Phenotype description
All patients in the family presented a characteristic lip shape at birth, with fish mouth-like, everted, thick lips, notably at the lip corners (Fig. 1b). No obvious abnormality of the arms, legs, or knee or elbow joints was observed at birth or before 1 year of age. After starting to walk, genu varum presented and became more serious with age, resulting in limited daily activities. The proband (V25) was the first child of his non-consanguineous parents, full-term normal delivery, birth weight 3.5 kg, length 50 cm. Everted lips were observed at birth. Developmental milestones were normal reached. Genu varum appeared when he started to walk at 1-year-old and became worse as height and weight increase. He received a high tibial osteotomy at age ten but the genu varum was not corrected. The space between the proband’s knees was 15 cm while standing naturally (Fig. 1c). This reduced significantly when standing forcibly by himself. Cubitus valgus was also noticed (Fig. 1d). He was 19 years old when he first came to our hospital, with height 165 cm and weight 52 kg. No dysmorphic feature was observed in his head, chest or spine. Psychomotor and intellectual development is normal. Physical examination showed muscle strength of V grade and normal muscle tension. No abnormality of bone metabolism was found by laboratory examination, with serum calcium 2.44 mmol/L, serum phosphate 1.4 mmol/L, serum alkaline phosphatase 46 U/L, ionized calcium 1.12 mmol/L, PTH 25.9 pg/mL, 24-h urine calcium 3.88 mmol, 24-h urine phosphate 4.86 mmol, 1,25(OH)2D3 51.17 pg/mL, and β-CTX 0.2 ng/mL. X-ray examination showed wide interspacing of the knee joint and patellar dislocation, and no obvious changes were observed in sclerotin, bone cortex, or bone trabecula of the bones composing the hip and knee joints (Additional file 2: Figure S1).
Disease locus mapping and coding region mutation screening in chromosome X
To determine the causative locus, linkage analysis and fine mapping was performed in the family. A candidate interval of 4 Mb (chrX: 136,218,962–140,388,078, hg19) containing 20 RefSeq genes, with LOD > 2, was identified (Fig. 1e, f). Whole exome sequencing was then performed in the proband to detect mutations in the coding region, especially in the candidate 4 Mb interval. Detected coding SNPs and small indels were sequenced in all available family members, yet no coding region mutations fully co-segregated with the phenotype in the family.
Structure variation and noncoding region mutational detection
To determine whether the rare X-linked compound syndrome was caused by an unknown microdeletion or microduplication, we performed a genome-wide high-resolution CNV scan. We did not detect any potential pathogenic CNVs which are absent in the DGV database in the critical region. A trio in the family (V25, IV29, and IV30) underwent WGS to detect complicated structural variation and SNPs or indels in the noncoding region. An average of 99.97 Gb of sequence was generated with a mean coverage depth of 34.54× across the whole genome for each individual. WGS of the trio implicated an intra-chromosomal translocation with one breakpoint embedded in the critical region. Two breakpoints implicated in WGS are at chrX: 733,365 and chrX: 139,502,956. The chrX: 139,502,956 breakpoint within the critical region was supported by four split reads with coverage of five in the male patient, two split reads with coverage of 12 in the obligate carrier mother, and no split reads with coverage of 13 in the unaffected father. The other breakpoint chrX: 733,365 outside the critical region, was supported by five split reads with coverage of 52 in the male patient, ten split reads with coverage of 44 in the obligate carrier mother, and no split reads supported in the unaffected father.
Identification of an insertion into the Xq27.1 palindrome from PAR1
To validate the identified structural variation, gap-PCR was performed spanning the two breakpoints. A 573 bp gap-PCR product was detected in all male patients and obligate female carriers in the family (Fig. 2a). Sanger Sequencing of the generated PCR products confirmed the junction of two breakpoints, considered to be the distal breakpoint junction (Fig. 2b). Genome walking from the locus-specific sequence distal to the breakpoint chrX: 139,502,956 also revealed the junction of two breakpoints. We noticed the chrX: 139,502,956 breakpoint was near the center of the palindromic sequence at Xq27.1, while the chrX: 733,365 breakpoint was located at Xp22.33, homologous with Yp11.32, in the pseudoautosomal region 1 (PAR1) of the sex chromosomes. CNVs in PAR were easily missed due to homology between the X and Y chromosomes, so we re-evaluated the SNP array data and found a > 100 kb copy number gain involving the identified chrX: 733,365 breakpoint in PAR1 in the proband (Fig. 3a). Copy number gains in the same region were also observed in four affected males (IV3, IV5, IV11 and IV23) and one obligate female carrier (III9), but not in unaffected familial males (IV25 and IV30) or 42 unrelated controls. To confirm this, we designed four qPCR assays to detect CNVs in four informative family members and two unrelated controls. Three qPCR assays within the implicated duplication region confirmed a copy number gain in the male patients and female carriers in the family, while one qPCR assay with primers spanning the breakpoints showed no CNV changes in the family (Fig. 3b). Combining qPCR and SNP array data, a ~ 105 kb duplication in PAR1 was confirmed with full co-segregation with the disease phenotype in the family. Considering the breakpoint junction identified by WGS, we speculated that the duplicated PAR1 fragment could insert in the palindrome at Xq27.1 (Fig. 2c). We further amplified the breakpoint junctions with primers from the sequences flanking the palindrome at Xq27.1 and the boundary of the Xp22.33 duplication. Sequence analysis of the resultant amplicons by Sanger sequencing verified the distal junction pinpointed in WGS (chrX: 733,365/chrY: 683,365 and chrX: 139,502,956) and showed the proximal junction between the lower boundary of the Xp22.33 duplication and the Xq27.1 palindrome (chrX: 628,417/chrY: 578,417 and chrX: 139,502,956; Fig. 2b). The junction sequencing results also indicated the duplicated fragment was inserted into Xq27.1 palindrome indirect orientation.
Expression detection of nearby genes
Since the 105 kb insertion contained no coding genes, we presumed the insertion could impact genes near the Xq27.1 palindrome via positional effects. We tested expression levels of three candidate genes within a topological associated domain on Xq27.1. As a result, we detected no expression of FGF13, SOX3, or FGF13-AS1 in peripheral blood of the proband or in unrelated controls.