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Niemann-Pick type C (NP-C) is a progressive autoso mal recessive disorder caused by mutations in the NPC1 or the NPC2 genes. An abnormal endosomal-lysosomal trafficking is described, resulting in multiple lipid accu mulation. NP-C onset might appear from prenatal life to adulthood. NP-C is a rare disease, with an estimated incidence of 1 case per 100 000 live births. It is pan-ethnic, and at least 95% of the cases are due to mutations in the NPC1 gene1.
The clinical signs of NP-C can be classified into viscer al, neurological and psychiatric. Because of the phenotypic heterogeneity, they could overlap with other metabolic diseases. Therefore, in order to confirm the diagnosis, it is mandatory to carry out specific laboratory tests2.
Previously, the diagnostic confirmation of NP-C in our hospital required sending the patient samples to be analyzed abroad, which implied a consequent delay in the diagnosis. Our following aim was to develop the NP-C se quence analysis and the filipin staining as functional study to confirm the diagnosis. Coding sequence (exons 1-25) and the flanking intronic regions of NPC1 gene (RefSeq NM_000271.5) were amplified by PCR using specific primers as already reported3. The PCR products were sequenced on a capillary DNA sequencer and the variants found were classified according to American College of Medical Genetics (ACMG) guidelines4. Filipin staining was performed on fibroblast cell culture from skin biopsies. The assay included, simultaneously, the cell culture of the patient, as well as one positive and one negative control. Cells with fluorescent perinuclear vesicles, after incubation with Streptomyces filipensis complex, were considered “positive”, as described by Vanier et al5. The filipin test was always performed in separate cultures in duplicate.
Find below a series of reported cases with infantile on set diagnosed in our hospital. The age at onset, expressed as median and range, was 0.2 (0.08-4.0) years and the age of diagnosis was 4.0 (2.5-8.9) years.
Clinical cases
Case 1
Female child who debuted at 0.08 years old with splenomegaly as a visceral sign, and having presented fetal hydrops. Hepa tomegaly was diagnosed at the age of 3.00. As neurological signs it was described a progressive vertical supranuclear gaze palsy and gelastic cataplexy. Frequent seizure episodes appeared at 12.5 years.
Her mother and father were aged 23.0 and 24.0, respec tively. Consanguinity was not reported. She had two siblings, a brother who was 3.0 years older and a two years younger sister. No family member presented symptoms.
Liver biopsy performed at 2 months of age described fatty vacuoles in hepatocytes and macrophages, canalicular thrombi, cholestasis, perisinusoidal fibrosis, mixed inflamma tory infiltrate and ductular proliferation. Skin biopsy reported a histiocytic proliferation with large foamy cytoplasm. Bone mar row biopsy showed hypercellularity with increased phagocytes (Table 1). NPC1 gene sequencing informed two known mis sense variations: c.3419G>T and c.3182T>C6. Both variants were classified as likely pathogenic (PS3, PM2, PP2, PP3, PP5). NP-C was confirmed by the filipin test on fibroblasts cell culture at the age of 3.00 (Fig. 1).
Table 1 Clinical findings, complementary studies and molecular results in Niemann-Pick disease type C patients
Fig. 1 Filipin test results: Niemann-Pick type C (NP-C) positive control (a, d). NPC negative control (b, e), and NP-C suspected sample with a filipin positive pattern (c, f). Arrow shows a filipin positive cell, with fluorescent perinuclear vesicles. Panel c and f, represent a 90% of filipin positive cells
Case 2
Male child, whose first hospitalization in the intensive care unit was at 0.08 years old because of respiratory distress. At 1.0 year of age, he presented vertical supranuclear gaze palsy. His parents were 25.0 years old. No more data about his family was available.
A molecular study of the NPC1 gene revealed a nonsense variant in homozygous or hemizygous state, c.2872C>T6. It was classified as pathogenic (PVS1, PS3, PM2, PP3, PP5). The filipin test confirmed the diagnosis of NP-C at 2.5 years old (Table 1).
Case 3
Male child, hospitalized for respiratory distress syndrome as newborn and for cholestasis at 0.3 years old. At the age of 4.0, he presented growth retardation, maturation delay and hepatosplenomegaly. As neurological manifestations, he pre sented development delay. The age of his parents was not reported, nor was a consanguinity. His family background also included three older sisters, one of them died at the age of 5.0, presumably because of a metabolic disease.
A bone marrow biopsy showed micro-megakaryocytes grouped in clusters. A NPC1 gene sequencing revealed a missense variant, c.3557G>A, present in the global database6. It was classified as likely pathogenic (PS3, PM2, PP2, PP3, PP5). A novel intronic variant c.287+1G>A was located at the 5’ donor splice site of intron 3. This location has a 0.98 score prediction as a splice site (Neural Network), and a confidence of 0.76 as donor splice site (NetGene2). This variant has not been reported, at least in ClinVar, The Human Gene Mutation Database, Ensembl, gnomAD, SIFT or in PROVEAN data bases, nor has it been published in Pubmed or Mastermind. In silico analysis predicts that this variant is one of the main causes of this disease, since it affects the splice site. In other words, as it is likely to disturb normal splicing, it alters the protein features (Mutation Taster). According to recommenda tions of the ACMG, this variant was classified as pathogenic (PVS1, PS3, PM2, PP3). NP-C was confirmed by a filipin test on fibroblasts cell culture at 4.5 years old (Table 1).
Case 4
A male child who presented ataxia and telangiectasia with dysphagia at the age of 4.0. A progressive encephalopathy was detected at 8.0 years old, with a generalized tonic-clonic seizure. His mother was 24.0 years old while his father was 20.0. Although consanguinity was not revealed, they had the same last name and lived in a small village. This patient had a 2.0-year-younger brother without symptoms. Further data about patient’s family involves a 28.0 years old maternal aunt’s death by a stroke and a cousin of his mother’s with congenital hydrocephalus.
By the molecular study of the NPC1 gene, the missense variant c.3557G>A was detected in homozygous or hemizy gous state. This was the same variant detected in the case 3 and it was classified as likely pathogenic (PS3, PM2, PP2, PP3, PP5). NP-C was confirmed by a filipin test at 8.9 years old (Table 1).
None of the cases facilitated parental samples for mo lecular analysis.
Discussion
A series of cases of Argentinian pediatric patients was presented, with 4.0 years old as median age at diagnosis. It is already known that the clinical manifestations mark edly vary according to the age of the disease onset1. An early infantile form, characterized by neurological mani festations and hepatosplenomegaly, was found in cases 1 and 3. While case 2 only presented neurological signs, the remaining patient exhibited a late infantile neurode generative form.
Molecular analysis of the NPC1 gene in the four patients identified the following variants: p.Gly1140Val, p.Ile1061Thr, p.Arg958Ter, p.Arg1186His and c.287+1G>A. Unfortunately, parental samples were not available at the moment of this study. Having access to them would have allowed us to assign each variable to a specific allele (cases 1 and 3) and to understand if the single variable was homozygous or hemizygous (cases 2 and 4). Even though the same variant was found in our patients 3 and 4, they presented different signs, mainly visceral in the former while neurological in last one. Case 3 not only had this variant, but also another mutation that affected the splicing site. In this case, a longitudinal follow-up would be recommended, taking into account that the clinical manifestations may change as he grows.
Case 4, on the other hand, had this variant in hemicigo sis (combined with a deletion) or in homocigosis, and the clinical picture included neurological damages.
To confirm the molecular diagnosis, our group devel oped the filipin staining as a functional study to detect intracellular lipid accumulation7. The filipin test resulted positive in the four cases, which corroborated a disorder in the intracellular cholesterol trafficking.
According to the Consensus of 2018 of the International Niemann-Pick Disease Registry, once NP-C is clinically suspected, a laboratory diagnosis algorithm proposes to test biochemical markers (as lyso-sphingomyelin) as screening tools. As a second step, a molecular analy sis of NPC1 and NPC2 genes is recommended if the biochemical markers profile is compatible with NP-C. In the presence of inconclusive results, and to assess the pathogenicity of novel genetic variants, a filipin test must be performed1.
In fact, the accuracy of these procedures tended our institution to offer both the molecular sequencing and the filipin staining, as tools for the NP-C diagnosis in pediatric patients all around Argentina.
Until this moment, there haven’t been any reports on the incidence of NP-C in Argentinian pediatric pa tients. Only one paper described two adult siblings with a choreic phenotype and diagnosed as NP-C through the measurement of lyso-SM-509 biomarker using high-performance liquid chromatography/tandem mass spectrometry and molecular analysis on NPC1 gene that were analyzed in a German company specialized in study of rare diseases8.
Many reports from Brazil discuss this topic. One of them is a study in a large cohort of 265 patients with a wide distribution of age9. Interestingly, among the variants they found in NPC1 gene, three of them match with ours: p.Gly1140Val; p.Ile1061Thr; p.Arg1186His. Furthermore, a review from Colombia focused on the link between psychiatric disorders and neurometabolic diseases10. Although no psychiatric disorder has been found in our patients, probably because of their young age, we do not discard an evolution to psychiatric disorders in adulthood. On their part, a group of Mexico reported three juvenile NP-C patients, but they do not coincide with ours in terms of molecular mutations11. Another Mexican group reported the case of a male patient with a juvenile form of NP-C that involved a “variant” filipin staining (different to the classical pattern found in our series) and a paternal germline mosaicism12.
In summary, there is a limited offer of reports about patients with NP-C in Latin-American countries, mainly published in Brazil. The aim of this paper is to enhance the knowledge about NP-C, which is quite difficult to diagnose.
Thus, our analysis and confirmation of this illness in four pediatric patients contribute to the characterization of this disease in Argentina, and consequently increase its casuistry.
