CPeroxisome is a ubiquitous cell organelle in human, which is essential for oxidation of branched-chain and very-long-chain fatty acids (VLCFAs), as well as the metabolism of plasminogens, amino acids, and ether lipids (1, 2). Peroxisome biogenesis disorders (PBDs) are a spectrum of diseases with peroxisomal defect and diverse peroxisomal metabolic dysfunctions due to mutations in the PEX genes (2,3). Reports demonstrated that the majority of Zellweger syndrome (ZS) cases have different mutations in the PEX1, PEX6, PEX10, PEX12 and PEX26 genes (4). ZS, also recognized as cerebrohepatorenal disorder, is a rare genetic condition which is defined by the absence or reduction of functional peroxisomes in the cells which are responsible for VLCFA beta-oxidation (5). Zellweger spectrum disorder (ZSD) and rhizomelic chondrodysplasia punctata type I (RCP) are classified into two categories. In addition, ZSD is categorized into three classes: neonatal adreno leukodystrophy (NALD) (the most common phenotype), infantile Refsum disease (IRD) and ZS (6). Epidemiological evidence shows that the ZS prevalence in the United States is estimated to be 1:50,000 births. Patients with serious disorders die in their first year of existence (7). However, a number of patients with milder phenotypes may survive until the second decade (8). A wide range of symptoms such as enlarged liver, renal cysts, seizures, retinal dystrophy, and hearing loss may be manifested (9).
Essential proteins for peroxisome biogenesis and conservation are called peroxins (PEX proteins). The most recognized peroxins promote protein entry from cytosol synthesis location to the peroxisome matrix. PEX1 ATPase promotes regeneration of protein receptor PEX5 peroxisome complex, which is the most frequently involved peroxin in biogenesis diseases of mammalian peroxisome. Mutations in the PEX1 are the frequent cause of PBDs in mammalian. In the lethal cases, the peroxisomes are not developed or the matrix proteins ingest ineffectively.
Therefore, a precise understanding of the molecular aspects of rare inherited disorders, such as ZS, requires genomic sequencing as a novel and effective strategy (10, 11). Presently, the whole-exome sequencing (WES) technology and the survey of protein-coding regions of genes in a genome, provide feasibilities for ZS investigation (12, 13). Herein, we aimed to present the molecular diagnosis of ZS in an Iranian family in the South Khorasan Province, using WES to detect the accurate etiology of idiopathic ZS.

 

Table 1. The primers sequence which were used to confirm PEX1 mutation

 
Figure 1. Identification of a missense PEX1 mutation. a) The proband indicated that specific characterize of Zellweger Syndrome in 8 years old including facial dysmorphology, speech difficulties and mental retardation; b) Consanguineous pedigree displayed affected members are II-4 and II-5 (twin). They are homozygous for the C.G2528A variant.

 
Table 2. The related Pathogenic variants of PEX1 gene

Discussion

We have detected a missense variation in PEX1, in an Iranian ZS patient. The proband had the common characteristics of ZS including facial dysmorphology, difficulty in speech and intellectual disability. Since the clinical appearance was not trustworthy, the affected individual was first distinguished based on the symptoms. The pedigree analysis demonstrated an autosomal recessive pattern (Figure 1). Subsequently, based on the WES findings of the proband, a missense variant (C.G2528A) was detected, with an autosomal recessive pattern in the second functional AAA domain (D2) (Figure 1).
PEX1 (MIM#602136) is a protein-coding gene located on chr7q21.2 location, which contains 24 exons. It produces a transcript of 4.4 kb. PEX1 mutations are detected in almost 70% of ZS patients. Evidence suggests that PEX1 can play a critical role as a matrix protein transporter in the peroxisomal membrane. The protein structure generally consists of four segments: N-terminal and C-terminal regions, and D1 and D2 domains. To date, 14 human PEX genes have been identified which encode peroxin proteins. They have function in different phases of peroxisome biogenesis, such as peroxisomal matrix protein importation, membrane formation, and peroxisome proliferation (17, 18). Peroxisomal membrane proteins are active as signaling sites for the autophagy, which are caused by antiviral immunity and reactive oxygen species (19). Glycine (Gly), a non-charged amino acid, is substituted with Aspartate (Asp) which is negatively-charged, and this could induce crucial functional and structural alterations. Phylogenetic protein sequence conservation analyses of PEX1 reveals that Glycine 843 is a protected amino acid in the protein sequence of human PEX1 (Figure 2). However, the exact protein function is not clearly known.
In a patient with Leber congenital amaurosis (LCA), the mutant PEX1 (p.Gly843Asp) which has a transformation (Gly to Asp) in cDNA bp 2528 has been documented once before (20). Reuber et al. reported that mutations in PEX1 could be the most common cause of PBDs occurrenceThey indicated that PEX1 deficient cells have different deficiencies in the peroxisomal targeting signal and protein matrix transfer. Figure 3 displays the projected functional partners of PEX1. Moreover, their results showed that the G843D allele is a common mutation in the PBDs (21). The new mutation attenuates the protein function and is associated with mild peroxisomal condition of group 1 complementation (Figures 4 and 5).

Figure 2. a) The Sanger sequencing analysis of homozygote affected. b) The Sanger sequencing analysis of heterozygote parent, an arrow shows the mutation; c) phylogenetic conservation studies of the PEX1 protein sequence and Glycine is conserved amino acids in the human PEX1 protein sequence

Figure 3. Predicted functional partners of PEX1

Figure 4. Prediction of PEX1 protein structure. To comprehend, the structural implications of mutations in PEX1, wild-type and mutant proteins were modeled using the online I-TASSER server.

Figure 5. Docking proteins to find the effect of mutation on PEX1

 
Meng-Meng Ge et al. reported four novel mutations (c.2416+1 G>T and c.2489delT) (c.1483+1 G>A and c.1727dupG) in the PEX1 gene using the WES technique (22). Ling Yu verified the homozygous mutations (c.818insCTTG) (p.Pro274Leufs*8) in the PEX6 gene by WES, which target gene panels of 'multiple congenital anomalies' and 'skeletal dysplasia’ in ZS patients. Evidence showed that the two novel mutations (p.Cys358* and p.Leu83Pro), have been known as pathogenic according to Genomics guideline. Phenotype-genotype associations were reported in the PBD-ZSD continuum patients (23). Barillari and coworkers recognized two specific variants in PEX1 by sequencing. One of them was the missense variant p.(Val92Leu);c.274 G>C which has been mentioned in the PBD cases before. The second one was p.(Ser714 Gln715dup);c.2140 2145dupa, which is a non-frameshift variant. It is not found in normal samples (24).
The researchers found that, in two Chinese newborns, all four mutations have critical control over the protein function. In addition, evidence indicates that the main causes of multiple organ dysfunction and neural disorder are β-oxidation deficiency, VLCFA accumulation, and docosahexaenoic acid (DHA) reduction (22). Bousfiha et al. detected a new homozygous PEX1 mutation (p. Leu1026Pro) in two deaf Moroccan siblings. Their reported symptoms included vision impairment, deep deafness and delay in development. They showed that this mutation can be because of ATP hydrolysis in the P-loop (25).
Recent developments in high‐throughput WES approaches and the next‐generation sequencing method have now revealed the genetic basis of disorders with unknown etiology. Furthermore, exome sequencing is progressively being used as an investigative device for precise genetic diseases diagnosis, especially in the context of substantial phenotypic and genetic heterogeneity disorders (26, 27). This result can be replicated as a result of consanguineous marriage in the future, which can be used for a more suitable customized treatment for ZS patients.

Conclusion

The results of the present study indicated a pathogenic heterozygous missense mutation in PEX1 at the codon 843 in ZS patients. This finding can be repeated in the future in the cited family. It may happen as a result of consanguineous marriage. This mutation may be used for ZS patients for a more desirable personalized medicine. Additionally, it is a missense variant (p. Gly843Asp) with an autosomal recessive pattern. It may be widespread among Iranian populations with intellectual disability. Taken together, the results of this study provided novel perceptions into the diagnostic power of WES, and revealed that it is possible to consider and treat the recognized mutations, individually.

Funding and support

This research resulted from an independent research without receiving any financial support.

 

Conflicts of Interest

Authors declared no conflict of interests.