Nonketotic hyperglycinemia (NKH), also known as glycine encephalopathy (#OMIM: 605899), is an inherited disorder characterized by abnormally elevated glycine levels. It is usually caused by the defect of the enzyme that breaks down glycine in the body, which causes its accumulation in tissues and organs, especially the brain, which can cause severe nervous system damage. According to epidemiological statistics (Applegarth et al., 1979; Coughlin et al., 2017), the incidence of newborns is 1/55,000 in Finland and 1/63,000 in British Columbia, Canada. The clinical manifestations of NKH are different and can be divided into classic and non-classical types (Feng et al., 2021). The classic type is more common (84%), with progressive encephalopathy manifestations, low response, lethargy, hypotonia, vomiting, myoclonic epilepsy, and symptoms such as hiccups or apneas. And the symptoms worsen in a short period of time, and most children need ventilator support. Approximately 80% of NKH is caused by mutations in the GLDC gene (Bayrak et al., 2021). The AMT gene mutation causes approximately 20% of cases (Cao et al., 2021). In this study, whole-exome sequencing (WES) combined with Sanger sequencing technology was used to detect the molecular pathogenicity of the proband. The data of patients combined with clinical manifestations were analyzed to clarify the possible causes of the disease and provide a theoretical basis for their clinical diagnosis and genetic counseling.

In this report, the compound heterozygous variation was found in the proband’s AMT gene, which was assessed as a pathogenic variation according to the latest guidelines and was newly developed. The paternal mutation site identified as c.982_983insG carries exon eight of the AMT gene [p. Ala328Glyfs*22]. According to the classification standards and guidelines of genetic variation of the American Society for Medical Genetics and Genomics (ACMG), this variation led to protein-truncating mutation, which might lead to nonsense-mediated mRNA decay (NMD) phenomenon, so PVS1 evidence was used. At the same time, the variation was not included in all normal population databases, and PM2 evidence could be used. Finally, the mutation was evaluated as a likely pathogenic mutation (LP). The patient’s other variant c.977delA [p. Glu326Glyfs*12] from the mother was also truncated, and PVS1 evidence could be used. This variation was also not included in any normal population database, and PM2 evidence could be used. And then, PM3 evidence was used when likely pathogenic mutations were detected at the transposition. The mutation was finally evaluated as pathogenic mutation (P). This compound heterozygous variation was verified by Sanger sequencing (Figure 1D). To obtain the theoretical structure of these gene mutants, 3D models were built on the public website SWISS-MODEL (Waterhouse et al., 2018) (https://swissmodel.expasy.org/; Figure 1E).

Nonketotic hyperglycinemia (NKH) has clinical heterogeneity, which is mainly divided into classic and non-classical types, and the classic type is more common (84%). The neonatal form of classic NKH involves genes that encode the glycine cleavage system and usually manifests as progressive encephalopathy from 6 h to 8 days after birth. The symptoms get worse in a short period of time, and most children need ventilator support (Kure et al., 1997; Chauke et al., 2016). Approximately 30% of the classical NHKs die in the neonatal period, and most of the children die within one year of age. Most of the survivors have severe brain development disorders and refractory epilepsy. We report a female patient with nonketotic hyperglycinemia, presenting with cerebral edema, respiratory failure, abnormal biochemical indicators including elevated glycine, elevated cerebrospinal fluid chloride, and abnormal MRI. This is caused by the compound heterozygous mutation in exon eight of the AMT gene, which has not been reported previously. The patient’s phenotype is classic glycine encephalopathy, which is onset in newborns, mainly due to the defect of amino methyltransferase in the glycine lyase system.

The metabolism of glycine is completed by the glycine lyase system (EC2.1.2.10) in the mitochondria in the body (Kure et al., 2001; Leung et al., 2020), which is composed of four protein components as follows: P protein (Kure et al., 2006), also known as pyridoxal phosphate-dependent glycine decarboxylase, which is produced by GLDC (OMIM: 238300) gene encoding; H protein (Poothrikovil et al., 2019), a lipoic acid-containing protein, encoded by the GCSH (OMIM: 238330) gene; T protein (Toone et al., 2003), also known as amino methyltransferase, is a kind of tetrahydrofolate-dependent transfer. The methyl enzyme is encoded by the AMT (OMIM: 238310) gene; the L protein (Bravo-Alonso et al., 2017), which is a lipoic acid dehydrogenase, is encoded by the DLD (OMIM: 238331) gene. It has been found that defects in T, P, and H proteins can lead to glycine encephalopathy. P protein defects are the most common, followed by T protein defects, and L protein defects are rare. The HGMD database includes 433 pathogenic mutation sites of P protein, 174 of which are clearly damaged by NKH; 98 pathogenic mutation sites of T protein are included, of which 44 are clearly damaged by NKH (Stenson et al., 2020). A compound heterozygous variant in the AMT gene of the proband was detected this time. It was assessed as a pathogenic variant according to the latest ACMG guidelines and was an unreported previous variant. There was an increase in glycine in the blood caused by a defect in the T protein of the glycine cleavage system. The inability to metabolize causes damage to the brain system, which can explain the clinical cause of the proband.

According to the results of genetic testing, the proband carries the paternal mutation site c.982_983insG in the eighth exon of the AMT gene, which is the G insertion between the 982nd and the 983rd position. Therefore, a frameshift occurs at the 328th amino acid of the protein sequence, so the translation is terminated prematurely at the 22nd amino acid position after that, resulting in protein truncation. According to the American Academy of Medical Genetics and Genomics (ACMG) genetic variation classification standards and guidelines (Richards et al., 2015; Abou Tayoun et al., 2018; Tavtigian et al., 2018), the variation is a protein-truncating mutation that may cause nonsense-mediated mRNA decay (NMD) phenomenon, so PVS1 is used as evidence. At the same time, if the mutation is not included in the gnomAD database (Karczewski et al., 2020), PM2 evidence can be used. Based on the evidence of PVS1 and PM2, this variant is assessed as likely pathogenic. The proband carries the maternal variant site c.977delA in the eighth exon of the AMT gene, which is the A deletion in the 977th position. The frameshift occurs at the 326th amino acid of the protein sequence, so the translation is terminated prematurely at the 12th amino acid position after that, resulting in protein truncation and forming a non-functional protein. Similarly, PVS1 evidence can be used; the gnomAD database does not include the mutation, and PM2 evidence can be used; at the same time, if the aforementioned possible pathogenic mutations are detected in the transposition, PM3 evidence can be combined with PVS1, PM2, and PM3 evidence, the variant is assessed as a pathogenic mutation (pathogenic).

Treatment of NKH is still achieved through symptomatic treatment (Korman et al., 2006; Magwebu et al., 2019). The use of low-dose strychnine nitrate and exchange blood therapy can reduce the blood glycine content, but these treatments have caused brain damage and further preventive treatments are invalid (von Wendt et al., 1980). Therefore, treatment is focused on reducing plasma glycine concentration by initiating sodium benzoate and utilizing N-methyl-D-aspartate receptor site antagonists (i.e., dextromethorphan and oral ketamine) to reduce glycinergic stimulation (Van Hove et al., 2005; Prasad et al., 2015). This therapy has been shown to be effective in controlling seizures and neurodevelopment in selected nonketotic hyperglycinemic populations (Bjoraker et al., 2016). So, if suspected NKH patients are found clinically, genetic diagnosis and corresponding treatment should be carried out as soon as possible so as not to delay the irreversible brain damage and bring a great burden to the whole family and society.

In conclusion, this study reports two novel compound heterozygous missense mutations in the AMT gene in the Han family of NKH: c.982dupG and c.977delA. The discovery and reporting of NKH-related gene mutations are helpful in analyzing genotypes. The correlation of phenotypes can clarify the etiology of patients. It provides a theoretical basis for clinical diagnosis and treatment and genetic counseling and expands the spectrum of pathogenic gene mutations in NKH. In addition, our research shows that whole-exome sequencing is very helpful for congenital disease screening, genetic diagnosis, and clinical genetic counseling.