abnormal splice site would result in the loss of 684 amino acids from the terminal end of ABCA1.20 Other splice-site mutations have been reported, but the mutation at 21 to exon 33 is novel.20 Plasma lipoproteins are believed to regulate ganglioside biosynthesis in the CNS. ABCA1 is widely expressed throughout the body, including cells within the CNS, but its role in brain lipid metabolism is not yet fully understood. In the brain, glia synthesize the apolipoproteins involved in CNS lipid metabolism.21 It has been demonstrated that glial ABCA1 is required for cholesterol efflux to apoA-I and also plays a key role in facilitating cholesterol efflux to apoE, which is the major apolipoprotein in the brain. In both astrocytes and microglia, ABCA1 deficiency reduces lipid efflux to exogenous apoE. The impaired ability to efflux lipids in ABCA12/2 glia results in lipid accumulation in both astrocytes and microglia.21 In addition, apoE secretion is compromised in ABCA12/2 astrocytes and microglia.21 In vivo, deficiency of ABCA1 results in a 65% decrease in apoE levels in whole brain, and a 75% to 80% decrease in apoE levels in hippocampus and striatum. More evidence is emerging that glial ABCA1 is a key influence on apoE metabolism in the CNS. Further research on the functions of ABCA1 in the CNS may lead to improved understanding of the plethora of clinical presentations of TD, including the reason for the atherogenicity associated with very low HDL. The transient neurologic symptoms in TD, similar to the relapsing-remitting CNS symptoms in this case, are seen in nearly 50% of TD cases.22 Histopathology and electron microscopy of biopsied peripheral nerves have revealed preferential lipid deposition in the paranodal regions of myelinating Schwann cells. It has been speculated that this accumulation of lipid may physically separate the myelin sheath from the axon at this site, and in turn, leads to paranodal malfunction, slowing of nerve conduction or conduction block, and the resulting clinical symptoms. Paranodal myelin remodelling eventually leads to recovery of paranodal function and to clinical recovery. The reconstituted paranodal region may be vulnerable to further accumulation of lipid deposits, leading to a cycle of breakdown and repair, consistent with the remitting-relapsing clinical course.22
Negi et al
Novel mutation in Tangier disease
Conclusion We present a unique phenotype of TD, related to a novel splice site mutation in ABCA1, that highlights an unusual constellation of neurologic symptoms mimicked multiple sclerosis, as well as evidence of premature atherosclerosis.
Acknowledgments The authors acknowledge Boston Heart Laboratories, Houston Eye Associates, and Yadollah Harati, MD, Department of Neuromuscular Sciences, Baylor College of Medicine, for their contribution in the preparation of the manuscript.
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