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NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

CHARGE syndrome is a rare genetic disorder mainly due to de novo and private truncating mutations of CHD7 gene. Here we report an intriguing hot spot of intronic mutations (c.5405-7G > A, c.5405-13G > A, c.5405-17G > A and c.5405-18C > A) located in CHD7 IVS25. Combining computational in silico analysis, experimental branch-point determination and in vitro minigene assays, our study explains this mutation hot spot by a particular genomic context, including the weakness of the IVS25 natural acceptor-site and an unconventional lariat sequence localized outside the common 40 bp upstream the acceptor splice site. For each of the mutations reported here, bioinformatic tools indicated a newly created 3' splice site, of which the existence was confirmed using pSpliceExpress, an easy-to-use and reliable splicing reporter tool. Our study emphasizes the idea that combining these two complementary approaches could increase the efficiency of routine molecular diagnosis.

Background: Intramedullary ependymomas are rare and benign tumors in the adult. Little is known about their physiopathology, but the implication of the NF2 gene is suspected because of their presence in a third of patients with type 2 neurofibromatosis (NF2), a disorder caused by mutation of the NF2 gene., Methods: We conducted a clinical and genetic study of a family in which 5 of 9 members suffered from intramedullary ependymoma. Karyotyping and CGH array analysis were performed on DNA from peripheral blood lymphocytes from affected participants. The NF2 gene sequences were then determined in DNA from 3 nonaffected and all 5 affected members of the family., Results: Karyotype and CGH array findings were normal. Sequencing of NF2 revealed a heterozygous deletion, c.811-39_841del69bp, at the intron 8/exon 9 junction, in all affected members that was absent from all nonaffected members. RT-PCR analysis and sequencing revealed a novel NF2 transcript characterized by a skipping of exon 9 (75 bp). This deletion is predicted to result in a 25-amino acid deletion in the N-terminal FERM domain of neurofibromin 2. Modeling of this mutant domain suggests possible disorganization of the subdomain C., Conclusion: We report the first family with an NF2 mutation associated with intramedullary ependymomas without other features of NF2 syndrome. This mutation, which has not been described previously, may particularly affect the function of neurofibromin 2 in ependymocytes leading to the development of intramedullary WHO grade II ependymomas. We propose that sporadic intramedullary ependymomas should also be analyzed for this region of NF2 gene.

Background: CHARGE syndrome is a rare, usually sporadic disorder of multiple congenital anomalies ascribed to a CHD7 gene mutation in 60% of cases. Although the syndrome is well characterised in children, only one series of 10 fetuses with CHARGE syndrome has been reported to date. Therefore, we performed a detailed clinicopathological survey in our series of fetuses with CHD7 mutations, now extended to 40 cases. CHARGE syndrome is increasingly diagnosed antenatally, but remains challenging in many instances., Method: Here we report a retrospective study of 40 cases of CHARGE syndrome with a CHD7 mutation, including 10 previously reported fetuses, in which fetal or neonatal clinical, radiological and histopathological examinations were performed., Results: Conversely to postnatal studies, the proportion of males is high in our series (male to female ratio 2.6:1) suggesting a greater severity in males. Features almost constant in fetuses were external ear anomalies, arhinencephaly and semicircular canal agenesis, while intrauterine growth retardation was never observed. Finally, except for one, all other mutations identified in our antenatal series were truncating, suggesting a possible phenotype-genotype correlation., Conclusions: Clinical analysis allowed us to refine the clinical description of CHARGE syndrome in fetuses, describe some novel features and set up diagnostic criteria in order to help the diagnosis of CHARGE syndrome after termination of pregnancies following the detection of severe malformations.

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions.

Objective: To document the phenotype associated with the p.[R74W;V201M;D1270N] and p.P841R mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene., Design: Case report., Setting: Biology and medicine of reproduction in a university hospital., Patient(s): A couple in which the man is carrier of the triple mutant p.[R74W;V201M;D1270N] allele in trans to p.P841R mutation and his spouse a heterozygous carrier for the severe p.F508del mutation of the CFTR gene, who became pregnant after intracytoplasmic sperm injection (ICSI) with twins., Intervention(s): Genetic counseling; CFTR gene sequencing; ICSI; children's follow-up., Main Outcome Measure(s): First report of a male phenotype associated with the p.P841R mutation., Result(s): The triple mutant p.[R74W;V201M;D1270N] allele associated with the unknown p.P841R mutations were detected in this man with congenital bilateral absence of the vas deferens, which may presume p.P841R as a severe mutation. After genetic counseling, the couple preferred prenatal diagnosis after ICSI than preimplantation genetic diagnosis, which revealed that the boys were both carriers of p.[R74W;V201M;D1270N] and p.F508del mutations. They are now 4 years old and show normal growth without nutritional deficiency., Conclusion(s): This case report documents for the first time a male phenotype associated with the p.P841R mutation and underlines the difficulties in counseling a man with congenital bilateral absence of the vas deferens carrying uncommon mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene before ICSI.

The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.