Wendy L. Flejter

A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779.

The genetic mechanisms involved in sex differentiation are poorly understood, and progress in identification of the genes involved has been slow. The fortuitous finding of chromosomal rearrangements in association with a sex-reversed phenotype has led to the isolation of SRY and SOX9, both shown to be involved in the sex-determining pathway. In addition, duplications of the X chromosome, deletions of chromosomes 9 and 10, and translocations involving chromosome 17 have been reported to be associated with abnormal testicular differentiation, leading to male-to-female sex reversal in 46,XY individuals. We present the cytogenetic and molecular analyses of four sex-reversed XY females, each with gonadal dysgenesis and other variable malformations, and with terminal deletions of distal chromosome 9p, resulting from unbalanced autosomal translocations. PCR amplification and DNA sequence analysis of SRY revealed no mutations in the high-mobility-group domain (i.e., HMG box) in any of the four patients. Conventional and molecular cytogenetic analyses of metaphase chromosomes from each patient suggest that the smallest region of overlap (SRO) of deletions involves a very small region of distal band 9p24. Loss-of-heterozygosity studies using 17 highly polymorphic microsatellite markers, as well as FISH using YAC clones corresponding to the most distal markers on 9p, showed that the SRO lies distal to marker D9S1779. These results significantly narrow the putative sex-determining gene to the very terminal region of the short arm of chromosome 9.

A YAC-, P1-, and cosmid-based physical Map of the BRCA1 region on chromosome 17q21

A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated. The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap. The majority of the interval has also been converted to a cosmid contig. Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones. This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al. (1995), Osborne-Lawrence et al. (1995), and Friedman et al. (1995).

Isolation and characterization of somatic cell hybrids with breakpoints spanning 17q22-->q24.

Somatic cell hybrid mapping panels have previously been constructed to assist in the regional assignment of anonymous DNA probes and cloned genes to human chromosome 17. While a substantial number of hybrids are available that subdivide the short arm of this chromosome and the proximal portion of its long arm into specific regions, relatively few exist with breakpoints in the distal portion of the long arm. To increase the resolution of this region, four additional human x rodent somatic cell hybrids have been constructed that include breakpoints spanning the region 17q22-->q24. Hybrid clones carrying the long-arm derivative of chromosome 17 were initially identified by fluorescence in situ hybridization. Hybrids were subsequently screened using the polymerase chain reaction with primer sets representing DNA markers previously mapped to chromosome 17. These hybrids expand the existing somatic cell hybrid panel for the distal portion of the long arm of chromosome 17.

Region-specific cosmids and STRPs identified by chromosome microdissection and FISH

A strategy for identifying short tandem repeat (STR)-containing cosmid clones from a specific chromosomal region is described. The approach is based on the use of uncloned, PCR-amplified DNA derived from chromosome microdissection and pooled groups of STR sequences as hybridization probes to screen a cosmid library. Cosmid clones that display a positive signal common to both hybridizations are then characterized for repeat length polymorphisms. This method has been applied to chromosome bands 17q12-q21, a region that includes a gene (BRCA1) involved in early onset familial breast and ovarian cancer. Of 1536 chromosome 17-specific cosmid clones tested, 38 were identified by the dual screening procedure. Fluorescence in situ hybridization revealed that 19 cosmids originated from the microdissected target region. Thirteen of the 19 cosmids were mapped between markers flanking the BRCA1 region and selected for further characterization. Tetranucleotide repeats were identified in 10 of these 13 cosmids. Primers designed for each marker were tested on a panel of 80 CEPH parents for allele sizes, frequencies, and observed heterozygosities. From these studies six polymorphic and one nonpolymorphic STRs were identified. A similar approach should be applicable for screening whole genomic or chromosome-specific cosmid libraries in efforts to isolate new polymorphic markers from any chromosomal region of interest.

868 A PRACTICAL METAPHASE MARKER OF THE INACTIVE X CHROMOSOME

The ability to identify the inactivated X chromosome with routine G- or Q-banding would have broad clinical and research applicability. We recently reported that the inactivated X frequently bends or folds in region Xql3Xq21 (Flejter et al, Am J Hum Genet 36:218, 1984). The fold occurs in about 88% of prometaphase, 50% of early metaphase, 30% of midmetaphase, and 10% of late metaphase inactive Xs. In prometaphase, the site of folding includes Xq11.2 and Xq13.3, infrequently extending to Xq21.1. An omega-shaped loop is frequently formed between sub-bands Xq11.2 and Xq13.3. It is paradoxical that the inactive X is the only chromosome identifiable in interphase, yet in metaphase it cannot be distinguished from its active homolog. The specific inactivation-associated fold at region Xq1 resolves that paradox and is a useful marker of the inactive X. 1. The KOP translocation, t(X; 14)(q13; q32), has nearly all of Xq translocated to 14q (Allerdice et al, Am J Med Genet 2:223, 1978). Cell line GM0074 has one normal 14, one Xq-, two der (14) chromosomes, and a Y. One der(14) folded in 10/18 cells scored, confirming translocation of the inactivation center adjacent to 14q distal. 2. Metaphase cells from other primates had a specific fold in the same region as in the human X: at Xq13-Xq21 in 2 gorillas, 1 chimp, 2 pygmy chimps, 1 orang, 1 baboon, 1 rhesus and 1 stump-tail monkey. This is further evidence for evolutionary conservation of the X chromosome. One chimpanzee exhibited the fold at Xq24; we suspect a pericentric inv in this individual. We have not seen a frequent fold in the X of a bat (Tadarida brasiliensis), Chinese hamster (Cricetulus griseus), or rat kangaroo (Potorous tridactylis). 3. Regarding the relationship of X inactivation and intelligence in fra(X)(q28) carriers, we observe that in cells of normal carriers the fold was mostly on the fragile X-positive chromosome, whereas in cells of an affected carrier the fold was mostly on the fragile X-negative chromosome. This is evidence that inactivation of the fragile X chromosome is positively correlated with intelligence in carriers.