Human Genetics

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

Purpose A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case by case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.

Structural variants (SVs) are larger genetic rearrangements such as deletions, duplications, and insertions and other forms of structural alterations. Given their large size, they can have an outsized effect on phenotype, however, the contribution of SVs to the genetic architecture of orofacial clefts (OFCs) is not well understood. In this study, we sought to better understand the contribution SVs to OFC risk by characterizing de novo SVs (dnSVs) in 1,549 probands with an OFC. Overall, we called 375 dnSVs in 307 probands (0.242 dnSVs per genome). The dnSV rate per genome was highest among individuals with cleft lip (CL, 0.29), followed by cleft lip and palate (CLP, 0.25) and cleft palate only (CP, 0.20), though differences across subtypes were not statistically significant. There were also no differences between sex or syndromic status, but we did identify a significant difference in the rate of duplications across OFC subtypes (p=0.005). Compared to CP (0.028), duplication rates were significantly higher for both CL (0.121; p = 0.003) and CLP (0.096; p = 0.002), suggesting a potential etiologic mechanism for CL/P. We systematically curated each dnSV to classify its potential for influencing OFC risk by examining the genes and regulatory elements disrupted by the SV and evidence for their role in craniofacial development or OFCs. We identified 22 dnSVs in regions associated with recurrent genomic disorders, including seven deletions and one duplication of the well-established OFC risk locus 22q11.2. Additionally, we identified 12 large (>1Mb) SVs, with eight meeting criteria for a likely pathogenic classification based on total gene involvement, and seven dnSVs disrupting well-known genes associated with autosomal dominant forms of OFC. The most notable was SATB2, for which we found four dnSVs, all in individuals with CP. Next, by combining evidence from recurrent dnSVs and de novo single nucleotide variants with Hi-C contact maps and predicted chromatin states from developing craniofacial tissues, we found new candidate genes for OFC risk including FHAD1, NBEA, NR3C2, and SPHKAP. Overall, dnSVs classified as likely pathogenic accounted for putative molecular diagnoses in approximately 1.67% of the cohort, with an additional 0.96% having variants of unknown significance with plausible risk for OFCs. Our results support dnSVs as an important component of OFC etiology and suggest their inclusion in analyses may improve diagnostic yield for affected individuals.

Abstract INTRODUCTION Despite major advances in genomic sequencing and analytical pipelines for rare disorders, more than 50% of affected individuals remain without a molecular diagnosis. One challenge is the interpretation of inherited variants exhibiting incomplete penetrance (IP). MATERIALS AND METHODS We developed a pipeline to systematically investigate inherited variants as the cause of rare disease in 4,325 families, consisting of affected probands and unaffected parents, from the Broad Center for Mendelian Genomics, part of the GREGoR Consortium. As a proof-of-concept of further omics follow-up, epigenomic and transcriptomic analyses were performed in one family harboring an inherited disease-causing frameshift variant in CHD8. RESULTS We identified 81 families where the affected proband harbored a putative disease-causing variant inherited from a reportedly unaffected parent. Clinical re-evaluation identified 30 families in which an inherited variant was considered diagnostic, including six previously missed diagnoses. In this cohort, inherited IP variants account for 1.6% of the diagnosed cases, with an overall diagnostic yield of 0.4% in the cohort. Methylation analysis of the CHD8 family confirmed a positive CHD8 clinical episignature in the male proband, but also in the reportedly unaffected mother. Transcriptomic analysis showed that despite similar CHD8 haploinsufficiency, the proband and mother displayed divergent downstream transcriptional effects. DISCUSSION This study highlights the importance of considering parentally inherited IP variants in rare disease, with at least 1 in 60 diagnostic variants inherited from a reportedly unaffected parent, and demonstrates the potential of integrating omics evaluation to improve variant interpretation.

Background: The inner ear is supplied by the labyrinthine artery, which is functionally close to an end artery with limited collateral reserve. Microvascular abnormalities have been implicated in sudden sensorineural hearing loss (SSNHL) and age-related hearing loss, yet inner-ear microvasculature cannot be assessed in vivo and current hearing genome-wide association studies (GWAS) are underpowered. We evaluated the shared genetic architecture of retinal arteriolar tortuosity (retina_AT) with blood pressure, cerebral small vessel disease (cSVD) and hearing phenotypes, and examined whether retinal microvasculature could serve as a candidate genetic window to inner-ear microvasculature. Methods: We used linkage-disequilibrium score regression (LDSC) to estimate SNP heritability for 19 phenotypes and genome-wide genetic correlations (rg) for 11 prespecified pairs. We ran approximate Bayes-factor colocalization (coloc.abf) between 19 independent retinal loci and systolic blood pressure (SBP), together with 95% credible-set fine-mapping, UCSC gene annotation, colocalization with GTEx aortic and tibial-artery expression QTL (eQTL) data and with white-matter hyperintensities (WMH), hearing-GWAS power calculations, and univariable and multivariable Mendelian randomization (MR) of blood pressure and heart rate on hearing. Results: Only retina_AT with SBP was significant genome-wide (rg = 0.10, P = 4.2x10, FDR q = 0.00042). The strongest retinal locus, 13q34, did not colocalize (PP.H3 = 0.996; peaks about 267 kb apart; allelic heterogeneity), whereas two secondary loci colocalized strongly--CSK at 15q24 (PP.H4 = 0.98; 3/3 credible variants within CSK) and PAWR at 12q21 (PP.H4 = 0.99; 3/5 within PAWR)--supported by tissue-matched, moderate eQTL evidence (PP.H4 = 0.71-0.75). At 13q34, retina_AT with WMH was an adequately powered null (PP.H4 = 0.019; only this region and phenotype were tested). On the hearing side, sharing of the blood-pressure magnitude (rg {approx} 0.10) was a near-powered null (about 1.5-fold larger sample needed for 80% power under that assumption, versus about 15-fold for weak rg {approx} 0.03 sharing), and rg [≥] 0.12 was excluded; MR found no robust causal effect. Conclusions: Retinal arteriolar tortuosity shows limited, distributed genetic sharing with blood pressure at the vascular-tone genes CSK and PAWR; no sharing with WMH was observed at 13q34 (only this region was tested). Retinal microvasculature may serve as a candidate genetic window to inner-ear microvasculature, but this analogy awaits direct testing by larger, vessel-oriented hearing GWAS and inner-ear tissue functional studies.

Neurodevelopmental disorders (NDDs) are clinically and genetically heterogeneous, and evidence linking additional genes to disease evolves rapidly. We developed SysNDD, an open-access, expert-curated resource that represents distinct gene-inheritance-disease associations as entities with standardized confidence, phenotype, and variant annotations. Each entity retains its curation history, so its evidence category can change as evidence accumulates. Currently, SysNDD contains 4,275 entities curated from 4,932 publications. Of these, 3,758 entities involving 3,271 genes describe NDDs; the remainder record non-NDD disorders, nearly all linked to NDD-associated genes, included for completeness. Regarding NDD evidence categories, 1,838 genes are Definitive, 179 Moderate, 1,248 Limited, and 6 Refuted. The entity model captures inheritance-specific disorders, including 220 genes with both autosomal dominant and autosomal recessive NDD associations. Comparison with seven other NDD gene resources showed complementary coverage, and evidence categories largely agreed with those of ClinGen, Gene2Phenotype, and PanelApp for shared gene-disease pairs. NDD genes formed distinct molecular modules, whereas associated disorders formed broad, overlapping phenotype groups that corresponded to those modules only selectively. SysNDD provides a continuously maintained, programmatically accessible framework for gene-disease evidence assessment, inheritance-aware interpretation, and systematic NDD research.

Background: Short-rib polydactyly syndrome type IV (Beemer-Langer syndrome) is a lethal skeletal ciliopathy that can result from biallelic pathogenic variants in KIAA0586 (TALPID3). Earlier clinical observations suggested an unusually high occurrence of this phenotype in Romani families from the Carpathian Basin. Methods: Whole-exome sequencing was performed in affected individuals from five independent families, and the frequency of KIAA0586 c.1815G>A was assessed in a Hungarian Romani genetic database of 500 individuals. Results: The affected individuals were homozygous for c.1815G>A, a splice-disrupting variant previously shown to cause exon 14 skipping. Four families self-identified as Roma from Hungary and one affected individual was from Turkey. Six heterozygous carriers were identified among 500 individuals in the Hungarian Romani cohort, corresponding to an allele frequency of 0.6%. This was markedly higher than the frequencies cited from ALFA (0.01%) and gnomAD (0.0014%). The Hungarian-Romani and Turkish affected individuals also shared genotypes at informative markers flanking the variant, supporting a shared ancestral haplotype. Conclusions: These observations strengthen evidence that KIAA0586 c.1815G>A is a founder allele enriched in at least some Romani populations. The data support consideration of ancestry-informed carrier testing in appropriately counseled families. Historical admixture may explain the occurrence of the same haplotype in a Turkish case, but this remains a hypothesis requiring broader population-genomic confirmation.

We investigated the genetic basis of a previously unclassified congenital vascular anomaly in a patient with multifocal cutaneous vascular tumors. Through genomic analysis, we identified a novel heterozygous de novo germline variant in GA-binding protein-alpha (GABPA), an ETS family transcription factor. Histopathologic evaluation demonstrated capillary-venous lesions lacking glucose transporter 1 expression, distinguishing this entity from common infantile hemangioma. Notably, the tumor vasculature exhibited a prominent alpha-SMA-positive perivascular cell layer, with nuclear localized GABPA detected in endothelial and perivascular cells. The GABPA c.509T>G (p.L170R) variant is absent from large population databases, including gnomAD, DeCAF, and RGC-MCPS databases, indicating it is extremely rare. The affected leucine residue is highly conserved across species and located within the Pointed (PNT) domain, a critical region mediating GABPA protein-protein interactions. AlphaMissense predicts this substitution to be highly damaging. Structure-based analysis using an AlphaFold-predicted approach, combined with in silico mutagenesis and molecular interaction analyses, revealed that the L170R substitution introduces new electrostatic interactions while disrupting native hydrophobic contacts within the PNT domain. Functional assessment in zebrafish demonstrated that mosaic stromal expression of GABPA-L170R caused abnormal vascular architecture compared with GABPA-wild-type controls, establishing a direct link between this variant and disrupted vascular development. Collectively, the genetic rarity, evolutionary conservation, predicted structural perturbation, and disruptive effects on vascular development establish GABPA-L170R as likely disease-causing variant. These findings define a new molecular mechanism underlying congenital vascular tumor formation and expand the role for ETS-family transcriptional regulation in human vascular anomalies.

Polygenic scores (PGS) predict an individual's genetic value for a complex trait of interest by aggregating effect size estimates from genome-wide association studies (GWAS). While powerful, this approach assumes that genetic variants affect the phenotype in an additive way, which might be problematic when dominance is part of the genetic architecture. To address this limitation, we developed PGSa,d, a simple polygenic scoring framework, rooted in quantitative genetics theory, that incorporates both additive (a) and dominance effects (d) directly. We evaluated PGSa,d against the standard GWAS-based PGS (PGS{beta}) in simulations. The results showed that PGSa,d consistently achieved lower root mean squared error (RMSE) and progressively higher R2 as dominance contributed more to the genetic architecture. We then applied the method to 16 blood cell traits, height, and body mass index (BMI) from UK Biobank. PGSa,d showed comparable R2 to PGS{beta} for all traits (including height and BMI whose architecture is predominantly additive) and significantly lower RMSE for four traits after strict Bonferroni correction. Across traits, RMSE improvement from PGSa,d was positively associated with the magnitude of the estimated dominance effects, whereby traits with larger dominance effects displayed lower RMSE from PGSa,d. As a byproduct of our analysis, we also identified 141 variants in 39 genomic regions with significant dominance effects after Bonferroni correction across traits, recapitulating known associations and identifying novel ones. Together, these results show that explicitly modeling additive and dominance effects is a robust strategy that can improve individual-level predictions, especially when dominance contributes to complex trait architecture.

Ascertaining the pathogenicity and clinical relevance of genetic mutations is a longstanding challenge across human disease. This is particularly relevant for genes with many variants of unknown significance (VUS), such as PRPH2-retinopathy, where complex variable phenotype patterns and limited genotype correlations hinder application of precision-medicine therapies. Recent AI-based pathogenicity tools, while powerful, may be limited by insufficient mechanistic or phenotypic training data. We established a three-dimensional proteoform-phenotype analysis framework by combining AI-generated protein structural models with clinical genomics data, in which expert consensus adjudicates model outputs through a human-in-the-loop AI approach, to evaluate 46 curated pathogenic missense variants against a background VUS pool. The framework was prospectively validated using an independently evaluated clinical patient cohort. This analysis resolved spatial biophysical signatures corresponding to distinct clinical phenotypes (i.e., retinitis pigmentosa, macular dystrophy, and pattern dystrophy) as well as complex blended presentations. Leveraging these confirmed structural signatures, we prioritized high-risk VUS. This mechanism-resolved framework can be extended to other therapeutically actionable genes to identify novel causal variants and improve diagnosis, prognosis, and better select rare, eligible patients for emerging molecular gene therapy trials.

Background: Dysfunction of the KCND3-encoded Kv4.3 channel, which carries the transient outward potassium current (Ito), has long been suspected to contribute to the pathophysiology of Brugada Syndrome (BrS). Although recent genome-wide association studies (GWAS) have implicated KCND3, the role of rare variation in this gene remains uncertain and clinically unactionable. Methods: Genome sequencing was performed in 983 BrS probands from Europe and Thailand. Putative gain-of-function (GoF) rare variants in KCND3 identified in patients were functionally characterized by patch-clamp analysis of Kv4.3 current properties using a HEK293 cell heterozygous model. Biobank datasets were utilized to evaluate rare-common variant interaction at the KCND3 locus. Results: Eight BrS cases carried six different rare KCND3 variants which clustered at the Kv4.3 C-terminus, with terminal exon rare variants significantly enriched in cases versus study (p=0.004) and population (p=2.0e-6) controls. Four of the five KCND3 case variants studied demonstrated significant GoF effects versus wild-type and putative benign variants, including increased Ito amplitude (p.Leu586Val, p.Ala634Thr) and slower inactivation (p.Gly600Arg, p.Ala654Thr, p.Ala634Thr). Analysis of p.Gly600Arg carriers in the UK Biobank and All of Us revealed a significant enrichment of the protective KCND3 expression-reducing allele of the GWAS lead single-nucleotide polymorphism rs12090194, suggesting a possible epistatic common-rare variant interaction modifies penetrance in carriers. Conclusions: GoF rare variants at the C-terminus of KCND3 are associated with BrS in a small proportion of cases. Determination of the rare and common variant status in BrS patients and their relatives may provide information about disease risk.

Background Short-read genome sequencing generates the signal necessary to detect balanced reciprocal translocations at single-nucleotide resolution, but standard structural variant callers represent them as breakend pairs. Structural variant callers produce thousands of breakend records per genome, the majority of which are false positives arising from segmental duplications, repetitive elements, and reference assembly artifacts. This specificity problem has prevented balanced translocation detection from entering routine genome sequencing workflows. We developed a highly specific pipeline to resolve balanced reciprocal translocations and applied it across five clinically distinct cohorts to characterize the prevalence and diagnostic contribution of balanced reciprocal translocations. Methods Short-read genome sequencing data from 16,131 individuals across five cohorts were analyzed: healthy participants from the 1000 Genomes Project (n=3,202), probands with structural birth defects and their family members (CHOP BDB cohort; n=2,875, including 1017 probands), probands enrolled in the GREGoR consortium (GREGoR cohort; n=3,574 probands), probands with undiagnosed disease and their family members enrolled in the Undiagnosed Diseases Network (UDN cohort; n=6,071 including 2,027 probands) and neonates who underwent rapid genome sequencing (CHOP Baby Eagle cohort; n=409). A computational pipeline was developed to characterize breakend events from standard structural variant callers to high-confidence balanced translocation calls by exploiting well-characterized properties of true balanced translocation signatures in short-read data. Translocation breakpoints were resolved to single-nucleotide precision and interrogated for disruption of known disease genes or potential regulatory consequence otherwise. A subset of the identified breakpoints was confirmed by Sanger sequencing. Results Balanced reciprocal translocations were identified in 0.196% of structural birth defect probands (2/1,017), 0.25% of the GREGoR probands (9/3,574), 0.39% of the UDN probands (8/2,027) and 0.24% of neonates who underwent rapid genome sequencing (1/409). Parental carrier frequency in the birth defects cohort was 0.269% (5/1,858), consistent with previously reported estimates in similarly ascertained families, and 0.098% (3/3,040) in the UDN cohort. All six translocations identified in the birth defects cohort were orthogonally validated using Sanger sequencing, confirming pipeline specificity. Approximately 31% of translocations across the three affected cohorts disrupted a known disease gene at the breakpoint, establishing or refining a molecular diagnosis in five cases previously unresolved. The single translocation identified in the newborn cohort disrupted no disease gene and was classified as a variant of uncertain significance.

Background Gene-level constraint and conservation scores are widely used to prioritise disease genes and to benchmark genomic language models. Purifying selection acts weakly on phenotypes manifesting after reproduction, so these scores are expected to carry less information about late-onset disease. An onset-dependent gradient has been demonstrated for human allele frequencies but not for the gene-level scores used in practice. Results We built an age-of-onset axis for 2,885 Mendelian diseases from Orphanet and fitted cross-species conservation (241-way phyloP) and human-population constraint (shet) on the same 5,322 gene-disease pairs against matched controls, to test whether one decays faster with onset. Their onset slopes differed by 0.23 logit units (97.5% confidence interval -0.10 to 0.56), equivalent to 0.024 units of area under the receiver operating characteristic curve (AUROC) in a design resolving 0.049; the interval excludes constraint losing discriminative power faster than conservation by more than 0.011 AUROC units, widening to 0.033 when somatic associations are dropped. The usual explanation, that early-onset disease genes are developmental, pleiotropic and haploinsufficient by construction, did not account for the contrast; conditioning barely moved it. The answer did, however, depend on the constraint metric: three alternatives yielded larger contrasts excluding zero, though paired comparison could not distinguish the estimates. The metrics also diverged on recessive disease genes, with shet ranked below the gene pool (AUROC 0.487) and loss-of-function depletion above it (0.533), though, against matched controls, both ranked them below (0.457 and 0.450). Evolutionary scores improved a non-evolutionary baseline by 17 to 23%, with no onset gradient. Conclusions Conservation and constraint do not lose their value at measurably different rates as disease onset rises, and with only 283 late-onset Mendelian diseases annotated genome-wide, the question cannot be settled more finely. The gene-class explanation does not account for the contrast. Metrics that rank genes near-identically can still yield opposite onset-stratified answers, so a result reported by one metric carries a sensitivity that its confidence interval does not show. No constraint metric identifies recessive disease genes against matched controls, so a pipeline with recessive disease in scope needs a correction that switching metric does not supply.

CUB and Sushi domains-containing CSMD proteins are involved in neuron development. Variants in the CSMD1-3 paralogs were GWAS-associated with cognition, attention-deficit hyperactivity disorder (ADHD), schizophrenia and brain structure, suggesting that these genes could be linked to neurodevelopmental disorders (NDD). Indeed, rare biallelic variants in all three CSMD paralogs were recently associated with epilepsy, while rare biallelic variants in CSMD1 were also linked with intellectual disability and cortical malformations. We identified nine individuals from eight families carrying CSMD2 biallelic missense variants in developmental delay/intellectual disability cohorts. Affected individuals presented with autism spectrum disorder (ASD), ADHD and brain anomalies. Consistent with gnomAD metrics that suggest CSMD2 haploinsufficiency, we also report two families segregating monoallelic CSMD2 frameshift variants, which complement a described de novo truncation variant in an ASD proband. We concomitantly report two phenotypically overlapping individuals with de novo CSMD2 missense variants and compare them to twelve de novo CSMD2 cases identified in published trio analyses. UK Biobank analyses showed nominally significant enrichment of ADHD, ASD and neuroticism in volunteers carrying at least one rare CSMD2 variant. In silico modelling and immunofluorescence assays showed that mutated residues in affected individuals with biallelic and de novo missense variants are clustering in two regions and that the encoded proteins showed impaired membrane localization and an increased cytoplasmic aggregation. Zebrafish crispant larvae for the orthologous csmd2 presented with microcephaly, smaller cerebellum, and reduced Purkinje cell size. Larvae also displayed decreased swimming velocity, diminished connectivity between the optic tecta, and abnormal peripheral neuronal branching. We demonstrate that rare, likely pathogenic variants in CSMD2 are associated with both autosomal recessive and dominant forms of NDD further substantiating the functional significance of CSMD paralogs in neurogenesis/synapse formation.

As the use of population descriptors such as race, ethnicity, and ancestry have become increasingly common in modern genetics research, there have been growing calls to critically examine their use. Most notably, in 2023, the National Academies of Science, Engineering, and Medicine (NASEM) published a report titled Using Population Descriptors in Genetics and Genomics Research: A New Framework for an Evolving Field, which included eight specific and actionable recommendations for researchers to implement the ethical and accurate use of population descriptors in genetic research. Here, we use the 2023 NASEM report as a benchmark to analyze the use of population descriptors in genome-wide association studies (GWAS). We develop a general toolkit for large language model-based bibliometrics, operationalize the report's recommendations into an evaluation framework, and apply this framework to evaluate all 4,007 papers from the GWAS Catalog published between 2007 and 2025 with full text available on PubMedCentral. We find significant improvements in adherence to NASEM report recommendations over time. However, most improvements predate the publication of the NASEM report itself, suggesting the report functioned primarily as a synthesis of existing best practices rather than a catalyst for change. We conclude by highlighting opportunities for growth in the field of human genetics.

To evaluate the clinical implementation of long-read genome sequencing (lrGS) and establish the population reference resources needed to support its use in diagnostics, we integrate two complementary initiatives: Genome of Sweden (GoS), Sweden's contribution to Genome of Europe (GoE), and the Genomic Medicine Sweden - Rare Diseases (GMS-RD) long-read sequencing pilot. The initial GoS release of 576 individuals provides population-scale maps of genetic and epigenetic variation. Among 485 GMS-RD probands, 161 (33.2%) receive a molecular diagnosis, with lrGS providing added clinical value in 40 (24.8%). As a first-line test, lrGS could have avoided at least 42 follow-up analyses and an additional testing cycle in 7.2% of probands, reducing costs and the burden on patients. By integrating population and clinical long-read genomics, Sweden establishes a national ecosystem for genomic research and clinical diagnostics. Our findings support the clinical implementation of lrGS and offer a scalable model for national collaboration.

Childhood apraxia of speech (CAS) is a neurodevelopmental disorder of speech-motor planning and programming, associated with literacy, academic, and psychosocial difficulties. We performed whole-exome sequencing in 11 families comprising 21 affected and 15 unaffected individuals with phenotyping spanning speech, language, cognition, literacy, and motor function. We identified 60 prioritized variant observations representing 22 unique variants across 20 genes with family-based segregation analysis assessed in informative relatives; 5 arose de novo. Our cohort included multiple affected siblings and extended relatives, with several families carrying multiple prioritized variants, suggesting cumulative or oligogenic susceptibility. Speech and literacy impairment co-occurred with oral-motor, feeding, and fine-motor difficulties. BrainSpan co-expression analysis identified MECP2 and ANK2 as the genes with the highest observed connectivity in the sampled postnatal network. Exploratory analysis of the KOLF2.1J perturbation atlas in undifferentiated human induced pluripotent stem cells identified a positive LMO3 expression-response score following NIPBL repression and opposing LMO3 and ANK2 response directions within a cohesin-associated perturbation cluster. These observations motivate testing in human neural cells. Functional enrichment, protein-association, and developmental-expression analyses supported shared biological themes involving chromatin regulation, neuronal signaling, and extracellular-matrix organization. Among six families, multiple affected relatives shared prioritized variants across a unique set of several genes, demonstrating multilocus candidate profiles whose individual and combined effects may contribute to CAS susceptibility. We propose four novel CAS candidate genes: LMO3 and PCDH10, previously linked to human accelerated regions, as well as MYCBP2 and PTPRS. Our work provides multiple testable hypotheses for future variant-specific functional validation in speech-motor development.

CACNA1A-related disorders arise from altered function of the P/Q-type calcium channel Cav2.1 and encompass a broad spectrum of neurological phenotypes, including developmental and epileptic encephalopathy, familial hemiplegic migraine type 1 and episodic ataxia type 2. Although loss-of-function and gain-of-function mechanisms have both been implicated, interpretation of rare CACNA1A missense variants remains challenging because clinical presentation is heterogeneous and many variants identified through diagnostic sequencing remain classified as variants of uncertain significance. To generate clinically interpretable functional evidence, we developed a calibrated high-throughput automated patch-clamp assay to assess the CACNA1A variants. Twenty-six curated benign/likely benign and 30 curated pathogenic/likely pathogenic missense variants were expressed in Flp-In HEK293 cells with CACNB4 and CACNA2D1. We quantified current density, {Lambda}V0.5 activation,{Lambda}V0.5 inactivation and recovery from inactivation (T0.5 RFI), and used benign controls to define parameter-specific normal ranges with {+/-}3 Z-score thresholds. This multi-parameter framework classified all benign controls as functionally normal and 29 of 30 pathogenic controls as abnormal, yielding 100% specificity and 96.7% sensitivity. Current density identified 19 pathogenic controls with loss of function, while gating and recovery from inactivation identified additional abnormal variants with preserved or partially preserved current density. The assay generated likelihood ratios of 0.033 for normal function and 25.13 for abnormal function, supporting BS3 and PS3 functional evidence, respectively. Application to 23 CACNA1A variants identified in Australian patients showed abnormal function in all four variants already classified as pathogenic or likely pathogenic. Among 19 variants of uncertain significance, 11 were functionally abnormal and nine were reclassified as likely pathogenic after integration of PS3 evidence with other ACMG/AMP criteria. These data show that CACNA1A missense variants can produce loss-of-function, gain-of-function or mixed effects, and that multi-parameter functional assessment is required for accurate interpretation. This calibrated automated patch-clamp framework provides clinically interpretable evidence for refining CACNA1A variant classification in diagnostic practice.

FBXO31 encodes the substrate receptor of an SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase that recognizes C-terminal amide-bearing proteins (CTAPs) and targets them for proteasomal degradation, a function of particular importance in neurons. A recurrent de novo missense variant, c.1000G>A p.(Asp334Asn), reprograms FBXO31 substrate specificity and causes an autosomal-dominant neurodevelopmental disorder with spastic cerebral palsy through a neomorphic gain-of-function mechanism. By contrast, biallelic loss of function has tentatively been linked to a recessive disorder. Here we report a female patient, born to consanguineous parents, with moderate intellectual disability, developmental delay and dysmorphic features, who carries a homozygous 54.3 kb deletion removing exons 2-8 and the coding part of exon 9 of FBXO31. The deletion retains only the exon 1-encoded F-box domain, while removing the coding sequence for the entire C-terminal substrate-recognition region. It is predicted to result in loss of function. Segregation is consistent with autosomal-recessive inheritance, with both parents and three of five unaffected siblings being heterozygous carriers. Together with two previously reported recessive families and the experimentally established neomorphic action of p.(Asp334Asn), the homozygous deletion supports an FBXO31 allelic series comprising dominant gain of function and recessive biallelic loss of function.

CBX1-related syndrome is characterized by developmental delay, hypotonia, autistic features, and mild dysmorphic features. This syndrome is caused by heterozygous missense variants in CBX1, which encodes heterochromatin protein 1 beta (HP1{beta}). These variants are situated within the chromodomain of HP1{beta}, a critical region that mediates the interaction between HP1{beta} and trimethylated H3K9. While a prior study identified three individuals with pathogenic CBX1 variants, the variant and clinical spectrum of CBX1-related syndrome remains largely unknown. Here we report three patients with novel de novo heterozygous variants in CBX1. Patients with CBX1 p.(Leu40Pro) and p.(Cys60Arg) manifested with global developmental delay, while the patient with CBX1 p.(Cys60Gly) primarily exhibited speech delay without concurrent motor developmental delay. All variants reside within chromodomain of the HP1{beta} protein. Interestingly, in vitro cellular assays revealed that CBX1 p.(Cys60Gly) variant showed an intermediate cellular phenotype between the wild type and the CBX1 p.(Leu40Pro) and p.(Cys60Arg) variants, suggesting a possible genotype-phenotype correlation. This report provides further evidence that missense variants within the chromodomain of HP1{beta} cause a syndromic neurodevelopmental disorder, CBX1-related syndrome.