Hemophilia A, an X-linked bleeding disorder caused by pathogenic variants in the F8 gene, requires precise genetic diagnosis for optimal management. Conventional stepwise sequence and copy number variation (CNV) analyses are time-consuming and may leave some cases unresolved. To evaluate the effectiveness of combining next-generation sequencing (NGS)-based sequence and CNV analysis in a single step for resolving previously undiagnosed Hemophilia A cases and identifying differential diagnoses. A cohort of 578 male patients with Factor VIII deficiency was assessed. Initial analyses included intron 22/intron 1 inversion testing and/or F8 sequencing. Twenty-seven unresolved cases underwent a targeted 57-gene coagulopathy panel using NGS with CNV analysis; CNVs were confirmed by multiplex ligation-dependent probe amplification (MLPA). The integrated NGS approach resolved 19 of 27 unresolved cases, increasing the overall diagnostic yield from 95.3% to 98.6%. Ten CNVs in F8 (8 deletions, 2 duplications) were identified and confirmed by MLPA. Variants in VWF and LMAN1 genes led to diagnoses of von Willebrand disease (n = 6) and combined Factor V/VIII deficiency (n = 1). One F8 intronic variant was detected, while 8 cases remained unresolved by the NGS-based approach. Integrating NGS-based CNV analysis into genetic diagnostic workflows enables simultaneous detection of sequence variants and CNVs, improves diagnostic yield, and facilitates identification of related bleeding disorders. This streamlined approach supports adoption as a first-line test for mild/moderate Hemophilia A and a secondary test for severe cases.

APC is the most frequently mutated gene in familial adenomatous polyposis (FAP). Recent advancements in sequencing technologies and the introduction of the 2024 APC-specific ACMG/AMP variant classification guidelines have reshaped the diagnostic landscape for hereditary polyposis syndromes. We retrospectively reviewed APC variant data from 425 Korean patients clinically suspected of FAP between 2006 and 2020. Genetic testing included direct sequencing, targeted next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and mutant enrichment with 3'-modified oligonucleotide PCR. Variants were reclassified using the 2024 APC-specific ACMG/AMP criteria. Pathogenic or likely pathogenic APC variants were detected in 166 of 425 patients (39.1%). Twenty-one novel truncating mutations were identified. The application of the 2024 APC-specific ACMG/AMP criteria led to the reclassification of 13 variants, with 2 pathogenic or likely pathogenic and 7 likely benign. Nine patients showed evidence of APC mosaicism. This is the first large-scale study to evaluate the clinical utility of the 2024 APC-specific ACMG/AMP variant interpretation guidelines, enabling the reclassification of previously designated variants of uncertain significance into more definitive categories. The detection of mosaic variants emphasizes the need for higher sequencing depth. Our results support the integration of NGS and further diagnostic evaluation into APC variant testing.

PMP22 duplications cause Charcot-Marie-Tooth disease (CMT), whereas PMP22 deletion results in hereditary neuropathy with liability to pressure palsies (HNPP). Although PMP22 point mutations can cause a spectrum of neuropathies, including hereditary motor and sensory neuropathy (HMSN) I, congenital hypomyelinating neuropathy, HMSN III, and HNPP, they are rare, and their features in Japanese patients have not been well characterized. Therefore, we compared the genetic, clinical, and electrophysiological characteristics of patients with PMP22 point mutations. This multicenter study enrolled 3352 Japanese patients clinically suspected of having inherited peripheral neuropathies/CMT who underwent fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, gene panel sequencing, or whole-exome analysis between 1995 and 2024. Clinical and electrophysiological data were obtained from patients' medical records. The study enrolled 30 patients with PMP22 point mutations and 57 patients with PMP22 duplications. Among the point mutation cases, 24 distinct variants were detected, including four novel variants (p.T23K, p.R95Sfs*16, p.M111R, and p.P144R). Two splice-site variants (c.78 + 3G > T and c.79-2A > G) were associated with HNPP, whereas the others were associated with polyneuropathy. Compared with patients with PMP22 duplications, those with point mutations experienced earlier disease onset (0 vs. 35.0years), were less likely to have a family history (14.3% vs. 61.7%), and were less frequently ambulatory (12/28 vs. 50/54). Upper limb compound muscle action potentials were more frequently undetectable in patients with point mutations (18/25 vs. 1/50). PMP22 point mutations-associated polyneuropathies were characterized by earlier disease onset and more severe effects than duplications, highlighting the broad clinical spectrum of PMP22-related neuropathies.

Third-generation sequencing (TGS) enables comprehensive detection of rare hemoglobin (Hb) variants due to its long-read capability. However, library preparation is often labor-intensive and time-consuming. This study developed a novel one-step library preparation (OSLP) method for TGS. By integrating barcode sequences into target-specific primers, the approach enables immediate barcoding on amplification, streamlining identification of hemoglobinopathy variants. A retrospective set of DNA samples (n = 455; 394 adult, 61 prenatal) underwent OSLP targeting of the HBA2/1, HBG2/1, HBD, and HBB genes on the Qitan Nanopore platform for variant and structural variation detection. Samples were divided into validation and application cohorts based on prior testing with multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing alongside Gap-polymerase chain reaction (GAP-PCR)/reverse dot blot (RDB). In the validation cohort, OSLP-TGS showed 100% concordance with routine methods (MLPA, Sanger, GAP-PCR, RDB), including 294 samples for α-globin genotyping [8 structural variants (SVs), 17 single nucleotide variants (SNVs)/insertions-deletions (indels)], 138 for β-globin genotyping (4 SVs, 22 SNVs/indels), and 61 prenatal samples for combined genotyping. The method directly spanned breakpoints and determined cis/trans configurations. In the application cohort, OSLP-TGS-Nanopore identified additional variants not covered by routine genotyping: 9 extra α-globin SNVs in 16/100 samples and 8 extra β-globin SNVs in 33/256 samples. Notably, 3 samples with the -SEA deletion harbored co-inherited novel variants, explaining their disproportionately low Hb A2 levels. The OSLP-TGS-Nanopore assay provides an efficient, comprehensive solution for detecting rare hemoglobinopathy variants, with a framework extendable to population screening for diverse genetic disorders.

Signal transduction and transcription activation5(STAT5) drives leukemic cell survival and treatment resistance in acute lymphoblastic leukemia (ALL), linking its activation to poor prognosis and highlighting it as an important biomarker and therapeutic target. We hypothesized that integrating STAT activation, cytokine receptor-like factor2 (CRLF2) overexpression, and copy number alterations (CNAs) affecting B-cell differentiation, cell-cycle control, and proliferation could define a high-risk ALL subtype, possibly including Philadelphia (Ph) like ALL. The study included 115 precursor-B-ALL patients, comprising 88 (76.5%) children and 27 (23.5%) adults, 66 males (57.4%) and 49 females (42.6%). Expressions of p-STAT3, p-STAT5, and CRLF2 were assessed by flow cytometry. Forty patients, including 29 with positive marker expression, of whom 18 lacked any of the recurrent genetic translocations, were further tested for CNAs by Multiplex Ligation-dependent Probe Amplification (MLPA). p-STAT3, p-STAT5, and/or CRLF2 were expressed in 36 (31.3%) patients. p-STAT3 was positive in 6 (5.2%) patients, p-STAT5 in 27 (23.4%) patients, and CRLF2 overexpression was detected in 15 (13.04%) patients. Thirty/forty patients (75%) had one or more CNAs abnormalities, including deletions or duplications. p-STAT5 was detected in 6/7 (85.7%) patients with IKZF1 deletion. Higher day 42 MRD was significantly associated with IKZF1 deletion (p = 0.04). Adult patients with positive p-STAT3, p-STAT5, and/or CRLF2 overexpression had shorter overall survival (P < 0.01) and disease-free survival (P < 0.001). The expression of p-STAT5 and/or CRLF2 is probably associated with dismal prognosis in adults. A combined assessment of CRLF2, p-STAT5, and CNAs might identify a high-risk subset of ALL, potentially encompassing Ph-like ALL.

The evaluation of Copy Number Variants in an unselected population of patients with Inherited Cardiac Conditions: The INTERACTION Study.

Objective: To analyze the genetic and clinical characteristics of patients with PAX6-associated ocular diseases (PAOD). Methods: This retrospective case series study recruited 40 PAOD patients from 26 families at Peking Union Medical College Hospital between January 2015 and March 2025. Medical history and ophthalmic examination results were collected, including visual acuity, color fundus photography, fundus autofluorescence, optical coherence tomography (OCT), and B-scan ultrasonography. Peripheral blood samples were obtained from patients and their family members for DNA extraction. The PAX6 pathogenic variants were detected using next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and quantitative real-time quantitative PCR (qPCR), followed by segregation analysis in pedigrees. Results: A total of 40 patients from 26 unrelated families were identified, with an equal distribution of 20 males and 20 females. The age at first visit ranged from 0.5 to 59.0 years (median: 25.5 years). Best corrected visual acuity ranged from no light perception to 0.4 (median: 0.1). Among the cases, 23 patients were diagnosed with congenital aniridia, while 17 patients had non-aniridia phenotypes, including anterior segment dysgenesis, Peters anomaly, and foveal hypoplasia. Foveal hypoplasia was a common clinical manifestation (100%, 23/23) in PAOD patients, with other common manifestations including nystagmus (94.9%, 37/39), cataract (80.0%, 28/35), and glaucoma (59.3%, 16/27). Twenty-five PAX6 pathogenic variants were identified, among which 9 novel variants were reported in this study, including 3 missense mutations, 3 large chromosomal rearrangements, 1 frameshift mutation, 1 splicing variant, and 1 small deletion. Truncating mutations (68.7%) were the most common variant type in aniridia patients, whereas missense mutations (70.0%) predominated in other PAX6-associated ocular diseases, showing a significant difference (P<0.001). Conclusion: PAOD exhibits high phenotypic heterogeneity, with foveal hypoplasia as a common feature and congenital aniridia being the most prevalent phenotype. Nystagmus combined with multiple ocular structural abnormalities are key clinical features suggestive of PAOD. This study expands the clinical and genetic spectrum of PAOD and provides further insights into genotype-phenotype correlations.

Kyphoscoliotic Ehlers-Danlos syndrome (kEDS, OMIM: #225400) is a rare subtype of Ehlers-Danlos syndrome characterized by joint hypermobility and spinal deformity, with occasional vascular complications. The rarity of vascular phenotypes in kEDS often leads to low clinical suspicion, contributing to delayed diagnosis and poor outcomes. We report a 13-year-old boy who presented with joint laxity, scoliosis, blue sclerae, and pectus excavatum, and who ultimately succumbed to aortic rupture. Initial whole-exome sequencing (WES) and copy number variation (CNV) analysis failed to identify a causative variant, and genes associated with Marfan syndrome were excluded by WES and multiplex ligation-dependent probe amplification (MLPA). Upon re-analysis of the WES data, however, a homozygous deletion spanning exons 15-16 of PLOD1 was detected and subsequently confirmed by quantitative PCR. This case demonstrates that small exon deletions in PLOD1 may not be reliably detected by routine WES/CNV pipelines, leading to diagnostic delay, and underscores the underrecognized vascular risk in kEDS. Clinicians should maintain vigilance for vascular complications in patients with kEDS and consider re-analysis strategies to ensure timely diagnosis and appropriate genetic counseling.

Objective: To investigate the genetic epidemiological characteristics of spinal muscular atrophy (SMA) among newborns in Foshan, and to evaluate the efficacy and clinical value of early disease-modifying therapy for affected infants. Methods: A retrospective cohort study was conducted on 132 850 newborns and 5 confinned SMA patients who underwent SMA genetic screening at the Department of Foshan Newborn Disease Screening Center, Foshan Women and Children Hospital from June 2020 to October 2025. Clinical data were collected of the 132 850 newborns and 5 confirmed SMA patients, including general characteristics, validation results via multiplex ligation-dependent probe amplification (MLPA), disease-modifying treatments, and motor function assessments. The carrier rate of survival motor neuron 1 (SMN1) gene exon 7 deletion and the incidence of SMA in Foshan were determined. Changes in muscle strength and motor milestones of confirmed SMA patients before and after early disease-modifying therapy were analyzed. Results: Of the 132 850 screened newborns, 2 378 cases were identified with heterozygous deletion of SMN1 exon 7, and 5 cases with homozygous deletion of SMN1 exon 7 (confirmed as SMA patients via MLPA). The carrier rate of SMN1 exon 7 deletion in Foshan newborns was 1/56 (2 383/132 850), and the SMA incidence (homozygous deletion of SMN1 exon 7) was 1/26 570 (5/132 850). Among the 5 diagnosed patients, 4 had 2 copies of SMN2 exon 7. One patient (case 1) did not receive disease-modifying therapy and died at 2 months of age, while 3\ other patients (case 2, 3, 4) all received disease-modifying therapy before 1.5 months of age. After treatment, their muscle strength improved, and they achieved the motor milestones of walking with assistance, sitting with support, and standing with support. Among the 5 confirmed patients, case 5 had 3 copies of SMN2 exon 7 and received disease-modifying therapy at 1.1 months of age. No obvious muscle strength abnormality was observed before or after treatment, and this patient achieved independent walking at 13 months of age. Conclusions: The carrier rate of SMN1 gene exon 7 deletion among newborns in Foshan is low, and the incidence of SMA (homozygous deletion type of SMN1) is also low. Early newborn screening for SMA, along with early diagnosis and treatment, can effectively improve the motor function in affected infants.

To investigate the molecular cause of choroideremia in two unrelated patients with no detectable mutations in the CHM gene. Two unrelated patients were examined by an ophthalmologist to obtain a clinical diagnosis. Patient and control cells were cultured and used as a source of DNA, RNA, and protein for analysis. Exonic regions of CHM were Sanger sequenced and copy number analysis was performed by multiplex ligation-dependent probe amplification. mRNA transcripts were analyzed by Sanger sequencing from synthesized cDNA. Protein expression was probed with western blot analysis. Suspecting an inversion, PCR in one case and inverse PCR in the second case were employed to locate the precise DNA breakpoints. Patient 1 and 2 were clinically diagnosed with choroideremia by experienced ophthalmologists. In both cases, sequencing of the promoter and coding regions of the CHM gene revealed no mutation and copy number analysis of the gene did not detect the presence of any large deletions or duplications. RNA obtained from the patient cells showed only a partial transcript in patient 1, and an abnormal CHM splice isoform in patient 2. The results from RNA and DNA analyses suggested that both patients' genomic DNA might contain an inversion mutation. In patient 1, a long-range PCR product amplified over two breakpoints confirmed an inversion event. This 6 kb inversion spanned from the 5'UTR to the first intron (NM_000390.4(CHM): c.[-836_-826del; 49 + 5526_49 + 5856del; -825_49 + 5525inv; insCGTCT].). In patient 2, inverse PCR revealed a different novel inversion of approximately 85 kb, spanning from intron 2 to intron 8 (NM_000390.4(CHM):c.116 + 6659_1166 + 20670inv). To detect these two inversions in future choroideremia samples, multiplex PCR assays were developed that produce distinct banding patterns that are diagnostic for these two mutations. We have identified inversion mutations in the CHM gene resulting in choroideremia. Though uncommon, inversions should be investigated as a possible cause of the disease in CHM patients, especially for those in whom no point mutations or copy number variants are found.

Hereditary spastic paraplegia (HSP) is a genetically neurodegenerative disorder with limited epidemiological data in Korea. This study aimed to characterize the genetic landscape of HSP in a large Korean cohort. We analyzed 657 patients with suspected HSP using Sanger sequencing and a targeted next-generation sequencing (NGS) panel covering 54 HSP-related genes. Variants were classified according to American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, and copy number variations (CNVs) were detected using in-house methods and multiplex ligation-dependent probe amplification. Pathogenic or likely pathogenic variants were identified in 121 patients (18%), with SPAST (83%) and ATL1 (6%) being the most frequently mutated genes. Notably, 5% of SPAST mutations were CNVs, underscoring the importance of CNV detection. Among the identified variants, 23 novel mutations were discovered, primarily in SPAST (19), REEP1 (3), and SACS (1), expanding the mutational spectrum of HSP. The diagnostic yield was higher with NGS (25%) compared to Sanger sequencing (16%), reflecting the panel's broader gene coverage. This study highlights the predominance of SPAST and ATL1 mutations in Korean HSP patients and emphasizes the utility of targeted NGS panels, particularly for detecting CNVs and novel variants. Despite these advances, the majority of cases remain unresolved, suggesting the need for broader sequencing approaches to uncover additional genetic factors. These findings provide a foundation for improved diagnostic strategies and personalized treatment in HSP.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder in which presymptomatic treatment substantially improves survival and motor outcomes, yet newborn screening for SMA remains unevenly implemented across Europe, and evidence from lower-resource health systems is needed to guide scale-up. In this study, we assessed the feasibility, diagnostic performance, and public health implications of implementing neonatal SMA screening in the Republic of Moldova within an established national newborn screening framework. A pilot genetic screening program was conducted using dried blood spot (DBS) samples collected through routine newborn screening workflows; SMN1 exon 7 deletion testing was performed by real-time polymerase chain reaction (qPCR), and positive findings were confirmed by multiplex ligation-dependent probe amplification (MLPA), alongside the evaluation of operational integration and system-level requirements. Screening was operationally feasible within existing DBS processes and demonstrated high analytical performance, consistent with published international experience, although performance results should be interpreted cautiously due to the limited sample size. Two SMA cases were confirmed in a small cohort, enabling early diagnosis and timely referral for disease-modifying therapy, and integration into the existing program was practical and resource-efficient. These findings support the incorporation of SMA into national newborn screening panels using DBS-based molecular methods, highlighting an implementable model for introducing advanced genetic testing within routine public health services.

Unmasking the genetic heterogeneity of cystic fibrosis in the Torbat Jam region: From common variants to large deletions and the challenge of missing heritability.

ABSTRACTResearch ObjectiveYoung‐onset Parkinson's disease (YOPD), defined by symptom onset at or before 50 years of age, has a strong genetic component. The mutation spectra vary markedly across ethnic groups. However, YOPD among the Hakka, a subgroup of the Han Chinese ethnicity, remains uncharacterized. We investigated the genetic and clinical profiles of YOPD in the Hakka population of western Fujian.Materials and MethodsA total of 33 unrelated patients with YOPD were included in the study. All patients underwent whole exome sequencing (WES) to screen all known Parkinson's disease (PD)‐related genes. Patients with a family history also received a spinocerebellar ataxia (SCA) gene panel test. If the SCA gene panel test result was negative, multiplex ligation‐dependent probe amplification (MLPA) for eight genes including DJ‐1, ATP13A2, PINK1, UCHL1, SNCA, LRRK2, PRKN, and GCH1 was performed. Potential pathogenic variants were confirmed by Sanger sequencing, and both the genetic spectrum and clinical characteristics of patients with YOPD were analyzed.ResultsAfter variant filtering, six variants in four YOPD‐related genes were identified in four unrelated patients. Of these patients, two harbored pathogenic ATXN2 repeat expansions. Four variants in VPS13C and PRKN, along with an SNCA exon 1–6 duplication, were classified as variants of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) criteria. A considerable proportion of patients harbored risk variants in LRRK2. Furthermore, nine unrelated patients harbored nine variants within six susceptibility genes associated with YOPD, including EIF4G1, COQ2, TENM4, NR4A2, UQCRC1, and GBA1.ConclusionThis study is the first to analyze the genetic spectrum and clinical characteristics of patients with YOPD in the Hakka population of western Fujian Province. Genetic testing of known pathogenic genes in patients with YOPD can facilitate more accurate diagnosis.

Birt–Hogg–Dubé syndrome (BHDS) is a hereditary cancer syndrome caused by pathogenic variants in the FLCN gene. BHDS is characterized by clinical heterogeneity and similarities with other non-hereditary diseases, which can complicate diagnosis. The aim of our study was to analyze FLCN variants in Russian patients and select the optimal diagnostic approach. We studied 121 unrelated patients suspected for BHDS and 29 of their relatives. Germline variants were analyzed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Variant annotation was performed according to the ACMG and AMP recommendations. Pathogenic and likely pathogenic (P/LP) FLCN variants were identified in 20.7% of patients, including six new variants. The distribution of FLCN variants in our cohort was consistent with data obtained from other authors. The mean age of patients with P/LP variants was higher than of those without: 46.91 versus 33.8 years (p < 0.05), suggesting the necessity to apply diagnostic criteria in young patients more carefully. The most common clinical manifestation of BHDS was pulmonary cysts/pneumothorax, while the most informative were alterations involving at least two of three organ systems, which was present in all patients with the P/LP variants, but only in 54% without them (p = 0.001). BHDS diagnostics involves sequencing exons 4–14 of the FLCN gene in patients with proposed clinical criteria. If the result is negative, extensive FLCN deletions are excluded using MLPA, and, in the absence of CNV, WGS is performed.

Atypical hemolytic uremic syndrome (aHUS) is a rare, complement-mediated disorder. Disease-associated variants have been found in complement factor H-related genes and complement regulatory genes. Particularly, homozygous deletions of CFHR1, CFHR3, or CFHR4 have been associated with aHUS. Generally, multiplex ligation-dependent probe amplification (MLPA) is used to detect copy number changes in the CFH and CFHR regions. We have developed a cost-effective droplet digital PCR (ddPCR) method for measuring copy number variations (CNVs) in these genes. A total of 41 DNA samples were analyzed using both ddPCR and MLPA to assess large deletions in the CFHR1, CFHR3, CFHR4 regions. The ddPCR primers were designed to avoid rare single nucleotide polymorphisms to enhance hybridization efficiency and accurate identification of deletions within the CFHR regions. The MLPA probe mix (Probemix P236 CFH Region) targeting CFHR regions was purchased from MRC Holland. Multiple heterozygous deletions were observed in the CFHR1, CFHR3, and CFHR4 genes. Homozygous deletions of CFHR1 and CFHR3 were seen in 2 cases, while 2 other cases showed homozygous CFHR1 and heterozygous CFHR3 deletions. All 41 samples demonstrated 100% concordance in copy number results between ddPCR and MLPA, as well as consistent reproducibility in both inter- and intratechnologist evaluations. Our findings demonstrate that ddPCR is a robust platform to identify large deletions in the CFHR regions. Future studies are needed to evaluate its ability to distinguish full vs partial deletions and to further establish its clinical diagnostic value.

Homozygous deletions of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) are recognized as poor prognostic markers in newly diagnosed IDH-mutant astrocytomas. However, a standardized definition of CDKN2A/B homozygous deletion has not yet been established. Furthermore, the prognostic significance of CDKN2A/B homozygous and hemizygous deletions at recurrence remains unclear. In this study, we investigated the prognostic impact of CDKN2A/B homozygous and hemizygous deletions in patients with recurrent IDH-mutant astrocytomas. We conducted a retrospective review of 32 patients treated at our institution between January 2006 and March 2023. CDKN2A/B homozygous and hemizygous deletions were defined as relative CDKN2A/B copy numbers of < 0.4 and < 0.7, respectively, as determined by multiplex ligation-dependent probe amplification. Univariate analysis demonstrated that both homozygous and hemizygous deletions at first recurrence were associated with shorter post-recurrence survival. Multivariable analysis identified both deletion types and histological grade at first recurrence as independent prognostic factors. Risk stratification based on histological grade and CDKN2A/B status effectively predicted survival outcomes following recurrence. In conclusion, CDKN2A/B status, alongside histological grading, represents an independent prognostic indicator in recurrent IDH-mutant astrocytoma.

This study aimed to analyze the most established genetic causes of Parkinson's disease (PD) in a cohort of patients from Czechia. PD is the second most common neurodegenerative disorder, with both genetic and environmental factors contributing to its pathogenesis. We examined 214 patients with PD and 186 controls. Each patient underwent an examination using the Montreal Cognitive Assessment to assess the severity of cognitive impairment. Genetic analysis was performed using next- -generation sequencing and multiplex ligation-dependent probe amplification, focusing on mutations in GBA1, LRRK2, PINK1, PRKN, and SNCA genes. Clinically relevant mutations in GBA1 (pathogenic/likely pathogenic or severe/mild/risk variants) were the most frequently observed variants (n = 30; 14.0% of the PD cohort in total), including both known pathogenic mutations and the mild risk allele p.Glu365Lys (n = 12; 5.6% of cases), which was associated with earlier disease onset. Pathogenic mutations were also detected in LRRK2 (n = 2; 0.9%), SNCA (n = 1; 0.5%), and compound heterozygous mutations in PRKN (n = 2; 0.9%). Carriers of heterozygous mutations in PRKN were equally represented in patients and controls. No pathogenic PINK1 mutations were identified. Cognitive impairment was not significantly more frequent in GBA1 mutation carriers (p = 0.320). A positive family history of PD was more common in patients than in healthy controls (n = 51; 24% vs. n = 6; 3%, p < 0.001). Our findings highlight notable genetic variability in Czech PD patients, particularly involving GBA1 mutations. However, these variants were not associated with early cognitive decline. The study underscores the value of genetic screening in PD and the need for further research into genotype-phenotype correlations.

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.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and genomic profiling has become increasingly vital for risk stratification. We conducted a retrospective single-center study including 96 newly diagnosed pediatric patients with ALL and mixed phenotype acute leukemia to characterize the genomic landscape using conventional and molecular cytogenetics, multiplex ligation-dependent probe amplification and targeted next-generation sequencing and to evaluate associations with treatment response and survival. Mutation co-occurrence analysis revealed a NRAS/KRAS/JAK cluster and an ETV6::RUNX1 cluster characterized by relative mutational exclusivity, with additional associations between NOTCH1–CDK4 and CDK4–CCND3 alterations. DelCDKN2A/CDKN2B alterations showed a trend toward higher day-15 minimal residual disease (MRD) levels and inferior 2-year event free survival rate (EFS). DelIKZF1-positive cases showed lower 2-year EFS. In contrast, ETV6::RUNX1 and high hyperdiploidy were associated with favorable early response and EFS. DelPAX5 did not independently influence outcome. ZNF384 positive cases showed higher early MRD levels despite excellent survival outcomes. NRAS/KRAS mutations were significantly associated with higher positive day-15 MRD (Wilcoxon, p = 0.0067) and remained independently associated after adjustment for white blood cell count and cytogenetic subgroup. Intermediate risk (IR) and high-risk groups showed comparable 2-year EFS, indicating limited discrimination by conventional risk stratification. The IR group displayed a heterogeneous genomic profile, with NRAS/KRAS, Ph-like mutations and delCDKN2A/CDKN2B among the most frequent alterations. These observations highlight the potential of integrated genomic profiling to refine risk stratification, particularly by identifying clinically relevant subgroups within the IR category.