Mustafa Naira M, Elabd Nevine E, Selim Laila A, Abdou Doaa M, Griffin Julian L
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK; Department of Chemical Pathology, Faculty of Medicine, Cairo University, Egypt.
Department of Chemical Pathology, Faculty of Medicine, Cairo University, Egypt.
Clin Chim Acta. 2022 Nov 1;536:70-76. doi: 10.1016/j.cca.2022.09.005. Epub 2022 Sep 18.
Cerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC-MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC-MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants.
The study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC-MS 2) Ultra high-pressure (or performance) liquid chromatography - Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR.
The linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C > G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G > A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing.
Both UHPLC-MS/MS and NMR spectroscopy are comparable to GC-MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites.
脑肌酸缺乏综合征（CCDS）是影响肌酸合成或转运的疾病。已经开发了几种方法来测量不同体液中的肌酸和胍基乙酸（GAA），包括基于气相色谱 - 质谱联用（GC-MS）和高压液相色谱 - 质谱联用（HPLC-MS）的方法。CCDS的诊断随后通过对肌酸生物合成基因胍基乙酸甲基转移酶（GAMT）、精氨酸：甘氨酸脒基转移酶（GATM）和肌酸转运基因溶质载体家族6成员8（SLC6A8）进行测序或通过功能性酶促测定来确认。本研究的目的是通过比较使用核磁共振波谱法（NMR）、GC-MS和HPLC-MS的方法，找到用于CCDS的最可靠和准确的筛查方法。此外，本研究旨在估计埃及患者队列中CCDS的患病率，并有可能发现新的变异。
本研究对150名具有与CCDS一致的临床症状和体征的受试者进行。使用三种技术对尿液样本进行代谢谱分析：1）GC-MS；2）超高压（或高效）液相色谱 - 串联质谱（UHPLC-MS/MS）；3）NMR。
UHPLC-MS/MS和NMR测定的肌酸和GAA峰面积的线性范围涵盖并超过了尿液中通常发现的范围。UHPLC-MS/MS测定肌酸和GAA的定量限和日内精密度结果比NMR更可靠。通过我们的分析方法鉴定出10例CCDS阳性病例，并对GAMT、GATM和SLC6A8基因进行了下一代测序（NGS）。进行了NGS，确认1例患者的GAMT基因有一个可能的致病变异：（NC_000019.10:g.1401317C > G，NP_000147.1:p.Ala54Pro）。此外，我们描述了GATM基因中的四个新的内含子变异：c.1043 - 357del和c.1043 - 357_1043 - 356insT，预计会激活潜在改变剪接的隐蔽受体位点，发现c.979 - 2,27G > A会显著改变外显子剪接增强子（ESE）/外显子剪接沉默子（ESS）基序比例，而c.1042 + 262del对剪接没有影响。
UHPLC-MS/MS和NMR波谱法在筛查CCDS方面与GC-MS相当。尽管如此，UHPLC-MS/MS方法的性能优于NMR波谱法。此外，需要对GATM、GAMT和SLC6A8基因的全长进行测序，以鉴定可能通过影响剪接位点导致CCDS的内含子变异。
