Ethics approval
This study was approved by the Ethics Committee of the Drum Tower Hospital, Nanjing University (Jiangsu, China). Informed consent was given by the family (Fig. 1), who agreed to joining this study and the use of the clinical data for scientific research and publication. The study was performed in the Prenatal Diagnosis Center of Nanjing Drum Tower Hospital (Jiangsu, China). The methods used in this study were performed in accordance with the approved guidelines.
Clinical Features
The proband was a 20-year-old Chinese woman (Ⅲ-5), she was short in stature (150 cm), physical examination showed no obvious symptoms except lower-extremity bowing (valgus deformities). She did not have musculoskeletal complaints, such as stress fractures, joint pain or osteomalacia, she could walk, run and jump without impaired mobility. Besides, her teeth and hearing were both normal, no dental abscesses or sensorineural hearing loss was found. Her intelligence and development were no different from healthy females. No medical treatment had been offered before. The X-ray of her lower extremities indicated knock knees, and a bilateral bowed femur, tibia and fibula (Fig. 2). The laboratory tests showed a low serum phosphate level (0.73 mmol/l), normal serum calcium level (2.3 mmol/l) and normal serum parathyroid hormone (22.33 pg/ml). Her family history was remarkable. The proband’s father (Ⅱ-10) was healthy, but her mother (Ⅱ-9) had similar symptoms, such as short stature(145 cm) and lower-extremity bowing (valgus deformities). The proband had three maternal aunts and two maternal uncles, except a maternal uncle (Ⅱ-11) with similar and more severe symptoms, the other maternal uncle, aunts and their offspring were all healthy. The symptomatic maternal uncle (Ⅱ-11) showed a significantly reduced height (135 cm) comparing to reference standards and more serious lower-extremity bowing (valgus deformities). In addition, joint pain and impair mobility had been occurred. The proband’s maternal grandfather (Ⅰ-1) died years ago because of other reasons, maternal grandmother (Ⅰ-2) was short in stature with lower-extremity bowing (valgus deformities). The laboratory tests and X-ray images were all lacked in the affected family members except proband.
DNA Extraction
Genomic DNA was extracted from peripheral whole blood samples, using the QIAamp DNA Blood kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA was stored at ‑20˚C prior to use.
Trio-based Whole-exome Sequencing
Collected the peripheral whole blood samples from proband and her parents, sent blood samples to Berry Genomics Corporation (Beijing, China) to perform the trio-based whole-exome sequencing (WES), and the BWA (Burrowa-Wheeler, v.0.5.9-r16) alignment algorithm was used to compare with the UCSC(http://genome.ucsc.edu/) hg19 human reference genome sequence, and the GATK (Genomic Analysis Tool Kit) was used to identify the mutation. To predict the outcomes of variants, the Human Gene Variant database (http://www.hgmd.cf.ac.uk), ExAC database, 1000 Genome database were used to annotate the detected mutation sites, and SIFT (https://sift.bii.a-star.edu.sg/), PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and other software were used to predict the effect of the nucleotide changes on protein function.
Sanger Sequencing Validation
A variant of the PHEX gene was identified in proband and her mother, the detected variants were confirmed using PCR. Information of the PHEX gene was obtained from the online NCBI database (https://www.ncbi.nlm.nih.gov/pubmed; RefSeq NM_000444.5). 1 pair of primer was designed for PCR amplification of the exon region that contains mutation and intron/exon boundaries of the PHEX gene using an online tool (http://www.primer3.com/). The following primer was used for a 582-base pair (bp) target sequence in exon 11: F:5′-CACTTTGACCGGCCTAAAAC-3′, R:5′-CCCCTGGAAAACTACACACC-3′. The PCR conditions were as follows: 94 ˚C for 3 min, 94 ˚C for 30 sec, 57˚C for 30 sec, 68˚C for 15sec, (39 cycles) and 68˚C for 6 min, 4˚C forever. The PCR-amplified DNA products were ectrophoresed on a 2% agarose gel, then were submitted to sanger sequencing in Sangon Biotech Co., Ltd. (Shanghai, China). Sequencing results were analyzed using Seqman software in the DNA star software package and aligned with the UCSC hg19 human standard sequence.