Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence.

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

A genome-wide association study identifies RNF213 as the first Moyamoya disease gene.

Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although ∼ 15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785,720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P = 5.3 × 10(-10)). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P = 1.2 × 10(-43), odds ratio = 190.8, 95% confidence interval = 71.7-507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.

Mutation analysis of the SHOC2 gene in Noonan-like syndrome and in hematologic malignancies.

Noonan syndrome is an autosomal dominant disease characterized by dysmorphic features, webbed neck, cardiac anomalies, short stature and cryptorchidism. It shows phenotypic overlap with Costello syndrome and cardio-facio-cutaneous (CFC) syndrome. Noonan syndrome and related disorders are caused by germline mutations in genes encoding molecules in the RAS/MAPK pathway. Recently, a gain-of-function mutation in SHOC2, p.S2G, has been identified as causative for a type of Noonan-like syndrome characterized by the presence of loose anagen hair. In order to understand the contribution of SHOC2 mutations to the clinical manifestations of Noonan syndrome and related disorders, we analyzed SHOC2 in 92 patients with Noonan syndrome and related disorders who did not exhibit PTPN11, KRAS, HRAS, BRAF, MAP2K1/2, SOS1 or RAF1 mutations. We found the previously identified p.S2G mutation in eight of our patients. We developed a rapid detection system to identify the p.S2G mutation using melting curve analysis, which will be a useful tool to screen for the apparently common mutation. All the patients with the p.S2G mutation showed short stature, sparse hair and atopic skin. Six of the mutation-positive patients showed severe mental retardation and easily pluckable hair, and one showed leukocytosis. No SHOC2 mutations were identified in leukemia cells from 82 leukemia patients. These results suggest that clinical manifestations in SHOC2 mutation-positive patients partially overlap with those in patients with typical Noonan or CFC syndrome and show that easily pluckable/loose anagen hair is distinctive in SHOC2 mutation-positive patients.