Objectives of the project

T h e previous section describes the information available on the location of deafness gen e(s) in the X q l 3-21 region at the beginning of this study. T h e objectives of this work were:

i) to identify the causative gene(s) by positional cloning so that accurate genetic counselling based on mutation detection could be offered to fam ilies,

ii) to learn more about how mutation of the gen e(s) causes deafness.

T h e decision w as taken to collaborate with the group in Nijm egen, Holland. Physical m apping of a critical region for deafness based on patients with large, overlapping deletions w as already underway in Nijm egen as a result of cloning of the choroiderem ia g en e (C rem ers e t al., 1 9 9 0 ). W e could benefit from the progress in physical mapping by choosing polymorphic m arkers likely to be most useful in term s of carrier detection and use these to rapidly screen for deletions in preselected patients. From the Dutch point of view the can did ate region rem ained large and the rarity of this condition necessitated a collaborative approach. T h e m olecular genetics group in Nijm egen working alone had to rely on colleagues in a clinical genetic setting providing D N A from large fam ilies independently mapping to this region, from m ales with contiguous g en e deletion syndrom es or from m ales with perilymphatic gusher at stapes surgery. How ever, following the work of Phelps and Reardon, close attention to the clinical phenotype of the patients enabled us to clinically screen a much larger num ber of d eaf boys than those seen in a clinical genetic setting; working with Dr. P e ter Phelps in the field of neuroradiology w e w ere able to select patients with the characteristic bony change of the inner e a r from a large num ber of patients undergoing C T scanning for a w ide variety of reasons. Audiological testing by Professor Linda Luxon and Dr. S u e Bellm an could confirm that the patients selected had the characteristic profile of absent stapedial reflexes and vestibular hypofunction, described in patients with D F N 3 mutations.

T h e approach taken in the identification of the causative gen e(s) w as

i) to identify further families and individuals with the sam e characteristic

subset of families mapping to Xq 13-21, in order to increase the pool of patients availab le for study at the molecular level. Careful clinical and radiological exam ination would permit the inclusion of small families and isolated cases in this group of patients.

ii) to test newly isolated polymorphic microsatellite repeats from the region in a genetic linkage study. T h e purpose w as to use closely linked m arkers for predictive carrier detection w hen requested by fem ales from fam ilies with X-linked deafness, prior to the availability of mutation detection. iii) to use the microsatellite and other markers to search for m icrodeletions by PG R -based techniques in order to further reduce the can did ate interval.

iv) in particular, to confirm the radiological phenotype of patient D20. T h e critical region for a gene m utated in X-linked deafness, w as defined by the breakpoints of patients 1/10 (mixed deafness and gusher) and patient D 20 (m ixed deafness) at the start of this study. As there w ere possibly two genes in this region, one causing deafness with morphological abnorm ality and gusher, and the other causing deafness alone with normal ear developm ent, it w as important to confirm that patient D 20 had the sam e phenotype as the other patients with microdeletions.

v) the positional cloning strategy used, involved the identification of overlapping deletions in unrelated patients to identify first one, and then a second region of deletion overlap. Cloning of one disease critical region (the sm aller of the two in which the proximal and distal deletion breakpoints had been m apped) in YA C s and subcloning the Y A C s into cosmids, w as done by th e Nijm egen group. This cosmid contig was incomplete over a small region of 150kb, which w as precisely the a re a thought to harbour a g en e responsible for deafness. O ur group adopted the com plem entary approach of screening a g ridded cosmid library to complete the contig. Contigging of these cosmids, by hybridisation and by characterisation with restriction enzym es, and identification of deletion breakpoints in our patients enabled us to screen a few cosmids for coding sequences using cross-species conservation of sequences and identification of rare cutting restriction enzym e sites. Completion of the contig enabled a positional candidate gene to be m apped by the Nijm egen group and both groups sought mutations in the candidate gene.

MATERIALS AND METHODS.