**Background:** Refsum disease is a peroxisomal disorder caused by biallelic pathogenic variants in PHYH, leading to accumulation of phytanic acid and a syndromic phenotype including retinitis pigmentosa (RP), peripheral neuropathy, cerebellar ataxia, and anosmia. The variant PHYH c.678+5G>T has conflicting interpretations in ClinVar (four uncertain, one likely pathogenic, one likely benign) and a relatively high allele frequency (0.0045 in South Asians, 0.001 overall), with five homozygotes in gnomAD v4.0.0. Previous work by Jansen et al. suggested this variant causes frameshifting exon 6 skipping, but the molecular effect had not been fully characterized. This study aimed to determine the splicing effect of c.678+5G>T using long-read cDNA amplicon sequencing.
**Methods:** Patients were recruited from Moorfields Eye Hospital (London, UK) and Buenos Aires, Argentina. Inclusion criteria: diagnosis of RP with biallelic PHYH variants, at least one being c.678+5G>T. Four patients (IDs 1-4) provided blood samples for RNA analysis; one carrier offspring and three healthy controls were also included. Total RNA was purified from PAXgene-stabilized whole blood, reverse transcribed to cDNA, and PCR amplified across the canonical PHYH transcript (NM_006214.4) from exon 1 to 3'UTR. Amplicons were barcoded and sequenced on Oxford Nanopore Technologies Flongle flowcells. Basecalling was performed with Guppy v5.0.16, reads filtered for 800-1800 bp and quality ≥Q10, aligned to GRCh38 with minimap2 v2.22, and splice junctions analyzed with IGV. Clinical data including best-corrected visual acuity (BCVA), fundus autofluorescence, spectral-domain OCT, and blood phytanic acid levels were collected from electronic health records.
**Key Results:** Four patients (IDs 1-4) provided blood samples. Patient 1 (c.678+5G>T / c.823C>T, p.Arg275Trp) had night blindness onset at age 12, BCVA 6/24 and 6/12 at first visit, lifelong anosmia, and mildly elevated phytanic acid (10.62 µmol/L, normal ≤10). Patient 2 (c.678+5G>T / c.103delT, p.Ser35ProfsTer19) had night blindness onset at age 10, BCVA 6/5 OU, no extraocular symptoms, and normal phytanic acid (11.12 µmol/L, normal ≤15). Patient 3 (c.678+5G>T / c.467C>A, p.Thr156Lys) had night blindness onset at age 20, BCVA 6/18 and HM, anosmia, peripheral neuropathy, and markedly elevated phytanic acid (259.2 µmol/L, normal ≤15). Patient 4 (homozygous c.678+5G>T) was asymptomatic at age 50, BCVA 6/6 OU, anosmia since childhood, and normal phytanic acid. Retinal imaging showed preserved ellipsoid zone and macular autofluorescence in patients 1, 2, 4, and 5; patient 3 had widespread atrophy.
Long-read RNA sequencing revealed that the c.678+5G>T allele primarily causes in-frame skipping of exons 5 and 6 (264 bp deletion, 88 amino acids). In heterozygotes, 31.1% to 55.1% of reads showed this skipping; in the homozygote (patient 4), 88.4% of reads skipped exons 5 and 6, with an additional 11.3% skipping only exon 6 (out-of-frame, p.Lys167GlyfsTer3). Controls showed 2-4.5% skipping of exons 5 and 6 (corresponding to an alternatively spliced isoform, NM_001323083.1) and no exon 6-only skipping. Phasing in patient 1 showed that 85.3% of reads from the c.678+5G>T allele skipped exons 5 and 6, while 14.7% included them, indicating incomplete loss of wild-type splicing. In patient 3, an estimated 90.3% of the c.678+5G>T allele underwent exon skipping.
**Clinical Implications:** This study demonstrates that PHYH c.678+5G>T is a hypomorphic allele causing in-frame skipping of exons 5 and 6, leading to a milder, often isolated retinal phenotype with normal or mildly elevated phytanic acid levels. The preserved macular structure and relatively late onset of visual symptoms in most patients contrast with classic Refsum disease. These findings support reclassifying the variant as pathogenic and emphasize the need for phytanic acid testing in patients with biallelic PHYH variants including c.678+5G>T, even if levels are normal, as dietary intervention may slow disease progression. Long-read RNA sequencing proved essential for characterizing this complex splicing variant, highlighting its utility in resolving conflicting variant interpretations.