Single Genetic Change May Strike Twice in Rare Anemia Case, Long-Read Study Suggests

In a rare glimpse into the hidden architecture of an inherited blood disorder, researchers have used cutting-edge long-read genome sequencing to dissect a single patient’s severe case of glucose-6-phosphate isomerase deficiency, an enzyme disorder that leaves red blood cells fragile and prone to destruction. The study, published in the Journal of Cellular and Molecular Medicine, goes beyond simply listing disease-causing mutations. Instead, it documents something unusual: a candidate “dual-effect” variant, a single letter change in the DNA that may simultaneously damage the enzyme it encodes and rewrite the epigenetic landscape around its own gene. The findings remain, by the authors’ own careful framing, a hypothesis rather than a proven mechanism, but they offer a striking preview of how whole-genome, single-molecule sequencing could reshape the diagnosis of rare enzymopathies.

Glucose-6-phosphate isomerase, or GPI, sits near the top of the glycolytic pathway, catalyzing the interconversion of glucose-6-phosphate and fructose-6-phosphate. Every cell in the body depends on glycolysis to some degree, but mature red blood cells are uniquely exposed. Once they extrude their nuclei during development, erythrocytes lose the ability to manufacture replacement enzymes, so their entire energy supply hangs on whatever GPI molecules they started with. When the GPI gene is knocked out on both copies of chromosome 19, the result is hereditary nonspherocytic hemolytic anemia, a condition whose severity ranges from mild, compensated hemolysis to life-threatening hydrops fetalis and, in some families, neurological impairment. More than 40 pathogenic variants have been described, yet clinicians still cannot reliably predict how sick a given patient will become from their genotype alone.

That genotype-phenotype discordance is precisely what motivated the new study. Traditional diagnostic workflows lean heavily on identifying coding-sequence mutations and assessing their likely structural damage to the encoded protein. But occasionally, patients with two defective alleles fare better than those with one, or genetically similar patients diverge sharply in clinical course. Several research groups have proposed that layers of regulation beyond the protein sequence, including allele-specific expression, cis-regulatory elements, and epigenetic modifications, might modulate disease severity in red cell enzymopathies. Direct evidence, however, has been scarce, largely because standard short-read sequencing struggles to resolve these features. Short reads often cannot phase variants across long stretches of DNA, and they cannot simultaneously capture the native methylation marks that decorate the genome.

The Polish-led team behind the new report turned to PacBio HiFi long-read sequencing to break through that barrier. Their patient was a 47-year-old woman with transfusion-dependent hemolytic anemia. Short-read Illumina whole-genome sequencing had already identified two rare heterozygous missense variants in the GPI gene: c.572A>G, which substitutes arginine for histidine at position 191, and c.1414C>T, which substitutes cysteine for arginine at position 472. Both variants are vanishingly rare in the European population, with frequencies below 0.0001 in the gnomAD database. Critically, short reads alone could not determine whether the two mutations sat on the same chromosome or on opposite ones, a distinction that matters enormously for interpreting residual enzyme function.

The long-read data settled the question directly. By phasing the variants across their shared haplotypes, the researchers showed that the mutations sit in trans, one on each parental copy of the gene. Haplotype 1 carries p.His191Arg, while Haplotype 2 carries p.Arg472Cys, and no wild-type allele remains. The team also catalogued 15 non-coding variants within the GPI locus, though deep-learning splice predictions from SpliceAI found no disruption of canonical splicing motifs above the standard reporting threshold. In parallel, full-length isoform sequencing of blood RNA, performed in two independent technical replicates, assembled the GPI transcriptome in unprecedented detail and revealed two previously undescribed minor isoforms alongside the canonical transcript, a descriptive bonus that the authors deliberately kept out of their mechanistic model.

The most provocative observation came from layering transcript counts and methylation calls onto the phased haplotypes. Quantifying allele-resolved reads at the discriminating c.1414 position, the researchers found that Haplotype 2, the one bearing p.Arg472Cys, was modestly over-represented in the transcript pool in both replicates: roughly 62 percent in the first and 55 percent in the second, pooling to about 60 percent overall. The direction was consistent, but the effect was statistically non-significant, and the authors are explicit that this constitutes a trend toward allele-specific expression, not an established one. Isoform-level allelic quantification was underpowered, with too few full-length reads to draw any conclusion at that resolution.

Methylation added a second intriguing thread. Using the polymerase kinetics of PacBio HiFi sequencing, the team obtained native 5-methylcytosine calls across the GPI locus without the bisulfite conversion that conventional methylation mapping requires. The wild-type cytosine at c.1414, preserved on Haplotype 1, sits within a CpG dinucleotide, the genomic context most prone to methylation in human cells. In the small number of informative reads spanning that position, the site was called as methylated on every read carrying the C allele. On Haplotype 2, the C-to-T transition physically destroys the CpG, so the methylated state there is abolished by definition. The co-occurrence of CpG loss on the same allele that shows a directional excess of transcripts is exactly the kind of correlation that fuels a hypothesis, and the authors are careful to label it as precisely that, noting that only a handful of reads support the methylation call.

What makes the model worth testing is what is already known about the protein on Haplotype 2. Prior biochemical work has established p.Arg472Cys as a thermally unstable variant that degrades rapidly, a property consistent with its comparatively low AlphaMissense pathogenicity score of 0.257, compared with 0.914 for p.His191Arg, since structure-based predictors do not necessarily capture instability effects. If the same allele that produces an unstable protein also carries a CpG whose loss slightly lifts transcriptional repression, then the cell might partially compensate by simply making more of a fragile enzyme from that allele. That compensation would depend on a nucleus and would be lost when red cells enucleate and stop transcribing, potentially explaining the age-dependent decline in GPI activity that earlier kinetic studies of hemolytic anemia have proposed. In nucleated tissues, by contrast, continuous transcription from the trans allele might explain why this patient, carrying a p.His191Arg variant previously linked to neurological features, presented with an exclusively hematological phenotype.

The authors are refreshingly transparent about the limits of a single-patient, correlative dataset. They did not measure GPI protein abundance, enzymatic activity, or stability in this patient, and the allelic imbalance never reached statistical significance. To convert their candidate dual-effect variant into an established mechanism, they lay out a demanding experimental agenda: allele-specific transcript quantification at far greater depth with formal correction for mapping bias, statistically powered haplotype-resolved methylation comparison across cell fractions, a direct test of whether the disrupted CpG carries regulatory activity through reporter assays or targeted demethylation, and direct measurement of GPI protein levels and enzymatic activity comparing reticulocyte-enriched and mature erythrocyte fractions. Only such evidence could show that transcriptional output, epigenetic state, and protein stability genuinely interact to shape disease.

Even as a hypothesis, the case marks a turning point in how rare enzymopathies can be examined. One sequencing workflow simultaneously phased compound heterozygous variants, mapped native methylation without chemical conversion, resolved novel transcript isoforms, and generated a specific, testable account of why one patient’s disease unfolded as it did. Short-read exomes and genomes, the workhorses of modern diagnostics, simply cannot deliver that integrated view of a locus. As long-read multi-omics becomes cheaper and more routine, the field will be able to ask systematically whether dual-effect variants, single changes that sabotage both a protein and its own regulatory context, recur across other patients and other genes. If they do, the humble one-letter mutation may turn out to be a far more layered act of molecular sabotage than anyone suspected.

Subject of Research: Integrative long-read multi-omics characterization of a compound heterozygous GPI deficiency patient revealing a candidate dual-effect coding and cis-regulatory variant

Article Title: Integrative Long‐Read Multi‐Omics of a Patient With GPI Deficiency: A Molecular Case Study of a Candidate Dual‐Effect GPI Variant

Article References: Stolarek, I., Delimata‐Raczek, J., Koralewska, N., Sikora, K., Rakoczy, M., Marcinkowska‐Swojak, M., Handschuh, L., Czyż, J., & Figlerowicz, M. (2026). Integrative Long‐Read Multi‐Omics of a Patient With GPI Deficiency: A Molecular Case Study of a Candidate Dual‐Effect GPI Variant. Journal of Cellular and Molecular Medicine, 30(17), Article e71338. https://doi.org/10.1111/jcmm.71338

Image Credits: AI Generated

DOI: 10.1111/jcmm.71338

Keywords: GPI deficiency, long-read sequencing, PacBio HiFi, hemolytic anemia, allele-specific expression, DNA methylation, glycolysis, red blood cells, compound heterozygosity, multi-omics, Iso-Seq, rare disease