case_images: PMC4152247_01
case_images: Identification of the (p.Met211ValfsX38) mutation in GCH1 gene for family 1. (A) Pedigree of family 1 is indicated. Black and open symbols denote affected and unaffected individuals, respectively. The proband is indicated by an arrow. (B) Verification by Sanger sequencing of the frameshift mutation in GCH1 gene (C) The mutation occurred at an evolutionarily conserved amino acid.
case_images: pone.0106388.g001.jpg
case_images: PMC4152247_02_pone.0106388.g001.jpg
case_images: pone-0106388-g001
case_images: The mutation was confirmed in the affected individuals (Fig. 1B) and not found in the unaffected family members by Sanger sequencing. We found that the variant resulted in frameshift mutation and a truncated protein, occurred in a highly conserved position (Fig. 1C), and was predicted to be disease-causing.
case_images: PMC4152247_02
case_images: Identification of the (p.Met211ValfsX38) mutation in GCH1 gene for family 1. (A) Pedigree of family 1 is indicated. Black and open symbols denote affected and unaffected individuals, respectively. The proband is indicated by an arrow. (B) Verification by Sanger sequencing of the frameshift mutation in GCH1 gene (C) The mutation occurred at an evolutionarily conserved amino acid.
case_images: pone.0106388.g001.jpg
case_images: PMC4152247_02_pone.0106388.g001.jpg
case_images: pone-0106388-g001
case_images: The mutation was confirmed in the affected individuals (Fig. 1B) and not found in the unaffected family members by Sanger sequencing. We found that the variant resulted in frameshift mutation and a truncated protein, occurred in a highly conserved position (Fig. 1C), and was predicted to be disease-causing.
case_images: Identification of the (p.Ala304Val and p.Arg453His) mutations in TH gene for family 2.. (A) Pedigree of family 2 is indicated. Black and open symbols denote affected and unaffected individuals, respectively. The proband is indicated by an arrow. (B) Verification by Sanger sequencing of the missense mutations in TH gene (C) The mutations occurred at two evolutionarily conserved amino acids.
case_images: pone.0106388.g002.jpg
case_images: PMC4152247_02_pone.0106388.g002.jpg
case_images: pone-0106388-g002
case_images: Exome sequencing was performed on her and her affected brother (II-1 and II-2 in Fig. 2A). Biopterin-dependent aromatic amino acid hydroxylase (Biopterin_H) domain which is rate-limiting catalyst for many important metabolic pathways and locates at amino acids 195-526 (http://pfam.sanger.ac.uk). Two novel heterozygous missense mutations (g. 2187929C>T, c.911C>T, p.Ala304Val and g. 2185599G>A, c.1358G>A, p.Arg453His) in domain of Biopterin_H of TH were identified and not reported in HGMD Database (http://www.hgmd.cf.ac.uk). Both mutations were confirmed as compound heterozygous mutations in the affected siblings (Fig. 2B) and as heterozygous state in their parents by Sanger sequencing. We found that the both variants occurred in a highly conserved position (Fig. 2C) and were predicted to be disease-causing.
case_images: The specific TH activity of the cell lysates after transfection with wild-type TH, mutant THA304V, and THR453H.. Compared with the wild-type, the specific TH activity with the mutant THA304V and THR453H were both significantly decreased (P<0.05). The average levels of TH activity were plotted, and error bars correspond to the standard deviation (SD). The t tests were used for statistics.
case_images: pone.0106388.g003.jpg
case_images: PMC4152247_02_pone.0106388.g003.jpg
case_images: pone-0106388-g003
case_images: Then, transiently expressed TH was analyzed for activity. The specific TH activity of the cell lysates after transfection with the mutant THA304V and THR453H were decreased to 74.1% and 62.7% of the wild-type (P = 0.03 and P = 0.01, respectively) (Fig. 3).