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 chromosomal translocations involving chromosome 17q24-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 17q24-q25 region. Through positional cloning, we isolated a 3.5 kb cDNA that is located at a close but distinct position from the SOX9 gene, from the region surrounding this breakpoint. Its mRNA, ~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 following 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.
Campomelic dysplasia (CMPD) is a rare congenital disorder characterized by shortening and bowing of the lower extremities, hypoplastic scapulae, cleft palate, micrognathia, tracheomalasia, talipes equinovalus and low-set ears. Sex reversal (SR) is a feature of nearly half of genotypical-male patients with CMPD (1 ,2 ). The frequent association of CMPD with chromosomal abnormalities involving distal 17q suggests that a mutant gene(s) responsible for CMPD and sex reversal is likely to be present at or around the breakpoints of the translocations commonly observed on chromosome 17q24.3-q25.1 in affected individuals (3 -5 ). Recently we reported a patient in whom acampomelic CMPD and sex reversal were associated with a de novo t(12;17) translocation (6 ); the breakpoint on 17q in this patient was located in the same region as that observed in the patients reported by others. For positional cloning of the putative CMPD gene(s), we mapped this patient's breakpoint on 17q by fluorescent in situ hybridization (FISH), using cosmid markers that had been localized to each chromosomal band in the candidate region as probes (7 ).
Foster et al. cloned a SRY-related gene, the SOX9, which mapped adjacent to chromosomal breakpoints of three patients on 17q (21 ) and described mutations of this gene in three patients without chromosomal aberrations. Its mouse homolog is suggested to play a role in chondrogenesis (22 ). On the basis of this evidence, the SOX9 gene is considered as a candidate gene for this syndrome. However, as chromosomal breakpoints are mapped 50 kb or more apart from the SOX9 gene and this syndrome may be a contiguous gene syndrome, it is unclear whether the other gene may be related to some phenotypes in this syndrome.
To consider a presence of the second gene associated with this syndrome, we cloned the breakpoint in our patient and isolated a cDNA adjacent to the breakpoint. Specific expression of this gene in testis suggested its candidacy for some role in CMPD and/or sex reversal.
The linear order of five cosmid loci previously mapped to chromosomal bands 17q24-q25 (7 ), as determined by two-color FISH on prophase chromosomes of normal individuals, was as follows: centromere/cCI17-591/cCI17-667/cCI17-509/cCI17-546/ cCI17-559/telomere. These clones were then examined on metaphase chromosomes of the patient to determine whether each marker locus was located proximally or distally to the breakpoint. Markers cCI17-591, cCI17-667 and cCI17-509 revealed signals on chromosome 17 and the derivative chromosome 17, while cCI17-546 and cCI17-559 showed signals on chromosome 17 and the derivative chromosome 12 (data not shown). These results indicated that the loci defined by cCI17-509 and cCI17-546 flank the chromosomal breakpoint.
To cover the region between the two flanking loci, we screened CEPH YAC libraries by Southern hybridization using cCI17-509 and cCI17-546 as probes. As YAC946e12 was found to contain both flanking marker loci, we constructed a cosmid library from this YAC clone in the standard manner (8 ). Cosmid clones containing human DNA inserts were selected by hybridization with labeled total human DNA. One of these clones, 3a, was mapped proximally to the breakpoint and closer than cCI17-509, by FISH (see Fig. 1 ). Pulsed field gel electrophoresis (PFGE) analyses revealed that clones 3a and cCI17-546 were located on the same genomic fragment whether the DNA was digested with ClaI (240 kb), Genotypical (420 kb), MluI (480 kb) or NruI (800 kb), indicating that the smallest of these fragments (240 kb) harbors the breakpoint. To isolate a smaller fragment containing the breakpoint, we performed cosmid walking from both flanking markers. The patient's DNA was examined by Southern analyses using each of three walking clones, 6E, 11F and 11-3-1, as a probe. Although no differences were detected by clones 6E or 11F, a 2.4 kb EcoRI fragment of cosmid 11-3-1 detected bands that were not observed in DNA from any of a large number of normal individuals, when the patient's DNA was digested with EcoRI or PvuII (Fig. 2 ). This result implied that the chromosomal breakage occurred within a 2.7 kb region of genomic DNA (as the 2.4 kb EcoRI fragment in 11-3-1 is at the end of the cosmid, the EcoRI fragment observed in Southern analysis is slightly larger than the probe).
We screened a cDNA library derived from adult testis using the 2.4 kb genomic EcoRI fragment as a probe and isolated a clone containing a 1.7 kb insert. By Southern analysis using this cDNA clone, a similar 2.7 kb band was observed in the patients DNA digested with EcoRI (data not shown). Northern blot analysis using this cDNA as a probe indicated that the transcript, ~3.7 kb long, was expressed specifically in testis among 16 different tissues examined (eight are shown in Fig. 3 ). We performed primer extension to clone the 5' region of the cDNA and obtained sequences accounting for 3.5 kb of the transcript (Fig. 4 ). We were unable to find any open reading frame longer than 150 bp within this 3.5 kb cDNA sequence, nor could we detect any peptide by in vitro translation experiments using RNA transcribed from this cDNA.
Studies of CMPD patients with chromosomal abnormalities, by us and others, have indicated that the genetic defect responsible for this syndrome is located at 17q24-q25 (5 ,6 ). The two distinct clinical features of CMPD, one a complex syndrome of skeletal malformations and the other sex reversal, suggest that CMPD involves contiguous genes (9 ). Though `campomelia' is one of its most common clinical features, cases without campomelia (acampomelic CMPD) have been reported (10 -13 ); the patient reported here belonged to the latter category. XY-female (SR) is also characteristic of CMPD; however, as molecular studies of SRY (testis determining factor, ref. 14 ), had failed to detect any mutations in six cases of CMPD including ours (6 ,15 ), we concluded that the sex reversal observed in CMPD patients is likely to involve a gene other than SRY.
Foster et al. cloned a SRY-related gene, the SOX9, which mapped adjacent to chromosomal breakpoints of three patients on 17q (21 ). They also described mutations of this gene in three patients without chromosomal abbreviation. The expression pattern of the corresponding mouse homolog of the SOX9 suggested that this gene plays a role in chondrogenesis (22 ). Together with these facts and positional candidacy, the SOX9 gene is considered as a candidate gene for this syndrome. However, as chromosomal breakpoints are mapped 50 kb or more apart from the SOX9 gene and this syndrome may be a contiguous syndrome, it is unclear whether the SOX9 gene can explain all the clinical phenotypes in the patients.
Using a positional cloning method, we cloned the breakpoint on 17q in the acampomelic CMPD patient with sex reversal and isolated a cDNA which was expressed specifically in testis. This gene is mapped centromeric side form cCI17-546 whereas the SOX9 gene is telomeric (23 ). The transcript contained no long open reading frame and we failed to detect any peptide by in vitro translation experiments using RNA transcribed from the cDNA. However, others have reported examples of X-inactivation gene, that are not translated to any peptide (16 ,17 ). The gene represented by our novel cDNA may encode a functional peptide in the 5' portion we have not yet cloned, but it is also possible that the RNA itself may play a significant role in differentiation or sex determination. In either case, the location of this gene at the chromosomal breakpoint and its specific expression in testis support a view that disruption of this gene could be responsible for the sex reversal observed in CMPD patients.
Clinical features of the patient used in this study were reported previously (6 ). The subsequent chromosome analysis revealed that this patient had de novo t(12;17) translocation, kariotypes of the parents were normal.
For two-color FISH, each of two probe DNAs was labeled with either biotin or digoxigenin using a nick-translation labeling kit (Boehringer-Mannheim). Hybridization was performed on prophase chromosomes of a karyotypically normal individual using 150 ng of biotin-labeled DNA, 50 ng of digoxigenin-labeled DNA and 20 ng of total human DNA (18 ). Hybridization signals were detected with avidin-FITC and antidigoxigenin-rhodamine.
Genomic DNA was prepared from nearly 107 cells embedded in each low-melting-point agarose plug. Electrophoresis was carried out in a Beckman GeneLineII system (19 ). DNA was electrophoresed in a 1.0% agarose gel at 350 mA with 1 min pulse time for 12 h, at 370 mA with 2 min pulse time for 12 h and at 390 mA constant current with 3 min pulse time for 12 h. After transfer to a nylon membrane, hybridization was performed as described elsewhere (19 ).
According to methods described previously (19 ), a cosmid library was constructed from a YAC DNA containing both of the markers most closely flanking the breakpoint. In brief, yeast genomic DNA was partially digested with Sau3A restriction endonuclease and fractionated by sucrose density-gradient centrifugation. After the ends of the 35-45 kb genomic DNA fragments were partially filled in with dATP and dGTP, the fragments were cloned into the XhoI site of a cosmid vector, pWEX15, that had been partially filled in with dCTP and dTTP.
After digestion with restriction enzymes, DNA samples were electrophoresed in a 0.8% agarose gel and transferred to nylon membrane. Genomic DNA fragments were labeled by the random-priming method and pre-annealed with human placental DNA to suppress hybridization of repetitive sequences. Hybridizations and washing were performed as described elsewhere (19 ).
We performed Northern analysis using multiple-tissue blots (Clontech) according to the manufacturer's instructions. The 1.7 kb cDNA insert isolated from the original cDNA clone was labelled by random-priming and used to probe the blots. Washes were done at 50oC in 0.1 * SSC and 0.1% SDS. The membrane was exposed to Kodak XAR-5 film at -80oC for a week.
We performed primer extension to clone the 5'-end of the cDNA, using a ZAP-cDNA synthesis kit (Toyobo, Japan) with testis RNA. The cDNA library was screened with oligonucleotides representing the 5' portion of the original cDNA clone.
Nucleotide sequences of cDNAs were determined by the dideoxy chain-termination method of Sanger et al. (20 ).
We thank Kiyoshi Noguchi for technical assistance. This work was supported in part by a grant-in-aid from the Japanese Ministry of Education, Culture and Science.
jnl.info{at}oup.co.uk
Human Molecular Genetics
Pages
Introduction
Results
Mapping the breakpoint on 17q
Cloning of the breakpoint
Isolation of cDNA
Discussion
Materials And Methods
Patient
FISH analysis
PFGE
Construction of a cosmid library
Southern hybridization
Northern blot analysis
Primer extension of cDNA cloning
Nucleotide sequencing
Acknowledgements
References
REFERENCES
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