Background
Citrullinemia type I (CTLN1, MIM# 215700) is a rare autosomal recessive disorder of the urea cycle caused by a deficiency of the argininosuccinate synthetase (ASS, EC 6.3.4.5) enzyme due to mutations in the
ASS1 gene [
1]. CTLN1 encompasses a spectrum of varying clinical phenotypes. Patients that present with fatal neonatal hyperammonemia are said to have classical citrullinemia, patients with late onset and/or mild symptoms are said to have mild citrullinemia, and a considerable number of asymptomatic individuals detected by expanded newborn screening (NBS) have only a biochemical phenotype [
2,
3].
Biochemically, CTLN1 with elevated citrulline concentrations can be detected by NBS. Nonetheless, increased levels of citrulline can also be found in other inherited metabolic disorders such as citrullinemia type II, argininosuccinate lyase deficiency, and pyruvate carboxylase deficiency [
4,
5]. Therefore, definitive diagnosis of CTLN1 mainly relies on an ASS enzyme assay and identification of
ASS1 gene mutations. However, determination of enzyme activity in liver tissue requires an invasive procedure, and direct measurement of ASS activity or indirect measurement using a C
14 incorporation assay in fibroblasts have not yet been evaluated in patients with mild CTLN1 [
6]. Therefore, molecular genetic testing is paramount, not only for clinical diagnosis but also for future prenatal testing and family member screening [
7].
The
ASS1 gene is located on chromosome 9q34.1 and contains 16 exons, with the translation start codon in exon 3, encoding 412 amino acids [
2,
8]. To date, At least 137 mutations that cause CTLN1 have been reported in the
ASS1 gene [
9]. However, only a few Chinese patients with CTLN1 have been reported, and
ASS1 gene mutations have been identified only sporadically in China [
10‐
17]. In this study, we present biochemical, clinical, and genetic characteristics of a new Chinese patient with CTLN1. In addition, we reviewed previous genotypes and phenotypes of Chinese patients with CTLN1, to help better understand the genetic background of this disease in the Chinese population.
Discussion and conclusions
In this study, we described a Chinese family with one child having a mild form of CTLN1. The patient had an elevated citrulline level, which was detected by MS-based NBS. No abnormalities were found in urinary organic acid analysis. The patient had normal growth and development during follow up, and the main clinical manifestation was a speech delay. A homozygous
ASS1 gene variant c.773 + 4A > C was identified in the patient. This variant has not been previously reported in the literature, and was predicted through bioinformatics analysis to cause a broken WT donor site and to affect splicing. Furthermore, it is likely to truncate the monomers, impairing the synthetase-binding domain. Similar variants leading to protein truncation have been reported previously [
13,
19,
20]. Therefore, we believe that the variant c.773 + 4A > C is associated with the pathogenesis of CTLN1. However, further functional studies are needed to validate the pathogenicity of this variant.
After combining our findings with those from previously reported Chinese patients with CTLN1, we performed mutation spectrum analysis. The results showed that the mutation spectrum of Chinese patients with CTLN1 was heterogeneous, with no high frequency or hot spot mutations. In comparison, many mutations have been documented at high frequencies in various other populations and the mutation spectra differ among different ethnic groups. p.Gly390Arg is by far the most common mutation and is widely distributed around the world [
9]. It has been proposed that a CpG dinucleotide in the coding region could be the cause of recurring mutations in this region [
21], and therefore the recurrent nature of this variant could be explained by its location in a CpG dinucleotide. For instance, the allelic frequencies of p.Gly390Arg in Indian and Turkish patients are 42.7 and 50%, respectively [
22,
23]. Likewise, 17.3% of German patients carry at least one p.Gly390Arg allele. p.Gly390Arg is also regarded as a recurrent mutation in a limited geographic area of Argentina [
24]. Similarly, the p.Val263Met variant seems to be common in the Pacific Island population [
25]. The most frequent mutations in Korean patients are c.421-2A > G, p.Gly324Ser, and c.1128-6_1188dup67, and in Japanese patients the predominant mutations are c.421-2A > G, p. Arg265His, and p.Arg304Trp [
26‐
30]. However, it is surprising that the particularly frequent mutation c.421-2A > G, reported in Korean and Japanese patients, has not yet been detected in Chinese patients.
To date, some mutations have been elucidated with clear genotype-phenotype correlations [
9], while most Chinese patients were in a compound heterozygous state, rendering it more difficult to investigate the relationship between genotype and phenotype. Though p.Arg127Gln was proven to be inactive in previous studies, in the neonatal CTLN1 group, a patient (no.2) in our study homozygous for p.Arg127Gln died shortly after birth, confirming previous reports and highlighting the severity of this mutation [
31]. Previous enzyme studies revealed that both p.Arg265Cys and p.Gly324Ser yielded < 2% of ASS wild-type activity, and both are known to be associated with a severe phenotype [
32]. Supporting these findings, a patient (no.4) who was compound heterozygous for p.Arg265Cys with a splicing mutation presented with early onset neonatal citrullinmia; of note is that an older sibling in this family progressed to severe encephalopathy and died 4 days after birth. A patient (no.10) homozygous for p.Gly324Ser presented with acute hyperammonemia and encephalopathy, again confirming previous studies. p.Arg363Trp was reported to be associated with neonatal CTLN1; consistent with this, a patient (no.9) with p.Arg363Trp in combination with a frameshift mutation died shortly after birth [
9]. Regarding mild/late-onset form CTLN1, a patient (no.1) homozygous for c.773 + 4A > C presented no clinical symptoms until 3 years of age, indicating that this variant may be related to mild symptoms. The remaining patients are all compound heterozygotes, and it is likely that the mutations p.Ser18Leu, p.Val141Gly, p.Pro144Arg, and p.Cys337Arg may allow for some residual ASS function, because the second allelic mutations in these patients are known to drastically impair ASS activity [
9,
13,
32].
In summary, we described one mild Chinese CTLN1 case with a novel splicing variant c.773 + 4A > C. We also reviewed previous genotypes and phenotypes of Chinese patients with CTLN1, hereby adding to our understanding of the molecular genetic background and clinical phenotype of CTLN1 in this population. The mutation spectrum of Chinese patients with CTLN1 was heterogeneous. More functional research is needed to elucidate the genotype-phenotype correlation in patients with CTLN1.
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