Analysis Of Clinical Features And Pathogenesis Of HTRA1-cerebral Small Vessel Disease

BackgroundCerebral small vessel disease(CSVD)is a syndrome characterized by recurrent strokes and vascular cognitive impairment caused by different etiologies,involving cerebral small vessels and surrounding brain parenchyma.Cerebral autosomal dominant arteriopathy with subcortical infarcts and white matter encephalopathy leukoencephalopathy(CADASIL)caused by NOTCH3 gene mutations was the most common monogenic inherited CSVDs.Migraines,recurrent ischemic strokes,cognitive impairment and mood disorders are common symptoms of CADASIL.However,it has been found that some patients with a similar clinical phenotype of CADASIL do not carry the NOTCH3 gene mutations,but heterozygous Htr A serine protease-1(HTRA1)gene mutations in clinical practice.This late-onset autosomal dominant CSVD caused by heterozygous HTRA1 mutations is called HTRA1-Cerebral small vessel disease(HTRA1-CSVD).Studies have shown that nearly 5% of CSVD with a family history are associated with heterozygous HTRA1 gene mutations.However,the clinical cognition of HTRA1-CSVD is limited,and its pathogenesis is not very clear.ObjectiveTo screen HTRA1 gene in patients with CSVD of Han nationality in Henan province suspected of CADASIL in clinical practice with negative pathogenic mutation of NOTCH3 gene exon 2-24,and analyze and summary the clinical features of patients with HTRA1-CSVD.To study the effect of heterozygous HTRA1 gene mutations on the cell phenotype,explore the possible pathogenesis of HTRA1-CSVD,which provides a theoretical basis for clarifying the pathogenesis of HTRA1-CSVD and searching for potential targeting drugs.MethodsFirstly,Sanger sequencing and/or whole exome sequencing were used to screen all exomes of HTRA1 gene in 130 CSVD patients of Han nationality in Henan province suspected of CADASIL in clinical practice with negative pathogenic mutation of NOTCH3 gene exon 2-24,and bioinformatics analysis and pathogenicity assessment of heterozygous HTRA1 gene mutations were performed.Then,the detailed clinical data of patients with HTRA1-CSVD were retrospectively analyzed and the clinical and radiological features were summarized.Finally,Human embryonic kidney 293T(HEK 293T)cells were transfected with wild type(WT)and mutant(G283R and S328A)HTRA1 overexpressed plasmids in vitro to observe the effects of heterozygous HTRA1 mutations on the HTRA1 protease activity,the TGF-β1signaling and the cell apoptosis.Graph Pad Prism 8.0 software was used to statistical analysis and draw the results.Data were compared by unpaired Student’s t test and one-way analysis of variance and expressed as the mean ± standard deviation.p<0.05 was considered as significant difference.Results6 heterozygous HTRA1 mutations were found [c.242G>A(p.G81D),c.412C>T(p.R138C),c.715G>A(p.E239K),c.847G>A(p.G283R),c.923G>C(p.G308A)and c.992C>T(p.P331L)] in this study,and five of which were reported for the first time.The 6 patients with heterozygous HTRA1 gene mutations were respectively from 6 unrelated pedigrees,including 2 females and 4 males.The age of first ischemic stroke was ranged from 42 to 71 years old,and all of whom have a family history of stroke or vascular dementia.The mainly clinical characteristics included recurrent ischemic strokes(5 cases),cognitive impairment(5 cases),mood disorder(4 cases),alopecia(2 cases)and spondylosis(4 cases).Magnetic resonance imaging(MRI)showed that all 6 patients had white matter hyperintensities and lacunar infarcts,5 patients had cerebral microbleeds,and 4 patients had spinal lesions.In vitro experiments,we found that the HTRA1 protease activity in the G283 R group was significantly decreased compared with WT group(p<0.0001),which was comparable to the HTRA1 protease activity in the S328 A group,and this was associated with a significant dominant negative effect of G283 R mutation on WT HTRA1 protease activity(p<0.05).Compared with WT group,the TGF-β1/Smad signaling was up-regulated(p<0.05),the expression of cleaved caspase3 and BAX were increased(p<0.05),the expression of BCL-2 was decreased(p<0.01),mitochondrial membrane potential in cells was significantly decreased(p<0.01),and the apoptosis was significantly increased(p<0.01)in G283 R group.These changes could be obviously ameliorated by SB431542(TGF-β1/Smad signaling pathway inhibitor)(p<0.05).Conclusion1.It is necessary to screen for HTRA1 gene when the pathogenic mutations in 2-24 exons of NOTCH3 gene are negative for CSVD patients suspected of CADASIL in clinical practice.2.G283 R heterozygous HTRA1 mutation leaded to the loss of HTRA1 protease activity through dominant negative effect,which leaded to the up-regulation of TGF-β1/Smad signaling,and may induce apoptosis through mitochondrial dysfunction.3.TGF-β1/Smad signaling pathway inhibitors may be a potential therapeutic drug for HTRA1-CSVD.