Inherited retinal dystrophies (IRDs) represent a diverse group of rare pathologies affecting vision, with significant genetic and clinical variability. Clinical exome sequencing was performed on 143 families clinically diagnosed with IRDs. The obtained variants were filtered and classified according to the American College of Medical Genetics and Genomics guidelines. Overall, a genetic diagnosis was achieved for 68.53% of the families in the cohort; 35 causative genes were identified, predominantly ABCA4 and USH2A. A total of 170 clinically relevant variants were identified, 45 (26.47%) of which were novel, with missense variants being the most common type (40.59%). This study reported aberrant splicing generated by the ABCA4 (NM_000350.2): c.1299A>G mutation through the functional assay of a minigene. Furthermore, the genes FAM161A and GUCY2D were associated with IRDs that are not typically linked to these genes. Consequently, this study expands the current understanding of IRDs and supports the use of clinical exome sequencing as an effective strategy for the genetic diagnosis of these pathologies.

BACKGROUND:Hereditary spastic paraparesis (HSP) encompasses a genetically and clinically heterogeneous group of neurodegenerative disorders, primarily characterized by progressive lower limb spasticity and weakness of the lower limbs. Although more than 80 genes have been associated with HSP, achieving definite genetic diagnosis remains challenging, limiting effective patient care, genetic counseling, and understanding of genotype-phenotype correlations. This study aimed to investigate the diagnostic yield of clinical exome sequencing (CES) in a cohort of Spanish individuals with suspected HSP and to explore genotype-phenotype correlations. METHODS:A total of 139 non-related Spanish individuals with HSP underwent standardized clinical evaluation and CES. Genetic analyses were performed using a virtual panel containing 129 HSP-associated genes, complemented by phenotype-driven filtering through the Human Phenotype Ontology. Statistical analyses were performed on core clinical and paraclinical features. RESULTS:After clinical review, 108 index cases were included. Male patients were slightly more represented and mean age at onset was 33 years. Pure HSP forms were more prevalent. The most frequent presenting symptoms were gait disturbance and recurrent falls. A genetic diagnosis was achieved in 57 patients (52,8%), with SPAST and SPG7 being the most frequently mutated genes. In total, pathogenic/likely pathogenic variants were identified across 21 genes, including 8 novel variants. HSP with autosomal recessive inheritance was more common than autosomal dominant (29 vs. 25 cases), while dominant/recessive X-linked disease forms were rare (3 cases). CONCLUSIONS:CES combined with HPO-based filtering is an effective strategy for achieving genetic diagnosis in patients with suspicion of HSP.

Anoctamin-5 gene (ANO5)-related myopathies are clinically heterogeneous, and predictors of progression remain poorly defined. This study aims to characterize their long-term clinical and radiological evolution and identify factors influencing disease severity. We conducted a retrospective observational study of patients with anoctaminopathies in the Valencian Community (Spain). Demographic, clinical, genetic, ancillary tests, and serial muscle MRI data were analyzed using univariate and Bayesian multivariate models. Thirty patients (21 males) from 23 families were included. Median disease duration was 13.5 years, and follow-up was 7 years (range 3–31). Median age at first assessment was 43.5 years (range 9–78). Most patients presented with pseudometabolic myopathy (47

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic disruption of the Survival Motor Neuron 1 (SMN1) gene. Accurate quantification of Survival Motor Neuron 2 (SMN2) copy number is essential for patient stratification, prognosis, and treatment decisions, yet remains challenging at high copy numbers and does not fully explain phenotypic variability. We compared complementary molecular approaches in 78 Spanish patients with genetically confirmed SMA. Multiplex ligation-dependent probe amplification (MLPA) and digital PCR (dPCR) showed complete concordance, supporting their reliability for SMN2 quantification. In contrast, the AmplideX PCR/CE SMN1/2 Plus Kit showed discrepancies in seven patients, mainly at clinically relevant thresholds (three vs. four SMN2 copies), indicating higher sensitivity to technical variability. Notably, dPCR resolved a case with >5 SMN2 copies, demonstrating superior resolution. Long-read sequencing (LRS) in ten patients with MLPA-detected rearrangements enabled high-resolution reconstruction of the SMN locus and revealed six previously undescribed SMN hybrid structures. Structural haplotype architecture, rather than copy number alone, may further determine SMN variability and contribute to phenotype heterogeneity. Overall, our findings support an integrated diagnostic strategy in which copy-number quantification is complemented by structural characterisation in complex cases, improving molecular resolution of the SMN locus.

BACKGROUND AND OBJECTIVE:Primary ciliary dyskinesia (PCD) is a rare genetic condition characterised by abnormal ciliary motility, primarily affecting the respiratory tract. Despite its clinical significance, there is currently no gold standard for PCD diagnosis. This study aims to address this diagnostic challenge by evaluating a comprehensive approach in a large cohort of patients with suspected PCD. METHODS:We conducted a retrospective analysis of 128 patients with suspected PCD at a specialised clinical reference unit. A thorough anamnesis was performed, followed by a triad of diagnostic tests: (i) a high-speed video analysis of ciliary beat pattern; (ii) transmission electron microscopy for ciliary ultrastructure examination; and (iii) a genetic analysis, primarily through clinical exome sequencing. Correlations between the clinical, morphological and genetic findings were studied. Functional assays on RNA were performed to assess new splicing variants. Pearson's chi-square test was used to compare categorical variables and comparisons of means were performed using the Student's t-test. RESULTS:A definitive PCD diagnosis was established in 72% of the studied patients. Notably, only 58% of the diagnosed cases showed positive results across all three diagnostic tests. Patients with immotile cilia have a higher frequency of neonatal respiratory distress and had a higher likelihood of receiving a genetic diagnosis. A high-speed video analysis was altered in 116 patients, 53 of them with immotile cilia. A transmission electron microscopy revealed ultrastructural alterations in 67 patients, with class 1 defects being more common. DNAH5, RSPH1 and DNAH11 were the most represented genes among the 18 causal genes found. Among the 71 causal genetic variants found, we highlight the overrepresentation of the c.85G>T in RSPH1 and describe the aberrant effect on RNA of the splicing variants DNAH11:c.11497-6T>G, DNAH9:c.2596-2dup, CCDC40:c.2597A>G and CCDC40:c.2832G>A. Finally, we describe a severe phenotype associated with the RSPH1 gene, contrary to previously reported data. CONCLUSIONS:This comprehensive analysis of a large cohort of patients with PCD underscores the challenges in achieving a definitive diagnosis and emphasises the need for a multi-faceted diagnostic approach. This study enhances our understanding of this rare condition, including the identification of new splicing variants and an unexpected severe phenotype associated with RSPH1, challenging previous assumptions about genotype-phenotype correlations in PCD.

BACKGROUND:Ciliopathies are characterized by the dysfunction of cilia, being inherited retinal dystrophies (IRDs) included in sensory ciliopathies. Besides, oro-facial-digital syndrome (OFD) is caused by mutations in ciliary genes, leading to dysmorphic features. Mutations in TBC1D32 were associated to retinal dystrophy and OFD, defining this form as OFD-IX. RESULTS:A clinical exome analysis performed on a patient presenting with OFD-IX and sensorineural hearing loss (SNHL) identified two variants in TBC1D32, one of which affects splicing, with its impact validated using a minigene assay. CONCLUSIONS:These results suggest that SNHL may represent a new clinical feature associated with this gene.

Distal myopathies (MPDs) are heterogeneous diseases of complex diagnosis whose prevalence and distribution in specific populations are unknown. Demographic, clinical, genetic, neurophysiological, histopathological and muscle imaging characteristics of a MPDs cohort from a neuromuscular reference center were analyzed to study their epidemiology, features, genetic distribution and factors related to diagnosis. The series included 219 patients (61

Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of the survival motor neuron 1 (SMN1) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carried out a screening test by quantitative PCR (qPCR) to amplify the exon seven of SMN1 using dried blood spot (DBS) samples. From October 2021 to August 2024, a total of 31,560 samples were tested in the Valencian Community (Spain) and 4 of them were positive for SMA, indicating an incidence of 1/7890. Genetic confirmation was performed using multiplex ligation-dependent probe amplification (MLPA) and AmplideX PCR/CE SMN1/2 Plus kit, in parallel obtaining concordant results in survival motor neuron 2 (SMN2) gene copy number. Within the first few weeks of their lives, two of the four patients detected by NBS showed signs of severe hypotonia, becoming ineligible for treatment. The other two patients were the first presymptomatic patients with two copies of SMN2 to receive treatment with Risdiplam in Spain. In order to treat positive cases in their early stages, we conclude that the official deployment of SMA newborn screening is necessary.

Intermediate CAG expansions in the gene ataxin-2 (ATXN2) are a known risk factor for ALS, but little is known about their role in FTD risk. Moreover, their contribution to the risk and phenotype of patients might vary in populations with different genetic backgrounds. The aim of this study was to assess the relationship of intermediate CAG expansions in ATXN2 with the risk and phenotype of ALS and FTD in the Spanish population. Repeat-primed PCR was performed in 620 ALS and 137 FTD patients in three referral centers in Spain to determine the exact number of CAG repeats. In our cohort, ≥27 CAG repeats in ATXN2 were associated with a higher risk of developing ALS (odds ratio [OR] = 2.666 [1.471–4.882]; p = 0.0013) but not FTD (odds ratio [OR] = 1.446 [0.558–3.574]; p = 0.44). Moreover, ALS patients with ≥27 CAG repeats in ATXN2 showed a shorter survival rate compared to those with <27 repeats (hazard ratio [HR] 1.74 [1.18, 2.56], p = 0.005), more frequent limb onset (odds ratio [OR] = 2.34 [1.093–4.936]; p = 0.028) and a family history of ALS (odds ratio [OR] = 2.538 [1.375–4.634]; p = 0.002). Intermediate CAG expansions of ≥27 repeats in ATXN2 are associated with ALS risk but not with FTD in the Spanish population. ALS patients carrying an intermediate expansion in ATXN2 show more frequent limb onset but a worse prognosis than those without expansions. In patients carrying C9orf72 expansions, the intermediate ATXN2 expansion might increase the penetrance and modify the phenotype.

Exploring non-coding regions is increasingly gaining importance in the diagnosis of inherited retinal dystrophies. Deep-intronic variants causing aberrant splicing have been identified, prompting the development of antisense oligonucleotides (ASOs) to modulate splicing. We performed a screening of five previously described USH2A deep-intronic variants among USH2A monoallelic patients with Usher syndrome (USH) or isolated retinitis pigmentosa. Sequencing of entire USH2A or USH genes was then conducted in unresolved or newly monoallelic cases. The splicing impact of identified variants was assessed using minigene assays, and ASOs were designed to correct splicing. The screening allowed to diagnose 30.95% of the studied patients. The sequencing of USH genes revealed 16 new variants predicted to affect splicing, with four confirmed to affect splicing through minigene assays. Two of them were unreported deep-intronic variants and predicted to include a pseudoexon in the pre-mRNA, and the other two could alter a regulatory cis-element. ASOs designed for three USH2A deep-intronic variants successfully redirected splicing in vitro. Our study demonstrates the improvement in genetic characterization of IRDs when analyzing non-coding regions, highlighting that deep-intronic variants significantly contribute to USH2A pathogenicity. Furthermore, successful splicing modulation through ASOs highlights their therapeutic potential for patients carrying deep-intronic variants.

Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of survival motor neuron 1 (SMN1) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carried out a screening test by quantitative PCR (qPCR) to amplify the exon seven of SMN1 using dried blood spot (DBS) samples. From October 2021 to August 2024 a total of 31,560 samples were tested and four of them were positive for SMA, indicating an incidence of 1/7,890. Genetic diagnosis was performed using multiplex ligation-dependent probe amplification (MLPA) and AmplideX PCR/CE SMN1/2 Plus kit in parallel obtaining concordant results in survival motor neuron 2 (SMN2) gene copy number. Within the first few weeks of their lives, two of the four patients detected by NBS showed signs of severe hypotonia, turning out to be not eligible for treatment. The other two patients were the first presymptomatic patients with two copies of SMN2 to get treatment with Risdiplam in Spain. In order to treat positive cases in early stages, we conclude that the official deployment of SMA newborn screening is necessary.

BACKGROUND:Spinocerebellar ataxia type 8 (SCA8) is a dominantly inherited expansion disorder with highly variable penetrance. ATXN8OS/ATXN8 expanded alleles have been identified in association with other types of hereditary ataxias, pointing to a possible genetic synergism. OBJECTIVES:We aimed to further investigate the molecular background of patients with SCA8 diagnosis. METHODS:Patients were selected from our cohort of 346 families. A total of 14 probands with SCA8 underwent additional investigation through exome sequencing. RESULTS:Pathogenic heterozygous STUB1 variants were found in 21.4% of SCA8 patients (3 of 14) compared to only 0.5% in the non-SCA8 group (1 of 222), indicating a statistically significant association (P < 0.05). CONCLUSIONS:The findings reported in this study might suggest a genetic synergism between STUB1 and ATXN8OS/ATXN8 expanded alleles. Further studies are needed to validate this observation and better define the clinical impact of this genetic interaction.

Autosomal dominant spinocerebellar ataxia 36 (SCA36) is caused by hexanucleotide repeat expansion in the NOP56 gene.