Biology subjects

Hoischen, A.

Publications and source records attributed to Hoischen, A..

16 recordsLinked to original sources

bioRxiv2026

Integrative optical genome mapping and long-read sequencing resolve constitutional complex rearrangements at nucleotide resolution

Complex rearrangements are one of the rarest types of structural variants (SVs) and can be divided into two categories: complex chromosomal rearrangements (CCRs) and complex genomic rearrangements (CGRs). CCRs include structural rearrangements that present at least three breakpoints and show exchange of genetic material between more than two chromosomes and CGRs are rearrangements that present more than one junction and/or more than one SV in cis. They are usually formed by one of the chromoanagenesis mechanisms, where a massive disruptive cellular event leads to multiple structural rearrangements. Classical cytogenomic techniques have been commonly applied for their characterization, but methodologies that involve longer DNA molecules, namely optical genome mapping (OGM) and long-read genome sequencing (lrGS), present a considerably higher SV detection resolution, revealing more details about the rearrangements, including precise breakpoint location. Here, we describe six patients with complex rearrangements investigated through a combination of different techniques: karyotyping, chromosomal microarray, and OGM were performed to characterize the rearrangements. Subsequently, lrGS was used to further resolve the alterations, refine their breakpoints' location, and sequence their junction points. Three patients presented CCRs involving three, four, and six chromosomes, while three exhibited CGRs involving one different chromosome each, providing a variety of complex SVs to show the importance of each technique and their combination in rearrangement resolution. In total, the complex rearrangements presented 127 breakpoints, 66 junction points and involved 14 of the 24 chromosomes. Higher-resolution techniques revealed additional complexity in all cases. Despite the advances provided by OGM and lrGS, conventional karyotyping remained indispensable for complete rearrangement resolution. In two patients, the findings supported a novel mechanism combining features of the different chromoanagenesis processes. Furthermore, evidence of inherited alterations was identified, and the comprehensive characterization of the rearrangements enabled more accurate genotype-phenotype correlations. Our findings indicate that an integrated approach combining karyotyping, OGM, and lrGS can completely resolve SVs, including complex rearrangements.

genomics↗

bioRxiv2026

Distinct mechanisms of CNV formation at the human 15q13.3 locus

Human chromosome 15q13.3 is a hotspot for recurrent pathogenic copy number variants (CNVs), which remain unresolved at the sequence level. We generated haplotype-resolved assemblies for 10 patient-parent trios and found that both the long ("BP4-BP5") and short ("CHRNA7") forms of 15q13.3 CNVs arise predominantly by non-allelic homologous recombination (NAHR) enabled by inversion polymorphisms. While most BP4-BP5 CNVs are structurally distinct, three breakpoints cluster in a 2 kbp PRDM9-enriched recombination hotspot. CHRNA7 CNVs originate from NAHR between CHRNA7-LCR repeats embedded within locus-spanning inversions and give rise to paired deletion/duplication events. Population analyses of 581 population haplotypes reveal at least 18 distinct structural haplotypes in 15q13.3 and more than 10-fold ancestry-stratification of BP4-BP5 CNV risk, where 68.4% of Europeans but only 5.1% of East Asians are predisposed. Comparison to six ape species indicates that the duplication architecture promoting instability expanded recently and is largely human-specific.

genetics↗

bioRxiv2025

Transcription-based identification of uncharacterized genes in the human immune response

Host-pathogen interactions are shaped by the nature of the pathogen and by host-related factors. The human host responses can be characterized in microbe-stimulated immune cells using transcriptomics. We set out to characterize gene expression changes as a result of microbial in vitro stimulation in RPMI-cultured human primary immune cells, using four pathogen ligands representing bacteria (LPS and S. aureus), viruses (Poly(I:C)) and fungi (C. albicans) for 4 and 24 hours, resulting in a total of 52 samples for analysis. We analyze the transcriptional changes on gene- and pathway level, highlighting common and distinct effects of pathogen stimulation. We highlight genes without a known function that were differentially expressed as a common effect of pathogen stimulation. We find uncharacterized genes such as KIAA0040 and CYRIA to be co-expressed with genes involved in innate immunity and downstream signaling from the PRRs, respectively. We identify 901 variants in uncharacterized genes in a cohort of patients with rare immune disorders, prioritizing 5 candidate variants in 4 genes that could underly these diseases. Our results therefore indicate the potential role of these genes the human immune response and provides candidate variants with potential relevance in rare immune-mediated diseases.

genomics↗

bioRxiv2024

Pushing the limits of single molecule transcript sequencing to uncover the largest disease-associated transcript isoforms in the human neural retina

Sequencing technologies have long limited the comprehensive investigation of large transcripts associated with inherited retinal diseases (IRDs) like Usher syndrome, which involves 11 associated genes with transcripts up to 19.6 kb. To address this, we used PacBio long-read mRNA isoform sequencing (Iso-Seq) following standard library preparation and an optimized workflow to enrich for long transcripts in the human neural retina. While our workflow achieved sequencing of transcripts up to 15 kb, this was insufficient for Usher syndrome-associated genes USH2A and ADGRV1, with transcripts of 18.9 kb and 19.6 kb, respectively. To overcome this, we employed the Samplix Xdrop System for indirect target enrichment of cDNA, a technique typically used for genomic DNA capture. This method facilitated the successful capture and sequencing of ADGRV1 transcripts as well as the full-length 18.9 kb USH2A transcripts. By combining algorithmic analysis with detailed manual curation of sequenced reads, we identified novel isoforms and alternative splicing events across the 11 Usher syndrome-associated genes, with implications for diagnostics and therapy development. Our findings demonstrate the Xdrop systems adaptability for cDNA capture and the advantages of integrating computational and manual transcript analyses. The full neural retina sequencing dataset is available via EGA under identifier EGAD50000000720.

genetics↗

bioRxiv2024

TRGT-denovo: accurate detection of de novo tandem repeat mutations

MotivationIdentifying de novo tandem repeat (TR) mutations on a genome-wide scale is essential for understanding genetic variability and its implications in rare diseases. While PacBio HiFi sequencing data enhances the accessibility of the genomes TR regions for genotyping, simple de novo calling strategies often generate an excess of likely false positives, which can obscure true positive findings, particularly as the number of surveyed genomic regions increases. ResultsWe developed TRGT-denovo, a computational method designed to accurately identify all types of de novo TR mutations--including expansions, contractions, and compositional changes-- within family trios. TRGT-denovo directly interrogates read evidence, allowing for the detection of subtle variations often overlooked in variant call format (VCF) files. TRGT-denovo improves the precision and specificity of de novo mutation (DNM) identification, reducing the number of de novo candidates by an order of magnitude compared to genotype-based approaches. In our experiments involving eight rare disease trios previously studied TRGT-denovo correctly reclassified all false positive DNM candidates as true negatives. Using an expanded repeat catalog, it identified new candidates, of which 95% (19/20) were experimentally validated, demonstrating its effectiveness in minimizing likely false positives while maintaining high sensitivity for true discoveries. Availability and implementationBuilt in Rust, TRGT-denovo is available as source code and a pre-compiled Linux binary along with a user guide at: https://github.com/PacificBiosciences/trgt-denovo.

bioinformatics↗

bioRxiv2024

Genome-wide profiling of highly similar paralogous genes using HiFi sequencing

Variant calling is hindered in segmental duplications by sequence homology. We developed Paraphase, a HiFi-based informatics method that resolves highly similar genes by phasing all haplotypes of a gene family. We applied Paraphase to 160 long (>10 kb) segmental duplication regions across the human genome with high (>99%) sequence similarity, encoding 316 genes. Analysis across five ancestral populations revealed highly variable copy numbers of these regions. We identified 23 families with exceptionally low within-family diversity, where extensive gene conversion and unequal-crossing over have resulted in highly similar gene copies. Furthermore, our analysis of 36 trios identified 7 de novo SNVs and 4 de novo gene conversion events, 2 of which are non-allelic. Finally, we summarized extensive genetic diversity in 9 medically relevant genes previously considered challenging to genotype. Paraphase provides a framework for resolving gene paralogs, enabling accurate testing in medically relevant genes and population-wide studies of previously inaccessible genes.

genomics↗

bioRxiv2024

Optical genome mapping enables accurate repeat expansion testing

Short tandem repeats (STRs) are amongst the most abundant class of variations in human genomes and are meiotically and mitotically unstable which leads to expansions and contractions. STR expansions are frequently associated with genetic disorders, with the size of expansions often correlating with the severity and age of onset. Therefore, being able to accurately detect the total repeat expansion length and to identify potential somatic repeat instability is important. Current standard of care (SOC) diagnostic assays include laborious repeat-primed PCR-based tests as well as Southern blotting, which are unable to precisely determine long repeat expansions and/or require a separate set-up for each locus. Sequencing-based assays have proven their potential for the genome-wide detection of repeat expansions but have not yet replaced these diagnostic assays due to their inaccuracy to detect long repeat expansions (short-read sequencing) and their costs (long-read sequencing). Here, we tested whether optical genome mapping (OGM) can efficiently and accurately identify the STR length and assess the stability of known repeat expansions. We performed OGM for 85 samples with known clinically relevant repeat expansions in DMPK, CNBP and RFC1, causing myotonic dystrophy type 1 and 2 and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), respectively. After performing OGM, we applied three different repeat expansion detection workflows, i.e. manual de novo assembly, local guided assembly (local-GA) and molecule distance script of which the latter two were developed as part of this study. The first two workflows estimated the repeat size for each of the two alleles, while the third workflow was used to detect potential somatic instability. The estimated repeat sizes were compared to the repeat sizes reported after the SOC and concordance between the results was determined. All except one known repeat expansions above the pathogenic repeat size threshold were detected by OGM, and allelic differences were distinguishable, either between wildtype and expanded alleles, or two expanded alleles for recessive cases. An apparent strength of OGM over current SOC methods was the more accurate length measurement, especially for very long repeat expansion alleles, with no upper size limit. In addition, OGM enabled the detection of somatic repeat instability, which was detected in 9/30 DMPK, 23/25 CNBP and 4/30 RFC1 samples, leveraging the analysis of intact, native DNA molecules. In conclusion, for tandem repeat expansions larger than [~]300 bp, OGM provides an efficient method to identify exact repeat lengths and somatic repeat instability with high confidence across multiple loci simultaneously, enabling the potential to provide a significantly improved and generic genome-wide assay for repeat expansion disorders.

genomics↗

bioRxiv2023

Multi-omic profiling of pathogen-stimulated primary immune cells

We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. RNA expression differences did not result in differences in the amounts of secreted proteins. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.

immunology↗

bioRxiv2022

SUsPECT: A pipeline for variant effect prediction based on custom long-read transcriptomes for improved clinical variant annotation

Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular in transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization. Our pipeline predicts the functional consequence and likely deleteriousness scores for missense variants in the context of novel open reading frames predicted from any transcriptome. We demonstrate the utility of SUsPECT by uncovering potential mutational mechanisms of pathogenic variants in ClinVar that are predicted to be benign using the reference transcript annotation. In further support of SUsPECTs utility, we identified an enrichment of immune-related variants predicted to have a more severe molecular consequence when annotating with a newly generated transcriptome from stimulated immune cells instead of the reference transcriptome. Our pipeline outputs crucial information for further prioritization of potentially disease-causing variants for any disease and will become increasingly useful as more long-read RNA sequencing datasets become available.

bioinformatics↗

bioRxiv2022

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing

Spinal muscular atrophy, a leading cause of early infant death, is caused by biallelic mutations of the SMN1 gene. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is impossible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across five ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations. Testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7-3.0%). Extending beyond simple copy number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers.

genetics↗

bioRxiv2021

Clone expansion of mutation-driven clonal hematopoiesis is associated with aging and metabolic dysfunction in individuals with obesity

AimsHaematopoietic clones caused by somatic mutations with [≥]2% variant allele frequency (VAF), known as clonal haematopoiesis of indeterminate potential (CHIP), increase with age and have been linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones are also associated with adverse clinical outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes, and to examine the development of clones over time in relation to age and metabolic dysregulation over up to 20 years in individuals with obesity. Methods and ResultsWe used an ultrasensitive single-molecule molecular inversion probe sequencing assay to identify clonal haematopoiesis driver mutations (CHDMs) in blood samples from individuals with obesity from the Swedish Obese Subjects study. In a single-timepoint dataset with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with VAF ranging from 0.01% to 31.15% and CHDM prevalence and clone sizes increasing with age. Longitudinal analysis over 20 years in CHDM-positive samples from 40 individuals showed that small clones can grow over time and become CHIP. VAF increased on average by 7% (range -4% to 27%) per year. Rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (HDL-C) (R=-0.68, P=1.74E-05). ConclusionOur results show that haematopoietic clones can be detected and monitored before they become CHIP and indicate that insulin resistance and low HDL-C, well-established cardiovascular risk factors, are associated with clonal expansion in individuals with obesity. Translational perspectivesClonal haematopoiesis-driver mutations are somatic mutations in haematopoietic stem cells that lead to clones detectable in peripheral blood. Haematopoietic clones with a variant allele frequency (VAF) [≥]2%, known as clonal haematopoiesis of indeterminate potential (CHIP), are recognized as an independent cardiovascular risk factor. Here, we show that smaller clones are prevalent, and also correlate with age. Our longitudinal observations in individuals with obesity over 20 years showed that more than half of all clone-positive individuals show growing clones and clones with VAF <2% can grow and become CHIP. Importantly, clone growth was accelerated in individuals with insulin resistance and low high-density lipoprotein-cholesterol (HDL-C). Translational outlook 1: Haematopoietic clones can be detected and monitored before they become CHIP. Translational outlook 2: The association between insulin resistance and low HDL-C with growth of haematopoietic clones opens the possibility that treatments improving metabolism, such as weight loss, may reduce growth of clones and thereby cardiovascular risk. One Sentence SummaryIn obesity, the growth rate of mutation-driven haematopoietic clones increased with insulin resistance and low HDL-C, both known risk factors for cardiovascular disease.

genomics↗

bioRxiv2020

Next generation cytogenetics: genome-imaging enables comprehensive structural variant detection for 100 constitutional chromosomal aberrations in 85 samples

Chromosomal aberrations and structural variations are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics, karyotyping and CNV-microarrays, in spite of the low resolution of the first one and the inability to detect neither balanced SVs nor to provide the genomic localization or the orientation of duplicated segments, of the latter. We here investigated the clinical utility of high resolution optical mapping by genome imaging for patients carrying known chromosomal aberrations in a context of constitutional conditions. For 85 samples, ultra-high molecular weight gDNA was isolated either from blood or cultured cells. After labeling, DNA was processed and imaged on the Saphyr instrument (Bionano Genomics). A de novo genome assembly was performed followed by SV and CNV calling and annotation. Results were compared to known aberrations from standard-of-care tests (karyotype, FISH and/or CNV-microarray). In total, we analyzed 100 chromosomal aberrations including 7 aneuploidies, 35 translocations, 6 inversions, 2 insertions, 39 copy number variations (20 deletions and 19 duplications), 6 isochromosomes, 1 ring chromosome and 4 complex rearrangements. High resolution optical mapping reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. Our study demonstrates the ability of high resolution optical mapping to detect almost all types of chromosomal aberrations within the spectrum of karyotype, FISH and CNV-microarray. These results highlight its potential to replace these techniques, and provide a cost-effective and easy-to-use technique that would allow for comprehensive detection of chromosomal aberrations.

genetics↗

bioRxiv2020

The impact of rare and common genetic variation in theInterleukin-1 pathway for human cytokine responses

BackgroundInterleukin(IL)-1 signaling is of major importance in human innate cytokine responses. Common variants in related genes have been linked to various inflammation-mediated diseases and stimulation-induced cytokine responses, but the role of rare variants remains to be elucidated. MethodsIn this study, we characterize the role of rare and common genetic variation, as identified by molecular inversion probe-based sequencing, in 48 genes related to the IL-1 pathway. Using a systems biology approach, we examined the inter-individual variability of in vitro stimulation-specific human cytokine responses from 463 healthy individuals of the Human Functional Genomics Project and assessed the role of rare and common genetic variants, separately and combined, by means of the Sequence Kernel Association Test. ResultsWe identified strong associations for rare genetic variants in NCF4 (adjP=7.2E-05) and CASP1 (adjP=3.0E-05) with IL-6 production in response to PHA and LPS stimulation, respectively. In addition, common variants in IL36A and IL38 were associated to both C. albicans-induced IL-1{beta} (IL36A adjP=0.0442; IL38 adjP=0.0092) and IL-6 production (IL36A adjP=0.0037; IL38 adjP=0.0082), an effect that was stronger at the subpathway level both for IL-1{beta} (adjP=0.0017) and IL-6 (adjP=1.8E-04). The common variant signature for the IL-1{beta} and IL-6 response to C. albicans was confirmed by an association with all anti-inflammatory genes (adjP=1.87E-03 and adjP=5.75E-04), and we validated this finding for non-coding common variants. Lastly, we identified a burden of rare variants in pro-inflammatory genes and LPS-induced IL-6 production (adjP=2.42E-04), and a new role for anti-inflammatory rare variants on S. aureus-stimulated IL-6 production (adjP=6.71E-03). ConclusionsIn conclusion, we show that both common and rare genetic variation in genes of the IL-1 pathway, separately and combined, differentially influence in vitro cytokine responses to various stimuli in healthy individuals. This study therefore accentuates potential mechanisms that are translatable into new hypothesis-driven characterization of common and rare variant involvement in a wide variety of inflammatory and immunological mechanisms and diseases.

systems biology↗

bioRxiv2020

Next generation cytogenetics: comprehensive assessment of 48 leukemia genomes by genome imaging

Somatic structural variants are important for cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all cytogenetic aberrations in a given sample still requires a combination of techniques, such as karyotyping, fluorescence in situ hybridization and CNV-microarrays. We hypothesize that the combination of these classical approaches could be replaced by high-resolution genome imaging. Bone marrow aspirates or blood samples derived from 48 patients with leukemia, who received a clinical diagnoses of different types of hematological malignancies, were processed for genome imaging with the Bionano Genomics Saphyr system. In all cases cytogenetic abnormalities had previously been identified using standard of care workflows. Based on these diagnostic results, the samples were divided into two categories: simple cases (<5 aberrations, n=37) and complex cases ([≥]5 aberrations or an unspecified marker chromosome, n=11). By imaging the labelled ultra-long gDNA molecules (average N50 >250kb), we generated on average [~]280-fold mapped genome coverage per sample. Chromosomal aberrations were called by Bionano Genomics Rare variant pipeline (RVP) specialized for the detections of somatic variants. Per sample, on average a total of 1,454 high confidence SVs were called, and on average 44 (range: 14-130) of those were rare i.e. not present in the population control database. Importantly, for the simple cases, all clinically reported aberrations with variant allele frequencies higher than 10% were detected by genome imaging. This held true for deletions, insertions, inversions, aneuploidies and translocations. The results for the complex cases were also largely concordant between the standard of care workflow and optical mapping, and in several cases, optical mapping revealed higher complexity than previously known. SV and CNV calls detected by optical mapping were more complete than any other previous single test and likely delivered the most accurate and complete underlying genomic architecture. Even complex chromothripsis structures were resolved. Finally, optical mapping also identified multiple novel events, including balanced translocations that lead to potential novel fusion-genes, opening the potential to discover new prognostic and diagnostic biomarkers. The full concordance with diagnostic standard assays for simple cases and the overall great concordance with (previously likely incompletely understood) complex cases demonstrates the potential to replace classical cytogenetic tests with genome imaging. In addition, this holds the potential to rapidly map new fusion genes and identify novel SVs and CNVs as novel potential leukemia drivers.

genomics↗

bioRxiv2020

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers critically implicated in human pathologies, with CHD6 being one of its least studied members. Here, we discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. We show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS-mutation affects CHD6 protein folding and impairs its ability to recruit co-factors in response to DNA damage or autophagy stimulation. This leads to an accumulation of DNA damage burden and to senescence-like phenotypes. By combining genomics and functional assays, we describe for the first time a molecular mechanism for the chromatin control of autophagic flux and genotoxic stress surveillance that applies broadly to human cell types and explains HSS onset.

cell biology↗

bioRxiv2019

Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

genetics↗