Overrepresentation of genetic variation in the AnkyrinG interactome is related to a range of neurodevelopmental disorders.

Upon the discovery of numerous genes involved in the pathogenesis of neurodevelopmental disorders, several studies showed that a significant proportion of these genes converge on common pathways and protein networks. Here, we used a reversed approach, by screening the AnkyrinG protein-protein interaction network for genetic variation in a large cohort of 1009 cases with neurodevelopmental disorders. We identified a significant enrichment of de novo potentially disease-causing variants in this network, confirming that this protein network plays an important role in the emergence of several neurodevelopmental disorders.

RNA-Seq detects a SAMD12-EXT1 fusion transcript and leads to the discovery of an EXT1 deletion in a child with multiple osteochondromas.

BACKGROUND: We describe a patient presenting with pachygyria, epilepsy, developmental delay, short stature, failure to thrive, facial dysmorphisms, and multiple osteochondromas. METHODS: The patient underwent extensive genetic testing and analysis in an attempt to diagnose the cause of his condition. Clinical testing included metaphase karyotyping, array comparative genomic hybridization, direct sequencing and multiplex ligation-dependent probe amplification and trio-based exome sequencing. Subsequently, research-based whole transcriptome sequencing was conducted to determine whether it might shed light on the undiagnosed phenotype. RESULTS: Clinical exome sequencing of patient and parent samples revealed a maternally inherited splice-site variant in the doublecortin (DCX) gene that was classified as likely pathogenic and diagnostic of the patient's neurological phenotype. Clinical array comparative genome hybridization analysis revealed a 16p13.3 deletion that could not be linked to the patient phenotype based on affected genes. Further clinical testing to determine the cause of the patient's multiple osteochondromas was unrevealing despite extensive profiling of the most likely causative genes, EXT1 and EXT2, including mutation screening by direct sequence analysis and multiplex ligation-dependent probe amplification. Whole transcriptome sequencing identified a SAMD12-EXT1 fusion transcript that could have resulted from a chromosomal deletion, leading to the loss of EXT1 function. Re-review of the clinical array comparative genomic hybridization results indicated a possible unreported mosaic deletion affecting the SAMD12 and EXT1 genes that corresponded precisely to the introns predicted to be affected by a fusion-causing deletion. The existence of the mosaic deletion was subsequently confirmed clinically by an increased density copy number array and orthogonal methodologies CONCLUSIONS: While mosaic mutations and deletions of EXT1 and EXT2 have been reported in the context of multiple osteochondromas, to our knowledge, this is the first time that transcriptomics technologies have been used to diagnose a patient via fusion transcript analysis in the congenital disease setting.

Clinical Presentation of a Complex Neurodevelopmental Disorder Caused by Mutations in ADNP.

BACKGROUND: In genome-wide screening studies for de novo mutations underlying autism and intellectual disability, mutations in the ADNP gene are consistently reported among the most frequent. ADNP mutations have been identified in children with autism spectrum disorder comorbid with intellectual disability, distinctive facial features, and deficits in multiple organ systems. However, a comprehensive clinical description of the Helsmoortel-Van der Aa syndrome is lacking. METHODS: We identified a worldwide cohort of 78 individuals with likely disruptive mutations in ADNP from January 2014 to October 2016 through systematic literature search, by contacting collaborators, and through direct interaction with parents. Clinicians filled in a structured questionnaire on genetic and clinical findings to enable correlations between genotype and phenotype. Clinical photographs and specialist reports were gathered. Parents were interviewed to complement the written questionnaires. RESULTS: We report on the detailed clinical characterization of a large cohort of individuals with an ADNP mutation and demonstrate a distinctive combination of clinical features, including mild to severe intellectual disability, autism, severe speech and motor delay, and common facial characteristics. Brain abnormalities, behavioral problems, sleep disturbance, epilepsy, hypotonia, visual problems, congenital heart defects, gastrointestinal problems, short stature, and hormonal deficiencies are common comorbidities. Strikingly, individuals with the recurrent p.Tyr719* mutation were more severely affected. CONCLUSIONS: This overview defines the full clinical spectrum of individuals with ADNP mutations, a specific autism subtype. We show that individuals with mutations in ADNP have many overlapping clinical features that are distinctive from those of other autism and/or intellectual disability syndromes. In addition, our data show preliminary evidence of a correlation between genotype and phenotype.

De novo variants in FBXO11 cause a syndromic form of intellectual disability with behavioral problems and dysmorphisms.

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.

Mutations in two large pedigrees highlight the role of ZNF711 in X-linked intellectual disability.

Intellectual disability (ID) affects approximately 1-2% of the general population and is characterized by impaired cognitive abilities. ID is both clinically as well as genetically heterogeneous, up to 2000 genes are estimated to be involved in the emergence of the disease with various clinical presentations. For many genes, only a few patients have been reported and causality of some genes has been questioned upon the discovery of apparent loss-of-function mutations in healthy controls. Description of additional patients strengthens the evidence for the involvement of a gene in the disease and can clarify the clinical phenotype associated with mutations in a particular gene. Here, we present two large four-generation families with a total of 11 males affected with ID caused by mutations in ZNF711, thereby expanding the total number of families with ID and a ZNF711 mutation to four. Patients with mutations in ZNF711 all present with mild to moderate ID and poor speech accompanied by additional features in some patients, including autistic features and mild facial dysmorphisms, suggesting that ZNF711 mutations cause non-syndromic ID.

New insights into the imprinted MEG8-DMR in 14q32 and clinical and molecular description of novel patients with Temple syndrome.

The chromosomal region 14q32 contains several imprinted genes, which are expressed either from the paternal (DLK1 and RTL1) or the maternal (MEG3, RTL1as and MEG8) allele only. Imprinted expression of these genes is regulated by two differentially methylated regions (DMRs), the germline DLK1/MEG3 intergenic (IG)-DMR (MEG3/DLK1:IG-DMR) and the somatic MEG3-DMR (MEG3:TSS-DMR), which are methylated on the paternal and unmethylated on the maternal allele. Disruption of imprinting in the 14q32 region results in two clinically distinct imprinting disorders, Temple syndrome (TS14) and Kagami-Ogata syndrome (KOS14). Another DMR with a yet unknown function is located in intron 2 of MEG8 (MEG8-DMR, MEG8:Int2-DMR). In contrast to the IG-DMR and the MEG3-DMR, this somatic DMR is methylated on the maternal chromosome and unmethylated on the paternal chromosome. We have performed extensive methylation analyses by deep bisulfite sequencing of the IG-DMR, MEG3-DMR and MEG8-DMR in different prenatal tissues including amniotic fluid cells and chorionic villi. In addition, we have studied the methylation pattern of the MEG8-DMR in different postnatal tissues. We show that the MEG8-DMR is hypermethylated in each of 13 non-deletion TS14 patients (seven newly identified and six previously published patients), irrespective of the underlying molecular cause, and is always hypomethylated in the four patients with KOS14, who have different deletions not encompassing the MEG8-DMR itself. The size and the extent of the deletions and the resulting methylation pattern suggest that transcription starting from the MEG3 promoter may be necessary to establish the methylation imprint at the MEG8-DMR.

Novel microdeletions on chromosome 14q32.2 suggest a potential role for non-coding RNAs in Kagami-Ogata syndrome.

In approximately 20% of individuals with Kagami-Ogata syndrome (KOS14, MIM 608149), characterized by a bell-shaped thorax with coat-hanger configuration of the ribs, joint contractures, abdominal wall defects and polyhydramnios during the pregnancy, the syndrome is caused by a maternal deletion of the imprinted gene cluster in chromosome 14q32.2. Most deletions reported so far included one or both of the differentially methylated regions (DMRs) - DLK1/MEG3 IG-DMR and MEG3-DMR. We present two unrelated families with two affected siblings each, presenting with classical KOS14 due to maternally inherited microdeletions. Interestingly, all four patients have lived through to adulthood, even though mortality rates for patients with KOS14 due to a microdeletion are relatively high. In the first family, none of the DMRs is included in the deletion and the methylation status is identical to that of controls. Deletions that do not encompass the DMRs in this region are thus sufficient to elicit the full KOS14 phenotype. In the second family, a partially overlapping deletion including both DMRs and MEG3 was detected. In summary, we show that patients with KOS14 can live into adulthood, that causal deletions do not have to include the DMRs and that consequently a normal methylation pattern does not exclude KOS14.

A robust protocol to increase NimbleGen SeqCap EZ multiplexing capacity to 96 samples.

Contemporary genetic studies frequently involve sequencing of a targeted gene panel, for instance consisting of a set of genes associated with a specific disease. The NimbleGen SeqCap EZ Choice kit is commonly used for the targeted enrichment of sequencing libraries comprising a target size up to 7 Mb. A major drawback of this commercially available method is the exclusive use of single-indexing, meaning that at most 24 samples can be multiplexed in a single reaction. In case of relatively small target sizes, this will lead to excessive amounts of data per sample. We present an extended version of the NimbleGen SeqCap EZ protocol which allows to robustly multiplex up to 96 samples. We achieved this by incorporating Illumina dual-indexing based custom adapters into the original protocol. To further extend the optimization of cost-efficient sequencing of custom target panels, we studied the effect of higher pre-enrichment pooling factors and show that pre-enrichment pooling of up to 12 samples does not affect the quality of the data. To facilitate evaluation of capture efficiency in custom design panels, we also provide a detailed reporting tool.

Five patients with a chromosome 1q21.1 triplication show macrocephaly, increased weight and facial similarities.

Recurrent rearrangements of chromosome 1q21.1 that occur as a consequence of non-allelic homologous recombination (NAHR) show considerable variability in phenotypic expression and penetrance. Chromosome 1q21.1 deletions (OMIM 612474) have been associated with microcephaly, intellectual disability, autism, schizophrenia, cardiac abnormalities and cataracts. Phenotypic features in individuals with 1q21.1 duplications (OMIM 612475) include macrocephaly, learning difficulties, developmental delay, intellectual disability and mild dysmorphic features. Half of these patients show autistic behavior. For the first time, we describe five patients, including monozygotic twins, with a triplication of the 1q21.1 chromosomal segment. Facial features common to all patients include a high, broad forehead; a flat and broad nasal bridge; long, downslanted palpebral fissures and dysplastic, low-set ears. Likely associated features include macrocephaly and increased weight. We observed that the triplications arose through different mechanisms in the patients: it was de novo in one patient, inherited from a triplication carrier in two cases, while the father of the twins is a 1q21.1 duplication carrier. The de novo triplication contained copies of both maternal alleles, suggesting it was generated by a combination of inter- and intrachromosomal recombination.