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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Genet Med. 2015 Mar 12;18(1):41–48. doi: 10.1038/gim.2015.25

High Incidence of Unrecognized Visceral/Neurological Late-onset Niemann-Pick Disease, type C1 Predicted by Analysis of Massively Parallel Sequencing Data Sets

Christopher A Wassif 1,2,#, Joanna L Cross 1,2,#, James Iben 1, Luis Sanchez-Pulido 3, Antony Cougnoux 1, Frances M Platt 2, Daniel S Ory 4, Chris P Ponting 3, Joan E Bailey-Wilson 5, Leslie G Biesecker 6, Forbes D Porter 1
PMCID: PMC4486368  NIHMSID: NIHMS658762  EMSID: EMS62276  PMID: 25764212

Abstract

Purpose

Niemann-Pick disease, type C (NPC) is a recessive, neurodegenerative, lysosomal storage disease caused by mutations in either NPC1 or NPC2. The diagnosis is difficult and frequently delayed. Ascertainment is likely incomplete due to both these factors and that the full phenotypic spectrum may not have been fully delineated. Given the recent development of a blood-based diagnostic test and development of potential therapies, it is important to understand the incidence of NPC and to define at risk patient populations.

Method

We evaluated data from four large massively parallel exome sequencing data sets. Variant sequences were identified and classified as pathogenic or non-pathogenic based on a combination of literature review and bioinformatic analysis. This methodology provided an unbiased approach to determining the allele frequency.

Results

Our data suggests an incidence rate for NPC1 and NPC2 of 1/92,104 and 1/2,858,998, respectively. However, evaluation of common NPC1 variants, suggests that there may be a late-onset NPC1phenotype with a markedly higher incidence on the order of 1/20,000-39,000.

Conclusions

We determined a combined incidence of classical NPC of 1/89,229 or 1.12 affected patients per 100,000 conceptions, but predict incomplete ascertainment of a late-onset phenotype of NPC1. This finding strongly supports the need for increased screening of potential patients.

Keywords: Next Generation sequence study, Niemann-Pick disease, type C, NPC, Allele frequency

Introduction

Niemann-Pick Type C (NPC) is an autosomal recessive, neurodegenerative lethal disorder with a clinical incidence of 1:104,000 1-3. This was considered to be a minimal estimate due to incomplete ascertainment of atypical phenotypes or limitations of current diagnostic testing. NPC is caused by disruption of either NPC1or NPC2 with mutations of NPC1 accounting for 95% of patients 1; 2. Loss of function of either NPC1 or NPC2 results in the accumulation of unesterified cholesterol and glycosphingolipids within the late-endosome/lysosome of all cells. Although the clinical presentation and progression of NPC is a continuous spectrum, patients can be classified into four general categories based on age of neurological onset. These categories are early-infantile, late-infantile, juvenile and adolescent/adult-onset 1. In the early infantile, late-infantile, and juvenile forms of the disease patients may initially present with neonatal cholestasis or hepatosplenomegaly. A small subset of NPC patients die of systemic liver disease usually during the neonatal period 1. However in the majority of NPC patients the liver disease frequently resolves, but neurological signs and symptoms follow 1; 2. Neurological symptoms are insidious and heterogeneous in nature, often initially manifesting in a non-specific manner (e.g., clumsiness or difficulty with school work) but commonly progress to include variable degrees of cerebellar ataxia, vertical supranuclear gaze palsy, gelastic cataplexy, seizures, and dementia. These neurological manifestations are invariably progressive 4; 5 and ultimately result in death.

The current diagnosis of NPC is based upon filipin staining of unesterified cholesterol in cultured fibroblasts or molecular testing. Filipin staining requires a skin biopsy, is performed in only a few specialized diagnostic laboratories worldwide and is not always conclusive. Molecular testing of NPC1 and NPC2 is also available; however, molecular testing in practice also has weaknesses. It is currently still inconclusive in 12-15% of the cases, because of unknown pathogenicity of the changes, lack of study of allele segregation, existence of one (possibly 2) unidentified mutant allele. Combined with the frequently nonspecific and insidious nature of the neurological disease onset, the difficulty of diagnosis contributes to a diagnostic delay on the order of 4-5 years 2 for the late infantile and juvenile forms of the disease. The diagnostic delay in the adolescent/adult-onset is likely greater but the full extent of that delay cannot be determined due to a limited number of reported cases. Recently a sensitive blood-based diagnostic test, which detects elevated oxysterols, has been developed and this blood-based test could economically and rapidly be used to screen potential patients6.

A number of therapies for NPC are actively being developed. Miglustat, a glycosphingolipid synthesis inhibitor, although not approved in the United States for treatment of NPC1, has been approved in the European Union and other countries for the treatment of NPC. 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) has shown significant promise in both mouse and feline (Charles Vite personal communication) models of NPC1 and is currently in a phase 1/2 trial (NCT01747135) at the NIH. The development of HP-β-CD for NPC1 has been reviewed by Ottinger et al.7 Other potential therapies under development include HDAC inhibitors 8-10, HSP70 (F. Platt), and delta-tocopherol11. Given the rapid development of potential therapeutic interventions, it is critical that the incidence of NPC and its full clinical spectrum be fully defined.

An increasing number of adult-onset NPC patients are being reported 1; 12-14. Psychiatric symptoms can be prominent 12-15 although affected adults without neurological manifestations have also been reported 16-18. The full phenotypic spectrum of adult-onset NPC disease has yet to be delineated. This led us to question whether the incidence of NPC might be greater than previous clinical estimates due to incomplete ascertainment. To estimate the incidence of NPC in a manner that is independent of clinical recognition of cases, we sought to determine a pathogenic carrier frequency of NPC1 and NPC2 variants utilizing data from four independent massively parallel exome sequencing projects, or next generation sequencing projects. Our data indicates that the classical incidence of NPC likely occurs at the clinically predicted rate of approximately 1:90,000, and suggest that there may be a late-onset phenotype or variant form with an incidence potentially as high as 1:19,000-36,000.

Material and Methods

We have recently reported the determination of the pathogenic allele frequency of the 7-dehydrocholesterol reductase gene (DHCR7)19. We utilized a similar approach for the determination of the variant frequency in NPC.

Data Sets

Four large independent massively parallel exome sequencing projects, or next generation sequencing projects were utilized. These data sets are the NHLBI GO Exome Sequencing Project (ESP) 20, V3 release of the 1000 Genomes Project 21, ClinSeq® 22, and a database from a NIH inter-institute collaboration on Autism (PIs: FD Porter, J Bailey-Wilson, E Tierney, A. Thurm). ESP contributed a maximum number of 13,006 chromosomes, 1000 Genome Project contributed 2,184 chromosomes, ClinSeq® contributed 1,902 chromosomes and the NIH inter-institute collaboration on Autism project contributed 662 chromosomes. Thus, a maximum total of 17,754 chromosomes were analyzed and this number was utilized as the denominator in total frequency calculations. None of these datasets included patients evaluated for NPC nor did we identify any individuals with two pathogenic mutations, so we considered them to be unbiased with respect to variation in NPC1 and NPC2.

Determination of Variant Calls and Annotation

Variant calls were downloaded for regions overlapping NPC1 and NPC2, by Perl script for every base of the coding exons plus/minus 5 base pairs of exon sequence when available. Mutations were annotated using SNPnexus 23 using Refseq annotations 24, pathogenicity predictions were performed using Polyphen-2, 25 SIFT,26 Mutation assessor 27. Intronic variations detected within 5 bases of intron exon boundaries were analyzed by MaxEntScan 28. Untranslated regions variations were excluded from the analysis of these data sets.

Determination of Pathogenicity of the Variant Call

Determination of the pathogenicity of a variant allele was a multistep process that utilized both bioinformatic tools and manual curation. We began by comparing the variants found in the data sets against the professional version of the Human Gene Mutation Database (HGMD®) 29 and the existing database of 78 patients with NPC1 in the NIH Cohort (PI: FD Porter) to determine which variants had been previously identified in patients known to have NPC. Since inclusion in HGMD does not require identification in a patient, primary literature was reviewed to determine the nature and manner in which the variations were detected. Variants were mapped onto known protein tertiary structures as part of the bioinformatic approach, identifying variable to conserved residues and possible interactions (Figures 1 and 2). Modeling of the variant NPC2 protein was performed using I-TASSER30.

Figure 1.

Figure 1

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Mapping of the coding variants onto the known structure of NPC2. Probably damaging mutations are labeled with red circles. The human NPC2 structural model (from positions 20 to 149) was created using Modeller based on the bovine NPC2 structure (PDB:2HKA) 37. Human NPC2 ribbon is colored according to evolutionary conservation using ConSurf server38; 39. Cholesterol sulfate (from PDB:2HKA)37 is shown in sticks. Beta strands are labeled (A to G).

Figure 2.

Figure 2

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Mapping human N-terminal domain (NTD)-NPC1 mutants. Probably and possibly damaging mutations are labeled with red circles. The human NTD-NPC1 (PDB:3GKI)40 ribbon was colored according to evolutionary conservation using the ConSurf server 39; 40. Cholesterol is shown in sticks. None of the NTD-NPC1 mutants is located at cholesterol interacting residues.

Single coding nucleotide variants were interrogated in silico by three different predictive software packages Polyphen-2, 25 SIFT, 26 Mutation assessor 27. Polyphen-2 provides a predicted assignment of “Benign”, “Possibly Damaging”, or “Probably Damaging” as well as a false discovery rate (FDR) for each single coding nucleotide variant call. For the determination of the pathogenesis of a single coding nucleotide variants Polyphen-2 calls of “Possibly Damaging”, or “Probably Damaging” were considered pathogenic. SIFT uses the same terminology as Polyphen-2 and the same approach was used. Mutation assessor has four predictive determinants predictive non-functional low, non-functional neutral, functional (medium), and functional (high). Mutation assessor predictions of functional (medium), and functional (high) where considered pathogenic. When the three predictive algorithms were discrepant and no published data supporting pathogenicity was available, we accepted the prediction of two of the three programs. Potential splice variants were processed in MaxEntScan 28 to provide a predictive determination of the variants affect on splicing; these were reported as “Strongly Negative”, “Negative”, or “Neutral”. Potential splice variants that were classified as Negative or Strongly Negative were considered to be pathogenic. All pathogenic variants were assumed to be fully penetrant.

Determination of predicted disease incidence

Once potential pathogenic variants were identified and a carrier frequency determined, the predicted disease incidence was calculated assuming a Hardy-Weinberg-Equilibrium (HWE). For this estimate we assumed that all pathogenic variants were fully penetrant. The HWE model also assumes that allelic variation is at equilibrium and thus not undergoing active selective pressure. Given that NPC1 is a receptor for filoviruses and its association with body mass, an assumption of neutral selection may not be correct. However, Al-Daghri et al 31 concluded selective pressure on NPC1 in humans is weak to neutral. We made the assumption that allelic frequencies were consistent across different ethnic groups represented in our dataset. The potential error making this assumption is greatest for the ESP cohort given that it includes large number of individuals of either European or African descent. We evaluated our data for reduction of heterozygosity due to ethnic difference (Wahlund effect) by determining a weighted frequency; however, only negligible changes were observed for any of the NPC1 or NPC2 pathogenic alleles (data not shown). Given the negligible effect the weighted frequencies were not applied to carrier frequency calculations.

Cloning and Sequence Analysis of the c.441+1G>A Variant Discovered in NPC2

Two heterozygous Epstein-Barr virus transformed lymphoblast cell lines for the c.441+1G>A variant, NIMH 42 and NIMH 77, were identified in the NIH inter-institute collaboration on Autism. These 2 lines and one control line were grown under standard growth conditions15% fetal bovine serum in RPMI (Life Technologies) for 3 days. Cell pellets were isolated and mRNA isolated as per manufactures protocol (Qiagen). Forward primer NPC2-F3 5′-GGTGGAGTGGCAACTTCAGG-3′ and Reverse primer NPC2-R2 5′-CACTGGATACCATTGGAGAGC-3′ were used to reverse transcribe the mRNA using Superscript III One-step RT-PCR System (Life Technologies). The cDNA was visualized on a 1.5% agarose gel. One band was observed for WT and two for NIMH 42 and NIMH 77. All bands were gel purified and cloned into the TOPO TA Cloning Kit for Sequencing (Life Technologies). Isolated colonies were grown overnight in LB-ampicillin and plasmid DNA was isolated (Qiagen). Sequencing was performed on a 3500×L Genetic Analyzer (Life Technologies) using BigDye sequencing kit as per manufacturer’s protocol.

Results

Analysis of exomic sequence data from 17,754 chromosomes as compared to the human reference sequence for NPC1 and NPC2, led to the identification of 16,455 and 271 nonsynonymous sequence variants in NPC1 and NPC2, respectively. The 16,455 variants identified in NPC1 were comprised of 147 distinct variants that included 129 coding single nucleotide base variants, 9 splice site changes, and 9 insertions/deletions (Table 1). The 271 nonsynonymous variants identified in NPC2 included 14 distinct changes consisting of 12 coding single nucleotide base variants and 2 splice site changes (Table 2).

Table 1.

This table summarizes the 16,455 distinct variants detected in NPC1. Each variant has a corresponding cDNA nucleotide number, protein change, and reference SNP “RS” number when available. The majority have been assigned either a Polyphen-2, SIFT, Mutation assessor, or MaxEntScan scores, as well variants that have been previously published are noted. Variants considered non-pathogenic are shaded grey. The number of alleles analyzed for each variant and the total number of times the variant was detected are noted in conjunction with the frequency of each variant in each of the four data sets and the carrier rate for each variant. The asterisk indicates the one novel variant detected in the NIH patient.

cDNA Protein rs# Polyphen-2/MaxEntScan/Published SIFT MutationAssessor Pred Total Alleles Total Variants NHLBI 1000 Genome Clinseq Autism Rate
c.110A>G p.D37G BENIGN BENIGN Predicted non-functional (low) 17754 1 0 0 1 0 0.006%
c.127G>A p.E43K rs138277307 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.180G>T p.Q60H rs145566943 BENIGN POSSIBLY DAMAGING Predicted functional (medium) 17754 5 4 1 0 0 0.028%
c.181-4A>C intronic rs374571310 Neutral 17754 2 2 0 0 0 0.011%
c.181-3A>G intronic rs371126954 Strong Negative Effect 17754 1 1 0 0 0 0.006%
c.209A>G p.N70S rs200291759 BENIGN BENIGN Predicted non-functional (low) 17754 1 0 1 0 0 0.006%
c.233G>A p.R78Q rs373274825 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.346C>T p.R116X rs144973225 PROBABLY DAMAGING/Published 17754 1 1 0 0 0 0.006%
c.347G>A p.R116Q rs140952850 BENIGN BENIGN Predicted non-functional (neutral) 17754 2 2 0 0 0 0.011%
c.410C>T p.T137M rs372947142 BENIGN/Published BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.424_425insGA p.K142Rfs 17266 1 1 0 0 0 0.006%
c.442G>C p.V148L rs200323346 BENIGN BENIGN Predicted non-functional (neutral) 17754 2 0 2 0 0 0.011%
c.445G>A p.G149R rs143205855 BENIGN BENIGN Predicted non-functional (low) 17754 2 1 1 0 0 0.011%
c.449A>G p.Q150R rs37594D577 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.466A>G p.M156V rs149074243 BENIGN BENIGN Predicted non-functional (low) 17754 6 6 0 0 0 0.034%
c.467T>C p.M156T rs147615070 BENIGN BENIGN Predicted non-functional (low) 17734 2 1 0 1 0 0.011%
c.481C>T p.R161W rs141243713 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 2 2 0 0 0 0.011%
c.520G>C P.G174R rs37009B528 PROBABLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.544G>A p.D182H rs201021988 BENIGN BENIGN Predicted non-functional (neutral) 17754 2 1 1 0 0 0.011%
c.547G>A p.A183T rs111256741 BENIGN BENIGN Predicted non-functional (neutral) 17754 7 4 3 0 0 0.039%
c.548C>T P.A183V rs192963719 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 0 1 0 0 0.006%
c.553A>G p.N185D rs139485263 BENIGN POSSIBLY DAMAGING Predicted functional (medium) 17752 4 3 0 1 0 0.023%
c.622G>C p.V208L rs372416248 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.631G>T p.D211Y rs367851289 PROBABLY DAMAGING BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.644A>G p.H215R rs1805081 BENIGN/Known polymorphism BENIGN Predicted non-functional (low) 17696 5250 3849 535 646 220 29.668%
c.665A>G p.N222S rs55680026 BENIGN/Published BENIGN Predicted non-functional (low) 17748 71 59 2 9 1 0.400%
c.688_693del p.S230_V231del Published 17226 3 3 0 0 0 0.017%
c.695A>G p.D232G rs201956601 BENIGN BENIGN Predicted functional (medium) 17754 1 0 1 0 0 0.006%
c.709C>T p.P237S rs80358251 BENIGN/Published benign BENIGN Predicted non-functional (low) 17730 183 150 14 15 4 1.032%
c.749A>C p.K250T BENIGN BENIGN Predicted non-functional (low) 17754 1 0 0 0 1 0.006%
cn.763C>T p.P255S rs373815982 POSSIBLY DAMAGING BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.764C>G p.P255R rs3710239B3 PROBABLY DAMAGING BENIGN Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.769C>T p.P257S rs368776731 BENIGN POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.782C>T p.T261M rs374169117 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.797A>G p.D266G rs370188327 POSSIBLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.806A>G p.Y269C POSSIBLY DAMAGING BENIGN Predicted non-functional (neutral) 17746 1 0 0 1 0 0.006%
c.811A>G p.1271V rs370810779 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.841C>T p.L281F rs377132020 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.873G>T p.W291C rs138151007 BENIGN 17754 10 7 2 1 0 0.056%
c.901G>A p.E301K rs150154006 POSSIBLY DAMAGING BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.962C>T p.A321V rs138079168 BENIGN BENIGN Predicted non-functional (low) 17750 2 1 1 0 0 0.011%
c.979G>A p.V3271 rs141361998 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.1001G>C p.C334S rs199693280 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 0 0 1 0 0.006%
c.1010G>A p.R337Q rs373390781 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
C.1022G>C p.R341P rs370181667 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.1039G>A p.V3471 rs376741451 BENIGN BENIGN Predicted non-functional (low) 17754 2 2 0 0 0 0.011%
c.1055G>T p.C352F rs149020783 BENIGN BENIGN Predicted non-functional (low) 17754 2 2 0 0 0 0.011%
c.1094C>T p.S365L rs200243024 POSSIBLY DAMAGING 17754 2 0 0 2 0 0.011%
c.1115G>A p.R372Q rs150053420 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.1166G>A p.R389H rs373751051 POSSIBLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.1208T>C p.F403S rs371234970 PROBABLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.1211G>A p.R404Q rs139751448 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (high) 17754 1 1 0 0 0 0.006%
c.1232G>A p.R411Q rs77080672 BENIGN BENIGN Predicted non-functional (neutral) 17754 34 26 5 1 2 0.192%
c.1270C>G p.P424A rs143797098 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.1274C>T p.S425L rs140149624 BENIGN 17754 1 1 0 0 0 0.006%
c.1300c>T p.P434S rs61731962 BENIGN/Known polymorphism BENIGN Predicted non-functional (low) 17752 212 189 18 3 2 1.194%
c.1346C>T p.A449V rs372289265 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.1348A>G p.1450V rs141892620 BENIGN BENIGN Predicted non-functional (low) 17752 5 3 0 2 0 0.028%
c.1367C>T p.5456F rs374159264 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.1412C>T p.P471L rs201226297 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 0 1 0 0 0.006%
c.1421C>T p.P4741 rs372445155 PROBABLY DAMAGING/Published POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.1472G>A p.S491N rs37075B521 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.1480G>A p.V494M rs199B12609 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.1506C>G p.D502E rs191537721 BENIGN BENIGN Predicted non-functional (low) 17754 1 0 1 0 0 0.006%
c.1532C>T p.T511M rs13381670 PROBABLY DAMAGING 17754 51 42 8 0 1 0.287%
c.1549G>A p.V517l rs201791992 BENIGN BENIGN Predicted non-functional (low) 17754 1 0 1 0 0 0.006%
c.1552C>T p.R518W rs377515417 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 2 2 0 0 0 0.011%
c.1561G>T p.A521S rs138184115 BENIGN/Published BENIGN Predicted non-functional (neutral) 17754 9 9 0 0 0 0.051%
c.1628C>T p.P543L rs369368181 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.1672G>T p.A558S rs201156397 POSSIBLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17704 1 0 0 1 0 0.006%
c.1756G>A p.E586K rs369753548 BENIGN BENIGN Predicted non-functional (low) 17754 2 2 0 0 0 0.011%
c.1766A>G p.N589S rs147021046 BENIGN BENIGN Predicted non-functional (low) 17732 8 7 0 1 0 0.045%
c.1780_1781insT p.Y594Lfs 17266 1 1 0 0 0 0.006%
c.1793A>G p.N598S rs201236716 BENIGN PROBABLY DAMAGING Predicted non-functional (low) 17754 1 0 1 0 0 0.006%
c.1870G>A p.V6241 rs76615690 BENIGN BENIGN Predicted non-functional (neutral) 17754 2 0 2 0 0 0.011%
c.1901A>G p.Y634C rs202140203 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17752 1 0 0 1 0 0.006%
c.1936C>T p.R646C rs368129141 POSSIBLY DAMAGING BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.1937G>A p.R646H rs112387550 BENIGN BENIGN Predicted non-functional (low) 17754 7 4 3 0 0 0.039%
c.1976C>T p.A659V rs140786703 POSSIBLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.1990G>A p.V664M rs376213990 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.2020_2021del p.V674Lfs 17266 319 319 0 0 0 1.848%
c.2027G>C p.5676T PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 0 0 0 1 0.006%
c.2083C>G p.L695V rs370323921 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (high) 17754 1 1 0 0 0 0.006%
c.2141G>A p.R714H rs375047023 PROBABLY DAMAGING PROBABLY DAMAGING Predicted non-functional (low) 17754 2 2 0 0 0 0.011%
c.2209C>G p.L737V rs201100763 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 0 0 1 0 0.006%
c.2257G>A p.V753M rs146874573 BENIGN POSSIBLY DAMAGING Predicted non-functional (low) 17754 2 2 0 0 0 0.011%
c.2338G>A p.V780M rs193182840 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 1 0 0 0.006%
c.2428G>T p.V810L rs145362908 BENIGN BENIGN Predicted non-functional (neutral) 17754 8 5 3 0 0 0.045%
c.2428G>C p.V810L rs145362908 BENIGN BENIGN Predicted non-functional (neutral) 17752 1 0 0 1 0 0.006%
c.2501T>C p.M834T rs373435883 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
*c.2524T>C p.F842L rs19029B665 PROBABLY DAMAGING 17754 1 0 1 0 0 0.006%
c.2525T>C p.F842S rs374068891 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.2551G>A p.A851T rs139297968 POSSIBLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 2 2 0 0 0 0.011%
c.2572A>G p.1858V rs1805082 BENIGN/Known polymorphism BENIGN Predicted non-functional (neutral) 17752 8005 5758 1100 864 283 45.094%
c.2605-6_2605-3del intronic Neutral 17266 1 1 0 0 0 0.006%
c.2621A>T p.D874V rs372030650 POSSIBLY DAMAGING/Published BENIGN Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.2705C>G p.S902C rs374656358 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.2731G>A p.G911S rs34302553 BENIGN POSSIBLY DAMAGING Predicted non-functional (low) 17754 69 69 3 0 3 0.389%
c.2796-4C>T intronic rs374406578 Neutral 17754 1 1 0 0 0 0.006%
c.2800C>T p.R934X rs370721218 PROBABLY DAMAGING/Published 17754 1 1 0 0 0 0.006%
c.2819C>T p.S940L rs143124972 PROBABLY DAMAGING/Published 17754 1 1 0 0 0 0.006%
c.2873G>A p.R958Q rs120074132 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.2882A>G p.N961S rs34084984 BENIGN/Published BENIGN Predicted non-functional (low) 17754 69 56 11 1 1 0.389%
c.2908_2909insTT p.S970Ffs 17266 1 1 0 0 0 0.006%
C.2911+4C>T intronic rs186588103 Neutral 17754 3 3 0 0 0 0.017%
c.2929+4C>T intronic rs186588103 Neutral 17754 1 0 1 0 0 0.006%
c.2972_2973del p.991_fs Published POSSIBLY DAMAGING Predicted functional (medium) 17724 2 0 0 1 1 0.011%
c.2974G>T p.G992W rs80358254 PROBABLY DAMAGING/Published POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3011C>T p.S1004L rs150334966 PROBABLY DAMAGING/Published 17750 11 8 0 2 1 0.062%
c.3019C>G p.P1007A rs80358257 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 3 2 1 0 0 0.017%
c.3028A>C p.K1010Q rs191876836 BENIGN BENIGN Predicted functional (medium) 17754 1 0 1 0 0 0.006%
c.3047A>G p.H1016R rs140211089 POSSIBLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3052G>A p.A1018T rs146666146 PROBABLY DAMAGING POSSIBLY DAMAGING Predicted functional (high) 17754 2 2 0 0 0 0.011%
c.3059G>C p.S1020T rs374719153 BENIGN BENIGN Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3182T>C p.11061T rs80358259 BENIGN/Published POSSIBLY DAMAGING Predicted functional (medium) 17754 5 5 0 0 0 0.028%
c.3184G>A p.A1062T rs369960141 POSSIBLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3217G>A p.G1073S rs141440861 BENIGN BENIGN Predicted non-functional (neutral) 17750 23 18 4 1 0 0.130%
c.3265G>A p.E1089K rs374526072 PROBABLY DAMAGING/Published POSSIBLY DAMAGING Predicted functional (high) 17754 1 1 0 0 0 0.006%
c.3343G>T p.V1115F rs34226296 BENIGN BENIGN Predicted non-functional (neutral) 17754 43 34 7 0 2 0.242%
c.3364T>C p.W1122R rs148571882 BENIGN BENIGN Predicted non-functional (low) 17754 2 1 0 0 1 0.011%
c.3422T>G p.V1141G rs144725473 PROBABLY DAMAGING/Published POSSIBLY DAMAGING Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.3477+4A>G intronic rs114073738 Neutral 17754 28 28 0 0 0 0.158%
c.3498+4A>G intronic Negative 17754 4 0 4 0 0 0.023%
c.3506G>T p.S11691 rs139612110 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 2 2 0 0 0 0.011%
c.3535A>G p.M1179V rs61731969 BENIGN BENIGN Predicted non-functional (neutral) 17694 61 54 5 2 0 0.345%
c.3548G>A p.R1183H rs148035987 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 8 7 0 0 1 0.045%
c.3550G>A p.V1184M POSSIBLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17676 1 1 0 1 0 0.006%
c.3556C>T p.R1186C rs145297180 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 2 2 0 0 0 0.011%
c.3557G>A p.R1183H rs200444084 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 2 1 1 0 0 0.011%
c.3560C>T p.A1187V rs113371321 POSSIBLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (high) 17754 1 0 1 0 0 0.006%
c.3566A>G p.E1189G rs369098773 POSSIBLY DAMAGING/Published BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.3577C>T p.H1193Y rs375309094 BENIGN BENIGN Predicted non-functional (neutral) 17754 1 1 0 0 0 0.006%
c.3598A>G p.S1200G rs35248744 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 63 61 1 0 1 0.355%
c.3611_3G14del p.L1204Qfs Published 17264 1 1 0 0 0 0.006%
c.3619T>C p.F1207L rs140827681 BENIGN POSSIBLY DAMAGING Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.3667A>G p.11223V rs368658600 BENIGN POSSIBLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3689T>C p.L12305 rs374150662 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3741_3745A 0 17748 1 0 0 1 0 0.006%
c.3742_3745del p.L1248Vfs Published 17266 1 1 0 0 0 0.006%
c.3755-5_3755-4insTC intronic Neutral 17266 4 4 0 0 0 0.023%
c.3796C>T p.R1266X rs376164368 PROBABLY DAMAGING 17754 1 1 0 0 0 0.006%
c.3797G>A p.R1266Q rs1805084 BENIGN/Known polymorphism BENIGN Predicted non-functional (low) 17754 1724 1275 307 101 41 9.710%
c.3799T>G p.Y1267D rs373435628 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.3811G>C p.E1271Q rs140527006 POSSIBLY DAMAGING BENIGN Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.3814C>T p.R1272C rs200264267 PROBABLY DAMAGING BENIGN Predicted non-functional (neutral) 17754 1 0 1 0 0 0.006%
c.3818A>G p.E1273G rs374032318 BENIGN BENIGN Predicted non-functional (low) 17754 1 1 0 0 0 0.006%
c.3821G>A p.R1274Q rs151305963 BENIGN BENIGN Predicted non-functional (neutral) 17754 5 4 0 0 1 0.028%
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Table 2.

This table summarizes the 271 distinct variants detected in NPC2. Each variant has a corresponding cDNA number; protein change and reference SNP “RS” number when available. The majority have been assigned either a Polyphen-2, SIFT, Mutation assessor, or MaxEntScan score, as well variants that have been previously published are noted. Variants considered non-pathogenic are shaded grey. The number of alleles analyzed for each variant and the total number of times the variant was detected is noted in conjunction with the frequency of each variant in each of the four data sets and the carrier rate for each variant.

cDNA Protein rs# Polyphen-2/MaxEntScan/Published SIFT MutationAssessor Pred Total Alleles Total Variants NHLBI 1000 Genome Clinseq Autism Rate
c.38T>C p.L13P rs147602717 PROBABLY DAMAGING POSSIBLY DAMAGING Predicted functional (medium) 17360 5 4 0 1 0 0.029%
c.49G>A p.A17T rs145302203 BENIGN BENIGN Predicted non-functional (low) 17748 1 1 0 0 0 0.006%
c.56C>A p.A19D rs369392502 PROBABLY DAMAGING PROBABLY DAMAGING Predicted functional (medium) 17748 1 1 0 0 0 0.006%
c.58G>T p.E20X rs80358260 Published PROBABLY DAMAGING Predicted functional (medium) 17750 1 1 0 0 0 0.006%
c.88G>A p.V30M rs151220873 POSSIBLY DAMAGING/Published POSSIBLY DAMAGING Predicted non-functional (low) 17252 34 25 1 4 4 0.197%
c.115G>A p.V39M rs80358261 PROBABLY DAMAGING/Published PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.212A>G p.K71R rs142075589 POSSIBLY DAMAGING BENIGN Predicted non-functional (neutral) 17754 3 2 1 0 0 0.017%
c.224A>T p.H75L rs369221608 PROBABLY DAMAGING Predicted functional (medium) 17754 1 1 0 0 0 0.006%
c.271G>A p.D91N rs148607507 POSSIBLY DAMAGING POSSIBLY DAMAGING Predicted non-functional (low) 17754 10 8 0 2 0 0.056%
c.278G>T p.C93F rs143960270 PROBABLY DAMAGING/Published Predicted functional (high) 17754 1 1 0 0 0 0.006%
c.292A>C p.N98H rs142858704 BENIGN BENIGN Predicted non-functional (low) 17754 12 11 1 0 0 0.068%
c.340C>G p.P114A rs371363324 PROBABLY DAMAGING Predicted functional (high) 17754 1 1 0 0 0 0.006%
c.441+1G>A intronic rs140130028 Strong Negative/Published 17752 96 83 0 6 7 0.541%
c.442-4A>C intronic rs114950106 Neutral 17754 104 104 0 0 0 0.586%
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The Human Gene Mutation Database HGMD® 29 was queried to establishing what observed variants in this data set might be pathogenic. For NPC1 (Table 1) and NPC2 (Table 2), 33 (32 pathogenic and one benign variant) of 147 (22.4%) and 5 out of 14 (35.7%) variants, respectively, had previously been reported in HGMD®. One additional novel NPC1 variant, c.2524T>C (p.F842L), was present in the NIH cohort (Table 1). The combination of Polyphen-2, SIFT, and Mutation assessor classified 53 NPC1 and 8 NPC2 coding nucleotide variants as pathogenic by our criteria. Of the predicted pathogenic variants 27 (51%) and 6 (75%) have not been reported in HGMD® for NPC1 and NPC2 respectively. Polyphen-2 also calculates a false discovery rate (FDR). For NPC1 and NPC2 predicted variants, the average FDR for a prediction of “Probably” or “Possibly” damaging were 0.04% and 0.03% respectively. These low mean FDRs had a negligible effect on the carrier incidence estimate and thus were not applied to either NPC1 or NPC2 carrier frequency calculations.

For NPC1 and NPC2, 2 out of 9 and 1 of 2, potential splice mutations were predicted to be pathogenic. Of the nine insertion/deletions (indels) identified in NPC1, a two base pair deletion, c.2020_2021del, was observed 319 times only in the ESP data set and thus was removed as a technical artifact unique to the ESP data set. The eight other NPC1 indels result in a frameshift, and thus were considered pathogenic. No indels were identified in NPC2.

Based on the above analysis, for NPC1 we initially considered the 68 distinct variants meeting the criteria of pathogenic (54 identified by predictive software to be pathogenic, 4 indicated by the predictive software as “benign” but known to be pathogenic, the 2 splice variants, and the 8 insertion/deletions). This accounted for 371 pathogenic alleles with an estimated carrier rate of 2.09% (371/17,754) and a predicted NPC incidence of 1/9,160. Given the order of magnitude difference between this number and clinical estimates, this prediction is likely a significant overestimation. Thus we applied manual curation to the NPC1 data set. Four variants, c.665A>G (p.N222S), c.1532C>T (p.T511M), c.2882A>G (p.N961S), and c.3598A>G (p.S1200G), accounted for 254 out of the 371 (68%) predicted pathogenic alleles. Allelic frequencies for these four alleles were 0.400, 0.287, 0.389 and 0.355 percent respectively. Given that their individual allelic frequencies exceed the allelic frequency of p.I1061T (0.028%), the most commonly reported mutant allele in patients with mutations in NPC1, by more than a factor of 10 (Table 1), it is not plausible that these alleles are associated with classical NPC disease. Excluding these four high frequency variants based on this assertion left 117 pathogenic alleles or a 0.659% (117/17,754) carrier rate. This carrier rate predicts an incidence of NPC attributable to NPC1 of 1/92,104.

We further evaluated the decision to exclude the four high frequency alleles based on lack of an association with classical NPC disease. Although, all three predictive packages indicate both p.N222S and p.N961S to be non-pathogenic these two variants have been reported in “visceral-only” or adult-onset NPC1 cases. The p.N222S variant was reported in combination with a p.I1061T mutation in a single adult onset (35 yr) patient with variant filipin staining 32. This patient initially presented with visceral disease (hepatosplenomegaly) and later manifested ataxia at 44 years of age. We have identified a p.N222S variant in combination with c.1402T>G, (p.C468G) in teenage sisters diagnosed based on splenomegaly. The second allele in this sib pair, p.C468G is predicted by Polyphen-2 to be “Probably” damaging. Pathological analysis of the spleen in the older sibling was suggestive of Niemann-Pick disease, but filipin staining was inconclusive. Neurological symptoms were absent and signs were very minor with deep tendon hyperreflexia and minor auditory brainstem response abnormalities noted on evaluation at 15 and 13 years of age respectively. NIH severity score for both was 1 4. Plasma oxysterol concentrations were consistent with a diagnosis of NPC in these two subjects. Mapping of p.N222S to the known tertiary structure provided no additional evidence for the pathogenicity of this residue (Figure 2). The p.N961S (c.2882A>C) variant has been reported in a compound heterozygous state with p.S666N, (c.1997G>A) (with a Polyphen-2 prediction of “Probably” damaging) in an adult case with subclinical hepatosplenamegaly and lymphadenopathy noted on autopsy following death due to acute pulmonary embolism and myocardial infarction 16. Although no neurological symptoms were reported, brain pathology was notable for distended neurons with increased lipofuscin granules. Assuming one or both of these variants are pathogenic, fully penetrant and associated with late onset NPC disease, the total disease incidence of NPC1 would range from 1/19,077-1/36,420.

Although predicted to be probably damaging by Polyphen-2, neither p.T511M nor p.S1200G have been reported in NPC1 patients. Millat et al 33 reported p.T511M as a novel nonpathological coding single nucleotide variant. The p.S1200G variant was reported in an “NPC uncertain” case in the recent ZOOM study 14. This subject, patient 5 in the ZOOM study, was a compound heterozygote for p.V664M, a known NPC1 mutation, but plasma cholestane-3β,5α,6β-triol testing6 was negative. Current data do not support classification of either p.T511M or p.S1200G variants as pathogenic alleles.

Sequence analysis of NPC2 (Table 2) identified 151 potential pathogenic alleles and calculated a pathogenic carrier frequency of 0.85% (151/17,754). Again the predicted disease incidence, 1/55,297 did not appear to be realistic unless one proposed an extreme degree of under-ascertainment. Thus, we similarly applied manual curation to the NPC2 data set. Review of the NPC2 data identified two high frequency variants that dominated the frequency calculation c.441+1G>A and c.88G>A (p.V30M), both variants are reported in HMGD®. The splice variant, c.441+1G>C, was predicted to be “Strongly Negative” by MaxEntScan. Molecular analysis of independent cell lines revealed multiple splicing events. The most prominent errant splicing event results in the insertion of 16 bases that leads to the alteration of the terminal 4 amino acids and the addition of 86 additional amino acids to the protein (supplemental figure1). Multiple lines of evidence strongly indicate that this errant splicing results in a functional protein. First, the variant has not been reported in association with a patient with NPC. Second, modeling of the variant protein using I-TASSER30 found no alterations to the cholesterol binding pocket or stability of the protein (data not shown). Finally, Huang et al. have demonstrated that generation of an NPC2 fusion protein with mCherry fused to the carboxy-terminal end of the protein is fully functional and is able to correct the NPC cellular phenotype in Npc2−/− mouse embryonic fibroblasts34. As such we have excluded c.441+1G>C as a pathogenic allele. The p.V30M variant, with a allelic frequency of 0.197%, is predicted to be possibly damaging by Polyphen-2 and SIFT but considered non-pathogenic by mutation assessor. The one reported NPC subject with the p.V30M variant was classified as a phenotypic NPC variant, a second mutation was not identified and near normal levels of cholesterol esterification was reported in skin fibroblasts 35. Inclusion of the p.V30M allele predicts a disease incidence of NPC attributable to NPC2 of 1/402,400 and that NPC2 should account for 18.6% of patients with NPC. This latter prediction conflicts with clinical data indicating that NPC2 account for only 2-5% of all patients with NPC 1; 2. Sequence alignment and structural analyses demonstrate that the p.V30 residue is not evolutionarily conserved and is present in a structurally variable region of the NPC2 protein well away from its binding pocket (Figure 1). Furthermore, p.V30M is found at a higher frequency than any known pathogenic NPC2 allele, coupled with the lack of evidence supporting functional importance and ultimately the lack of any clinical correlation, has lead us to exclude p.V30M as a pathogenic allele. We are, therefore, left with 21 pathogenic alleles (0.118% carrier frequency) and a predicted disease incidence of 1/2,858,998 conceptions for NPC2.

Based on the above analysis of both NPC1 and NPC2, the combined incidence is predicted to be 1/89,229 or 1.12 cases per 100,000 conceptions and the fraction of NPC2 cases is predicted to be 3.1%. The predicted number of cases is slightly more than the 0.96 cases per 100,000 conceptions reported by Vanier2 when she accounted for prenatal cases and the fraction of NPC2 cases is consistent with prior clinical observation of 2-5%1-3.

Discussion

The impact of NPC1 variation on human health may be significant. Work by multiple groups has demonstrated that c.644A>G (p.H215R) is associated with obesity36. In this analysis of NPC1 variants we identified the p.H215R variant in almost a third of the NPC1 alleles. Our work now demonstrates that two relatively common NPC1 variants with a combined carrier frequency approaching 0.8% may contribute, in compound heterozygous state, to a late-onset NPC1 phenotype for which the phenotypic spectrum and clinical significance remains to be defined. This late-onset NPC1 phenotype may represent a milder manifestation of NPC1 deficiency with predominately visceral manifestations. The degree to which this late-onset NPC1 phenotype is associated with high frequency NPC1 alleles and the adult-onset NPC1 phenotype that includes significant neurological and psychological symptoms also remains to be defined.

Failure to ascertain certain alleles in patients such as the p.V30M in NPC2 or the p.T511M and p.S1200G in NPC1 could be due to prenatal lethality; however, as NPC is an autosomal recessive disorder, it is difficult to hypothesize a plausible mechanism, such as a dominant inhibitory function, by which these alleles would uniquely result in prenatal lethality.

Based on clinical case reports, one needs to consider the possibility that p.N222S and p.N961S maybe pathogenic with allelic frequencies of 0.400 and 0.389 percent respectively. The evidence for clinical relevance is strongest for p.N222S, which has been observed in two independent cases with similar visceral and delayed neurological manifestations, variant filipin staining in fibroblasts, and positive plasma oxysterol testing in the siblings. Assuming pathogenicity is related to a compound heterozygous state and full penetrance, the combined frequency of p.N222S with another pathogenic NPC1 mutation would be 1/35,667. Although only limited data are available, if one includes p.N961S based on a single report with no supporting diagnostic testing, the incidence of a late onset variant of NPC1 disease would increase to 1/19,077. Another possible to explanation of these high predicted incidences is that some individuals harboring these variants either in combination with another pathogenic allele or in the homozygous state may be asymptomatic or only manifest subclinical signs.

Leveraging existing “whole exome sequence” data we have estimated the disease incidence of NPC utilizing both bioinformatic tools and manual curation. With respect to classical NPC disease, we estimate that the incidence of NPC1 and NPC2 are on the order of 1/92,000 and 1/2,900,000, respectively, with a combined incidence of approximately 1/89,000. These estimates are in agreement with previous clinical estimates. Thus, our data does not support significant under-ascertainment of classical NPC cases. Concurrence with clinical data also suggests that we are not missing a significant number of alleles, such as large indels or intronic mutations that are not detected by “whole exome sequencing.” However, our data suggests that there may be significant under-ascertainment of a late-onset NPC1 phenotype. This late-onset phenotype may present as visceral-only or neurological mild NPC1, and with a potential incidence of 1/19,000-1/39,000. Further work is necessary to fully delineate this late-onset NPC1 phenotype, but the current study suggests that NPC should be considered in individuals with visceral lipidosis or unexplained neurological and psychiatric symptoms.

Supplementary Material

Supplementary _Appendix_ online only material_ etc._

Supplemental Figure 1: Provides the molecular characterization of the c.441+1G>C NPC2 splice variant. A) Shows the products of the reverse transcription-PCR amplification of the region surrounded the exon 4/5 junction, visualized on a 1.8% agarose gel. The WT cell line presents a single 192 base band while both line heterozygous for the splice variant present with the control band and an alternately spliced prominent slightly larger band. B) Sequencing of the larger prominent alternative splice revealed the insertion of 16 nucleotides. C) Resulting sequence and frameshift as a result of the 16 base insertion.

Acknowledgements

J. L. C., J. I., C.L., A. T., S. S., J.E.B-W., L. G. B., F. D. P., and C. A. W. supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development or the National Human Genome Research Institute intramural research programs, DHHS. J.L.C. C.A.W. is a NIH/Oxford Scholar and J.L.C is a Wellcome Trust/NIH Scholar. F.M.P. is a Royal Society Wolfson Research Merit Award holder.

Footnotes

L. G. B. is an uncompensated advisor to the Illumina Corporation and receives royalties from the Genentech Corporation; all other authors report no conflict of interest.

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Supplementary Materials

Supplementary _Appendix_ online only material_ etc._

Supplemental Figure 1: Provides the molecular characterization of the c.441+1G>C NPC2 splice variant. A) Shows the products of the reverse transcription-PCR amplification of the region surrounded the exon 4/5 junction, visualized on a 1.8% agarose gel. The WT cell line presents a single 192 base band while both line heterozygous for the splice variant present with the control band and an alternately spliced prominent slightly larger band. B) Sequencing of the larger prominent alternative splice revealed the insertion of 16 nucleotides. C) Resulting sequence and frameshift as a result of the 16 base insertion.

NIHMS62276-supplement-Supplementary___Appendix__online_only_material__etc__.tiff (2.6MB, tiff)

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