Transcription factor SOX3 is involved in X-linked mental retardation with growth hormone deficiency

Abstract

Physical mapping of the breakpoints of a pericentric inversion of the X chromosome (46,X,inv[X][p21q27]) in a female patient with mild mental retardation revealed localization of the Xp breakpoint in the IL1RAPL gene at Xp21.3 and the Xq breakpoint near the SOX3 gene (SRY [sex determining region Y]-box 3) (GenBank accession number NM_005634) at Xq26.3. Because carrier females with microdeletion in the IL1RAPL gene do not present any abnormal phenotype, we focused on the Xq breakpoint. However, we were unable to confirm the involvement of SOX3 in the mental retardation in this female patient. To validate SOX3 as an X-linked mental retardation (XLMR) gene, we performed mutation analyses in families with XLMR whose causative gene mapped to Xq26-q27. We show here that the SOX3 gene is involved in a large family in which affected individuals have mental retardation and growth hormone deficiency. The mutation results in an in-frame duplication of 33 bp encoding for 11 alanines in a polyalanine tract of the SOX3 gene. The expression pattern during neural and pituitary development suggests that dysfunction of the SOX3 protein caused by the polyalanine expansion might disturb transcription pathways and the regulation of genes involved in cellular processes and functions required for cognitive and pituitary development.

Figure 1

Physical map of the breakpoints of the pericentric inversion of the X chromosome. a, Physical map of the pericentric inversion 46,X,inv(X)(p21.3q27.1). The positions of the two breakpoint areas are indicated by diagonally striped boxes. The markers and STS are indicated (dashes). Their mapping showed that the IL1RAPL gene was disrupted by the Xp breakpoint near the 6th exon. Moreover, the Xq breakpoint area (100 kb) was localized 100 kb centromeric from the SOX3 gene. b, Results of in situ hybridization analyses on metaphases of the patient with pericentric inversion, showing FISH signals obtained on normal and inverted X chromosomes with YAC clones 453B11 and 905E10 (green) for the Xp and Xq breakpoints, respectively, and with a chromosome-X–alphasatellite probe (red) (Qbiogen). Note the hybridization signals on the inverted X chromosome on both the Xp and Xq sides, indicating that the two YAC clones spanned the breakpoints.

Figure 2

Expansion of polyalanine tract detected in the family with mental retardation and GHD (N3) and deletion in the same polyalanine tract in family T84. A, Family N3. a, Family pedigree. b, Two percent agarose gel electrophoresis of PCR products corresponding to amplification of part of SOX3 containing the 33-bp duplication. PCR amplification of SOX3 was performed using 50–100 ng of genomic DNA, 0.5 μM primers described elsewhere by Xiang et al. (2000), and 2.5 U of Taq polymerase (Promega). The PCR cycles consisted of an initial denaturation step of 95°C for 5 min, followed by 30 cycles of amplification (95°C for 1 min, 60°C for 1 min, 72°C for 1 min) and a final cycle at 72°C for 7 min. Fifteen family members, including six affected males, were available for this study who showed cosegregation of the duplication with the phenotype. c, The resulting amino acid sequence of 711–743dup (33 bp) is indicated in italics. The PCR products were purified by gel electrophoresis and QIAquick gel extraction kit (QIAGEN) and sequenced by ABI BigDye terminator cycle sequencing chemistry on an ABI377 automated sequencer. B, Family T84. a, Family pedigree. b, Two percent agarose gel electrophoresis of PCR products corresponding to amplification of part of SOX3 containing the 27-bp deletion. The deletion was observed for the two affected males, their healthy mother, and their maternal grandfather. c, The resulting amino acid sequence of 718del27 (27 bp) is indicated in italics.

Figure 3

Position in the SOX3 gene of the mutations identified in the MR family with GHD and in family T84. The polymorphic deletion is indicated in italics.