The patient first came to medical attention at birth with a striking constellation of findings. He presented with hypotonia, meaning diminished muscle tone, along with bilateral talipes equinovarus, the clubfoot deformity in which both feet are twisted inward and downward. His hands showed adducted thumbs, held drawn in toward the palm, and camptodactyly of the index fingers, a permanent flexion contracture of those fingers. Craniofacial dysmorphic features completed the picture of a multisystem congenital syndrome. Taken together, these findings fall within the spectrum of arthrogryposis-like presentations, in which multiple joints are fixed in abnormal positions from before birth, typically reflecting reduced fetal movement or abnormalities of the neuromuscular system during development.

The clinical course that followed was demanding. The boy experienced delayed motor and speech milestones, meaning he reached the stages of sitting, walking, and speaking significantly later than typically developing children. He developed an abnormal gait and suffered from chronic constipation severe enough to require long-term laxative use, a detail that hints at involvement of the autonomic or enteric nervous system. Over the years he underwent multiple orthopedic and ophthalmologic surgeries to address the structural consequences of his condition, and he continues to receive supportive therapies. Notably, his cognitive development remains preserved, a feature that distinguishes his presentation from many other multisystem developmental syndromes in which intellectual disability is a core component.

The genetic explanation emerged through whole exome sequencing, the technique at the heart of modern rare disease diagnosis. Rather than reading the entire three-billion-letter genome, exome sequencing targets the roughly one to two percent of DNA that encodes proteins, where the majority of known disease-causing variants reside. This approach allows laboratories to scan thousands of genes simultaneously in a single test, and it has transformed the diagnosis of congenital disorders by uncovering variants in genes that no one would have suspected from the clinical picture alone. In this patient, sequencing revealed not one but two different variants in ERBB2, one inherited on each of the two copies of the gene, a configuration known as compound heterozygosity.

The two variants are molecularly distinct in ways that matter for interpreting their effects. The first, designated c.1018C>T, converts a cytosine to a thymine at position 1018 of the coding sequence, changing the codon for arginine at position 340 into a premature stop signal, an alteration annotated as p.Arg340Ter. A premature termination codon of this kind typically triggers nonsense-mediated mRNA decay, a cellular quality-control pathway that destroys transcripts containing early stop signals, or else produces a truncated protein lacking essential functional domains. The second variant, c.3412+1_3412+13del, is a deletion that removes the first nucleotide of intron 3412 together with twelve following intronic nucleotides, destroying the canonical splice donor site at that exon-intron boundary. Splice donor mutations of this type generally cause the splicing machinery to skip the preceding exon or retain intronic sequence, either outcome disrupting the reading frame and abolishing functional protein production. The predicted consequence of this second alteration is annotated as uncertain, denoted p.?, reflecting the difficulty of predicting the exact aberrant transcript.

ERBB2 is far better known to the public under another name: HER2, the growth factor receptor famous as a driver and drug target in certain breast cancers. The gene encodes a receptor tyrosine kinase that sits in the cell membrane and partners with the other three members of the ERBB family, EGFR, ERBB3, and ERBB4. When signaling molecules called neuregulins or other ligands engage these receptors, ERBB2 forms dimers with its partners and switches on intracellular cascades, including the MAP kinase and PI3K-AKT pathways, that tell cells to grow, differentiate, and survive. Unlike its relatives, ERBB2 is a preferred dimerization partner with no known soluble ligand of its own, making it a central hub of the signaling network. In developing tissues, particularly the nervous system and the heart, this signaling is essential, which helps explain why germline variants that cripple the gene can produce effects across multiple organ systems.

Before this report, the clinical literature on ERBB2-related congenital disease rested on a remarkably thin foundation: a single family in which two siblings carried a homozygous missense variant, meaning an identical single-letter protein-altering change on both gene copies. That first description established ERBB2 as a gene implicated in rare multisystem developmental disorders, but a phenotype defined by one family is inherently provisional. The new case changes the picture in two ways at once. First, it adds a second, unrelated family, confirming that ERBB2-associated disease is not confined to a single pedigree. Second, it demonstrates that the disorder can arise from a different genetic mechanism, compound heterozygosity for a nonsense variant and a splice-site deletion, rather than a homozygous missense change, and that the resulting phenotype can include features not observed in the original siblings.

The classification of the variants followed established standards. The report references the criteria jointly published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, the framework used worldwide to grade sequence variants as pathogenic, likely pathogenic, uncertain, or benign. Under this system, evidence from population frequency databases such as dbSNP, curated mutation collections such as the Human Gene Mutation Database, computational predictions, and the observed segregation of variants within families all feed into the final classification. The authors’ conclusion that this case represents a possible phenotypic extension of the known disorder is deliberately measured: with only a handful of patients described, distinguishing which features are core to the syndrome and which are variable remains an ongoing challenge that each new case helps to resolve.

The diagnostic journey also illustrates why exome sequencing has become the decisive tool for conditions like this one. A presentation combining clubfoot, contractures, hypotonia, and dysmorphic features could plausibly be attributed to dozens of different genes, and for many years such children accumulated diagnoses of unspecified arthrogryposis or syndromic developmental delay. Sequencing collapses that diagnostic odyssey into a single analysis, and in this case it pinpointed a gene whose connection to congenital disease had been established only once before. The Lithuanian team, based at Vilnius University and Vilnius University Hospital Santaros Klinikos, conducted the work with ethics approval from the Vilnius Regional Biomedical Research Ethics Committee and with written informed consent from the patient’s parents for both participation and publication.

For the emerging field of ERBB2-related medicine, the case carries implications in two directions. In oncology, ERBB2 is one of the most intensively targeted genes in human disease, with engineered antibodies and small-molecule inhibitors transforming outcomes for HER2-positive cancers. In genetics, the same gene is now firmly on the map of recessive congenital disorders, and the new report suggests that the clinical spectrum may be wider than the first family implied, potentially including preserved cognition, chronic gastrointestinal involvement, and a pattern of contractures and deformities requiring serial surgical intervention. Every additional patient described sharpens the genotype-phenotype correlations that clinicians will need to counsel families, interpret future sequencing results, and, eventually, evaluate whether therapies aimed at ERBB2 pathways might one day extend from cancer wards to the care of children with rare developmental disease.

Subject of Research: ERBB2-associated multisystem congenital disorder and the expansion of its phenotypic and genetic spectrum through a novel case report

Article Title: Broadening the phenotypic and genetic spectrum of ERBB2-associated multisystem congenital disorder

Article References: Jakubėnaitė, A., Gurskaitė, G., Morkūnienė, A., & Burnyte, B. (2026). Broadening the phenotypic and genetic spectrum of ERBB2-associated multisystem congenital disorder. BMC Pediatrics. https://doi.org/10.1186/s12887-026-07773-8

Image Credits: AI Generated

DOI: 10.1186/s12887-026-07773-8

Keywords: ERBB2, HER2, whole exome sequencing, congenital anomalies, arthrogryposis, compound heterozygosity, rare disease, craniofacial dysmorphic features, receptor tyrosine kinase, hypotonia, genetics, pediatrics

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

VPS41 encodes a component of the HOPS complex, a six-part molecular machine that acts as a tether, physically bringing lysosomes together with late endosomes and autophagosomes so these membrane-bound compartments can fuse. This fusion step is central to autophagy, the cellular recycling program that clears damaged proteins and organelles, and to endolysosomal trafficking, the logistics network that shuttles cargo through the cell’s degradation compartments. When this pathway falters, cellular waste accumulates, and the consequences are felt most severely in neurons, which are long-lived cells with limited capacity for self-renewal. Disruption of lysosomal function has been implicated in lysosomal storage disorders, neurodevelopmental syndromes, and neurodegenerative conditions, making genes like VPS41 prime suspects in unexplained neurological disease. Since VPS41-related disorders were first reported in 2020, the handful of documented patients have shown cerebellar ataxia, cognitive impairment, and dystonia, inherited in an autosomal recessive pattern, meaning both copies of the gene must be impaired for disease to emerge.

The patient at the center of the new report presented a clinical picture that was both characteristic and perplexing. He had cerebellar ataxia, hypotonia, and intellectual disability dating from early childhood, together with a full-scale IQ of 49 on formal cognitive assessment. Brain magnetic resonance imaging revealed anterior-predominant atrophy of the cerebellum, the movement-coordination structure at the back of the brain. Yet he also displayed features never before recorded in this syndrome: progressive swan-neck deformities of the second to fourth fingers on both hands and pes cavus, a high-arched foot deformity. Because pes cavus often signals peripheral nerve disease, clinicians repeatedly investigated that possibility. Motor nerve conduction studies performed at ages ten, fifteen, and twenty years consistently showed preserved conduction velocities in both arms and legs, and spinal cord imaging of the cervical and upper thoracic regions showed no abnormalities suggestive of posterior column degeneration. A 103-gene sequencing panel covering Charcot-Marie-Tooth disease and related inherited neuropathies, along with chromosomal microarray analysis, all came back unrevealing.

Exome sequencing eventually flagged four variants in VPS41, each confirmed by Sanger sequencing. One was a splice-site variant, c.385-2A>G, at a canonical position where the splicing machinery recognizes an exon boundary. The other three were missense variants that each change a single amino acid: p.Val137Met, p.Thr294Met, and p.Arg416His. Segregation analysis in the family added a complication. The healthy older brother carried none of the variants, and the father carried two of them, Thr294Met and Arg416His, on what was presumed to be one chromosome. But the inheritance pattern of the splice-site variant and the Val137Met variant could not be established, because the patient’s mother had died and her DNA was unavailable. Under the American College of Medical Genetics and Genomics classification framework, the splice-site variant was judged likely pathogenic based on its predicted severe effect on splicing and its rarity in population databases, while the three missense variants remained variants of uncertain significance. Without knowing which variants sat on which parental chromosomes, the team could not confirm that the patient had one damaging mutation on each of his two VPS41 copies, the configuration required for recessive disease.

That is where long-read genome sequencing made the decisive difference. Unlike standard short-read sequencing, which chops DNA into fragments of a few hundred bases and struggles to bridge complex haplotypes, long-read technology can span entire haplotype blocks in single DNA molecules, directly revealing which variants travel together on the same chromosome. The analysis showed that Val137Met sat in cis with the splice-site variant, meaning both occupied the same maternal allele, while Thr294Met and Arg416His lay in trans, on the opposite paternal copy. Because the splice-site variant disrupts splicing and the missense variants on the other chromosome were candidates for functional damage, the phasing narrowed the field to two alleles requiring laboratory validation. In addition, computational splicing prediction with SpliceAI assigned the Arg416His variant a high probability of disrupting the adjacent exon boundary, with delta scores of 0.96 for donor loss and 0.93 for acceptor loss, elevating it to the primary suspect on the paternal allele. With maternal DNA absent, this resolution would have been impossible using conventional approaches.

The researchers then examined RNA extracted from patient-derived lymphoblastoid cell lines, immortalized white blood cells that provide a renewable window into the patient’s gene expression. Transcriptome analysis revealed an abnormal splice junction consistent with skipping of exon 7, a defect the team attributed to the c.385-2A>G variant on the maternal copy. Quantitative RT-PCR showed that total VPS41 messenger RNA was significantly reduced in the patient’s cells compared with controls, and targeted RT-PCR with sequencing confirmed the exon 7 skip, which deletes 66 bases while preserving the reading frame. Because the deletion keeps triplets intact, the transcript evades nonsense-mediated decay, the cellular quality-control system that normally destroys messages carrying premature stop codons, allowing a shortened protein to be made. The paternal allele told a different story. A second primer set detected a low-abundance transcript lacking exon 15, and when the researchers treated the cells with cycloheximide, a drug that indirectly blocks nonsense-mediated decay, this aberrant band grew clearly visible. Exon 15 skipping removes 62 bases and shifts the reading frame, creating a premature stop codon, which explains why the defective message is normally degraded almost completely.

The protein-level consequences were equally informative. Western blot analysis showed that VPS41 protein was present in the patient’s cells but reduced to roughly 30 percent of control levels. Simple loss of one allele through nonsense-mediated decay would be expected to halve expression, so the deeper reduction suggests the exon-7-skipped protein produced by the maternal allele is itself partially unstable or targeted for accelerated degradation. To probe that idea, the team built structural models of the HOPS complex with AlphaFold and compared the wild-type machine against a version carrying the 22-amino-acid in-frame deletion, p.Ile129_Lys150del, that the exon 7 skip produces. Although the deleted region falls outside the annotated WD40 repeat domain spanning roughly amino acids 302 to 747, it lies near TPR-like and CHCR motifs that help shape the scaffold-like beta-propeller architecture of VPS41. The modeling showed a reshaped interaction landscape: some intersubunit interfaces, such as the A-E pairing, gained contact area and predicted stability, while the B-E and B-F interfaces weakened substantially, with destabilizing free-energy changes of 6.6 and 4.3 kilocalories per mole. The deletion, in other words, does not simply amputate part of the protein; it redistributes stress across the entire six-subunit complex and may undermine its overall integrity.

Transmission electron microscopy of the patient’s lymphoblastoid cells then provided the cellular corroboration. The images revealed characteristic endolysosomal abnormalities, including multiple multivesicular bodies and multilamellar bodies, precisely the kind of membrane-compartment pileup expected when the HOPS tethering machinery cannot complete fusion events efficiently. Taken together, the RNA findings, the protein reduction, the structural perturbation, and the ultrastructural phenotype supplied functional evidence for both alleles. Applying the American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines alongside Clinical Genome Resource sequence variant interpretation recommendations, the team reclassified the maternal splice-site variant as likely pathogenic based on PVS1-moderate, PM2, and PM3 evidence, and the paternal Arg416His variant as likely pathogenic based on PVS1 and PM2 evidence, formally establishing a compound heterozygous diagnosis of autosomal recessive spinocerebellar ataxia 29.

The case carries lessons that reach well beyond a single family. It demonstrates that long-read sequencing can resolve haplotypes in rare disease diagnostics even when parental samples are unavailable, a situation that arises frequently given the age at which many such patients are evaluated. It also confirms that RNA-based functional assays, performed directly on patient-derived cells, can convert variants of uncertain significance into actionable diagnoses by revealing the exact molecular consequence of each change. The newly reported swan-neck deformities and pes cavus expand the phenotypic spectrum of VPS41-related disease, though the authors caution that, in a single case, it remains uncertain whether these features are specific to the syndrome or coincidental, and additional cases will be needed to settle that question. Limitations acknowledged by the team include the single-patient design, the current cost and limited clinical availability of long-read sequencing, and the absence of updated spinal cord imaging in adulthood. Even so, the study marks a clear demonstration of how third-generation sequencing and transcript-level analysis together can close diagnostic gaps that once seemed permanent, offering a template for the thousands of rare disease patients still waiting for an answer.

Subject of Research: Biallelic VPS41 variants causing autosomal recessive spinocerebellar ataxia 29 resolved by long-read sequencing and RNA analysis

Article Title: Biallelic VPS41 Variants in Autosomal Recessive Spinocerebellar Ataxia 29 Resolved by Long‐Read Sequencing and RNA Analysis

Article References: Nakamura, N., Nishio, Y., Nyuzuki, H., Fukushima, A., Miura, M., Kobayashi, Y., Ishioka, R., Tsukada, K., Oka, Y., Tsujikawa, K., Morinaga, H., Inaba, M., Tohyama, J., Nakazawa, Y., Ikeuchi, T., Ono, T., Saitoh, S., & Ogi, T. (2026). Biallelic VPS41 Variants in Autosomal Recessive Spinocerebellar Ataxia 29 Resolved by Long‐Read Sequencing and RNA Analysis. Molecular Genetics & Genomic Medicine, 14(9), Article e70285. https://doi.org/10.1002/mgg3.70285

Image Credits: AI Generated

DOI: 10.1002/mgg3.70285

Keywords: VPS41, HOPS complex, spinocerebellar ataxia, long-read sequencing, haplotype phasing, lysosomal trafficking, autophagy, splicing defect, nonsense-mediated decay, rare disease genetics, compound heterozygosity, exon skipping