The two known complementation groups of Niemann-Pick Type C disease, NPC1 and NPC2, result from non-allelic protein defects. Both the NPC1 and NPC2 (HE1) gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. We describe mutation analysis on samples from 143 unrelated affected NPC patients using conformation sensitive gel electrophoresis and DNA sequencing as the primary mutation screening methods for NPC1 and NPC2, respectively. These methods are robust, sensitive, and do not require any specialized laboratory equipment. Analyses identified two NPC1 mutations for 115 (80.4%) patients, one NPC1 mutation for 10 (7.0%) patients, two NPC2 mutations for five (3.5%) patients, one NPC2 mutation for one (0.7%) patient, and no mutations for 12 (8.4%) patients. Thus, mutations were identified on 251 of 286 (88%) disease alleles, including 121 different mutations (114 in NPC1 and seven in NPC2), 58 of which are previously unreported. The most common NPC1 mutation, I1061T, was detected on 18% of NPC alleles. Other NPC1 mutations were mostly private, missense mutations located throughout the gene with clustering in the cysteine-rich luminal domain. Correlation with biochemical data suggests classification of several mutations as severe and others as moderate or variable. The region between amino acids 1038 and 1253, which shares 35% identity with Patched 1, appears to be a hot spot for mutations. Additionally, a high percentage of mutations were located at amino acids identical to the NPC1 homolog, NPC1L1. Biochemical complementation analysis of cases negative for mutations revealed a high percentage of equivocal results where the complementation group appeared to be non-NPC1 and non-NPC2. This raises the possibilities of an additional NPC complementation group(s) or non-specificity of the biochemical testing for NPC. These caveats must be considered when offering mutation testing as a clinical service.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

Objective: To assess the efficiency of target-enrichment next-generation sequencing (NGS) with copy number assessment in inherited neuropathy diagnosis.Methods: A 197 polyneuropathy gene panel was designed to assess for mutations in 93 patients with inherited or idiopathic neuropathy without known genetic cause. We applied our novel copy number variation algorithm on NGS data, and validated the identified copy number mutations using CytoScan (Affymetrix). Cost and efficacy of this targeted NGS approach was compared to earlier evaluations.Results: Average coverage depth was ;7603 (median 5 600, 99.4% . 1003). Among 93 patients, 18 mutations were identified in 17 cases (18%), including 3 copy number mutations: 2 PMP22 duplications and 1 MPZ duplication. The 2 patients with PMP22 duplication presented with bulbar and respiratory involvement and had absent extremity nerve conductions, leading to axonal diagnosis. Average onset age of these 17 patients was 25 years (2-61 years), vs 45 years for those without genetic discovery. Among those with onset age less than 40 years, the diagnostic yield of targeted NGS approach is high (27%) and cost savings is significant (;20%). However, the cost savings for patients with late onset age and without family history is not demonstrated. Conclusions:Incorporating copy number analysis in target-enrichment NGS approach improved the efficiency of mutation discovery for chronic, inherited, progressive length-dependent polyneuropathy diagnosis. The new technology is facilitating a simplified genetic diagnostic algorithm utilizing targeted NGS, clinical phenotypes, age at onset, and family history to improve diagnosis efficiency. Our findings prompt a need for updating the current practice parameters and payer guidelines. Neurology ® 2016;86:1762-1771 GLOSSARY CIAP 5 chronic idiopathic axonal polyneuropathy; CMT 5 Charcot-Marie-Tooth; CNV 5 copy number variation; DGV 5 Database of Genomic Variants; HMSN 5 hereditary motor and sensory polyneuropathy; MAF 5 minor allele frequency; NGS 5 next-generation sequencing; VUS 5 variants of unclear significance; WES 5 whole exome sequencing.Polyneuropathies are common, with overall prevalence of 1.7%, and 6.6% among persons over 60 years old.1 The high prevalence, multiple impairments, and complicated diagnostic procedures lead to high health care cost.1,2 Separating inherited from acquired causes is often difficult for insidious adult-onset cases, due to overlapping phenotypes.3,4 Furthermore, inherited and acquired polyneuropathies may coexist, resulting in worse severity. [5][6][7][8] One large prospective study indicated that as many as 42% of patients with previously undiagnosed polyneuropathy coming to tertiary care referral had familial occurrence. 9Pragmatic diagnostic strategies have long been sought to improve testing effectiveness and reduce costs in neuropathy diagnosis. [10][11][12] In Charcot-Marie-Tooth clinics, algorithms have been developed for selecting candidate genes.13 This approach is helpful for patients who have c...