Newborn screening and variant analysis for methionine adenosyltransferase I/III deficiency / 中华医学遗传学杂志

OBJECTIVE@#To summarize newborn screening for methionine adenosyltransferase I/III (MAT I/III) deficiency in Quanzhou region of Fujian Province.@*METHODS@#A total of 364 545 neonates were screened for inherited metabolic diseases by tandem mass spectrometry. High-throughput next generation sequencing combined with Sanger sequencing was used to detect potential variants in newborns with MAT I/III deficiency. Pathogenicity of suspected variants was predicted by using MutationTaster and HSF software.@*RESULTS@#Three newborns were identified with MAT I/III deficiency by newborn screening, which yielded an incidence rate of 1 in 121 515. Amino acid and acylcarnitine analysis suggested that the serum methionine of the three patients have increased to various extents. All patients showed normal growth and development during follow-up, and were found to carry MAT1A gene variants including two missense variants [c.776C>T (p.Ala259Val) and c.791G>A (p.Arg264His)] and a synonymous variant [c.360C>T (p.Cys120Cys)]. Among these, c.776C>T (p.Ala259Val) and c.791G>A (p.Arg264His) were known to be pathogenic, whereas c.360C>T (p.Cys120Cys) was a novel variant. Bioinformatics analysis suggested that this variant may alter RNA splicing and affect the structure and function of the MAT1A protein.@*CONCLUSION@#A systematic review of newborn screening for MAT I/III deficiency was provided. Discovery of the novel variant has enriched the variant profile of the MAT1A gene and provided a basis for the diagnosis of this disease.

Methionine Adenosyltransferase 1: A Proteomic Surrogate Marker of Early Hepatocellular Carcinoma in Cirrhotic Patients

BACKGROUND/AIMS: Because there is a lack of effective biomarkers, we aimed to discover proteomic candidate markers for hepatocellular carcinoma (HCC) in cirrhotic patients at the highest-risk of HCC, and to validate the markers. METHODS: We collected tumor tissue from 5 cirrhotics with HCC, and from 5 cirrhotics without HCC, who underwent liver resection or transplantation. These tissue samples were analyzed by 2-dimensional difference gel electrophoresis coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and potential markers were validated at the transcriptional and translational levels. We also performed western blot assays using other blood samples from 10 cirrhotics with HCC and 10 without HCC. RESULTS: Among the 66 distinguishable spots on 2-D gel images, we identified 15 proteins overexpressed more than 1.5 fold in terms of volume ratio in the tumors. Ten of the over-expressed proteins were identified by MALDI-TOF MS; of those, only methionine adenosyltransferase 1 (MAT1), a protein specific for liver, and acyl-CoA dehydrogenase were significantly up-regulated in tumors in further immunoblotting analyses (Ps<0.05). There was no between-pair difference in MAT1 mRNA measured by real-time polymerase chain reaction (P=0.96). However, in western blots of serum samples, distinct MAT1 bands were observed in all 10 HCC patients, but in only 2 of the non-HCC patients. CONCLUSIONS: MAT1 is a potential marker for surveillance in cirrhotic patients with and without prior HCC.

Analysis of MAT1A gene mutations in a child affected with simple hypermethioninemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect potential mutations of MAT1A gene in a child suspected with simple hypermethioninemia by MS/MS neonatal screening.</p><p><b>METHODS</b>Clinical data of the child was collected. Genomic DNA was extracted by a standard method and subjected to targeted sequencing using an Ion AmpliseqInherited Disease Panel. Detected mutations were verified by Sanger sequencing.</p><p><b>RESULTS</b>The child showed no clinical features except evaluated methionine. A novel compound mutation of the MAT1A gene, i.e., c.345delA and c.529C>T, was identified in the child. His father and mother were found to be heterozygous for the c.345delA mutation and c.529C>T mutation, respectively.</p><p><b>CONCLUSION</b>The compound mutation c.345delA and c.529C>T of the MAT1A gene probably underlie the disease in the child. The semi-conductor sequencing has provided an important means for the diagnosis of hereditary diseases.</p>

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

S-Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies.

RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells.</p><p><b>METHODS</b>The four siRNA against MAT 2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6. We inserted the target sequence of MAT 2A gene into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucA-MAT 2A. The recombinant plasmid and siRNA-producing plasmid were co-transfected into 293 T cells using this construct via lipofectamine methods. The inhibition effect was detected by measuring luciferase activity in the cell lysate to screen the effective siRNA, and then, the effective siRNA was transfected into Bel-7402 cells. The effect of siRNA treatment on the MAT 2A mRNA level and the MAT activity of hepatoma cells were measured. In order to study the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells, the tumor cell killing rate was analyzed by MTT method and the rate of apoptosis of hepatoma cells was evaluated by flow cytometry.</p><p><b>RESULTS</b>The two siRNA among the four siRNA displayed inhibitory effect on the lucifermase expression with the inhibitory rates of 81% and 89% respectively. The expression of MAT 2A mRNA in Bel-7402 cells was specifically inhibited and the MAT activity in Bel-7402 cells was decreased. Furthermore, silencing of the MAT 2A gene by RNAi significantly inhibited hepatoma cell growth and led to induction of apoptosis.</p><p><b>CONCLUSION</b>RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells; MAT 2A is an ideal target of gene-specific therapy for liver cancer.</p>

Production of SAM by recombinant Pichia pastoris / 生物工程学报

To utilize Pichia pastoris to produce S-adenosyl-L-methionine (SAM), an intracellular expression vector harboring S. cerevisiae SAM2 was transformed into GS115. A recombinant strain having 2 copies of expression cassette was obtained through G418 resistance screening. This strain had higher SAM synthetase activity and higher SAM production capacity than the original strain, when cultured in medium containing methanol and methionine. The carbon source and nitrogen source of medium was optimized. The results showed SAM production by this strain was closely related to carbon metabolism. With supplementation of 0.2% glycerol every day from the beginning of 3rd day, this strain produced 1.58 g/L SAM when cultured in a medium containing 0.75% L-methionine and optimized carbon and nitrogen source after 6 days.