Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum.

CASK is a multi-domain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development such as RELN. Here we describe a previously unreported X-linked brain malformation syndrome caused by mutations of CASK. All five affected individuals with CASK mutations had congenital or postnatal microcephaly, disproportionate brainstem and cerebellar hypoplasia, and severe mental retardation.

Thrombin generation in two families with MYH9-related platelet disorder.

MYH9-related platelet disorders are inherited macrothrombocytopenias with additional clinical manifestations including renal failure, hearing loss, pre-senile cataract, and inclusion bodies in leucocytes that are present in different combinations. The MYH9 gene codes for the cytoplasmic contractile protein non-muscular myosin heavy chain IIA, present in several tissues. The bleeding tendency is usually mild to moderate but rarely, thrombotic complications are also seen. We report on the thrombin generation potential (ETP) in patients with MYH9-related disease with and without arterial thrombosis. In family A, four affected members [c.5521G>A mutation causing p.(Glu1841Lys)] were evaluated. Three of them had a moderate bleeding tendency and in two renal insufficiency and hearing loss were already present. These two patients had an arterial thrombosis (myocardial infarction and pons infarction, respectively) before 50 years of age. In family B, two members were affected [c.4679T>G, resulting in p.(Val1560Gly)]. Their bleeding tendency was mild (bleeding scores 4 and 3, respectively). Thrombelastography (ROTEM) was normal in all six individuals. ETP was below the normal range in family B. However, in family A, the two members affected by thrombosis had a normal ETP, indicating that other factors compensated for the low platelet count and might have contributed to the arterial thrombosis.

The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module.

Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.

Predictive genetic testing of at-risk relatives requires analysis of all CCM genes after identification of an unclassified CCM1 variant in an individual affected with cerebral cavernous malformations.

The mutation detection rate for familial cerebral cavernous malformations (CCM) is extremely high, being about 90 % if direct sequencing of the three genes, CCM1, CCM2, and CCM3, is used in conjunction with quantitative analyses to detect larger CCM1-3 deletions/duplications. We here report on an individual who had presented with more than 30 cerebral and spinal cavernous malformations, two intracranial meningiomas, and disease manifestation only in the mid-forties. A CCM1 missense variant of unclear relevance was found during the first sequencing step. Thereafter, direct sequencing of all three CCM genes revealed the typical pathogenic loss-of-function mutation c.598C > T/p.Q200* in the CCM3 gene. Our results demonstrate that mutation analyses of all three CCM genes in the index patient regardless of previous identification of an unclassified CCM1 variant is crucial for reliable predictive testing of at-risk relatives.

Disruption of neurexin 1 associated with autism spectrum disorder.

Autism is a neurodevelopmental disorder of complex etiology in which genetic factors play a major role. We have implicated the neurexin 1 (NRXN1) gene in two independent subjects who display an autism spectrum disorder (ASD) in association with a balanced chromosomal abnormality involving 2p16.3. In the first, with karyotype 46,XX,ins(16;2)(q22.1;p16.1p16.3)pat, NRXN1 is directly disrupted within intron 5. Importantly, the father possesses the same chromosomal abnormality in the absence of ASD, indicating that the interruption of alpha-NRXN1 is not fully penetrant and must interact with other factors to produce ASD. The breakpoint in the second subject, with 46,XY,t(1;2)(q31.3;p16.3)dn, occurs approximately 750 kb 5' to NRXN1 within a 2.6 Mb genomic segment that harbors no currently annotated genes. A scan of the NRXN1 coding sequence in a cohort of ASD subjects, relative to non-ASD controls, revealed that amino acid alterations in neurexin 1 are not present at high frequency in ASD. However, a number of rare sequence variants in the coding region, including two missense changes in conserved residues of the alpha-neurexin 1 leader sequence and of an epidermal growth factor (EGF)-like domain, respectively, suggest that even subtle changes in NRXN1 might contribute to susceptibility to ASD.

Diagnostic Single Gene Analyses Beyond Sanger.

Molecular testing of congenital coagulation and platelet disorders offers confirmation of clinical diagnoses, supports genetic counselling, and enables predictive and prenatal diagnosis. In some cases, genotype-phenotype correlations are important for predicting the clinical course of the disease and adaptation of individualized therapy. Until recently, genotyping has been mainly performed by Sanger sequencing. While next generation sequencing (NGS) enables the parallel analysis of multiple genes, the cost-value ratio of custom-made panels can be unfavorable for analyses of specific small genes. The aim of this study was to transfer genotyping of small genes involved in congenital coagulation and platelet disorders from Sanger sequencing to an NGS-based method. A LR-PCR approach for target enrichment of the entire genomic regions of the genes F7, F10, F11, F12, GATA1, MYH9, TUBB1 and WAS was combined with high-throughput sequencing on a MiSeq platform. NGS detected all variants that had previously been identified by Sanger sequencing. Our results demonstrate that this approach is an accurate and flexible tool for molecular genetic diagnostics of single small genes.

High mutation detection rates in cerebral cavernous malformation upon stringent inclusion criteria: one-third of probands are minors.

Cerebral cavernous malformations (CCM) are prevalent vascular malformations occurring in familial autosomal dominantly inherited or isolated forms. Once CCM are diagnosed by magnetic resonance imaging, the indication for genetic testing requires either a positive family history of cavernous lesions or clinical symptoms such as chronic headaches, epilepsy, neurological deficits, and hemorrhagic stroke or the occurrence of multiple lesions in an isolated case. Following these inclusion criteria, the mutation detection rates in a consecutive series of 105 probands were 87% for familial and 57% for isolated cases. Thirty-one novel mutations were identified with a slight shift towards proportionally more CCM3 mutations carriers than previously published (CCM1: 60%, CCM2: 18%, CCM3: 22%). In-frame deletions and exonic missense variants requiring functional analyses to establish their pathogenicity were rare: An in-frame deletion within the C-terminal FERM domain of CCM1 resulted in decreased protein expression and impaired binding to the transmembrane protein heart of glass (HEG1). Notably, 20% of index cases carrying a CCM mutation were below age 10 and 33% below age 18 when referred for genetic testing. Since fulminant disease courses during the first years of life were observed in CCM1 and CCM3 mutation carriers, predictive testing of minor siblings became an issue.