Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest.Here, we present a short amplicon reverse transcription-polymerase chain reaction (RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.
Introduction
A growing number of studies have examined the link between inflammatory markers (IM) and the pathophysiology of obsessive-compulsive disorder (OCD). However, this association has yet to be fully identified.
Objectives
This review aims to systematically evaluate evidence from studies examining peripheral IM in adult participants with OCD compared to controls. IM included: CRP, TNFa, IFNγ, IL1/4/6/10.
Methods
Databases used for literature searching: Medline, Embase, PsycINFO (until October 2021). Studies that examined IM in the blood of adult OCD and control groups were included. Screening and data extraction adhered to PRISMA guideline standards. The quality assessment utilised funnel plots and the approach developed by Hawker et al. 2002. A random-effects meta-analysis model was adopted. PROSPERO reference number: CRD42021284766.
Results
The systematic review (19 studies, 1,225 participants) and meta-analysis (12 studies, 796 participants) had an average quality assessment score of 28.3 (medium quality) and 30.7 (high quality), respectively. The average heterogeneity of each IM analysed was 76.6%. Totalled, each study and IM analysis showed more insignificant differences (n=35) than significant differences (n=25). The
meta-analysis revealed no significant difference for overall IM assessments. However, a sub-analysis of IL6 (excluding studies using serum or lipopolysaccharide stimulation) found significantly lower levels of IL6 within the OCD group (effect size: 3.98 and 95% CI: 0.43,7.53).
Conclusions
This is an up-to-date systematic review examining IM in OCD. Insignificant results found may have resulted from the relatively high heterogeneity or varied study designs. One sub-analysis of IL6 identified an association, although further studies are required with larger sample sizes and fewer disparities.
Disclosure
No significant relationships.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.