Positional Cloning of Genes Responsible for Diseases through Analysis of Patients with Chromosomal Translocation.

Abstract

We have isolated a part of candidate gene responsible for Klippel-Feil syndrome (KFS) and a second candidate gene responsible for Campomelic syndrome using a patient with chromosomal translocation. We have isolated a part of a candidate gene responsible for KFS by positional cloning using a patient witht (5 ; 17) (q13 ; q25) . Analyses of the patient's chromosomes with seven cosmid markers on 17q24-25.1, which were linearly ordered, indicated that the breakpoint is located between two loci defined by cosmids, cCI17-509 and cCI17-546. Subsequently, a cosmid library was constructed from a YAC clone containing both flanking loci. By Southern hybridization using cosmid markers as probes, we detected extra bands in the patient's DNA digested with EcoRI+HindIII, PvuII, PstI, BglII or SadI when a cosmic clone, 3a was used. This result implied that the chromosomal breakage on 17q occurred within genomic DNA contained by cosmid 3a. We further isolated exon-like fragments by an exon-amplification method which may be a part of a candidate gene responsible for KFS. Campomelic dysplasia (CMPD), a rare congenital disorder, is characterized by a variety of skeletal anomalies, low-set ears and, in nearly half of genotypical-male patients, sex reversal. Observations of chromo-somal translocations involving chromosome 17824-q25 in several CMPD patients have implied that disruption of one or more genes in the breakpoint region is responsible for this disease. Using fluorescence in situ hybridization, we mapped the chromosome-17 breakpoint in a patient with acampomelic CMPD and sex reversal, who carries a de novo constitutional t (12; 17) translocation, between two known cosmid markers in the 17824-q25 region. Through positional cloning, we isolated a 3.5-kb cDNA that is located at a close but distinct position form the SOX9 gene, from the region surrounding this breakpoint. Its mRNA, approximately 3.7 kb long, was expressed specifically in testis among 16 adult tissues examined by northern-blot analysis. As we were unable to find any long open reading frame in the 3.5-kb cDNA sequence or to detect any peptide fol-lowing an in vitro translation experiment using RNA transcribed from this cDNA, we speculate that this gene may play a critical role in differentiation or sex determination as a functional RNA.