September/October 2001 ⅐ Vol. 3 ⅐ No. 5 article Clinical-biochemical correlation in molecularly characterized patients with Niemann-Pick type C Vardiella Meiner, MD1, Shoshi Shpitzen, BSc2, Hanna Mandel, MD3, Aharon Klar, MD4, Ziva Ben-Neriah, MD1, Jol Zlotogora, MD, PhD5, Michal Sagi, PhD1, Alex Lossos, MD6, Ruth Bargal, MSc1, Vivy Sury, BSc1, Rivka Carmi, MD7, Eran Leitersdorf, MD2, and Marsha Zeigler, PhD1 Purpose: Niemann-Pick disease type C (NP-C) is an autosomal recessive lipid storage disease manifested by an impairment in cellular cholesterol homeostasis. The clinical phenotype of NP-C is extremely variable, ranging from an acute neonatal form to an adult late-onset presentation. To facilitate phenotype-genotype studies, we have analyzed multiple Israeli NP-C families. Methods: The severity of the disease was assessed by the age at onset, hepatic involvement, neurological deterioration, and cholesterol esterification studies. Screening of the entire NPC1 coding sequence allowed for molecular characterization and identification of disease causing mutations. Results: A total of nine NP-C index cases with mainly neurovisceral involvement were characterized. We demon- strated a possible link between the severity of the clinical phenotype and the cholesterol esterification levels in fibroblast cultures following 24 hours of in vitro cholesterol loading. In addition, we identified eight novel mutations in the NPC1 gene. Conclusions: Our results further support the clinical and allelic heterogeneity of NP-C and point to possible association between the clinical and the biochemical phenotype in distinct affected Israeli families. Genet Med 2001:3(5):343–348. Key Words: lipid storage disease, cholesterol esterification, mutation analyses, NPC1 gene, consanguineous marriage Niemann-Pick disease type C (NP-C) is an autosomal reces- of NP-C is extremely variable ranging from an acute neonatal sive lipid storage disease manifested by an impairment in cel- form, showing mainly liver involvement and rapid neurologic lular cholesterol homeostasis (OMIM number 257220). The deterioration, to an adult late-onset presentation, with slowly prevalence has been estimated at 1:150,000 in Europe, yet the progressive ataxia and a movement disorder. The late infantile incidence in early life is probably underestimated, because of and juvenile forms are considered to be the most common nonspecific presentations and possible under-diagnosis. Aca- classical presentations with the insidious onset of ataxia, verti- dians in Nova Scotia, individuals of Hispanic descent in Colo- cal supranuclear gaze palsy, and cognitive impairment in up to rado and New Mexico, and a Bedouin group in Israel represent 80% of the patients.1 genetic isolates each with a common founder. In the Acadians The underlying cellular defect is a disruption in the sterol of Nova Scotia, a milder variant of the disease that has been at trafficking, which leads to accumulation of free cholesterol first erroneously considered to be a distinct entity termed Ni- within the lysosomal and late-endosomal compartment of the emann Pick disease type D (NPD), proved later by molecular cell. The biochemical nature of NP-C became clear following analysis to be allelic to NP-C.1 the identification of a natural murine NP-C model presenting The disease is clinically characterized by progressive degen- with degenerative lipid storage disease.2 The affected mice had eration of the central nervous system with visceral accumula- elevated levels of unesterified cholesterol in various tissues. tion of cholesterol and sphingomyelin. The clinical phenotype Although a defect in cholesterol esterification was shown in NP-C patients,3 acyl CoA:cholesterol acyltransferase (ACAT) 4 From the 1Department of Human Genetics; 2Center for Research, Prevention, and Treatment deficiency was ruled-out. The accumulation of lysosomal un- of Atherosclerosis, Department of Medicine; 6Department of Neurology, Hebrew University- esterified cholesterol was found to be due to reduced release of Hadassah Medical School and Hadassah University Hospital, Jerusalem, Israel; 3Department free cholesterol from lysosomes to other cellular organelles of Pediatrics, Rambam Medical Center, Technion Faculty of Medicine, Haifa, Israel; 4Pedi- such as plasma membrane and the Golgi apparatus.5,6 Hence, atric Gastroenterology Unit, Bikur Cholim General Hospital, Hebrew University-Hadassah the definitive diagnosis of NP-C is based on the demonstration Medical School, Jerusalem, Israel; 5Department of Community Genetics, Public Health Ser- vices, Ministry of Health, Israel; and 7Clinical Genetics Unit, Soroka Medical Center, Ben- of abnormal cholesterol esterification in skin fibroblasts cul- Gurion University of the Negev, Beer-Sheva, Israel. tured with LDL-derived tritiated oleate. This assay provides the Vardiella Meiner, MD, Department of Human Genetics, Hadassah University Hospital, PO means for diagnosis of affected individuals, yet it cannot be Box 12000, Jerusalem, 91120 Israel. utilized for the identification of carriers. Attempts to identify a Received: May 9, 2001. correlation between the clinical phenotype and the severity of Accepted: June 28, 2001. the disruption of cellular cholesterol homeostasis have been Genetics IN Medicine 343 Meiner et al. inconclusive.7–10 Although a “variant” biochemical phenotype midified incubator using minimal essential medium (MEM) was described for patients with the adult-onset presentation, supplemented with 10% (v/v) fetal calf serum and antibiotics no definitive correlation has been observed for the late infan- according to standard protocols. [3H]Oleic acid mixture was tile and juvenile forms. prepared by sonicating 5 mCi [3H]oleic acid and 16.75 mg of The NPC1 gene has been cloned following its mapping to the sodium oleate in 4.4 mL of 14% fatty acid free bovine serum. long arm of chromosome 18 using linkage and positional clon- Cholesteryl ester synthesis was assayed as follows. Normal and ing.11,12 The NPC1 cDNA sequence predicts a protein of 1,278 patients’ cells (in 4th passage) were grown in T-25 flasks con- amino acids with an estimated molecular mass of 142 kDa. taining Eagles’ MEM medium with 5% bovine lipoprotein- Recently, topological analysis of the NPC1 protein has revealed deficient serum (LPDS) obtained from Sigma (Medium A). the existence of 13 transmembrane domains, 7 luminal loops, 6 After 4 days, the medium was replaced by either 2 mL of me- cytoplasmic loops, and a cytoplasmic tail.13 To date, more than dium A ϩ 0.02 mL of [3H]oleic acid mixture (2 flasks), or 100 NPC1 mutations, mostly missense, have been identified medium A ϩ 0.02 mL of [3H]oleic acid mixture ϩ 100 ␮gof throughout the gene, with no apparent hot spots.14–16 Most human LDL (Biomedical Technologies)/1 mL of Eagles’ MEM patients with NPC1 mutations are compound heterozygotes medium (Medium B). Cells were incubated for 4.5 and 24 with private unique mutations. The two known exceptions are hours at 37ЊC after which the cells were harvested and homog- a common I1061T mutation found in the Hispanic population enized by ultrasonication in 0.5 mL of distilled water. For lipid in the upper Rio Grande Valley in the southwestern United extraction, 0.1 mL of the homogenate was added to 1 mL of States, and in the United Kingdom, and France15 and the mu- chloroform:methanol (2:1). The lipid phase was dried under tation G992W in the Acadian population of Nova Scotia.17 nitrogen and dissolved in 0.1 mL of chloroform:methanol According to accumulated data so far, NPC1 is associated (2:1) of which 0.025 mL were spotted on plastic Silica Gel 60 with approximately 80% of known cases; however, both link- thin layer chromatography plates. The plates were developed age techniques and cellular complementation studies have in hexane:ethyl ether:glacial acetic acid (90:10:1, v/v/v). The shown the existence of genetic heterogeneity, providing proof radioactive cholesteryl ester was visualized by exposing the for the presence of an additional disease-causing gene.18 Re- plates to iodine vapors. The cholesteryl ester spot was scraped cently, HE1 encoding a ubiquitously expressed lysosomal cho- from the plate, and radioactivity was counted. Cholesteryl ester lesterol binding protein was identified as a second gene for formation was determined by subtracting the counts obtained NP-C.19 NP-C patients from both complementation groups from the extracted lipids incubated in medium A from the demonstrate similar clinical and biochemical phenotypes, sug- counts obtained from the extracted cells that were incubated in gesting that NPC1 and HE1 may interact or function sequen- medium B. The values were expressed as cholesteryl 3H- tially in a common metabolic pathway. oleate/mg protein for 4.5 and 24 hours of incubation. NP-C is observed in a relatively high frequency in Israel,20 especially among the Israeli non-Jewish population compris- ing Muslim Arabs, Bedouins, Druze, and Samaritans sub- Molecular studies groups. A relatively high rate of autosomal recessive disorders Total RNA was extracted from fibroblast monolayers using is observed as a consequence of the high consanguinity rate the TRI Reagent᭨ (Molecular Research Center, Inc.) accord- among these various groups. In this study, we characterized the ing to manufacturer’s instructions. Reverse transcription was clinical, biochemical, and molecular features of NP-C in Israel. performed using AMV Reverse Transcriptase (Promega) and standard protocols. The NPC1 cDNA was amplified in over-