Maria Josep Coll

Diagnostic tests for Niemann-pick disease type C (NP-C) : a critical review

Niemann-Pick disease type C (NP-C) is a neurovisceral lysosomal cholesterol trafficking and lipid storage disorder caused by mutations in one of the two genes, NPC1 or NPC2. Diagnosis has often been a difficult task, due to the wide range in age of onset of NP-C and clinical presentation of the disease, combined with the complexity of the cell biology (filipin) laboratory testing, even in combination with genetic testing. This has led to substantial delays in diagnosis, largely depending on the access to specialist centres and the level of knowledge about NP-C of the physician in the area. In recent years, advances in mass spectrometry has allowed identification of several sensitive plasma biomarkers elevated in NP-C (e.g. cholestane-3β,5α,6β-triol, lysosphingomyelin isoforms and bile acid metabolites), which, together with the concomitant progress in molecular genetic technology, have greatly impacted the strategy of laboratory testing. Specificity of the biomarkers is currently under investigation and other pathologies are being found to also result in elevations. Molecular genetic testing also has its limitations, notably with unidentified mutations and the classification of new variants. This review is intended to increase awareness on the currently available approaches to laboratory diagnosis of NP-C, to provide an up to date, comprehensive and critical evaluation of the various techniques (cell biology, biochemical biomarkers and molecular genetics), and to briefly discuss ongoing/future developments. The use of current tests in proper combination enables a rapid and correct diagnosis in a large majority of cases. However, even with recent progress, definitive diagnosis remains challenging in some patients, for whom combined genetic/biochemical/cytochemical markers do not provide a clear answer. Expertise and reference laboratories thus remain essential, and further work is still required to fulfill unmet needs.

Antisense oligonucleotide treatment for a pseudoexon-generating mutation in the NPC1 gene causing Niemann-Pick type C diseaseb

Niemann‐Pick type C disease is an autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. While most of the mutations are missense, a few splicing mutations have also been described. We identified and characterized a novel point mutation c.1554‐1009G>A located in intron 9 of the NPC1 gene in a Spanish patient. Sequencing of the cDNA from the patient showed that this intronic mutation creates a cryptic donor splice site resulting in the incorporation of 194 bp of intron 9 as a new exon (pseudoexon) in the mRNA. This new transcript bears a premature termination codon and is degraded by the nonsense‐mediated mRNA decay mechanism. Experimental confirmation that the point mutation generates the inclusion of a pseudoexon in the mRNA was obtained using a minigene. A specific antisense morpholino oligonucleotide targeted to the cryptic splice site was designed and transfected into fibroblasts from the patient. Using this approach, normal splicing was restored. These results demonstrate the importance of screening deep intronic regions and support the efficacy of antisense therapeutics for the treatment of diseases caused by pseudoexon‐generating mutations.

Cholestane-3β,5α,6β-triol: high levels in Niemann-Pick type C, cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3β,5α,6β-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.

Molecular analysis of 30 Niemann-Pick type C patients from Spain.

Macías‐Vidal J, Rodríguez‐Pascau L, Sánchez‐Ollé G, Lluch M, Vilageliu L, Grinberg D, Coll MJ, the Spanish NPC Working Group. Molecular analysis of 30 Niemann–Pick type C patients from Spain.

Hunter disease in the Spanish population : Molecular analysis in 31 families

Mucopolysaccharidosis type II (Hunter disease) is an X-linked disorder due to deficiency of the lysosomal enzyme iduronate 2-sulphatase. Here we report an update of molecular studies in 31 Spanish families with Hunter disease. We found a total of 22 novel small mutations (7 reported previously by our group), and 4 large deletions or rearrangements. Particularly relevant are two mutations, one showing an alternatively spliced product although the normal splice site is conserved; the other mutation results in an amino acid change that most likely modifies regulation of expression of the IDS gene. Except for large gene alterations and for the G374sp mutation already described, we could not establish a clear phenotype–genotype correlation. Mutation G374sp is the point mutation most frequent in our population (10%) and is always associated with mild phenotype. Our molecular analyses carried out in a relatively large series of patients with Hunter disease contribute to the identification of new mutations and reinforce the conclusions drawn in other populations about the genotype–phenotype correlation and the gene distribution of mutations.

Mutation 1091delC is highly prevalent in Spanish Sanfilippo syndrome type A patients

The gene resposible for Sanfilippo syndrome type A, a lysosomal disorder caused by deficiency of sulfamidase, was recently cloned and more than 40 mutations were identified. This paper presents the mutation analysis and clinical findings in 11 Spanish patients in whom 19 of the 22 mutant alleles have been identified. This is the first report on mutations in Spanish Sanfilippo A patients. Seven different mutations were found, four of which (Q85R, R206P, A354P, and L386R) were not previously described. Mutation 1091del C was the most prevalent, accounting for nearly one‐half of the mutated alleles, while mutations R245H and R74C were not found. Haplotype analysis suggests a founder effect as the cause of the high frequency of 1091del C in this population. Hum Mutat 12:274–279, 1998.

Exome sequencing identifies a new mutation in SERAC1 in a patient with 3-methylglutaconic aciduria

3-Methylglutaconic aciduria (3-MGA-uria) is a heterogeneous group of syndromes characterized by an increased excretion of 3-methylglutaconic and 3-methylglutaric acids. Five types of 3-MGA-uria (I to V) with different clinical presentations have been described. Causative mutations in TAZ, OPA3, DNAJC19, ATP12, ATP5E, and TMEM70 have been identified. After excluding the known genetic causes of 3-MGA-uria we used exome sequencing to investigate a patient with Leigh syndrome and 3-MGA-uria. We identified a homozygous variant in SERAC1 (c.202C>T; p.Arg68*), that generates a premature stop codon at position 68 of SERAC1 protein. Western blot analysis in patients fibroblasts showed a complete absence of SERAC1 that was consistent with the prediction of a truncated protein and supports the pathogenic role of the mutation. During the course of this project a parallel study identified mutations in SERAC1 as the genetic cause of the disease in 15 patients with MEGDEL syndrome, which was compatible with the clinical and biochemical phenotypes of the patient described here. In addition, our patient developed microcephaly and optic atrophy, two features not previously reported in MEGDEL syndrome. We highlight the usefulness of exome sequencing to reveal the genetic bases of human rare diseases even if only one affected individual is available.

Nonsense-mediated mRNA decay process in nine alleles of Niemann-Pick type C patients from Spain

Mutations in NPC1 or NPC2 genes are responsible of Niemann-Pick type C disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by a non-regulation of intracellular lipid trafficking. Alterations such as nonsense or frame shift mutations generate a premature termination-codon (PTC). Nonsense-mediated mRNA decay (NMD) is a natural cellular process that degrades mRNAs that encode a prematurely truncated protein. In this study we have analyzed 9 NPC1 mutations which generate a PTC (p.R116X, p.Q119VfsX8, p.W260X, p.S425X, p.A558GfsX12, p.Q775X, p.G993EfsX4, p.R1059X and p.I1061NfsX4), in order to determine if their mRNAs suffer NMD process. To achieve this objective we compared fibroblasts of patients carrying these alleles with and without cycloheximide (CHX) treatment using conventional PCR and real-time PCR. The results of conventional PCR of untreated fibroblasts showed a reduction of the amount of NPC1 mRNA compared to control in all patients. After CHX-treatment, a recovery of mRNA was detected but not in all the alleles. However, when real-time PCR was used, the recovery was observed including those alleles that qualitatively showed no apparent increase in mRNA level. In conclusion, we confirmed that NMD process is responsible for the mRNA decay for all the analyzed NPC1 PTC-encoding mutations.

Characterisation of two deletions involving NPC1 and flanking genes in Niemann–Pick Type C disease patients

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal disorder characterised by the accumulation of a complex pattern of lipids in the lysosomal-late endosomal system. More than 300 disease-causing mutations have been identified so far in the NPC1 and NPC2 genes, including indel, missense, nonsense and splicing mutations. Only one genomic deletion, of more than 23 kb, has been previously reported. We describe two larger structural variants, encompassing NPC1 and flanking genes, as a cause of the disease. QMPSF, SNP inheritance and CytoScan® HD Array were used to confirm and further characterise the presence of hemizygous deletions in two patients. One of the patients (NPC-57) bore a previously described missense mutation (p.T1066N) and an inherited deletion that included NPC1, C18orf8 and part of ANKRD29 gene. The second patient (NPC-G1) had a 1-bp deletion (c.852delT; p.F284Lfs*26) and a deletion encompassing the promoter region and exons 1-10 of NPC1 and the adjacent ANKRD29 and LAMA3. This study characterised two novel chromosomal microdeletions at 18q11-q12 that cause NPC disease and provide insight into missing NPC1 mutant alleles.

The proteasome inhibitor bortezomib reduced cholesterol accumulation in fibroblasts from Niemann-Pick type C patients carrying missense mutations

Niemann–Pick disease type C (NPC) is a lipid storage disorder mainly caused by mutations in the NPC1 gene. Approximately 60% of these mutations are missense changes that may induce reduced NPC1 protein levels by increased degradation via ubiquitin–proteasome. This is the case for the most prevalent worldwide mutation, p.Ile1061Thr, as well as for other three missense changes. In the present study, we analyzed the NPC1 levels in fibroblasts from eighteen NPC patients presenting missense mutations. We found that fourteen of these cells lines showed decreased levels of NPC1. Six of these cell lines were homozygous, whereas the other eight were associated with a frame shifting mutation. We focused our attention in the NPC homozygous samples and demonstrated that, in most of the cases, NPC1 reduction was a consequence of a decrease of its half‐life. NPC cells were treated not only with the proteasome inhibitors carbobenzoxy‐l‐leucyl‐l‐leucyl‐l‐leucinal or N‐acetyl‐leucyl‐leucyl‐norleucinal, both widely used as a research tools, but also with bortezomib, the first proteasome inhibitor to reach clinical applications, although it has never been used in NPC disease. We observed that, after treatment, the mutant NPC1 protein levels were partially recovered in most of the cell lines. Importantly, these mutant proteins partially recovered their activity and substantially reduced free cholesterol levels. These results suggest that by enhancing the NPC1 protein stability with the use of proteasome inhibitors, their functionality might be recovered and this might represent a therapeutical approach for future treatments of NPC disease resulting from specific missense mutations.