A patient with Beta-Propeller Protein-Associated Neurodegeneration: a new missense mutation of the WDR45 gene

Beta-propeller protein-associated neurodegeneration (BPAN), first described by Haack et al. in 2012, is the only known X-linked NBIA form (NBIA5, OMIM: 300894) caused by mutations in the WDR45 gene (Haack et al., 2013). Neurodegeneration with brain iron accumulation (NBIA) presents a group of inherited neurological diseases with progressive extrapyramidal symptoms and focal iron accumulation generally observed in the globus pallidus and substantia nigra (Hayflick et al., 2013). BPAN is the most common NBIA disorder; its estimated prevalence is 2 to 3 per million individuals (Wilson et al., 2021). Not more than 100 BPAN cases carrying WDR45 mutation have been described worldwide by 2021 (Cong et al., 2021). All published cases of BPAN have been sporadic, with a clear female predominance and mutations in various exons of the WDR45 gene (Wilson et al., 2021; Cong et al., 2021). Here we report a 9-year-old girl with BPAN having de novo mutation c.755T>C not formerly detected in WDR45. The patient presents several typical features of BPAN as well as some RLS peculiarities.

The patient is a 9-year-old girl delivered after a full-term pregnancy to non-consanguineous parents. Her birth weight was 3750 g, and Apgar scores were 8/9. She had no family history of mental or neurological disorders. The girl developed with a delay in motor skills: she sat independently at 10–11 months, began to crawl at 12–13 months, and walked unsupported at two years of age. Her gait was unstable. She also had a developmental speech disorder: babbling at the age of one year without further progress.

Intellectual disability became apparent around the age of two. Stereotypical hand movements in the form of “twisting objects in hands”, and “putting in the mouth” appeared after one year. Despite the presence of constant stereotyped movements, the girl has not lost the ability to use her hands. Since the age of 6, flexion contractures have been detected in the elbow joints, and a year later, scoliosis was diagnosed.

Epilepsy started at one year and seven months in the form of febrile tonic-clonic seizures. Three years later non-febrile tonic-clonic seizures appeared, as well as shuddering in the limbs on awakening with a partial impairment of consciousness. Epileptic seizures stopped at the age of 5 years whilst the patient was taking valproic acid 30 mg/kg and levetiracetam 50 mg/kg. At the age of 7, antiepileptic drugs were withdrawn, and there has been no relapse of epilepsy to date.

According to EEG data, at the age of 4, during passive wakefulness, the main rhythm was 4.4–5 Hz and an amplitude of up to 110 μV in the central-parietal-occipital leads. With the onset of sleep, epileptiform activity in the form of sharp-slow wave complexes arose in the right frontotemporal-parietal leads. As sleep deepened, the high-amplitude spike-polyspike-wave epileptiform activity of a high index was recorded in the frontotemporal leads maximum under F8–T4. (Fig. 1a). At 9 years after four years of epileptic seizures termination and two years since the discontinuation of antiepileptic drugs, according to EEG data, epileptiform activity persisted in the form of sharp-slow wave complexes in the right frontal centrotemporal leads, periodically with diffuse spread (Fig. 1b).

Figure 1.

At the age of nine, the girl had severe mental retardation and lack of speech. Self-service skills were missing. The patient had autistic symptoms – 19 points on the CASD scale. Constant stereotypical movements of the hands (twisting objects, putting hands in the mouth) were typical. Neither oculomotor nor bulbar abnormalities were detected. Muscle tone in the limbs was slightly dystonic. Tendon reflexes were increased, and Babinsky’s symptom was positive on both sides. There were slight flexion contractures in the elbow joints and scoliotic spine deformity. She walked on her own, with support on a wide base. Some notable features of the phenotype were evident: low hair growth on the forehead, hypertelorism, fused eyebrows, low-set ears, and a protruding lower jaw, but there was no microcephaly (Fig. 2). Her cardiovascular and respiratory systems, as well as abdominal organs, were normal. There were no changes in biochemical laboratory tests.

Figure 2.

When the patient was 5 years old, signs of peripheral pigmentary retinal dystrophy of both eyes were detected: pigment disorganisation and many small grouped linearly pigmented foci along the fundus periphery.

MRI scans at the age of eight years (Fig. 3) revealed iron deposits in the substantia nigra and globus pallidus. Neither abnormal myelination nor atrophy of the corpus callosum and the brainstem was identified.

Figure 3.

Considering the iron accumulation in the basal nuclei and substantia nigra, epilepsy and impaired intellectual disabilities, we clinically suspected the Beta-propeller protein-associated neurodegeneration in the proband. Therefore, the patient was referred for a genetic examination to look for mutations in the WDR45 gene.

Genomic DNA was isolated from the buccal epithelium of the proband and parents by phenol/chloroform extraction.

We performed the next-generation sequencing (NGS) of proband’s DNA using Illumina’s TruSight One Expanded sequencing panel on NextSeq 550 (Illumina Inc., USA). Sample preparation was fulfilled using the Illumina DNA Prep with Enrichment kit. Quality filtering, alignment against the reference genome NCBI build 37 (UCSC hg19), and variant calling were carried out with DRAGEN applications. Variants were annotated by ANNOVAR software using RefGene, InterVar, NHLBI Exome Sequencing Project (ESP), gnomAD, 1000 Genomes Project, REVEL, ClinVar, dbSNP, and dbNSFP databases. Annotated variants were filtered based on their frequency, location, and in silico score of pathogenicity and further investigated for compliance with the studied clinical phenotype.

All putative variants were classified in accordance with the guidelines of the American College of Medical Genetics and Genomics (Richards et al., 2015). The Sanger sequencing (ABI 3500 Genetic Analyzer) was performed for variant confirmation and family genotyping.

A heterozygous missense variant c.755T>C in exon 9 of the WDR45 gene (NM_001029896.2, Xp11.23) was detected in proband. The variant leads to amino acid substitution p.Leu252Pro and has not been previously described in the literature or the genetic databases. It is likely to change an evolutionarily conserved amino acid residue (PhyloP100way = 8.37) and highly impact protein function (class C65 by Align-GVGD). Multiple lines of computational evidence support a deleterious effect of c.755T>C on the gene product: SIFT = 0; PolyPhen-2 HumVar = 0.996; Mutation Taster = 1.000; CADD = 27.6; Revel = 0.739.

Family genotyping revealed that the variant appeared de novo (Fig. 4).

Figure 4.

A diagnosis of NBIA5 (OMIM: 300894) is proposed for a patient with a set of special clinical features and definite MRI pictures. So far, 10 different genes are associated with various NBIA subtypes (Tello et al., 2018). At the same time, overlapping clinical features exist between some of the subtypes (Cong et al., 2021; Wang et al., 2019). Taking into account these items, it is no wonder that most individuals suffering from BPAN, or NBIA type 5, had, on average, a 16-years delay from the moment of clinical presentation to diagnosis (Adang et al., 2020). To avoid misdiagnosis and to speed up diagnostics, genetic testing has been widely used since 2013. BPAN was associated with WDR45 pathogenic mutations (Hayflick et al., 2013).

WDR45 encodes a WD40-repeat-containing phosphatidylinositol-3-phosphate-binding protein, also called WIPI4. It is predicted to fold into a β-propeller structure containing 7 WD40-repeat regions. The group of this protein family is involved in various essential biological processes, including signal transduction, cell cycle progression, gene regulation, etc. WIPI4 is especially important for autophagy, an intracellular degradation system (Li, Roberts, 2001). This process consists of packing cytoplasmic components into autophagosomes and delivering them to lysosomes for subsequent degradation. WIPI4 is thought to be primarily involved in phosphatidylinositol 3-phosphate binding and phagophore expansion and/or closure (Cong et al., 2021). In addition, it has been shown to regulate autophagy through the AMPK and mTOR upstream pathways and possibly to control the size and maturation of nascent autophagosomes (Ji et al., 2021).

It is assumed that in patients with mutations in WDR45, the autophagic flow is impaired; consequently, aberrant autophagic structures accumulate in cells (Saitsu et al., 2013). Although loss-of-function WDR45 mutations are considered causative for BPAN, cases of missense changes with various disease severity were described (Cong et al., 2021). To date, nearly 20 missense variants in the WDR45 have been reported as pathogenic or likely pathogenic; they provoke either BPAN, ID or DDE. The currently known pathogenic variants leading to BPAN are not concentrated in a specific region or domain but are localised throughout the WDR45 gene and often occur in the area of WD40-repeats. WD40-repeats are known to act as scaffolds for protein interactions and reversible formation of protein complexes (Smith, 2008) therefore pathogenic variants in the WDR45 may prevent normal action of WD40 and thus result in the WDR45-associated disease phenotype (Li, Roberts, 2001; Ji et al., 2021; Chard et al., 2019).

The missense variant c.755T>C is localised in the area of WD40-repeats of WDR45 – the WD6 domain. This variant can be considered pathogenic based on the following ACMG criteria: de novo variant in the family without a history of BPAN or other NBIA (PS2); absence in population databases Exome Sequencing Project, 1000 Genomes Project, and Exome Aggregation Consortium (PM2); the variant is located in a region of the WDR45, where benign changes are extremely rare (PM1); the results of more than three programs for predicting pathogenicity in silico confirm the pathogenic effect of missense change on protein (PP3); missense variant in a gene with a low rate of benign missense variation and in which benign missense variants are implicated in disease mechanisms (PP2); typical clinical phenotype observed in WDR45 mutation carriers (PP4).

In this case report, we describe a nine-year-old girl with profound ID, epilepsy, Rett-like stereotypical limb movements, pigmentary retinopathy combined with iron accumulation in globus pallidus and substantia nigra. Specific clinical features observed led us to the presumed diagnosis of NBIA type 5, which was confirmed during exome sequencing. The data currently available indicate that the c.755T>C (p.Leu252Pro) missense de novo variant (not reported before) leads to the disabling disease phenotype and adds one more patient to the growing BPAN cohort.