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Determining pathogenicity of genomic variation identified by next-generation sequencing techniques can be supported by recurrent disruptive variants in the same gene in phenotypically similar individuals. However, interpretation of novel variants in a specific gene in individuals with mild-moderate intellectual disability (ID) without recognizable syndromic features can be challenging and reverse phenotyping is often required. We describe 24 individuals with a de novo disease-causing variant in, or partial deletion of, the F-box only protein 11 gene (FBXO11, also known as VIT1 and PRMT9). FBXO11 is part of the SCF (SKP1-cullin-F-box) complex, a multi-protein E3 ubiquitin-ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. Twenty-two variants were identified by next-generation sequencing, comprising 2 in-frame deletions, 11 missense variants, 1 canonical splice site variant, and 8 nonsense or frameshift variants leading to a truncated protein or degraded transcript. The remaining two variants were identified by array-comparative genomic hybridization and consisted of a partial deletion of FBXO11. All individuals had borderline to severe ID and behavioral problems (autism spectrum disorder, attention-deficit/hyperactivity disorder, anxiety, aggression) were observed in most of them. The most relevant common facial features included a thin upper lip and a broad prominent space between the paramedian peaks of the upper lip. Other features were hypotonia and hyperlaxity of the joints. We show that de novo variants in FBXO11 cause a syndromic form of ID. The current series show the power of reverse phenotyping in the interpretation of novel genetic variances in individuals who initially did not appear to have a clear recognizable phenotype.

Contains fulltext : 204872.pdf (Publisher’s version ) (Open Access) We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.

Recurrent somatic variants in SPOP are cancer specific; endometrial and prostate cancers result from gain-of-function and dominant-negative effects toward BET proteins, respectively. By using clinical exome sequencing, we identified six de novo pathogenic missense variants in SPOP in seven individuals with developmental delay and/or intellectual disability, facial dysmorphisms, and congenital anomalies. Two individuals shared craniofacial dysmorphisms, including congenital microcephaly, that were strikingly different from those of the other five individuals, who had (relative) macrocephaly and hypertelorism. We measured the effect of SPOP variants on BET protein amounts in human Ishikawa endometrial cancer cells and patient-derived cell lines because we hypothesized that variants would lead to functional divergent effects on BET proteins. The de novo variants c.362G>A (p.Arg121Gln) and c. 430G>A (p.Asp144Asn), identified in the first two individuals, resulted in a gain of function, and conversely, the c.73A>G (p.Thr25Ala), c.248A>G (p.Tyr83Cys), c.395G>T (p.Gly132Val), and c.412C>T (p.Arg138Cys) variants resulted in a dominant-negative effect. Our findings suggest that these opposite functional effects caused by the variants in SPOP result in two distinct and clinically recognizable syndromic forms of intellectual disability with contrasting craniofacial dysmorphisms.

Contains fulltext : 206417.pdf (Publisher’s version ) (Open Access) BACKGROUND: Wnt and Wnt-associated pathways play an important role in the genetic etiology of oligodontia, a severe form of tooth agenesis. Loss-of-function mutations in LRP6 , encoding a transmembrane cell-surface protein that functions as a coreceptor in the canonical Wnt/b-catenin signaling cascade, also contribute to genetic oligodontia. METHODS AND RESULTS: We describe a three-generation family with hereditary thrombocytopenia and oligodontia. Genome wide array analysis was performed. The array results from the index patient revealed an interstitial loss of 150 kb in 8p23.1 (chr8:6,270,299-6,422,558; hg19) encompassing MCPH1 and ANGPT2 and an interstitial loss of 290 kb in 12p13.2 (chr12:12,005,720-12,295,290; hg19) encompassing ETV6, BCL2L14 and LRP6. CONCLUSION: This case report shows a three-generation family with hereditary thrombocytopenia and oligodontia with a heterozygous 290 kb novel contiguous gene deletion in band p13.2 of chromosome 12, encompassing LRP6 and ETV6. In this report we discuss the clinical relevance of the deletion of both genes and illustrate the importance of thorough examination of oligodontia patients. Comprising not only the oral status but also the medical history of the patients and their relatives.

Contains fulltext : 169850.pdf (Publisher’s version ) (Closed access) Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

Contains fulltext : 154935.pdf (Publisher’s version ) (Closed access) Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome. 9 p.

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.

The oral-facial-digital (OFD) syndromes comprise a group of related disorders with a combination of oral, facial and digital anomalies. Variants in several ciliary genes have been associated with subtypes of OFD syndrome, yet in most OFD patients the underlying cause remains unknown. We investigated the molecular basis of disease in two brothers with OFD type II, Mohr syndrome, by performing single-nucleotide polymorphism (SNP)-array analysis on the brothers and their healthy parents to identify homozygous regions and candidate genes. Subsequently, we performed whole-exome sequencing (WES) on the family. Using WES, we identified compound heterozygous variants c.[464G>C];[1226G>A] in NIMA (Never in Mitosis Gene A)-Related Kinase 1 (NEK1). The novel variant c.464G>C disturbs normal splicing in an essential region of the kinase domain. The nonsense variant c.1226G>A, p.(Trp409*), results in nonsense-associated alternative splicing, removing the first coiled-coil domain of NEK1. Candidate variants were confirmed with Sanger sequencing and alternative splicing assessed with cDNA analysis. Immunocytochemistry was used to assess cilia number and length. Patient-derived fibroblasts showed severely reduced ciliation compared with control fibroblasts (18.0 vs 48.9%, P

Item does not contain fulltext OBJECTIVES: A novel, 3D technique to measure the differences in tooth crown morphology between the MSX1 cases and non-affected controls was designed to get a better understanding of dental phenotype-genotype associations. MATERIALS AND METHODS: Eight Dutch subjects from a single family with tooth agenesis, all with an established nonsense mutation c.332 C > A, p. Ser 111 Stop in exon 1 of MSX1, were compared with unaffected controls regarding several aspects of tooth crown morphology of incisor and molar teeth. A novel method of quantitative three-dimensional analysis was used to detect differences. RESULTS: Statistically significant shape differences were observed for the maxillary incisor in the MSX1 family compared with the controls on the following parameters: surface area, buccolingual dimension, squareness, and crown volume (P

Background Dental agenesis is the most common, often heritable, developmental anomaly in humans. Mutations in MSX1, PAX9, AXIN2 and the ectodermal dysplasia genes EDA, EDAR and EDARADD have been detected in familial severe tooth agenesis. However, until recently, in the majority of cases (similar to 90%) the genetic factor could not be identified, implying that other genes must be involved. Recent insights into the role of Wnt10A in tooth development, and the finding of hypodontia in carriers of the autosomal recessive disorder, odontooncychodermal dysplasia, due to mutations in WNT10A (OMIM 257980; OODD), make WNT10A an interesting candidate gene for dental agenesis.Methods In a panel of 34 patients with isolated hypodontia, the candidate gene WNT10A and the genes MSX1, PAX9, IRF6 and AXIN2 have been sequenced. The probands all had isolated agenesis of between six and 28 teeth.Results WNT10A mutations were identified in 56% of the cases with non-syndromic hypodontia. MSX1, PAX9 and AXIN2 mutations were present in 3%, 9% and 3% of the cases, respectively.Conclusion The authors identified WNT10A as a major gene in the aetiology of isolated hypodontia. By including WNT10A in the DNA diagnostics of isolated tooth agenesis, the yield of molecular testing in this condition was significantly increased from 15% to 71%.

Kabuki Syndrome (KS) is a rare syndrome characterized by intellectual disability and multiple congenital abnormalities, in particular a distinct dysmorphic facial appearance. KS is caused by mutations in the MLL2 gene, encoding an H3K4 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. Direct sequencing of all 54 exons of the MLL2 gene in 45 clinically well-defined KS patients identified 34 (75.6%) different mutations. One mutation has been described previously, all others are novel. Clinically, all KS patients were sporadic, and mutations were de novo for all 27 families for which both parents were available. We detected nonsense (n=11), frameshift (n=17), splice site (n=4) and missense (n=2) mutations, predicting a high frequency of absent or non-functional MLL2 protein. Interestingly, both missense mutations located in the C-terminal conserved functional domains of the protein. Phenotypically our study indicated a statistically significant difference in the presence of a distinct facial appearance (p=0.0143) and growth retardation (p=0.0040) when comparing KS patients with an MLL2 mutation compared to patients without a mutation. Our data double the number of MLL2 mutations in KS reported so far and widen the spectrum of MLL2 mutations and disease mechanisms in KS.© 2010 Wiley-Liss, Inc. PMID: 21280141 [PubMed - in process]

The triad of micrognathia, glossoptosis, and concomitant airway obstruction defined as "Robin sequence" (RS) is caused by oropharyngeal developmental events constrained by a reduced stomadeal space. This sequence of abnormal embryonic development also results in an anatomical configuration that might predispose the fetus to a cleft palate. RS is heterogeneous and many different etiologies have been described including syndromic, RS-plus, and isolated forms. For an optimal diagnosis, subsequent treatment and prognosis, a thorough understanding of the embryology and pathogenesis is necessary. This manuscript provides an update about our current understanding of the development of the mandible, tongue, and palate and possible mechanisms involved in the development of RS. Additionally, we provide the reader with an up-to-date summary of the different etiologies of this phenotype and link this to the embryologic, developmental, and genetic mechanisms.

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14-18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs. published online 9 October 2013

We performed a clinical, biochemical, and genetic study in 16 patients from 11 families with adult-onset acid maltase deficiency. All patients were compound heterozygotes and carried the IVS1(-13T --G) transversion on one allele; the second allele harbored either a deletion of a T at position 525 in exon 2 (7 probands, 64%) or a deletion of exon 18 (1 proband, 9%). Deterioration of handicap was related to age, and decrease in vital capacity to duration of the symptomatic stage. Respiratory insufficiency was never the first manifestation. The levels of activity of serum creatine kinase and of alpha-glucosidase in peripheral blood cells or muscle were helpful for the diagnosis, but did not have prognostic value. The adult form of acid maltase deficiency appears to be both clinically and genetically rather homogeneous; decrease of alpha-glucosidase activity is the final common pathway leading to destruction of muscle fibers and progression of muscle weakness over a period of years.

BACKGROUND: We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype.METHODS AND RESULTS: We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated.CONCLUSIONS: HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Nonsyndromic orofacial clefts (OFC) are common birth defects caused by certain genes interacting with environmental factors. Mutations and association studies indicate that the homeobox gene MSX1 plays a role in human clefting. In a Dutch case-control triad study (mother, father, and child), we investigated interactions between MSX1 and the parents' periconceptional lifestyle in relation to the risk of OFC in their offspring. We studied 181 case- and 132 control mothers, 155 case- and 121 control fathers, and 176 case- and 146 control children, in which there were 107 case triads and 66 control triads. Univariable and multivariable logistic regression analyses were applied, and odds ratios (OR), 95% confidence intervals (CI) were calculated. Allele 4 of the CA marker in the MSX1 gene, consisting of nine CA repeats, was the most common allele found in both the case and control triads. Significant interactions were observed between allele 4 homozygosity of the child with maternal smoking (OR 2.7, 95% CI 1.1-6.6) and with smoking by both parents (OR 4.9, 95% CI 1.4-18.0). Allele 4 homozygosity in the mother and smoking showed a risk estimate of OR 3.2 (95% CI 1.1-9.0). If allele 4 homozygous mothers did not take daily folic acid supplements in the recommended periconceptional period, this also increased the risk of OFC for their offspring (OR 2.8, 95% CI 1.1-6.7). Our findings show that, in the Dutch population, periconceptional smoking by both parents interacts with a specific allelic variant of MSX1 to significantly increase OFC risk for their offspring. Possible underlying mechanisms are discussed.

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.