Transaldolase deficiency is a rare metabolic disease caused by pathogenic variants in the TALDO1 gene. Transaldolase plays an important role in the ribose-5-phosphate production, maintaining the NADPH-dependent lipid biosynthesis and cellular redox homeostasis. A small number of patients, predominantly children, have been reported, with a wide range of phenotypic presentations, including liver and kidney disease, involvement of the hematopoietic and endocrine systems, as well as possible early death. We aim to provide further insight into the clinical progression of transaldolase deficiency in adolescence and adulthood. We report on three adult patients with genetically confirmed transaldolase deficiency, including two novel genetic variants in TALDO1. Although the patients have been symptomatic since newborn age, initially with hepatomegaly and cytopenias, they were only diagnosed during adolescence or adulthood. Genetic analysis was performed only at 17, 26, and 32 years, respectively, which, however, did not reveal any genetic variants that would be expected to cause a milder disease course. In adulthood, the dominant clinical features were hypergonadotropic hypogonadism, osteopenia, renal and hepatic involvement. In conclusion, when reporting three new adult cases and comparing them with 47 accessible cases from the literature, our findings suggest that, even if clinical manifestations begin in the neonatal period, the overall phenotype may remain relatively mild, with gradual progression. This means that patients presenting with otherwise unexplained progressive liver disease, kidney dysfunction, cytopenia, and hypergonadotropic hypogonadism should be tested for transaldolase deficiency. We recommend closely monitoring patients with known transaldolase deficiency regarding the above-mentioned problems.

OBJECTIVE:Thioredoxin-interacting protein (TXNIP) is a protein involved in redox metabolism, but also a key regulator of glucose and lipid metabolism in preclinical models. To date, four patients with biallelic loss-of-function variants in TXNIP have been described, presenting with lactic acidosis and variable hypoglycemia, hepatomegaly, developmental delay and seizures. However, the role of TXNIP in human metabolism and its mechanistic effects across different organs are not fully understood. METHODS:We clinically, biochemically and genetically characterized a cohort of six additional individuals with biallelic pathogenic variants in TXNIP. Organ specimens from patients and mice were analyzed by gene expression, histology, and lipidomic and proteomic profiling. RESULTS:We confirmed lactic acidosis as the main clinical sign and added adult-onset cardiomyopathy, skeletal muscle weakness, and dyslipidemia to the extended disease spectrum. Heart, liver and muscle patient specimens showed pathological lipid accumulation, and mechanistic studies uncovered increased fatty acid synthesis markers and complex rearrangements of the lipidome and proteome. In Txnip-deficient mice, restricting dietary carbohydrates partially rescued fatty acid synthesis markers and lipid storage in the heart but led to dyslipidemia. CONCLUSIONS:Our studies show that TXNIP is an important metabolic modifier in cardiac and skeletal muscle as well as in lipoprotein metabolism and that biallelic pathogenic variants in TXNIP lead to a pleiotropic disease affecting cellular lipid metabolism in multiple organ systems, with potentially fatal adult-onset cardiomyopathy.

DNA replication is carried out by the replisome and is essential for maintaining genome integrity and cell proliferation. Pathogenic variants in genes encoding various replisome components cause microcephalic primordial dwarfism (MPD), characterized by growth retardation, microcephaly, and developmental abnormalities. Here, we report bi-allelic hypomorphic variants in WDHD1 as a cause of MPD with a broad spectrum of additional abnormalities, including acute liver failure, in 17 subjects from 14 families. WDHD1 encodes a replisome scaffolding protein (also known as AND-1 and Ctf4), which is essential for replisome assembly, replication fork stability, and sister chromatid cohesion. We found aberrant splicing of WDHD1 pre-mRNAs for all intronic variants tested and markedly reduced WDHD1 protein levels in subject-derived fibroblasts. Fibroblasts with bi-allelic WDHD1 variants showed globally reduced replication fork speed and impaired replication control, accompanied by spontaneous DNA damage and a G1-to-S transition defect. Using various cell biology approaches, we show that subject fibroblasts displayed reduced proliferation, abnormal nuclear morphology, including micronuclei, multilobed, and enlarged nuclei, as well as an increased number of metaphases with premature sister chromatid separation. Together, our findings establish WDHD1 as a protein required for normal organismal growth and development in humans and underscore its multiple functions in maintaining genome integrity.

BACKGROUND:Long-read sequencing and multi-omic analytical frameworks are increasingly being adopted in rare disease diagnostics. However, clinical workflows comprehensively integrating these methodologies remain uncommon. OBJECTIVE:This study aimed to assess the potential and limitations of integrating long-read genomic, transcriptomic, and proteomic analyses to characterize complex structural variants. METHODS:Two unrelated patients presenting with dystonia and comorbid neurological features underwent nanopore-based long-read DNA sequencing. In patient 1, complementary transcriptomic and proteomic analyses were performed. RESULTS:The workflow enabled the identification and characterization of two pathogenic complex structural variants: a homozygous AluY-mediated inversion disrupting PANK2, underlying neurodegeneration with brain iron accumulation (patient 1), and a heterozygous de novo 16p13.3 duplication-triplication event associated with an atypical dystonia-parkinsonism phenotype (patient 2). CONCLUSIONS:Our findings underscore the diagnostic potential of integrated long-read and multi-omic approaches for complex structural variant characterization, while illustrating persistent limitations of automated pipelines and highlighting unpredictable relationships between genomic, transcriptomic, and proteomic findings. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

OBJECTIVE:Genomic sequencing leaves >50% of dystonia-affected individuals without a diagnosis. Where DNA-oriented approaches remain insufficient, integrating multiomics is essential to advance genome interpretation. Herein, we incorporated RNA sequencing (RNA-seq) data from 167 patients with dystonia across a range of ages and presentations. METHODS:We leveraged an RNA-seq analysis pipeline, focused on the identification of expression and splicing aberrations, on RNA-seq from skin biopsies. The recruited patients had early-onset dystonia in 85.0%, non-focal dystonia in 92.2%, and coexisting features in 76.0%. Thirty-six patient samples with pre-identified variants (36/167, 21.6%) and 131 samples with no previously prioritized diagnostic candidates from genomic sequencing (131/167, 78.4%) were evaluated. RESULTS:We found that >80% of dystonia-associated genes were detected by fibroblast RNA-seq. Expression and splicing aberration analyses produced a manageable number of significant RNA defects affecting dystonia-associated genes. The approach was especially successful in validating pathogenic effects of loss-of-function variants, with disease-relevant RNA-underexpression detected for 66.7% (10/15). Studying aberrant expression and splicing in the context of other pre-identified variant types yielded relevant results in 28.6% (6/21 samples). We obtained a 6.9% (9/131) diagnostic uplift for patients without prior candidates, all of whom exhibited combined dystonia with autosomal recessive inheritance. The new diagnoses from RNA-seq and genomic reanalysis were based on previously neglected splice-region (3/9) and deep(er) intronic (6/9) variants. For the observed events, integration of new machine-learning scores predicted corresponding aberrant gene expression in the brain. INTERPRETATION:Fibroblast-based RNA-seq in our selected cohort improved variant interpretation and offered a modest yield in patients without prior candidate variants. ANN NEUROL 2026;99:1363-1378.

BACKGROUND:Diabetes mellitus is a common but incompletely characterised manifestation of mitochondrial diseases (MD). Data on risk factors, clinical course, and treatment recommendations are lacking. METHODS:In this multinational cohort study, we analysed longitudinal data of patients with a genetically confirmed MD from the GENOMIT registry included at German, Austrian, and Italian sites between 07/2009-01/2025. Our objectives were to (1) expand the genetic spectrum of mitochondrial diabetes mellitus (mDM), (2) identify risk factors, (3) delineate the clinical course, and (4) characterise real-world use of antidiabetic therapies. FINDINGS:Of 2399 patients, 1225 (51%) were female, and 281 (12%; 172 female) had mDM. Diabetes occurred across 31 genotypes and exhibited marked genotype dependence, with the highest prevalence in m.3243A>G carriers (177/360 [49%]). Only the m.3243A>G variant was associated with a significantly increased risk of mDM (HR = 10.3; 95% CI 5.2-20.4, p < 0.0001), whereas single mtDNA deletions, multiple mtDNA deletions, and primary LHON variants, as well as sex, BMI, ethnicity, smoking, hypertension and dyslipidaemia did not show a significant association. Median diabetes onset in patients with the m.3243A>G variant was at 47.7 years (SD 45.2-52.0). Among patients with mDM, 140/281 (50%) used insulin, and 111/281 (40%) received non-insulin antidiabetic drugs, most commonly metformin, which was discontinued in 8/50 users. Literature review revealed neurological events temporally linked to metformin application in m.3243A>G carriers, though long-term use without adverse events was likewise reported. INTERPRETATION:mDM is frequent in patients with MD, and the individual risk is strongly genotype dependent. While caution is warranted, our data do not justify universal avoidance of metformin; prospective, genotype-informed studies are needed to guide management. FUNDING:German Ministry of Research, Technology and Space; Italian Ministry of Health; European Union.

Abstract Background Pleuroparenchymal fibroelastosis (PPFE) is a rare, fibrotic lung disease with poor prognosis, usually affecting adults which most commonly occurs idiopathically. Biallelic pathogenic variants in DGUOK cause mitochondrial DNA (mtDNA) depletion syndrome, predominantly affecting infants with severe hepatic and neurological symptoms. Detailed description of pulmonary manifestations with late-onset presentation have not been reported. Methods We describe nine patients with PPFE and DGUOK -associated mitochondriopathy. Clinical, radiological, histopathological, and genetic data were systematically collected from all patients. Functional studies, single nucleus RNA sequencing (snRNAseq), immunofluorescence staining, transmission electron microscopy and respiratory chain enzyme activity assays were conducted on patient-derived fibroblasts, muscle or lung tissues. mtDNA content quantification was performed on whole genome sequencing (WGS) data. Results All patients (ages 5–36) presented with progressive dyspnea, weight loss and some with spontaneous pneumothoraces. Chest computed tomography and lung biopsies showed features of PPFE. Biallelic pathogenic DGUOK variants were identified in all patients, seven of them carry an unreported intronic variant leading to mtDNA depletion. snRNAseq of lung tissue from four pediatric patients identified Aberrant Basaloid cells and intermediate cells as their precursor localized at the fibrotic edge. Mitochondrial alterations were identified by electron microscopy. Conclusion PPFE in children and young adults is associated with DGUOK -related mitochondriopathy. For the first time, we demonstrate Aberrant Basaloid cells in pediatric fibrotic lung tissue. Since pulmonary involvement may be underrecognized or misinterpreted and the clinical presentation may not always be typical of a mitochondriopathy, we recommend genetic testing in all patients with PPFE of unknown origin.

Hypertension (HTN) has been linked to changes in DNA methylation. However, longitudinal epigenome-wide analyses are still limited. We analyzed data from the KORA F4 and FF4 studies, conducted approximately 7 years apart. The dataset included 2614 participants, each with DNA methylation measured at least once. Leucocyte DNA methylation was profiled using the Illumina 450 k and EPIC arrays. Linear mixed-effects models were employed to identify associations between methylation sites and HTN status, systolic (SBP) and diastolic blood pressure (DBP). Interaction terms with follow-up time captured longitudinal methylation trajectories. We further examined CpG sites related to reversed, persistent, or progressive HTN and assessed their correlations with gene expression. One CpG site was associated with SBP and four with DBP, all representing novel loci, including RILP (cg08625564) and SVIL (cg15298791). Differential annual methylation changes were observed for 2, 23, and 12 CpG sites by HTN status, SBP, and DBP, respectively, highlighting genes such as RHPN2, CLDND1, ZNF69, and FKBP1B. Twenty CpG sites were associated with persistent HTN, including PLCB2 and MPPE1. In whole blood, 22 significant CpG–transcript pairs were detected, involving 14 CpG sites and 19 gene transcripts. This longitudinal epigenome-wide study identified novel CpG sites associated with blood pressure and persistent HTN. We observed differential DNA methylation trajectories over time linked to HTN, SBP, and DBP, with several changes correlating with gene expression, suggesting functional relevance. These findings underscore the dynamic role of DNA methylation in blood pressure regulation and provide new insights into epigenetic mechanisms of HTN.

Background and Objectives:Mitochondrial DNA (mtDNA) disorders exhibit striking clinical variability that is poorly explained by known factors such as variant heteroplasmy, age, or sex. Nuclear genetic modifiers likely play a significant role in this heterogeneity. We aimed to characterize the nature of nuclear genetic involvement for 2 common syndromic presentations of the common pathogenic mtDNA variant, m.3243A>G: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and maternally inherited diabetes and deafness (MIDD). Methods:We assembled a multicenter cohort of clinically ascertained carriers of m.3243A>G (total n = 488), identifying 198 individuals across 76 pedigrees suitable for genetic linkage analysis. We investigated 4 clinical features characteristic of MELAS and MIDD: diabetes, hearing impairment, stroke-like episodes, and encephalopathy. Haseman-Elston regression-based genetic linkage analysis was performed to identify regions of the nuclear genome cosegregating with these features. The effects of m.3243A>G heteroplasmy, age, and sex were accounted for using logistic regression; empirical significance thresholds were determined through feature-specific gene-dropping simulations. Association analyses were performed in 247 individuals using single-variant (SAIGE) and gene-based approaches (SAIGE-GENE+ and MAGMA) to refine candidate loci within a significant linkage region. Results:We identified significant genetic linkage to encephalopathy (chromosome 7q22; LOD = 3.72), and regions suggestive of genetic linkage on chromosomes 1, 5, 6, 11, and 13, for encephalopathy and stroke-like episodes. No linkage was identified for diabetes or hearing impairment. Association analysis within the chromosome 7 region identified variant rs62500792 (intergenic between SDHAF3 and TAC1) with the lowest p value (3.7 × 10-5), yet no variants reached the proportional significance threshold (5.3 × 10-6). Gene-based analyses highlighted PLOD3 (p = 3.9 × 10-3) and IMMP2L (p = 6.4 × 10-3) as candidates, as each showed the strongest gene-level signals within the linkage region across complementary burden-testing methods, although neither reached corrected significance thresholds. Discussion:The nuclear genetic architecture modifying m.3243A>G differs across clinical features. Severe neurologic features (encephalopathy and stroke-like episodes) may be influenced by a small number of nuclear genes with relatively large effect sizes, whereas the nuclear contribution to diabetes and hearing impairment appears more polygenic. This study highlights the value of large, well-characterized patient cohorts in identifying modifier loci and advancing knowledge of the mechanisms underlying phenotypic variability in mtDNA disease.

Patients with suspected monogenic disorders often remain undiagnosed after exome sequencing. We report a family with two sisters affected by a complex spastic paraplegia. Initial exome sequencing had identified monoallelic pathogenic nonsense variants in AP4S1 and AP4B1, subunits of the adaptor protein complex 4 (AP-4), suggesting digenic inheritance. As digenic inheritance has not been established for AP-4-associated disorders, we applied a multiomics approach including genome sequencing, RNA sequencing and proteomics to clarify the genetic cause. By RNA sequencing a predicted synonymous variant (NM_006594.5:c.969G > A), compound heterozygous to the nonsense variant in AP4B1 and previously considered as benign, was re-prioritized as aberrant splicing was demonstrated. Proteomics showed reduced abundance of AP-4 components AP4B1 and AP4M1 and an upregulation of the cargo protein ATG9A, confirming AP-4 deficiency. Although the AP4S1 variant resulted in nonsense-mediated decay, the identification of biallelic causative variants in AP4B1 established the diagnosis of monogenic "Spastic paraplegia 47, autosomal recessive" while the initial hypothesis of digenic inheritance was refuted. This study illustrates the value of multiomics approaches in the diagnostic workflow of rare diseases and the potential for pathogenicity of synonymous variants.

AIMS:The study aimed to assess the effectiveness of three clinical diagnostic criteria [Simon Broome (SB), MEDPED (MP), and guideline-derived (GL-EAS)] in identifying children with familial hypercholesterolaemia (FH) compared with genetic testing. The evaluation involved 1337 children with elevated LDL cholesterol (LDL-C) levels, focusing on the sensitivity and specificity of these clinical scores in detecting genetically confirmed FH cases. METHODS AND RESULTS:Clinical data were gathered by a self-reporting questionnaire. Clinical FH was defined in accordance with the tested FH score. Genetically confirmed heterozygous FH (HeFH) was defined by a (likely) pathogenic variant. Of the 1337 children undergoing genetic analysis, 211 showed a pathogenic FH mutation. Applying SB, MP, and GL-EAS criteria resulted in 210/1337, 125/1337, and 112/835 children being categorized to have FH clinically. The sensitivity of the clinical scores ranged from 0.44 to 0.54 with a positive predictive value (PPV) of 0.51-0.79. The specificity was 0.91-0.97 with a negative predictive value (NPV) of 0.89-0.91. Similar results were observed for the three clinical scores regarding sensitivity, specificity, PPV, and NPV in subgroup analyses defined by gender, age (<10 years vs. ≥10 years), or weight [≥90th BMI (body mass index) percentile vs. <90th BMI percentile]. CONCLUSION:Clinical FH scores offer a high degree of specificity for FH diagnosis in children, but at the expense of low sensitivity. Specifically, half of the mutation-positive children in this study would have been missed for early diagnosis and preventive treatment. Given the widespread availability of affordable genetic testing, such analysis should be performed at a lower threshold than that indicated by these clinical scores.

OBJECTIVE:Mitochondrial diseases are the most common inherited metabolic disorders, characterized by pronounced clinical and genetic heterogeneity that complicates molecular diagnosis. Although DNA-based sequencing approaches have become standard in genetic testing, up to half of patients remain without a definitive diagnosis. We aimed to perform RNA sequencing (RNA-seq) of patient-derived skin fibroblasts to enhance the molecular diagnostic efficacy of mitochondrial disease in undiagnosed cases in China. METHODS:We performed RNA-seq on skin fibroblasts from 140 pediatric patients with suspected mitochondrial disease who remained genetically undiagnosed after whole exome sequencing (WES). Aberrant RNA expression and splicing were identified using the detection of RNA outliers pipeline (DROP). Based on WES findings, patients were stratified into a candidate group (n = 28), in which RNA-seq evaluated the pathogenicity of WES-identified variants of uncertain significance and an unsolved group (n = 112), in which RNA-seq was used to pinpoint candidate genes. In six cases where RNA-seq identified the aberrant RNA event but WES did not detect the causative variants, whole genome sequencing (WGS) was performed. RESULTS:Integrative RNA-seq, WES, and WGS analysis resulted in a genetic diagnosis in 25% of patients overall (20/28 [71%] in the candidate group; 15/112 [13%] in the unsolved group). Aberrant splicing explained most candidate-group diagnoses, including variants misclassified by in silico predictors such as SpliceAI. 14% of protein-truncating variants predicted to undergo nonsense-mediated decay (NMD) escaped degradation, highlighting the functional limits of current predictions. The variants identified in the unsolved cohort included synonymous, missense, deep intronic, near-splice-site variants, and large deletions. The most frequent among them was a recurrent synonymous East Asian founder mutation in ECHS1, accounting for seven cases. Interestingly, across 233 pathogenic variants associated with aberrant RNA phenotypes compiled from this study and prior reports, half were noncoding and half were coding variants. CONCLUSION:RNA-seq substantially enhances molecular diagnosis in mitochondrial disease by exposing cryptic splicing, regulatory, and NMD-escape events invisible to DNA sequencing alone. These data advocate transcriptome analysis as an essential component of comprehensive genomic diagnostics in neurometabolic disease.

Protein-truncating variants in the 3' region of a transcript, evading mRNA degradation and giving rise to aberrant truncated proteins, are an underrecognized cause in Mendelian diseases. Here, we report two individuals with heterozygous de novo nonsense variants in the penultimate and last exon of NUSAP1, both presenting with early-onset refractory epilepsy, global developmental delay, congenital microcephaly, and a recognizable facial gestalt. RNA sequencing performed in one individual did not show a reduction in expression, compatible with escape of aberrant transcripts from nonsense mediated mRNA decay (NMD). We systematically analyzed gnomAD population data to delineate a critical region at the 3' region of NUSAP1, where nonsense variants introduce a premature termination codon and escape NMD. Such variants are absent from healthy controls, while frameshift variants producing C-terminal elongations appear tolerated. This position-dependent model provides guidance for diagnostic variant interpretation.

Many non-coding variants influence complex traits and diseases through gene regulation, yet the mechanisms linking these variants to downstream biology remain poorly understood. Here, we present eQTLGen Phase 2, a comprehensive genome-wide analysis of gene expression quantitative trait loci (eQTLs) in 43,301 blood samples from 52 datasets. Beyond local ciseffects, this sample size enabled the first systematic mapping of trans-eQTLs at scale. We identify cis-eQTLs for nearly all expressed genes (94.7%) and trans-eQTLs for over half (56.2%). Second, by colocalizing cis-eQTLs with trans-eQTLs, we infer a directed gene regulatory network comprising 47,554 directed gene regulatory relationships. These networks reveal how genetic perturbations in upstream regulators produce dose-dependent downstream effects, supported by Perturb-seq and ChIP-seq data. Third, integrating this network with 87 genome-wide association studies allows us to systematically prioritize trait-relevant pathways and candidate genes. Variants exerting both cis- and trans-effects are markedly more likely to colocalize with trait associations than cis-only variants, delineating a subset of functionally active cis-eQTLs from a large group with limited downstream impact. This distinction provides a conceptual framework for identifying regulatory variants that truly mediate complex trait biology. Together, these results provide a publicly available resource of cis- and trans-eQTLs and an in vivo scaffold for human gene-regulatory networks, elucidating how propagation of cis-effects modulates complex disease.

Our understanding of the genetic landscape of inherited optic neuropathies has grown significantly over the past decades, and it is now known to involve many genes found in both the nuclear and mitochondrial genomes, exhibiting all possible inheritance patterns. Furthermore, pathogenic variants in nuclear genes of mitochondrial respiratory Complex I (CI) subunits have been identified in some cases of ION, in addition to the more common severe presentation of CI deficiencies, usually with early onset. We conducted NGS screening of CI genes to identify potential causative variants in patients with optic atrophy, also performing comprehensive clinical assessments, including neuroimaging studies (MRI) and neurological evaluations. Detailed molecular structure modeling was performed to better evaluate the damaging effects of both novel and previously reported variants in the relevant CI subunits. We identified and characterized candidate causative variants in 31 patients from 23 unrelated families, with biallelic or hemizygous variants in 11 different nuclear CI-related genes encoding polypeptides involved in the structure of CI, including 3 core subunits (NDUFS7, NDUFV1, NDUFV2), 4 accessory subunits (NDUFA1, NDUFA10, NDUFA12, NDUFB11), and 4 assembly factors (NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF8). Notably, defects in core CI subunits in this cohort lead to isolated optic atrophy, while defects in accessory CI subunits and assembly factors resulted in a spectrum of phenotypes, from isolated to syndromic optic atrophy. For 12 cases, the subacute onset of vision loss enabled us to associate or confirm novel genes (NDUFS7, NDUFV1, NDUFAF2, NDUFAF4, NDUFAF8) with the autosomal recessive Leber Hereditary Optic Neuropathy (arLHON) phenotype. Moreover, in the NDUFS7 subunit a partial spatial segregation was noted for missense variants causing either Leigh syndrome or isolated optic atrophy, hinting at possible disease-specific molecular defects. Our case series broadens the genetic spectrum of inherited optic neuropathies, emphasizing the crucial role of nuclear CI genes in its pathogenesis. The arLHON phenotype emerges as linked to numerous nuclear CI genes for which an insidious onset of optic atrophy is also reported, and in some cases the same variant may underlie both phenotypes. Overall, we highlight the possibly so far underestimated prevalence of CI nuclear subunits in the molecular diagnosis of ION, prompting to include all CI-related genes in the standard diagnostic screening.

BACKGROUND AND AIMS:The role of genetic testing as part of universal screening programmes for familial hypercholesterolaemia (FH) in children is not well defined. Here, a two-step approach to identify children carrying FH-causing variants was investigated. METHODS:In this study from Southern Germany, paediatricians were invited to offer FH screening to all children aged 4.8-14.9 years at routine paediatric examinations. The FH screening programme began in September 2020 in Bavaria and has involved up to 480 paediatricians. It included biochemical and genetic testing using 0.2 mL of blood taken from a fingertip. In case of low-density lipoprotein cholesterol (LDL-C) serum concentration ≥3.36 mmol/L (≥130 mg/dL), FH-causing variants were determined in the same sample with a focused panel covering most frequent variants (n = 48) and sequencing of relevant genes. RESULTS:Out of 25 431 children screened so far, 1689 children had an LDL-C ≥ 3.36 mmol/L (>130 mg/dL), which defined this concentration as the 93rd percentile. Pathogenic variants were identified by the focused panel in 157 and by next-generation sequencing in 283 children, respectively. While 17% (283/1670) of all genetically analysed children tested positive, the fraction of individuals with FH-causing variants increased across the spectrum of LDL-C serum concentrations from 4.7% (23/492) at 3.36-3.49 mmol/L (130-135 mg/dL) to 78.6% (81/103) above 5.17 mmol/L (200 mg/dL). Overall, the prevalence of FH-causing variants was high (1:90). One reason was a founder variant (n = 63) within the LDLR gene, found 40 times more frequent than European average. The analysis of recruitment data revealed significant ascertainment bias, with lower recruitment rate practices exhibiting higher prevalence. After adjustment for the bias using a generalized linear mixed model, the predicted prevalence was 1 in 163 (0.61%), which is highly consistent with large-scale genomic benchmarks as gnomAD (1:165, n = 622 057) and the UK Biobank (1:176, n = 48 741). CONCLUSIONS:The prevalence of FH determined in this study is significantly higher than previously published estimates (∼1:250), highlighting the importance of this condition for public health and supporting calls for a national paediatric screening programme, given the availability of effective treatment options. For children between 5 and 15 years, biochemical screening is an effective way to select patients for genetic testing, with sequencing of candidate genes being superior to variant screening. In summary, the VRONI study demonstrates the feasibility and efficacy of a combined biochemical and genetic screening for FH in children.

This retrospective study on X-linked PDHA1-related pyruvate dehydrogenase complex (PDHc) deficiency combined a systematic literature review with a multicentre survey exploring genotypes, phenotypes and survival. Data from 891 individuals (45% unpublished) were included. Of note, 53% of cases were females. Median age at last assessment was 6 years (range 0-80 years, n = 622). We detected 331 different (118 unpublished) PDHA1 variants, of which 75% (305/405) had occurred de novo. Variants in this study were uploaded to ClinVar (SCV006297015-SCV006297345). The 10 most frequent variants accounted for 36% of the diagnoses. Sixty-nine per cent of the variants were private; missense (50%) and frameshift (20%) variants were most common. Frameshift/nonsense (FS/N) variants in males (44/401, 11%) were confined to regions escaping nonsense-mediated decay (NMD) and were significantly less frequent than in females (151/461, 33%). Neonatal or infantile (405/529, 77%) presentations were most frequent, with pre/perinatal abnormalities reported in 47% (159/342). FS/N variants in the NMD-predicted region 3.9 [95% confidence interval (CI) 1.54-11.04] times increased the odds of fetal findings. Females presented significantly earlier [2 months, interquartile range (IQR) 7.0, n = 224] than males (8 months, IQR 16.6, n = 233), with increased risk of neonatal presentation [odds ratio (OR) 3.01 (95% CI 1.279-7.616)] when harbouring FS/N variants in the NMD-predicted region. The overall (n = 242) mean survival time was 10.9 (95% CI 9.9-11.9) years. On average, females survived 4.5 (95% CI 2.62-6.40) years longer than males despite presenting more severe phenotypes. Poor survival was associated with male sex [hazard ratio (HR) 3.3 (95% CI 1.95-5.62)], neonatal presentation [HR 5.5 (95% CI 2.17-14.09)], FS/N variants in the NMD-predicted region [HR 4.0 (95% CI 1.78, 9.16)] and splice variants [HR 2.3 (95% CI 1.15, 4.59)]. More severe clinical phenotypes were predicted by neonatal or infantile presentations and by female sex. Developmental delay (DD), intellectual disability (ID), muscle hypotonia, abnormal movements, seizures, feeding difficulties and microcephaly were the most frequent phenotypes, all occurring in more than half. Corpus callosum or basal ganglia alterations and cerebral atrophy were common. Four per cent (36/891) were reported to have mild phenotypes with no DD nor ID (25/36 males). This is the largest dataset on a nuclear-encoded defect of mitochondrial energy metabolism. The genotypic and phenotypic details further defines the disease landscape and can be used for variant interpretation. The correlations between genotypes, sex, phenotypes and survival, adds substantial improvement to counselling.