title: Functional analysis of novel splicing and missense mutations identified in the ASS1 gene in classical citrullinemia patients.
abstract: BACKGROUND
  Classical citrullinemia (CTLN1) is an inborn error of the urea cycle caused by reduced/abolished activity of argininosuccinate synthetase due to mutations in the ASS1 gene. To determine the pathogenicity of novel variants detected in patients is often a huge challenge in molecular diagnosis. The purpose of our study was to characterize novel ASS1 gene mutations identified in CTLN1 patients.
  
  
  METHODS
  Exon trapping assay with pSPL3 was used to confirm splice aberrations while bioinformatics structural analysis predicted the possible effects of missense mutations.
  
  
  RESULTS
  Novel donor site (c.174+1G>A) and missense (p.V141G) mutations were detected in a patient exhibiting a biochemical phenotype only. The splice mutation provoked exon skipping hence the truncated product. The mutation p.V141G, is predicted to disturb a hydrophobic pocket in the ATP binding domain in the ASS. Both mutations are predicted to lower binding of ATP. The second patient presented with early onset neonatal citrullinemia marked by an elevated biochemical profile and a clinical phenotype. Analysis revealed a donor site (c.773+1G>A) mutation leading to both exon skipping and intron retention. Subsequent introduction of premature stop codons would result in severely truncated products likely to be degraded. A previously reported R265C is predicted to distort the citrulline binding site.
  
  
  CONCLUSIONS
  Three novel mutations are reported in this study. They expand the spectrum of genetic pathology underlying CTLN1. Overall this study provides new insight of CTLN1 and illustrates a comprehensive protocol investigating inborn errors of metabolism at the molecular level.
authors.affiliation: Affiliated Children's Hospital, School of Medicine, Zhejiang University, 57 Zhugan Lane, Hangzhou 310003, China; Department of Cell Biology and Medical Genetics, Research Building A 713, School of Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, China.
authors.forename: Joseph Kagunda
authors.initials: JK
authors.lastname: Kimani
authors.affiliation: Department of Cell Biology and Medical Genetics, Research Building A 713, School of Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, China.
authors.forename: Tianying
authors.initials: T
authors.lastname: Wei
authors.affiliation: Pyongyang College of Medicine, Pyongyang, DPR Republic of Korea.
authors.forename: Kim
authors.initials: K
authors.lastname: Chol
authors.affiliation: Center for Structural Biology, Research Building A 210, Life Sciences Institute, Zhejiang University, Yuhangtang Road 866, Hangzhou, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Cell Biology and Medical Genetics, Research Building A 713, School of Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.affiliation: Center for Structural Biology, Research Building A 210, Life Sciences Institute, Zhejiang University, Yuhangtang Road 866, Hangzhou, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ye
authors.affiliation: Affiliated Children's Hospital, School of Medicine, Zhejiang University, 57 Zhugan Lane, Hangzhou 310003, China. Electronic address: xinwenhuang@126.com.
authors.forename: Xinwen
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Cell Biology and Medical Genetics, Research Building A 713, School of Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, China; Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, USA. Electronic address: mingqi@zju.edu.cn.
authors.forename: Ming
authors.initials: M
authors.lastname: Qi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001124:Argininosuccinate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism; D020159:Citrullinemia / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D005091:Exons; D005260:Female; D006801:Humans; D007223:Infant; D007438:Introns; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D011485:Protein Binding; D012326:RNA Splicing
chemical_list: D002956:Citrulline; D000255:Adenosine Triphosphate; D001124:Argininosuccinate Synthase
keywords: ASS1; Citrullinemia type I; Hyperammonemia; Mutation; Splicing