We established a detection system for GLDC
deletions by using the MLPA method, and showed that deletions within this gene are a common cause of NKH. Fourteen different types of GLDC
deletions were identified in screening 65 patients with neonatal‐onset NKH. GLDC
deletions were identified in 21 of 65 patients with NKH (32.3%), and in 25 of 130 NKH alleles (19.2%) by MLPA analysis. The MLPA method provides a good first‐line screen in a condition where there are no common mutations and full sequencing of 25 exons of the GLDC
gene is a lengthy process. The deletion detection rates by MLPA analysis were 18% and 22.5% in the first and second cohorts, respectively. In our previous study, the exon‐sequencing analysis has shown GLDC
mutations in 41 of 90 alleles (45%).21
Thus, this MLPA test improved the sensitivity of mutation detection from 45% to 63%. Mutations for NKH are highly heterogeneous: the prevalent mutations previously reported are Finnish S564I mutation (70%)14
and Caucasian R515S mutation (5%),15
hampering the genetic testing in diagnosis of NKH. In contrast, GLDC
deletions seem to be prevalent in different ethnic groups. In a previous study, we analysed the relative allele number of the GLDC
exon 1 by using GLDCP
as a copy number control.22
As MLPA analysis covers the whole gene in one simple assay, it is highly recommended for the first screening in the genetic testing of NKH.
Point mutations in MLPA‐probe binding sites may cause false positives in MLPA analysis, notably where a single exon is deleted. A mismatching in the binding site of the MLPA probes is known to reduce the ligation efficiency. In our study, we encountered four single‐exon deletions in the analysis of families P41, B3, B5 and B7. Subsequent sequencing analysis of the probe binding sites showed that the patient in family P41 had a 1‐bp deletion and that the patient from B5 carried a 1‐base substitution at the splicing accepter site of intron 4. Both mutations are predicted to be disease causing. No base change was found in the patient from B3. In the patient from B7, exon 9 of the patient failed to be amplified by PCR and a single‐exon deletion was confirmed by subsequent sequencing across the breakpoint (fig 2A). As the MLPA probes for GLDC were designed to bind an exon–intron boundary to avoid detection of the pseudogene of GLDC, GLDCP, the MLPA method can also detect some mutations that cause aberrant splicing. Sequencing the probe‐binding regions of the GLDC gene where MLPA analysis suggests a single‐exon deletion is therefore necessary before making a diagnosis of GLDC deletion.
- A screening system for genomic deletions within GLDC has been developed by the multiplex‐ligation‐dependent probe amplification (MLPA) method.
- GLDC deletions were identified in approximately 20% of non‐ketotic hyperglycinaemia (NKH) mutant alleles.
- The MLPA analysis is useful for first‐line screening in the genetic testing of NKH.
In a previous study, we diagnosed the patient of family P32 as a homozygote of a nonsense mutation, c.1786C→T (p.R596X), although there was no history of consanguinity.21
A familial study was not possible because no parental DNA was available. The present study showed that he was heterozygotic for a deletion containing all 25 GLDC
exons (table 3, fig 1G), indicating that he was a compound heterozygote of the nonsense mutation c.1786C→T and the deletion of exons 1–25. As this deletion was the biggest one so far identified, we looked to see whether it involved any adjacent genes. We performed a microarray analysis to determine the genotypes of many single‐nucleotide polymorphisms (SNPs) by using the GeneChip Human Mapping 100 k Set (Affymetrix, Santa Clara, California, USA). GLDC
is located between base positions 6635650 and 6522467 bp in chromosome 9 (GenBank, NT_008413). The JMJD2C gene (6748083–7165647 bp) is located 5′ upstream of GLDC
whereas the UHRF2 gene (6403151–6497051 bp) lies 3′ downstream of GLDC
. The SNP at the base position 6606648 bp, which is located within the GLDC
gene, was indeed homozygotic in this patient (data not shown). In contrast, two SNPs at the base positions of 6513056 and 6759229 bp were heterozygotic, suggesting that the deletion is <246 kb, and thus that the two adjacent genes are unlikely to be involved in the deletion.
We determined flanking sequences of interstitial deletions in five patients, and Alu
‐mediated recombination was identified in three of five patients. The Alu
elements, approximately 300 bp in length, compose about 10% of the whole human genome.25
There are several inherited disorders in which Alu
‐mediated recombination/deletion is a common cause: hereditary angioedema, C1‐INH
α‐thalassemia, α‐globin gene;27
and Ehlers–Danlos syndrome, PLOD
‐mediated genomic recombination has also been reported in non‐inherited human cancer, hepatoma.29
A total of 120 copies of Alu
repeats are present in the GLDC
gene, which has a length of 113 kb, resulting in one Alu
of 1.1 kb on average. This is much higher than the average density of one Alu
every 3–4 kb over the whole human genome.30
deletions tend to be located in the 5′ end of the GLDC
gene, which may be explained by the fact that the region contains a high number of Alu
The diagnosis of NKH is difficult to establish on clinical and biochemical grounds alone, and typically requires a liver biopsy for enzyme analysis or DNA studies to confirm a diagnosis. However, the complex nature of the genetics of NKH (three genes and no common mutations) makes DNA analysis a lengthy and difficult process. Our finding that deletions within the GLDC gene are one of the most common causes of NKH and the development of a simple assay for such mutations will make genetic analysis for this disorder much more straightforward. Such analysis will reduce the need for a liver biopsy in a sick child, make diagnosis easier, and improve the ease and reliability of antenatal diagnosis.