Molecular Genetics of Niemann-Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants

Abstract

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. In 2009, the molecular characterization of 44 NPC Italian patients has been published. Here, we present an update of the genetic findings in 105 Italian NPC patients belonging to 83 unrelated families (77 NPC1 and 6 NPC2). NPC1 and NPC2 genes were studied following an algorithm recently published. Eighty-four different NPC1 and five NPC2 alleles were identified. Only two NPC1 alleles remained non detected. Sixty-two percent of NPC1 alleles were due to missense variants. The most frequent NPC1 mutation was the p.F284Lfs*26 (5.8% of the alleles). All NPC2 mutations were found in the homozygous state, and all but one was severe. Among newly diagnosed patients, 18 novel NPC1 mutations were identified. The pathogenic nature of 7/9 missense alleles and 3/4 intronic variants was confirmed by filipin staining and NPC1 protein analysis or mRNA expression in patient's fibroblasts. Taken together, our previous published data and new results provide an overall picture of the molecular characteristics of NPC patients diagnosed so far in Italy.

Keywords: NPC1; NPC2; Niemann–Pick C disease; mutations.

Figure 1

Relative distribution of the clinical phenotypes of Niemann–Pick type C1 (NPC1) patients based on the age at onset of first neurological signs. Severe infantile (SI): age at onset <2 years; Late infantile (LI): age at onset 2–6 years; Juvenile (J): age at onset 6–15 years; and Adult (A): age at onset ≥15 years. Patients who died during the first month of life due to liver or respiratory insufficiency without signs of neurological involvement were classified as early infantile systemic lethal form (EISL). NC: not classifiable.

Figure 2

Spectrum of NPC1 type of mutation mutations.

Figure 3

NPC1 protein abundance in patients carrying seven novel missense mutations. (A) Representative western blot analysis of NPC1 protein expression in NPC patients and normal control fibroblast cell lines. (B) The intensity of the NPC1 signals was normalized against actin. The NPC1 protein content in NPC fibroblasts was expressed as a percentage of the NPC1 protein content found in fibroblasts from a normal control.

Figure 4

Functional analysis of four NPC1 splicing mutations. (A) The c.181-2A>G mutation caused the creation of a novel acceptor splice site leading to the insertion of a nucleotide in the mRNA transcript and the alteration of the reading frame; (B) the c.3591+121C>T and (C) the c.3591+105A>T mutations led to the generation of a cryptic acceptor splice site within intron 23, causing the retention of 119 and 103 nt, respectively, the shifting in the open reading frame, and the generation of a premature stop codon. The abnormal transcript resulting from the presence of c.3591+121C>T mutation was degraded via non-sense mediated decay (NMD). (D) In patient NP67, the skipping of exon 14 was observed (r.2131_2245del). The abnormal transcript was degraded via NMD.

Figure 5

NPC1 protein structure and variants. The structure of NPC1 (PDB 5U73) showed the location of the NPC1 variants identified in this study. Functional domains containing NPC1 variants are indicated in different colors. MLD, middle luminal domain = green; CTD, C-terminal domain = yellow; TM5 = blue; Cytosolic loop = red; lumenal loop between TM5 and TM6 = pink.

Figure 6

Distribution of genotypes among NPC phenotypes. Genotypes were categorized as homozygote (null/null) or compound heterozygote (null/missense) for severe mutations and homozygote for a missense mutation (missense/missense). 75% and 65% of EISL (early infantile systemic lethal) and SI (severe infantile) patients, respectively, were homozygote for severe mutations, while 66.7% of LI (Late infantile) 84.2% of J (juvenile) , and 80% of A (adult) patients presented missense mutations in both alleles. No homozygote or compound heterozygote for severe mutations was detected in J or A patients.