Exclusion of the 750‐kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1‐linked families

Abstract

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for non-annotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. To summarize, our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.

TAR Cloning: Perspectives for Functional Genomics, Biomedicine, and Biotechnology

“…(B) For TAR cloning experiments, the vector DNA is linearized by a unique endonuclease located between the hooks to expose targeting sequences. Recombination between the vector hooks and homologous sequences in the co-transformed human DNA fragment results in rescue of the desired region as a circular yeast artificial chromosome (YAC) that replicates and segregates properly 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. The same TAR vector can be applied to multiple DNA samples.…”

“…Many genes in the human genome reside within segmental duplications (SDs) with a high level of similarity, which prevents their mutational analysis by routine PCR methods. A TAR cloning strategy has been successfully applied to recover and analyze several duplicated regions, and, therefore, this approach is applicable to all gene families whose analysis is prohibited by the presence of SDs 29, 32, 34, 35, 36. Accumulating information shows a plethora of structural variations (such as deletions, insertions, inversions, translocations, and copy number variations [CNVs]) in genomes of different individuals, some of which may lead to disease 43, 44, 45, 46, 47.…”

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

“…For example, by transfecting the isolates into mammalian cells, the functionality of several genes was confirmed, including the breast cancer gene BRCA1 (Annab et al 2000; Kononenko et al 2014), the human HPRT gene (Kouprina et al 1998), the tumor suppressor gene KAI1 (Kouprina and Larionov 2006), the metastasis suppressor gene TEY1 (Nihei et al 2002), the human TERT gene coding for a catalytic subunit of human telomerase (Leem et al 2002), the cancer-associated genes VHL (mutated in von Hippel–Lindau syndrome) and NBS1 (mutated in Nijmegen breakage syndrome (Kim et al 2011). Further evidence of the accuracy of the TAR cloning was obtained by isolation and shot-gun-sequencing of the genomic regions containing the SPANX ( S perm P rotein A ssociated with the N ucleus on the X chromosome) genes (Kouprina et al 2004a; 2005a; 2007a; 2007b; 2012). The sequences of one of the SPANX gene family members, SPANX-C , isolated from several individuals differed only by a single nucleotide substitution due to natural polymorphism (Kouprina et al 2005a).…”

“…An example of the application of TAR cloning for long-range molecular haplotypying is isolation of the SPANX genes from different individuals (Kouprina et al 2007a; 2007b; 2012). This case is very complicated because sequences of these gene members have a high level of homology and reside within large segmental duplications with >95% identity.…”

“…Direct isolation of a set of overlapping genomic segments of the 750 kb region in X-linked families with predisposition to prostate cancer revealed no disease-specific alterations within the SPANX gene cluster (Kouprina et al 2012). Thus, TAR cloning allowed exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy.…”