Analysis Of Clinical Features And Arsb Gene Mutation In Children With Mucopolysaccharidosis Type Ⅵ

Bachground Mucopolysaccharidosis type Ⅵ（MPS Ⅵ） or Maroteaux-Lamysyndrome（MIM#253200）is an autosomal recessive lysosomal storage disorderdescribed in1963by Dr. Pierre Maroteaux and Dr. Maurice Lamy. The disease iscaused by the deficiency of the enzyme N-acetylgalactosamine-4-sulfatase, also calledarylsulfatase B（ARSB）, which is encoded by ARSB gene. The main function ofARSB is to degrade the glycosaminoglycan（GAG）, dermatan sulfate（DS） andchondroitin sulfate（CS）. Because of the deficiency of ARSB, dermatan sulfate andchondroitin sulfate can’t be degraded and then accumulate in the lysosome, leading tothe damage of cells in the body. Also the undegraded dermatan sulfate and chondroitinsulfate excrete from the urine. The birth prevalence of MPS Ⅵ is range from1in43261to505160births in different ethnic populations in the world. The birthprevalence of MPS Ⅵ of Taiwan is1in833000births and there is noepidemiological statistics in Chinese mainland. The clinical presentation of MPS Ⅵvaries greatly with respect to age of onset and rate of disease progression. And thereare three types of MPS Ⅵ, they are severe form, intermediate form and mild form.The severe form is characterized in most cases by onset before3years of age andsuffers from short stature, skeleton deformity, anchylosis, hepatosplenomegaly,corneal clouding and so on. But the intelligence of MPS Ⅵ patients is normal, witchis different from other types of MPS. Patients with the severe form were frequentlyreported to die from cardiopulmonary complications in the2nd or3rd decades. Themild form is characterized by later onset of symptoms and the long time survival. The skeletal X-ray shows “dysostosis multiplex” and the highlevel of unine GAG are theuseful diagnostic clues of MPS Ⅵ, and the level of unine GAG is associated withthe clinical phenotype. High uGAG levels (>200μg/mg creatinine) is associated withsevere form and uGAG <100μg/mg is associated with mild form. The assay ofARSB enzyme activity in peripheral blood leukocytes or cultured fibroblasts andARSB genetic mutations are the reliable method to make the definite diagnosis ofMPS Ⅵ. ARSB gene locates on chromosome5q13-q14, comprising8exons. ThecDNA of ARSB gene is222bp in length encoding533amino acids. Approximately150ARSB gene mutations have been identified thus far, including missen mutations,nonsense mutations, splice-site mutations, small deletions and insertions, largedeletions and indels. Missen mutations and nonsense mutations are the most commonmutations, about73%. The more common mutations identified in patients from NorthAmerica,South America, Europe, and Australasia are p.Y210C、 p.S384N、c.1143-1G>C and c.1143-8T>G. These mutations present frequency are higher than10%. And in Brazil is the mutation1533del123, about23%. The mutation p.F399Lhas a high frequency about27.8%founded in9MPS Ⅵ patients from Taiwanreported by Wei-De Lin. The relationship between genotype and phenotype is notcompletely clear. Mutations like p.L72R、p.Y255X are associated with severe form.Mutations like p.D83Y、p.R434I are associated with mild form. And mutations likep.R102H、p.T442M are associated with intermediate form. HSCT and ERT are themain treatment for MPS Ⅵ patients. Early diagnosis and treatment will preventthe irreversible damage of vital organs like skeleton, heart, lung, eye and so on.Prenatal diagnosis is an effective way to prevent the birth of MPS Ⅵ patients.Objective To explore the clinical features and molecular analysis of ARSB gene inchildren with mucopolysaccharidosis type Ⅵ and make further analysis of therelationship between the genotype and phenotype of ARSB gene.Method Thirteen children, who visited the Guangzhou Women and Children’sMedical Centre from January2009to December2013because of “short stature orskeleton deformity”, were diagnosed as MPS VI based on the examinations of bone X-ray, urinary GAG levels and ARSB enzyme activity determination and their clinicalfeatures were retrospectively reviewed. The mutation analysis of ARSB gene wasperformed by polymerase chain reaction（PCR）and direct sequencing. When themutations were detected, the mutations assay was performed on the parentsofprobands. SIFT, PolyPhen-2software and f analysis of amino acid conservation inten different species were used to predict the possible impact of novel missensemutation on the structure and function of ARSB protein.Result Thirteen patients were diagnosed at the average age of（3.9±2.2）years with6male and7female，who came from13unrelated families in Guangdong province（9patients）,Jiangxi province（2patients）,Hunan province（1patients）and Shandongprovince（1patients）. All patients were presented with severe form. The main clinicalfeatures were coarse facial features, short stature, skeleton deformity, corneal cloudingand hepatosplenomegaly with normal intelligence. The radiological findings in allchildren are characteristic of dysostosis multiplex, like abnormal development ofvertebral bodies of the spine, campylorrhachia and paddle-shaped widened ribs. TheMRI in one patient showed spinal cord compression and multiple cystis degenerationin the corona radiate, cella lateralis and callosum. High uGAG levels were detected,(307.10±112.14) mg/L (Normal below70mg/L). The ARSB enzyme activity inisolated leukocytes was low,(13.29±6.22) nmol/(mg·h)[Normal range (47~169)nmol/(mg·h)] by fluorogenic assay and (0.24±0.18) U/g [Normal range (1.01~11.47) U/g] by colorimetric assay. Eleven kinds of mutation in ARSB gene wereidentified and the detectable rate was76.9%（20/26）. Eleven mutations included6missense mutation（sp.L72R、p.G167R、p.Y175D、p.G303E、p.F399L and p.T442M）,4nonsense mutations（p.Y255X、p.R327X、p.S403X and p.S464X）,1large deletion（incl. ex.2、3）.Among these mutations,7mutations（p.L72R、p.G167R、p.G303E、p.F399L、p.T442M、p.Y255X and p.R327X）were previously reported, and the other4mutation（sp.Y175D, p.S403X, p.S464X and large deletion incl. ex.2、3）were novel.The pathogenic mutation p.F399L was the most common mutation in this study(30.8%). The amino acid of mutant position of the novel p.Y175D mutation was highly conserved in ten different species and was predicted to impact the structure andfunction of ARSB protein possibly according to the SIFT and PolyPhen-2software.The large deletion incl. ex.2、3would lead to structural modification of the proteinand loss of activity.Conclusion By retrospectively reviewing the clinical features and analyzing ARSBgene of thirteen MPS Ⅵ patients, and reviewing of the literature at the same time,the following conclusions can be made.1、The severe form patients onset before3years of age and the progress of thedisease was quick. The main clinical features were progressive coarse facialfeatures, short stature, skeleton deformity, corneal clouding andhepatosplenomegaly with normal intelligence.2、Although the intelligence was normal with MPS Ⅵ patients, there were abnormalfindings in brain MRI, like multiple cystis degeneration and so on. Also, highattention should be paid to spinal cord compression in MPS Ⅵ patients.3、Urinary GAG level is associated with clinical classification of MPS Ⅵ patients.High uGAG levels (>200μg/mg) was associated with severe form.4、Eleven different mutations of ARSB gene were identified in13children with MPSⅥ. Four of them（p.Y175D, p.S403X, p.S464X and large deletion incl. ex.2、3）were novel. The large deletion incl. ex.2、3possibly related to severe form.5、The c.1197C>G（p.F399L） mutation was a hot-spot mutation of ARSB gene in thisstudy and related to severe form.