Mutation Report

Mutation Analysis in a Patient with Succinic Semialdehyde Dehydrogenase Deficiency: A Compound Heterozygote with 103-121del and 1460T > A of the ALDH5A1 Gene
Tsutomu Aoshima^a, Mitsuharu Kajita^a, Yoshitaka Sekido^b, Yoshiko Ishiguro^a, Ikuya Tsuge^a, Masahiko Kimura^c, Seiji Yamaguchi^c, K.Kazuyoshi Watanabe^a, Kaoru Shimokata^b, Toshimitsu Niwa<sup>b

a</sup>Department of Pediatrics, Nagoya University School of Medicine, Nagoya,
^bDepartment of Clinical Preventive Medicine, Nagoya University Hospital, Nagoya, and
^cDepartment of Pediatrics, Shimane Medical College, Shimane, Japan

Address of Corresponding Author

Hum Hered 2002;53:42-44 (DOI: 10.1159/000048603)

 Outline

• Key Words
• Abstract
• Description of the Mutation
• Source of the Material
• Methods
• Comments
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Mutation
• Succinic semialdehyde dehydrogenase deficiency
• 4-Hydroxybutyric aciduria
• 4-Aminobutyric acid

  Abstract

We saw a 17-month-old boy with moderate psychomotor retardation, and enzymatically diagnosed succinic semialdehyde dehydrogenase (SSADH) deficiency. After extracting mRNA and genomic DNA from his cultured lymphoblasts, we analyzed the entire coding region of the ALDH5A1 gene using reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. He was demonstrated to be a compound heterozygote with two novel mutations (103-121 del and 1460T>A). The former leads to a frameshift and premature termination, and the latter is a missense mutation, V487E. Both mutations were also detected in the genomic DNA. Taken together with previous mutation reports, genetic heterogeneity was suspected for SSADH deficiency, and may account for the wide range of its phenotype.

Copyright © 2002 S. Karger AG, Basel

 Description of the Mutation

A patient with succinic semialdehyde dehydrogenase (SSADH) deficiency was found to be a compound heterozygote. One mutation is a 19-bp deletion at nucleotides 103-121 of the ALDH5A1 gene (GenBank accession No. NM_001080) that leads to a frameshift and premature termination. Another mutation in another allele is a T to A substitution at nucleotide 1460, which leads to an amino acid change from valine to glutamic acid at codon 487. Both mutations were also seen in the genomic DNA, 38752-38770del and 77217T > A (GenBank accession No. AL031230).

 Source of the Material

We previously reported a 17-month-old boy with SSADH (EC 1.2.1.24) deficiency (MIM 271980) [1]. He was the second child of nonconsanguineous parents. His 4-year-old brother showed normal development. A developmental delay was diagnosed in early infancy. He controlled his head at the age of 6 months and crawled at 14 months. When he was brought to our hospital, he could not sit without support nor speak meaningful words. Although he showed marked nonparalytic hypotonia, he was hyperactive, crawling around and hitting his head against the wall of the consultation room. Physical examination revealed no other abnormalities. His development quotient according to Tsumori and Inage was decreased to 45. The ECG and head MRI showed no abnormalities. Analysis of organic acids using gas chromatography-mass spectrometry revealed an increase in urinary excretion of 4-hydroxybutyric acid (GHB), glutaric acid and adipic acid. GHB levels were increased in serum (7,450 µmol/l), cerebrospinal fluid (CSF) (200 µmol/l) as well as urine (107.4 mmol/mol Cr). 4-Aminobutyric acid (GABA) levels were also increased in both serum (1,626 pmol/ml) and CSF (393 pmol/ml). Based on these data, he was suspected to suffer from SSADH deficiency. Then we measured the activity of SSADH in his cultured lymphoblasts according to the method of Gibson et al. [2], and found that it was decreased to 5% of the normal control level (0.06 nmol/mg protein/min). Thus, we enzymatically diagnosed SSADH deficiency. The activity of SSADH in his mother's cultured lymphoblasts was 60% of normal control (0.83 nmol/mg protein/min), indicating that she is a heterozygote for the disease. We could not get a sample from the patient's father.

 Methods

After extraction of mRNA from his cultured lymphoblasts using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech), the ALDH5A1 gene was analyzed by standard reverse transcription-polymerase chain reaction (RT-PCR). Then, we synthesized a pair of PCR primers (5'-TTGCCTGTTTCCTGTCGCCGTCGTT-3' and 5'-AGAATCCTACAAGCCCCCG-3') to cover the entire coding region (1,608 bp), and performed PCR using TaKaRa LA Taq with the GC buffer kit (Takara). The 1,648-bp amplified fragments were subcloned into pGEM-T easy plasmid (Promega), and sequenced. Further, genomic DNA was extracted from his cultured lymphoblasts using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals). The mutations detected in the cDNA were confirmed by both genomic sequencing and PCR-restriction fragment length polymorphism analysis.

 Comments

SSADH deficiency is a rare autosomal recessive disorder. The deficient activity of SSADH in the GABA degradative pathway results in accumulation of GHB. GHB is a neuropharmacologically active compound used intravenously as an anesthetic drug, which acts as a direct or indirect GABA receptor agonist. Patients with SSADH deficiency show nonspecific neurological abnormalities, such as psychomotor retardation, speech delay and seizures. High levels of GHB in CSF probably reflect its abnormally high concentration in the brain; it may show neurotoxicity and impair the neurophysiological role of GABA in the development and function of the brain. GHB has also been found in nonneural tissues of animals, although its role is unclear.

The boy was demonstrated to be a compound heterozygote with a 19-bp deletion at nucleotides 103-121 (fig. 1a) and 1460T > A in the cDNA of the ALDH5A1 gene, which were also detected in the genomic DNA, 38752-38770del and 77217T > A. The deletion mutation leads to a frameshift and premature termination, which may cause a loss of SSADH activity. His mother did not carry this deletion mutation. Since another mutation 1460T > A creates an MboII restriction site, PCR digestion was performed. His mother was heterozygous for the 1460T > A mutation (fig. 1b). This substitution was not seen in 104 alleles of normal Japanese controls. It leads to an amino acid change from valine to glutamic acid at codon 487. Since we did not perform an expression analysis for this change, we cannot deny that the 1460T > A is a rare polymorphism. However, because no other substitution was seen in the same allele, we suspect that the V487E caused a loss of SSADH activity in the patient and his mother. Taken together with previous mutation reports, genetic heterogeneity was suspected for SSADH deficiency, and may account for the wide range of its phenotype.

Fig. 1.a Electropherograms of sequence analyses with the cDNAs of the ALDH5A1. A 19-bp deletion at nucleotide 103-121 exhibiting a heterozygous pattern was seen. b 3% agarose gel electrophoresis of the MboII-digested PCR fragments amplified with the genomic DNAs using a pair of primers, 5'-GTCATCAATGGTGCCCTCATC-3' and 5'-AGAATCCTACAAGCCCCCG-3'. The amplified 296-bp fragments were digested to 251- and 45-bp in the wild type, whereas 159- and 92-bp bands were detected in the patient. The patient showed a heterozygous mutation pattern. MW: X174/HaeIII digest; C: normal control; Pt: patient; M: patient's mother.

The cDNA sequence of the ALDH5A1 gene and mutational analyses were reported by Chambliss et al. [3] in two families with SSADH deficiency in 1998. However, full-length cDNA clones of the ALDH5A1 gene had not been isolated, because of the high G + C content in the 5' end. The sequence data of the 5' end of the gene at nucleotides 1-257 was that inferred from amino acid sequence and the genomic clone sequence. Their recombinant human SSADH was considerably less active (22-fold less) than recombinant rat SSADH. They could not exclude a possibility that there may be another intron somewhere within this region. However, in the present study, we analyzed the full-length cDNA which contains the GC-rich region, and confirmed that the sequence data were correct. Cloning of the full-length cDNA of the ALDH5A1 gene permitted a further mutational study and understanding of the relationship between the phenotype and the genotype of SSADH deficiency.

  References

1.

Ishiguro Y, Kajita M, Aoshima T, Watanabe K, Kimura M, Yamaguchi S: The first case of 4-hydroxybutyric aciduria in Japan. Brain Dev 2001;23:128-130.

2.

Gibson KM, Lee CF, Chambliss KL, Kamali V, Francois B, Jaeken J, Lakobs: 4-Hydroxybutyric aciduria: Applicaton of a fluorometric assay to the determination of succinic semialdehyde dehydrogenase activity in extracts of cultured human lymphoblasts. Clin Chim Acta 1991;196:219-221.

3.

Chambliss KL, Hinson DD, Trettel F, Malaspina P, Novelletto A, Jakobs C, Gibson KM: Two exon-skipping mutation as the molecular basis of succinic semialdehyde dehydrogenase deficiency (4-hydrobutyric aciduria). Am J Hum Genet 1998;63:399-408.

  Author Contacts

Toshimitsu Niwa
Department of Clinical Preventive Medicine
Nagoya University Hospital
65 Tsuruma-cho, Showa-ku, Nagoya 466-8560 (Japan)
Tel. +81 52 744 1980, Fax +81 52 744 1954, E-Mail tniwa@med.nagoya-u.ac.jp

  Article Information

Received: Received: August 4, 2001
Revision received: August 29, 2001
Number of Print Pages : 3
Number of Figures : 1, Number of Tables : 0, Number of References : 3

  Publication Details

Human Heredity (International Journal of Human and Medical Genetics)
Founded 1950 as Acta Genetica et Statistica Medica by Gunnar Dahlberg; Continued by M. Hauge (1965-1983)

Vol. 53, No. 1, Year 2002 (Cover Date: Released March 2002)

Journal Editor: J. Ott, New York, N.Y.
ISSN: 0001-5652 (print), 1423-0062 (Online)

For additional information: http://www.karger.ch/journals/hhe

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.