The aim of this study was to identify the causative gene in patients with overlapping NS phenotype who had the 12q12-13.11 deletion. T2-weighted axial images of mice taken at 7 to 8 months in each of ten wild-type mice. Intellectual disability (ID), which is a neurodevelopmental condition associated with impaired intellectual and adaptive function, has a prevalence of 2–3% in the general population (1, 2).
The molecular pathology underlying NS and NS-related disorders is a functional change, or gain-of-function, in the Ras-Mitogen-activated protein kinase (MAPK) signaling pathway, which is involved in growth factor-mediated cell proliferation, differentiation, and apoptosis (7). Mutations in genes encoding the proteins of the SWI/SNF complex cause neurodevelopmental disorders, including ID and autism spectrum disorders. The expression of IFITM1 (MIM, 604456), an interferon-inducible gene, is dependent on ARID2 expression, and IFITM1 expression is inversely related to ERK activation (27).
The aim of this study was to identify the causative gene in a patient with an overlapping NS phenotype who had a 3.7 Mb microdeletion in the 12q1-13.11 region and to investigate the downstream transcriptional effects of the deleted genes.
Patient
Microarray-based comparative genomic hybridization study
Whole exome sequencing (WES)
Target Enrichment System (Aligent Technologies, Santa Clara, CA, USA) and then sequenced on the Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA). The single nucleotide variants (SNVs) and short insertion-deletion variants were called with SAMtools software with reference to dbSNP and 1,000 genomes.
In vitro transfection with small hairpin RNA (shRNA)
ERK activation assay
Analysis of IFITM1 and CAV-1 expression in skin fibroblast
The Western blot analysis was performed with anti-IFITM1 (Genetex Inc., Irvine, CA, USA), anti-CAV1 (Genetex Inc., Irvine, CA, USA) and anti-β-Actin antibodies (Bioworld, Dublin, OH , USA). vol/vol) bisacrylamide-acrylamide 37.5:1] and transferred to nitrocellulose membranes, which were immunoblotted with specific antibodies. Goat polyclonal to rabbit IgG (Genetex Inc., Irvine, CA, USA), or mouse IgG (Genetex Inc., Irvine, CA, USA) was used as a secondary antibody.
Ras-GTP activity assay
Generation of Arid2 knockout mice using CRISPR/Cas9
C57BL/6N and ICR mice (Orient Bio Inc., Republic of Korea) were used as embryo donors and foster mothers, respectively, and CRISPR/Cas9-mediated gene targeting in mice was performed as previously described ( 31 ). Founder mice with frameshift mutations were identified by Sanger sequencing of the target region of Arid2 gene using PCR products amplified from genomic DNA samples isolated from the tail biopsy samples of mouse pups with the following primer pair: 5-GCGTTTGAACCGCGATCT-3 and 5 - CAGGGATGGCTTTAA -3. Selected founders with null mutations were crossed to wild-type mice, and the null mutant alleles were confirmed in F1 progeny by Sanger sequencing.
The parental Arid2+/- mice were backcrossed into C57BL/6 for over three generations. The Arid2+/- mice were tail genotyped. Ten adult Arid2+/- and wild-type (WT) mice were selected to identify dysmorphology. Magnetic resonance imaging (MRI) of the brain was performed on the mice aged 7 to 8 months.
Morris water maze (MWM) test
Open Field Test
Statistical analysis
Clinical and genetic characteristics of six patients with 12q12 deletion
Genomic alterations at 12q12 in six patients with Noonan-like disorders and list of genes in this region. Bilateral ptosis, hypertelorism, downward-sloping palpebral fissures, epicanthus, long philtrum, micrognathia, high arched palate, clefts. Inclined palpebral fissures, short broad nose with upturned nostril, small mouth, low-set ears, high arched palate.
Hypertelorism, downslanting palpebral fissures, bilateral ptosis, thin eyebrows, low and posteriorly rotated ears, mild micrognathia. Hypertelorism, ptosis, downslanting palpebral fissures, high arched palate, thick lips, micrognathia, set low and posterior.
Haploinsufficiency of ARID2 increases Ras-MAPK pathway activity
The Ras-MAPK pathway activity, represented by the ratio of phosphorylated ERK1 (P-ERK1) to total ERK1 and P-ERK2 to total ERK2, was significantly higher in those treated with shRNA-ARID2 under EGF (10 ng/mL) stimulation. No significant changes were observed in the activity of AKT and the mTOR signaling pathway, which was represented by Akt and RSP6, respectively, after with shRNA-ARID2.
Haploinsufficiency of ARID2 is related to decreased expression of IFITM1 and CAV-1
In the fibroblast of the patient with ARID2 haploinsufficiency, the expression of IFITM1 and CAV-1 was decreased, which are inversely related to the activation of ERK. There were no significant differences in brain size, distances between orbits and between babies, as measured by MRI. Frontal cortex thickness and the ratio of frontal cortex thickness to total brain thickness were significantly reduced compared to WT.
The volume of the hippocampus and the ratio of the volume of the hippocampus to total brain weight were greater in Arid2+/− compared to WT (Fig. 7). Axial T2-weighted images of the mice taken at 7 to 8 months in each of ten wild-type and Arid2+/- mice. However, there were no differences in total distances traveled in the Morris water maze vs.
The phenotype of this mouse model recapitulated that of a patient with ARID2 haploinsufficiency, which is short in stature and ID. Because pathogenic variants in SWI/SNF-associated genes result in classic CSS, NBS, and many overlapping phenotypic spectra ranging from syndromic ID to severe atypical CSS, the term “SWI/SNF-associated intellectual disability ( SSRIDD)" ( 36, 37). NS and NS-related disorders display a unique phenotype, but because a common mechanism of enhanced Ras-MAPK pathway signaling results in ERK activation, they share many clinical features, including craniofacial dysmorphism, cardiac malformations, ocular, skin, and musculoskeletal abnormalities, and increased risk of cancer.
In the present study, we demonstrated that ARID2 haploinsufficiency was selectively associated with increased ERK activation. This inhibition of the Ras-MAPK pathway was expected to be mediated through its interaction with IFITM1 and CAV-1 ( Fig. 8 ). There were no neuroanatomical abnormalities except for reduced cerebral cortex size and large hippocampus volume, but we did not perform a detailed histological examination.
Maximal induction of a subset of interferon target genes requires the chromatin remodeling activity of the BAF complex. Clinical correlates of mutations affecting six components of the SWI/SNF complex: detailed description of 21 patients and review of the literature.
Arid2 +/- mice had reduced body size, thin frontal cortex, and large hippocampus
Arid2 +/- mice show deficits in spatial learning and memory
Pórke umi variante patógena umi genes asociados SWI/SNF-pe oreko resultado CSS clásico, NBS ha heta espectro fenótipo oñembojoajúva ohóva ID síndrómico guive CSS atípico severo peve. Mbyky, avei ID, ha’e ambue mba’e ojehecharamovéva haploinsuficiencia ARID2 ha mutaciones SWI/SNF rehegua. Detalles: ARID2 haꞌehína peteĩ ingrediente activo Ras-MAPK rehegua oĩva serie ARID2-pe.
목적: 지적 장애는 지능과 적응 행동이 손상되는 발달 장애로 단일 유전자 이상부터 염색체 이상까지 다양한 유전적 돌연변이로 인해 발생할 수 있습니다. 본 연구에서는 경도 지적 장애, 저신장, 안면 기형 등 누난증후군군과 유사한 임상양상을 보인 12q11 미세결실 환자의 표현형이 반수체 결손과 연관되어 있음을 밝히고자 하였다. ARID2 유전자의 방법: 삭제된 유전자와 관련된 신호 전달 경로를 조사하기 위해 HeLa 세포의 작은 헤어핀 RNA를 사용하여 녹아웃 연구를 수행했습니다.
또한 CRISPR/Cas9 시스템을 사용하여 Arid2 반수체 결실이 있는 마우스 모델을 구축하여 이 유전자가 환자의 임상 특성과 관련이 있는지 조사했습니다. 결과: 유전자가 결실된 HeLa 세포에서 작은 헤어핀 RNA를 통해 ARID2 유전자의 기능이 일시적으로 소멸되었을 때 ERK1과 ERK2의 활성은 증가되었으나 Akt와 RSP6의 활성은 변화하지 않았다. 또한 IFITM1은 ERK 활성을 억제하는 것으로 알려져 있으며, 그 발현은 ARID2에 의해 조절되고 CAV-1과 상호 작용하여 ERK를 억제합니다.
이는 ARID2의 반수체 결실이 IFITM1 및 CAV-1의 발현을 감소시킴으로써 Ras-MAPK 신호 전달 시스템을 향상시킨다는 것을 시사합니다. Arid2의 일배체형이 결실된 마우스는 체중과 키가 작았으며 학습 및 기억 장애가 동반되었습니다. 결론: 본 연구에서는 12q12 결실의 임상 증상의 원인이 ERK 증진과 연관된 ARID2 반수체 결실임을 확인하였다.
또한 Arid2 반수체 결핍 마우스는 성장 및 학습 장애를 보였으며 환자와 유사한 임상 증상을 나타내는 것으로 확인되었습니다. 이 마우스 모델을 사용하여 Arid2 반수체 삭제로 관찰된 지적 장애와 관련된 추가 기능 연구를 수행할 수 있습니다.
