Driving mosaicism: somatic variants in reference population databases and effect on variant interpretation in rare genetic disease.

BACKGROUND: Genetic variation databases provide invaluable information on the presence and frequency of genetic variants in the 'untargeted' human population, aggregated with the primary goal to facilitate the interpretation of clinically important variants. The presence of somatic variants in such databases can affect variant assessment in undiagnosed rare disease (RD) patients. Previously, the impact of somatic mosaicism was only considered in relation to two Mendelian disease-associated genes. Here, we expand the analyses to identify additional mosaicism-prone genes in blood-derived reference population databases. RESULTS: To identify additional mosaicism-prone genes relevant to RDs, we focused on known/previously established ClinVar pathogenic and likely pathogenic single-nucleotide variants, residing in genes associated with early onset, severe autosomal dominant diseases. We asked whether any of these variants are present in a higher-than-expected frequency in the reference population databases and whether there is evidence of somatic origin (i.e., allelic imbalance) rather than germline heterozygosity (~ half of the reads supporting alternative allele). The mosaicism-prone genes identified were further categorized according to the processes they are involved in. Beyond the previously reported ASXL1 and DNMT3A, we identified 7 additional autosomal dominant RD-associated genes with known pathogenic single-nucleotide variants present in the reference population databases and good evidence of allelic imbalance: BRAF, CBL, FGFR3, IDH2, KRAS, PTPN11 and SETBP1. From this group of 9 genes, the majority (n = 7) was important for hematopoiesis. In addition, 4 of these genes were involved in cell proliferation. Further assessment of the known 156 hematopoietic genes led to identification of 48 genes (21 not yet associated with RDs) with at least some evidence of mosaicism detectable in reference population databases. CONCLUSIONS: These results stress the importance of considering genes involved in hematopoiesis and cell proliferation when interpreting the presence and frequency of genetic variants in blood-derived reference population databases, both public and private. This is especially important when considering new variants of uncertain significance in known hematopoietic/cell proliferation RD genes and future novel gene-disease associations involving this class of genes.

Transcriptome profiling revealed early vascular smooth muscle cell gene activation following focal ischemic stroke in female rats - comparisons with males.

BACKGROUND: Women account for 60% of all stroke deaths and are more often permanently disabled than men, despite their higher observed stroke incidence. Considering the clinical population affected by stroke, an obvious drawback is that many pre-clinical and clinical studies only investigate young males. To improve therapeutic translation from bench to bedside, we believe that it is advantageous to include both sexes in experimental models of stroke. The aims of this study were to identify early cerebral vascular responses to ischemic stroke in females, compare the differential gene expression patterns with those seen in males, and identify potential new therapeutic targets. RESULTS: Transient middle cerebral artery occlusion (tMCAO) was used to induce stroke in both female and male rats, the middle cerebral arteries (MCAs) were isolated 3 h post reperfusion and RNA was extracted. Affymetrix whole transcriptome expression profiling was performed on female (n = 12) MCAs to reveal differentially expressed genes. In total, 1076 genes had an increased expression and 879 genes a decreased expression in the occluded MCAs as compared with the control MCAs from female rats. An enrichment of genes related to apoptosis, regulation of transcription, protein autophosphorylation, inflammation, oxidative stress, and tissue repair and recovery were seen in the occluded MCA. The high expression genes chosen for qPCR verification (Adamts4, Olr1, JunB, Fosl1, Serpine1, S1pr3, Ccl2 and Socs3) were all shown to be upregulated in the same manner in both females and males after tMCAO (p < 0.05; n = 23). When comparing the differentially expressed genes in female MCAs (occluded and non-occluded) with our previous findings in males after tMCAO, a total of 297 genes overlapped (all groups had 32 genes in common). CONCLUSIONS: The cascades of processes initiated in the vasculature following reperfusion are complex. Dynamic gene expression alterations were observed in the occluded MCAs, and to a less pronounced degree in the non-occluded MCAs. Dysregulation of inflammation and blood-brain barrier breakdown are possible pharmacological targets. The sample of genes (< 1% of the differentially expressed genes) validated for this microarray did not reveal any sex differences. However, sex differences might be observed for other gene targets.

Promote Biomarker Discovery by Identifying Homogenous Primary Headache Subgroups.

Within- and between-study heterogeneity impede identification of valid primary headache biomarkers. Homogenous subgroup identification and investigation of differential biochemical profiles and networks within and across headache categories, based on statistical techniques, might promote biomarker discovery. When studying common primary headaches with a multifactorial etiology, variability might be captured at different levels (eg, genetics, clinical features, comorbidities, triggers). Moreover, focus on biochemical profiles instead of single compounds is crucial to develop strategies for accurate differential diagnosis.

Expression of Pituitary Adenylate Cyclase-activating Peptide, Calcitonin Gene-related Peptide and Headache Targets in the Trigeminal Ganglia of Rats and Humans.

Neurotransmitter and headache target localization in the trigeminal ganglia (TG) might increase the understanding of sites of action, and mechanisms related to headache therapy. The overall aim of the study was to investigate the presence of migraine targets in the TG with particular emphasis on pituitary adenylate cyclase-activating peptide (PACAP) and calcitonin gene-related peptide (CGRP), known to be involved in cranial pain processing, and selected headache targets. Rat- and human TG were processed for immunohistochemistry. PACAP-38, CGRP and the headache targets were expressed in rat and human TG. PACAP receptors were confined to neurons and satellite glial cells (SGCs), however with variability between the receptor subtypes PACAP type I receptor (PAC1) and vasoactive intestinal peptide/PACAP receptors 1/2 (VPAC1/2). 5-Hydroxytryptamine (5-HT) receptors were expressed in neuronal somas in rat and human TG (human TG frequency: 5-HT1D > 5-HT1B/1F). Synaptosomal-associated protein 25 kDa (SNAP25) was primarily expressed in SGCs in humans, and neurons in rats, while synaptic vesicle glycoprotein 2A (SV2-A) was confined to SGCs and some neurons in rats and humans. Occasionally, PACAP-38-positive cells also expressed VPAC1, SNAP25 and SV2-A. VPAC1 was generally detected in SGCs enveloping PACAP-38-positive and -negative neuronal somas. Our study revealed potential sites of actions for anti-headache drugs such as PACAP receptor antagonists, Lasmiditan (5-HT1F agonist) and Botox (blocks exocytosis through SV2-A/SNAP25) in rat and human TG and considerable overlap between species in expression to specific cell types, except for VPAC1 and SNAP25.

Cerebrovascular Gene Expression in Spontaneously Hypertensive Rats After Transient Middle Cerebral Artery Occlusion.

Hypertension is a major risk factor for stroke, which is one of the leading global causes of death. In the search for new and effective therapeutic targets in stroke research, we need to understand the influence of hypertension in the vasculature following stroke. We used Affymetrix whole-transcriptome expression profiling as a tool to address gene expression differences between the occluded and non-occluded middle cerebral arteries (MCAs) from spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats after transient middle cerebral artery occlusion (tMCAO), to provide clues about the pathological mechanisms set in play after stroke. Verified by quantitative PCR, expression of Ccl2, Edn1, Tgfß2, Olr1 and Serpine1 was significantly increased in the occluded compared to non-occluded MCAs from both SHRs and WKY rats. Additionally, expression of Mmp9, Icam1, Hif1α and Timp1 was increased in the occluded compared to non-occluded MCAs isolated from WKY rats. In comparison between occluded MCAs from SHRs versus occluded MCAs from WKY rats, expression of Ccl2, Olr1 and Serpine1 was significantly increased in SHR MCAs. However, the opposite was observed regarding expression of Edn1. Thus these data suggest that Ccl2, Edn1, Tgfß2, Olr1 and Serpine1 may be possible mediators of the vascular changes in the occluded MCAs from both SHRs and WKY rats after tMCAO. The aforementioned genes possess biological functions that are consistent with early stroke injuries. In conclusion, these genes may be potential targets in future strategies for acute stroke treatments that can be used in patients with and without hypertension.

Cerebrovascular gene expression in spontaneously hypertensive rats.

Hypertension is a hemodynamic disorder and one of the most important and well-established risk factors for vascular diseases such as stroke. Blood vessels exposed to chronic shear stress develop structural changes and remodeling of the vascular wall through many complex mechanisms. However, the molecular mechanisms involved are not fully understood. Hypertension-susceptible genes may provide a novel insight into potential molecular mechanisms of hypertension and secondary complications associated with hypertension. The aim of this exploratory study was to identify gene expression differences in the middle cerebral arteries between 12-week-old male spontaneously hypertensive rats and their normotensive Wistar-Kyoto rats using an Affymetrix whole-transcriptome expression profiling. Quantitative PCR and western blotting were used to verify genes of interest. 169 genes were differentially expressed in the middle cerebral arteries from hypertensive compared to normotensive rats. The gene expression of 72 genes was decreased and the gene expression of 97 genes was increased. The following genes with a fold difference ≥1.40 were verified by quantitative PCR; Postn, Olr1, Fas, Vldlr, Mmp2, Timp1, Serpine1, Mmp11, Cd34, Ptgs1 and Ptgs2. The gene expression of Postn, Olr1, Fas, Vldlr, Mmp2, Timp1 and Serpine1 and the protein expression of LOX1 (also known as OLR1) were significantly increased in the middle cerebral arteries from spontaneously hypertensive rats compared to Wistar-Kyoto rats. In conclusion, the identified genes in the middle cerebral arteries from spontaneously hypertensive rats could be possible mediators of the vascular changes and secondary complications associated with hypertension. This study supports the selection of key genes to investigate in the future research of hypertension-induced end-organ damage.