| BackgroundWith the improvement of living standards, more attention is being given to the height of children throughout development. Short stature brings a heavy burden of psychological and economic stress on individuals and families. Short stature is defined as a height more than two standard deviations below the mean for the age, gender, and ethnic group established standards （below the 3rd percentile） for the child, and can be divided into proportionate and disproportionate types. Notably, patients with disproportionate body proportions usually have skeletal dysplasia （SD）. The incidence of SD is approximately 1/5,000 at birth, which represents 5% of children with birth defects. However,the incidence might be higher because of under-diagnosis. Moreover,children with SD usually display musculoskeletal abnormalities leading to severe physical disabilities and other system function abnormalities involving hearing, vision,neurological,respiratory,cardiac,renal, and psychological problems,which may result in the patient not being able to carry out daily activities by themselves and/or having a shortened lifespan. Owing to the genetic and physical heterogeneity of SDs, early discovery, diagnosis, and intervention may be the most effective way to reduce the harm it causes.However, SDs include hundreds of highly genetically and clinically heterogeneous disorders, which presents a huge challenge in the accurate diagnosis and treatment of the diseases. According to the 2015 report of the Nosology and Classification of Genetic Skeletal Disorders, the total number of genetic skeletal diseases is 436, which can be divided into 42 groups according to the features of molecular, biochemical, and radiographic findings. The number disease-related genes has increased from 226 to 364 since the 2010 report. Common genetic skeletal diseases include avascular necrosis of the femoral head （ANFH）,achondroplasia （ACH）,spondyloepiphyseal dysplasia （SED）,multiple epiphyseal dysplasia （MED）, and osteogenesis imperfecta （OI）.Over the last 10 years, remarkable progress has been made in understanding the molecular basis of numerous SD diseases. However, the genotype-phenotype relationship is still poorly understood. Therefore, more complete clinical data through diagnosis and identification of individual gene mutations will reveal molecular pathogenic mechanisms, which will provide clinicians with an improved means for accurate differential diagnosis as well as allow scientific researchers to better understand gene functions. However, to date, such studies of SDs are still very scarce.Objectives1. To analyze the clinical manifestations and expression profiles of the 7 recruited families of disproportionate short stature with skeletal dysplasia（SD）.2. To perform the genetic analysis in all the patients by detecting related pathogenic genes, pinpoint causative genes and confirm the precise mutant site and type of each mutation.3. To predict the protein functions of all the novel missense mutations detected in this study, and conduct bioinformatics analysis.4. To explore the correlations between the phenotype and genotype of disproportionate short stature with SD.5. To provide information necessary to improve diagnosis and treatment of disproportionate short stature with SD for clinicians.Subjects and Methods1. Subjects: From September 2015 to October 2016, 7 disproportionate short stature with SD patients were enrolled at department of Pediatrics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China.2. Methods: （1）We analyzed the inheritance patterns, clinical manifestations and radiographic features of the probands from 7 recruited families of disproportionate short stature with SD. （2） Mutations in a short stature custom panel （495 curated genes）were detected, utilizing targeted exome sequencing （TES） technology. （3） The protein functions of all the novel mutations were predicted and bioinformatics analysis was conducted. （4） We diagnosed the seven patients based on clinical data, radiographic features, and genetic testing results, and further analyzed the correlations between the gene mutations and various clinical and radiographic findings. （5）We provided the clinical thought for disproportionate short stature with SD through literature review.Results1. Clinical characteristics analysis: Seven probands in the present study all presented with disproportionate short stature with SD such as short neck （P1,P2, P4,P5）, pectus carinatum （PI, P4, P5, P6,P7）, barrel chest （P2）, kyphosis and scoliosis （P4）,swollen interphalangeal joints （P3）, and ’trident’ hands （P6）. The seven patients included three males and four females and the mean age was 8.1 （2 y 3 m-12 y） years.The age of onset age for three patients （P1, P6, P7） was at birth; for three patients （P3,P4, P5） in early childhood; and for one patient （P2）, in late childhood. The heights of all patients were more than two standard deviations below the median value for the child’s age and sex. According to the upper-to-lower body-segment ratio （U/L） and arm spans,five patients （P1-P5） were diagnosed as short-trunk short stature, while two patient （P6,P7） was diagnosed as short-limb type. The severity of the clinical phenotypes and the affected family members were different. In general, according to physical examination and anthropometric measurements, the seven subjects all conform with the diagnosis of disproportionate short stature with SD.2. Biochemical analysis: Samples from the seven patients were assayed for general biochemical indicators. Levels of alkaline phosphatase （ALP）, which is related to the growth and development of children, were found to be increased, but the rest of the factors tested were all within the normal ranges. Bone metabolism index was subsequently tested. Levels of 25-hydroxy vitamin D （25（OH）D） were found to be significantly decreased, whereas parathyroid hormone （PTH） levels were normal. The seven patients were initially suspected to have inflammatory rheumatic diseases.However, laboratory findings for inflammation and rheumatoid factor were within the normal ranges. None of the laboratory results supported a diagnosis of connective tissue disease.3. Radiological findings: Radiographic examinations were performed on the seven patients. Radiographic results of the patients all showed deformity of the spine, pelvis,and extremities, though different features were prominent in each patient. The key radiological features of the seven patients are platyspondyly, dysplasia of epiphysis and metaphyseal changes （PI）, platyspondyly with hump-shaped posterior portions （P2）,’bullet-shaped’ vertebral bodies and swollen interphalangeal joints with joint space narrowing （P3）,scoliosis, platyspondyly with ’bullet-shaped’ appearance and femoral head and acetabulum hypoplasia （P4）, platyspondyly, narrow disc spaces, irregular and broadening metaphyses of the long bones, dumbbell-shaped short tubular bones of hand （P5）, and narrowing of the interpediculate distance from L1-L5 （P6）, short long bones of the limbs and broadening metaphyses （P7）.4. Mutation detection: To further confirm the diagnoses,we subsequently applied TES technology for the seven patients and their family members. We found mutations in six different genes. We identified a heterozygous mutation （c.1636G>A） in COL2A1 in P1, causing the substitution of glycine to serine in codon 546; an intragenic deletion in SEDL in P2, which was approximately 1661 bp in size, involving 806 bp in intron 5, 99 bp in exon 6, and 756 bp in the 3’ portion of exon 6, resulting in truncated Sedlin proteins; compound heterozygous mutations （c.730G>C/c.589+2T>C） in WISP3 in P3,resulting in a glycine to arginine substitution at p.244 and altered splicing, respectively;compound heterozygous mutations （c.516518delCAA/c.719A>G） in GALNS in P4,resulting in an asparagine deletion at p.172 and a tyrosine to cysteine substitution at p.240, respectively; a heterozygous mutation （c.2395C>G） in TRPV4 in P5, causing the substitution of proline to alanine at codon 799; a heterozygous mutation （c.1138G>A） in FGFR3 in P6, resulting in a glycine to arginine substitution at p.380; a heterozygous mutation c.1284C>A in FGFR3 in P7, resulting in a asparagine to lysine substitution at p.428. Using the Human Gene Mutation Database （HGMD）, we determined that these mutations included three novel mutations (1661-bp deletion （in5/ex6del） in SEDL,c.730G>C （p.G244R） in WISPS, c.516518delCAA （p.172deIN） in GALNS, and the rest of the mutations that had previously been identified.5. Bioinformatic analysis: To clarify whether the three novel mutations in the present study are pathogenic, we conducted bioinformatics analysis. The human SEDL gene consists of six exons which span approximately 20 kb of genomic DNA and encodes a 140-amino acid protein. The 423-bp coding region encompasses exons 3-6 and the untranslated regions comprises exons 1, 2, the 5’ portion of exon 3, and the 3’portion of exon 6. The start （ATG） and stop （TGA） codons are in exons 3 and 6,respectively. The sites of 1661 bp deletion involve 806 bp in intron 5, 99 bp in exon 6,and 756 bp in the 3’ portion of exon 6 identified in P2. The human SEDL conserved domain is depicted with Sedlin and the N-terminal conserved region （SedlinN; amino acids 9-136） （Fig.5A）. Gross deletions involving the splice site of exon 6 can cause abnormal transcription and translation, resulting in an incomplete protein structure and affecting the protein function of the mutant SEDL gene.The human WISP3 gene consists of five exons which span approximately 15 kb of genomic DNA and encodes a 372-amino acid protein. The 1119-bp coding region encompasses exons 1-5 and the untranslated regions consists of the 5’ portion of exon 1 and the 3’ portion of exon 5. The Start （ATG） and Stop （TAA） codons are in exons 1 and 5, respectively. The human WISP3 conserved domain is depicted with insulin-like growth factor binding-like module （IGFBP; amino acids 46-116）, von Willebrand type C repeat module （VWC; amino acids 117-180）, thrombospondin type 1 repeat module（TSPl;amino acids 207-261）, and C-terminal cysteine knot （CTCK; amino acids 262-354）. The missense mutation c.730G>C （p.G244R） was predicted to be damaging by Polyphen v.2, with a score of 1.00. Multiple amino acid sequence alignments show that p.Gly244 is conserved across various species. The novel mutations found in WISP3 are located in the TSP-1 domain, which binds heparin or sulphated proteoglycans to modulate cell adhesion and maintain extracellular matrix （ECM） composition, thereby possibly leading to abnormal protein aggregation in the cytoplasm.The human GAINS gene consists of 14 exons which span approximately 50 kb of genomic DNA and encodes a 522-amino acid protein. The human GALNS conserved domain is depicted with N-acetylgalactosamine-6-sulphatase （GALNS; amino acids 30-494）. Multiple sequence alignments of amino acid residues across different species suggests that p.N172 and p.Y240 are highly conserved. The mutations in GALNS are predicted to destroy the protein’s hydrophobic core,thereby leading to misfolding and affecting its catalytic activity.6. Definitive diagnosis: In conclusion, we diagnosed the seven patients based on clinical data, radiographic features, and genetic testing results. The seven patients in our study were diagnosed as follows: spondyloepiphyseal dysplasia congenital （SEDC）（P1）,X-linked spondyloepiphyseal dysplasia tarda （SEDT） （P2）,spondyloepiphyseal dysplasia tarda with progressive arthropathy （SEDT-PA）（P3）, Mucopolysaccharidosis type IVA （MPS IVA）（P4）, metatropic dysplasia （MD）（P5）, ACH（P6）, hypochondro-plasia（HCH）（P7）.Conclusions1. The phenotype of disproportionate short stature with SD is associated with its genotype.2. TES technology is of important value for the diagnosis of disproportionate short stature with SD.3. This study found three new pathogenic gene mutations, which will provide clues for its functional research.4. Establish systematic clinical diagnosis process of disproportionate short stature with SD. |
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