Abstract Background Pathogenic variants in AGO2 , encoding a central component of the RNA-induced silencing complex (RISC), cause the neurodevelopmental disorder Lessel-Kreienkamp syndrome (LESKRES). The variant spectrum and associated molecular mechanisms underlying phenotypic variability and disease severity remain incompletely understood. Methods We investigated 45 newly identified individuals carrying 33 distinct AGO2 variants, 30 of which were previously unreported. Phenotypic data from these and previously reported cases ( n = 70) were integrated to delineate the LESKRES-associated clinical spectrum and genotype–phenotype correlations. Functional studies included shRNA-based silencing, co-immunoprecipitation, subcellular localization, and sequencing of AGO2-bound miRNAs. Results All individuals presented with a neurodevelopmental disorder of variable severity. Delayed speech and language development (97%), intellectual disability (97%), and motor delay (93%) were the most consistent features, frequently accompanied by muscular hypotonia, autistic traits, attention deficit hyperactivity disorder, visual impairment and structural brain anomalies. Systemic manifestations, including skeletal, craniofacial, cardiac, and male urogenital anomalies were common, underscoring AGO2’s multisystemic role. Moreover, we report occurrence of gonadal mosaicism and reveal the presence of interfamilial and variant-specific clinical heterogeneity. Variants clustered in defined regions of AGO2, including the L1 loop, helix-7, and multiple loops of the PIWI domain, highlight structural hotspots critical for RISC activity. Not all pathogenic variants impaired shRNA-mediated silencing; this was restricted to p.(Arg714Trp) and p.(Asn729His). Biochemical analyses revealed that p.(Asp619Asn) impaired GW182 binding and P-body assembly. Variants p.(Arg506Gln), p.(Glu531Gln) p.(Gly604Arg) and p.(Asp619Asn), reduced C-terminal phosphorylation, implicating defective AGO2 recycling. AGO2–miRNA co-immunoprecipitation and sequencing demonstrated variant-specific perturbations in miRNA association, strand selectivity, and isomiR generation. Variants near the hinge of the helix-7 region, especially p.(Phe182del), induced extensive changes in miRNA association and 3′-end modification, suggesting impaired anchoring within the miRNA-binding pocket. Conclusions Our findings substantially broaden the clinical and molecular landscape of LESKRES, establishing AGO2 as a pivotal regulator of neurodevelopment whose structural integrity is essential for precise miRNA-mediated gene regulation. Pathogenic variants disrupt distinct interconnected processes: P-body association, phosphorylation-dependent turnover, and miRNA interactions, culminating in dysregulated post-transcriptional gene silencing. These mechanistic insights link specific structural perturbations in AGO2 to graded clinical outcomes and underscore the critical role of AGO2 conformational dynamics in human neurodevelopment.

IntroductionEpilepsy is a common central nervous system disorder characterized by abnormal brain electrical activity. Diagnosing epilepsy can be challenging, as it has various causes and different clinical manifestations. We aimed to analyze whether there are similarities in the metabolic profiles of patients with epilepsy despite different etiology, seizure frequency, seizure type, and patient age.MethodsUntargeted metabolomics analysis results were analyzed in three cohorts. The pediatric cohort (PED-C) consisted of 110 pediatric individuals with suspicion of a genetic disorder with unclear etiology; the adult cohort (AD-C) of 250 adults from the Estonian Biobank (EstBB), and the elderly cohort (ELD-C) of 583 adults ≥ 69 years from the EstBB. Epilepsy was diagnosed in 35, 11 and 26 cases, respectively.ResultsSignificant differences (FDR corrected p-value <0.05) were detected in the PED-C in eight metabolites, mainly for lipids. Restricting the analysis to individuals with epilepsy and without any changes in brain magnetic resonance imaging increased the number of significant metabolites to 16. Nine significantly altered lipids were found in the AD-C, mainly triacylglycerides (TAGs). In the ELD-C, significant changes in 20 metabolites, including multiple TAGs, were observed in the metabolic profile of participants with previously diagnosed epilepsy.DiscussionSeveral components of cell membranes were among the altered metabolites, indicating that cell membrane fluidity may have a significant role in the mechanism of epilepsy, and changes in lipid balance may indicate epilepsy. However, further studies are needed to evaluate whether untargeted metabolomics analysis could prove helpful in diagnosing epilepsy earlier.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.

BACKGROUND:Pathogenic variants in AXIN2 have been associated with tooth agenesis, colon polyps, and colon cancer. Given the rare nature of this phenotype, we set out to collect additional genotypic and phenotypic information.METHODS:Data were collected via a structured questionnaire. Sequencing was performed in these patients mostly due to diagnostic purpose. A little more than half of the AXIN2 variant carriers were identified by NGS; other six were family members.RESULTS:Here, we report 13 individuals with a heterozygous AXIN2 pathogenic/likely pathogenic variant who have a variable expression of oligodontia-colorectal cancer syndrome (OMIM 608615) or oligodontia-cancer predisposition syndrome (ORPHA 300576). Three individuals from one family also had cleft palate, which might represent a new clinical feature of AXIN2 phenotype, also given the fact that AXIN2 polymorphisms have been found in association with oral clefting in population studies. AXIN2 has already been added to multigene cancer panel tests; further research should be conducted to determine whether it should be added to cleft lip/palate multigene panels.CONCLUSION:More clarity about oligodontia-colorectal cancer syndrome, about the variable expression, and associated cancer risks is needed to improve clinical management and to establish guidelines for surveillance. We collected information about the surveillance that was advised, which might support clinical management of these patients.

OBJECTIVE:To assess the prevalence of congenital anomalies (CAs), chromosomal abnormalities and monogenic diseases among births and terminated pregnancies due to fetal anomalies (TOPFA) in 2020 in Estonia. Up to 2020 no data on prevalence of CAs in Estonia is reported.METHODS:For retrospective observational study data of all births and terminations of pregnancies after 12th gestational week from (i) the Estonian Medical Birth Registry, (ii) Abortion Registy, (iii) Health Insurance Fund and (iv) hospital records were linked. To calculate the total, live birth, stillbirth and TOPFA prevalence of CAs with 95% confidence intervals (CI), guidelines issued by EUROCAT, European network for the epidemiological surveillance of CAs, https://eu-rd-platform.jrc.ec.europa.eu/eurocat_en were followed.RESULTS:In 2020 the total prevalence of CAs, chromosomal abnormalities and monogenic diseases in Estonia was 378.6 per 10,000 births (95% CI 346.0, 413.5). The most prevalent CAs were heart defects, 163.7 cases per 10,000 births (95%CI 142.5, 187.2). The prevalence of chromosomal abnormalities and genetic diseases was 92.6 per 10,000 births (95%CI 76.8, 110.6), 80% of cases were among TOPFAs. No newborns with major aneuploidies (Trisomy 21, 18, 13, polyploidy) were reported in 2020. Live birth prevalence of CAs, including chromosomal abnormalities and genetic diseases was 258.4 per 10,000 live births (95%CI 231.5, 287.5) and stillbirth prevalence of CAs 0.8 per 10,000 births.CONCLUSIONS:The prevalence of CAs and genetic disorders in Estonia is one of the highest compared to prevalence reported by other European regions. It indicates to high population coverage with prenatal diagnostics in Estonia. Low number of major aneuploidies among live births may reflect good detection rate of major chromosomal abnormalities and cultural preferences.

Accurate diagnosis for patients living with neurodevelopmental disorders is often met with numerous challenges, related to the ambiguity of findings and lack of specificity in genetic variants leading to pathology. Genome-wide DNA methylation analysis has been used to develop highly sensitive and specific 'episignatures' as biomarkers capable of differentiating and classifying complex neurodevelopmental disorders. In this study we describe distinct episignatures for KAT6A syndrome, caused by pathogenic variants in the lysine acetyltransferase A gene (KAT6A), and for the two neurodevelopmental disorders associated with lysine acetyl transferase B (KAT6B). We demonstrate the ability of our models to differentiate between highly overlapping episignatures, increasing the ability to effectively identify and diagnose these conditions.

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WBP4 (WW Domain Binding Protein 4) is part of the early spliceosomal complex, and was not described before in the context of human pathologies. Ascertained through GeneMatcher we identified eleven patients from eight families, with a severe neurodevelopmental syndrome with variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal and gastrointestinal abnormalities. Genetic analysis revealed overall five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including enrichment for abnormalities of the nervous system and musculoskeletal system genes, suggesting that the overlapping differentially spliced genes are related to the common phenotypes of the probands. We conclude that biallelic variants in WBP4 cause a spliceosomopathy. Further functional studies are called for better understanding of the mechanism of pathogenicity.

Introduction:Epilepsy is a common central nervous system disorder characterized by abnormal brain electrical activity. We aimed to compare the metabolic profiles of plasma from patients with epilepsy across different etiologies, seizure frequency, seizure type, and patient age to try to identify common disrupted pathways.Material and methods:We used data from three separate cohorts. The first cohort (PED-C) consisted of 31 pediatric patients with suspicion of a genetic disorder with unclear etiology; the second cohort (AD-C) consisted of 250 adults from the Estonian Biobank (EstBB), and the third cohort consisted of 583 adults ≥ 69 years of age from the EstBB (ELD-C). We compared untargeted metabolomics and lipidomics data between individuals with and without epilepsy in each cohort.Results:In the PED-C, significant alterations (p-value <0.05) were detected in sixteen different glycerophosphatidylcholines (GPC), dimethylglycine and eicosanedioate (C20-DC). In the AD-C, nine significantly altered metabolites were found, mainly triacylglycerides (TAG), which are also precursors in the GPC synthesis pathway. In the ELD-C, significant changes in twenty metabolites including multiple TAGs were observed in the metabolic profile of participants with previously diagnosed epilepsy. Pathway analysis revealed that among the metabolites that differ significantly between epilepsy-positive and epilepsy-negative patients in the PED-C, the lipid superpathway (p = 3.2*10-4) and phosphatidylcholine (p = 9.3*10-8) and lysophospholipid (p = 5.9*10-3) subpathways are statistically overrepresented. Analogously, in the AD-C, the triacylglyceride subclass turned out to be statistically overrepresented (p = 8.5*10-5) with the lipid superpathway (p = 1.4*10-2). The presented p-values are FDR-corrected.Conclusion:Our results suggest that cell membrane fluidity may have a significant role in the mechanism of epilepsy, and changes in lipid balance may indicate epilepsy. However, further studies are needed to evaluate whether untargeted metabolomics analysis could prove helpful in diagnosing epilepsy earlier.

Inherited metabolic disorders (IMD) are a group of hereditary diseases wherein the impairment of a biochemical pathway is intrinsic to the pathophysiology of the disease. Estonia's small population and nationwide digitalised healthcare system make it possible to perform an epidemiological study that covers the whole population. A study was performed in Tartu University Hospital, which is the only tertiary care unit in Estonia for diagnosing patients with IMD, to define the prevalence and live birth prevalence of IMDs and the effectiveness of new diagnostic methods on the diagnosis of IMD. During the retrospective study period from 1990 to 2017, 333 patients were diagnosed with IMD. Statistical analysis showed a significant increase in IMD diagnoses per year from 0.47 to 2.51 cases per 100 000 persons (p < 0.0001) during the study period. Live birth prevalence of IMD in Estonia was calculated to be 41.52 cases per 100 000 live births. The most frequently diagnosed IMD groups were disorders of amino acid metabolism, disorders of complex molecule degradation, mitochondrial disorders, and disorders of tetrapyrrole metabolism. Phenylketonuria was the most frequently diagnosed disorder of all IMD (21.6%). Our results correlated well with data from other developed countries and, along with high birth prevalence, add confidence in the effectiveness of our diagnostic yield. Implementation of new diagnostic methods during study period may largely account for the significant increase in the number of IMD diagnoses per year. We conclude that the implementation of new diagnostic methods continues to be important and contributes to better diagnosis of rare diseases.

Abstract KBG syndrome (KBGS) is characterized by distinctive facial gestalt, short stature and variable clinical findings. With ageing, some features become more recognizable, allowing a differential diagnosis. We aimed to better characterize natural history of KBGS. In the context of a European collaborative study, we collected the largest cohort of KBGS patients (49). A combined array- based Comparative Genomic Hybridization and next generation sequencing (NGS) approach investigated both genomic Copy Number Variants and SNVs. Intellectual disability (ID) (82%) ranged from mild to moderate with severe ID identified in two patients. Epilepsy was present in 26.5%. Short stature was consistent over time, while occipitofrontal circumference (median value: −0.88 SD at birth) normalized over years. Cerebral anomalies, were identified in 56% of patients and thus represented the second most relevant clinical feature reinforcing clinical suspicion in the paediatric age when short stature and vertebral/dental anomalies are vague. Macrodontia, oligodontia and dental agenesis (53%) were almost as frequent as skeletal anomalies, such as brachydactyly, short fifth finger, fifth finger clinodactyly, pectus excavatum/carinatum, delayed bone age. In 28.5% of individuals, prenatal ultrasound anomalies were reported. Except for three splicing variants, leading to a premature termination, variants were almost all frameshift. Our results, broadening the spectrum of KBGS phenotype progression, provide useful tools to facilitate differential diagnosis and improve clinical management. We suggest to consider a wider range of dental anomalies before excluding diagnosis and to perform a careful odontoiatric/ear-nose-throat (ENT) evaluation in order to look for even submucosal palate cleft given the high percentage of palate abnormalities. NGS approaches, following evidence of antenatal ultrasound anomalies, should include ANKRD11.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

Background: Rare diseases are an important population health issue and many promising therapies have been developed in recent years. In light of novel genetic treatments expected to significantly improve spinal muscular atrophy (SMA) patients' quality of life and the urgent need for SMA newborn screening (NBS), new epidemiological data were needed to implement SMA NBS in Estonia.Objective: We aimed to describe the birth prevalence of SMA in the years 1996-2020 and to compare the results with previously published data.Methods: We retrospectively analyzed clinical and laboratory data of SMA patients referred to the Department of Clinical Genetics of Tartu University Hospital and its branch in Tallinn.Results: Fifty-seven patients were molecularly diagnosed with SMA. SMA birth prevalence was 1 per 8,286 (95% CI 1 per 6,130-11,494) in Estonia. Patients were classified as SMA type 0 (1.8%), SMA I (43.9%), SMA II (22.8%), SMA III (29.8%), and SMA IV (1.8%). Two patients were compound heterozygotes with an SMN1 deletion in trans with a novel single nucleotide variant NM_000344.3:c.410dup, p.(Asn137Lysfs*11). SMN2 copy number was assessed in 51 patients.Conclusion: In Estonia, the birth prevalence of SMA is similar to the median birth prevalence in Europe. This study gathered valuable information on the current epidemiology of SMA, which can guide the implementation of spinal muscular atrophy to the newborn screening program in Estonia.