Clinical evidence supports a novel PEX5 stop-loss variant associated w

Introduction

Zellweger syndrome (ZS) is a rare autosomal recessive condition of neonatal onset characterized by severe dysfunction of the central nervous system, liver, and kidneys. ZS was first described in 1964 in children with failure to thrive, congenital glaucoma, craniofacial dysmorphism, and early death.¹ Then, in 1965, polycystic kidneys and intrahepatic biliary dysgenesis were added as additional features,² leading to the designation cerebro-hepato-renal dysgenesis, which was later renamed as ZS.³ The causal association between ZS and the absence of peroxisomes in hepatocytes and renal proximal tubules was established in 1973.⁴

ZS is clinically characterized by neuronal migration disorders, early-onset seizures, dysmorphic facial features, skeletal abnormalities resembling chondrodysplasia punctata, muscle weakness, developmental delay, renal cysts, and severe liver disease. Death usually occurs within the first year of life.^1,5–8 The metabolic dysfunction is expressed by the accumulation of very long-chain fatty acids, pristanic acid, phytanic acid, and total bile acids; along with reduced plasmalogens in erythrocytes.^9,10 ZS is caused by mutations in at least one of several PEX genes, which encode peroxisome assembly proteins involved in complex catabolic and anabolic pathways. ZS represents the predominant type of peroxisome biogenesis disorders (PBDs); it is mainly caused by mutations in the PEX1 gene, which accounts for approximately 60% of all PBDs cases, but it can be caused by any of the ZS-PEX genes, regardless of their specific phenotype.^{5,6,8,11–13} This spectrum entails a clinical continuum of various phenotypes, ranging from the most severe manifestation known as Zellweger syndrome; to milder forms such as neonatal adrenoleukodystrophy, infantile Refsum disease, and Heimler syndrome.^5,6,11–13

PTS1R (Peroxisomal Target Signal 1 Receptor, also known as PEX5), is a peroxin, a group of proteins that are essential for the formation of functional peroxisomes, cellular organelles derived from the endoplasmic reticulum that are involved in multiple metabolic pathways. PTS1R is located in the cytosol and peroxisomes and is part of both the formation and degradation of these organelles. First, it recognizes and binds matrix proteins containing the C-terminal tripeptide peroxisome target sequence (PTS) to import them into the peroxisome through an ATP-requiring action, acting as a receptor.¹⁴ On the other hand, during peroxisome degradation known as pexophagy, PTS1R is phosphorylated by ATM protein and then ubiquitinated at L209 by the peroxisomal E3-ligase, PEX2/10/12, to be recognized by the autophagic adaptor SQSTM1/p62.^15,16

PTS1R also participates in autophagy regulation outside the peroxisome through inhibition of the mTORC1 pathway, and it has been shown that its absence impairs the cell’s ability to start this process under stress situations.¹⁶ It is encoded by the PEX5 gene located at 12p13.31, containing 16 exons. It has multiple isoforms, with two main coding for functional proteins derived from alternative splicing of exon 7: PEX5S (short) and PEX5L (long). The short isoform only imports PTS-1 sequences while the long one also recognizes PTS-2.¹ Even though the testis and brain are the tissues with the highest expression, PEX5 is ubiquitous in human samples, explaining the multiorgan dysfunction seen in patients with peroxisomal biogenesis disorders caused by PEX5 deleterious variants: Peroxisome Biogenesis Disorder 2A (PBD2A), Peroxisome Biogenesis Disorder 2B (PBD2B) and Rhizomelic Chondrodysplasia Punctata type 5 (RCDP5).^14–17 Variants are found in the entire gene, however, exons 12 and 14 have a particularly high number of them, with the most common variant being c.1578T>G p.(Asn526Lys).¹⁸

There is no data regarding the incidence of ZS in Colombia, however worldwide data indicates that the highest incidence was estimated to be 1 in 12.000 in the French-Canadian region of Quebec.¹⁹ The incidence in the United States is around 1 in 50.000 newborns⁷ and in Japan, is estimated to be 1 in 500.000 births.²⁰

We describe a highly consanguineous family with two affected siblings, one of whom was found to carry a novel variant in the PEX5 gene. Our clinical findings, together with the molecular analysis of the variant, enable us to propose it as the causative mutation of the disease.

Case Report

Index case (VI-4) is a male, born from the second pregnancy of healthy consanguineous parents. He was delivered via cesarean section following premature rupture of membranes. Birth weight was 2.590 g (5th percentile) and height was 49 cm (32nd percentile). At 18 days of life, he debuted with gaze deviation and tonic seizure; at two months, the seizures had progressed to generalized tonic-clonic seizures with cyanosis. Treatment with oxcarbazepine and levetiracetam was initiated, leading to seizure remission. At 4 months, the patient developed social smiling, could bring his hands to his mouth, and later grasp objects with one hand and produce monosyllables, but he was unable to support his head and maintain sitting position. The patient presented global hypotonia and distinctive facial features, as described in Figure 1. A magnetic resonance imaging (MRI) scan revealed asymmetric polymicrogyria with more extensive involvement of the left hemisphere, along with additional cortical alterations indicative of cortical encephalopathy. Shortly after the first consultation, the patient was hospitalized due to his severe neurological phenotype. Given that his neuroimaging was indicative of a neuronal migration disorder, all studies performed were aimed primarily towards the diagnosis of a neurological pathology, without suspecting a disease that would involve other systems.

Figure 1 Clinical and Molecular Findings; (A) Phenotype: brachycephaly, broad forehead with frontal bossing, medially sparse eyebrows, telecanthus, ocular proptosis, midfacial hypoplasia, depressed nasal bridge, small nose, short and deep philtrum, cupid’s bow upper lip and tent-shaped mouth, open book posture due to severe hypotonia. (B) Pedigree. The proband (VI-4) is described with cortical abnormality and epilepsy, his brother (VI-3) with epilepsy and leukodystrophy and a paternal cousin (V-4) with epilepsy. Two consanguinity unions have been identified. (C) Sanger sequencing. The mother and father show a heterozygous duplication of 4 nucleotides. The proband displays a homozygous duplication pattern in the electrophoretogram, red arrow indicates the position of the duplication in PEX5:c.1897_1900ACTA.

The patient’s older brother (VI-3) showed severe global developmental delay, focal epilepsy and leukodystrophy. He died at the age of five, remaining undiagnosed with a suspected rare genetic disorder.

Their parents, aged 37 (mother) and 29 years (father), were healthy and consanguineous with two endogamous matings in their family history (Figure 1B). The couple had three pregnancies: the patient, his older brother who died at the age of five due to respiratory complications during ICU hospitalization, and a spontaneous abortion at eight weeks. The mother has another healthy daughter from a previous non-consanguineous union. Additionally, there is a history of epilepsy in a maternal second-degree relative.

Molecular Analyses

The DNA of the patient and both parents was extracted from a whole-blood sample using the Quick-DNA 96 plus kit (Zymo Research). After assessing the quality and quantity of the DNA, a genomic library was prepared using MGIEasy FS DNA Library Prep Kit and fragments ranging from 200 to 400pb were obtained. The regions of interest were captured through the Exome Capture V5 probe and streptavidin beads. The final PCR reaction was enriched and employed specific primers. Sequencing and library preparation were performed using MGI DNBSEQ-G50 platform for Gencell Pharma (Bogotá, Colombia). The reads obtained were mapped and aligned to the reference genome (hg19) and variant calling was performed using the GATK v4.0.5.1 tool.¹¹ Analyses of coverage and depth were conducted using the BAMBA tool, and 50X was the acceptable threshold. The variants obtained in VCF format (variant call format) were imported into VarSeq software (Golden Hélix v2.2.3) for annotation and filtering according to different criteria. Filtered candidate variants were assessed using the American College of Medical Genetics and Genomics guidelines (ACMG). Bioinformatics procedures were performed in the laboratory of the Genetics and Genomics Research Center of the Universidad del Rosario (CIGGUR), Bogotá (Colombia).

Through trio exome sequencing the homozygous variant (NM_001131025.2):c.1897_1900dupACTA p.(Met634Asnfs*16) in the PEX5 gene was identified. Despite its classification as a variant of uncertain significance (VUS), given the history of parental consanguinity and the diagnosis of leukodystrophy in a sibling, the diagnosis of Zellweger syndrome, an autosomal recessive peroxisome biogenesis disorder, was made. The identified molecular variant was confirmed by Sanger sequencing (SS) in the patient and both parents (Figure 1C), demonstrating parental segregation.

Discussion

We identified two siblings with epilepsy, congenital hypotonia, developmental delay, dysmorphic facies, and different neuroimaging abnormalities. In the oldest sibling adrenoleukodystrophy vs metabolic disease was suspected and in the youngest one, our index case, cortical encephalopathy diagnosis was proposed. Despite both children showing similar symptoms and neuroimaging findings suggesting a shared clinical landscape, a clear consensus on the diagnosis could not be reached.

We performed an exome trio analysis identifying the homozygous variant (NM_001131025.2):c.1897_1900dupACTA p.(Met634Asnfs*16) in the PEX5 gene. This variant is a four-nucleotide duplication at position 1897 of the cDNA, located within exon 16 of the gene, classified as a VUS by the ACMG criteria PM4, PM2 and PP1. It induces a stop-loss mutation which results in the downstream extension of 9 amino acids in the protein. The allelic frequency of this variant remains unknown in the GnomAD population database, and it has not been reported in clinical databases or in scientific literature reviewed.

A series of frameshift variants located in the last exon of different PEX genes, which result in the extension of the protein by a stop codon loss mechanism, have been identified in the literature in association with ZS. Ebberink et al (2010) provided a comprehensive overview of all variants identified in their genetic complementation studies of more than 600 skin fibroblast cell lines from patients with Zellweger syndrome spectrum disorders, diagnosed based on metabolite analysis in plasma and/or detailed studies in fibroblasts. To identify which PEX gene was defective in each cell line, the researchers employed functional analyses, including a polyethylene glycol (PEG)-mediated cell fusion assay and a PEX cDNA transfection assay. Once the defective PEX gene was identified, researchers performed Sanger sequencing of the coding region and flanking intronic sequences, or alternatively, sequenced PEX genes cDNAs prepared from RNA extraction and RT-PCR to characterize their molecular variants. They identified stop-loss variants across PEX1, PEX2, PEX10 and PEX16 genes, highlighting the contribution of this type of molecular variants in the etiology of ZS.²¹ Similarly, Régal et al (2010) reported a 8.5 year-old child with cerebellar atrophy, slowly progressive ataxia, axonal motor neuropathy and posterior column dysfunction. Two mutations in PEX10 were found in the child, one of them was the pathogenic variant (NM_153818.1):c.764_765insA p.(Leu256Alafs*103), previously reported in patients with Zellweger syndrome, which is predicted to extend the protein product.^22–25 In addition, Ebberink et al (2010) described two siblings with progressive spastic paraparesis and ataxia, who developed cataracts and peripheral neuropathy. They showed a characteristic pattern of progressive leukodystrophy and brain atrophy on MRI scan. The subsequent sequencing of all known PEX genes revealed the homozygous frameshift variant (NM_004813):c.984delG p.(Ile330Serfs*27) in PEX16 gene, which results in a stop-codon loss, introducing a termination codon at amino acid position 357 in a protein with 337 amino acids.²⁶ These findings suggest that stop-loss is a common mechanism within the PEX gene family by which frameshift variants elongate, rather than truncate, functional protein products. This phenomenon plays a significant role in the molecular pathogenesis of Zellweger spectrum disorders.

In addition to existing evidence regarding the impact of terminal frameshift mutations in PEX genes, it is important to highlight that the mutation identified in our patient is located at the C-terminal end of the PEX5 protein, potentially affecting the last 7th tetratricopeptide repeat (TPR) motif within the TPR domain, which spans residues 321 to 639. The TPR domain is essential for binding to the peroxisomal targeting signal type 1 (PTS1) present on peroxisomal matrix proteins. This interaction is critical for the recognition and import of these proteins into peroxisomes.^27,28

Additionally, the C-terminal region of PEX5 is involved in forming complexes with other peroxisomal proteins, such as PEX14, a key component of docking and translocation machinery at the peroxisomal membrane. This interaction is essential for the translocation of the PEX5–cargo complex into the peroxisomal matrix.²⁹ The dual role of the C-terminal domain in both PTS1 recognition and interaction with peroxisomal membrane components underscores its functional relevance in the peroxisomal protein import cycle and its potential link to Zellweger spectrum disorders.³⁰

Molecular variants affecting this critical functional domain, are expected to impair peroxisomal function, leading to the accumulation of very long chain fatty acids (VLCFA) and other metabolic abnormalities.³¹ In our patient, biochemical profiling that could confirm the dysregulation of these metabolites could not be performed due to the unavailability of specialized testing in the rural area from which the patient originated. Nevertheless, as in previously reported cases, the combination of clinical features and the molecular finding was sufficient to support the diagnosis of the syndrome.

There have been many case reports on pathogenic variants in PEX5. First, Baroy et al, described the first cases of rhizomelic chondrodysplasia punctata 5 (RCDP5); they found two families in Pakistan, both consanguineous, with children affected by severe global developmental delay, multiple skeletal anomalies, epilepsy and congenital cataracts.³² Later, Ali et al also described a Pakistani family with two consanguineous marriages; 12 individuals were studied, with 5 affected by severe global developmental delay, epilepsy, hypotonia and congenital cataracts.³³ On the other hand, Pronicka et al sequenced 113 patients with possible mitochondrial diseases due to clinical manifestations and found one with a pathogenic mutation in PEX5 that led to developmental regression, deafness and leukoencephalopathy, classifying it as ZS,³⁴ showing the wide range of differential diagnosis of this disease (Table 1).

Table 1 Comparison of Index Case (VI-4) with Reported Patients with PEX5 Pathogenic Variants

Based on the patient’s genetic findings, the family history of consanguinity, the spectrum of neurological disorders between the siblings and the literature review about stop-loss variants associated with different peroxisomal disorders in PEX5 and other ZS genes (PEX6, PEX10, PEX12, PEX16, PEX19), wee can assert that the novel homozygous stop-loss variant PEX5 (NM_001131025.2):c.1897_1900dupACTA p.(Met634Asnfs*16), is causing an autosomal recessive peroxisome biogenesis disorder that explains the phenotype observed in both siblings. In this context, it is important to consider that in certain cases, genetic variants classified as VUS according to ACMG may be considered causative of the observed phenotype based on clinical findings. Although these variants do not fulfill all the established criteria for pathogenicity, their correlation with the patient’s clinical presentation, family history, and, when available, functional studies, supports their role in the disease etiology. Such cases highlight the importance of integrating clinical judgment with genetic findings to provide a more comprehensive interpretation of rare disorders.

Limitations

After the first clinical consultation, the patient suffered significant neurological deterioration leading to hospitalization in a local hospital, where advanced biochemical studies were not available. Although the results of the genetic test were obtained during the hospitalization, the patient died shortly after, before further studies could be carried out in search of abnormalities in other systems, such as X-rays and ultrasound scans, or new samples could be taken to carry out functional studies.

Conclusion

We report a novel molecular variant in PEX5 associated with Zellweger Spectrum Disorder (ZSD). Although the variant meets ACMG criteria for being classified as a VUS, it is unequivocally responsible for the disease. This conclusion is supported by the clinical phenotype of the patient and his sibling, parental segregation, and the established role of PEX5 in peroxisomal disorders. In addition, the PEX5 variant identified may impair the TPR domain, which is essential for mediating PTS1 cargo recognition. A mutation at position 634 could therefore result in defective import of matrix proteins into peroxisomes.

Also, the same type of variants (stop loss) have been associated with the disease in other ZSD-related genes. This case highlights the importance of considering genetic diseases as potential diagnoses in the context of severe neurological pathology with parental consanguinity history. Early genetic tests can allow the diagnosis of specific genetic pathologies, improve the medical approach by identifying other systems that may be compromised, and provide timely genetic counseling that can prevent the occurrence of new cases of the disease.

Abbreviations

ZS, Zellweger syndrome; MRI, A magnetic resonance imaging; ACMG, American College of Medical Genetics and Genomics guidelines; VUS, variant of uncertain significance; SS, Sanger sequencing; PBDs, peroxisome biogenesis disorders; PTS1R, Peroxisomal Target Signal 1 Receptor; PBD2A, Peroxisome Biogenesis Disorder 2A; PBD2B, Peroxisome Biogenesis Disorder 2B; RCDP5. Rhizomelic Chondrodysplasia Punctata type 5.

Data Sharing Statement

All the used data are included in this article.

Consent for Publication and Ethics Approval

Written informed consent and medical photographs parental approval for publication was obtained from the patient’s parent. The study was approved by the Ethics Committee of Universidad del Rosario (Approval DVO005-1614-CV1441, June 2021).

Acknowledgments

We thank our colleagues from the Universidad Nacional de Colombia’s population genetics team for opening a space with the community and supporting us with data collection. We also thank the laboratory team, especially Maria Alejandra Coronel from the Universidad Nacional de Colombia, who helped us with sample collection and processing, and Sophya Villamil from the Universidad del Rosario clinical team, who provided feedback on the manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This project was supported by the Ministry of Science, Technology, Innovation, Minciencias. Project in health promoting personalized medicine and translational research, Grant 632-2021 (November 2021), Universidad Nacional de Colombia, Medisens IPS and Universidad del Rosario (Grant QAN BG273).

Disclosure

The authors report no conflicts of interest in this work.

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