Publications

Abstract

Why do some children fail to acquire speech and language skills despite adequate environmental input and overtly normal neurological and anatomical development? It has been suspected for several decades, based on indirect evidence, that the human genome might hold some answers to this enigma. These suspicions have recently received dramatic confirmation with the discovery of specific genetic changes which appear sufficient to derail speech and language development. Indeed, researchers are already using information from genetic studies to aid early diagnosis and to shed light on the neural pathways that are perturbed in these inherited forms of speech and language disorder. Thus, we have entered an exciting era for dissecting the neural bases of human communication, one which takes genes and molecules as a starting point. In the current article I explain how this recent paradigm shift has occurred and describe the new vistas that have opened up. I demonstrate ways of bridging the gaps between molecules, neurons and the brain, which will provide a new understanding of the aetiology of speech and language impairments.

Abstract

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Abstract

Disruptions of the human FOXP2 gene cause problems with articulation of complex speech sounds, accompanied by impairment in many aspects of language ability. The FOXP2/Foxp2 transcription factor is highly similar in humans and mice, and shows a complex conserved expression pattern, with high levels in neuronal subpopulations of the cortex, striatum, thalamus, and cerebellum. In the present study we generated mice in which loxP sites flank exons 12-14 of Foxp2; these exons encode the DNA-binding motif, a key functional domain. We demonstrate that early global Cre-mediated recombination yields a null allele, as shown by loss of the loxP-flanked exons at the RNA level and an absence of Foxp2 protein. Homozygous null mice display severe motor impairment, cerebellar abnormalities and early postnatal lethality, consistent with other Foxp2 mutants. When crossed to transgenic lines expressing Cre protein in a spatially and/or temporally controlled manner, these conditional mice will provide new insights into the contributions of Foxp2 to distinct neural circuits, and allow dissection of roles during development and in the mature brain.

Abstract

Specific language impairment (SLI) is defined as an inability to develop appropriate language skills without explanatory medical conditions, low intelligence or lack of opportunity. Previously, a genome scan of 98 families affected by SLI was completed by the SLI Consortium, resulting in the identification of two quantitative trait loci (QTL) on chromosomes 16q (SLI1) and 19q (SLI2). This was followed by a replication of both regions in an additional 86 families. Both these studies applied linkage methods to one phenotypic trait at a time. However, investigations have suggested that simultaneous analysis of several traits may offer more power. The current study therefore applied a multivariate variance-components approach to the SLI Consortium dataset using additional phenotypic data. A multivariate genome scan was completed and supported the importance of the SLI1 and SLI2 loci, whilst highlighting a possible novel QTL on chromosome 10. Further investigation implied that the effect of SLI1 on non-word repetition was equally as strong on reading and spelling phenotypes. In contrast, SLI2 appeared to have influences on a selection of expressive and receptive language phenotypes in addition to non-word repetition, but did not show linkage to literacy phenotypes.

Abstract

Mutations in FOXP2, a member of the forkhead family of transcription factor genes, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system. The identification of FOXP2 targets in the developing human brain, therefore, provides a unique tool with which to explore the development of human language and speech. Here, we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) by use of chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro. ChIP-chip identified 285 FOXP2 targets in fetal human brain; statistically significant overlap of targets in BG and IFC indicates a core set of 34 transcriptional targets of FOXP2. We identified targets specific to IFC or BG that were not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of central nervous system patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study to use human brain tissue, making the FOXP2-target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlighting genes likely to be involved in the development of human higher-order cognitive processes.

Abstract

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.

Abstract

The human Xp11.23-p11.22 interval has been implicated in several inherited diseases including Wiskott-Aldrich syndrome; three forms of X-linked hypercalciuric nephrolithiaisis; and the eye disorders retinitis pigmentosa 2, congenital stationary night blindness, and Aland Island eye disease. In constructing YAC contigs spanning Xp11. 23-p11.22, we have previously shown that the region around the synaptophysin (SYP) gene is refractory to cloning in YACs, but highly stable in cosmids. Preliminary analysis of the latter suggested that this might reflect a high density of coding sequences and we therefore undertook the complete sequencing of a SYP-containing cosmid. Sequence data were extensively analyzed using computer programs such as CENSOR (to mask repeats), BLAST (for homology searches), and GRAIL and GENE-ID (to predict exons). This revealed the presence of 29 putative exons, organized into three genes, in addition to the 7 exons of the complete SYP coding region, all mapping within a 44-kb interval. Two genes are novel, one (CACNA1F) showing high homology to alpha1 subunits of calcium channels, the other (LMO6) encoding a product with significant similarity to LIM-domain proteins. RT-PCR and Northern blot studies confirmed that these loci are indeed transcribed. The third locus is the previously described, but not previously localized, A4 differentiation-dependent gene. Given that the intron-exon boundaries predicted by the analysis are consistent with previous information where available, we have been able to suggest the genomic organization of the novel genes with some confidence. The region has an elevated GC content (>53%), and we identified CpG islands associated with the 5' ends of SYP, A4, and LMO6. The order of loci was Xpter-A4-LMO6-SYP-CACNA1F-Xcen, with intergenic distances ranging from approximately 300 bp to approximately 5 kb. The density of transcribed sequences in this area (>80%) is comparable to that found in the highly gene-rich chromosomal band Xq28. Further studies may aid our understanding of the long-range organization surrounding such gene-enriched regions.

Abstract

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.

Abstract

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones

Abstract

A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization techniques was used to map YACs, cosmids and DNA markers from the Xp11.2 region relative to the X chromosome breakpoint of the renal cell carcinoma-associated t(X;1)(p11;q21). The position of the breakpoint could be determined as follows: Xcen-OATL2-DXS146-DXS255-SYP-t(X;1)-TFE 3-OATL1-Xpter. Fluorescence in situ hybridization experiments using TFE3-containing YACs and cosmids revealed split signals indicating that the corresponding DNA inserts span the breakpoint region. Subsequent Southern blot analysis showed that a 2.3-kb EcoRI fragment which is present in all TFE3 cosmids identified, hybridizes to aberrant restriction fragments in three independent t(X;1)-positive renal cell carcinoma DNAs. The breakpoints in these tumors are not the same, but map within a region of approximately 6.5 kb. Through preparative gel electrophoresis an (X;1) chimaeric 4.4-kb EcoRI fragment could be isolated which encompasses the breakpoint region present on der(X). Preliminary characterization of this fragment revealed the presence of a 150-bp region with a strong homology to the 5' end of the mouse TFE3 cDNA in the X-chromosome part, and a 48-bp segment in the chromosome 1-derived part identical to the 5' end of a known EST (accession number R93849). These observations suggest that a fusion gene is formed between the two corresponding genes in t(X;1)(p11;q21)-positive papillary renal cell carcinomas.