X-linked congenital hypertrichosis syndrome is associated with interchromosomal insertions mediated by a human-specific palindrome near SOX3

Abstract

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.

Figure 1

Phenotypes and Genetic Locus of a Distinct X-Linked Congenital Hypertrichosis Syndrome (A) Pedigree of the five-generation Chinese family with eleven affected individuals. For V1, the diagnosis was made from a chorionic villus sample. Individuals whose DNA was available are indicated by “+.” (B) Photograph of the proband showing severe CGH. (C) Photograph and MRI image showing a cervical meningocele in the proband. (D) X-ray image of the proband showing scoliosis. (E) Schematic diagram of Xq26.3-q27.2 showing the RefSeq genes in the critical region for the X-linked congenital hypertrichosis syndrome. A solid blue bar represents the critical region. The positions of all genetic markers used in linkage analysis are shown.

Figure 2

Identification of an Inherited Interchromosomal Insertion at Xq27.1 in the Chinese Family with a Distinct X-Linked Congenital Hypertrichosis Syndrome (A) Copy-number state of a 600 kb genomic region on chromosome 5q35.3 showing the presence of a microduplication in the proband. CN, copy number. (B) Validation of the microduplication and its segregation with the disease phenotype by qPCR. RCN, relative copy number. Error bars represent SD. (C and D) Two-color FISH signals on a representative interphase nucleus (C) and typical metaphase chromosomes (D) demonstrating the insertion event. BAC probes are CTD-2507I18 (red), RP11-55E17 (green), and RP11-671F22 (green). See Figure 4A for the positions of BAC clones. (E and F) Sequence analysis of the proximal (E) and distal (F) insertion junctions. Reference sequences on Xq27.1 and 5q35.3 are indicated in red and blue, respectively. The proximal junction contains a microinsertion from the X chromosome (black) and a 2 bp microinsertion (green) of unknown origin. (G) PCR amplification of the distal insertion junction showing segregation of the insertion with the phenotype. All genomic positions correspond to the February 2009 human reference sequence (GRCh37).

Figure 3

Identification of an Inherited Interchromosomal Insertion at Xq27.1 in the Mexican Family with X-Linked CGH (A) Pedigree of the five-generation Mexican family with X-linked CGH. Individuals whose DNA was available are indicated by “+.” (B) Copy-number state of a 600 kb genomic region on chromosome 4q31 showing the presence of a microduplication in an affected individual. CN, copy number. (C) Validation of the microduplication by qPCR. RCN, relative copy number. Error bars represent SD. (D and E) Sequence analysis of the proximal (D) and distal (E) insertion junctions. Reference sequences on Xq27.1 and 4q31 are indicated in red and blue, respectively. The distal junction contains a 25 bp microinsertion. (F) PCR amplification of the proximal insertion junction showing segregation of the insertion with the phenotype. All genomic positions correspond to the February 2009 human reference sequence (GRCh37).

Figure 4

Interchromosomal Insertions Mediated by the Same Human-Specific Palindrome (A) Schematic diagram depicting the two independent insertions found in the present study. Red solid bars represent the inserted fragments, and indicated sizes correspond to base pairs (bp). Red lines display the orientation of insertions. Positions of the BAC probes used in two-color FISH and of the RefSeq genes on the corresponding chromosomal regions are shown. (B) Schematic diagram of the 180 bp human-specific palindrome with a summary of the breakpoints identified in the present study. Solid triangles indicate insertion breakpoints in the two study families (Chinese in red and Mexican in green). JBPcn, junction breakpoint in the Chinese family; JBPmx, junction breakpoint in the Mexican family. Open triangles represent the deletion breakpoints in normal individuals (Chinese in red, Mexican in green, and Yoruban in black). A black solid bar represents the LINE-1 element that contains the proximal JBPcn. The precise position of the JBPcn is given. (C) Schematic diagram showing the small human-specific palindrome at Xq27.1 and its flanking sequences. The 180 bp palindromic sequence is boxed. A chimp has two halves of the palindrome but in direct orientation. All other three nonhuman primates only have one half of the palindrome. All genomic positions correspond to the February 2009 human reference sequence (GRCh37).