FRA2A is a CGG repeat expansion associated with silencing of AFF3.

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5-12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship.

Detection of a case of chronic myeloid leukaemia with deletions at the t(9;22) translocation breakpoints by a genome-wide non-invasive prenatal test.

OBJECTIVE: Non-invasive prenatal tests (NIPTs) interrogating the complete genome are able to detect not only fetal trisomy 13, 18 or 21 but additionally provide information on other (sub)chromosomal aberrations that can be fetal or maternal in origin. We demonstrate that in a subset of cases, this information is clinically relevant and should be reported to ensure adequate follow-up. METHOD: Genome-wide NIPT was carried out and followed by a software analysis pipeline optimized to detect subchromosomal aberrations. RESULTS: The NIPT profile showed deletions on chromosomes 9 and 22: NIPT 9q33.3q34.12(129150001-133750000)x1,22q11.23(23550001-25450000)x1,22q13.1(37850001-39600000)x1. This result was confirmed by single nucleotide polymorphism array on maternal genomic DNA, which also demonstrated that the deletions were somatic in nature. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction revealed that the deletions were flanking the translocation breakpoint on the derivative chromosome 9 as the result of a t(9;22)(q34;q11.2) translocation with BCR-ABL1 fusion typical for chronic myeloid leukaemia (CML). Multidisciplinary counselling, together with complete blood count, taught that the woman was in an early chronic phase CML. The woman was followed up closely, and treatment could be postponed until after delivery. CONCLUSION: Genome-wide NIPT identified a CML in chronic phase caused by the typical t(9;22)(q34;q11.2) translocation and accompanied by deletions flanking the translocation breakpoints. © 2016 John Wiley & Sons, Ltd.

Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders.

AnkyrinG, encoded by the ANK3 gene, is involved in neuronal development and signaling. It has previously been implicated in bipolar disorder and schizophrenia by association studies. Most recently, de novo missense mutations in this gene were identified in autistic patients. However, the causative nature of these mutations remained controversial. Here, we report inactivating mutations in the Ankyrin 3 (ANK3) gene in patients with severe cognitive deficits. In a patient with a borderline intelligence, severe attention deficit hyperactivity disorder (ADHD), autism and sleeping problems, all isoforms of the ANK3 gene, were disrupted by a balanced translocation. Furthermore, in a consanguineous family with moderate intellectual disability (ID), an ADHD-like phenotype and behavioral problems, we identified a homozygous truncating frameshift mutation in the longest isoform of the same gene, which represents the first reported familial mutation in the ANK3 gene. The causality of ANK3 mutations in the two families and the role of the gene in cognitive function were supported by memory defects in a Drosophila knockdown model. Thus we demonstrated that ANK3 plays a role in intellectual functioning. In addition, our findings support the suggested association of ANK3 with various neuropsychiatric disorders and illustrate the genetic and molecular relation between a wide range of neurodevelopmental disorders.

A CGG-repeat expansion mutation in ZNF713 causes FRA7A: association with autistic spectrum disorder in two families.

We report de novo occurrence of the 7p11.2 folate-sensitive fragile site FRA7A in a male with an autistic spectrum disorder (ASD) due to a CGG-repeat expansion mutation (∼450 repeats) in a 5' intron of ZNF713. This expanded allele showed hypermethylation of the adjacent CpG island with reduced ZNF713 expression observed in a proband-derived lymphoblastoid cell line (LCL). His unaffected mother carried an unmethylated premutation (85 repeats). This CGG-repeat showed length polymorphism in control samples (five to 22 repeats). In a second unrelated family, three siblings with ASD and their unaffected father were found to carry FRA7A premutations, which were partially or mosaically methylated. In one of the affected siblings, mitotic instability of the premutation was observed. ZNF713 expression in LCLs in this family was increased in three of these four premutation carriers. A firm link cannot yet be established between ASD and the repeat expansion mutation but plausible pathogenic mechanisms are discussed.

The transcriptional regulator ADNP links the BAF (SWI/SNF) complexes with autism.

Mutations in ADNP were recently identified as a frequent cause of syndromic autism, characterized by deficits in social communication and interaction and restricted, repetitive behavioral patterns. Based on its functional domains, ADNP is a presumed transcription factor. The gene interacts closely with the SWI/SNF complex by direct and experimentally verified binding of its C-terminus to three of its core components. A detailed and systematic clinical assessment of the symptoms observed in our patients allows a detailed comparison with the symptoms observed in other SWI/SNF disorders. While the mutational mechanism of the first 10 patients identified suggested a gain of function mechanism, an 11th patient reported here is predicted haploinsufficient. The latter observation may raise hope for therapy, as addition of NAP, a neuroprotective octapeptide named after the first three amino acids of the sequence NAPVSPIQ, has been reported by others to ameliorate some of the cognitive abnormalities observed in a knockout mouse model. It is concluded that detailed clinical and molecular studies on larger cohorts of patients are necessary to establish a better insight in the genotype phenotype correlation and in the mutational mechanism.

CNV-WebStore: online CNV analysis, storage and interpretation.

BACKGROUND: Microarray technology allows the analysis of genomic aberrations at an ever increasing resolution, making functional interpretation of these vast amounts of data the main bottleneck in routine implementation of high resolution array platforms, and emphasising the need for a centralised and easy to use CNV data management and interpretation system. RESULTS: We present CNV-WebStore, an online platform to streamline the processing and downstream interpretation of microarray data in a clinical context, tailored towards but not limited to the Illumina BeadArray platform. Provided analysis tools include CNV analsyis, parent of origin and uniparental disomy detection. Interpretation tools include data visualisation, gene prioritisation, automated PubMed searching, linking data to several genome browsers and annotation of CNVs based on several public databases. Finally a module is provided for uniform reporting of results. CONCLUSION: CNV-WebStore is able to present copy number data in an intuitive way to both lab technicians and clinicians, making it a useful tool in daily clinical practice.

Array-based MLPA to detect recurrent copy number variations in patients with idiopathic mental retardation.

Microdeletions, either subtelomeric or interstitial, are responsible for the mental handicap in approximately 10-20% of all patients. Currently, Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used to detect these small aberrations in a routine fashion. Although cost-effective, the throughput is low and the degree of multiplexing is limited to maximally 40-50 probes. Therefore, we developed an array-based MLPA method, with probes identified by unique tag sequences, allowing the simultaneous analysis of 180 probes in a single experiment thereby covering all known mental retardation loci with at least two probes. We screened 120 patients with idiopathic mental retardation. In this group we detected 6 aberrations giving a detection rate of 5%, consistent with similar studies. In addition we tested 293 patients with mental retardation who were negative for fragile X syndrome and commercially available subtelomeric MLPA. We found seven causative rearrangements in this group (detection rate of 2.4%) thereby illustrating the value of including probes for interstitial microdeletion syndromes and additional probes in the telomeric regions in targeted screening sets for mental retardation. Array-based MLPA may thus be a good candidate to develop probe sets that rapidly detect copy number changes of disease associated loci in the human genome. This method may become a valuable tool in a routine diagnostic setting as it is a fast, user-friendly and relatively low-cost technique providing straightforward results requiring only 125 ng of genomic DNA.

Advances in understanding fragile X syndrome and related disorders.

PURPOSE OF REVIEW: Fragile X syndrome is the most common form of inherited intellectual disability. Over the past 2 decades, insights into the cause of this disease have increased tremendously. This review will highlight recent discoveries with an emphasis on biochemical pathways affected in the disorder that are potentially amenable to treatment. RECENT FINDINGS: Recent work in the field demonstrated that multiple pathways are deregulated as a consequence of the FMR1 gene inactivation in patients with fragile X syndrome. In fragile X patients, no fragile X mental retardation protein is formed and thereby protein translation is compromised. As a consequence, a variety of biological pathways are disturbed. These pathways include mainly the metabotropic glutamate receptor and gamma-aminobutyric acid (GABA)ergic pathways, but recently potassium channels and the muscarinic cholinergic receptor have also been implied in fragile X syndrome. An overview is given of the potential therapeutic targets and clinical studies that have been performed. SUMMARY: The gene defect underlying fragile X syndrome was discovered back in 1991. Since then, there has been enormous progress in our understanding of the molecular basis of the disease. Excitingly, our insights have now reached a next phase in which therapy specifically targeting the underlying molecular defect becomes feasible.

Involvement and therapeutic potential of the GABAergic system in the fragile X syndrome.

Many drugs have been developed that are able to modulate the GABAergic system, which is involved in anxiety, depression, epilepsy, insomnia, and learning and memory. The recent observation that the GABA(A) receptor is underexpressed in the fragile X syndrome, an inherited mental retardation disorder, therefore raised hopes for targeted therapy of the disorder. This review summarizes the lines of evidence that demonstrate a malfunction of the GABAergic system. The GABAergic system clearly emerges as an attractive target for therapy of the fragile X syndrome, and thus provides an excellent example of how genetic research can lead to unique opportunities for treatment.

Identification of non-recurrent submicroscopic genome imbalances: the advantage of genome-wide microarrays over targeted approaches.

Genome-wide analysis of DNA copy-number changes using microarray-based technologies has enabled the detection of de novo cryptic chromosome imbalances in approximately 10% of individuals with mental retardation. So far, the majority of these submicroscopic microdeletions/duplications appear to be unique, hampering clinical interpretation and genetic counselling. We hypothesised that the genomic regions involved in these de novo submicroscopic aberrations would be candidates for recurrent copy-number changes in individuals with mental retardation. To test this hypothesis, we used multiplex ligation-dependent probe amplification (MLPA) to screen for copy number changes at eight genomic candidate regions in a European cohort of 710 individuals with idiopathic mental retardation. By doing so, we failed to detect additional submicroscopic rearrangements, indicating that the anomalies tested are non-recurrent in this cohort of patients. The break points flanking the candidate regions did not contain low copy repeats and/or sequence similarities, thus providing an explanation for its non-recurrent nature. On the basis of these data, we propose that the use of genome-wide microarrays is indicated when testing for copy-number changes in individuals with idiopathic mental retardation.

Diverse chromosome breakage mechanisms underlie subtelomeric rearrangements, a common cause of mental retardation.

Subtelomeric rearrangements are an important cause of both isolated and familial idiopathic mental retardation. A variety of different rearrangements such as pure truncations, unbalanced translocations, interstitial deletions, and inverted duplications have been detected throughout various screening studies. The cause of these aberrations is poorly understood as only few of the breakpoints have been determined and studied. We molecularly characterized the breakpoints of three rearrangements including a 1p subtelomeric deletion, a 1q subtelomeric deletion, and an unbalanced translocation between chromosomes 11q and 20q; we propose that diverse chromosome breakage mechanisms underlie subtelomeric rearrangements. The breakpoint sequences suggest that unusual non-B-DNA structures including triplex, tetraplex, and hairpin structures may be involved. In addition, we saw that the seemingly pure truncations of chromosomes 1p and 1q were in fact more complex rearrangements as highly repetitive sequences were joined to the chromosome end at the site of breakage.

A de novo subtelomeric monosomy 11q (11q24.2-qter) and trisomy 20q (20q13.3-qter) in a girl with findings compatible with Jacobsen syndrome: case report and review.

We report on a 2-year-old dysmorphic girl with prenatal and postnatal growth deficiency, cardiopathy, left-sided hydronephrosis due to pyelourethral junction stenosis, frequent respiratory infections and psychomotor retardation, in whom a de novo unbalanced submicroscopic translocation (11q;20q) was detected by subtelomeric multiplex ligation-dependent probe amplification and fluorescence in situ hybridization analyses. Additional fluorescence in situ hybridization studies with locus-specific BAC probes and analyses with microsatellite markers revealed that this translocation resulted in a paternal chromosome 11q terminal deletion of approximately 8.9 Mb and a subtelomeric 20q duplication of approximately 3.7 Mb. A subtelomeric 20q trisomy has only been reported in four cases so far. A subtelomeric 11q deletion has been clinically reported in 18 patients. We review the clinical phenotype of these patients. We suggest that patients with a subterminal (11q24.2/25-qter) deletion may present with features of the well-known phenotype of terminal 11q deletion or Jacobsen syndrome.

Impaired GABAergic inhibition in the hippocampus of Fmr1 knockout mice.

Many clinical and molecular features of the fragile X syndrome, a common form of intellectual disability and autism, can be modeled by deletion of the Fmr1 protein (Fmrp) in mice. Previous studies showed a decreased expression of several components of the GABAergic system in Fmr1 knockout mice. Here, we used this mouse model to investigate the functional consequences of Fmrp deletion on hippocampal GABAergic inhibition in the CA1-region of the hippocampus. Whole-cell patch-clamp recordings demonstrated a significantly reduced amplitude of evoked inhibitory postsynaptic currents (eIPSCs) and a decrease in the amplitude and frequency of spontaneous IPSCs. In addition, miniature IPSCs were reduced in amplitude and frequency and decayed significantly slower than mIPSCs in controls. Quantitative real-time PCR revealed a significantly lower expression of α2, ß1 and δ GABAA receptor subunits in the hippocampus of the juvenile mice (P22) compared to wild-type littermates. Correspondingly, we found also at the protein level reduced amounts of α2, ß1 and δ subunits in Fmr1 knockout mice. Overall, these results demonstrate that the reduction in several components of the GABAergic system is already present at young age and that this reduction results in measurable abnormalities on GABAA receptor-mediated phasic inhibition. These abnormalities might contribute to the behavioral and cognitive deficits of this fragile X mouse model.

TBP as a candidate gene for mental retardation in patients with subtelomeric 6q deletions.

Monozygotic twin brothers with a subtelomeric 6q deletion presented with mental retardation, microcephaly, seizures, an enlarged cisterna magna, dimpling at elbows, a high arched palate and a thin upper lip. The same subtelomeric deletion was detected in the mother of the patients, presenting with a milder phenotype. We narrowed down the breakpoint to a region of approximately 100 kb and estimated the size of the terminal deletion to be 1.2 Mb. This region contains four known and seven putative genes. Comparison of the deletion with other reported patients showed TBP was the most plausible candidate gene for the mental retardation in this syndrome. We verified that the TBP gene expression was halved in our patients using real-time PCR. Cognitive and behavioural tests performed on previously described heterozygous tbp mice suggested that TBP is potentially involved in cognitive development.

A de novo subterminal trisomy 10p and monosomy 18q in a girl with MCA/MR: case report and review.

We report on a 3-year-old girl with psychomotor retardation, cardiopathy, strabismus, umbilical hernia, and facial dysmorphism in whom a de novo unbalanced submicroscopic translocation (10p;18q) was found by MLPA (Multiplex Ligation dependent Probe Amplification) and FISH analyses. Additional FISH studies with locus specific RP11 BAC probes and analyses with microsatellites revealed that the translocation resulted in a deletion estimated between 6 and 9 Mb on the maternal chromosome 18 and a subtelomeric 10p duplication of approximately 6.9 Mb. The proband's karyotype is 46,XX.ish der(18) t(10;18)(18pter-->18q23:10p15 --> 10pter). A subterminal duplication of 10p, as well as a subterminal deletion of 18q have been rarely reported so far. The clinical phenotype of this patient is reviewed and discussed.

Subtelomeric rearrangements in the mentally retarded: a comparison of detection methods.

In recent years, subtelomeric rearrangements, e.g., chromosome deletions or duplications too small to be detected by conventional cytogenetic analysis, have emerged as a significant cause of both idiopathic and familial mental retardation. As mental retardation is a common disorder, many patients need to be tested on a routine basis. In this review, we will discuss the different methods that have been applied in laboratories worldwide, including multiprobe fluorescence in situ hybridization (FISH), multiallelic marker analysis, multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, comparative genomic hybridization (CGH), and multicolor FISH, including spectral karyotyping (SKY), subtelomeric combined binary ratio labeling FISH (S-COBRA FISH), multiplex FISH telomere integrity assay (M-TEL), telomeric multiplex FISH (TM-FISH), and primed in situ labeling (PRINS).

Positron Emission Tomography (PET) Quantification of GABAA Receptors in the Brain of Fragile X Patients.

Over the last several years, evidence has accumulated that the GABAA receptor is compromised in animal models for fragile X syndrome (FXS), a common hereditary form of intellectual disability. In mouse and fly models, agonists of the GABAA receptor were able to rescue specific consequences of the fragile X mutation. Here, we imaged and quantified GABAA receptors in vivo in brain of fragile X patients using Positron Emission Topography (PET) and [11C]flumazenil, a known high-affinity and specific ligand for the benzodiazepine site of GABAA receptors. We measured regional GABAA receptor availability in 10 fragile X patients and 10 control subjects. We found a significant reduction of on average 10% in GABAA receptor binding potential throughout the brain in fragile X patients. In the thalamus, the brain region showing the largest difference, the GABAA receptor availability was even reduced with 17%. This is one of the first reports of a PET study of human fragile X brain and directly demonstrates that the GABAA receptor availability is reduced in fragile X patients. The study reinforces previous hypotheses that the GABAA receptor is a potential target for rational pharmacological treatment of fragile X syndrome.

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA).

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.

A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP.

Despite the high heritability of autism spectrum disorders (ASD), characterized by persistent deficits in social communication and interaction and restricted, repetitive patterns of behavior, interests or activities, a genetic diagnosis can be established in only a minority of patients. Known genetic causes include chromosomal aberrations, such as the duplication of the 15q11-13 region, and monogenic causes, as in Rett and fragile-X syndromes. The genetic heterogeneity within ASD is striking, with even the most frequent causes responsible for only 1% of cases at the most. Even with the recent developments in next-generation sequencing, for the large majority of cases no molecular diagnosis can be established. Here, we report ten patients with ASD and other shared clinical characteristics, including intellectual disability and facial dysmorphisms caused by a mutation in ADNP, a transcription factor involved in the SWI/SNF remodeling complex. We estimate this gene to be mutated in at least 0.17% of ASD cases, making it one of the most frequent ASD-associated genes known to date.

A de novo balanced t(2;6)(p15;p22.3) in a patient with West Syndrome disrupts a lnc-RNA.

In a male patient with West Syndrome we identified a perfectly balanced, de novo balanced translocation 46,XY,t(2;6)(p15;p22.3). No known protein coding genes were disrupted by the translocation and positional effects on nearby genes were excluded by expression studies. A putative long non-coding RNA, BX118339, spans the breakpoint on chromosome 6. It can be hypothesized that disruption of this non-coding transcript plays a role in the pathogenesis of the patient.