Gijs van Haaften

The ABCC9 gene encodes the widely expressed SUR2 subunit of ATP-sensitive potassium (K_ATP) channels. Autosomal recessive loss-of-function variants in ABCC9 cause ABCC9-related Intellectual disability and Myopathy Syndrome (AIMS). Here, we sought to compile multiple case reports from previously unidentified individuals with the primary objective of further establishing the clinical consequences of ABCC9 variants. We combine multiple case reports with genetic diagnoses and functional tests of recombinant K_ATP channels. We report 5 cases of AIMS, including a neonate, and a woman who presented as a sexagenarian with signs of dementia. All variants are predicted to lead to nonsense mediated decay of ABCC9 transcripts and/or drastic truncation of SUR2. Functional tests of recombinant channels confirm that disease-associated SUR2 truncations cause a complete loss-of-function. These new cases further demonstrate the prominence of white matter abnormalities resembling periventricular leukomalacia or small vessel disease as a key hallmark of the disorder, alongside developmental delay, intellectual impairment, seizures, and fatigability. These latest findings also highlight neonatal presentation of disease, deterioration following surgical procedures, and the potential for motor and cognitive decline, which should be monitored in older individuals. These findings provide new insights into the spectrum of pathology and natural history of AIMS. This new cohort underscores that AIMS is characterized by the combination of periventricular leukomalacia, developmental delay and intellectual disability, and muscle weakness and fatigability - and is driven by biallelic loss-of-function variants in ABCC9.

Genetic cardiomyopathies consist of a heterogeneous group of myocardial disorders caused by variants that disrupt key regulators of cardiac structure and function. Variants in PLN, encoding phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a), have been linked to both dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Among these, the PLN Arg14del (R14del) variant is the most prevalent. PLN R14del cardiomyopathy is characterized by the accumulation of large perinuclear PLN aggregates in cardiomyocytes of end-stage heart failure tissue. However, the mechanisms driving PLN aggregate formation and their role in disease progression remain unresolved. Using a humanized plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients and pharmacological modeling in wild type (WT) cardiac slices, we demonstrate that previously described PLN aggregates represent accumulated sarcoplasmic reticulum (SR)-derived PLN-containing vesicles that form due to impaired SERCA2a activity and increased cytosolic Ca²⁺ levels. Furthermore, these SR-derived vesicles often localize adjacent to lysosomes. Interestingly, Ca ²⁺ dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in SR-derived vesicle accumulation at the microtubule organizing center (MTOC). This perinuclear accumulation induces microtubule aster formation and subsequent cellular disorganization, including sarcomere misalignment and nuclear deformation. Strikingly, reactivation of lysosomal function through fasting reduces SR-derived vesicle accumulation, restores microtubule integrity, and rescues cellular organization in plna R14del zebrafish hearts. Together, these findings identify impaired lysosomal clearance of SR-derived vesicles and the resulting microtubule disorganization as key pathological mechanisms driving PLN R14del cardiomyopathy. Additionally, our results highlight lysosomal reactivation as a promising potential therapeutic strategy to halt or reverse PLN R14del cardiomyopathy progression. Main findings: PLN aggregates in PLN R14del cardiomyopathy represent SR-derived vesicles formed due to Ca²⁺ dysregulation. These SR-derived vesicles often localize perinuclearly at the microtubule organizing center (MTOC), where they are positioned adjacent to lysosomes. Ca ²⁺ dysregulation leads to lysosomal dysfunction which drives vesicle accumulation responsible for microtubule remodeling and pathological cellular rearrangements. Lysosomal reactivation restores vesicle clearance and rescues cardiomyocyte structure.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated alpl^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl^-/- compared to alpl^+/+ embryos. The survival of alpl^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl^-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl^-/- embryos. Metabolic profiling of alpl^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.

The complex 2 Mb survival motor neuron (SMN) locus on chromosome 5q13, including the spinal muscular atrophy (SMA)-causing gene SMN1 and modifier SMN2, remains incompletely resolved due to numerous segmental duplications. Variation in SMN2 copy number, presumably influenced by SMN1 to SMN2 gene conversion, affects disease severity, though SMN2 copy number alone has insufficient prognostic value due to limited genotype–phenotype correlations. With advancements in newborn screening and SMN-targeted therapies, identifying genetic markers to predict disease progression and treatment response is crucial. Progress has thus far been limited by methodological constraints. To address this, we developed HapSMA, a method to perform polyploid phasing of the SMN locus to enable copy-specific analysis of SMN and its surrounding genes. We used HapSMA on publicly available Oxford Nanopore Technologies (ONT) sequencing data of 29 healthy controls and performed long-read, targeted ONT sequencing of the SMN locus of 31 patients with SMA. In healthy controls, we identified single nucleotide variants (SNVs) specific to SMN1 and SMN2 haplotypes that could serve as gene conversion markers. Broad phasing including the NAIP gene allowed for a more complete view of SMN locus variation. Genetic variation in SMN2 haplotypes was larger in SMA patients. Forty-two percent of SMN2 haplotypes of SMA patients showed varying SMN1 to SMN2 gene conversion breakpoints, serving as direct evidence of gene conversion as a common genetic characteristic in SMA and highlighting the importance of inclusion of SMA patients when investigating the SMN locus. Our findings illustrate that both methodological advances and the analysis of patient samples are required to advance our understanding of complex genetic loci and address critical clinical challenges.

Spinal muscular atrophy (SMA) is an autosomal recessive disease most commonly caused by homozygous deletion of the SMN1 gene. Parents of affected children are typically carriers, with a recurrence risk of 25% for future pregnancies. Their close relatives have up to 50% chance of being carriers. Carriers typically possess a single copy of the SMN1 gene; however, some parents carry two copies of SMN1. Current standard diagnostic carrier tests are unable to distinguish between silent carriers with two copies on one chromosome (2 + 0 genotype) and non-carriers (1 + 1 genotype), where a de novo deletion occurred. This distinction is crucial for recurrence risk assessment, which highlights the unsolved challenge to carrier testing and genetic counseling. We combined microsatellite marker analysis, SMN copy number analysis, Sanger sequencing, long-read sequencing and de novo assembly to investigate the cause of the absence of SMN1 in a pedigree with an SMA patient identified through newborn screening, whose parents each carried two SMN1 copies. Our analysis revealed that the father is a silent carrier, while de novo assembly of the SMN locus showed a 1.4 megabase (Mb) de novo deletion between mother and child. This deletion encompasses SMN1 and SMN2 and represents the first reported nucleotide-level resolved SMA-causing deletion to date. Our findings allowed informed counseling of at-risk relatives and illustrate the complexity of SMA carrier testing and counseling. This case underscores the feasibility of and need for advanced genetic testing for SMA carriership in select cases, to improve genetic counseling practices, risk assessment, and family planning.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic var-iants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization and in severe cases from vitamin B6-responsive sei-zures. To study HPP we generated alpl-/- zebrafish line using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf) no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl-/- compared to alpl+/+ embryos. The survival of alpl-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl-/- embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl-/- embryos. Accumulation of d3-pyridoxal 5’-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl-/- embryos incu-bated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl-/- embryos. Metabolic profiling of alpl-/- larvae using direct-infusion high-resolution mass spectrome-try showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated first zebrafish model of HPP that shows multi-ple features of human disease and is suitable to study pathophysiology of HPP and to test novel treatments.

Spinal muscular atrophy (SMA) is a severe neurodegenerative disease caused by defects in the survival motor neuron 1 ( SMN1 ) gene. The wide variability in SMA severity is partially explained by an inverse correlation with copy number variation of the second human SMN gene ( SMN2 ). Nevertheless, significant variability in severity and treatment response remains unexplained, prompting a search for accessible biomarkers that could explain and predict this variability. DNA methylation of SMN2 has been proposed as one such biomarker, but comprehensive evidence and analyses are lacking. Here, we combined long-read nanopore sequencing with targeted bisulfite sequencing to enable high-resolution analysis of SMN2 -specific methylation patterns. We observed tissue-specific variation in DNA methylation across the entire 30 kb SMN2 gene in 29 patients analyzed by long-read nanopore sequencing, identifying variable methylation patterns in the promoter, introns, and 3’ UTR. Subsequent targeted analysis of these regions by bisulfite sequencing of blood-derived DNA in 365 SMA patients showed no association between SMN2 methylation and disease severity or treatment response, excluding blood methylation patterns as predictive biomarkers. However, we discovered significant age-associated variation in SMN2 methylation, particularly in intron 1 and the 3’ UTR, highlighting DNA methylation as a possible modifier of SMN expression during development and aging. Our approach provides a broadly applicable strategy for detailed but cost-effective and high-throughput characterization of DNA methylation in other genes and diseases, including complex genetic regions.

The complex 2 Mb survival motor neuron (SMN) locus on chromosome 5q13, including the spinal muscular atrophy (SMA)-causing gene SMN1 and modifier SMN2 , remains incompletely resolved due to numerous segmental duplications. Variation in SMN2 copy number, presumably influenced by SMN1 to SMN2 gene conversion, affects disease severity, though SMN2 copy number alone has insufficient prognostic value due to limited genotype-phenotype correlations. With advancements in newborn screening and SMN -targeted therapies, identifying genetic markers to predict disease progression and treatment response is crucial. Progress has thus far been limited by methodological constraints. To address this, we used targeted nanopore long-read sequencing to analyze copy-specific variation in SMN and neighboring genes. In 25 healthy controls, we identified single nucleotide variants (SNVs) specific to SMN1 and SMN2 haplotypes that could serve as gene conversion markers. In 31 SMA patients, 45% of haplotypes showed varying SMN1 to SMN2 gene conversion breakpoints, serving as direct evidence of gene conversion as a common genetic characteristic in SMA and prompting further investigation into gene conversion markers as disease modifiers. Our findings illustrate that both methodological advances and the analysis of patient samples are required to advance our understanding of complex genetic loci and address critical clinical challenges.