Importance of the biochemical investigations for the functional characterization of a NPC1 variant identified by exome sequencing

Niemann–Pick disease type C (NPC) is one of the lysosomal storage disorders. It is caused by biallelic pathogenic variants in NPC1 or NPC2, which results in a defective cholesterol trafficking inside the late endosome and lysosome. There is a high clinical variability in the age of presentation and the phenotype of this disorder making the diagnosis challenging. Here, we report a patient with an infantile onset global developmental delay, microcephaly and dysmorphic features, homozygous for c.3560C>T (p.A1187V) variant in NPC1. His plasma oxysterol levels were normal on two occasions. His lyso‐sphingomyelin‐509 (lyso‐SM 509) and urinary bile acid levels were normal. Based on the phenotype and biochemical features, the diagnosis of NPC was excluded in this patient. We emphasize the importance of functional characterization in the classification of novel variants to prevent a misdiagnosis. Matching the phenotype and biochemical evidence with the molecular genomic tests is crucial for the confirmation of genetic diagnoses.


| INTRODUCTION
Niemann-Pick disease type C (NPC) (OMIM #257220) is one of the lysosomal storage diseases.It is inherited autosomal recessively and is due to biallelic pathogenic variants in either NPC1 (OMIM #607623), which encodes NPC1 protein (accounting for 95% of the NPC patients) or NPC2 (OMIM #601015), which encodes NPC2 protein, (accounting for 5% of patients) (Burton et al., 2021).The NPC1 is a late-endosomal membrane protein, and NPC2 is a soluble protein that primarily localizes to the lysosomes.Both proteins are responsible for the transport of cholesterol and other lipids (e.g., sphingolipids) across the hydrophobic endosomal bilayer membrane (Blom et al., 2003;Li et al., 2017).NPC is characterized by a defective intracellular lipid trafficking resulting in the accumulation of cholesterol and glycosphingolipids in the late endosomes or lysosomes of the cells (Patterson et al., 2012).
NPC has high variable phenotypes.The disease onset can be neonatal, early infantile, late infantile, juvenile and adult onset.Cholestasis and hepatomegaly are characteristic phenotypes for neonatal onset form, whereas motor delay, hypotonia, and clumsiness are prominent phenotypic features for early and late infantile forms.
Ataxia, dystonia, and psychiatric problems are phenotypic features of the juvenile and adult-onset forms (Patterson et al., 2012).
Miglustat, a substrate reduction therapy, is the only approved drug for the treatment of NPC.It acts by inhibiting glucosylceramide synthase, which catalyzes the initial step in the synthesis of glycosphingolipids (Neßlauer et al., 2019).It has been used for the treatment of the progressive neurological manifestations of the disease.It has been proven to be effective in slowing down neurological progression including ambulation, speech, dysmetria dystonia and swallowing (Patterson et al., 2020).
We report a 7-year-old male patient referred to our metabolic genetic clinic for the management of NPC due to homozygous likely pathogenic variant in NPC1.However, his plasma oxysterols were normal.We report this patient to highlight that even if the variants were originally classified as likely pathogenic and segregated in the parents, the functional studies ruled out the genetic diagnosis of NPC.It is important to remember that phenotypes and disease specific biomarkers are crucial to confirm genetic diagnoses.

| PATIENT REPORT AND RESULTS
We received a signed case report consent form from the parents.This 7-year-old boy had a remarkable pregnancy due to a single fetal umbilical artery during the pregnancy.He was referred to the Maternal Fetal Medicine Clinic and his fetal echocardiogram was normal.He was delivered by cesarean section for fetal heart rate abnormalities at 37 + 5 weeks gestation after induction.His Apgar scores were 7 and 9 at 1 and 5 min respectively.His birth weight and head circumference were at the 10th percentile.He had respiratory distress, poor feeding and hypoglycemia and was transferred to the neonatal intensive care unit (NICU) at the age of 1 day.He had dysmorphic features including a prominent forehead, small jaw, hypertelorism, low-set and posteriorly rotated ears, penile chordee, and bilateral fifth finger clinodactyly.He required supplemental oxygen but was able to wean off a few days after birth.He progressed to full bottle feeds using a slowflow nipple.He passed his hearing screening.His newborn screening was normal.He was discharged home at age 2 weeks.
He has severe global developmental delay that he is not able to sit independently, crawl, walk, or speak at the age of 7 years.He acquired head control by age 3-4 years.He started babbling at age 3 years but had no words.His receptive language is severely delayed, and he does not understand simple commands.He is not toilet trained.There was no history of developmental regression or seizures.
He was reported to be a quiet and calm boy who is very sensitive to noise.He had a circumcision and repair of the penile chordee within the first month of age.Due to failure to thrive, he received g-tube insertion at the age of 20 months.
His height was below the first percentile (Z = À2.42),his weight was at the fifth percentile (Z = À1.68) and his head circumference was below the second percentile cm (Z < À2.05) at the age of 6.5 years.He had dysmorphic features including brachycephaly, full-arched eyebrows, long palpebral fissures, long eyelashes, a broad nasal bridge and nasal tip, downturned corners of his mouth and fetal pads on his fingertips.
His family history was remarkable for four pregnancy losses in his mother (3 and 6 months of gestation due to spontaneous abortion, polyhydramnios, and spontaneous abortion by 2-3 months of gestation and due to an ectopic pregnancy).He has an older healthy brother.There is a paternal relative with developmental delay.Parents are non-consanguineous.
His extensive investigations were normal for blood count, alanine transaminase, aspartate transaminase, gamma-glutamyl transpeptidase, albumin, conjugated and unconjugated bilirubin, international normalized ratio, partial thromboplastin time, urea, creatinine, and chromosomal microarray.Brain magnetic resonance imaging (MRI) (at age of 2 years and 3 months) revealed diffuse white matter volume loss, and a small incidental arachnoid cyst within the anteromedial left temporal lobe and brachycephalic head shape (Figure 1).His echocardiography was normal.
A clinical exome sequencing trio was performed using patients' and parents' DNA samples in the Blueprint Genetics laboratory according to their methods and identified a homozygous likely pathogenic variant (c.3560C>T; p.Ala1187Val) in NPC1 at the age of 6 years.Both parents were heterozygous for the same variant.This variant replaces alanine with valine at position 1187.Both amino acids have the same biophysical characteristics (neutral and non-polar).Alanine is a conserved amino acid in nine out of nine species.The p.-Ala1187Val NPC1 variant was classified as uncertain significance in gnomAD v4.0.0.It was reported in 74 out of 1,613,908 alleles and there was one homozygote (accessed in February 2024) (Karczewski et al., 2020).It is predicted to be damaging by MutationTaster (Schwarz et al., 2014) and Polyphen2, but predicted to be tolerated by SIFT (Vaser et al., 2016).His plasma oxysterols were normal on two occasions.His cholestane-3beta,5alpha,6beta-triol was normal at 0.055 nmol/mL (reference range ≤0.07 nmol/mL), 7-ketocholesterol was normal at 0.038 nmol/mL (reference range ≤0.1 nmol/mL), and lyso-sphingomyelin was normal at 0.006 nmol/mL (reference range ≤0.1 nmol/mL).His lyso-sphingomyelin 509 (Lyso-SM-509) was normal (0.8 ng/mL; reference range <0.9).Urinary bile acid metabolites were normal.Unfortunately, he does not have any confirmed underlying genetic diagnosis currently.

| DISCUSSION
We present a patient who had homozygous p.Ala1187Val variant in NPC1 identified by clinical exome sequencing which was initially characterized as likely pathogenic leading to a referral to our metabolic genetics clinic.Patient's phenotype was not supportive of NPC as he had early infantile onset severe global developmental delay, failure to thrive, and dysmorphic features.There was no history of developmental regression, and epilepsy in late infancy to childhood nor hepatosplenomegaly, and jaundice in the neonatal period.For these reasons, we performed extensive biochemical investigations for NPC.Plasma oxysterol levels, Lyso-SM-509, and urinary bile acid metabolites were all normal which served as functional characterization of this variant and proved that there was no damaging effect of this variant on the NPC1 protein function in the homozygous state.We report this patient to emphasize the importance of biomarkers for functional characterization of variants in inherited metabolic disorders.
Interestingly, the p.Ala1187Val NPC1 variant was previously reported in a patient who had a second NPC1 variant (c.665A>G; p.-Asn222Ser) in trans.The phenotype showed epilepsy and psychotic symptoms in an adult.Unfortunately, there were no reports of biochemical investigations and parental segregation results in the study (Fancello et al., 2009).The same research group performed filipin staining for several patients with NPC including the above-mentioned patient in their study (Dardis et al., 2020).The compound heterozygous NPC1 variants resulted in a defective cholesterol trafficking, however, it was less severe than in the classic NPC profile.Another study reported that patients with heterozygous NPC1 variants may have a mildly positive filipin staining result that is similar to the variant NPC profile (Vanier et al., 2016).Monies et al., (Monies et al., 2019) reported homozygous p.Ala1187Val NPC1 variant in a 25-month-old female with global developmental delay, intellectual disability, spasticity and white matter changes in brain MRI.However, no biochemical investigations were performed.It is possible that the p.Ala1187Val NPC1 variant is probably not damaging if it is homozygous and is a hypomorph variant.However, it might be a disease causing variant if it is compound heterozygous with a second pathogenic NPC1 variant.
We are reporting biochemical investigations in a patient for this homozygous variant for the first time.
The c.3560C>G (p.Ala1187Gly) variant was reported with the c.2050C>T (p.Leu684Phe) variant in NPC1 in a 23 year old patient who had elevation of cholestane-3β,5α,6β-triol-concentration in plasma (Reunert et al., 2016).In our patient alanine at 1187 was replaced with valine, and the results of the functional studies showed that this substitution does not affect the NPC1 protein function.It was previously reported that Lyso-SM 509 is a specific biomarker for NPC and was elevated in all patients with NPC independent of their age (Boenzi et al., 2021;Deodato et al., 2018).The performance of several urinary bile acid metabolites was previously investigated as a diagnostic biomarker for patients with NPC (Maekawa et al., 2019).3β-Sulfooxy-7β-hydroxy-5-cholenoic acid level was elevated in all patients with NPC compared to controls with 100% sensitivity and specificity (Maekawa et al., 2019).For these reasons, we performed plasma Lyso-SM 509 and urinary bile acid measurements in our patient which were normal and excluding the diagnosis of NPC.
In summary, clinical exome sequencing reported homozygous likely pathogenic variant in NPC1 in our patient who had early infantile onset global developmental delay, and dysmorphic features.Subsequent biochemical investigations including plasma oxysterol, C-triol, lyso-SM 509, and urinary bile acid measurements were normal.These biomarkers served as functional characterization of the p.Ala1187Val NPC1 variant identified in our patient in homozygous state leading to downgrading of this variant from likely pathogenic to variant of unknown significance.Functional assessments using several biomarkers are necessary to validate the impact of rare or novel variants.

Human
NPC1 consists of 1278 amino acid residues and includes three luminal domains including N-terminal domain, middle luminal domain, C-terminal domain (cysteine-rich domain) and 13 transmembrane helices.Alanine1187 is in the cytoplasm between transmembrane 11 and 12 helices (amino acid residues 1182-1199) within a putative alpha helix.In an internal helical position, alanine is regarded as the most stabilizing residue.It seems that the Ala1187 to Gly1187 amino acid change at the same position resulted an abnormal protein function, whereas homozygous Ala1187 to Val1187 amino acid change at the same position may result normal protein function.
AUTHOR CONTRIBUTIONS Conceptualization, project administration, supervision, validation: Saadet Mercimek-Andrews.Data curation, investigation, writing, review and F I G U R E 1 Brain MRI of the patient at 2 years and 8 months of age is depicted in Figure 1.(a) T2 weighted axial view of the brain showing dilatation of both lateral ventricles secondary to diffuse white matter volume loss.(b) T1 flair sagittal view of the brain showing thin caliber of corpus callosum and brachycephalic head shape.editing: Nihal Almenabawy, Clara Hung, Iveta Sosova, and Saadet Mercimek-Andrews.Formal analysis: Nihal Almenabawy and Saadet Mercimek-Andrews.Methodology: Nihal Almenabawy and Saadet Mercimek-Andrews.Writing original draft: Nihal Almenabawy and Saadet Mercimek-Andrews.