Clinical and Investigative Medicine

 

Limiting the Niemann­Pick Type C critical region to a 1-cM interval

Tanya L. Gillan
David M. Byers, PhD
D. Christie Riddell, PhD
Paul E. Neumann, MD
Wenda L. Greer, PhD

Clin Invest Med 1997;20(5):339-343

[résumé]

Tanya Gillan is a graduate student and Dr. Greer is an Associate Professor in the Department of Pathology; Dr. Byers is an Associate Professor in the Departments of Pediatrics and Biochemistry; Dr. Riddell is an Associate Professor in the Departments of Pathology and Biochemistry; and Dr. Neumann is a Professor in the Departments of Anatomy and Neurobiology and Pathology, Dalhousie University, Halifax, NS.

(Original manuscript submitted June 27, 1997; received in revised form July 18, 1997; accepted July 24, 1997)

Reprint requests to: Dr. Wenda Greer, Queen Elizabeth II Health Sciences Centre -- VG Site, DNA Laboratory, Hematology, Rm. 223B, Mackenzie Bldg., 5788 University Ave., Halifax NS B3H 1V8

Contents

Abstract

 Objective: To refine the position of and isolate the gene responsible for Niemann­Pick Type II (NP Type II) disease, an autosomal, recessive neurodegenerative disorder usually affecting children. The underlying biochemical defect results in an impairment in transport of intracellular cholesterol. This disease has been classified into two subtypes, NPC and NPD. NPD and the major complementation group of NPC both map to chromosome 18q11-12; therefore, they are likely allelic variants. The NP Type II gene was previously localized between microsatellite markers D18S44 and D18S1108.

Design: Linkage analysis.

Setting: Pathology department of a university-associated hospital.

Patients: An NPC family, including proband, parents and sister.

Outcome measures: NP Type II disease phenotype and biochemical phenotype (cholesterol esterification).

Results: DNA from the individuals in the NPC family was genotyped at 12 microsatellite loci from the critical region. The deduced haplotypes identify a meiotic recombinant that has allowed the distal limit of the critical region to be moved from D18S1108 to D18S1101.

Conclusion: The NP Type II gene lies proximal to the microsatellite marker D18S1101, within the 1-cM interval between D18S1101 and D18S1398. This represents approximately 1.1 mb on the physical map.

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Résumé

Objectif : Préciser la position du gène qui cause la maladie de Niemann­Pick de type II (NP de type II), trouble neurodégénératif récessif autosomique qui atteint les enfants, et l'isoler. Le défaut biochimique sous-jacent entraîne un déficit du transport du cholestérol intracellulaire. Cette maladie a été subdivisée en 2 sous-types, NPC et NPD. Le type NPD et le principal groupe de complémentation du NPC pointent tous 2 vers le chromosome 18q11-12 et il s'agit donc probablement de gènes allélomorphes. Le gène NP de type II a été localisé auparavant entre les marqueurs microsatellites D18S44 et D18S1108.

Conception : Analyse des liens.

Contexte : Service de pathologie d'un hôpital affilé à une université.

Patients : Une famille NPC, y compris le proposant, les parents et la soeur.

Mesures des résultats : Phénotype de la maladie NP de type II et phénotype biochimique (estérification du cholestérol).

Résultats : On a établi le génotype de l'ADN des individus de la famille NPC à 12 locimicrosatellites à partir de la région critique. Les haplotypes déduits identifient un recombinant méiotique qui a permis de déplacer de D18S1108 à D18S1101 la limite distale de la région critique.

Conclusion : Le gène NP de type II se trouve à un site proximal du marqueur microsatellite D18S1101 à l'intérieur de l'intervalle 1 cM entre D18S1101 et D18S1398, ce qui représente environ 1,1 mb sur la carte physique.

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Introduction

 Niemann­Pick Type II (NP Type II) disease was described by Crocker and Farber1 as an autosomal, recessive neurodegenerative disorder that usually occurs during childhood. Clinical features include ataxia, dysarthria, dystonia, vertical supranuclear gaze palsy, dementia and hepatosplenomegaly.2 There is marked phenotypic heterogeneity among individuals with this disease, both with respect to severity of symptoms and age of onset, which can be any age between less than 1 year and 20 years. The fundamental genetic defect associated with NP Type II is still unknown. It has been shown to be a lysosomal storage disorder; however, the underlying biochemical defect is unknown. The biochemical hallmark of NP Type II cells is abnormal processing of intracellular cholesterol, which results in accumulation of unesterified low-density-lipoprotein (LDL)-derived cholesterol within lysosomes.3 In normal cells, LDL-derived cholesterol is rapidly transported from lysosomes to the endoplasmic reticulum, where it is esterified by acyl-CoA:cholesterol acyl-transferase (ACAT). In NP Type II cells, the level of ACAT activity in response to LDL cholesterol is reduced or absent. Studies of numerous NP Type II cell lines have demonstrated considerable biochemical heterogeneity with respect to LDL cholesterol esterification.4

NP Type II disease has been subclassified into NPC and NPD.5,2 NPC is of panethnic origin and is clinically heterogeneous, whereas NPD has been identified only in descendants of an Acadian population from Yarmouth County, Nova Scotia, and is more homogeneous. NPC has been found to comprise at least 2 complementation groups.6,7 NPD and the major complementation group of NPC were localized to chromosome 18q11-12 between microsatellite markers D18S44 and D18S66;8­10 thus, they are likely allelic variants.10 The biochemical phenotype of NPD appears to be a milder variant of NPC.11,12 Our previous linkage disequilibrium analysis narrowed the critical region by placing the distal limit at locus D18S1108. Here we report, as part of the effort to positionally clone the NPC/NPD locus, the identification of a recombinant NPC chromosome between markers D18S44 and D18S1101, which redefines the NPC/NPD critical region.

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Materials and methods

Subjects

An NPC family was brought to our attention by the Department of Medical Genetics at the IWK Grace Health Sciences Centre in Halifax, NS. Peripheral blood was collected from the proband, parents and sister with ethical approval from the IWK Grace Health Sciences Centre. The proband is a 17-year-old young man with a phenotype from the mild end of the NPC clinical spectrum.

Microsatellite analysis

We used 12 polymorphic microsatellite markers that span a 15-cM interval including the NPC/NPD critical region of chromosome 18. These markers included D18S40, D18S869, D18S44, D18S1101, D18S1108, D18S480, D18S975, D18S66, D1S478 and D18S1151, which were obtained from the Genome Data Base (http://gdbwww.gdb.org) or Whitehead Institute Data Base (www.genome.we.mit.edu), as well as D18S1397 and D18S1396 (unpublished observations). Total genomic DNA was extracted with the use of a standard high-salt extraction protocol. Alleles at each locus were amplified by polymerase chain reaction (PCR) with 32P dCTP, using conditions and primers as we have described elsewhere.10 PCR products were separated electrophoretically on an 8% polyacrylamide gel and detected by autoradiography with exposure from O/N to 3 days at room temperature or -70°C.

Measurement of cholesterol esterification

Cholesterol esterification in Epstein­Barr-virus-transformed lymphoblasts was determined by measuring incorporation of [3H]oleic acid into cholesteryl-[3H]oleate according to the protocol outlined by Byers and associates.13 In brief, 12 × 106 lymphoblasts were seeded into 150 cm2 flasks and grown for 4 days at 37°C with 5% carbon dioxide in RPMI 1640 containing 5% delipidated serum. On the day of the experiment, cells were seeded in 35-mm dishes at a density of 3.5 × 106 cells with 2 mL of this medium, with or without 10% fetal bovine serum (FBS). Four hours later, [3H]oleic acid (3.6 µCi, 0.2 µmol) was added to each dish. Cells were pulsed with [3H]oleate for 2 hours and harvested at 4°C by centrifugation (1500 × g for 5 minutes), washed once with 2 mL ice-cold phosphate-buffered saline solution and resuspended in 2 mL of hexane:isopropanol (3:2 v/v) for lipid extraction.14 Lipid extracts were separated by thin-layer chromatography and identified by comparison with standard mixtures.13

Bayesian analysis

The carrier risk of individual II-2 (Fig. 1) was estimated using Bayes theorem. The a priori probability of being a carrier is 2/3. The conditional probability for reduction of cholesterol esterification (below the 95% confidence interval for normal values) is 94% (D.M. Byers, unpublished data).

Complementation studies

Cultured fibroblasts from the proband were fused with NPD fibroblasts using 40% polyethylene glycol according to a protocol previously described by Watkins and Rosenblatt.15 Fusions of proband to proband and proband to normal served as negative and positive controls, respectively. Levels of cholesterol storage were assessed by staining with fluorescent filipin, as described by Byers and associates.16 A reduction in filipin staining in tri- and tetra-nucleated cells, comparable to that seen in normal fibroblasts, was considered evidence of complementation.

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Results

Results Fig. 1 illustrates the segregation pattern of 12 microsatellite marker loci from the centromeric region of chromosome 18 in the family, which included a son affected with NPC disease (II-1) and an unaffected daughter (II-2). Although the proband presents clinically with a relatively mild phenotype, resembling that of the Nova Scotian NPD patients, the diagnosis is NPC, since neither of his haplotypes are similar to those associated with NPD. We have shown, with complementation studies in which his fibroblasts were fused to NPD fibroblasts, that he belongs to the major NPC complementation group that maps to human chromosome 18. Levels of stored cholesterol returned to normal when his fibroblasts were fused with normal, but not with NPD, fibroblasts (data not shown).

The deduced haplotypes indicate that one or the other of the children has inherited a recombinant maternal chromosome with the breakpoint within the NPC/NPD critical region between microsatellite markers D18S1397 and D18S1101. The mother's DNA is uninformative at D18S1396, which is located between D18S1397 and D18S1101. The daughter has arbitrarily been represented as the recombinant; however, this does not affect the mapping of NPC relative to the recombination point. She and her affected brother share the same maternal haplotype proximal to D18S1101, but they differ at D18S1101 and distal loci. To position the gene with respect to the breakpoint, it was necessary to determine the carrier status of individual II-2. The cholesterol esterification assay indicates that individual II-2 is a carrier of the NP Type II gene (Fig. 2). The level of esterification in her cells more closely resembled that of her heterozygous mother, from whom she received the recombinant chromosome, than that of the normal control.

On the basis of these results, we conclude that the NP Type II gene lies above the microsatellite marker D18S1101 and within the 1-cM interval between D18S1101 and D18S44.

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Discussion

Research efforts of our laboratory are directed toward isolating and characterizing the NPC/NPD genetic defect through a strategy of positional cloning. Previous studies by Carstea and associates8,9 localized the NPC gene to a 5-cM region on chromosome 18 between microsatellite markers D18S44 and D18S66. More recently, we have mapped NPD within this region, which suggests that NPC and NPD are likely allelic variants.10 We subsequently placed the NPD locus between a newly isolated marker, D18S1398, and D18S1108 (unpublished observations). We now report a recombinant chromosome that redefines the NPC critical region to a 1-cM interval between markers D18S44 and D18S1101. If NPC and NPD are allelic, the NPC/NPD locus resides between D18S1398 and D18S1101. This represents approximately 1.1 mb on the physical map. A significant portion of this interval could be excluded from the critical region by further delineation of the recombination breakpoint reported here through the development and use of additional polymorphic markers. Identification of the NP Type II gene through positional cloning will ultimately provide insight into the pathologic process and biochemical characteristics of this disease and lead to better therapies.

Any conclusion concerning gene location derived from this meiotic recombination depends on accurate diagnosis of the carrier status of individual II-2. As with all biochemical carrier assays, measurement of ACAT activity in NP families shows some overlap between heterozygotes and normals. The mean and standard deviation (SD) for this assay was previously reported as 0.79 (SD 0.21) nmol/h/mg protein for normal individuals and 0.45 (SD 0.18) nmol/h/mg protein for heterozygotes.13 Thus, the value of 0.41 nmol/h/mg protein in II-2 is well below normal. Her mother, from whom she has probably inherited the gene defect, shows a similar level. On the basis of Bayesian analysis, which takes ACAT activities into account, her carrier risk of 67%, estimated a priori, is now estimated at 98% a posteriori. It is interesting to note that ACAT activity in her father was well within the normal range. Heterozygote levels that deviate from expected values have been reported previously by Roff and associates.17

As this article was going to press, Carstea and associates18 reported the identification of the NPC gene.
The gene was found within the interval defined here.

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Acknowledgements

 We would like to thank Gabrielle Girouard, Susan Sparrow, Robert Zwicker and Heather Keith for excellent technical assistance. This work was supported by the Ara Parseghian Medical Research Foundation, the National Niemann­Pick Foundation and the Medical Research Council of Canada.

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References
  1. Crocker AC, Farber S. Niemann­Pick disease: a review of 18 patients. Medicine 1958;37:1-95.
  2. Pentchev PG, Vanier MT, Suzuki K, Patterson MC. Niemann­Pick disease type C: a cellular cholesterol lipidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular basis of inherited disease. 7th ed. New York: McGraw-Hill; 1995. p. 2626-39.
  3. Pentchev PG, Comly ME, Kruth HS, Vanier MT, Wenger DA, Patel S, et al. A defect in cholesterol esterification in Niemann­Pick disease (type C) patients. Proc Natl Acad Sci U S A 1985;82:8247-51.
  4. Vanier MT, Rodriguez-Lafrasse C, Rousson R, Gazah N, Juge MC, Pentchev PG, et al. Type C Niemann­Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing. Biochem Biophys Acta 1991;1096:328-37.
  5. Spence MW, Callahan JW. Sphingomyelin-cholesterol lipidoses: the Niemann­Pick group of diseases. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic basis of inherited disease. 6th ed. New York: McGraw-Hill; 1989. p. 1655-76.
  6. Steinberg SJ, Ward CP, Fensom AJ. Complementation studies in Niemann­Pick disease type C indicate the existence of a second group. J Med Genet 1994;31:317-20.
  7. Vanier MT, Duthel S, Rodriguesz C, Pentchev P, Carstea ED. Genetic heterogeneity in Niemann­Pick C disease: a study using cell hybridization and linkage analysis. Am J Hum Genet 1996;58:118-25.
  8. Carstea ED, Polymeropoulos MH, Parker CC, Detera-Wadleigh SD, O'Neill RR, Patterson MC, et al. Linkage of Niemann­Pick disease type C to human chromosome 18. Proc Natl Acad Sci U S A 1993;90:2002-4.
  9. Carstea ED, Parker CC, Fandino LB, Vanier MT, Overhauser J, Weissenbach J, et al. Localizing the human Niemann­Pick gene to 18q11-12 [abstract]. Am J Hum Genet 1994;(55 suppl):A182.
  10. Greer WL, Riddell DC, Byers DM, Welch JP, Girouard GS, Sparrow SM, et al. Linkage of Niemann­Pick disease type D to the same region of human chromosome 18 as Niemann­Pick disease type C. Am J Hum Genet 1997;61:139-42.
  11. Byers DM, Rastogi SR, Cook HW, Palmer FBStC, Spence MW. Defective activity of acyl-CoA: cholesterol O-acyltransferase in Niemann­Pick type C and type D fibrolasts. Biochem J 1989;262:713-9.
  12. Sidhu H, Rastogi SAR, Byers DM, Guernsey DL, Cook HW, Palmer FBStC, et al. Regulation of low density lipoprotein receptor and 3-hydroxy-3methyl-glutaryl-CoA reductase activities are differentially affected in Niemann­Pick type C and type D fibroblasts. Biochem Cell Biol 1993;71:467-74.
  13. Byers DM, Douglas J, Cook HW, Palmer FBStC, Ridgeway ND. Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann­Pick type C and type D patients. Biochim Biophys Acta 1994;1226:173-80.
  14. Liscum L, Faust JR. Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann­Pick type C fibroblasts. J Biol Chem 1987;262:17002-8.
  15. Watkins D, Rosenblatt D. Failure of lysosomal release of vitamin B12: a new complementation group causing methylmalonic aciduria (cbIF). Am J Hum Genet 1986;39:404-8.
  16. Byers DM, Douglas JA, Cook HW, Palmer FBSC, Ridgway ND. Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann­Pick type C and type D patients. Bichem Biophys Acta 1994;1226:173-80.
  17. Roff CF, Goldin E, Comly ME, Blanchette-Mackie J, Cooney D, Brady RO, et al. Niemann­Pick type-C disease: deficient intracellular transport of exogenously derived cholesterol. Am J Med Genet 1992;42:593-8.
  18. Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, et al. Niemann­Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 1997;277:232-5.
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