Improvement of diagnostic yield in carbamoylphosphate synthetase 1 (CPS1) molecular genetic investigation by RNA sequencing

Abstract

Carbamoylphosphate synthetase 1 (CPS1) deficiency is a rare inborn error of metabolism leading often to neonatal onset hyperammonemia with coma and high mortality. The biochemical features of the disease are nonspecific and cannot distinguish this condition from other defects of the urea cycle, namely N‐acetylglutamate synthase deficiency. Therefore, molecular genetic investigation is required for confirmation of the disease, and nowadays this is done with increasing frequency applying next‐generation sequencing (NGS) techniques. Our laboratory has a long‐standing interest in CPS1 molecular genetic investigation and receives samples from centers in Europe and many other countries. We perform RNA‐based CPS1 molecular genetic investigation as first line investigation and wanted in this study to evaluate our experience with this approach as compared to NGS. In the past 15 years, 297 samples were analyzed, which were referred from 37 countries. CPS1 deficiency could be confirmed in 155 patients carrying 136 different genotypes with only a single mutation recurring more than two times. About 10% of the total 172 variants comprised complex changes (eg, intronic changes possibly affecting splicing, deletions, insertions, or deletions_insertions), which would have been partly missed if only NGS was done. Likewise, RNA analysis was crucial for correct interpretation of at least half of the complex mutations. This study gives highest sensitivity to RNA‐based CPS1 molecular genetic investigation and underlines that NGS should be done together with copy number variation analysis. We propose that unclear cases should be investigated by RNA sequencing in addition, if this method is not used as the initial diagnostic procedure.

Long-term follow-up of children with carbamoyl phosphate synthase 1 deficiency detected in newborn screening

“…CPS1缺乏症属于常染色体隐性遗传病，其致病基因 CPS1 位于染色体2q35，包含38个外显子和4500个编码核苷酸，以错义突变多见，其他无义、剪接点突变、框移突变及小片段缺失亦有报道 ［ 15 - 16 ］ 。LOVD数据库（ http：//www.LOVD.nl/CPS1 ）、HGMD数据库（ http：//www.hgmd.org/ ）等显示，超过240个 CPS1 致病性变异广泛分布在各个外显子中，多数为家族特有变异，这给CPS1缺乏症患儿的基因诊断带来了巨大挑战。本文资料6例患儿检测到10种不同的基因型，以点突变为主，包括1个无义突变、1个同义突变及5个错义突变，另有2个剪接突变，1个移码变异，体现 CPS1 基因变异具有高度遗传异质性，与既往文献报道一致 ［ 15 ］ 。患儿最终经长期随访联合生化表型获得诊断。本文资料中基因改变以错义突变为主，如c.3031G>A（p.V1011M）、c.2441G>A（p.R814Q），通过SIFT、Polyphen-2和MutationTaster等多个软件对以上位点进行蛋白功能预测，均提示为“高度危害”。例1检测到c.2359C>T变异，即第2359位碱基胞嘌呤突变为胸腺嘌呤，导致过早产生终止密码子，可能导致编码蛋白的截短或由于无义介导的衰变而导致蛋白缺失。Rapp等 ［ 17 ］ 报道过此位点纯合变异的患者资料，并对其做了肝细胞活检酶学测定，发现该患者肝脏中检测的CPS1活性完全丧失，且在9个月后死亡。例1在随访10年期间出现多次血氨升高，但经积极治疗后生长发育正常。例3在新生儿期死于高氨血症，基因检测显示存在c.3075T>C和c.3405-1G>T变异，其中c.3075T>C虽为同义突变，推测也存在改变mRNA剪切、蛋白质空间位置，最终产生有害变异的可能 ［ 18 ］ ；而c.3405-1G>T为经典剪接突变，往往影响mRNA剪切，导致蛋白质功能丧失 ［ 19 ］ 。例4存在c.1549+1G>T变异，该变异亦为经典剪接突变，经HSF软件预测，该变异影响mRNA剪切，进而可能影响蛋白质合成，该患儿目前一般状况良好。例6检测到的c.390-403del为移码突变，预测可导致多肽链合成提前终止。由于 CPS1 变异分散，多数为家系特有，本文资料未发现基因型与表型的相关性，有待进一步积累资料挖掘。…”