Single immunoglobulin interleukin-1 receptor related (sigirr) variants and uses thereof
A technology of protein and nucleic acid molecules, applied in the field of genetics, can solve problems such as insufficient understanding of inflammatory bowel disease and patients not being genetically diagnosed
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Embodiment 1
[0207] Example 1: Patient recruitment and phenotyping
[0208] Whole-exome sequencing and trio-based variant analysis were performed on a 13-year-old patient with EO-IBD, his IBD-affected mother, and his unaffected father. The family has been probed for genetic evaluation of the patient for EO-IBD.
Embodiment 2
[0209] Example 2: Genomic samples
[0210] Genomic DNA was extracted from peripheral blood samples and transferred to the Regeneron Genetics Center (RGC) for whole exome sequencing and stored at -80°C in an automated biobank. Fluorescence-based quantification is performed to ensure DNA quantity and quality suitable for sequencing purposes.
[0211] 1 μg of DNA was sheared to an average fragment length of 150 base pairs (Covaris LE220) and prepared for exome capture with a custom kit from Kapa Biosystems. Samples were captured using NimbleGen SeqCap VCRome2.1 or Integrated DNA Technologies xGen Exome Target Design. Samples were barcoded, pooled and multiplexed for sequencing on an Illumina HiSeq 2500 using 75bp paired-end sequencing with v4 chemistry. Captured fragments were sequenced to achieve a minimum of 85% coverage of target bases at 20-fold or greater coverage. Following sequencing, data were processed using a cloud-based pipeline developed at RGC that uses DNAnexus a...
Embodiment 3
[0212] Example 3: Genomic Data Analysis
[0213]Standard quality control filters for minimum read depth (>10), genotype quality (>30), and allelic balance (>20%) were applied to the identified variants. Annotation and analysis pipelines developed using RGCs classify and annotate passing variants (whether synonymous, non-synonymous, spliced, frameshifted, or non-frameshifted) based on their potential functional impact. Family relationships were verified by measures derived from ancestry identity from genetic data using PRIMUS (Staples et al., Amer. J. Human Genet., 2014, 95, 553-564) and cross-referencing with pedigrees reported for this family to Infer associations and relationships within groups.
[0214] Given the reported family history, pedigree-based variant analysis and segregation were performed to identify candidate disease genes under an autosomal dominant pattern of inheritance. Variants that were shared between the affected proband and his affected mother but not ...
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