June 1, 2021  |  

Screening and characterization of causative structural variants for bipolar disorder in a significantly linked chromosomal region onXq24-q27 in an extended pedigree from a genetic isolate

Bipolar disorder (BD) is a phenotypically and genetically complex and debilitating neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition in BD with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BP susceptibility, however no disease genes have been identified to date.


June 1, 2021  |  

Screening for causative structural variants in neurological disorders using long-read sequencing

Over the past decades neurological disorders have been extensively studied producing a large number of candidate genomic regions and candidate genes. The SNPs identified in these studies rarely represent the true disease-related functional variants. However, more recently a shift in focus from SNPs to larger structural variants has yielded breakthroughs in our understanding of neurological disorders.Here we have developed candidate gene screening methods that combine enrichment of long DNA fragments with long-read sequencing that is optimized for structural variation discovery. We have also developed a novel, amplification-free enrichment technique using the CRISPR/Cas9 system to target genomic regions.We sequenced gDNA and full-length cDNA extracted from the temporal lobe for two Alzheimer’s patients for 35 GWAS candidate genes. The multi-kilobase long reads allowed for phasing across the genes and detection of a broad range of genomic variants including SNPs to multi-kilobase insertions, deletions and inversions. In the full-length cDNA data we detected differential allelic isoform complexity, novel exons as well as transcript isoforms. By combining the gDNA data with full-length isoform characterization allows to build a more comprehensive view of the underlying biological disease mechanisms in Alzheimer’s disease. Using the novel PCR-free CRISPR-Cas9 enrichment method we screened several genes including the hexanucleotide repeat expansion C9ORF72 that is associated with 40% of familiar ALS cases. This method excludes any PCR bias or errors from an otherwise hard to amplify region as well as preserves the basemodication in a single molecule fashion which allows you to capture mosaicism present in the sample.


June 1, 2021  |  

Structural variant in the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene found to be linked to bipolar disorder

Bipolar disorder (BD) is a phenotypically and genetically complex neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BD susceptibility, however no disease genes have yet to be identified. Here, we have fully characterized a ~12 Mb significantly linked (lod score=3.54) genomic region on chromosome Xq24-q27 in an extended family from a genetic isolate that was using long-read single molecule, real-time (SMRT) sequencing. The family segregates BD in at least 4 generations with 16 individuals out of 61 affected. Thus, this family portrays a highly elevated reoccurrence risk compared to the general population. It is expected that the genetic complexity would be reduced in isolated populations, even in genetically complex disorders such as BD, as in the case of this extended family. We selected 16 key individuals from the X-chromosomally linked family to be sequenced. These selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed a Nimblegen capture array enriching for 5-9 kb fragments spanning the entire 12 Mb region that were then sequenced using long-read SMRT sequencing to screen for causative structural variants (SVs) explaining the increased risk for BD in this extended family. Altogether, 192 SVs were detected in the critically linked region however most of these represented common variants that could be seen across many of the family members regardless of the disease status. One SV stood out that showed perfect segregation among all affected individuals that were carriers of the disease haplotype. This was a 330bp Alu deletion in intron 4 of the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene that has previously been shown to play a central role in brain development and function. Moreover, Alu elements in general have also previously been associated with at least 37 neurological and neurodegenerative disorders. In order to validate the finding and the functionality of the identified SV further studies like isoform characterization are warranted.


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