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.

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.

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.

A Novel SLC6A8 Mutation in a Large Family with X-Linked Intellectual Disability: Clinical and Proton Magnetic Resonance Spectroscopy Data of Both Hemizygous Males and Heterozygous Females.

X-linked creatine transport (CRTR) deficiency, caused by mutations in the SLC6A8 gene, leads to intellectual disability, speech delay, epilepsy, and autistic behavior in hemizygous males. Additional diagnostic features are depleted brain creatine levels and increased creatine/creatinine ratio (cr/crn) in urine. In heterozygous females the phenotype is highly variable and diagnostic hallmarks might be inconclusive. This survey aims to explore the intrafamilial variability of clinical and brain proton Magnetic Resonance Spectroscopy (MRS) findings in males and females with CRTR deficiency. X-chromosome exome sequencing identified a novel missense mutation in the SLC6A8 gene (p.G351R) in a large family with X-linked intellectual disability. Detailed clinical investigations including neuropsychological assessment, measurement of in vivo brain creatine concentrations using quantitative MRS, and analyses of creatine metabolites in urine were performed in five clinically affected family members including three heterozygous females and one hemizygous male confirming the diagnosis of CRTR deficiency. The severe phenotype of the hemizygous male was accompanied by most distinct aberrations of brain creatine concentrations (-83% in gray and -79% in white matter of age-matched normal controls) and urinary creatine/creatinine ratio. In contrast, the heterozygous females showed varying albeit generally milder phenotypes with less severe brain creatine (-50% to -33% in gray and -45% to none in white matter) and biochemical urine abnormalities. An intrafamilial correlation between female phenotype, brain creatine depletion, and urinary creatine abnormalities was observed. The combination of powerful new technologies like exome-next-generation sequencing with thorough systematic evaluation of patients will further expand the clinical spectrum of neurometabolic diseases.

Parental origin of de novo cytogenetically balanced reciprocal non-Robertsonian translocations.

De novo cytogenetically balanced reciprocal non-Robertsonian translocations are rare findings in clinical cytogenetics and might be associated with an abnormal phenotype. Knowledge of the parental origin and mechanisms of formation is still limited. By microdissection of the derivative chromosomes and their normal homologs from metaphases followed by microsatellite-mediated marker analysis we identified 7 cases of paternal and 3 cases of maternal origin in a cohort of 10 patients with de novo cytogenetically balanced reciprocal non-Robertsonian translocations. Neither in the maternal nor in the paternal group of our study parental age seems to be increased. Together with the data from the literature our results confirm that the majority of de novo cytogenetically balanced reciprocal translocations are of paternal origin, but the preponderance does not appear to be as distinct as previously thought and the paternal age does not seem to be necessarily a major contributing factor.

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.

Hypergonadotropic hypogonadism in a patient with inv ins (2;4).

We report on a 30-year-old man with azoospermia, primary hypogonadism and minor dysmorphic features who carried a balanced insertional chromosome translocation inv ins (2p24;4q28.3q31.22)de novo. Molecular cytogenetic analyses of the chromosome breakpoints revealed the localization of the breakpoint in 4q28.3 between BACs RP11-143E9 and RP11-285A15, an interval that harbours the PCDH10 gene. In 4q31.22, a breakpoint-spanning clone (RP11-6L6) was identified which contains the genes LSM6 and SLC10A7. On chromosome 2, BACs RP11-531P14 and RP11-360O18 flank the breakpoint in 2p24, a region void of known genes. In conclusion, the chromosome aberration of this patient suggests a gene locus for primary hypogonadism in 2p24, 4q28.3 or 4q31.2, and three possible candidate genes (LSM6, SLC10A7 and PCDH10) were identified by breakpoint analyses.

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.

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.

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.

Delineation of an interstitial 9q22 deletion in basal cell nevus syndrome.

Basal cell nevus syndrome (Gorlin syndrome) is an autosomal dominant disorder characterized by the presence of multiple basal cell carcinomas (BCC), odontogenic keratocysts, palmoplantar pits, and calcification in the falx cerebri caused by mutational inactivation of the PTCH gene. In few cases, the syndrome is due to a microdeletion at 9q22. Using high-resolution chromosome analysis we have identified a patient with the karyotype, 46,XY,del(9)(q21.3q31) de novo. He had typical clinical features consistent with basal cell nevus syndrome, but also additional features likely to be caused by loss of additional chromosomal material in this region. The deletion breakpoints were characterized with fluorescence in situ hybridization (FISH) analysis using BAC clones. The 15 Mb long deletion includes 87 RefSeq genes including PTCH. Hemizygosity of one or more genes might contribute to the additional symptoms observed in this patient.

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.

Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus.

Interspecific hybridization in the rodent genera Peromyscus and Mus results in abnormal placentation. In the Peromyscus interspecies hybrids, abnormal allelic interaction between an X-linked locus and the imprinted paternally expressed Peg3 locus was shown to cause the placental defects. In addition, loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placental size. As in extreme cases this placental dysplasia constitutes a post-zygotic barrier against interspecies hybridization, this finding was the first direct proof that imprinted genes may be important in speciation and thus in evolution. In the Mus interspecies hybrids, a strong role of an X-linked locus in placental dysplasia has also been detected. However, here we show by backcross and allele specific expression analyses that neither LOI of Peg3 nor abnormal interactions between Peg3 and an X-linked locus are involved in generating placental dysplasia in Mus hybrids, although the placental phenotypes observed in the two genera seem to be identical. In contrast to this, another dysgenesis effect common to Peromyscus and Mus hybrids, altered foetal growth, is caused at least in part by the same X-chromosomal regions in both genera. These findings first underline the strong involvement of the X-chromosome in the genetics of speciation. Secondly, they indicate that disruption of epigenetic states, such as LOI, at specific loci may be involved in hybrid dysgenesis effects in one group, but not in another. Thus, we conclude that even in closely related groups divergent molecular mechanisms may be involved in the production of phenotypically similar post-zygotic barriers against hybridization.

High rate of constitutional chromosomal rearrangements in apparently sporadic ALS.

Of 85 patients with ALS, the authors identified 3 patients with balanced translocations and 2 patients with pericentric inversions, all affecting distinct chromosomal loci. The high rate of constitutional aberrations (5.9%) suggests that ALS is, in part, associated with recombination-based rearrangements of genomic sequences.