Introduction

Moyamoya disease (MMD) is a rare cerebrovascular disease of unknown etiology, characterized by stenosis or occlusion of the end of the internal carotid artery and the formation of an abnormally thin network of blood vessels near the occlusion artery1. MMD exhibits primary clinical symptoms of ischemic and/or hemorrhagic stroke and cognitive impairment, affecting both children and adults, thereby increasing the likelihood of disability and death due to the unavailability of effective treatments addressing its pathogenesis2,3.

In recent times, scholars have conducted extensive research on the etiology and pathogenesis of MMD, unearthing numerous promising genes. However, despite these advancements, the precise etiology of moyamoya disease remains indeterminate, underscoring the necessity for in-depth investigation of MMD genes4. The emergence of high-throughput sequencing technology identified potential genes associated with moyamoya disease susceptibility, notably RNF213, ACTA2 and MYH115,6,7. Unfortunately, none of this has been successfully tested in mouse models. Mammalian homolog of Drosophila diaphanous 1 (DIAPH1) is a formin protein encoding the RhoA GTPase mDia, which plays an important role in vascular remodeling and thrombosis. A study showed that the expression of DIAPH1 gene was significantly down-regulated in patients with ischemic stroke8, furthermore, a recent genetic association study using whole-gene exon sequencing identified rare damaging variants of DIAPH1 in 24-proband discovery cohort and an 84-proband validation cohort of non–East Asian patients with MMD. These mutations may related to thrombocytopenia and clinical symptoms such as ischemic stroke and angiographical stage9. But at present, there is no study on DIAPH1 gene mutation in Asian population, so we report DIAPH1 gene mutation in the Asian patient and further investigate its associated clinical features, in particular the angiography results of the posterior cerebral artery (PCA). Our study will help clarify the genetic background of moyamoya disease in different ethnic groups and provide opportunities for early diagnosis and surgical treatment of moyamoya disease.

Results

A total of 50 patients were included in our study (Table 1). There were 25 females and 25 males. The median age at diagnosis was 24 years. Of the 50 patients, 4 (8%) had a history of hypertension, 3 (6%) had a history of smoking, almost all participants were found not to have any underlying diseases that could affect the results of the study. Infarction was the most common initial symptom (25, 50%). The others were hemorrhage (17, 34%), TIA (8, 16%). PCA involvement was observed in 12 (23.8%) patients (Table 1).

Table 1 Sample demographics of patients with MMD.
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DNA sequencing results

Nine gene mutations were detected in 50 moyamoya disease patients, including 7 synonymous mutations and 2 missense mutations. Of the 7 synonymous mutations, 5 are rs2302102 (p.Gly1193=, c.3579C>T in exon27), one is rs199683670(p.Asp210=, c.630T>C in exon 7), another one is no datebase rs number (p.D349=, c.1047C>T in exon 11). Of the 2 missense mutations, one is rs142480526 (p.A67V, c.200C>T in exon3), another one is no datebase rs number (p.E1120K, c.3358G>A on exon 25). The same mutations in non-east Asians as previously described were not reported, more details are in supplementary materials 1.

Clinical and DSA features between different group

The DSA image of 9 MMD patients with DIAPH1 gene mutation is shown in Fig. 1, with each image is named by their exon fragments. Among them, 7 cases had typical PCA involvement (Fig. 2), while only 5 cases in the group without genetic mutation were involved, the constituent ratio of the former (77.8%) was significantly higher than that of the latter (12.2%) (p = 0.001), but there was no significant statistical difference in age, sex, initial symptoms, platelet count, BMI, Suzuki stage and so on (Table 2).

Figure 1
figure 1

The anteroposterior (AP) view of internal carotid artery (ICA) angiography of 9 MMD patients with DIAPH1 mutation. What red arrows indicate is the typical MMD vascular manifestation.

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Figure 2
figure 2

Six anteroposterior and one lateral (No. 6) vertebral arteriography showed typical PCA involvement in 7 patients in the group of DIAPH1mutation. The red arrow shows the involved posterior cerebral artery. Areas under the ROC curve of DIAPH1 mutation as a independent risk factor.

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Table 2 Characteristics between different group.
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Logistic regression analysis of variables associated with PCA involvement

Between groups with and without PCA involvement, there were significant statistical differences in DIAPH1 mutation and compensation of leptomeningeal from posterior to anterior. DIAPH1 mutation was more frequently observed in the PCA involvement than in the PCA non-involvement group (58.3% versus 5.3%; p = 0.001). Compensation of leptomeningeal from posterior to anterior was more frequently observed in the PCA non-involvement than in the PCA involvement group (58.3% versus 5.3%; p = 0.001). But there were no significant differences in sex, age, first symptom and clinical features. Multivariate logistic regression analysis of risk factors for PCA involvement showed that after adjusting for sex, age, platelet count, there is an association between DIAPH1 mutation and PCA involvement (OR 29.483; 95% CI 3.920–221.736; p = 0.001) (Table 3), areas under the ROC curve is 82.79 (Fig. 2).

Table 3 Logistic regression analysis of predictors for PCF.
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Discussion

The main purpose of this study is to explore the genetic background of moyamoya disease in different ethnic groups. We have identified a different Asian Moyamoya disease and European DIAPH1 mutation with a site associated with posterior circulation involvement.

As reported by Kundishora et al.9, DIAPH1 is a novel moyamoya disease risk gene in non-East Asian patients with moyamoya disease. They conducted a genetic association study using whole-exome sequencing. We found two new mutations (p.A67V, c.200C>T in exon3; p.E1120K, c.3358G>A on exon 25) of the DIAPH1 gene in two Asian MMD. DIAPH1 is a formin protein encoding the RhoA GTPase mDia10, which can stimulates actin filament assembly at the barbed ends after being activated by GTP-bound RhoA11. DIAPH1 induces platelet formation in megakaryocytes by regulating actin and microtubule cytoskeleton12, which could causes macrothrombocytopenia and extends the spectrum of DIAPH1-related disease13. DIAPH1 also mediates vascular remodeling by integrating oxidative stress and signal transduction pathways in smooth muscle cells. In cardiovascular and cerebrovascular diseases, DIAPH1 gene mutations increase the genetic susceptibility to the risk of cerebral ischemic stroke8. Several studies have proposed the effect of DIAPH1 on angiogenesis from different perspectives14. Studies published in mice lacking DIAPH1 have shown that the mouse is protected from hyperdilation of the femoral intima/medium after endothelial stripping, and has reduced infarct size and loss of cardiac function after ligation of the coronary artery in the left anterior descending branch of the heart15. And the deletion of DIAPH1 can reduce infarct size after the occurrence of myocardial infarction16.

The Receptor for Advanced Glycation End Products (RAGE) is expressed by multiple cell types in the brain and spinal cord that are associated with the mechanism of neurovascular and neurodegenerative disorders, such as vascular cells (endothelial cells, smooth muscle cells and pericytes), neurons and glia (microglia and astrocytes)17. Current structural and functional evidence points to the interaction of the RAGE cytoplasmic domain with the forming protein DIAPH1 as a key cytoplasmic hub for RAGE ligand-mediated cell signaling activation. A SNP rs1035798 gene variant of RAGE was found to be significantly associated with the subtype of small vessel disease (SVD) (OR 1.56, 95% CI 1.16–2.09), adjusted p-value < 0.05), and the association was independent of hypertension, diabetes, and smoking18.

We found a very interesting phenomenon that the probability of PCA involvement in the DIAPH1 gene mutation group was significantly higher than that in the non-mutation group (77.8% versus 12.2%, p = 0.000). PCA involvement could affect the leptomeningeal collateral flow from the PCA to anterior circulation, which could influence clinical outcomes19. These patients often had more severe anterior circulation stenosis and more likely to have cerebral infarction, which often led to bad prognosis20. About 1/3 of MMD patients had PCA involvement21. Previous studies have proved that genetic factors can affect the clinical manifestations of moyamoya patients, for example, the pR4810K polymorphism of RNF213 gene is closely related to the risk of MMD. Patients with AA genotype have earlier onset age and PCA involvement than those with GG and GA genotypes22,23, and the first symptoms are more likely to be cerebral infarction24. DIAPH1 is a key effector of actin remodeling in vascular cells and platelets13, which mediates vascular remodeling by integrating oxidative stress and signal transduction pathways in smooth muscle cells8. Furthermore, it has been reported that to a lesser extent DIAPH1 silencing has disrupted stress fibers in endothelial cells. And miR-24 targets on DIAPH1 downstream of Rho signaling, therefore repressing the actin polymerization through Profilin14. Therefore, similar to the effect of RNF213 on angiogenesis, we speculate that MMD patients with DIAPH1 gene deficiency can produce vascular intima and media thinning induced by ischemia, so blood vessels are vulnerable to hemodynamic stress and secondary injury, which may promote the involvement of posterior cerebral artery.

In addition, there were limitations in this study. The relatively small sample size may deviate the interpretation of cerebrovascular changes, so it is necessary to improve the statistical efficiency to determine the accuracy of the results. In future studies, a larger, multi-ethnic sample size is needed to determine the significance of DIAPH1 mutations in MMD. Nevertheless, this study has certain clinical significance to clarify the cerebrovascular damage of MMD under different genetic backgrounds.

Conclusion

In conclusion, it is premature to conclude an association between DIAPH1 gene and MMD, especially in Asian patients. However, we found a strong association between DIAPH1 gene mutation and PCA involvement.

Methods

The data that support the findings of this study are available from the corresponding author on reasonable request.

Study design and inclusion and exclusion criteria

The whole blood samples used in this study were obtained from patients admitted to Beijing Tiantan Hospital from October 2020 to August 2022. Patients were eligible if (1) they were diagnosed with unilateral or bilateral MMD by digital subtraction angiography (DSA), following the lastest Japanese guidelines25; (2) under the age of 50; (3) agree to participate in the study and provide a blood sample; (4) without serious underlying diseases that could affect the results of the study. Patients were not eligible if (1) they refused to participate in the study; (2) without DSA; (3) lack of laboratory data; (4) Moyamoya syndrome with thalassemia, Down syndrome and systemic immune system disease. The study was approved by the Institutional Review Board of Beijing Tiantan Hospital. This experiment was approved by the Ethics Committee of Beijing Tiantan Hospital (KY 2020-045-02) and all methods were performed in accordance with the relevant guidelines and regulations. Informed consents were obtained from patients or their representatives.

Baseline data collection

Information on sex, age, first symptoms, ethnicity, place of birth, BMI, platelet count, DSA data were collected. The first symptoms were transient ischemic attack (TIA), cerebral infarction and cerebral hemorrhage. Besides, peripheral blood samples were collected after admission for all patients. Levels of routine and biochemical blood tests were measured using enzymatic methods.

Classification and evaluation of angiographic variables

Suzuki stage, posterior cerebral artery (PCA) involvement, compensation of leptomeningeal from posterior to anterior were evaluated. If the occlusion or stenosis of P1–P3 segments of unilateral or bilateral PCA is confirmed by digital subtraction angiography, PCA involvement is considered26. Two experienced neurosurgeons (Y.Z. and R.W.) independently evaluated the angiographic findings.

Variant detection of DIAPH1

Refer to Relax Gene Blood DNA system Instruction Book, genomic DNA from patients was extracted from peripheral blood samples using a Magnetic bead method genome extraction kit (NMG0121-100, CHINA). Design primers for DIAPH1 gene using Primer Premier 5 software, all primers are in supplementary materials 2. The primer sequence of DIAPH1 gene was amplified by polymerase chain reaction (PCR) amplification instrument, the PCR reaction conditions are as follows: pre-denaturation at 96 °C last for 5 min; denaturation at 96 °C for 20 s; annealing at 60 °C for 20 s; chain extension at 72 °C for 30 s, amplification for 35 cycles, and finally supplementary extension at 72 °C for 10 min. The PCR system is 25 μL. 3 μL PCR products were detected by 1.0% agarose gel electrophoresis, and then purified according to the standard operating procedure of magnetic bead purification. The purified product was sequenced by ABI3730xL sequencer. The data was processed using the phred\phrap software.

Statistical analysis

Statistics analyses were performed using SPSS (version 26.0) and GraphPad Prism (version 9.4). Measurements that matched the normal distribution were presented as X ± SD, the independent sample t test was used for the comparison between groups. Measurements that did not matched the normal distribution were presented as median and interquartile range; the Mannwhitney U test was used for the comparison between groups. The counting data was expressed as frequency and percentage, and the comparison between groups was compared with χ2 test or Fisher accurate test. Multivariate logistic regression analysis was used to determine the independent risk factors of PCA involvement. Two-sided test p < 0.05 was statistically significant.