title: Identification and functional analysis of a novel PRKAG2 mutation responsible for Chinese PRKAG2 cardiac syndrome reveal an important role of non-CBS domains in regulating the AMPK pathway.
abstract: BACKGROUND
  PRKAG2 gene encodes the γ2 regulatory subunit of AMP-activated protein kinase (AMPK) that acts as a sensor of cellular energy status, and its germline mutations are responsible for PRKAG2 cardiac syndrome (PCS). The majority of missense mutations of cystathionine beta-synthase (CBS) domains found in PCS impair the binding activity of PRKAG2 to adenosine derivatives, and therefore lead to PRKAG2 function impairment and AMPK activity alteration, resulting in a familial syndrome of ventricular preexcitation, conduction defects, and cardiac hypertrophy. However, it is unclear about the PRKAG2 mutation in the non-CBS domain. Here, a Chinese family exhibiting the cardiac syndrome associated with a novel heterozygous PRKAG2 mutation (Gly100Ser) mapped to exon 3 encoding a non-CBS domain is described and the function of this novel mutation was investigated in vitro.
  
  
  METHODS
  The PRKAG2 G100S and R302Q mutations were constructed by a two-step polymerase chain reaction and then transfected into CCL13 cells by lentivirus vectors. Wild-type PRKAG2 gene transfection was used as a negative control. PRKAG2 expression was determined by Western blot. Immunofluorescence was used to localize the intracellular PRKAG2 proteins. MTT assay was performed to explore the effect of mutations on cell proliferation. Periodic acid-Schiff staining was used for detecting glycogen accumulation. AMPK concentration was measured with enzyme-linked immunosorbent assay.
  
  
  RESULTS
  Our results showed neither intracellular localization of PRKAG2 nor cell growth was altered. In contrast, PRKAG2 protein expression levels were significantly reduced by this mutation. Furthermore, PRKAG2-mediated activity of AMPK was attenuated, resulting in glycogen metabolism dysregulation. These findings revealed that non-CBS domains of PRKAG2 were essential to the regulation of AMPK activity, similar to CBS.
  
  
  CONCLUSIONS
  Our study ascribes a crucial regulatory role to the novel PRKAG2 G100S mutation, and reiterates that PCS occurs as a consequence of AMPK signaling abnormality caused by PRKAG2 gene mutations.
authors.affiliation: Department of Cardiovascular Diseases, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Bi-li
authors.initials: BL
authors.lastname: Zhang
authors.forename: Rong-liang
authors.initials: RL
authors.lastname: Xu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Xian-xian
authors.initials: XX
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Li-ping
authors.initials: LP
authors.lastname: Ma
authors.forename: Jian-qiang
authors.initials: JQ
authors.lastname: Hu
authors.forename: Jian-liang
authors.initials: JL
authors.lastname: Zhang
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ye
authors.forename: Xing
authors.initials: X
authors.lastname: Zheng
authors.forename: Yong-wen
authors.initials: YW
authors.lastname: Qin
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000293:Adolescent; D000328:Adult; D044466:Asian People; D006332:Cardiomegaly / Q000235:genetics*; D003541:Cystathionine beta-Synthase; D005091:Exons / Q000235:genetics; D005260:Female; D006327:Heart Block / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology*; D013577:Syndrome; D014927:Wolff-Parkinson-White Syndrome / Q000235:genetics*; D055815:Young Adult
chemical_list: C526223:PRKAG2 protein, human; D055372:AMP-Activated Protein Kinases; D003541:Cystathionine beta-Synthase
keywords: AMPK; CBS; Familial cardiac syndrome; Gene mutation; PRKAG2