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The majority of rare diseases are genetic, and regardless of advanced high-throughput genomics-based investigations, 60% of patients remain undiagnosed. A major factor limiting our ability to identify disease-causing alterations is a poor understanding of the morbid and normal human genome. A major genomic contributor of which function and distribution remain largely unstudied are the transposable elements (TE), which constitute 50% of our genome. Here we aim to resolve this knowledge gap and increase the diagnostic yield of rare disease patients investigated with clinical genome sequencing. To this end we characterized TE insertions in 1000 Swedish individuals from the SweGen dataset and 2504 individuals from the 1000 Genomes Project (1KGP), creating seven population-specific TE insertion databases. Of note, 66% of TE insertions in SweGen were present at >1% in the 1KGP databases, proving that most insertions are common across populations. Focusing on the rare TE insertions, we show that even though ~0.7% of those insertions affect protein coding genes, they rarely affect known disease casing genes (<0.1%). Finally, we applied a TE insertion identification workflow on two clinical cases where disease causing TE insertions were suspected and could verify the presence of pathogenic TE insertions in both. Altogether we demonstrate the importance of TE insertion detection and highlight possible clinical implications in rare disease diagnostics. [ABSTRACT FROM AUTHOR]

Optic nerve hypoplasia (ONH) is a congenital malformation with a reduced number of retinal ganglion cell axons in a thin optic nerve. It is a common cause of visual impairment in children and ONH is associated with neurodevelopmental disorders, pituitary hormone deficiencies, and brain malformations. In most cases, the aetiology is unknown, but both environmental factors and genetic causes have been described. This study aimed to identify genetic variants underlying ONH in a well-characterised cohort of individuals with ONH. We performed array comparative genomic hybridization and whole genome sequencing in 29 individuals with ONH. Rare variants were verified by Sanger sequencing and inheritance was assessed in parental samples. We identified 11 rare single nucleotide variants (SNVs) in ten individuals, including a homozygous variant in KIF7 (previously associated with Joubert syndrome), a heterozygous de novo variant in COL4A1 (previously described in an individual with porencephaly), and a homozygous variant in COL4A2. In addition, one individual harboured a heterozygous variant in OPA1 and a heterozygous variant in COL4A1, both were inherited and assessed as variants of unknown clinical significance. Finally, a heterozygous deletion of 341 kb involving exons 7–18 of SOX5 (associated with Lamb-Schaffer syndrome) was identified in one individual. The overall diagnostic yield of pathogenic or likely pathogenic variants in individuals with ONH using whole genome sequencing was 4/29 (14%). Our results show that there is a genetic heterogeneity in ONH and indicate that genetic causes of ONH are not rare. We conclude that genetic testing is valuable in a substantial proportion of the individuals with ONH, especially in cases with non-isolated ONH. [ABSTRACT FROM AUTHOR]

Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated. [ABSTRACT FROM AUTHOR]

Copy number variations (CNVs) within the human genome have been linked to a diversity of inherited diseases and phenotypic traits. The currently used methodology to measure copy numbers has limited resolution and/or precision, especially for regions with more than 4 copies. Whole genome sequencing (WGS) offers an alternative data source to allow for the detection and characterization of the copy number across different genomic regions in a single experiment. A plethora of tools have been developed to utilize WGS data for CNV detection. None of these tools are designed specifically to accurately estimate copy numbers of complex regions in a small cohort or clinical setting. Herein, we present AMYCNE (automatic modeling functionality for copy number estimation), a CNV analysis tool using WGS data. AMYCNE is multifunctional and performs copy number estimation of complex regions, annotation of VCF files, and CNV detection on individual samples. The performance of AMYCNE was evaluated using AMY1A ddPCR measurements from 86 unrelated individuals. In addition, we validated the accuracy of AMYCNE copy number predictions on two additional genes (FCGR3A and FCGR3B) using datasets available through the 1000 genomes consortium. Finally, we simulated levels of mosaic loss and gain of chromosome X and used this dataset for benchmarking AMYCNE. The results show a high concordance between AMYCNE and ddPCR, validating the use of AMYCNE to measure tandem AMY1 repeats with high accuracy. This opens up new possibilities for the use of WGS for accurate copy number determination of other complex regions in the genome in small cohorts or single individuals. [ABSTRACT FROM AUTHOR]

The detection of recurrent somatic chromosomal rearrangements is standard of care for most leukemia types. Even though karyotype analysis—a low-resolution genome-wide chromosome analysis—is still the gold standard, it often needs to be complemented with other methods to increase resolution. To evaluate the feasibility and applicability of mate pair whole genome sequencing (MP-WGS) to detect structural chromosomal rearrangements in the diagnostic setting, we sequenced ten bone marrow samples from leukemia patients with recurrent rearrangements. Samples were selected based on cytogenetic and FISH results at leukemia diagnosis to include common rearrangements of prognostic relevance. Using MP-WGS and in-house bioinformatic analysis all sought rearrangements were successfully detected. In addition, unexpected complexity or additional, previously undetected rearrangements was unraveled in three samples. Finally, the MP-WGS analysis pinpointed the location of chromosome junctions at high resolution and we were able to identify the exact exons involved in the resulting fusion genes in all samples and the specific junction at the nucleotide level in half of the samples. The results show that our approach combines the screening character from karyotype analysis with the specificity and resolution of cytogenetic and molecular methods. As a result of the straightforward analysis and high-resolution detection of clinically relevant rearrangements, we conclude that MP-WGS is a feasible method for routine leukemia diagnostics of structural chromosomal rearrangements. [ABSTRACT FROM AUTHOR]

Background: The salivary α-amylase locus (AMY1) is located in a highly polymorphic multi allelic copy number variable chromosomal region. A recent report identified an association between AMY1 copy numbers and BMI in common obesity. The present study investigated the relationship between AMY1 copy number, BMI and serum amylase in childhood-onset obesity. Patients: Sixty-one subjects with a history of childhood-onset obesity (mean age 19.1 years, 54% males) and 71 matched controls (19.8 yrs, 45% males) were included. All anthropometric measures were greater in the obese; their mean BMI was 40 kg/m2 (range 25-62 kg/m2) compared with 23 kg/m2 in the controls (15-32 kg/m2). Results: Mean AMY1 copy numbers did not differ between the obese and control subjects, but gender differences were observed; obese men showed the highest and obese women the lowest number of AMY1 copies (p=0.045). Further, only in affected females, AMY1 copy number correlated significantly with whole body fat percent (r=-0.512, p=0.013) and BMI (r=-0.416, p=0.025). Finally, a clear linear association between AMY1 copy number and serum salivary amylase was observed in all subgroups but again differences existed between obese males and females. Conclusions: In conclusion, our findings suggest that AMY1 copy number differences play a role in childhood-onset obesity but the effect differs between males and females. Further studies in larger cohorts are needed to confirm these observations. [ABSTRACT FROM AUTHOR]

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements., Competing Interests: The authors have declared that no competing interests exist.