A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform

Radiographs of patient FA.II-6. (A) Radiograph of the knees at 8 months showing extensive punctate calcification (indicated by arrow). (B) Pelvis at 6 years showing narrow ilii, small and fragmented capital femoral epiphyses (indicated by arrow) with subtle calcifications. (C and D) Frontal and lateral view of the spine at 11 years showing slight s-shaped scoliosis, osteopenia and anisospondyly.

Barøy T, Koster J, Strømme P, Ebberink MS, Misceo D, Ferdinandusse S, Holmgren A, Hughes T, Merckoll E, Westvik J, Woldseth B, Walter J, Wood N, Tvedt B, Stadskleiv K, Wanders RJA, Waterham HR, and Frengen E. (2015) A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform. Human Molecular Genetics, 24(20)

Peroxisomes are organelles that are involved in many metabolic functions including fatty acid oxidation and synthesizing plasmalogens, a class of lipids that contain a vinyl-ether bond at sn-1. A crucial part of peroxisome function is the recognition of proteins carrying peroxisome targeting signals by receptors encoded by peroxisome biogenesis factor (PEX) 5 or 7, allowing these matrix proteins to be imported into the peroxisome where they are functional. Mutations in PEX genes cause peroxisome biogenesis disorders (PBDs), including Zellweger spectrum disorder (ZSD), infantile Refsum disease, and rhizomelic chondrodysplasia punctata (RCDP). RCDP is a class of genetic disorders that are caused by the inability to produce plasmalogens and results in severe developmental defects and cognitive deficits (more information about RCDP can be found here), caused by a deficiency in one of the early enzymes in plasmalogen biosynthesis. Barøy et al identified and described a novel and ultra-rare type of RCDP, type 5.  This was possible through identifying mutations in PEX5 long isoform (PEX5L), and describing the clinical phenotype.

The four patients studied were from two families; Family A had 3 affected siblings and the fourth patient was from Family B. All four patients were born at normal weight with congenital cataracts that were operated on within the first 6 months of life. Two of the patients from Family A were found to have delayed growth and microencephaly by 2 years old. All four were found to have severe intellectual disability at assessments around 5-7 years of age, and this diagnosis was consistent at later assessments. The first two individuals from Family A were unable to walk independently, but the youngest from Family A and the patient from Family B could take steps independently by 3 and 4 years, respectively. All affected individuals from Family A developed seizures at between 8 and 17 years of age. The first two affected patients from Family A had muscle atrophy in the extremities at 27 and 8, respectively. Overall, all individuals had delayed physical and cognitive development and were dependent upon their parents for nearly all care needs throughout the study.

To determine the novel variant causing the above pathology, whole-exome sequencing of DNA from the 3 siblings was performed. As multiple siblings were affected in the first family while both parents were not, it was assumed that the variant was recessive. When compared against the 1000 Genomes Project, any variant with a frequency above 0.01 was removed as it was too common, or if they were predicted by snpEff, SIFT, or PolyPhen-2 (tools used to predict the possible impact of amino acid substitutions) to have low impact. Five candidate genes (PEX5, TMEM82, TMCO4, SMC1B, and CLDN17) were found with homozygous variants and of these only c.722dupA in PEX5 was thought to be likely disease-causing due to the known role of PEX5 in peroxisome biogenesis and the phenotypes that this can cause. PEX5 encodes a long (L) and a short (S) isoform and the mutation found in these patients is only a part of the long isoform. The fourth patient was independently referred due to symptoms similar to that of a peroxisome disorder, and was confirmed to share this mutation.

Using fibroblasts from the patients, Barøy et al were able to investigate the effect that this variant has on expression of PEX5L. Both the long and short isoforms were detected in regular fibroblast, but only the short isoform (PEX5S) was able to be detected in the samples from the patients, indicating that the variant caused a loss of the long isoform. To confirm this loss in level of protein and to confirm whether PTS1- or PTS2-tagged proteins are reduced due to this genetic loss, immunofluorescence was used to look for PTS1-tagged calatase as a marker for peroxisomal targeting signal 1 (PTS1) protein import. Peroxisomal staining was found in control cells as well as all the fibroblast cell lines from the patients, indicating that PTS1 import is not affected. Import of PTS2-tagged phytanolyl-CoA 2-hydroxylase (PHYH) was measured by determining the phytanic acid level in plasma from the patients and the α-oxidation level of phytanic acid in the fibroblasts. The levels of phytanic acid in the plasma was increased, while the phytanic acid α-oxidation was reduced, indicating a deficiency in PHYH import and that the peroxisomal dysfunction in the patients in due to PTS2-tagged proteins.

Barøy et al discovered a novel peroxisomal disorder through identifying a specific mutation that causes a rare form of RCDP. Most patients with PEX5L mutations are misdiagnosed with ZSD although they do not exhibit some of the characteristics symptoms of a typical ZSD diagnosis including hepatic dysfunction and sensorineural hearing loss. Increased awareness of this novel form of RCDP will hopefully lead to improved diagnosis and clinical management of the disease.