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Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of genetic neuropathies, with more than 90 associated genes. While the majority of individuals with genetic diagnosis have demyelinating CMT, a significant proportion of individuals with axonal CMT remain genetically unsolved. Aminoacyl-tRNA synthetases (aaRS) have been implicated in various neurological disorders, including CMT. Here, we hypothesize that hypomorphic variations of DARS2 , which encodes the mitochondrial aspartyl-tRNA synthetase, are associated with axonal CMT. We investigated four individuals from two unrelated families with axonal CMT. We conducted clinical assessments, genetic testing, and functional studies in fibroblasts from two affected individuals. Two pairs of siblings, now adults, presented with progressive distal muscle weakness with onset during childhood, absent lower limb reflexes and normal cognitive function. Brain MRI scans showed no abnormalities. Electrophysiological studies revealed positive waves and fibrillations and was consistent with a diagnosis of axonal motor and sensory neuropathy. Genetic testing identified compound heterozygous variants in DARS2 in both families (c.713C>T; p.Ser238Phe and c.1006C>T; p.Arg336Cys in Family 1; and c.74del; p.Ile25Thrfs*38 and c.1006C>T; p.Arg336Cys in Family 2). The four individuals share the variant DARS2 : c.1006C>T, p.Arg336Cys, located in the insertion subdomain of DARS2, where very few variants have been previously reported. In Family 1, this variant is in trans with another missense variant (c.713C>T; p.Ser238Phe) located in the dimerization domain of DARS2, which is an essential region for the correct functionality of the protein, as in other aaRS. In Family 2, it is combined with a truncating variant. Functional analyses in fibroblasts revealed reduced DARS2 protein levels and altered mitochondrial localization. Additionally, we observed a decrease in both mitochondrial mass and fragmentation, consistent with a disrupted mitochondrial network morphology, suggesting the involvement of DARS2 in the disease pathophysiology. The identification of fully penetrant variants (p.Ser238Phe and p.Ile25Thrfs*38) combined with a hypomorphic variant (p.Arg336Cys) expands the phenotypic spectrum of DARS2-related disorders, establishing a novel association between DARS2 and axonal CMT. The combination of penetrant variants and hypomorphic variants distinguishes these cases from previously reported with DARS2-related leukoencephalopathy. We propose that the genotypic combination of a hypomorphic and a penetrating allele for DARS2 explains the symptoms in these two families with a pure axonal CMT. These findings underscore the importance of mitochondrial aspartyl-tRNA synthetase in peripheral nerve function and highlight potential targets for therapeutic intervention in CMT. [ABSTRACT FROM AUTHOR]

Next-generation sequencing has enhanced the rate of genetic diagnosis in neuromuscular diseases. Nevertheless, half of the patients remain without a molecular diagnosis. One factor that can impede the acquisition of a genetic diagnosis is the presence of mosaicism: the coexistence of at least two different genetic cell populations within an organism. We present two cases of neuromuscular pediatric patients in which a phenotype-driven analysis guided the careful interrogation of candidate genes. Patient A showed a phenotype of congenital myopathy (CM). Trio genome sequencing detected a heterozygous likely pathogenic variant in RYR1 (c.14566G>A; p.Ala4856Thr), inherited from his unaffected mother. It was considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother. The comparison between alternative allele frequencies (AAF) showed that, while the affected child had an AAF of 0.60, the mother had an AAF of only 0.23. A quantification through ddPCR from blood-derived DNA revealed a 17% of the alternative allele in the mother's sample, whereas in the individual's sample, 49% was observed. Patient B was born with congenital hip dislocation, hypotonia and distal hyperlaxity. Upon clinical examination of the parents, it was detected a left hemicorporal hypotrophy and hand retractions in the father. A neuromuscular gene panel in patient B and his father was inconclusive. RNAseq performed on the muscle sample of the father revealed an exon 9 skipping of COL6A1 in 15% of the transcripts. In genome sequencing it was detected an exon 9 deletion of COL6A1 in 10% of the reads in the father and 50% in patient B (p.Gly269_Pro286del), which was validated through a custom MLPA. Our study highlights the importance of phenotype-driven analysis and careful interrogation of the likely relevant genes and variants and illustrates the genetic mosaicism as a possible explanation behind genetically unsolved affected individuals. [ABSTRACT FROM AUTHOR]

The Taq IA polymorphism is a marker of both the Ankyrin Repeat and Kinase Domain containing I gene ( ANKK1 ) encoding a RIP-kinase, and the DRD2 gene for the dopamine receptor D2. Despite a large number of studies of Taq IA in addictions and other psychiatric disorders, there is difficulty in interpreting this genetic phenomenon due to the lack of knowledge about ANKK1 function. In SH-SY5Y neuroblastoma models, we show that ANKK1 interacts with the synapse protein FERM ARH/RhoGEF and Pleckstrin Domain 1 (FARP1), which is a guanine nucleotide exchange factor (GEF) of the RhoGTPases RAC1 and RhoA. ANKK1-FARP1 colocalized in F-ACTIN-rich structures for neuronal maturation and migration, and both proteins activate the Wnt/PCP pathway. ANKK1, but not FARP1, promotes neuritogenesis, and both proteins are involved in neuritic spine outgrowth. Notably, the knockdown of ANKK1 or FARP1 affects RhoGTPases expression and neural differentiation. Additionally, ANKK1 binds WGEF, another GEF of Wnt/PCP, regulating its interaction with RhoA. During neuronal differentiation, ANKK1-WGEF interaction is downregulated, while ANKK1-FARP1 interaction is increased, suggesting that ANKK1 recruits Wnt/PCP components for bidirectional control of F-ACTIN assembly. Our results suggest a brain structural basis in Taq IA-associated phenotypes.

Establishing a molecular diagnosis remains challenging in half of individuals with childhood-onset neuromuscular diseases (NMDs) despite exome sequencing. This study evaluates the diagnostic utility of combining genomic approaches in undiagnosed NMD patients. We performed deep phenotyping of 58 individuals with unsolved childhood-onset NMDs that have previously undergone inconclusive exome studies. Genomic approaches included trio genome sequencing and RNASeq. Genetic diagnoses were reached in 23 out of 58 individuals (40%). Twenty-one individuals carried causal single nucleotide variants (SNVs) or small insertions and deletions, while 2 carried pathogenic structural variants (SVs). Genomic sequencing identified pathogenic variants in coding regions or at the splice site in 17 out of 21 resolved cases, while RNA sequencing was additionally required for the diagnosis of 4 cases. Reasons for previous diagnostic failures included low coverage in exonic regions harboring the second pathogenic variant and involvement of genes that were not yet linked to human diseases at the time of the first NGS analysis. In summary, our systematic genetic analysis, integrating deep phenotyping, trio genome sequencing and RNASeq, proved effective in diagnosing unsolved childhood-onset NMDs. This approach holds promise for similar cohorts, offering potential improvements in diagnostic rates and clinical management of individuals with NMDs., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: Data were collected and analyzed following the ethics guidelines of Hospital Sant Joan de Déu (PIC 98-20). The family provided written informed consent for the study., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Congenital myopathies (CMs) are rare genetic disorders for which the diagnostic yield does not typically exceed 60% . We performed deep phenotyping, histopathological studies, clinical exome and trio genome sequencing and a phenotype-driven analysis of the genomic data, that led to the molecular diagnosis in a child with CM. We identified a heterozygous variant in RYR1 in the affected child, inherited from her asymptomatic mother. Given the alignment of the clinical and histopathological phenotype with RYR1-CM, we considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother, as subsequently demonstrated. Our study is an example of how heterozygous variants inherited from asymptomatic parents are frequently dismissed. When the genotype-phenotype correlation is strong, it is recommended to consider a parental mosaicism.