X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.

cFlavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ⌬cslA and ⌬cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (⌬cslA ⌬cslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G 4 and of the chondroitin lyase-deficient ⌬cslA ⌬cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. C olumnaris disease can affect almost all species of freshwater fish, including cultured and wild fish, often resulting in heavy economic losses in aquaculture worldwide (1, 2). Its causative agent, Flavobacterium columnare, has been the focus of microbiological as well as aquaculture research for decades, with respect to diagnosis, epidemiology, pathology, preventive measures, and possible virulence factors (2-4). The genetic diversity of F. columnare has been recognized by the presence of at least three distinct genomovars (5-7). However, the pathogenesis of this important pathogen is still rather unclear. Although a few molecules, such as chondroitin AC lyase, have been reported as possible virulence factors (8-11), the lack of genetic manipulation systems suitable for F. columnare has been an obstacle in understanding the mechanisms involved in the pathogenesis of this bacterium. Staroscik et al. (12) developed techniques to introduce replicative plasmids into F. columnare by conjugation and also to construct mutants by insertional mutagenesis and transposon-mediated mutagenesis. However, both processes remain inefficient, and few subsequent reports utilizing these techniques have been published (12,13).A technique to construct in-frame deletions was recently developed for the genetically tractable bacterium Flavobacterium j...