Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family

We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (32) close to marker D11S4107. Sequencing of the exons and exon-intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383 + 5delGTGA in the 5' end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383 + 5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331 G<A), was also shown to affect splicing resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331 G<A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c5383 + 5delGTGA mutation does not have an inactivating effect on the protein. (C) 2012 Elsevier B.V. All rights reserved.

Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzymes function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches.