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Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS., Author summary Smith-Kingsmore Syndrome is a rare disease caused by damage in a gene named MTOR that is associated with excessive growth of the head and brain, delays in development and deficits in intellectual functioning. We report 7 patients who have changes in MTOR that have never been reported before. We describe new medical findings in these patients that may be common in Smith-Kingsmore Syndrome more broadly. We then identify how these new gene changes impact the function of the MTOR protein and thus cell function downstream. Lastly, we show that changes in the gene that lie deep inside the 3D structure of the MTOR protein are more likely to cause disease than those changes that lie on the surface of the protein. We may be able to use the 3D properties of MTOR gene changes to predict if future changes we see are likely to cause disease or not.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.

Context Prader-Willi syndrome (PWS) is a complex hypothalamic disorder, combining hyperphagia, hypotonia, intellectual disability, and pituitary hormone deficiencies. Annual mortality of patients with PWS is high (3%). In half of the patients, the cause of death is obesity related and/or of cardiopulmonary origin. Health problems leading to this increased mortality often remain undetected due to the complexity and rareness of the syndrome. Objective To assess the prevalence of health problems in adults with PWS retrospectively. Patients, Design, and Setting We systematically screened 115 PWS adults for undiagnosed health problems. All patients visited the multidisciplinary outpatient clinic for rare endocrine syndromes at the Erasmus University Medical Center, Rotterdam, Netherlands. We collected the results of medical questionnaires, interviews, physical examinations, biochemical measurements, polygraphy, polysomnography, and radiology. Main outcome measures Presence or absence of endocrine and nonendocrine comorbidities in relation to living situation, body mass index, genotype, and demographic factors. Results Seventy patients (61%) had undiagnosed health problems, while 1 in every 4 patients had multiple undiagnosed health problems simultaneously. All males and 93% of females had hypogonadism, 74% had scoliosis, 18% had hypertension, 19% had hypercholesterolemia, 17% had type 2 diabetes mellitus, and 17% had hypothyroidism. Unfavorable lifestyles were common: 22% exercised too little (according to PWS criteria) and 37% did not see a dietitian. Conclusions Systematic screening revealed many undiagnosed health problems in PWS adults. Based on patient characteristics, we provide an algorithm for diagnostics and treatment, with the aim to prevent early complications and reduce mortality in this vulnerable patient group.

Crigler-Najjar syndrome presents as severe unconjugated hyper-bilirubinemia and is characteristically caused by a mutation in the UGT1A1 gene, encoding the enzyme responsible for bilirubin glucuronidation. Here we present a patient with Crigler-Najjar syndrome with a completely normal UGT1A1 coding region. Instead, a homozygous 3 nucleotide insertion in the UGT1A1 promoter was identified that interrupts the HNF1 alpha binding site. This mutation results in almost complete abolishment of UGT1A1 promoter activity and prevents the induction of UGT1A1 expression by the liver nuclear receptors CAR and PXR, explaining the lack of a phenobarbital response in this patient. Although animal studies have revealed the importance of HNF1 alpha for normal liver function, this case provides the first clinical proof that mutations in its binding site indeed result in severe liver pathology stressing the importance of promoter sequence analysis. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Predictive genetic testing for familial adenomatous polyposis (FAP) is routinely offered to children at-risk from the age of 10 years onwards. Predictive testing for FAP at a younger age is debatable, because of absence of medical benefits. However, circumstances may arise when testing at a younger age (< 10 years) is appropriate. Currently, there is a lack of published experience with predictive testing of children at this young age. We evaluated 13 children who were tested for FAP at the age younger than 10 years; 7 mutation-carriers and 6 non-carriers. Parents of these children were re-contacted and open-ended semi-structured interviewed. None of the contacted parents regretted the timing of genetic testing. The major reasons for testing at the young age were (1) testing of all children in the family at the same moment; (2) certainty for the future; and (3) preparing the child for future surveillance. None of the parents observed changes in mental or physical health in their child after testing. Also, young genetic testing did not lead to colon surveillance before it was indicated. Genetic testing for FAP at a young age is experienced as causing no harm by parents. Future studies should evaluate children's own experiences with early genetic testing.

Mutations in WDR62 are associated with primary microcephaly; however, they have been reported with wide phenotypic variability. We report on six individuals with novel WDR62 mutations who illustrate this variability and describe three in greater detail. Of the three, one lacks neuromotor development and has severe pachygyria on MRI, another has only delayed speech and motor development and moderate polymicrogyria, and the third has an intermediate phenotype. Weobserved a rare copy number change of unknown significance, a 17q25qter duplication, in the first severely affected individual. The 17q25 duplication included an interesting candidate gene, tubulin cofactor D (TBCD), crucial in microtubule assembly and disassembly. Sequencing of the non-duplicated allele showed a TBCD missense mutation, predicted to cause a deleterious p. Phe1121Val substitution. Sequencing of a cohort of five patients with WDR62 mutations, including one with an identical mutation and different phenotype, plus 12 individuals with diagnosis of microlissencephaly and another individual with mild intellectual disability (ID) and a 17q25 duplication, did not reveal TBCD mutations. However, immunostaining with tubulin antibodies of cells from patients with both WDR62 and TBCD mutation showed abnormal tubulin network when compared to controls and cells with only the WDR62 mutation. Therefore, we propose that genetic factors contribute to modify the severity of the WDR62 phenotype and, although based on suggestive evidence, TBCD could function as one of such factors. (C) 2014 Wiley Periodicals, Inc.

BACKGROUND: Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer.METHODS: Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X).RESULTS: Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less 'BRCA1-like' than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (pA p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%.CONCLUSIONS: Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.

Objective: Desmoid tumours are rare mesenchymal tumours with unpredictable progression and high recurrence risk. They can occur sporadically or in association with Familial Adenomatous Polyposis (FAP), which is caused by germline APC mutations. The Wnt/beta-catenin pathway has a central role in the pathogenesis of desmoid tumours. These tumours can occur due to either a somatic CTNNB1 or APC mutation but can also be the first manifestation of FAP. Because germline APC analysis is not routinely performed in children with desmoid tumours, the diagnosis FAP may escape detection. The aim of this study is to form guidelines for the identification of possible APC germline mutation carriers among children with desmoid tumours, based on CTNNB1 mutation analysis and immunohistochemical analysis (IHC) for beta-catenin. Patients and methods: We performed IHC of beta-catenin and mutation analysis of CTNNB1 and APC in 18 paediatric desmoid tumours, diagnosed between 1990 and 2009 in the Erasmus MC, Rotterdam. Results: In 11 tumours, IHC showed an abnormal nuclear beta-catenin accumulation. In this group a CTNNB1 mutation was detected in seven tumours. In two tumours with an abnormal nuclear beta-catenin accumulation and no CTNNB1 mutation, an APC mutation was identified, which appeared to be a germline mutation. Conclusions: Aberrant staining of beta-catenin in paediatric desmoids helps to identify children at risk for FAP. We recommend to screen paediatric desmoid tumours for nuclear localisation of beta-catenin and consequently for CTNNB1 mutations. For patients with nuclear beta-catenin expression and no CTNNB1 mutations, APC mutation analysis should be offered after genetic counselling. (C) 2012 Elsevier Ltd. All rights reserved.

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene,SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carryingSATB1variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression and a severe phenotype. Contrastingly, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay and encode truncated proteins, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.