J. Perinat. Med. 2014; aop
Ning Liu, Xiang-dong Kong*, Quan-cheng Kan, Hui-rong Shi, Qing-hua Wu, Zhi-hong Zhuo, Qiao-ling Bai and Miao Jiang
Mutation analysis and prenatal diagnosis in a Chinese family with succinic semialdehyde dehydrogenase and a systematic review of the literature of reported ALDH5A1 mutations Abstract Aims: Succinic semialdehyde dehydrogenase (SSADH) deficiency is a neurometabolic disease in which the degradation of γ-aminobutyric acid (GABA) is impaired. The purpose of this study was to report two novel ALDH5A1 mutations responsible for SSADH deficiency in a Chinese family and the prenatal diagnosis of an at-risk fetus with DNA sequencing. Results: Genetic analysis of ALDH5A1, in a child with SSADH deficiency, parents, and 10 weeks’ gestation at-risk fetus and 100 healthy unrelated volunteers, was performed. The coding sequence and the intron/exon junctions of ALDH5A1 were analyzed by bidirectional DNA sequencing. The proband was identified to have a compound heterozygous mutations with c.496T > C (p.W166R) and c.589G > A (p.V197M). Each of his parents carried a deleterious mutation. DNA sequencing of chorionic villus revealed the fetus was a carrier, but not affected, and this was confirmed after birth by genetic analysis of umbilical cord blood and urine organic acid analysis. A study in 2003 described 35 mutations of ALDH5A1 in 54 unrelated families, and the current study and systematic literature review identified nine additional novel mutations in eight
*Corresponding author: Xiang-dong Kong, MD, PHD, Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Jianshe Rd, Erqi District, Zhengzhou, Henan 450052, P. R. China, Tel.: +86-0371-66862729; +86-15037133788, Fax: +86-0371-66862729, E-mail: [email protected]; and Henan Center for Translational Medicine, Zhengzhou 450052, China Ning Liu and Quan-cheng Kan: Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China; and Henan Center for Translational Medicine, Zhengzhou 450052, China Hui-rong Shi, Qing-hua Wu, Qiao-ling Bai and Miao Jiang: Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Zhi-hong Zhuo: Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
unrelated families bringing the total number of unique mutations of ALDH5A1 resulting in SSADH deficiency to 44, and the 44 mutations occur from exon 1 to exon 10. No mutational hotspots or prevalent mutations were observed, and all mutations appeared vital for the function of SSADH. Conclusions: Two novel ALDH5A1 mutations likely responsible for SSADH deficiency were identified, and DNA sequencing provided an accurate diagnosis for an atrisk fetus whose sibling had SSADH deficiency. Keywords: γ-Aminobutyric acid; γ-hydroxybutyric acid; ALDH5A1; DNA sequencing; GABA; GHB; SSADH; succinic semialdehyde dehydrogenase deficiency. DOI 10.1515/jpm-2014-0164 Received May 13, 2014. Accepted October 23, 2014. Previously published online xx.
Introduction Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare neurometabolic disease with autosomal recessive inheritance in which the degradation of γ-aminobutyric acid (GABA) is impaired. GABA is a major inhibitory neurotransmitter in the brain, and is formed from glutamate by the action of glutamic acid decarboxylase [1]. It is converted to succinic semialdehyde (SSA) by the GABA transaminase, and SSA is metabolized in the mitochondria by SSADH to succinic acid, which then enters the Krebs cycle for energy production or can be converted back to GABA [1]. In the absence of SSADH, GABA is not broken down into succinic acid, but is converted into γ-hydroxybutyric acid (GHB), which results in accumulation of GABA and GHB in the urine and cerebrospinal fluid [1].
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2 Liu et al., Prenatal genetic diagnosis of SSADH
SSADH deficiency often presents in infancy or early childhood with nonspecific clinical manifestations. The clinical phenotype is highly variable, and ranges from mild retardation in language and motor development to severe neurological defects including seizures, hypotonia, ataxia, and behavioral problems, especially in older patients [2, 3]. It is unclear whether elevated GABA, GHB, or other neurometabolic change accounts for the phenotype, but the primary metabolic abnormality is an excessive concentration of GHB in the physiological fluids, with elevations up to 800-fold in the plasma and 1200-fold in the cerebrospinal fluid [4]. Magnetic resonance imaging (MRI) of the brain may show abnormalities in the globus pallidus and the subcortical white matter, but the radiological features are not pathognomonic [1, 3]. Treatment of SSADH deficiency is difficult, and no consistently successful therapy has been found [1, 5]. Treatment of symptoms is the primary method of management, though clinical trials of taurine, the GABA B receptor antagonist SGS742, and other drugs are underway [5, 6]. The ALDH5A1 gene is located on chromosome 6p22, and consists of 10 exons encompassing over 38 kb [7]. The open reading frame consists of 1605 bp, encoding 535 amino acids, of which the first 47 residues form the mitochondrial targeting sequence [7]. ALDH5A1 is the only gene known to be associated with SSADH deficiency [8]. Since the first reported case of SSADH deficiency in 1981 [9], mutation analyses in patients of different geographic origins have revealed a wide spectrum of mutations, and consanguinity is present in approximately 40% of cases [10]. However, evidence for a genotype-phenotype correlation is scanty [11]. While diagnosis is typically made by organic acid analysis of urine or plasma, followed by determination of SSADH enzyme activity in leukocytes [1], DNA sequence analysis provides the most exact method of diagnosis and is especially useful for a prenatal diagnosis of SSADH [1, 11, 12]. The purpose of this report is to describe an infant with SSADH deficiency with two novel pathogenic mutations of ALDH5A1 and the use of DNA sequencing to determine the status of the mother’s fetus of 10 weeks’ gestation. In addition, we review the literature of reported ALDH5A1 mutations associated with SSADH deficiency.
Subjects and methods This study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Written informed consent from the guardian of the proband, and of all other subjects included in the study, for all testing and procedures performed was obtained. The control participants were
aware that the blood specimens were to be used for research in the genetic laboratory in our hospital, but were not aware of this particular study. Blood specimens were taken exclusively for this study.
Proband A 1-year 11-month-old boy was referred to our hospital because of repetitive aspiration pneumonia and intractable seizures since the age of 4 months. The boy was the first child of nonconsanguineous healthy parents, delivered full term by a normal vaginal delivery with no evidence of fetal asphyxia. The child started to have epileptic seizures 2 weeks after birth, accompanied with frequent choking coughs and poor appetite. Muscle strength was average, but reduced muscle tone was noted. By 3 months, head movement was still unstable, and he was experiencing intermittent febrile episodes and was susceptible to pneumonia. He controlled his head at the age of 10 months, and crawled at 14 months. When first seen at our hospital, he could not walk without support or speak meaningful words. Blood tests including blood gas analysis, blood chemistry, thyroid function, and karyotypic chromosome analysis were unremarkable. Subarachnoid space enlargement and ventriculomegaly was found in cranial MRI. No abnormal findings were noted on the electroencephalography (EEG). A marked increase in GHB, which is crucial for the diagnosis of SSADH deficiency, was found using gas chromatography-mass spectrometry: GHB levels were increased in the urine (997 mmol/mol creatinine) and cerebrospinal fluid (CSF; 576 μmol/L), and GABA levels were also increased in both serum and CSF. Based on these data, a clinical diagnosis of SSADH deficiency was made. The mother of the boy was 10 weeks pregnant with her second child and was seen at the genetic counseling clinic because of the concern that the fetus may be affected with SSADH deficiency.
Collection of samples and genomic DNA extraction Peripheral blood samples using ethylenediamine tetraacetic acid (EDTA) for anticoagulation were collected from the patient and his parents. Transabdominal chorionic villi sampling was performed at 10 weeks’ gestation for evaluation of the at-risk fetus. In addition, blood samples were obtained from 100 unrelated healthy Chinese individuals visiting the hospital for routine health examinations to serve as normal controls. Genomic DNA was isolated from each sample using a commercial kit (TIANamp DNA Kit, Tiangen Biotech, Beijing, China).
Polymerase chain reaction (PCR) and DNA sequencing PCR amplification and DNA sequencing of 10 exons of the ALDH5A1 gene were performed as previously described [11]. The amplifications included all of the exons and exon/intron boundaries of the ALDH5A1 gene. PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) with a total volume of 25 μL using 20–50 ng of DNA, 1 μL of each primer, and 13 μL 2 × Taq PCR MasterMix (containing a cocktail of dNTP, Tris-HCl, Taq polymerase, KCl, and MgCl2), and ddH2O. The PCR conditions were denaturation at 94°C for 4 min followed by 32 cycles [94°C for 30 s, then 58°C–64°C
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Liu et al., Prenatal genetic diagnosis of SSADH 3
(depending on the primer) for 40 s, extension at 72°C for 1 min], followed by a further extension at 72°C for 7 min. The PCR products were examined by agarose gel (1.5%) electrophoresis followed by staining the gel in ethidium bromide (0.5 g/mL), then visualization under ultraviolet (UV) light in a gel documentation system (BioSpectrum 310 Imaging System; UVP, Upland, CA, USA). PCR amplicons were bidirectionally sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) to detect gene mutations.
Nomenclature and sequence analysis Mutation nomenclature follows the guidelines of den Dunnen and Antonarakis [13]. Numbering of nucleotide and amino acid positions referred to the complete cDNA sequence (GenBank accession Y11192), including the mitochondrial leader sequence and starting from the first ATG or amino acid. Mutations were named according to Human Genome Variation Society (HGVS) naming conventions [14].
Bioinformatic analysis of the mutation sequences Evolutionary conservation of a non-synonymous variant was investigated with protein sequence alignment generated by ClustalW (http://www.ebi.ac.uk/clustalw/) and compared with that presented by the Ensembl Database (http://www.ensembl.org). The functional consequences of the missense variants were predicted using Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen). Protein Variation Effect Analyzer (PROVEAN) scores, which predict whether a protein sequence variation affects protein function, were also determined (http://provean.jcvi.org/about.php).
Prenatal genetic diagnoses and maternal pollution exclusion and follow-up Prenatal genetic diagnosis of the 10 weeks’ gestation at-risk fetus was performed by transabdominal chorionic villus sampling after the genotypes of the proband were identified. Maternal pollution was ruled out from fetal samples using a PowerPlex 16 HS System Kit (Promega Corporation, Madison, WI, USA) and GeneMapper ID v3.2 Soft. Umbilical cord blood was collected at birth for genetic analysis, and urine organic acid analysis using gas chromatography-mass spectrometry was detected concurrently after birth. Developmental assessment of the newborn was performed with age.
Systematic literature review
Figure 1 Sequencing diagrams showing W166R and V197M mutations. (A, B) Forward/reverse, respectively, sequencing diagrams showing a TC heterozygous peak for the c.496T > C (p.W166R) heterozygous mutation. (C, D) Forward/reverse, respectively, sequencing diagrams of the proband showing a GA heterozygous peak for the c.589G > A (p.V197M) heterozygous mutation.
Searches of Medline, Cochrane, EMBASE, and Google Scholar were conducted until November 1, 2013 using combinations of the following keywords: succinic semialdehyde dehydrogenase, SSADH, γ-aminobutyric acid, GABA, ALDH5A1, mutational analysis, gene analysis. Inclusion criteria were descriptive studies, case reports, and case series designed for pre-/postnatal diagnosis of SSADH deficiency and analysis of the ALDH5A1 gene. Non-English language
publications were excluded. The references lists of studies identified by the literature searches were also examined for other potentially relevant studies. Studies were identified by the search strategy by two independent reviewers. Where there was uncertainty regarding eligibility, a third reviewer was consulted. The following information/
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4 Liu et al., Prenatal genetic diagnosis of SSADH
data were extracted from studies that met the inclusion criteria: the name of the first author, year of publication, patient’s age and gender, family history, diagnostic method, and mutation analysis and results.
the allelic changes were likely pathological rather than polymorphism. The frequencies of p.H180Y and p.P182L were 18% and 6%, respectively, in healthy individuals.
Results
Bioinformatic analysis
ALDH5A1 mutation analysis
The tryptophan (W) to arginine (R) exchange at position 166 and the valine (V) to methionine (M) exchange at position 197 affect amino acids, which are evolutionally highly conserved in orthologous proteins of chickens, horses, and chimpanzees (Figure 5). The missense variants p.W166R and p.V197M were considered putative pathogenic mutations because SIFT and PolyPhen predicted that the functional consequences of them are “not tolerated” and “probably damaging”. In addition, the PROVEAN scores were –12.947 and –2.912, respectively (cutoff = –2.5).
The proband was identified to have a compound heterozygous mutations with c.496T > C (p.W166R) and c.589G > A (p.V197M) (Figure 1). The former mutation is a T to C substitution at nucleotide 496, which leads to an amino acid change from tryptophan to arginine at codon 166, and the latter mutation is a G to A substitution at nucleotide 589, which leads to an amino acid change from valine to methionine at codon 197. The proband also exhibited a c.538C > T and c.545C > T homozygous nucleotide substitution, which results in a p.H180Y and p.P182L amino acid substitution (Figure 2), a confirmed single nucleotide polymorphism. Three heterozygotes, p.V197M, p.H180Y, and p.P182L, were derived from his maternal genome (Figure 3), and three heterozygotes, p.W166R, p.H180Y, and p.P182L, were derived from his paternal genome (Figure 4). The two missense variants, p.W166R and p.V197M, were not detected in any of the 100 healthy subjects, which indicated that
Prenatal genetic diagnoses and follow-up Analysis of ALDH5A1 of the at-risk fetus revealed heterozygous mutations of maternal deleterious p.V197M, p.H180Y, and p.P182L polymorphisms (Figure 6), which indicated the fetus is a carrier but not affected. Umbilical cord blood
Figure 2 Sequencing diagrams showing H180Y and P182L mutations. Forward sequencing diagram showing CT homozygous peaks for c.538C > T (p.H180Y) and c.545C > T (p.P182L) indicating they are homozygous polymorphisms.
Figure 3 Sequencing diagrams of the proband’s mother showing V197M heterozygous mutation and H180Y/P182L heterozygous polymorphisms. (A, B) Forward/reverse, respectively, sequencing diagram of the proband’s mother showing GA heterozygous peaks for c.589G > A (p.V197M) indicating a heterozygous mutation. (C) c.538C > T heterozygous peak and c.545C > T heterozygous peak, which indicate heterozygous polymorphisms of p.H180Y and p.P182L.
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Liu et al., Prenatal genetic diagnosis of SSADH 5
et al. [11] in 2003, and studies included in that review were not selected for inclusion in the current study. A total 121 studies were identified in the initial literature search, and ultimately seven studies meeting the inclusion criteria were included in the analysis [15–21]. The clinical characteristics of the patients in the included studies as well as the present study are summarized in Table 1. A total of 11 patients were included, with ages ranging from 4 months to 27 years, and consanguinity was reported in four studies. Developmental delay, hypotonia, speech delay, and intellectual disability were common clinical symptoms, and seizures were reported in four studies. The causative mutations identified are summarized in Table 2, and included missense, deletions, frameshift/ insertions, and frameshift/deletions. In six studies, patients were homozygous, and in two compound heterozygous. In all studies that reported mutant SSADH activity, the enzyme activity was 5% or less than normal or undetectable. Table 3 summarizes all of the currently known disease-causing ALDH5A1 mutations, including those reported by Akaboshi et al. [11]. A total of 42 mutation sites, not including those in the current study, have been identified including 22 point mutations, eight deletions, five insertions, and seven splice site mutations.
Discussion
Figure 4 Sequencing diagrams of the proband’s father showing W166R heterozygous mutation and H180Y/P182L heterozygous polymorphisms. (A, B) Forward/reverse, respectively, sequencing diagram of the proband’s father showing TC heterozygous peaks for c.496T>C (p.W166R) indicating a heterozygous mutation. (C) c.538C>T heterozygous peak and c.545C>T heterozygous peak, which indicate heterozygous polymorphisms of p.H180Y and p.P182L.
collected for genetic analysis was consistent with prenatal diagnosis, and urine organic acid analysis using gas chromatography-mass spectrometry of the infant after birth was normal. The psychomotor development of the infant was appropriate for age, and no symptoms of SSADH were noted.
Systematic literature review A flow diagram of study selection is shown in Figure 7. A prior comprehensive review was conducted by Akaboshi
SSADH deficiency was first reported in 1981 as γ-hydroxybutyric (GHB) aciduria, in relation to the biochemical hallmark of an accumulation of GHB in urine, plasma, and CSF [9]. The clinical picture of the disorder is a heterogeneous phenotype ranging from mild to severe psychomotor retardation, delayed speech development, muscular hypotonia, and ataxia [2, 3]. Other associated features include hyporeflexia, seizures, aggressive behavior, hyperkinesis, choreoathetosis, and nystagmus. The clinical features of our patient correspond to the typical manifestations of SSADH deficiency, a marked increase in GHB was found in the urine and CSF using gas chromatography-mass spectrometry, and subarachnoid space enlargement and ventriculomegaly was detected on cranial MRI confirming a diagnosis of SSADH deficiency. Many factors may influence the clinical features in patients with SSADH deficiency. There is no correlation between GHB levels in physiological fluids and the severity of the disease, yet the heterogeneity in clinical presentation in siblings is less extensive than between unrelated patients [2, 3].
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6 Liu et al., Prenatal genetic diagnosis of SSADH
Figure 5 Genetic conservation of the W166 and V197. Conservation of the tryptophan (W) in position 166 and the valine (V) in position 197 in different species.
Figure 6 Sequencing diagram of the fetus showing V197M mutation and H180Y/P182L polymorphism. (A, B) Forward/reverse, respectively, sequencing diagram of the fetus showing GA heterozygous peaks for c.589G > A (p.V197M) indicating a heterozygous mutation. (C) c.538C > T (p.H180Y) heterozygous peak and c.545C > T (p.P182L) heterozygous peak.
Possible relevant studies identified through database search after duplicates removed (n=121)
Non-relevant studies excluded (n=111)
Full-text articles assessed for eligibility (n=12)
Studies excluded (n=3) Research article (n=1) Not disease of interest (n=1) Insufficient information reported (n=1)
Studies included in review (n=9)
Figure 7 Flow diagram of study selection.
Owing to nonspecific clinical symptoms, the differential diagnosis of SSADH deficiency can be challenging, and diagnostic difficulties include the variable and
occasionally low urinary excretion of GHB, which may hamper detection using routine organic acid analyses. Diagnosis can also be hampered because GHB levels in
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Jung (2006) [19]
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1 1 1 1
1
23 months
3.5 years
9 months
3 years
13 years
4 months
6 months
M
F
F
M
F
M
M
2F/2M
Gender
Russian N/A Tunisian Chinese
Greek
Japanese
Pakistani
Iranian
Ethnic origin
N/A Nonconsanguineousd Nonconsanguineous
Consanguineous
Consanguineous
Nonconsanguineous
Consanguineous
Consanguineous
Family history
Organic acid analysis (urine) Enzymatic assay (lymphocyte), MRI (brain) Organic acid analysis (urine, CSF) Enzymatic assay (fibroblast) Organic acid analysis (urine) Enzymatic assay (lymphocyte) Organic acid analysis (urine, CSF)
Organic acid analysis (urine) Enzymatic assay (lymphocyte) MRI (brain)
Organic acid analysis (urine)
Linkage analysis and mutation screening (blood DNA) Enzymatic assay (lymphoblast) Organic acid analysis (urine) MRI (brain)
Diagnostic criteria (sample source)
Developmental delay, hypotonia, speech delay, severe intellectual disability Development delay, gross and fine motor development delay Development delay, severe neurological deficits, psychomotor delay Development delay, neurodevelopmental disorder, hypotonia, speech delay, intellectual deficiency Development delay, psychomotor delay, speech delayc Psychomotor delay, hypotonia Psychomotor delay, hypotonia, speech delay, delayed cognition Development delay, speech delay
Clinical symptoms
CSF = cerebrospinal fluid, MRI = magnetic resonance imaging, CSF = cerebrospinal fluid, NR=not reported. a Patients were from one family. Age of patients shown as mean (range). b No history of seizures, and seizures were not observed during the follow-up period either. c Patient had SSADH deficiency and also exhibited features of partial Wilms’ tumor, aniridia, genital abnormalities, and mental retardation (WAGR) syndrome with obesity. d Parents were from the same village.
Present study
Bekri (2004) [21]
Blasi (2006) [20]
1
Knerr (2010) [18]
Yamakawa (2012) [17]
1
24.5 (21–27) yearsa
4a
Püttmann (2013) [15]
Kwok (2012) [16]
Patient age
Patient number
First author (year of publication)
Table 1 Clinical characteristics of case reports with succinic semialdehyde dehydrogenase deficiency.
Yes
NR
Nob
NR
Yes
Yes
Nob
Yes
History of seizures
Liu et al., Prenatal genetic diagnosis of SSADH 7
Bekri (2004) [21] Present study
PCR = polymerase chain reaction, RT = reverse transcription, NAD = nicotinamide adenine dinucleotide, NR = not reported. a All sample sources were DNA or RNA from patient lymphocytes or lymphoblasts. b Nucleotides and amino acid residues are numbered from the start codon according to the cDNA sequence of GenBank accession Y11192. c In-frame exon-skipping deletion of exon 7.
PCR PCR PCR and RT-PCR Homozygous PCR Compound heterozygous PCR
Yamakawa (2012) [17] Knerr (2010) [18] Jung (2006) [19] Blasi (2006) [20]
Compound heterozygous Homozygous Homozygous Homozygous
NR
Frameshift/deletion c.1466_1472delTGGTTGG (exon 10) p.Met489ThrfsX4 Premature termination NR Missense c.496T > C (exon 3) p.Trp166Arg NR NR Missense c.589G > A (exon 3) p.Val197Met
< 1% Undetectable Reduced
NR
Premature termination NR NR NR Truncation p.Trp112Xfs p.Met432Leu p.Gly409Asp p.Gly196Asp p.339_391del c.336delG (exon 1) c.1294A > C (exon 8) c.1226G > A (exon 8) c.587G > A (exon 3) c.1015_1173del (exon 7)
Loss of NAD+binding Undetectable Premature termination NR p.Lys301Glu p.Glu486fs c.901A > G (exon 6) c.1456_1457ins34 (exon 10)
Missense Frameshift/ insertion Frameshift/deletion Missense Missense Missense Deletionc PCR PCR Püttmann (2013) [15] Homozygous Kwok (2012) [16] Homozygous
Type of mutation Methoda Homozygosity First author (year of publication)
Table 2 Summary of ALDH5A1 mutations in the included studies.
Change in nucleotide (exon)b
Change in proteinb
Effect of mutation
Mutant SSADH activity compared to normal
8 Liu et al., Prenatal genetic diagnosis of SSADH
body fluids can be a result of sedation with GHB, as well as the illicit use of GHB as a recreational drug [22, 23]. In addition, GHB aciduria has been associated with the use of certain catheters [24]. Enzyme deficiency can be demonstrated in leucocytes isolated from whole blood [1]. Genetic analysis, however, is the most exact method to define the disorder. The ALDH5A1 gene is located on chromosome 6p22 and consists of 10 exons encompassing over 38 kb. The open reading frame consists of 1605 bp, encoding 535 amino acids, of which the first 47 residues form the mitochondrial targeting sequence. We performed genetic analysis of the proband, parents, and the at-risk fetus, and the proband was demonstrated to be a compound heterozygote with p.W166R and p.V197M pathogenic mutations and two single nucleotide polymorphisms (SNPs), p.H180Y and p.P182L, of the ALDH5A1 gene. Three heterozygotes, p.V197M, p.H180Y, and p.P182L, were derived from his maternal genome and three heterozygotes, p.W166R, p.H180Y, and p.P182L were derived from his paternal genome. As we did not perform an expression analysis for this change, we cannot deny that p.W166R and p.V197M are rare polymorphisms. However, because no other substitution was seen in the same allele, we suspect that p.W166R and p.V197M caused a loss of SSADH activity in the patient. Furthermore, although p.H180Y and p.P182L are known polymorphism [4, 9, 25, 26], it is suggested that the effects of the two amino acid substitutions appear to be multiplicative, without any further synergistic effect [11]. They may be considered to be pathogenic polymorphisms, which means they cooperatively affect the enzyme activity in mutant alleles in patients and also reduce the enzyme activity in normal individuals. In a report published in 2003, Akaboshi et al. [11] identified 27 novel mutation sites of the ALDH5A1 gene and summarized 35 SSADH deficiency-causing mutations in 54 unrelated families. The current systematic literature review including cases since 2003, together with the present case, describes 10 mutations in eight unrelated families. Apart from one mutation site described by Knerr et al. [18] that was already reported by other studies [10 –12], the other mine mutations are novel. This brings the total number of unique mutations of ALDH5A1 (including those in the present study) resulting in SSADH deficiency to 44, and the 44 mutations occur from exon 1 to exon 10. No mutational hotspots or prevalent mutations were observed, and all mutations appear vital for the function of SSADH. As SSADH deficiency is inherited in an autosomal recessive manner, a child of an affected individual has a 25% chance of being affected, a 50% chance of being
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Liu et al., Prenatal genetic diagnosis of SSADH 9
Table 3 Summary of all known disease-causing ALDH5A1 mutations. Exon
Change in nucleotidea
Change in proteina
Type of mutation
Mutant SSADH activityb
1 1 1 1 1 1 1 Exon 2/exon 5 2 Exon 2/intron 2 3 3 3 3 3 Intron 3/exon 4
p.A12fsX123 p.S35fsX49 p.S55fsX79 p.Q79X p.C93F p.C93_R99dup p.W112Xfs p.E119_K290del p.Y128X ? p.A153fsX12 p.H154fsX10 p.G176R p.K192X p.G196D p.K148fsX16
n.d. < 5% n.d. n.d. 3%c n.d. n.d. n.d. n.d. n.d. n.d. n.d. < 1%c n.d. Undetectable n.d.
p.W204X p.S208fsX2 p.C223Y p.T233M p.N255S p.R261X p.G268E p.L243_K289del
n.d. n.d. 5%c 4%c 17%c n.d. < 1%c Undetectable
Exon 5/intron 5
IVS5+1G > T (r.EX5del)d
p.L243 > K289del
n.d.
6 6 7 Intron 6/exon 7 7 7 8 8 8 8 Exon 8/intron 8
p.K301E p.N335K p.339_391del ? p.P382L p.P382Q p.G409D p.R412X p.M432L p.P442fsX18 p.V392fsX10
Undetectable 1%c Reduced n.d. 2%c n.d. < 1%; < 1%c n.d. n/a n.d. n.d.
Exon 9/intron 9
c.901A > G c.1005C > A c.1015_1173del IVS6-2A > C (r.spl?) c.1145C > T c.1145C > A c.1226G > A c.1234C > T c.1294A > C c.1323dupG IVS8+1delG; IVS8+3A > T (r.EX8del)d IVS9+1G > T (r.EX9del)d
Insertion Deletion Deletion Nonsense Missense Insertion Frameshift/deletion ? Nonsense Splice donor site Insertion Deletion Missense Nonsense Missense Deletion/splice acceptor site Nonsense Deletion Missense Missense Missense Nonsense Missense Deletion/splice donor site Deletion/splice donor site Missense Missense Deletion Splice acceptor site Missense Missense Missense Nonsense Missense Insertion Deletion
4 4 4 4 5 5 5 Exon 5/intron 5
c.34dupG c.103_121del c.160_161delCT c.235C > T c.278G > T c.278_298dup c.336delG r.EX2_EX5del c.384C > G IVS2+1G > A (r.spl?) c.455_456dupAG c.460_473del c.526G > A c.574A > T c.587G > A IVS3-2 A > G (r.439_452del)d c.612G > A c.621delC c.668G > A c.698C > T c.764A > G c.781C > T c.803G > A IVS5+1G > A (r.EX5del)d
p.K448fsX52
Undetectable
10 10 10 10 10
c.1456_1457ins34 c.1460T > A c.1466_1472delTGGTTGG c.1540C > T c.1597G > A
p.E486fs p.V487E p.M489TfsX4 p.R514X p.G533R
Frameshfit/deletion/ splice donor site Frameshift/insertion Missense Frameshift/deletion Nonsense Missense
n.d. < 5% n.d. n.d. < 1%c
Mutations presented in the current study are not included in the table. n.d. = not determined, ? = unconfirmed. a Nucleotides and amino acid residues are numbered from the start codon according to the cDNA sequence of GenBank accession Y11192. b SSADH activity measured from patients’ leukocytes and compared to normal, unless otherwise stated. c SSADH expressed and enzyme activity measured in mammalian cells. d Splice site mutations resulting in the change of RNA and were confirmed by sequencing RT-PCR products.
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10 Liu et al., Prenatal genetic diagnosis of SSADH
an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Biochemical testing for determination of carrier status is not reliable [1, 5]. Prenatal diagnosis is typically made by measurement of GHB in amniotic fluid or assay of SSADH enzyme activity in chorionic villus tissue and/or cultured amniocytes [1]. However, as Hogema et al. [12] noted, there can be discordance between the enzyme activity in chorionic villus and metabolite analysis of amniotic fluid, likely a result of SSADH enzyme stability, illustrating the importance of DNA analysis for the prenatal diagnosis of SSADH deficiency. Prenatal diagnosis of SSADH deficiency has been performed in a limited number of at-risk pregnancies using a combination of GHB quantitation in amniotic fluid in conjunction with SSADH activity determination in biopsied chorionic villi and/or cultured amniocytes early in gestation [27]. The current study has provided evidence that genetic analysis is sufficient for the prenatal diagnosis of SSADH deficiency. The primary limitation of this study is that only one family was examined. However, the results showed two novel mutations that are likely to be responsible for SSADH deficiency and the value of DNA sequencing for prenatal diagnosis.
Conclusions This study identified two novel mutations of ALDH5A1 that are likely responsible for SSADH deficiency and brings the total number of reported unique mutations to 44. As the mutations are novel, confirmation that the specific genetic changes are pathological and disease causing in additional unrelated family members is necessary. DNA sequencing provided an accurate diagnosis for an at-risk fetus whose sibling was affected by SSADH deficiency. DNA sequencing may become the preferred method of prenatal diagnosis for this condition. Competing interest: The author(s) declare that they have no competing interests. Authors’ contributions: Ning Liu was the guarantor of the integrity of the entire study and carried out the study design. Xiang-dong Kong participated in literature research and manuscript review. Hui-rong Shi carried out the data analysis. Qing-hua Wu participated in the data acquisition and manuscript preparation. Zhi-hong Zhuo carried out clinical studies. Qiao-ling Bai carried out the manuscript editing. Miao Jiang participated in the experimental studies. All authors have read and approved the manuscript.
References [1] Pearl PL, Dorsey AM, Barrios ES, Gibson KM. Succinic semialdehyde dehydrogenase deficiency. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2013. 2004 May 05 [updated 2013 Sep 19]. [2] Pearl PL, Gibson KM, Acosta MT, Vezina LG, Theodore WH, Rogawski MA, et al. Clinical spectrum of succinic semialdehyde dehydrogenase deficiency. Neurology. 2003;60:1413–7. [3] Pearl PL, Novotny EJ, Acosta MT, Jakobs C, Gibson KM. Succinic semialdehyde dehydrogenase deficiency in children and adults. Ann Neurol. 2003;6(54 Suppl):S73–80. [4] Gibson KM, Aramaki S, Sweetman L, Nyhan WL, DeVivo DC, Hodson AK, et al. Stable isotope dilution analysis of 4-hydroxybutyric acid: an accurate method for quantification in physiological fluids and the prenatal diagnosis of 4-hydroxybutyric aciduria. Biomed Environ Mass Spectrom. 1990;19:89–93. [5] Kim KJ, Pearl PL, Jensen K, Snead OC, Malaspina P, Jakobs C, et al. Succinic semialdehyde dehydrogenase: biochemicalmolecular-clinical disease mechanisms, redox regulation, and functional significance. Antioxid Redox Signal. 2011;15:691–718. [6] Vogel KR, Pearl PL, Theodore WH, McCarter RC, Jakobs C, Gibson KM. Thirty years beyond discovery – clinical trials in succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism. J Inherit Metab Dis. 2013;36:401–10. [7] Trettel F, Malaspina P, Jodice C, Novelletto A, Slaughter CA, Caudle DL, et al. Human succinic semialdehyde dehydrogenase. Molecular cloning and chromosome localization. Adv Exp Med Biol. 1997;414:253–60. [8] Chambliss KL, Hinson DD, Trettel F, Malaspina P, Novelletto A, Jakobs C, et al. Two exon-skipping mutations as the molecular basis of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria). Am J Hum Genet. 1998;63:399–408. [9] Jakobs C, Bojasch M, Monch E, Rating D, Siemes H, Hanefeld F. Urinary excretion of gamma-hydroxybutyric acid in a patient with neurological abnormalities: the probability of a new inborn error of metabolism. Clin Chim Acta. 1981;111:169–78. [10] Gibson KM, Christensen E, Jakobs C, Fowler B, Clark MA, Hammersen G, et al. The clinical phenotype of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria): case reports of 23 new patients. Pediatrics. 1997;99:567–74. [11] Akaboshi S, Hogema BM, Novelletto A, Malaspina P, Salomons GS, Maropoulos GD, et al. Mutational spectrum of the succinate semialdehyde dehydrogenase (ALDH5A1) gene and functional analysis of 27 novel disease-causing mutations in patients with SSADH deficiency. Hum Mutat. 2003;22:442–50. [12] Hogema BM, Akaboshi S, Taylor M, Salomons GS, Jakobs C, Schutgens RB, et al. Prenatal diagnosis of succinic semialdehyde dehydrogenase deficiency: increased accuracy employing DNA, enzyme, and metabolite analyses. Mol Genet Metab. 2001;72:218–22. [13] den Dunnen JT, Antonarakis SE. Nomenclature for the description of human sequence variations. Hum Genet. 2001;109:121–4.
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Liu et al., Prenatal genetic diagnosis of SSADH 11
[14] Human Genome Variation Society. Nomenclature for the description of sequence variants. 2013. http://www.hgvs.org/ mutnomen. Accessed March 11, 2013. [15] Püttmann L, Stehr H, Garshasbi M, Hu H, Kahrizi K, Lipkowitz B, et al. A novel ALDH5A1 mutation is associated with succinic semialdehyde dehydrogenase deficiency and severe intellectual disability in an Iranian family. Am J Med Genet A. 2013;161A:1915–22. [16] Kwok JS, Yuen CL, Law LK, Tang NL, Cherk SW, Yuen YP. A novel ALDH5A1 mutation in a patient with succinic semialdehyde dehydrogenase deficiency. Pathology. 2012;44:280–2. [17] Yamakawa Y, Nakazawa T, Ishida A, Saito N, Komatsu M, Matsubara T, et al. A boy with a severe phenotype of succinic semialdehyde dehydrogenase deficiency. Brain Dev. 2012;34:107–12. [18] Knerr I, Gibson KM, Murdoch G, Salomons GS, Jakobs C, Combs S, et al. Neuropathology in succinic semialdehyde dehydrogenase deficiency. Pediatr Neurol. 2010;42:255–8. [19] Jung R, Rauch A, Salomons GS, Verhoeven NM, Jakobs C, Michael Gibson K, et al. Clinical, cytogenetic and molecular characterization of a patient with combined succinic semialdehyde dehydrogenase deficiency and incomplete WAGR syndrome with obesity. Mol Genet Metab. 2006;88:256–60. [20] Blasi P, Palmerio F, Caldarola S, Rizzo C, Carrozzo R, Gibson KM, et al. Succinic semialdehyde dehydrogenase deficiency: clinical, biochemical and molecular characterization of a new patient with severe phenotype and a novel mutation. Clin Genet. 2006;69:294–6. [21] Bekri S, Fossoud C, Plaza G, Guenne A, Salomons GS, Jakobs C, et al. The molecular basis of succinic semialdehyde
[22]
[23]
[24]
[25]
[26]
[27]
dehydrogenase deficiency in one family. Mol Genet Metab. 2004;81:347–51. Wolf NI, Haas D, Hoffmann GF, Jakobs C, Salomons GS, Wevers RA, et al. Sedation with 4-hydroxybutyric acid: a potential pitfall in the diagnosis of SSADH deficiency. J Inherit Metab Dis. 2004;27:291–3. Wong CG, Chan KF, Gibson KM, Snead OC. Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug. Toxicol Rev. 2004;23:3–20. Wamelink MM, Roos B, Jansen EE, Mulder MF, Gibson KM, Jakobs C. 4-Hydroxybutyric aciduria associated with catheter usage: a diagnostic pitfall in the identification of SSADH deficiency. Mol Genet Metab. 2011;102:216–7. Gibson KM, Hoffmann GF, Hodson AK, Bottiglieri T, Jakobs C. 4-hydroxybutyric acid and the clinical phenotype of succinic semialdehyde dehydrogenase deficiency, an inborn error of GABA metabolism. Neuropediatrics. 1998;29:14–22. De Rango F, Leone O, Dato S, Novelletto A, Bruni AC, Berardelli M, et al. Cognitive functioning and survival in the elderly: the SSADH C538T polymorphism. Ann Hum Genet. 2008;72:630–5. Gibson KM, Baumann C, Ogier H, Rossier E, Vollmer B, Jakobs C. Pre- and postnatal diagnosis of succinic semialdehyde dehydrogenase deficiency using enzyme and metabolite assays. J Inherit Metab Dis. 1994;17:732–7.
The authors stated that there are no conflicts of interest regarding the publication of this article.
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Ning Liu, Xiang-dong Kong*, Quan-cheng Kan, Hui-rong Shi, Qing-hua Wu, Zhi-hong Zhuo, Qiao-ling Bai and Miao Jiang
Mutation analysis and prenatal diagnosis in a Chinese family with succinic semialdehyde dehydrogenase and a systematic review of the literature of reported ALDH5A1 mutations Abstract Aims: Succinic semialdehyde dehydrogenase (SSADH) deficiency is a neurometabolic disease in which the degradation of γ-aminobutyric acid (GABA) is impaired. The purpose of this study was to report two novel ALDH5A1 mutations responsible for SSADH deficiency in a Chinese family and the prenatal diagnosis of an at-risk fetus with DNA sequencing. Results: Genetic analysis of ALDH5A1, in a child with SSADH deficiency, parents, and 10 weeks’ gestation at-risk fetus and 100 healthy unrelated volunteers, was performed. The coding sequence and the intron/exon junctions of ALDH5A1 were analyzed by bidirectional DNA sequencing. The proband was identified to have a compound heterozygous mutations with c.496T > C (p.W166R) and c.589G > A (p.V197M). Each of his parents carried a deleterious mutation. DNA sequencing of chorionic villus revealed the fetus was a carrier, but not affected, and this was confirmed after birth by genetic analysis of umbilical cord blood and urine organic acid analysis. A study in 2003 described 35 mutations of ALDH5A1 in 54 unrelated families, and the current study and systematic literature review identified nine additional novel mutations in eight
*Corresponding author: Xiang-dong Kong, MD, PHD, Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Jianshe Rd, Erqi District, Zhengzhou, Henan 450052, P. R. China, Tel.: +86-0371-66862729; +86-15037133788, Fax: +86-0371-66862729, E-mail: [email protected]; and Henan Center for Translational Medicine, Zhengzhou 450052, China Ning Liu and Quan-cheng Kan: Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China; and Henan Center for Translational Medicine, Zhengzhou 450052, China Hui-rong Shi, Qing-hua Wu, Qiao-ling Bai and Miao Jiang: Prenatal Diagnosis Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Zhi-hong Zhuo: Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
unrelated families bringing the total number of unique mutations of ALDH5A1 resulting in SSADH deficiency to 44, and the 44 mutations occur from exon 1 to exon 10. No mutational hotspots or prevalent mutations were observed, and all mutations appeared vital for the function of SSADH. Conclusions: Two novel ALDH5A1 mutations likely responsible for SSADH deficiency were identified, and DNA sequencing provided an accurate diagnosis for an atrisk fetus whose sibling had SSADH deficiency. Keywords: γ-Aminobutyric acid; γ-hydroxybutyric acid; ALDH5A1; DNA sequencing; GABA; GHB; SSADH; succinic semialdehyde dehydrogenase deficiency. DOI 10.1515/jpm-2014-0164 Received May 13, 2014. Accepted October 23, 2014. Previously published online xx.
Introduction Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare neurometabolic disease with autosomal recessive inheritance in which the degradation of γ-aminobutyric acid (GABA) is impaired. GABA is a major inhibitory neurotransmitter in the brain, and is formed from glutamate by the action of glutamic acid decarboxylase [1]. It is converted to succinic semialdehyde (SSA) by the GABA transaminase, and SSA is metabolized in the mitochondria by SSADH to succinic acid, which then enters the Krebs cycle for energy production or can be converted back to GABA [1]. In the absence of SSADH, GABA is not broken down into succinic acid, but is converted into γ-hydroxybutyric acid (GHB), which results in accumulation of GABA and GHB in the urine and cerebrospinal fluid [1].
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2 Liu et al., Prenatal genetic diagnosis of SSADH
SSADH deficiency often presents in infancy or early childhood with nonspecific clinical manifestations. The clinical phenotype is highly variable, and ranges from mild retardation in language and motor development to severe neurological defects including seizures, hypotonia, ataxia, and behavioral problems, especially in older patients [2, 3]. It is unclear whether elevated GABA, GHB, or other neurometabolic change accounts for the phenotype, but the primary metabolic abnormality is an excessive concentration of GHB in the physiological fluids, with elevations up to 800-fold in the plasma and 1200-fold in the cerebrospinal fluid [4]. Magnetic resonance imaging (MRI) of the brain may show abnormalities in the globus pallidus and the subcortical white matter, but the radiological features are not pathognomonic [1, 3]. Treatment of SSADH deficiency is difficult, and no consistently successful therapy has been found [1, 5]. Treatment of symptoms is the primary method of management, though clinical trials of taurine, the GABA B receptor antagonist SGS742, and other drugs are underway [5, 6]. The ALDH5A1 gene is located on chromosome 6p22, and consists of 10 exons encompassing over 38 kb [7]. The open reading frame consists of 1605 bp, encoding 535 amino acids, of which the first 47 residues form the mitochondrial targeting sequence [7]. ALDH5A1 is the only gene known to be associated with SSADH deficiency [8]. Since the first reported case of SSADH deficiency in 1981 [9], mutation analyses in patients of different geographic origins have revealed a wide spectrum of mutations, and consanguinity is present in approximately 40% of cases [10]. However, evidence for a genotype-phenotype correlation is scanty [11]. While diagnosis is typically made by organic acid analysis of urine or plasma, followed by determination of SSADH enzyme activity in leukocytes [1], DNA sequence analysis provides the most exact method of diagnosis and is especially useful for a prenatal diagnosis of SSADH [1, 11, 12]. The purpose of this report is to describe an infant with SSADH deficiency with two novel pathogenic mutations of ALDH5A1 and the use of DNA sequencing to determine the status of the mother’s fetus of 10 weeks’ gestation. In addition, we review the literature of reported ALDH5A1 mutations associated with SSADH deficiency.
Subjects and methods This study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Written informed consent from the guardian of the proband, and of all other subjects included in the study, for all testing and procedures performed was obtained. The control participants were
aware that the blood specimens were to be used for research in the genetic laboratory in our hospital, but were not aware of this particular study. Blood specimens were taken exclusively for this study.
Proband A 1-year 11-month-old boy was referred to our hospital because of repetitive aspiration pneumonia and intractable seizures since the age of 4 months. The boy was the first child of nonconsanguineous healthy parents, delivered full term by a normal vaginal delivery with no evidence of fetal asphyxia. The child started to have epileptic seizures 2 weeks after birth, accompanied with frequent choking coughs and poor appetite. Muscle strength was average, but reduced muscle tone was noted. By 3 months, head movement was still unstable, and he was experiencing intermittent febrile episodes and was susceptible to pneumonia. He controlled his head at the age of 10 months, and crawled at 14 months. When first seen at our hospital, he could not walk without support or speak meaningful words. Blood tests including blood gas analysis, blood chemistry, thyroid function, and karyotypic chromosome analysis were unremarkable. Subarachnoid space enlargement and ventriculomegaly was found in cranial MRI. No abnormal findings were noted on the electroencephalography (EEG). A marked increase in GHB, which is crucial for the diagnosis of SSADH deficiency, was found using gas chromatography-mass spectrometry: GHB levels were increased in the urine (997 mmol/mol creatinine) and cerebrospinal fluid (CSF; 576 μmol/L), and GABA levels were also increased in both serum and CSF. Based on these data, a clinical diagnosis of SSADH deficiency was made. The mother of the boy was 10 weeks pregnant with her second child and was seen at the genetic counseling clinic because of the concern that the fetus may be affected with SSADH deficiency.
Collection of samples and genomic DNA extraction Peripheral blood samples using ethylenediamine tetraacetic acid (EDTA) for anticoagulation were collected from the patient and his parents. Transabdominal chorionic villi sampling was performed at 10 weeks’ gestation for evaluation of the at-risk fetus. In addition, blood samples were obtained from 100 unrelated healthy Chinese individuals visiting the hospital for routine health examinations to serve as normal controls. Genomic DNA was isolated from each sample using a commercial kit (TIANamp DNA Kit, Tiangen Biotech, Beijing, China).
Polymerase chain reaction (PCR) and DNA sequencing PCR amplification and DNA sequencing of 10 exons of the ALDH5A1 gene were performed as previously described [11]. The amplifications included all of the exons and exon/intron boundaries of the ALDH5A1 gene. PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) with a total volume of 25 μL using 20–50 ng of DNA, 1 μL of each primer, and 13 μL 2 × Taq PCR MasterMix (containing a cocktail of dNTP, Tris-HCl, Taq polymerase, KCl, and MgCl2), and ddH2O. The PCR conditions were denaturation at 94°C for 4 min followed by 32 cycles [94°C for 30 s, then 58°C–64°C
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Liu et al., Prenatal genetic diagnosis of SSADH 3
(depending on the primer) for 40 s, extension at 72°C for 1 min], followed by a further extension at 72°C for 7 min. The PCR products were examined by agarose gel (1.5%) electrophoresis followed by staining the gel in ethidium bromide (0.5 g/mL), then visualization under ultraviolet (UV) light in a gel documentation system (BioSpectrum 310 Imaging System; UVP, Upland, CA, USA). PCR amplicons were bidirectionally sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) to detect gene mutations.
Nomenclature and sequence analysis Mutation nomenclature follows the guidelines of den Dunnen and Antonarakis [13]. Numbering of nucleotide and amino acid positions referred to the complete cDNA sequence (GenBank accession Y11192), including the mitochondrial leader sequence and starting from the first ATG or amino acid. Mutations were named according to Human Genome Variation Society (HGVS) naming conventions [14].
Bioinformatic analysis of the mutation sequences Evolutionary conservation of a non-synonymous variant was investigated with protein sequence alignment generated by ClustalW (http://www.ebi.ac.uk/clustalw/) and compared with that presented by the Ensembl Database (http://www.ensembl.org). The functional consequences of the missense variants were predicted using Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen). Protein Variation Effect Analyzer (PROVEAN) scores, which predict whether a protein sequence variation affects protein function, were also determined (http://provean.jcvi.org/about.php).
Prenatal genetic diagnoses and maternal pollution exclusion and follow-up Prenatal genetic diagnosis of the 10 weeks’ gestation at-risk fetus was performed by transabdominal chorionic villus sampling after the genotypes of the proband were identified. Maternal pollution was ruled out from fetal samples using a PowerPlex 16 HS System Kit (Promega Corporation, Madison, WI, USA) and GeneMapper ID v3.2 Soft. Umbilical cord blood was collected at birth for genetic analysis, and urine organic acid analysis using gas chromatography-mass spectrometry was detected concurrently after birth. Developmental assessment of the newborn was performed with age.
Systematic literature review
Figure 1 Sequencing diagrams showing W166R and V197M mutations. (A, B) Forward/reverse, respectively, sequencing diagrams showing a TC heterozygous peak for the c.496T > C (p.W166R) heterozygous mutation. (C, D) Forward/reverse, respectively, sequencing diagrams of the proband showing a GA heterozygous peak for the c.589G > A (p.V197M) heterozygous mutation.
Searches of Medline, Cochrane, EMBASE, and Google Scholar were conducted until November 1, 2013 using combinations of the following keywords: succinic semialdehyde dehydrogenase, SSADH, γ-aminobutyric acid, GABA, ALDH5A1, mutational analysis, gene analysis. Inclusion criteria were descriptive studies, case reports, and case series designed for pre-/postnatal diagnosis of SSADH deficiency and analysis of the ALDH5A1 gene. Non-English language
publications were excluded. The references lists of studies identified by the literature searches were also examined for other potentially relevant studies. Studies were identified by the search strategy by two independent reviewers. Where there was uncertainty regarding eligibility, a third reviewer was consulted. The following information/
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4 Liu et al., Prenatal genetic diagnosis of SSADH
data were extracted from studies that met the inclusion criteria: the name of the first author, year of publication, patient’s age and gender, family history, diagnostic method, and mutation analysis and results.
the allelic changes were likely pathological rather than polymorphism. The frequencies of p.H180Y and p.P182L were 18% and 6%, respectively, in healthy individuals.
Results
Bioinformatic analysis
ALDH5A1 mutation analysis
The tryptophan (W) to arginine (R) exchange at position 166 and the valine (V) to methionine (M) exchange at position 197 affect amino acids, which are evolutionally highly conserved in orthologous proteins of chickens, horses, and chimpanzees (Figure 5). The missense variants p.W166R and p.V197M were considered putative pathogenic mutations because SIFT and PolyPhen predicted that the functional consequences of them are “not tolerated” and “probably damaging”. In addition, the PROVEAN scores were –12.947 and –2.912, respectively (cutoff = –2.5).
The proband was identified to have a compound heterozygous mutations with c.496T > C (p.W166R) and c.589G > A (p.V197M) (Figure 1). The former mutation is a T to C substitution at nucleotide 496, which leads to an amino acid change from tryptophan to arginine at codon 166, and the latter mutation is a G to A substitution at nucleotide 589, which leads to an amino acid change from valine to methionine at codon 197. The proband also exhibited a c.538C > T and c.545C > T homozygous nucleotide substitution, which results in a p.H180Y and p.P182L amino acid substitution (Figure 2), a confirmed single nucleotide polymorphism. Three heterozygotes, p.V197M, p.H180Y, and p.P182L, were derived from his maternal genome (Figure 3), and three heterozygotes, p.W166R, p.H180Y, and p.P182L, were derived from his paternal genome (Figure 4). The two missense variants, p.W166R and p.V197M, were not detected in any of the 100 healthy subjects, which indicated that
Prenatal genetic diagnoses and follow-up Analysis of ALDH5A1 of the at-risk fetus revealed heterozygous mutations of maternal deleterious p.V197M, p.H180Y, and p.P182L polymorphisms (Figure 6), which indicated the fetus is a carrier but not affected. Umbilical cord blood
Figure 2 Sequencing diagrams showing H180Y and P182L mutations. Forward sequencing diagram showing CT homozygous peaks for c.538C > T (p.H180Y) and c.545C > T (p.P182L) indicating they are homozygous polymorphisms.
Figure 3 Sequencing diagrams of the proband’s mother showing V197M heterozygous mutation and H180Y/P182L heterozygous polymorphisms. (A, B) Forward/reverse, respectively, sequencing diagram of the proband’s mother showing GA heterozygous peaks for c.589G > A (p.V197M) indicating a heterozygous mutation. (C) c.538C > T heterozygous peak and c.545C > T heterozygous peak, which indicate heterozygous polymorphisms of p.H180Y and p.P182L.
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Liu et al., Prenatal genetic diagnosis of SSADH 5
et al. [11] in 2003, and studies included in that review were not selected for inclusion in the current study. A total 121 studies were identified in the initial literature search, and ultimately seven studies meeting the inclusion criteria were included in the analysis [15–21]. The clinical characteristics of the patients in the included studies as well as the present study are summarized in Table 1. A total of 11 patients were included, with ages ranging from 4 months to 27 years, and consanguinity was reported in four studies. Developmental delay, hypotonia, speech delay, and intellectual disability were common clinical symptoms, and seizures were reported in four studies. The causative mutations identified are summarized in Table 2, and included missense, deletions, frameshift/ insertions, and frameshift/deletions. In six studies, patients were homozygous, and in two compound heterozygous. In all studies that reported mutant SSADH activity, the enzyme activity was 5% or less than normal or undetectable. Table 3 summarizes all of the currently known disease-causing ALDH5A1 mutations, including those reported by Akaboshi et al. [11]. A total of 42 mutation sites, not including those in the current study, have been identified including 22 point mutations, eight deletions, five insertions, and seven splice site mutations.
Discussion
Figure 4 Sequencing diagrams of the proband’s father showing W166R heterozygous mutation and H180Y/P182L heterozygous polymorphisms. (A, B) Forward/reverse, respectively, sequencing diagram of the proband’s father showing TC heterozygous peaks for c.496T>C (p.W166R) indicating a heterozygous mutation. (C) c.538C>T heterozygous peak and c.545C>T heterozygous peak, which indicate heterozygous polymorphisms of p.H180Y and p.P182L.
collected for genetic analysis was consistent with prenatal diagnosis, and urine organic acid analysis using gas chromatography-mass spectrometry of the infant after birth was normal. The psychomotor development of the infant was appropriate for age, and no symptoms of SSADH were noted.
Systematic literature review A flow diagram of study selection is shown in Figure 7. A prior comprehensive review was conducted by Akaboshi
SSADH deficiency was first reported in 1981 as γ-hydroxybutyric (GHB) aciduria, in relation to the biochemical hallmark of an accumulation of GHB in urine, plasma, and CSF [9]. The clinical picture of the disorder is a heterogeneous phenotype ranging from mild to severe psychomotor retardation, delayed speech development, muscular hypotonia, and ataxia [2, 3]. Other associated features include hyporeflexia, seizures, aggressive behavior, hyperkinesis, choreoathetosis, and nystagmus. The clinical features of our patient correspond to the typical manifestations of SSADH deficiency, a marked increase in GHB was found in the urine and CSF using gas chromatography-mass spectrometry, and subarachnoid space enlargement and ventriculomegaly was detected on cranial MRI confirming a diagnosis of SSADH deficiency. Many factors may influence the clinical features in patients with SSADH deficiency. There is no correlation between GHB levels in physiological fluids and the severity of the disease, yet the heterogeneity in clinical presentation in siblings is less extensive than between unrelated patients [2, 3].
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6 Liu et al., Prenatal genetic diagnosis of SSADH
Figure 5 Genetic conservation of the W166 and V197. Conservation of the tryptophan (W) in position 166 and the valine (V) in position 197 in different species.
Figure 6 Sequencing diagram of the fetus showing V197M mutation and H180Y/P182L polymorphism. (A, B) Forward/reverse, respectively, sequencing diagram of the fetus showing GA heterozygous peaks for c.589G > A (p.V197M) indicating a heterozygous mutation. (C) c.538C > T (p.H180Y) heterozygous peak and c.545C > T (p.P182L) heterozygous peak.
Possible relevant studies identified through database search after duplicates removed (n=121)
Non-relevant studies excluded (n=111)
Full-text articles assessed for eligibility (n=12)
Studies excluded (n=3) Research article (n=1) Not disease of interest (n=1) Insufficient information reported (n=1)
Studies included in review (n=9)
Figure 7 Flow diagram of study selection.
Owing to nonspecific clinical symptoms, the differential diagnosis of SSADH deficiency can be challenging, and diagnostic difficulties include the variable and
occasionally low urinary excretion of GHB, which may hamper detection using routine organic acid analyses. Diagnosis can also be hampered because GHB levels in
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Jung (2006) [19]
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1 1 1 1
1
23 months
3.5 years
9 months
3 years
13 years
4 months
6 months
M
F
F
M
F
M
M
2F/2M
Gender
Russian N/A Tunisian Chinese
Greek
Japanese
Pakistani
Iranian
Ethnic origin
N/A Nonconsanguineousd Nonconsanguineous
Consanguineous
Consanguineous
Nonconsanguineous
Consanguineous
Consanguineous
Family history
Organic acid analysis (urine) Enzymatic assay (lymphocyte), MRI (brain) Organic acid analysis (urine, CSF) Enzymatic assay (fibroblast) Organic acid analysis (urine) Enzymatic assay (lymphocyte) Organic acid analysis (urine, CSF)
Organic acid analysis (urine) Enzymatic assay (lymphocyte) MRI (brain)
Organic acid analysis (urine)
Linkage analysis and mutation screening (blood DNA) Enzymatic assay (lymphoblast) Organic acid analysis (urine) MRI (brain)
Diagnostic criteria (sample source)
Developmental delay, hypotonia, speech delay, severe intellectual disability Development delay, gross and fine motor development delay Development delay, severe neurological deficits, psychomotor delay Development delay, neurodevelopmental disorder, hypotonia, speech delay, intellectual deficiency Development delay, psychomotor delay, speech delayc Psychomotor delay, hypotonia Psychomotor delay, hypotonia, speech delay, delayed cognition Development delay, speech delay
Clinical symptoms
CSF = cerebrospinal fluid, MRI = magnetic resonance imaging, CSF = cerebrospinal fluid, NR=not reported. a Patients were from one family. Age of patients shown as mean (range). b No history of seizures, and seizures were not observed during the follow-up period either. c Patient had SSADH deficiency and also exhibited features of partial Wilms’ tumor, aniridia, genital abnormalities, and mental retardation (WAGR) syndrome with obesity. d Parents were from the same village.
Present study
Bekri (2004) [21]
Blasi (2006) [20]
1
Knerr (2010) [18]
Yamakawa (2012) [17]
1
24.5 (21–27) yearsa
4a
Püttmann (2013) [15]
Kwok (2012) [16]
Patient age
Patient number
First author (year of publication)
Table 1 Clinical characteristics of case reports with succinic semialdehyde dehydrogenase deficiency.
Yes
NR
Nob
NR
Yes
Yes
Nob
Yes
History of seizures
Liu et al., Prenatal genetic diagnosis of SSADH 7
Bekri (2004) [21] Present study
PCR = polymerase chain reaction, RT = reverse transcription, NAD = nicotinamide adenine dinucleotide, NR = not reported. a All sample sources were DNA or RNA from patient lymphocytes or lymphoblasts. b Nucleotides and amino acid residues are numbered from the start codon according to the cDNA sequence of GenBank accession Y11192. c In-frame exon-skipping deletion of exon 7.
PCR PCR PCR and RT-PCR Homozygous PCR Compound heterozygous PCR
Yamakawa (2012) [17] Knerr (2010) [18] Jung (2006) [19] Blasi (2006) [20]
Compound heterozygous Homozygous Homozygous Homozygous
NR
Frameshift/deletion c.1466_1472delTGGTTGG (exon 10) p.Met489ThrfsX4 Premature termination NR Missense c.496T > C (exon 3) p.Trp166Arg NR NR Missense c.589G > A (exon 3) p.Val197Met
< 1% Undetectable Reduced
NR
Premature termination NR NR NR Truncation p.Trp112Xfs p.Met432Leu p.Gly409Asp p.Gly196Asp p.339_391del c.336delG (exon 1) c.1294A > C (exon 8) c.1226G > A (exon 8) c.587G > A (exon 3) c.1015_1173del (exon 7)
Loss of NAD+binding Undetectable Premature termination NR p.Lys301Glu p.Glu486fs c.901A > G (exon 6) c.1456_1457ins34 (exon 10)
Missense Frameshift/ insertion Frameshift/deletion Missense Missense Missense Deletionc PCR PCR Püttmann (2013) [15] Homozygous Kwok (2012) [16] Homozygous
Type of mutation Methoda Homozygosity First author (year of publication)
Table 2 Summary of ALDH5A1 mutations in the included studies.
Change in nucleotide (exon)b
Change in proteinb
Effect of mutation
Mutant SSADH activity compared to normal
8 Liu et al., Prenatal genetic diagnosis of SSADH
body fluids can be a result of sedation with GHB, as well as the illicit use of GHB as a recreational drug [22, 23]. In addition, GHB aciduria has been associated with the use of certain catheters [24]. Enzyme deficiency can be demonstrated in leucocytes isolated from whole blood [1]. Genetic analysis, however, is the most exact method to define the disorder. The ALDH5A1 gene is located on chromosome 6p22 and consists of 10 exons encompassing over 38 kb. The open reading frame consists of 1605 bp, encoding 535 amino acids, of which the first 47 residues form the mitochondrial targeting sequence. We performed genetic analysis of the proband, parents, and the at-risk fetus, and the proband was demonstrated to be a compound heterozygote with p.W166R and p.V197M pathogenic mutations and two single nucleotide polymorphisms (SNPs), p.H180Y and p.P182L, of the ALDH5A1 gene. Three heterozygotes, p.V197M, p.H180Y, and p.P182L, were derived from his maternal genome and three heterozygotes, p.W166R, p.H180Y, and p.P182L were derived from his paternal genome. As we did not perform an expression analysis for this change, we cannot deny that p.W166R and p.V197M are rare polymorphisms. However, because no other substitution was seen in the same allele, we suspect that p.W166R and p.V197M caused a loss of SSADH activity in the patient. Furthermore, although p.H180Y and p.P182L are known polymorphism [4, 9, 25, 26], it is suggested that the effects of the two amino acid substitutions appear to be multiplicative, without any further synergistic effect [11]. They may be considered to be pathogenic polymorphisms, which means they cooperatively affect the enzyme activity in mutant alleles in patients and also reduce the enzyme activity in normal individuals. In a report published in 2003, Akaboshi et al. [11] identified 27 novel mutation sites of the ALDH5A1 gene and summarized 35 SSADH deficiency-causing mutations in 54 unrelated families. The current systematic literature review including cases since 2003, together with the present case, describes 10 mutations in eight unrelated families. Apart from one mutation site described by Knerr et al. [18] that was already reported by other studies [10 –12], the other mine mutations are novel. This brings the total number of unique mutations of ALDH5A1 (including those in the present study) resulting in SSADH deficiency to 44, and the 44 mutations occur from exon 1 to exon 10. No mutational hotspots or prevalent mutations were observed, and all mutations appear vital for the function of SSADH. As SSADH deficiency is inherited in an autosomal recessive manner, a child of an affected individual has a 25% chance of being affected, a 50% chance of being
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Liu et al., Prenatal genetic diagnosis of SSADH 9
Table 3 Summary of all known disease-causing ALDH5A1 mutations. Exon
Change in nucleotidea
Change in proteina
Type of mutation
Mutant SSADH activityb
1 1 1 1 1 1 1 Exon 2/exon 5 2 Exon 2/intron 2 3 3 3 3 3 Intron 3/exon 4
p.A12fsX123 p.S35fsX49 p.S55fsX79 p.Q79X p.C93F p.C93_R99dup p.W112Xfs p.E119_K290del p.Y128X ? p.A153fsX12 p.H154fsX10 p.G176R p.K192X p.G196D p.K148fsX16
n.d. < 5% n.d. n.d. 3%c n.d. n.d. n.d. n.d. n.d. n.d. n.d. < 1%c n.d. Undetectable n.d.
p.W204X p.S208fsX2 p.C223Y p.T233M p.N255S p.R261X p.G268E p.L243_K289del
n.d. n.d. 5%c 4%c 17%c n.d. < 1%c Undetectable
Exon 5/intron 5
IVS5+1G > T (r.EX5del)d
p.L243 > K289del
n.d.
6 6 7 Intron 6/exon 7 7 7 8 8 8 8 Exon 8/intron 8
p.K301E p.N335K p.339_391del ? p.P382L p.P382Q p.G409D p.R412X p.M432L p.P442fsX18 p.V392fsX10
Undetectable 1%c Reduced n.d. 2%c n.d. < 1%; < 1%c n.d. n/a n.d. n.d.
Exon 9/intron 9
c.901A > G c.1005C > A c.1015_1173del IVS6-2A > C (r.spl?) c.1145C > T c.1145C > A c.1226G > A c.1234C > T c.1294A > C c.1323dupG IVS8+1delG; IVS8+3A > T (r.EX8del)d IVS9+1G > T (r.EX9del)d
Insertion Deletion Deletion Nonsense Missense Insertion Frameshift/deletion ? Nonsense Splice donor site Insertion Deletion Missense Nonsense Missense Deletion/splice acceptor site Nonsense Deletion Missense Missense Missense Nonsense Missense Deletion/splice donor site Deletion/splice donor site Missense Missense Deletion Splice acceptor site Missense Missense Missense Nonsense Missense Insertion Deletion
4 4 4 4 5 5 5 Exon 5/intron 5
c.34dupG c.103_121del c.160_161delCT c.235C > T c.278G > T c.278_298dup c.336delG r.EX2_EX5del c.384C > G IVS2+1G > A (r.spl?) c.455_456dupAG c.460_473del c.526G > A c.574A > T c.587G > A IVS3-2 A > G (r.439_452del)d c.612G > A c.621delC c.668G > A c.698C > T c.764A > G c.781C > T c.803G > A IVS5+1G > A (r.EX5del)d
p.K448fsX52
Undetectable
10 10 10 10 10
c.1456_1457ins34 c.1460T > A c.1466_1472delTGGTTGG c.1540C > T c.1597G > A
p.E486fs p.V487E p.M489TfsX4 p.R514X p.G533R
Frameshfit/deletion/ splice donor site Frameshift/insertion Missense Frameshift/deletion Nonsense Missense
n.d. < 5% n.d. n.d. < 1%c
Mutations presented in the current study are not included in the table. n.d. = not determined, ? = unconfirmed. a Nucleotides and amino acid residues are numbered from the start codon according to the cDNA sequence of GenBank accession Y11192. b SSADH activity measured from patients’ leukocytes and compared to normal, unless otherwise stated. c SSADH expressed and enzyme activity measured in mammalian cells. d Splice site mutations resulting in the change of RNA and were confirmed by sequencing RT-PCR products.
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10 Liu et al., Prenatal genetic diagnosis of SSADH
an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Biochemical testing for determination of carrier status is not reliable [1, 5]. Prenatal diagnosis is typically made by measurement of GHB in amniotic fluid or assay of SSADH enzyme activity in chorionic villus tissue and/or cultured amniocytes [1]. However, as Hogema et al. [12] noted, there can be discordance between the enzyme activity in chorionic villus and metabolite analysis of amniotic fluid, likely a result of SSADH enzyme stability, illustrating the importance of DNA analysis for the prenatal diagnosis of SSADH deficiency. Prenatal diagnosis of SSADH deficiency has been performed in a limited number of at-risk pregnancies using a combination of GHB quantitation in amniotic fluid in conjunction with SSADH activity determination in biopsied chorionic villi and/or cultured amniocytes early in gestation [27]. The current study has provided evidence that genetic analysis is sufficient for the prenatal diagnosis of SSADH deficiency. The primary limitation of this study is that only one family was examined. However, the results showed two novel mutations that are likely to be responsible for SSADH deficiency and the value of DNA sequencing for prenatal diagnosis.
Conclusions This study identified two novel mutations of ALDH5A1 that are likely responsible for SSADH deficiency and brings the total number of reported unique mutations to 44. As the mutations are novel, confirmation that the specific genetic changes are pathological and disease causing in additional unrelated family members is necessary. DNA sequencing provided an accurate diagnosis for an at-risk fetus whose sibling was affected by SSADH deficiency. DNA sequencing may become the preferred method of prenatal diagnosis for this condition. Competing interest: The author(s) declare that they have no competing interests. Authors’ contributions: Ning Liu was the guarantor of the integrity of the entire study and carried out the study design. Xiang-dong Kong participated in literature research and manuscript review. Hui-rong Shi carried out the data analysis. Qing-hua Wu participated in the data acquisition and manuscript preparation. Zhi-hong Zhuo carried out clinical studies. Qiao-ling Bai carried out the manuscript editing. Miao Jiang participated in the experimental studies. All authors have read and approved the manuscript.
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[14] Human Genome Variation Society. Nomenclature for the description of sequence variants. 2013. http://www.hgvs.org/ mutnomen. Accessed March 11, 2013. [15] Püttmann L, Stehr H, Garshasbi M, Hu H, Kahrizi K, Lipkowitz B, et al. A novel ALDH5A1 mutation is associated with succinic semialdehyde dehydrogenase deficiency and severe intellectual disability in an Iranian family. Am J Med Genet A. 2013;161A:1915–22. [16] Kwok JS, Yuen CL, Law LK, Tang NL, Cherk SW, Yuen YP. A novel ALDH5A1 mutation in a patient with succinic semialdehyde dehydrogenase deficiency. Pathology. 2012;44:280–2. [17] Yamakawa Y, Nakazawa T, Ishida A, Saito N, Komatsu M, Matsubara T, et al. A boy with a severe phenotype of succinic semialdehyde dehydrogenase deficiency. Brain Dev. 2012;34:107–12. [18] Knerr I, Gibson KM, Murdoch G, Salomons GS, Jakobs C, Combs S, et al. Neuropathology in succinic semialdehyde dehydrogenase deficiency. Pediatr Neurol. 2010;42:255–8. [19] Jung R, Rauch A, Salomons GS, Verhoeven NM, Jakobs C, Michael Gibson K, et al. Clinical, cytogenetic and molecular characterization of a patient with combined succinic semialdehyde dehydrogenase deficiency and incomplete WAGR syndrome with obesity. Mol Genet Metab. 2006;88:256–60. [20] Blasi P, Palmerio F, Caldarola S, Rizzo C, Carrozzo R, Gibson KM, et al. Succinic semialdehyde dehydrogenase deficiency: clinical, biochemical and molecular characterization of a new patient with severe phenotype and a novel mutation. Clin Genet. 2006;69:294–6. [21] Bekri S, Fossoud C, Plaza G, Guenne A, Salomons GS, Jakobs C, et al. The molecular basis of succinic semialdehyde
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The authors stated that there are no conflicts of interest regarding the publication of this article.
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