Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.

Lymphatic disease represents a well-described manifestation of Noonan syndrome (NS), yet the full phenotypic spectrum remains incompletely characterized, especially in asymptomatic individuals. We conducted a cross-sectional study including 10 individuals with NS (four with peripheral lymphedema and six without) and 10 age- and sex-matched cardiovascularly healthy controls. Central lymphatic anatomy was visualized using non-contrast T2-weighted MR-lymphangiography, and peripheral lymphatic morphology and function were assessed with near-infrared fluorescence (NIRF) imaging and strain gauge plethysmography (SGP). Variations in thoracic duct morphology were observed in 89% (8/9) of NS individuals compared to 20% (2/10) of controls. Thoracic lymphatic classifications (Biko/Dori scale) differed significantly in NS individuals, both with and without lymphedema, compared with healthy controls (p < 0.05). Central lymphatic abnormalities were observed in five of nine NS individuals, including three without clinical signs of lymphatic disease. Abnormal peripheral conduction in collective vessels, characterized by retrograde or absent flow, was detected in all four NS individuals with lymphedema (p < 0.05), while normal conduction was observed in asymptomatic NS individuals and controls. Findings from this study indicate that central lymphatic abnormalities may be present in NS regardless of symptom status, with abnormal peripheral conduction developing later in association with clinically manifest lymphedema. Central lymphatic abnormalities extend beyond clinically apparent disease and broaden the recognized phenotype, highlighting the value of lymphatic imaging for diagnosis and monitoring.

Whole-genome sequencing identifies intronic variants whose pathogenicity can be predicted with tools like SpliceAI. However, an actionable classification of such variants may require RNA-based validation, which can be limited by low expression in clinically accessible tissues. We report two fetuses from one family with Arthrogryposis multiplex congenita 6 (AMC6 [OMIM # 619334]) and biallelic NEB variants: a paternally inherited likely pathogenic frameshift variant, Chr2(GRCh38):g.151579391del; NM_001164508.2:c.16653del; NP_001157980.2:p.(Asp5552ThrfsTer5), and a maternally inherited intronic variant of uncertain clinical significance, Chr2(GRCh38):g.151496267G>A; NM_001164508.2:c.24486+9C>T; NP_001157980.2:p.(?). Because NEB is poorly expressed in fibroblasts, we used CRISPR activation to induce its expression in fibroblasts from the heterozygous mother. RNA-sequencing subsequently confirmed that the intronic variant generated a novel splice donor site associated with inferred loss of splicing at the canonical donor site. After NMD-inhibition, we could thus identify 45.5% of NEB transcripts with a 7 bp exon extension, predicted to result in a protein-coding frameshift. The intronic variant was classified as likely pathogenic, allowing a genetic diagnosis.

Structural variations (SVs) play important roles in genetic diversity, evolution, and carcinogenesis and are, as such, important for human health. However, it remains unclear how spatial proximity of double-strand breaks (DSBs) affects the formation of SVs. To investigate if spatial proximity between two DSBs affects DNA repair, we used data from 3C experiments (Hi-C, ChIA-PET, and ChIP-seq) to identify highly interacting loci on six different chromosomes. The target regions correlate with the borders of megabase-sized topologically associated domains (TADs), and we used CRISPR-Cas9 nuclease and pairs of single guide RNAs (sgRNAs) against these targets to generate DSBs in both K562 cells and H9 human embryonic stem cells (hESCs). Droplet digital PCR (ddPCR) was used to quantify the resulting recombination events, and high-throughput sequencing was used to analyze the chimeric junctions created between the two DSBs. We observe a significantly higher formation frequency of deletions and inversions with DSBs in proximity compared with deletions and inversions with DSBs not in proximity in K562 cells. Additionally, our results suggest that DSB proximity may affect the ligation of chimeric deletion junctions. Taken together, spatial proximity between DSBs is a significant predictor of large-scale deletion and inversion frequency induced by CRISPR-Cas9 in K562 cells. This finding has implications for understanding SVs in the human genome and for the future application of CRISPR-Cas9 in gene editing and the modeling of rare SVs.

Objective This study presents several cases of mosaicism for STS gene deletions in uncultured chorionic villus samples analyzed with chromosomal microarray without prior trypsinization. We aimed to confirm these results with MLPA on the chorionic villus samples and to evaluate the presence of mosaicism in follow-up amniocentesis. Methods We retrospectively collected cases of prenatally identified STS gene deletions in chorionic villus samples and amniocenteses at Aarhus University Hospital. A subgroup with mosaic microarray results was analyzed with MLPA. Results Four non-mosaic (of which three were inherited) and 16 mosaic STS gene deletions were identified. Mosaicism was confirmed with MLPA in all cases suitable for MLPA analysis. All 10 mosaic cases with follow-up amniocentesis showed normal results. In general, STS gene deletions in a mosaic state were smaller in size and had breakpoints located within the common fragile site FRAXB, whereas non-mosaic STS deletions were larger with breakpoints located close to VCX genes. Deletion size differed significantly between mosaic cases of this study and STS gene deletions in patients with X-linked Ichthyosis reported in ClinVar. Conclusion We report and confirm several cases of placental mosaicism for STS gene deletions. All mosaic cases with follow-up amniocentesis were confined to the placenta. Mosaic deletions likely arose from strand breaks at the common fragile site FRAXB, whereas the classical non-mosaic genotype found in patients with X-linked Ichthyosis arises from non-allelic homologous recombination during meiosis. These results support the existing hypothesis that placental mosaicism for copy number variants likely arise in common fragile sites.

Hailey-Hailey disease (OMIM#169600) is an autosomal dominantly inherited genodermatosis characterized by erosions in the flexural areas of the body. Hailey-Hailey disease is caused by variants in ATP2C1 , but for ~10% of the patients, no causative variant is found in the coding region of ATP2C1 . We aimed to determine the genetic cause of Hailey-Hailey disease in a family without a variant in the coding areas of ATP2C1 . By genome sequencing and analysis of all exon and intron sequences of ATP2C1 , we identified the variant c.532-560 T>G (NM_014382.5) in intron 7 of ATP2C1 . The variant is predicted by in silico tools to create a new deep intronic donor splice site. Segregation analysis detected the variant in the three affected family members. RNA sequencing confirmed that the variant creates a new deep intronic donor splice site that gives rise to an alternative exon. The identified deep-intronic variant in ATP2C1 is the likely cause of Hailey-Hailey disease. This is to our knowledge the first report of a deep-intronic variant as the cause of Hailey-Hailey disease, which shows that the analysis of the intronic sequences of ATP2C1 could increase the genetic diagnostic yield for Hailey-Hailey disease patients.

Hypohidrotic Ectodermal Dysplasia is a syndrome with hypotrichosis, hypohidrosis, and hypodontia as the main symptoms. The prevalence is estimated to one in 5000-10,000 persons. In 10-15% the disease is caused by pathogenic variants in EDAR, and most of the known causal variants to date are missense or nonsense variants. We present a patient with classic Hypohidrotic Ectodermal Dysplasia and mammary gland aplasia with a duplication within EDAR as the likely cause. The duplication is de novo in the patient, and genome sequencing of DNA extracted from blood has revealed that the duplication is in tandem conformation, most likely entailing an altered EDAR protein with a dominant negative effect. This is to our knowledge the first report of an intragenic duplication in EDAR as causal for Hypohidrotic Ectodermal Dysplasia.

The growth and development of the skeleton is regulated by bone morphogenetic proteins of which several are linked to genetic skeletal disorders. So far, no human skeletal malformations have been associated with variants in BMP5. Here, we report a patient with biallelic loss of function variants in BMP5 and a syndromic phenotype including skeletal dysostosis, dysmorphic features, hypermobility, laryngo-tracheo-bronchomalacia and atrioventricular septal defect. We discuss the phenotype in relation to the known tissue-specific expression of Bmp5 and similar morphological abnormalities previously reported in experimental animal models. Our findings suggest a new association between BMP5 variants and a range of developmental anomalies, involving ears, heart and skeleton, thereby increasing understanding of BMP5's role in human development.

Background Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. Methods We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. Results Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. Conclusions This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1- associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.

Genetic research has identified a large number of genetic variants, both rare and common, underlying neurodevelopmental disorders (NDD) and major psychiatric disorders. Currently, these findings are being translated into clinical practice. However, there is a lack of knowledge and guidelines for psychiatric genetic testing (PsychGT) and genetic counseling (PsychGC). The European Union-funded COST action EnGagE (CA17130) network was started to investigate the current implementation status of PsychGT and PsychGC across 35 participating European countries. Here, we present the results of a pan-European online survey in which we gathered the opinions, knowledge, and practices of a self-selected sample of professionals involved/interested in the field. We received answers from 181 respondents. The three main occupational categories were genetic counselor (21.0%), clinical geneticist (24.9%), and researcher (25.4%). Of all 181 respondents, 106 provide GC for any psychiatric disorder or NDD, corresponding to 58.6% of the whole group ranging from 43.2% in Central Eastern Europe to 66.1% in Western Europe. Overall, 65.2% of the respondents reported that genetic testing is offered to individuals with NDD, and 26.5% indicated the same for individuals with major psychiatric disorders. Only 22.1% of the respondents indicated that they have guidelines for PsychGT. Pharmacogenetic testing actionable for psychiatric disorders was offered by 15%. Interestingly, when genetic tests are fully covered by national health insurance, more genetic testing is provided for individuals with NDD but not those with major psychiatric disorders. Our qualitative analyses of responses highlight the lack of guidelines and knowledge on utilizing and using genetic tests and education and training as the major obstacles to implementation. Indeed, the existence of psychiatric genetic training courses was confirmed by only 11.6% of respondents. The question on the relevance of up-to-date education and training in psychiatric genetics on everyday related practice was highly relevant. We provide evidence that PsychGC and PsychGT are already in use across European countries, but there is a lack of guidelines and education. Harmonization of practice and development of guidelines for genetic counseling, testing, and training professionals would improve equality and access to quality care for individuals with psychiatric disorders within Europe.

Supplementary Table 3. Results of the statistical analyses of SNPs from project 12 in BRCA-mutation carriers affected or non-affected with breast cancer and according to their estrogen receptor status.

Supplementary Table 2. Results of the statistical analyses in BRCA mutation carriers.

Supplementary Methods, Tables 1-3, Figure 1 from Common Breast Cancer Susceptibility Alleles and the Risk of Breast Cancer for <i>BRCA1</i> and <i>BRCA2</i> Mutation Carriers: Implications for Risk Prediction

BOADICEA is a comprehensive risk prediction model for breast and/or ovarian cancer (BC/OC) and for carrying pathogenic variants (PVs) in cancer susceptibility genes. In addition to BRCA1 and BRCA2 , BOADICEA version 6 includes PALB2 , CHEK2 , ATM , BARD1 , RAD51C and RAD51D . To validate its predictions for these genes, we conducted a retrospective study including 2033 individuals counselled at clinical genetics departments in Denmark. All counselees underwent comprehensive genetic testing by next generation sequencing on suspicion of hereditary susceptibility to BC/OC. Likelihoods of PVs were predicted from information about diagnosis, family history and tumour pathology. Calibration was examined using the observed-to-expected ratio (O/E) and discrimination using the area under the receiver operating characteristics curve (AUC). The O/E was 1.11 (95% CI 0.97–1.26) for all genes combined. At sub-categories of predicted likelihood, the model performed well with limited misestimation at the extremes of predicted likelihood. Discrimination was acceptable with an AUC of 0.70 (95% CI 0.66–0.74), although discrimination was better for BRCA1 and BRCA2 than for the other genes in the model. This suggests that BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing for hereditary susceptibility to BC/OC despite suboptimal calibration for individual genes in this population.

Supplementary Table 1. The 3,248 SNPs included in the study