国家神经系统疾病临床医学研究中心数据管理与共享系统
Data Management And Sharing System of China National Clinical Research Center for Neurological Diseases
A novel KRIT1/CCM1 gene insertion mutation associated with cerebral cavernous malformations in a Chinese family.
第一作者：hu wang
通讯作者：wei li ,yongjun wang
2017-02-03
已发表文章
主要研究背景及目的
Cerebral cavernous malformation (CCM) is a vascular anomaly with defects consisting of dilated, thin-walled, and leaky capillaries without intervening nervous tissue. The clinical phenotype of CCM is highly variable. Approximately, 40% of CCM patients can be clinically asymptomatic, while the rest of them may suffer from recurrent headache, focal neurological deficits, cerebral hemorrhage, and seizures (Labauge et al. 2007). The disease can occur in sporadic or autosomal dominant inherited familial form. Most of CCM cases are sporadic while a small part of them are familial cerebral cavernous malformation (FCCM) (Lanfranconi et al. 2014; Rigamonti et al. 1988). Its true prevalence remains unknown. However, one large study of autopsy stated that the CCM prevalence was between 0.1–0.5% of the general population (Otten et al. 1989). And the FCCM is often characterized by incomplete penetrance and multiple lesions (Bacigaluppi et al. 2013). Moreover, more than 50% of familial cases were found to carry the KRIT1/CCM1 mutations (Denier et al.2006) which is the highest mutations among the three identified genes (KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3). As we know, the KRIT1/CCM1 encodes for the krev interaction trapped 1 (Krit1) protein, which is involved in maintaining the integrity of endothelial junctions, and may play an important role in microtubule targeting (Fischer et al. 2013; Sahoo et al. 1999). In previous studies, several cases have been reported from Italian, French, and Japanese families (D’Angelo et al. 2013; Denier et al. 2006; Tsutsumi et al. 2013). However, less than ten KRIT1/CCM1 mutations have been reported from Chinese families (Yang e t al. 2016) . Here, we present the clinical, neuroradiological, and molecular features of a Chinese family with CCM harboring a novel KRIT1 /CCM1 insertion mutation. This type of mutation is extremely rare , and no reports were found from Chinese families. Moreover, the CCM lesions in expanded ventricles were shown in an affected subject, and similar symptoms of hydrocephalus were observed, which were extremely rare in previous reports.
方法
Mutation Analysis Genomic DNA was extracted from peripheral blood lymphocytes of the proband (II-1) and three affected relatives (I-1, II-2, and II-3). KRIT1/CCM1, MGC4607/CCM2,and PDCD10/CCM3 coding exons and their intronic boundaries were amplified using polymerase chain reaction (PCR). PCR products underwent sequencing using the BigDye Terminator 3.1 protocol on a 3500Dx Genetic Analyzer (Applied Biosystems). The sequencing results were compared by NCBI/BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). KRIT1 mRNA Detection The total RNA of four affected subjects and four healthy controls were isolated from white blood cells with TRIzol® Reagent (Ambion™) according to the manufacturer’s instructions. To prepare first strand cDNA, mRNA was reverse transcribed with PrimeScript™ RT reagent Kit with gDNA Eraser (Takara) in a total 20 μl reaction mixture. To analyze mRNA level, real time PCR was performed with SYBR PCR Master Mix (Power® SYBR green, Applied Biosystems, Foster, CA) and the StepOne Plus™ Real-Time PCR System (Applied Biosystems). The primer of KRIT1(Foreword: GACCCGGAAGCTCTTTCACC; Reverse: CTCCTCCT GTTTCCCGAAGG) and GADPH (Foreword: CTATAAATTGAGCCCGCAGCC; Reverse: GCGCCCAATACGAC CAAATC) were used. After normalization with GAPDH mRNA, the target gene mRNA level in a sample was quantified by relative quantification method. Statistical Analysis Data were expressed as mean±SD. Statistical differences among the groups were assessed by independent samples t test. Statistical significance was set at P<0.05.
结论
The study demonstrates this Chinese FCCM with the novel KRIT1/CCM1 insertion mutation that has not been reported previously. Moreover, their mRNA level of KRIT1/CCM 1 was significantly decreased. Our finding expands the number of molecular defects underlying familial CCM and enhances our understanding of CCM mutations and the associated clinical phenotypes. It also helps to provide appropriate genetic counseling for CCM patients.
主要结果
Clinical Investigation Four members affected by FCCM were identified as shown in the three generation Chinese pedigree (Fig. 1). The proband (II-1) is a 32-year-old woman. She had complained of sudden faint with loss of consciousness while running at the age of 16. Then a definitive diagnosis was obtained by radiological evidence of cerebral space occupying lesion ( diameter, 3 cm) in the left superior temporal and the pathologic evidence. The lesion was surgically removed in 1999. And she was treated with prophy-lactic antiepileptic drug (AED) after operation. However, the seizures occurred 1 year later. It may be due to sudden drug withdrawal. The seizure semiology was described as a feeling of uncomfortable and tiredness before the epileptic seizure started. And it was followed by loss of awareness, head swinging, eyes closed jittering, tonic posturing of the limbs, and clonic movements. The sound and light could cause fear in the proband. Then she has infrequent seizures by taking carbamazepine regularly until now. The last neurological examination was performed in September 2016. The MRI (SWI sequence) results suggested that multiple areas of hypointense lesions were typically characterized by mixed signal intensity, and the hypointense region in left temporal was the evidence of postoperative changes (Fig. 2 a, b). Proband’s 52-year-old mother (I-1) had complained of intermittent headache and dizziness for 10 years. Onset time lasted from several hours to several days. And she suffered from diplopia after impacting her head recently. Cerebral MRI (SWI sequence) revealed severe multiple CCM lesions in the cerebrum, brainstem, and cerebellum, especially a few lesions were found in the lateral ventricle and the third ventricle (Fig. 2f–g), which probably originated from the choroid plex-us. Moreover, enlargement of the lateral ventricle was observed. The Evans’ index was 0.33. The hundreds of lesions were presented in MRI as mixed signal intensity, which were surrounded by hypointense rims. However, she was otherwise asymptomatic (last neurological examination performed in September 2016). Proband’s 26-year-old brother (II-2) (Fig. 2k–o) and 29-year-old sister (II-3) (Fig. 2c, d) claimed no symptoms despite of their MRI imaging showing the similar CCM lesions with different severities. Pathological Findings H&E staining showed the dilated vascular channels without intervening brain parenchyma. The muscularis of thin-walled capillaries are predominantly absent. Moreover, some fibrosis thrombosis can be found (Fig. 3a). The hemosiderin-laden macrophages were accumulated to the surrounding tissue (Fig. 3b). Molecular Genetic Study We found that in the proband, a heterozygous novel insertion mutation defined as c.1896_1897insT was found in the KRIT1/CCM1. Conversely, sequence analysis of CCM2 and CCM3 did not disclose pathogenetic mutations. KRIT1/CCM1 sequences of her mother, brother, and sister were also analyzed, and same mutation was found in all subjects. This c.1896_1897insT is a novel frameshift mutation within patients’ DNA (Fig.4). It changes the KRIT1/CCM1 gene reading frame, resulting in a premature stop codon (p.Pro633SerfsTer22). We checked this variant and it is not present at ExAC database. Evaluation of KRIT1/CCM1 mRNA Expressional Level To investigate the nonsense-mediated mRNA decay and function loss, we analyzed the mRNA expression of KRIT1/CCM1 gene. Our real-time PCR results revealed that, in comparison with the healthy control group, mRNA level of KRIT1/CCM1 was significantly decreased in CCM subjects (CCM family = 0.42 ± 0.20 vs. healthy control = 1.01 ± 0.16, P = 0.004, n = 4; Fig. 5).