Acknowledgements This research was supported by grants DC00496 and DC02746 from the National Institute on Deafness and O

1. Characterization of a balanced translocation breakpoint to within the FOXP2 gene in a two-generation family with language impairment J.B. Bjork1, J.B. Tomblin2, C.A. Williams3, S.R. Patil1, M.R. O'Brien2, J.C. Murray1 1) Dept Pediatrics, U of Iowa, Iowa City, IA; 2) Dept Speech Pathology and Audiology, U of Iowa, Iowa City, IA; 3) Dept Pediatrics, U of Florida, Gainesville, FL. • Introduction • We previously reported on a balanced 7;13 chromosomal translocation within the forkhead transcription factor gene, FOXP2, in a mother-daughter pair. Both individuals present with a developmental speech and language disorder that persists despite adequate intelligence and opportunity for language learning. • The human capacity to acquire language seems to be unique, setting humans apart from other primates. FOXP2 was first identified in a large kindred (the KE family) with multiple individuals exhibiting developmental speech and language disorders. Since then, it has consistently been implicated in the etiology of speech and language. • Several cases with developmental speech and language disorder have been reported to be associated with chromosomal abnormalities • Studies have shown altered ultrasonic vocalizations in mice null for FOXP2, as well as upregulation of FOXP2 during song learning for adult songbirds. • As such, FOXP2 has also been proposed to be important in the evolution of speech and language as complex human traits Figure 2. Results of sequencing at breakpoints on chromosomes 7 and 13 for BT translocation. The chromosomes involved in the translocation are identified as derivative chromosomes (der) and are the two lower sequences whereas the normal sequence for each chromosome are at the top. A region of 6 bases common to both chromosomes is identified as a homologous region. Chromosome position data is consistent with the UCSC Genome Browser (March 2006 build). • Results • Sequence analysis revealed the translocation breakpoint to be within intron 9-10 of FOXP2 on chromosome 7 (7q31.1), between 114064876 and 114064883 • The breakpoint on chromosome 13 was in intron 7-8 of the replication factor C subunit 3 gene (RFC3) (13q13.2), between 33306028 and 33306035 • A 6bp homologous sequence was found to comprise the locus of the break point. This sequence was common to the breakpoint region on both chromosomes 7 and 13. • Figure 2 shows the DNA sequences for the normal and derived chromosomes and identifies the specific location of the breakpoint. Neither of these breakpoints involved deletion or transposition of DNA material. Background to the Current Study We recently described the speech characteristics of a mother (B) and daughter (T) with a balanced 7;13 translocation involving FOXP2. The pattern of speech, language, and cognitive performance for both individuals is similar to that of the affected members of the KE family (Figure 1). That is, they exhibit very poor speech and language abilities despite largely normal non-verbal cognition. It is hypothesized that mutations of FOXP2 variants may be implicated in speech and language disorders in these individuals. The precise nature of these are outlined in this poster. Discussion A frameshift mutation is anticipated for each fusion protein transcribed from the FOXP2 gene variants, FOXP2-RFC3 and RFC3-FOXP2, resulting in the premature truncation of gene transcripts. Vernes et al. (2006) described the functional properties of a severe truncation of FOXP2, resulting in a protein product R328X. This protein was limited to the cytoplasm, and had no nuclear activity, leading to haploinsufficiency of FOXP2 and the loss of repression/enhancement on a target DNA promoter. It is likely that the substantial truncation of FOXP2 in the current study has similar functional consequences. That is, B and T have a reduced dose of FOXP2 but no active mutant protein variants, such as occurs with the R553H mutation in the KE family. In addition, the proximity of RFC3, a gene involved in elongation of primed DNA templates, to NBEA, a gene implicated in autism, cannot be ignored. RFC3 lies ~1 Mb upstream from NBEA, such that variation in RFC3 potentially influences the phenotype of B and T. Figure 1. Standard scores for affected members of the KE family and TB (mean 1 , SD 3). • Methods • BAC and fosmid clones were utilized in fluorescent in-situ hybridization (FISH) analysis on G-banded metaphase chromosome spreads to map the breakpoint. • Once the breakpoint on each chromosome was localized to within one fosmid clone, long-range PCR was used to fine-map the breakpoint to within thousands of base pairs • This was continued until successful products were amplified spanning the breakpoints on both the der(7) and der(13) chromosomes, as well as the normal chromosomes 7 and 13 • A standard PCR approach was then used to amplify products for direct sequencing using standard protocols. • Key Conclusions • The likely effect of the BT chromosomal translocation is loss of the repressor or enhancer activity of the FOXP2 protein on its downstream DNA promoter target sequence(s) • It is therefore probable that haplosufficiency of FOXP2 cosegregates with language difficulties. • It is hypothesized that the disruption of RFC3, and its proximity to a potential autism locus, has an effect on the BT phenotype Acknowledgements This research was supported by grants DC00496 and DC02746 from the National Institute on Deafness and Other Communicative Disorders.