Background
Non-ketotic hyperglycinemia (NKH; OMIM 605899), also known as glycine encephalopathy, is an autosomal recessive metabolic disorder caused by a deficiency in the glycine cleavage system (GCS), resulting in a massive accumulation of glycine in body fluids [
1]. Most patients present with lethargy and hypotonia in the first week of life, and often progress to apnea requiring ventilation [
2]. Some patients die during the neonatal period. The majority of survivors exhibit severe mental retardation and intractable seizures typical of severe NKH [
3]. One-sixth of NKH patients have an attenuated form of the disease, half of whom present in early-to-mid infancy with seizures, hypotonia, and developmental delay and/or cognitive impairments, behavioral problems, and impaired work or school performance [
4]. Diagnosis is based on the detection of elevated glycine concentrations in cerebrospinal fluid (CSF) together with an increased CSF/plasma glycine ratio.
The GCS consists of the enzymes glycine decarboxylase (P-protein), amino-methyltransferase (T-protein), hydrogen carrier protein (H-protein), and dihydrolipoamide dehydrogenase (L-protein) [
5]. The P, T, and H proteins are encoded by
GLDC (OMIM 238300),
AMT (OMIM 238310), and
GCSH (OMIM 238330) genes, respectively. Approximately 70~75% of affected individuals carry disease-causing mutations in
GLDC gene, whereas 20% and <1% NKH patients have mutations in
AMT and
GCSH genes, respectively [
6]. In addition, approximately 5% of patients with enzyme-proven glycine encephalopathy do not have a pathogenic variant in
GLDC,
AMT or
GCSH genes; these cases therefore represent a variant form of NKH [
7]. Although forming only a relatively small percentage of NKH cases, in a populous country, such as China, it is possible that NKH is much more prevalent in China than it has been realized. Only a few studies have, however, examined the genetic profile of NKH patients in China, with the result that only two Chinese NKH patients have been diagnosed using molecular genetic analyses [
8,
9]. We here report the clinical and genetic features of a Chinese family including three siblings with NKH.
Discussion
Clinically, the patients in this Chinese family are representative of neonatal NKH patients. The proband, along with his younger siblings, exhibited severe and progressive manifestation such as lethargy, hypotonia and seizures, with greatly elevated glycine levels in their plasma and CSF. Molecular genetic analysis by Sanger sequencing and MLPA, identified compound heterozygous variants in GLDC in these three patients, namely a c.2680A > G (p.Thr894Ala) variant in exon 23 and a heterozygous deletion of exon 3, which were inherited respectively from their parents. Although these three siblings have the same compound heterozygous variants in GLDC gene, and similar clinical phenotypes, the severity of the disease, as well as the outcome, were variable. Because of this, we speculate that other genetic and environmental factors may also be responsible for the clinical phenotype including the severity and the outcome, which requires further research.
NKH is primarily caused by mutations in
GLDC gene. The 113.15 kb
GLDC gene located on chromosome 9p24.1, encodes a 1021 amino acid protein called glycine decarboxylase. To date, more than 122 mutations in
GLDC have been reported to cause NKH, including a multitude of missense mutations and different deletions involving multiple
GLDC exons [
19]. The mutations found in NKH are highly heterogeneous, although recurrent missense mutations reported include the p.R515S mutation found in Caucasians and the p.S564I mutation observed in Finnish populations [
6,
20]. Intragenic copy number variations (CNVs) have been noted in approximately 20% of
GLDC alleles, the majority of which are multi-exon deletions [
21]. These mutated alleles caused by multi-exon deletions or duplications occur in various haplotypes and in different ethnic groups. In China, to the best of our knowledge, only two NKH patients with compound heterozygous mutations in
GLDC gene have been reported to date [
8,
9].
In this work, we describe the novel variant c.2680A > G, which leads to the substitution of the polar amino acid threonine with the nonpolar animo acid alanine at position 894 of the P-protein. Computational analysis predicted that the variant is likely to have pathogenic significance and a conservation analysis in different species showed that this amino acid was highly conserved across a broad range of species, which again strongly suggests that the variant at this site might be deleterious. The deletion of exon 3 of the
GLDC gene is a frameshift variant resulting in the premature termination of the P-protein, and is also likely to be clinically significant. This CNV has previously been reported by Coughlin et al. in a systematic study of 578 families, among which one child had a homozygous deletion of exon 3 [
22], and this is the first time this variant has been found in the Chinese population. Although the MLPA technique fails to pinpoint the exact location of this deletion, based on MLPA probes and subsequent qPCR experiments, we know that the upstream breakpoint was from the MLPA probe binding site of exon 2 to qPCR upstream primer 5′ site of exon 3, and the downstream breakpoint was from qPCR downstream primer 3′ site of exon 3 to the MLPA probe binding site of exon 4 (Additional file
3: Figure S1). According to the HGVS nomenclature, the break point can be expressed as NG_016397.1 (NM_000170.2): c. (261_335-95) _ (470 + 111_476) del. In short, in view of the fact that this CNV might be overlooked by conventional sequencing strategies, MLPA or another technique that detects deletions should therefore also be utilized to analyze the presence of potential CNVs in
GLDC gene.
Conclusions
In this study, we describe the clinical and genetic features of a Chinese family with three siblings affected with NKH. A novel variant, as well as a previously reported deletion in GLDC gene, were identified in the three affected siblings. Thus, our findings suggest that these two variants in GLDC gene probably underlie the pathogenesis of NKH in this family, and also enrich the mutational spectrum of GLDC gene.
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