Clinical, biological and evolutionary analyses of pendrin 716T > A (V239D) from Family Y. (a) CT scan of the temporal bones of a child from Family Y who is homozygous for 716T > A (V239D) reveals enlargement of the vestibular aqueduct, or EVA (red arrows). EVA is characteristic of Pendred syndrome. All four homozygous relatives in Family Y have prelingual, severe hearing loss, with thresholds > 85 decibels at all frequencies. There was no clinical indication of goitre and serum thyroid-stimulating hormone and thyroxine levels were normal. (b) Intracellular localisation and trafficking of YFP-tagged mutant (V239D) and wild-type (PDS) pendrin, prepared as previously described [12, 23]. Living COS7 cells were transiently transfected with YFP-V239D mutant pendrin (green) and with CFP-GPI (glycosylphosphatidylinositol [GPI] tagged with crimson fluorescent protein [CFP], red), which localises to the Golgi apparatus and the plasma membrane . After incubation, cells were visualised by confocal microscopy . Merged confocal images (yellow) indicate that pendrin V239D is retained in the endoplasmic reticulum, whereas wild-type pendrin (PDS) colocalises with GPI to the Golgi apparatus and plasma membrane. (c) The predicted severity of V239D in Family Y was compared with other disease-associated mis-sense mutations in pendrin (red diamonds) and to wild-type sequences of other human SLC26A anion transporters at homologous sites (blue symbols) using Grantham's amino acid difference formula . Some, but not all, pendrin mis-sense mutations are more diverged from wild-type pendrin than are other SLC26A transporters. By this measure, V239D is among the most divergent mis-sense mutations of pendrin, particularly given the conservation of other SLC26A proteins at this site. (d) Palestinian and Turkish individuals with the 716A (239D) mutation share an extended haplotype of 126 -260 kilobases flanking pendrin (yellow boxes), based on markers polymorphic among wild-type (716T) haplotypes. Pendrin 716T > A has not been observed in any other Palestinian families with inherited hearing loss or Turkish individuals with Pendred syndrome, suggesting that these two families may share a recent common ancestor.