There are different types of genetic bone diseases with serious consequences，such as deformity and even death. According to the latest classification criteria of the international genetic bone diseases revised in 2010, bone diseases are classified into 40 groups and 456 conditions . Common bone diseases that can manifest short stature and short limb malformations include achondroplasia (ACH), pseudoachondroplasia (PSACH), hypochondroplasia (HCH), thanatophoric dysplasia (TD), metaphyseal chondrodysplasia Schmid type, achondrogenesis(ACG), Ellis-Van Creveld syndrome(EVC), osteogenesis imperfecta(OI), asphxiating thoracic dyaplasia (ATD), short-rib polydactylia syndrome, pycnodysostosis among others . The genetic bone diseases our department has encountered in recent ten years involve more than 20 conditions including ACH, HCH, TD, PSACH, MED, SEDC, SEDT, XLH, ACG, mucopolysaccharidosis (MPS) type I, type II, type IIIB, type IVA, type VI, osteogenesis imperfecta type I, type II and type IV（OI-I, OI-II, OI-IV） . The case reported in this paper is a very rare, complicated bone disease, of which the etiology is unknown. We would like to communicate the case detail to the community.
The proband is male, Han nationality. His birth weight was 3 kg and birth height was 48 cm. The funnel chest was noticed at the age of 5 months and the retardation of growth and development was suspected at the age of 1 year old. He was with slightly low muscle tension; both growth hormone and thyroid hormone levels have been normal. The frontal and lateral CR of the hands / lumbar / thoracic vertebrae showed short stubby fingers and metacarpal bones, pointy distal part of the proximal interphalangeal joints, wide and concave phalange base and metacarpal bone distal metaphysis, pointed distal part of the ulna bilaterally. We also found 1~2 vertebral kyphosis and the pointed anterior border of the T11~L3 vertebra with tongue-like processes. The iliac bones were deformed and irregularly-shaped and the acetabulums were irregular and with abnormal shape (Fig.1, 2). The MRI of the skull（FSE/T2WI/AXL; FLAIR/AXL; SE/T1WI/AXL、SAG）showed no obvious abnormalities, clearly distinguished cortex and the medulla of the cerebra, normal sulci, gyri, brain ventricles and midline structures, no abnormal flow void signal in the skull. Bilateral maxillary sinus, ethmoid sinus and mastoid cells showed circular isometric T2WI signal (Fig.3).
Because PSACH was suspected, COMP gene mutations was examined in detail and no pathogenic mutation was found. Other related genes and FGFR3 gene of the similar ACH disease (Note: some doctors suspected ACH according to the CR film of the proband’s forearm, palm, finger bone, Fig.4) were examined and no mutation was found. MPSIVA was suspected as well because of similar symptoms and signs (short, severe bone deformities, but normal intelligence) (Fig.5, 6) and weakly positive urine GAGs test result. GALNS gene was examined in detail and two new base substitutions were identified: c.1459 A>T, p.N487Y heterozygous missense mutation (paternal) and IVS13 DS (+26) G>T heterozygous mutation (maternal).
To further understand the role of these two mutations, we screened more than 50 cases of the normal control group, conducted bioinformatics analyses, and examined the GALNS activity in the proband and his father (Note: the mother's was not measured due to pregnancy). Although these two mutations have not been reported in the HGMD and SNP databases, the pathological mutation of the same codon (c.1460 A>G, p.N487S) is included in the HGMD database . The two new mutations were not found in 70 normal controls. Protein structure changes were not noticeable in the tertiary structure of swiss-modeling prediction or in the secondary and tertiary structure of phyre prediction. The amino acids at the mutation sites were highly conserved across species. Two GALNS enzyme assays were conducted in Guangzhou maternal and child center and Peking Union Medical College Hospital separately. The first test results 77.19 (in units: nmol/mg.17h), 102.32 for the child and father, respectively, (normal range of 40 ~ 170). The second test results were 60.3 for the child and the father's was in the normal range (normal range: 69.8 ~ 159.2). Multiple q-PCR experiments were inconclusive and failed to establish whether these two mutations could lead to changes at the transcription level. Sequencing of GALNS cDNA confirmed that there was "c.1459 A>T" heterozygous missense mutation, and there was no splicing site changes induced by IVS13 ds(+26)G>T.
The child has been in treatment for 8 years since 2006 when he was 1 year old in several hospitals and research institutions including Hubei Shiyan Taihe Hospital, HUST, Tongji Hospital, Peking Union Medical College Hospital, Guangdong Provincial Maternity and Child Care Center, Dongguan Shiqiao hospital, Guangzhou JINYU Med Center for clinical laboratory, the First Affiliated Hospital of SUN Yat-sen University, Zhongshan Medical College Department of medical genetics. Besides the routine clinical examinations and imaging scans, we tested urinary GAGs (negative or weakly positive), GC-MS profile (without obvious characteristic abnormal organic acid metabolites), and MPS enzymatic activity, and examined COMP, DTDST, MATN3, FGFR3, GALNS gene mutations. Two new variants were identified in the GALNS gene: a heterozygous missense mutation (c.1459 A>T, p.N487Y, paternal), the other one is IVS13 ds (+26) G>T heterozygous variation (maternal), both of which have not been reported either in literature or in HGMD (//www.hgmd.cf.ac.uk/ac/index.php) and SNP databases.
Based on the fact that the mutations of p.N487S are pathological from the literatures , we suspect that the heterozygous missense mutation (p.N487Y) of the GALNS gene is involved. Both mutations are in codon 487 and both yield similar polar neutral amino acids. The IVS13 ds(+26) G>T variation is not a SNP according to the SNP database and the screening from nearly 70 cases in the control group. Mucopolysaccharidosis type IVA and MPSIVA disease (Morquio Disease) seem not supported as well. The symptoms are also not completely consistent with congenial spondyloepihyseal dysplasia (MIM 184250, also known as Strudwick syndrome) with joint relaxation, whose mode of inheritance is AR or AD  . We speculate that the proband’s disease may not be a single disease, and there may be structure or function abnormality of several related genes or enzymes. The parents declined our suggestion to do a whole genome scan or exome sequencing .
This article is the translated version of an article published at RESEARCH in Chinese, titled "一罕见疑难骨病", on 2014-04-21.
Thanks to Professor Aihua Yin from the prenatal diagnosis center of Guangdong Provincial Maternity and Child Care Center for introducing the patient to our department; to Professor Yonglan Huang, doctor Xiaoyuan Zhao from the endocrine and metabolic laboratory of the Guangzhou women and children's medical center for doing the quantitative examination of urinary GAGAs and enzyme examination of each type of the mucopolysaccharidosis (MPS) for the child; to Dr. Weimin Zhang from the Central Laboratory of the Peking Union Medical College Hospital，China Academy of Medical Sciences for reexamining the GALNS activity of the MPSIVA type; to Professor Yan Meng from the basic medical research of the Chinese Academy of Medical Sciences, the department of medical genetics of Beijing Union Medical College，WHO community control of hereditary diseases Cooperation Center for reexamining and organizing the expert consultation; to Professor Huiwen Zhang from the endocrine and the genetic metabolic disease research department of the Affiliated Xinhua Hospital of Shanghai Jiaotong University for giving guidance and valuable advice. Thanks to Professor Jingxin Pan, Professor Chunmiao Guo from the Second Affiliated Hospital of Fujian Medical University for putting forward many valuable comments and suggestions. Thanks to the patient and his family for cooperation and providing a variety of valuable medical records and photographs!
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