Exome Analysis of a New Disease-causing Mutation in a Preterm Neonate with NP-C Disease

Niemann-Pick C disease (NP-C) is an ultra-rare, autosomal recessive, neurovisceral lysosomal lipid storage disorder (LSD) with impaired intracellular lipid trafficking, and an estimated incidence of approximately 1:/90.000 live births [Wassif CA et al. Genet Med 2016; 18(1): 41–48]. Disease severity in most patients is dependent on the degree of neurological involvement, but neurological abnormalities are typically preceded by systemic signs (i.e., cholestatic jaundice in the neonatal period or isolated spleno- or hepatosplenomegaly in infancy or childhood) as well non-immune hydrops [Whybra C et al. Orphanet J Rare Dis 2012; 8; 7: 86]. The first neurological symptoms vary with age of onset and include delay in developmental motor milestones, gait disturbances, falls, clumsiness, cataplexy, school problems, and ataxia. The most characteristic sign is vertical supranuclear gaze palsy. Cataplexy, seizures and dystonia as well as dementia are other common features; in adults, psychiatric problems may occur as well [Rohanizadegan M et al., Cold Spring Harb Mol Case Stud 2017; 21; 3: a002147]. Due to the extreme rarity of NP-C disease, establishing a correct diagnosis is challenging, and is often realized only with a substantial delay [Vanier MT., Orphanet J Rare Dis 2010; 5: 16]. Here, we report a case of neonatal NP-C disease diagnosed by prenatal exome analysis, demonstrating a new disease-causing mutation.

This is a male, preterm infant born by caesarean section because of pathological CTG at 33² weeks of gestation (birth weight: 2110 grams) to a G1P0 mother. Antenatal scans at 25 weeks of gestation demonstrated moderate ascites, but no further fluid collections. On follow-up scans, increasing amounts of ascites as well as marked bilateral hydroceles were noted. Prenatal genetic testing was performed in the 31^st week of pregnancy by trio-exome sequencing (enrichment: Roche/NimbleGen-Technology; Sequencing: Ilumina-System) after amniocentesis yielded normal cytogenetic results.

In the immediate postnatal period, the infant suffered from severe respiratory distress, and required intubation as well as drainage of ascites. On clinical examination and on X-ray, profound hepatosplenomegaly was noted as well as bilateral hydroceles ([Fig. 1a], [b]), but no neurological abnormalities. Due to anemia, thrombocytopenia and coagulation abnormalities, multiple red blood cell, platelet and fresh frozen plasma transfusions were required. Clinical chemistry demonstrated direct hyperbilirubinemia (max. direct serum bilirubin concentration: 11 mg/dL). The clinical picture was compatible with LSD, which was confirmed by the results from prenatal exome analysis (available in the second week of life), yielding the diagnosis of NP-C disease. Molecular analysis revealed compound-heterozygosity of the pathogenic variants c.3294dup,p.(Ile1099Tyrfs*22) and c.352_353del,p.(Gln119Valfs*22) in the NPC1-gene. Both variants are frameshift mutations generating a new stop codon resulting either in nonsense-mediated mRNA-decay or premature truncation of the protein. The variant c.352_353del has not yet been reported in a patient with NP-C disease. The parents were identified to be heterozygous carriers of one of the variants, thus providing strong evidence for autosomal recessive NP-C disease. Metabolic analysis also demonstrated elevated levels of chitotriosidase (890.4 nmol/h/ml (normal ≤150 nmol/h/ml) and reactive oxidative species/oxysterols (cholestan-3β,5α,6β-triol (C-triol): 0.937 ng/µL; normal ≤ 0.05 ng/µL) in this infant.

Fig. 1 a Abdominal and chest X-ray on admission demonstrating profound hepatosplenomegaly, ascites. b Main clinical features during the further course included jaundice, severe hepatosplenomegaly (ascites drainage in situ), bilateral inguinal hydrocele, and respiratory failure.

Based on these results, the infant was started on miglustat therapy in the first month of life (with a final dose of 10 mg/kg/d). After start of treatment, the neonate demonstrated transient clinical stabilization, and was temporarily weaned from the ventilator. However, the further clinical course was characterized by recurrent episodes of respiratory failure, progressive hepatosplenomegaly, ascites, and bilateral inguinal hydroceles, and failure to thrive. On day 74 of life, after multiple consultations with the parents, intensive care treatment was withdrawn.

In animal models of NP-C disease, miglustat has been shown to reduce neuronal glycosphingolipid accumulation, delay the onset of neurological dysfunction, and prolong survival. A clinical proof-of-concept study showed improved lipid trafficking in peripheral blood B lymphocytes after miglustat treatment [Pineda M et al., Orphanet J Rare Dis 2018; 15; 13(1): 140]. Subsequently, clinical trials demonstrated that miglustat stabilized neurological disease progression in pediatric patients with NP-C disease, with comparable safety and tolerability to that observed in adults and juveniles. [Patterson MC et al., J Child Neurol 2010; 25: 300–305.] Historically, technically difficult and time-consuming Filipin staining formed the basic diagnostic method in establishing the diagnosis of NP-C. Nowadays, chitotriosidase, the macrophage activation associated plasma-borne enzyme, has been introduced as a biomarker for a number LSDs, including Gaucher disease as well as NP-C, but lacks specificity for NP-C disease. Furthermore, chitotriosidase is not informative in a substantial proportion of patients because of a frequent loss-of-function variant in the normal population (deficient activity in homozygous individuals), and therefore false negative results may be seen in NPC patients.

Recently the reactive oxidative species, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been established as reliable and convenient diagnostic biomarkers for NP-C [Degtyareva AV et al., BMC Med Genet 2019; 20(1): 123]. Of note, in our patient the diagnosis of NP-C was made on the basis of prenatal exome analysis – demonstrating a new disease-causing mutation with elevated levels of ChT and oxysterols.

We conclude that early diagnosis of rare and ultra-rare metabolic diseases (eg, LSDs) may be facilitated by using (prenatal) exome analysis, thus establishing an early definite diagnosis [Friedmann JM et al., JAMA 2020; doi: 10.1001/jama.2020.11126. Online first; Whu AC et al. JAMA Pediatrics 2020; 1; 174(9): 821–822]. A recent study in 127 consecutive unexplained cases of non-immune hydrops fetalis demonstrated that exome sequencing identified a diagnostic variant in 29% of cases (most frequently disorders affecting the RAS–MAPK cell-signaling pathway) unexplained by standard genetic testing [Sparks TN et al., N Engl J Med 2020; 383(18): 1746–1756]. These data support the use of exome sequencing for non-immune hydrops fetalis cases with non-diagnostic results of chromosomal microarray analysis or karyotype analysis to inform prognosis, establish recurrence risk, and direct prenatal and postnatal clinical care as done in our patient (eg, initiation of miglustat therapy) [Sparks TN et al., N Engl J Med 2020; 383(18): 1746–1756]. The integration of NP-C testing into standardized newborn screening may provide another feasible tool in establishing an early diagnosis [Sobrido MJ et al., Orphanet Journal Rare Diseases 2019; 14(1): 20].

Publication History

Article published online:
08 June 2021