De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.

The power of the Mediator complex-Expanding the genetic architecture and phenotypic spectrum of MED12-related disorders.

MED12 is a member of the large Mediator complex that controls cell growth, development, and differentiation. Mutations in MED12 disrupt neuronal gene expression and lead to at least three distinct X-linked intellectual disability syndromes (FG, Lujan-Fryns, and Ohdo). Here, we describe six families with missense variants in MED12 (p.(Arg815Gln), p.(Val954Gly), p.(Glu1091Lys), p.(Arg1295Cys), p.(Pro1371Ser), and p.(Arg1148His), the latter being first reported in affected females) associated with a continuum of symptoms rather than distinct syndromes. The variants expanded the genetic architecture and phenotypic spectrum of MED12-related disorders. New clinical symptoms included brachycephaly, anteverted nares, bulbous nasal tip, prognathism, deep set eyes, and single palmar crease. We showed that MED12 variants, initially implicated in X-linked recessive disorders in males, may predict a potential risk for phenotypic expression in females, with no correlation of the X chromosome inactivation pattern in blood cells. Molecular modeling (Yasara Structure) performed to model the functional effects of the variants strongly supported the pathogenic character of the variants examined. We showed that molecular modeling is a useful method for in silico testing of the potential functional effects of MED12 variants and thus can be a valuable addition to the interpretation of the clinical and genetic findings.

New insights into Brunner syndrome and potential for targeted therapy.

We report two families with Brunner syndrome living in one state of Australia. The first family had a predicted protein-truncating variant of monoamine oxidase A (MAOA) (p.S251KfsX2). Affected males had mild intellectual disability (ID), obsessive behaviour, limited friendships and were introverted and placid during clinical interview. The family disclosed episodic explosive aggression after a diagnosis was made. The second family had a missense variant in MAOA (p.R45W). Affected males had borderline-mild ID, attention deficit disorder and limited friendships. One had a history of explosive aggression in childhood and episodic symptoms of flushing, headaches and diarrhoea. Their carrier mother had normal intelligence but similar episodic symptoms. Characteristic biochemical abnormalities included high serum serotonin and urinary metanephrines and low urinary 5-hydroxyindoleacetic acid (5-HIAA) and vanillylmandelic acid (VMA). Symptomatic individuals in the second family had particularly high serotonin levels, and treatment with a serotonin reuptake inhibitor and dietary modification resulted in reversal of biochemical abnormalities, reduction of 'serotonergic' symptoms and behavioural improvement. Brunner syndrome should be considered as a cause of mild ID with paroxysmal behavioural symptoms. It can be screened for with serum/urine metanephrine and serotonin measurement. Cautious treatment with a serotonin reuptake inhibitor, dietary modifications and avoidance of medications contraindicated in patients on monoamine oxidase inhibitors can improve symptoms.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

The X-chromosome-linked intellectual disability protein PQBP1 is a component of neuronal RNA granules and regulates the appearance of stress granules.

The polyglutamine-binding protein 1 (PQBP1) has been linked to several X-linked intellectual disability disorders and progressive neurodegenerative diseases. While it is currently known that PQBP1 localizes in nuclear speckles and is engaged in transcription and splicing, we have now identified a cytoplasmic pool of PQBP1. Analysis of PQBP1 complexes revealed six novel interacting proteins, namely the RNA-binding proteins KSRP, SFPQ/PSF, DDX1 and Caprin-1, and two subunits of the intracellular transport-related dynactin complex, p150(Glued) and p27. PQBP1 protein complex formation is dependent on the presence of RNA. Immunofluorescence studies revealed that in primary neurons, PQBP1 co-localizes with its interaction partners in specific cytoplasmic granules, which stained positive for RNA. Our results suggest that PQBP1 plays a role in cytoplasmic mRNA metabolism. This is further supported by the partial co-localization and interaction of PQBP1 with the fragile X mental retardation protein (FMRP), which is one of the best-studied proteins found in RNA granules. In further studies, we show that arsenite-induced oxidative stress caused relocalization of PQBP1 to stress granules (SGs), where PQBP1 co-localizes with the new binding partners as well as with FMRP. Additional results indicated that the cellular distribution of PQBP1 plays a role in SG assembly. Together these data demonstrate a role for PQBP1 in the modulation of SGs and suggest its involvement in the transport of neuronal RNA granules, which are of critical importance for the development and maintenance of neuronal networks, thus illuminating a route by which PQBP1 aberrations might influence cognitive function.

The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans.

In mouse, four genes have been found to undergo genomic imprinting resulting in differential expression of maternally and paternally inherited alleles. To determine whether the cognate genes are also subject to imprinting in humans, we have studied allele-specific expression patterns of insulin-like growth factor 2, IGF2-receptor and H19 in human fetal and adult tissues. In keeping with previous findings in mice, our results indicate that in human fetal tissues the paternal H19 alleles is inactive. IGF2 is monoallelically expressed in various tissues but surprisingly not in adult human liver. The human IGF2R gene, another classic example of imprinting in mice, was found to be expressed from both alleles. We provide the first direct evidence for differential imprinting in the human and murine genome.

Balanced translocation in a patient with craniosynostosis disrupts the SOX6 gene and an evolutionarily conserved non-transcribed region.

Craniosynostosis is a congenital developmental disorder involving premature fusion of cranial sutures, which results in an abnormal shape of the skull. Significant progress in understanding the molecular basis of this phenotype has been made for a small number of syndromic craniosynostosis forms. Nevertheless, in the majority of the approximately 100 craniosynostosis syndromes and in non-syndromic craniosynostosis the underlying gene defects and pathomechanisms are unknown. Here we report on a male infant presenting at birth with brachycephaly, proptosis, midfacial hypoplasia, and low set ears. Three dimensional cranial computer tomography showed fusion of the lambdoid sutures and distal part of the sagittal suture with a gaping anterior fontanelle. Mutations in the genes for FGFR2 and FGFR3 were excluded. Standard chromosome analysis revealed a de novo balanced translocation t(9;11)(q33;p15). The breakpoint on chromosome 11p15 disrupts the SOX6 gene, known to be involved in skeletal growth and differentiation processes. SOX6 mutation screening of another 104 craniosynostosis patients revealed one missense mutation leading to the exchange of a highly conserved amino acid (p.D68N) in a single patient and his reportedly healthy mother. The breakpoint on chromosome 9 is located in a region without any known or predicted genes but, interestingly, disrupts patches of evolutionarily highly conserved non-genic sequences and may thus led to dysregulation of flanking genes on chromosome 9 or 11 involved in skull vault development. The present case is one of the very rare reports of an apparently balanced translocation in a patient with syndromic craniosynostosis, and reveals novel candidate genes for craniosynostoses and cranial suture formation.

Breakpoints around the HOXD cluster result in various limb malformations.

BACKGROUND: Characterisation of disease associated balanced chromosome rearrangements is a promising starting point in the search for candidate genes and regulatory elements. METHODS: We have identified and investigated three patients with limb abnormalities and breakpoints involving chromosome 2q31. Patient 1 with severe brachydactyly and syndactyly, mental retardation, hypoplasia of the cerebellum, scoliosis, and ectopic anus, carries a balanced t(2;10)(q31.1;q26.3) translocation. Patient 2, with translocation t(2;10)(q31.1;q23.33), has aplasia of the ulna, shortening of the radius, finger anomalies, and scoliosis. Patient 3 carries a pericentric inversion of chromosome 2, inv(2)(p15q31). Her phenotype is characterised by bilateral aplasia of the fibula and the radius, bilateral hypoplasia of the ulna, unossified carpal bones, and hypoplasia and dislocation of both tibiae. RESULTS: By fluorescence in situ hybridisation, we have mapped the breakpoints to intervals of approximately 170 kb or less. None of the three 2q31 breakpoints, which all mapped close to the HOXD cluster, disrupted any known genes. CONCLUSIONS: Hoxd gene expression in the mouse is regulated by cis-acting DNA elements acting over distances of several hundred kilobases. Moreover, Hoxd genes play an established role in bone development. It is therefore very likely that the three rearrangements disturb normal HOXD gene regulation by position effects.

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation.

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

A cryptic deletion of 2q35 including part of the PAX3 gene detected by breakpoint mapping in a child with autism and a de novo 2;8 translocation.

We report a de novo, apparently balanced (2;8)(q35;q21.2) translocation in a boy with developmental delay and autism. Cross species (colour) paint (Rx) and SKY FISH, forward and reverse chromosome painting, and FISH with subtelomeric probes were used to examine the patient's karyotype, but further rearrangements were not detected. FISH with region specific clones mapping near 2q35 and 8q21.2 breakpoints and STS mapping performed on the isolated derivative chromosomes were used to refine the location of the breakpoints further. A cryptic deletion of between 4.23 and 4.41 Mb in extent and involving at least 13 complete genes or transcription units was found at the breakpoint on 2q35. The deletion includes the promoter and 5' untranslated region of the paired box 3 (PAX3) gene. The child has very mild dystopia canthorum which may be associated with the PAX3 haploinsufficiency. The 8q21.2 breakpoint is within MMP16 which encodes matrix metalloproteinase 16. We postulate that the cryptic deletion and rearrangement are responsible for the patient's phenotype and that a gene (or genes) responsible for autism lies at 2q35 or 8q21.2. The results present a step towards identifying genes predisposing to autism.

Evidence against a major role of PEG1/MEST in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterised by interauterine and postnatal growth retardation, with or without additional dysmorphic features. Most cases are sporadic but a few familial cases have been described. A subset of patients exhibit maternal uniparental disomy for chromosome 7 (mUPD7) strongly suggesting that genomic imprinting plays a role in the aetiology of the disease. We and others have recently characterised the human PEG1/MEST gene, the first imprinted gene known to be located on chromosome 7. Although the function of PEG1/MEST is unknown, the paternal-specific expression of this gene and its location at 7q32, render it a promising candidate for SRS. As a prerequisite for mutation screening in 49 patients with SRS and 9 with primordial growth retardation (PGR), we determined the complete genomic structure of the PEG1/MEST gene which consists of 12 exons. Apart from one silent mutation and two novel polymorphisms, nucleotide changes were not detected in any of these patients. Moreover, methylation patterns of the 5' region of PEG1/MEST were found to be normal in 35 SRS and 9 PGR patients and different from the pattern seen in patients with mUPD7. These findings strongly argue against a role of PEG1/MEST in the majority of Silver-Russell syndrome cases.

Gene dosage analysis in Silver-Russell syndrome: use of quantitative competitive PCR and dual-color FISH to estimate the frequency of duplications in 7p11.2-p13.

Silver-Russell syndrome (SRS) describes a heterogeneous malformation syndrome mainly characterized by intrauterine and postnatal growth retardation (IUGR/PNGR). Approximately 10% of SRS cases have been associated with maternal uniparental disomy (matUPD) 7. This suggests the involvement of at least one imprinted gene on chromosome 7 in the pathogenesis of SRS. Additionally, two familial and one single SRS patients have been published with an interstitial duplication in 7p11.2-p13, including the genes GRB10 and IGFBP1; IGFBP3 was investigated in only one case revealing duplication; conversely, double gene dosage of EGFR was excluded in all 3 patients. Two further cytogenetically abnormal cases, one with a paracentric inversion (7)(p14p12) and one with matUPD7/partial trisomy for 7p13-q11, confirmed that the proximal short arm of chromosome represents an interesting region possibly harboring (a) candidate gene(s) for SRS. Although previously published investigations on the genes GRB10, IGFBP1, IGFBP3, and EGFR report neither disease-relevant mutations nor abnormal imprinting patterns, the SRS cases with chromosomal duplications suggest that variation of gene copy number might be a further type of mutation. To obtain meaningful results on the frequency of duplications in proximal 7p, we screened 32 SRS patients using quantitative PCR assays for GRB10, IGFBP1, IGFBP3, and EGFR. The data were confirmed by dual-color fluorescence in situ hybridization (FISH) of spot check samples. Results obtained by both methods exclude duplications in all analyzed patients and indicate an overall percentage of duplication among SRS patients between 2.4% (GRB10) and 5% (IGFBP1). By testing and evaluating quantitative competitive PCR for various loci, we developed a practical approach for gene dosage analysis which can be easily established for routine purposes.

gamma2-COP, a novel imprinted gene on chromosome 7q32, defines a new imprinting cluster in the human genome.

We describe a novel imprinted gene, gamma 2-COP (nonclathrincoatprotein), identified in a search for expressed sequences in human chromosome 7q32 where the paternally expressed MEST gene is located. gamma 2-COP contains 24 exons and spans >50 kb of genomic DNA. Like MEST, gamma 2-COP is ubiquitously transcribed in fetal and adult tissues. In fetal tissues, including skeletal muscle, skin, kidney, adrenal, placenta, intestine, lung, chorionic plate and amnion, gamma 2-COP is imprinted and expressed from the paternal allele. In contrast to the monoallelic expression observed in these fetal tissues, biallelic expression was evident in fetal brain and liver and in adult peripheral blood. Biallelic expression in blood is supported by the demonstration of gamma 2-COP transcripts in lymphoblastoid cell lines with maternal uniparental disomy 7. Absence of paternal gamma 2-COP transcripts during embryonic development may contribute to Silver-Russell syndrome. However, on mutation scanning the only gamma 2-COP mutation detected was maternally derived. Amino acid comparison of gamma2-COP protein revealed close relation to gamma-COP, a subunit of the coatomer complex COPI, suggesting a role of gamma2-COP in cellular vesicle traffic. The existence of distinct coatomer complexes could be the basis for the functional heterogeneity of COPI vesicles in retrograde and anterograde transport and/or in cargo selection. Together, gamma 2-COP and MEST constitute a novel imprinting cluster in the human genome that may contain other, as yet unknown, imprinted genes.

Preferential inactivation of a dupX(q23 --> q27-28) chromosome in a girl with mental retardation and dysmorphy.

We report on an 18-year-old female with de novo tandem duplication Xq23-->Xq27-28. The breakpoints of the duplication segment have been mapped by FISH using a panel of locus specific YACs. Despite selective inactivation of the aberrant X chromosome, proven by a combination of molecular and cytogenetic studies, the patient exhibits mental retardation, dysmorphic features and short stature. Possible mechanisms explaining this unexpected finding are discussed.

Molecular cloning and characterization of the Fugu rubripes MEST/COPG2 imprinting cluster and chromosomal localization in Fugu and Tetraodon nigroviridis.

We isolated Fugu genomic clones using the human MEST (Mesoderm-Specific Transcript) cDNA as probe. Sequence analysis revealed the presence of MEST and three additional genes which show homology to plant DNBP (DNA-Binding Protein), vertebrate COPG2 (Coat Protein Gamma 2), as well as to human and mouse UCN (Urocortin). Structures of Fugu and human MEST, COPG2 and UCN genes are very similar. Since MEST and COPG2 are neighboring genes on human chromosome 7q32, we can conclude that we identified their orthologs and that linkage of these genes is evolutionarily conserved in vertebrates. Unlike human MEST which underlies isoform-specific imprinting and is methylated in a parent-of-origin-specific fashion, the CpG island of the Fugu ortholog is completely methylated. The translation start of Fugu MEST is identical to the non-imprinted human isoform which is in good agreement with the assumption that genomic imprinting is restricted to mammals. Comparative mapping of these genes by fluorescence in-situ hybridization to metaphase chromosomes of Fugu rubripes and Tetraodon nigroviridis showed clear signals on one of the smallest acrocentric chromosomal pairs, which in Fugu, can be easily classified by its unique triangular shape.

Molecular cytogenetic characterization of ring chromosome 15 in three unrelated patients.

We report molecular cytogenetic characterization of ring chromosome 15 in three unrelated male patients with the karyotype 46,XY,r(15). One was a stillborn child with several malformations, and the other two cases showed pre- and postnatal growth retardation and developmental delay, common features for ring chromosome 15 syndrome. One of these patients also displayed clinical features resembling Prader-Willi syndrome (PWS). To delineate the extent of the deletion on chromosome 15, we have carried out fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) mapping to the distal long arm of chromosome 15. The deletion breakpoints clustered within a 4.5-6.5 Mb region proximal to the 15q telomere. Two deletions involved the same known genes, while the largest deletion observed in the stillborn child involved three additional genes, including the COUP-TFII gene, which has been suggested to play a role in heart development. The heart malformations, which are observed in this patient, are thus likely to be due to hemizygosity/haploinsufficiency of the COUP-TFII gene. In all three patients, the insulin-like growth factor I receptor gene (IGF1R) gene was deleted supporting the association between IGF1R and growth retardation seen in ring chromosome 15 syndrome.

Genomic structure and comparative analysis of nine Fugu genes: conservation of synteny with human chromosome Xp22.2-p22.1.

The pufferfish Fugu rubripes has a compact 400-Mb genome that is approximately 7.5 times smaller than the human genome but contains a similar number of genes. Focusing on the distal short arm of the human X chromosome, we have studied the evolutionary conservation of gene orders in Fugu and man. Sequencing of 68 kb of Fugu genomic DNA identified nine genes in the following order: (SCML2)-STK9, XLRS1, PPEF-1, KELCH2, KELCH1, PHKA2, AP19, and U2AF1-RS2. Apart from an evolutionary inversion separating AP19 and U2AF1-RS2 from PHKA2, gene orders are identical in Fugu and man, and all nine human homologs map to the Xp22 band. All Fugu genes were found to be smaller than their human counterparts, but gene structures were mostly identical. These data suggest that genomic sequencing in Fugu is a powerful and economical strategy to predict gene orders in the human genome and to elucidate the structure of human genes.