Whole-exome sequencing reveals the genetic causes and modifiers of moyamoya syndrome

Abstract

Moyamoya vasculopathy secondary to various genetic disorders is classified as moyamoya syndrome (MMS). Recent studies indicate MMS occurs due to a combination of genetic modifiers and causative mutations for the primary genetic disorders. We performed whole-exome sequencing (WES) in 13 patients with various genetic disorders who developed MMS. WES successfully revealed the genetic diagnoses of neurofibromatosis type 1 (NF-1), Down syndrome, multisystemic smooth muscle dysfunction syndrome, Noonan syndrome, and alpha thalassemia. The previously reported modifier genes, RNF213 and MRVI1, were confirmed in the NF-1 and Down syndrome cases. Further analysis revealed rare hypomorphic variants in the causative genes of the primary disorders underlying MMS, such as Alagille syndrome and Rasopathies, conferred susceptibility to MMS. Genes involved in the development of pulmonary arterial hypertension (PAH), such as ABCC8 and BMPR2, were also identified as potential modifiers. The rare variants in the MMS and PAH genes were significantly enriched in the eight Japanese patients with MMS compared with the 104 Japanese individuals from the 1000 Genomes Project. Disease genes associated with the arterial occlusive conditions represented by those of Rasopathies and PAH may provide novel diagnostic markers and future therapeutic targets for MMS as well as moyamoya disease with an unknown cause.

Fig. 1

Minigene assays for the splicing mutations in NF1. (A) The pET01 constructs used in this study. Stretches of the exon–intron sequences containing the wild type or mutant alleles of c.4578-1G>A, c.3974G>A, and c.6474+1G>T in NF1 (NM_001042492) were cloned into the multiple cloning site of the pET01 vector. (B) RT- PCR analysis using HeLa cells transfected with an empty (mock), wild-type, or mutant pET01 vectors. The primer pair used for RT-PCR was located within the 3′ and 5′ exons. The RT-PCR products were separated on a 2% agarose gel and stained with ethidium bromide. (C) Sequencing chromatograms of the RT-PCR products. The product from the mutant c.4578-1A allele contained two types of abnormal intron retentions, whereas those from the mutant c.3974A and c.6474+1T alleles exhibited total skipping of the affected exons.

Fig. 2

Structural variations detected from the WES data. (A) Results of XHMM in the Down syndrome cases. Z-scores transformed from principal component analysis-normalized read depths shifted to positive throughout chromosome 21, indicating chromosomal amplification. Red dots represent amplifications with high quality scores above the threshold (Q = 30). A total of 53, 115, and 68 BAM files were used for the calculations for patients 7, 8, and 9, respectively. (B) XHMM of the patient with alpha thalassemia. Green dots represent deletions with quality scores above the threshold (Q = 30). A total of 40 BAM files were used for the calculation. (C) Copy number profile around the HBA1 and HBA2 loci from EXCAVATOR2. Log2R denotes the log-transformed ratio (log2 ratio) between the Window Mean Read Count (WMRC, window width = 20,000 bp) of the patient and 39 control samples. SegMean denotes the log2 ratio segment means obtained using the Heterogeneous Shifting Level Model (HSLM) segmentation algorithm.

Fig. 3

Minigene assay of the c.4412-2A>G mutation in ABCC8. (A) The sequence around the c.4478-2A>G mutation in ABCC8 (NM_000352) was cloned into multiple cloning sites of the pET01 vector. (B) RT-PCR analysis using HEK293T and COS7 cells transfected with an empty (mock), wild-type, or mutant pET01 vector. The RT-PCR products were separated on a 3% agarose gel and stained with ethidium bromide. (C) Sequencing chromatograms of the RT-PCR products. The products from the mutant vector contained three patterns of aberrant splicing, which were separated by cloning into the pT7 blue t-vector and BamHI-digested pGEM-3Zf(+) vector.

Fig. 4

Nluc-based bioluminescence analyses. (A) Intracellular expression of the HiBiT-tagged SMAD9. (B) TGF-beta/BMP signaling luciferase assay for SMAD9 p.E30K. (C) Cell-surface expression of the HiBiT-tagged BMPR2. p.R147X was used as a positive control because it is a known pathogenic mutation lacking transmembrane domain (ClinVar Accession: VCV000222513.13, rs869025366). (D) TGF-beta/BMP signaling luciferase assay for BMPR2 p.M365I. Error bars indicate the standard deviation. BRE, BRE-driven luciferase vector; *, p < 0.05; **, p < 0.01.

Fig. 5

Functional analyses of the p.P500S variant in PTGIS using HCtAECs. (A) Intracellular expression of the HiBiT-tagged PTGIS. (B) Concentration of 6-keto-PGF1alpha in the culture supernatant of HCtAECs. The horizontal lines represent the median values. **, p < 0.01.