Publications

Abstract

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Abstract

Rare mutations of the FOXP2 transcription factor gene cause a monogenic syndrome characterized by impaired speech development and linguistic deficits. Recent genomic investigations indicate that its downstream neural targets make broader impacts on common language impairments, bridging clinically distinct disorders. Moreover, the striking conservation of both FoxP2 sequence and neural expression in different vertebrates facilitates the use of animal models to study ancestral pathways that have been recruited towards human speech and language. Intriguingly, reduced FoxP2 dosage yields abnormal synaptic plasticity and impaired motor-skill learning in mice, and disrupts vocal learning in songbirds. Converging data indicate that Foxp2 is important for modulating the plasticity of relevant neural circuits. This body of research represents the first functional genetic forays into neural mechanisms contributing to human spoken language.

Abstract

Heterozygous mutations of the human FOXP2 gene cause a developmental disorder involving impaired learning and production of fluent spoken language. Previous investigations of its aetiology have focused on disturbed function of neural circuits involved in motor control. However, Foxp2 expression has been found in the cochlea and auditory brain centers and deficits in auditory processing could contribute to difficulties in speech learning and production. Here, we recorded auditory brainstem responses (ABR) to assess two heterozygous mouse models carrying distinct Foxp2 point mutations matching those found in humans with FOXP2-related speech/language impairment. Mice which carry a Foxp2-S321X nonsense mutation, yielding reduced dosage of Foxp2 protein, did not show systematic ABR differences from wildtype littermates. Given that speech/language disorders are observed in heterozygous humans with similar nonsense mutations (FOXP2-R328X), our findings suggest that auditory processing deficits up to the midbrain level are not causative for FOXP2-related language impairments. Interestingly, however, mice harboring a Foxp2-R552H missense mutation displayed systematic alterations in ABR waves with longer latencies (significant for waves I, III, IV) and smaller amplitudes (significant for waves I, IV) suggesting that either the synchrony of synaptic transmission in the cochlea and in auditory brainstem centers is affected, or fewer auditory nerve fibers and fewer neurons in auditory brainstem centers are activated compared to wildtypes. Therefore, the R552H mutation uncovers possible roles for Foxp2 in the development and/or function of the auditory system. Since ABR audiometry is easily accessible in humans, our data call for systematic testing of auditory functions in humans with FOXP2 mutations.

Abstract

Specific language impairment (SLI) is a common developmental disorder haracterized by difficulties in language acquisition despite otherwise normal development and in the absence of any obvious explanatory factors. We performed a high-density screen of SLI1, a region of chromosome 16q that shows highly significant and consistent linkage to nonword repetition, a measure of phonological short-term memory that is commonly impaired in SLI. Using two independent language-impaired samples, one family-based (211 families) and another selected from a population cohort on the basis of extreme language measures (490 cases), we detected association to two genes in the SLI1 region: that encoding c-maf-inducing protein (CMIP, minP = 5.5 × 10−7 at rs6564903) and that encoding calcium-transporting ATPase, type2C, member2 (ATP2C2, minP = 2.0 × 10−5 at rs11860694). Regression modeling indicated that each of these loci exerts an independent effect upon nonword repetition ability. Despite the consistent findings in language-impaired samples, investigation in a large unselected cohort (n = 3612) did not detect association. We therefore propose that variants in CMIP and ATP2C2 act to modulate phonological short-term memory primarily in the context of language impairment. As such, this investigation supports the hypothesis that some causes of language impairment are distinct from factors that influence normal language variation. This work therefore implicates CMIP and ATP2C2 in the etiology of SLI and provides molecular evidence for the importance of phonological short-term memory in language acquisition.

Abstract

It has long been hypothesised that the human faculty to acquire a language is in some way encoded in our genetic program. However, only recently has genetic evidence been available to begin to substantiate the presumed genetic basis of language. Here we review the first data from molecular genetic studies showing association between gene variants and language disorders (specific language impairment, speech sound disorder, developmental dyslexia), we discuss the biological function of these genes, and we further speculate on the more general question of how the human genome builds a brain that can learn a language.

Abstract

Neurodevelopmental disorders that disturb speech and language are highly heritable. Isolation of the underlying genetic risk factors has been hampered by complexity of the phenotype and potentially large number of contributing genes. One exception is the identification of rare heterozygous mutations of the FOXP2 gene in a monogenic syndrome characterised by impaired sequencing of articulatory gestures, disrupting speech (developmental verbal dyspraxia, DVD), as well as multiple deficits in expressive and receptive language. The protein encoded by FOXP2 belongs to a divergent subgroup of forkhead-box transcription factors, with a distinctive DNA-binding domain and motifs that mediate hetero- and homodimerisation. FOXP1, the most closely related member of this subgroup, can directly interact with FOXP2 and is co-expressed in neural structures relevant to speech and language disorders. Moreover, investigations of songbird orthologues indicate that combinatorial actions of the two proteins may play important roles in vocal learning, leading to the suggestion that human FOXP1 should be considered a strong candidate for involvement in DVD. Thus, in this study, we screened the entire coding region of FOXP1 (exons and flanking intronic sequence) for nucleotide changes in a panel of probands used earlier to detect novel mutations in FOXP2. A non-synonymous coding change was identified in a single proband, yielding a proline-to-alanine change (P215A). However, this was also found in a random control sample. Analyses of non-coding SNP changes did not find any correlation with affection status. We conclude that FOXP1 mutations are unlikely to represent a major cause of DVD.

Abstract

Childhood syndromes disturbing language development are common and display high degrees of heritability. In most cases, the underlying genetic architecture is likely to be complex, involving multiple chromosomal loci and substantial heterogeneity, which makes it difficult to track down the crucial genomic risk factors. Investigation of rare Mendelian phenotypes offers a complementary route for unravelling key neurogenetic pathways. The value of this approach is illustrated by the discovery that heterozygous FOXP2 (where FOX is forkhead box) mutations cause an unusual monogenic disorder, characterized by problems with articulating speech along with deficits in expressive and receptive language. FOXP2 encodes a regulatory protein, belonging to the forkhead box family of transcription factors, known to play important roles in modulating gene expression in development and disease. Functional genetics using human neuronal models suggest that the different FOXP2 isoforms generated by alternative splicing have distinct properties and may act to regulate each other's activity. Such investigations have also analysed the missense and nonsense mutations found in cases of speech and language disorder, showing that they alter intracellular localization, DNA binding and transactivation capacity of the mutated proteins. Moreover, in the brains of mutant mice, aetiological mutations have been found to disrupt the synaptic plasticity of Foxp2-expressing circuitry. Finally, although mutations of FOXP2 itself are rare, the downstream networks which it regulates in the brain appear to be broadly implicated in typical forms of language impairment. Thus, through ongoing identification of regulated targets and interacting co-factors, this gene is providing the first molecular entry points into neural mechanisms that go awry in language-related disorders

Abstract

Family and twin studies of developmental dyslexia have consistently shown that there is a significant heritable component for this disorder. However, any genetic basis for the trait is likely to be complex, involving reduced penetrance, phenocopy, heterogeneity and oligogenic inheritance. This complexity results in reduced power for traditional parametric linkage analysis, where specification of the correct genetic model is important. One strategy is to focus on large multigenerational pedigrees with severe phenotypes and/or apparent simple Mendelian inheritance, as has been successfully demonstrated for speech and language impairment. This approach is limited by the scarcity of such families. An alternative which has recently become feasible due to the development of high-throughput genotyping techniques is the analysis of large numbers of sib-pairs using allele-sharing methodology. This paper outlines our strategy for conducting a systematic genome-wide search for genes involved in dyslexia in a large number of affected sib-pair familites from the UK. We use a series of psychometric tests to obtain different quantitative measures of reading deficit, which should correlate with different components of the dyslexia phenotype, such as phonological awareness and orthographic coding ability. This enable us to use QTL (quantitative trait locus) mapping as a powerful tool for localising genes which may contribute to reading and spelling disability.

Abstract

Recent application of nonparametric-linkage analysis to reading disability has implicated a putative quantitative-trait locus (QTL) on the short arm of chromosome 6. In the present study, we use QTL methods to evaluate linkage to the 6p25-21.3 region in a sample of 181 sib pairs from 82 nuclear families that were selected on the basis of a dyslexic proband. We have assessed linkage directly for several quantitative measures that should correlate with different components of the phenotype, rather than using a single composite measure or employing categorical definitions of subtypes. Our measures include the traditional IQ/reading discrepancy score, as well as tests of word recognition, irregular-word reading, and nonword reading. Pointwise analysis by means of sib-pair trait differences suggests the presence, in 6p21.3, of a QTL influencing multiple components of dyslexia, in particular the reading of irregular words (P=.0016) and nonwords (P=.0024). A complementary statistical approach involving estimation of variance components supports these findings (irregular words, P=.007; nonwords, P=.0004). Multipoint analyses place the QTL within the D6S422-D6S291 interval, with a peak around markers D6S276 and D6S105 consistently identified by approaches based on trait differences (irregular words, P=.00035; nonwords, P=.0035) and variance components (irregular words, P=.007; nonwords, P=.0038). Our findings indicate that the QTL affects both phonological and orthographic skills and is not specific to phoneme awareness, as has been previously suggested. Further studies will be necessary to obtain a more precise localization of this QTL, which may lead to the isolation of one of the genes involved in developmental dyslexia.

Abstract

The murine homologue of the human chloride channel gene, CLCN5, defects in which are responsible for Dent disease, has been cloned and characterized. We isolated the entire coding region of mouse Clcn5 cDNA and approximately 45 kb of genomic sequence embracing the gene. To study its transcriptional control, the 5' upstream sequences of the mouse Clcn5 gene were cloned into a luciferase reporter vector. Deletion analysis of 1.5 kb of the 5' flanking sequence defined an active promoter region within 128 bp of the putative transcription start site, which is associated with a TATA motif but lacks a CAAT consensus. Within this sequence, there is a motif with homology to a purine-rich sequence responsible for the kidney-specific promoter activity of the rat CLC-K1 gene, another member of the chloride-channel gene family expressed in kidney. An enhancer element that confers a 10- to 20-fold increase in the promoter activity of the mouse Clcn5 gene was found within the first intron. The organization of the human CLCN5 and mouse Clcn5 gene structures is highly conserved, and the sequence of the murine protein is 98% similar to that of human, with its highest expression seen in the kidney. This study thus provides the first identification of the transcriptional control region of, and the basis for an understanding of the regulatory mechanism that controls, this kidney-specific, chloride-channel gene.

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.