Molecular characterization of CPS1 deletions by array CGH

Abstract

CPSI deficiency usually results in severe hyperammonemia presenting in the first days of life warranting prompt diagnosis. Most CPS1 defects are non-recurrent, private mutations, including point mutation, small insertions and deletions. In this study, we report the detection of large deletions varying from 1.4 kb to >130 kb in the CPS1 gene of 4 unrelated patients by targeted array CGH. These results underscore the importance of analysis of large deletions when only one mutation or no mutations are identified in cases where CPSI deficiency is strongly indicated.

1. Introduction

Urea cycle disorders are inherited defects in the disposal of nitrogenous waste from protein. Carbamoyl phosphate synthetase I (CPSI; MIM *608307; EC 6.3.4.16), the first and the rate-limiting step, synthesizes carbamoyl phosphate from ammonia, bicarbonate, and ATP. The reaction requires a cofactor, N-acetylglutamate (NAG), synthesized by N-acetylglutamate synthase. CPSI deficiency represents a continuum of symptoms ranging from neonatal onset, which is often lethal, to late onset at any age in life. The neonatal presentation of CPSI deficiency usually results in severe hyperammonemia presenting in the first days of life warranting prompt diagnosis [1–3]. CPS1 gene consists of 38 exons and encodes a polypeptide of 1500 amino acids [4]. To date, nearly 100 mutations have been reported and documented in Human Gene Mutation Database (HGMD, http://www.biobase-international.com). In addition, about 200 mutations have been identified in a multicenter CPS1 study of a large patient cohort (Häberle et al., unpublished data). Most of the CPS1 mutations are non-recurrent, private mutations. Although small deletions and duplications are relatively common and account for about 20% of total mutations in the above-mentioned study, only a few large deletions and insertions have been reported in CPS1 gene [5–7]. This is due to the limitation of direct DNA sequencing analysis, which does not detect large deletions. Southern blot, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR (qPCR), and fluorescence in situ hybridization (FISH) are used to detect large deletions. However, these methods are tedious and only useful for the detection of known deletions.

In this study, we report the detection of large deletions in CPS1 gene by targeted oligonucleotide array CGH, MitoMet®, in 4 unrelated cases. The size of deletion varies from 1.4 kb to >130 kb.

2. Clinical descriptions

Case 1 is a 3-year old girl who initially presented with hyperammonemia, low citrulline, and respiratory alkalosis. PCR failed to amplify exons 9 and 10 of the CPS1 gene. Deleterious point mutations were not detected in the remaining exons by sequencing analysis. These results suggested a possible homozygous deletion involving exons 9 and 10.

Case 2 was a newborn infant girl presenting in critical condition due to hyperammonemia. Sequence analysis of CPS1 revealed a heterozygous novel variant, c.2945G>T (p.G982V). Glycine at amino acid position 982 is evolutionarily conserved from yeast to human, and is predicted to be deleterious by computer algorithms, SIFT [10] and PolyPhen [11]. Two other variants at the same amino acid position, c.2945G>A (p.G982D) and c.2944G>A (p.G982S), have been previously reported in patients with CPSI deficiency [12,13]. Collectively, these data suggested that the c.2945G>T (p.G982V) variant was likely pathogenic. However, the second mutant allele could not be identified by sequencing analysis.

Case 3 presented on the second day of life with irritability, feeding problems, and presumed sepsis. Hyperammonemia and low plasma citrulline were noted later. The child died on day of life 9. Postmortem liver analysis showed CPSI deficiency. Sequence analysis of the CPS1 cDNA derived from cultured fibroblasts revealed an apparently homozygous mutation c.1760G>A (p.R587H) in exon 16 [14]. This mutation has been previously reported in patients with CPSI deficiency [5,12]. Sequence analysis of the parents revealed that the mother was a carrier of the c.1760G>A (p.R587H) mutation, but the father was not. cDNA analysis using RNA from phytohaemagglutinine (PHA)-stimulated white blood cells from the father revealed a deletion of exon 24 in a heterozygous state, suggesting that mRNA from the father's allele was unstable (T. Edkins, personal communication).

Case 4 presented with early-onset hyperammonemia and was referred for the analysis of the CPS1 gene based on the low plasma citrulline and urine orotic acid. The patient died from metabolic crisis at age 7 months. Genetic analysis revealed an apparently homozygous mutation, c.3484delC in both cDNA and genomic DNA. However, the mutation was only found in the mother, therefore paternal DNA was analyzed by array CGH for the detection of intragenic deletions in this region.

3. Materials and methods

3.1. MitoMet® array CGH

A custom designed clinical oligonucleotide array CGH (MitoMet®) was manufactured using the Agilent microarray platform (Agilent Technologies, Santa Clara, CA). This is a clinically validated 44 K oligonucleotide array with a complete coverage of the mitochondrial genome (6400 probes for the 16.6-kb genome) and 192 nuclear genes related to mitochondrial structure/function and metabolic diseases, including CPS1, NAGS, OTC, ASS1, ASL, and ARG1 genes in the urea cycle, with an average spacing of approximately 250 bp/probe [8,9].

4. Results

4.1. Case 1

PCR failed to amplify exons 9 and 10 of the CPS1 gene. MitoMet® array CGH analysis detected a homozygous deletion measuring at least 1.3 kb (nucleotide position 211,163,672–211,164,925, NCBI36/hg18) in the long arm of chromosome 2, encompassing exons 9 and 10 of the CPS1 gene (Fig. 1A). PCR across the putative breakpoints and sequence analysis identified junction points to be positions c.841–194 and c.1086+77, corresponding to a 1440 bp deletion including exons 9–10 with a direct repeat of 3 nucleotides (TTG) at the breakpoints (Fig. 1A). The deletion of exons 9–10 is predicted to result in an inframe deletion of the CPSI protein (p.I281_E362del).

Fig. 1.

MitoMet® array CGH results. A. A homozygous deletion encompassing exons 9–10 of the CPS1 gene was detected. PCR across the deletion junction and sequencing confirmed a 1440 bp deletion from c.841–194 to c.1086+77, which is predicted to be an in-frame deletion (p.I281_E362del). B. A heterozygous deletion encompassing exons 9–11 of the CPS1 gene was detected. A 2395 bp deletion was confirmed by PCR and sequencing, which is predicted to be an in-frame deletion (p.I281_E388del). C. A heterozygous deletion of exon 24 of CPS1 gene was detected by MitoMet® array CGH and confirmed by PCR and sequencing. The 5′ end of the deletion junction included a poly-A tract, the size of the deletion is 2559 bp. The deletion of exon 24 is predicted to be a frameshift deletion (p.E966AfsX27). D. A large heterozygous deletion encompassing intron 2 to 3′ UTR was detected by MitoMet® array CGH. The size of deletion ranges from 134 kb to 767 kb based on backbone probe positions. The deletion does not include any known genes at the 3′ end of CPS1 gene.

4.2. Case 2

Sequencing analysis of the CPS1 gene revealed a heterozygous mutation, c.2945G>T (p.G982V). Array CGH analysis demonstrated an intragenic deletion encompassing exons 9 to 11 of the CPS1 gene. Sequence analysis of the PCR products of the deletion junction revealed a deletion of 2395 bp (c.841–212_c.1164+26) with a direct repeat of 5 nucleotides (TATTA) flanking the breakpoints (Fig. 1B) and resulting in an in-frame deletion, p.I281_E388del, of 108 amino acids of the CPS1 protein. These results are consistent with the diagnosis of CPSI deficiency.

4.3. Case 3

The deletion of CPS1 in this case was first detected by cDNA analysis from PHA-stimulated white blood cells and later defined by standard sequencing of the flanking regions of the deletion breakpoint. MitoMet® array CGH confirmed the deletion (Fig. 1C). The 5′ end of the breakpoint involved a poly-A tract of greater than 20 bp in size. The deletion size of 2559 bp, including exon 24, is predicted to result in an out-of-frame deletion (p.E966AfsX27).

4.4. Case 4

DNA samples from the deceased child were not available. MitoMet® array CGH analysis of CPS1 of the proband's father revealed a heterozygous deletion of exons 3–38. The size of deletion was estimated to range from 134 kb to 767 kb based on the backbone probe positions including the extended CPS1 3′-UTR region (Fig. 1D). No other known gene is located in the 3′ side of the deleted region.

5. Discussion

Oligonucleotide array-based CGH is a powerful tool to detect large intragenic rearrangements [8,9]. The high-density probe coverage of the target coding regions allowed us to identify the approximate break-points (Figs. 1A–D). In this study, the size of deletions ranged from 1.4 kb to >134 kb. Each targeted coding exon is covered on average by 5 probes. The immediate flanking introns are also densely covered. To score a deletion a cut-off of a minimum of 5 consecutively absent probes was set. In our experience, an intragenic deletion as small as 240 bp has been detected (Wong L-J, unpublished observation).When the deletion breakpoints fall outside of the densely probed coding region, it may be difficult to estimate the breakpoint location due to the sparse coverage of backbone region by probes (Fig. 1D).

In cases 1–3, the sequences at the breakpoints were identified by PCR across the deletion junctions, followed by sequencing. Short two to five nucleotide microhomologies at the breakpoints were identified in case 1 (TTG), case 2 (TATTA) and case 3 (AA). Common or recurrent breakpoints were not detected in this study. Analysis of the junction sequences using RepeatMasker (http://www.repeatmasker.org/) web tool did not reveal any interspersed or simple repeat sequence homology. ClustalW (http://www.clustal.org/) analysis revealed no significant increase of sequence homology in the breakpoint regions compared to homology in a randomly selected CPS1 gene region. The lack of recurrent deletion breakpoints, a paucity of repeat elements in the CPS1 genomic environment indicates that nonalleleic homologous recombination (NHAR) unlikely to be the mechanism involved in CPS1 large deletion. On the other hand, the presence of microhomology regions (TTG, TATTA and AA) suggests the possible involvement of microhomology-mediated break-induced replication (MMBIR) or a single event of fork stalling and template switching (FoSTeS X1) in our cases [15–17]. However, the alternative mechanism microhomology-mediated end joining (MMEJ, also known as Ku-independent NHEJ) could not be excluded [18].

Although CPS1 is larger than OTC, large OTC deletions account for 5–10% of all OTC deficiency cases [9], compared to about 1–2% of CPSI deficiency cases (Häberle et al., unpublished data). This may be explained by the unique genomic environment of chromosome X surrounding the OTC gene and extend to include the DMD and GK (glycerol kinase) gene regions [9,19]. All four patients in this study presented with hyperammonemia in early infancy, suggesting that CPS1 deletions confer risk of the early-onset CPSI deficiency. The possibility of large intragenic deletions in the CPS1 gene should be considered if the clinical presentation, biochemical and/or enzymatic studies strongly support CPSI deficiency, but only one or no deleterious mutations were identified. This report demonstrates the great utility of a targeted oligonucleotide array CGH for this purpose [8].