Williamson Robin E, Darrow Keith N, Michaud Sebastien, Jacobs Julie S, Jones Marilyn C, Eberl Daniel F, Maas Richard L, Liberman M Charles, Morton Cynthia C
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Am J Med Genet A. 2007 Jul 15;143A(14):1630-9. doi: 10.1002/ajmg.a.31724.
Genes with a role in the auditory system have been mapped by genetic linkage analysis of families with heritable deafness and then cloned through positional candidate gene approaches. Another positional method for gene discovery is to ascertain deaf individuals with balanced chromosomal translocations and identify disrupted or disregulated genes at the site(s) of rearrangement. We report herein the use of fluorescence in situ hybridization (FISH) to map the breakpoint regions on each derivative chromosome of a de novo apparently balanced translocation, t(8;9)(q12.1;p21.3)dn, in a deaf individual. Chromosomal breakpoints were assigned initially by GTG-banding of metaphase chromosomes and then BAC probes chosen to map precisely the breakpoints by FISH experiments. To facilitate cloning of the breakpoint sequences, further refinement of the breakpoints was performed by FISH experiments using PCR products and by Southern blot analysis. The chromosome 9 breakpoint disrupts methylthioadenosine phosphorylase (MTAP); no known or predicted genes are present at the chromosome 8 breakpoint. Disruption of MTAP is hypothesized to lead to deafness due to the role of MTAP in metabolizing an inhibitor of polyamine synthesis. Drosophila deficient for the MTAP ortholog, CG4,802, were created and their hearing assessed; no hearing loss phenotype was observed. A knockout mouse model for MTAP deficiency was also created and no significant hearing loss was detected in heterozygotes for Mtap. Homozygous Mtap-deficient mice were embryonic lethal.
通过对遗传性耳聋家族进行遗传连锁分析，已定位了在听觉系统中起作用的基因，然后通过定位候选基因方法进行克隆。另一种用于基因发现的定位方法是确定具有平衡染色体易位的耳聋个体，并鉴定重排位点处被破坏或失调的基因。我们在此报告使用荧光原位杂交（FISH）来定位一名耳聋个体中一条新发的明显平衡易位t(8;9)(q12.1;p21.3)dn的每条衍生染色体上的断点区域。染色体断点最初通过中期染色体的GTG显带进行定位，然后选择BAC探针通过FISH实验精确绘制断点。为了便于克隆断点序列，通过使用PCR产物的FISH实验和Southern印迹分析对断点进行了进一步细化。9号染色体断点破坏了甲硫腺苷磷酸化酶（MTAP）；8号染色体断点处不存在已知或预测的基因。由于MTAP在代谢多胺合成抑制剂中的作用，推测MTAP的破坏会导致耳聋。构建了MTAP直系同源基因CG4,802缺失的果蝇，并对其听力进行评估；未观察到听力损失表型。还构建了MTAP缺陷的基因敲除小鼠模型，在Mtap杂合子中未检测到明显的听力损失。纯合Mtap缺陷小鼠胚胎致死。
