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Prenatal Diagnosis of Succinic Semialdehyde Dehydrogenase Deficiency: Increased Accuracy Employing DNA, Enzyme, and Metabolite Analyses
Molecular Genetics and Metabolism 72, 218 –222 (2001) doi:10.1006/mgme.2000.3145, available online at http://www.idealibrary.com on
Prenatal Diagnosis of Succinic Semialdehyde Dehydrogenase Deficiency: Increased Accuracy Employing DNA, Enzyme, and Metabolite Analyses B. M. Hogema,* ,† ,‡ S. Akaboshi,* M. Taylor,* G. S. Salomons,† C. Jakobs,† Ruud B. Schutgens,† B. Wilcken,§ S. Worthington,㛳 G. Maropoulos, ¶ M. Grompe,* ,** and K. M. Gibson* ,** ,1 *Department of Molecular and Medical Genetics and **Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon; †Department of Clinical Chemistry, Academic Hospital, Free University, Amsterdam, The Netherlands; ‡Department of Biochemistry, Cardiovascular Research Institute COEUR, Medical Faculty, Erasmus University, Rotterdam, The Netherlands; §Biochemical Genetics Laboratory, New Children’s Hospital, Parramatta, New South Wales, Australia; 㛳Department of Clinical Genetics, Liverpool Hospital, Sydney, Australia; and ¶Department of Chemical Pathology, Children’s Hospital Kyriakou, Athens, Greece Received November 29, 2000, and in revised form December 27, 2000; published online February 27, 2001
Key Words: GABA (4-aminobutyric acid); succinic semialdehyde dehydrogenase; ␥-hydroxybutyric acid (GHB); 4-hydroxybutyric aciduria; prenatal diagnosis; autosomal recessive inheritance; chorionic villi; amniocytes; amniocentesis.
Inherited succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24; McKusick 271980) deficiency is a defect of GABA degradation which leads to accumulation of 4-hydroxybutyric acid (␥-hydroxybutyric acid; GHB) in physiologic fluids of patients. Prenatal diagnosis (PND) was performed in three at-risk pregnancies employing combinations of: (1) reverse-transcription-polymerase chain reaction (RT-PCR) and genomic DNA amplification followed by sequencing using isolated leukocytes or cultured human lymphoblasts; (2) GHB quantitation in amniotic fluid; or (3) SSADH enzyme assay in chorionic villus (CV) and/or amniocytes. In two pregnancies, all analyses were concordant for prediction of disease status in the fetus. In the third case, enzyme activity in CV (deficient) and metabolite analysis in amniotic fluid (normal) were discordant. For clarification, mutation analysis was undertaken in CV, confirming heterozygosity for the mutation previously identified in the proband. We hypothesize that delayed transit time for shipment of CV between Greece and the United States (8 days) led to enhanced degradation of heterozygous SSADH enzyme activity. Our data demonstrate the importance of combined metabolite, enzyme, and DNA analysis for increased accuracy in the PND of SSADH deficiency. © 2001 Academic Press
Inherited succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare defect of GABA degradation, presents with a broad spectrum of heterogeneous, nonspecific neurologic sequalae which can range from mild to quite severe (1,2). Thus, prenatal diagnosis (PND) in at-risk families has become an important diagnostic option. PND of SSADH deficiency has been performed in a limited number of at-risk pregnancies using a combination of 4-hydroxybutyric acid (GHB) quantitation in amniotic fluid in conjunction with SSADH activity determination in biopsied chorionic villus (CV) and/or cultured amniocytes (3). Although to date metabolite analysis and enzyme determination in at-risk pregnancies have been concordant for prediction of fetal disease status, we have observed increased excretion of GHB in patients for whom SSADH enzyme activity in peripheral cells was normal (4). In addition, Thorburn and co-workers (5) have noted that SSADH activity in cultured amniocytes can be highly variable. DNA-based PND of SSADH deficiency would circumvent difficulties associated with SSADH enzyme stability and delayed transit times
1
To whom correspondence should be addressed at Biochemical Genetics Laboratory, Oregon Health Sciences University, Genetics Laboratories, 2525 SW 3rd Avenue, Portland, OR 97210. Fax: (503) 494-6922. E-mail: [email protected]. 218 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
PRENATAL DIAGNOSIS OF SSADH DEFICIENCY
for shipment of tissue. Enzyme instability and delayed transit may have a more significant impact in those instances in which the fetus is heterozygous for a disease-associated mutation. Availability of the human SSADH gene sequence (6,7) and preliminary mutation screening in patient samples (8,9) should enable molecular PND, which represents a valuable addition to the accurate PND of SSADH deficiency. In the current report, we present the first combined enzyme, metabolite, and molecular PND of SSADH deficiency in three pregnancies at risk for SSADH deficiency. In one pregnancy, amniotic fluid GHB level was normal whereas SSADH enzyme activity in CV was deficient; molecular analysis was critical to determine the correct heterozygous status of the fetus. Thus, DNA-based PND represents a valuable addition to current methodology available for PND of human SSADH deficiency (10). MATERIALS AND METHODS 4-Hydroxybutyric acid in amniotic fluid was determined by stable-isotope-dilution gas chromatographymass spectrometry (11). SSADH activity was determined in tissue homogenates using a fluorometric assay quantifying NADH in relation to succinic semialdehyde consumption (12). Propionyl- coenzyme A carboxylase (PCC) activity was determined as a control enzyme to establish tissue viability (3). Genomic DNA was isolated from Ficoll- gradient-purified lymphocytes or from cultured lymphoblasts using a salting out method (13). RNA was isolated using RNA-STAT60 (Tel-Test Inc., Friendwood, TX) as recommended by the manufacturer. RT-PCR was performed using Gibco (Rockville, MD) reverse transcriptase and Taq polymerase. Betaine (1 M) was added to the reactions to improve PCR yield of the GC-rich 5⬘ end of the SSADH gene (14). Taq polymerase was added after the temperature had reached 94°C, or the reaction was done in “easystart” tubes (MBP, San Diego, CA). Otherwise, the PCR mix was made as specified by the manufacturer. PCR conditions were set at 94°C, 4 min; 94°C, 45 s; 53°C, 1 min; and 72°C, 2 min for 30 cycles, followed by a further extension at 72°C for 5 min. Primers used in the RT-PCR were 5⬘-TCTGGGCATGGTAGCCGACTG-3⬘ (foreward) and 5⬘GGACTGGATGAGTTCTGAAAAATTC-3⬘ (back). For amplification of exons 4, 5, and 8 from genomic DNA, primer sequences were based on the submitted sequence (NCBI Accession No. AL031230)
219
and were as follows: exon 4, 5⬘-GGTTTGTCAATCAGTTGTGC-3⬘ (foreward) and 5⬘-ATAGATACCATTTACAGTAGG-3⬘ (back); exon 5, 5⬘-GTAAATTGTTGGCACATGTTTG-3⬘ (foreward) and 5⬘-TGGTGATCAGGATGAAATAG-3⬘ (back); exon 8, 5⬘-CTGAATCTCTGCAAATGTGGTTCC-3⬘ (foreward) and 5⬘-CTCTTTCAGGGTTTCCTATG-3⬘ (back). The temperature profile was identical to that of the RT-PCR, except for a shorter elongation time (1 min). PCR products were purified using a Qiagen (Valencia, CA) PCR cleanup kit. The purified PCR products were sequenced using BigDye terminators (Perkin Elmer, Boston, MA) and an ABI (Foster City, CA) 370 automated sequencer. To compare conservation of amino acid residues in the SSADH polypeptide, multiple protein alignments were done using the Clustal method. SSADH protein alignment included the following species: Agrobacterium tumefaciens, Rhizobium sp. NGR234, Synechocystis PCC6803, Streptomyces coelicolor, Sphingonomas aromaticivorans, Schizosaccharomyces pombe (two different SSADH proteins), Saccharomyces cerevisiae, Zymomonas mobilis, Mycobacterium leprae, Ralstonia eutropha, Rattus norvegicus, Emericella nidulans, Escherichia coli, Deinococcus radiodurans (two different SSADH proteins), Clostridium kluyveri, Caenorhabditis elegans, Bacillus subtilis, and Arabidopsis thaliana. RESULTS AND DISCUSSION Mutations in patients and parents were detected using a combination of RT-PCR, genomic PCR, and sequencing. All mutations found with RT-PCR and sequencing were verified by genomic PCR and sequencing (Fig. 1; Table 1). The mutations identified were not detected in a total of 140 control chromosomes, suggesting that these mutations were not simply common polymorphisms. The patient from the first family has been described (1); patient IC. Sequencing the RT-PCR product suggested the patient was homozygous for a 803G ⬎ A mutation in exon 5, leading to a glycine to glutamate substitution at position 268 (G268E, numbering according to Chambliss et al. (8)), but sequencing genomic DNA showed that the patient was heterozygous for this mutation and that the mother was a carrier. Further analysis showed that the father and the patient were heterozygous for a 612G ⬎ A substitution in exon 4, leading to the introduction of a stop codon at position 204 (W204X). Analysis of genomic DNA
220
HOGEMA ET AL.
FIG. 1. Schematic diagram of the human SSADH gene showing mutations described in the current report. Solid boxes represent exons, while straight lines represent introns. Nonsense alleles are shown on the top, while missense alleles are depicted on the bottom. The 3⬘-untranslated region of the gene is depicted by the shaded box (gene structure is not drawn to scale).
from amniocytes and chorionic villi showed that the at-risk fetus carried both mutations. Whereas it is highly likely that the introduction of a stop codon will affect the function of the protein, the G268E substitution could be a polymorphism that is not pathogenic. Comparing human SSADH with other SSADH proteins and other human aldehyde dehydrogenases from the GenBank database showed that the glycine at position 268 is conserved in 15 out of 22 (putative) SSADH proteins in different species, and in 13 out of 16 human aldehyde dehydrogenases. Other amino acids at this position have aliphatic side chains (alanine in two cases, valine in three, isoleucine in one) or a hydroxyl group (threonine in three cases, serine in one). The strong conservation of the glycine, even in species as distantly related to humans as bacteria and yeast, suggests that a substitution to glutamate would affect protein
function. Determination of GHB in amniotic fluid and SSADH activity measurement in amniocytes and a chorionic villus sample suggested an affected pregnancy (Table 1). The family elected to terminate the pregnancy and analysis of fetal DNA showed the expected two mutations. For the second family (Table 1), genomic PCR and sequencing showed that the proband (5) was heterozygous for two mutations (612G ⬎ A and 1234C ⬎ T) that introduce stop codons in exons 4 and 8, respectively, at positions 204 and 412 (W204X and R412X). Further analysis showed that the mother was a carrier of the 612G ⬎ A mutation and the father carried the 1234C ⬎ T mutation. Father and fetus also shared an interesting polymorphism (a deletion/insertion of 1 bp approximately 75 bp upstream of exon 8 never encountered in other patient or control samples). DNA from the proband did
TABLE 1 Prenatal Diagnosis of SSADH Deficiency in Three Unrelated Families SSADH activity Family 1 2 3
Proband mutations
GHB AF
G268E W204X W204X R412X G409D G409D
4.01
Mutations in at-risk pregnancy
AC
CV
ND
0 (0.4) —
0.04 (1.1) 0.4
1.12
0.14
0.01
AC Both present — —
CV
Fetal DNA
Both Both present present Neither — present Heterozygote —
Prediction
Outcome
Affected
Terminated
Unaffected
Miscarriage
Unaffected carrier
Healthy newborn
Note. Control GHB levels in amniotic fluid (AF) 0.42–2.2 mol/L (n ⫽ 30). All enzyme activities in units of nmol/min/mg protein. Parenthetic values represent parallel controls of the same tissue type, when available. Additional abbreviations: AC, amniocytes; CV, chorionic villus; GHB, 4-hydroxybutyric acid. The CV sample for Family 3 was delayed 8 days in transit. Marker enzyme activity in this sample (propionyl-CoA carboxylase) was 0.24 (control values 2.2, 5.2 nmol/min/mg protein, n ⫽ 2), suggesting tissue compromise in transit. ND, not determined.
PRENATAL DIAGNOSIS OF SSADH DEFICIENCY
not show this polymorphism. For prenatal diagnosis, DNA was isolated from a chorionic villus sample taken at 9 weeks gestation. PCR and sequence analysis showed no 612G ⬎ A or 1234C ⬎ T mutations; the paternal insertion/deletion polymorphism upstream of exon 8 was present, however, representing a useful internal control verifying that fetus and proband inherited different chromosomes from their father. Determination of the SSADH activity in the chorionic villus sample also suggested an unaffected pregnancy (Table 1). In the third family presenting for prenatal diagnosis (Table 1; Fig. 1), RT-PCR and genomic PCR verified that the proband was homozygous for a 1226G ⬎ A mutation, leading to substitution of aspartic acid for glycine at position 409 (G409D) of the polypeptide sequence. Analysis of SSADH activity in biopsied chorionic villus tissue revealed deficiency, yet the marker enzyme activity was also quite low, suggesting tissue compromise in transit (Table 1). Due to these results, amniocentesis was pursued which revealed a normal level of GHB in amniotic fluid and demonstrable SSADH enzyme activity in amniocytes cultured from the spun amniotic fluid specimen (Table 1). Mutation analysis of the chorionic villus sample using genomic PCR revealed heterozygosity for the G409D mutation. Analysis of position 409 in 13 other aldehyde dehydrogenases from different species revealed conservation of this glycine, suggesting that this residue is critical for proper function of the protein. Our results from the current study verify the importance and utility of having DNA testing available for prenatal diagnosis of SSADH deficiency. This is especially important in those instances in which samples are transported over long distances, as there may be significant delays in transit time. The data also suggest that delays in transit may have an even more pronounced deleterious effect in instances in which the fetal genotype is heterozygous. In the latter case, there is the possibility that deleterious mutations may lead to aberrant protein folding or aggregation. DNA testing will become an increasingly important part of the testing capabilities for SSADH deficiency, especially as the number of affected probands continues to grow, and should enable accurate PND as early as 8 –9 weeks of gestation.
human SSADH gene, and Ms. Wendy Guerand, Danielle Schor, and Virginia Perreira for determination of GHB quantities in amniotic fluid specimens. We are indebted to Dr. Daniel Bluestone and Dr. Seymour Packman who provided clinical evaluation of the proband in Family 1, and Drs. A. Skartoutsou, K. Boudris, and O. Papandreou who provided clinical assessment and follow-up in Family 3.
REFERENCES 1.
Gibson KM, Christensen E, Jakobs C, Fowler B, Clarke MA, Hammersen G, Raab K, Kobori J, Moosa A, Vollmer B, Rossier E, Iafolla AK, Matern D, Brouwer OF, Finkelstein J, Aksu F, Weber H-P, Bakkeren JAJM, Gabreels FJM, Bluestone D, Barron TF, Beauvais P, Rabier D, Santos C, Umansky R, Lehnert W. The clinical phenotype of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria): Case reports of 23 new patients. Pediatrics 99: 567–574, 1997.
2.
Gibson KM, Jakobs C. Disorders of  and gamma-amino acids in free and peptide linked forms. In The Metabolic and Molecular Bases of Inherited Disease, 8th ed. Vol 2, Chapter 91 (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds.). New York: McGraw-Hill, pp 2079 –2105, 2001.
3.
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 17:732–737, 1994.
4.
Gibson KM, Sweetman L, Kozich V, Pijackova A, Tscharre A, Cortez A, Eyskens F, Jakobs C, Duran M, Poll-The BT. Unusual enzyme findings in five patients with metabolic profiles suggestive of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria). J Inherit Metab Dis 21:255–261, 1998.
5.
Thorburn DR, Thompson GN, Howells DW. A fluorimetric assay for succinic semialdehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency. J Inherit Metab Dis 16:942–949, 1993.
6.
Chambliss KL, Caudle DL, Hinson DD, Moomaw CR, Slaughter CA, Jakobs C, Gibson KM. Molecular cloning of the mature NAD(⫹)-dependent succinic semialdehyde dehydrogenase from rat and human. cDNA isolation, evolutionary homology, and tissue expression. J Biol Chem 270:461– 467, 1995.
7.
Trettel F, Malaspina P, Jodice C, Novelletto A, Slaughter CA, Caudle DL, Hinson DD, Chambliss KL, Gibson KM. Human succinic semialdehyde dehydrogenase. Molecular cloning and chromosomal localization. Adv Exp Med Biol 414:253–260, 1997.
8.
Chambliss KL, Hinson DD, Trettel F, Malaspina P, Novelletto A, Jakobs C, Gibson KM. Two exon-skipping mutations as the molecular basis of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria). Am J Hum Genet 63:399 – 408, 1998.
9.
Hogema BM, Jakobs C, Oudejans CBM, Schutgens RB, Grompe M, Gibson KM. Mutation analysis in succinic semialdehyde dehydrogenase (SSADH) deficiency (4-hydroxybutyric aciduria). Am J Hum Genet 65:A238 (abstract), 1999.
10.
Jakobs C, Hogema BM, Taylor M, Akaboshi S, Schutgens RBH, Wilcken B, Worthington S, Maropolous G, Grompe M,
ACKNOWLEDGMENTS The authors are indebted to Dr. Patrizia Malaspina and Dr. Andrea Novelletto for supplying genomic sequence data on the
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Gibson KM. Prenatal diagnosis (PND) of succinic semialdehyde dehydrogenase (SSADH) deficiency: Increased accuracy using DNA, enzyme and metabolite analysis. J Inherit Metab Dis 23:110 (abstract), 2000. 11. Gibson KM, Aramaki S, Sweetman L, Nyhan WL, DeVivo DC, Hodson AK, Jakobs C. 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 Spectrometry 19:89 –93, 1990. 12. Gibson KM, Lee CF, Chambliss KL, Kamali V, Francois B,
Jaeken J, Jakobs C. 4-Hydroxybutyric aciduria: Application of a fluorometric assay to the determination of succinic semialdehyde dehydrogenase activity in extracts of cultured human lymphoblasts [letter]. Clin Chim Acta 196:219 –221, 1991. 13. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. 14. Henke W, Herdel K, Jung K, Schnorr D, Loening SA. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res 25:3957–3958, 1997.
Prenatal Diagnosis of Succinic Semialdehyde Dehydrogenase Deficiency: Increased Accuracy Employing DNA, Enzyme, and Metabolite Analyses B. M. Hogema,* ,† ,‡ S. Akaboshi,* M. Taylor,* G. S. Salomons,† C. Jakobs,† Ruud B. Schutgens,† B. Wilcken,§ S. Worthington,㛳 G. Maropoulos, ¶ M. Grompe,* ,** and K. M. Gibson* ,** ,1 *Department of Molecular and Medical Genetics and **Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon; †Department of Clinical Chemistry, Academic Hospital, Free University, Amsterdam, The Netherlands; ‡Department of Biochemistry, Cardiovascular Research Institute COEUR, Medical Faculty, Erasmus University, Rotterdam, The Netherlands; §Biochemical Genetics Laboratory, New Children’s Hospital, Parramatta, New South Wales, Australia; 㛳Department of Clinical Genetics, Liverpool Hospital, Sydney, Australia; and ¶Department of Chemical Pathology, Children’s Hospital Kyriakou, Athens, Greece Received November 29, 2000, and in revised form December 27, 2000; published online February 27, 2001
Key Words: GABA (4-aminobutyric acid); succinic semialdehyde dehydrogenase; ␥-hydroxybutyric acid (GHB); 4-hydroxybutyric aciduria; prenatal diagnosis; autosomal recessive inheritance; chorionic villi; amniocytes; amniocentesis.
Inherited succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24; McKusick 271980) deficiency is a defect of GABA degradation which leads to accumulation of 4-hydroxybutyric acid (␥-hydroxybutyric acid; GHB) in physiologic fluids of patients. Prenatal diagnosis (PND) was performed in three at-risk pregnancies employing combinations of: (1) reverse-transcription-polymerase chain reaction (RT-PCR) and genomic DNA amplification followed by sequencing using isolated leukocytes or cultured human lymphoblasts; (2) GHB quantitation in amniotic fluid; or (3) SSADH enzyme assay in chorionic villus (CV) and/or amniocytes. In two pregnancies, all analyses were concordant for prediction of disease status in the fetus. In the third case, enzyme activity in CV (deficient) and metabolite analysis in amniotic fluid (normal) were discordant. For clarification, mutation analysis was undertaken in CV, confirming heterozygosity for the mutation previously identified in the proband. We hypothesize that delayed transit time for shipment of CV between Greece and the United States (8 days) led to enhanced degradation of heterozygous SSADH enzyme activity. Our data demonstrate the importance of combined metabolite, enzyme, and DNA analysis for increased accuracy in the PND of SSADH deficiency. © 2001 Academic Press
Inherited succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare defect of GABA degradation, presents with a broad spectrum of heterogeneous, nonspecific neurologic sequalae which can range from mild to quite severe (1,2). Thus, prenatal diagnosis (PND) in at-risk families has become an important diagnostic option. PND of SSADH deficiency has been performed in a limited number of at-risk pregnancies using a combination of 4-hydroxybutyric acid (GHB) quantitation in amniotic fluid in conjunction with SSADH activity determination in biopsied chorionic villus (CV) and/or cultured amniocytes (3). Although to date metabolite analysis and enzyme determination in at-risk pregnancies have been concordant for prediction of fetal disease status, we have observed increased excretion of GHB in patients for whom SSADH enzyme activity in peripheral cells was normal (4). In addition, Thorburn and co-workers (5) have noted that SSADH activity in cultured amniocytes can be highly variable. DNA-based PND of SSADH deficiency would circumvent difficulties associated with SSADH enzyme stability and delayed transit times
1
To whom correspondence should be addressed at Biochemical Genetics Laboratory, Oregon Health Sciences University, Genetics Laboratories, 2525 SW 3rd Avenue, Portland, OR 97210. Fax: (503) 494-6922. E-mail: [email protected]. 218 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
PRENATAL DIAGNOSIS OF SSADH DEFICIENCY
for shipment of tissue. Enzyme instability and delayed transit may have a more significant impact in those instances in which the fetus is heterozygous for a disease-associated mutation. Availability of the human SSADH gene sequence (6,7) and preliminary mutation screening in patient samples (8,9) should enable molecular PND, which represents a valuable addition to the accurate PND of SSADH deficiency. In the current report, we present the first combined enzyme, metabolite, and molecular PND of SSADH deficiency in three pregnancies at risk for SSADH deficiency. In one pregnancy, amniotic fluid GHB level was normal whereas SSADH enzyme activity in CV was deficient; molecular analysis was critical to determine the correct heterozygous status of the fetus. Thus, DNA-based PND represents a valuable addition to current methodology available for PND of human SSADH deficiency (10). MATERIALS AND METHODS 4-Hydroxybutyric acid in amniotic fluid was determined by stable-isotope-dilution gas chromatographymass spectrometry (11). SSADH activity was determined in tissue homogenates using a fluorometric assay quantifying NADH in relation to succinic semialdehyde consumption (12). Propionyl- coenzyme A carboxylase (PCC) activity was determined as a control enzyme to establish tissue viability (3). Genomic DNA was isolated from Ficoll- gradient-purified lymphocytes or from cultured lymphoblasts using a salting out method (13). RNA was isolated using RNA-STAT60 (Tel-Test Inc., Friendwood, TX) as recommended by the manufacturer. RT-PCR was performed using Gibco (Rockville, MD) reverse transcriptase and Taq polymerase. Betaine (1 M) was added to the reactions to improve PCR yield of the GC-rich 5⬘ end of the SSADH gene (14). Taq polymerase was added after the temperature had reached 94°C, or the reaction was done in “easystart” tubes (MBP, San Diego, CA). Otherwise, the PCR mix was made as specified by the manufacturer. PCR conditions were set at 94°C, 4 min; 94°C, 45 s; 53°C, 1 min; and 72°C, 2 min for 30 cycles, followed by a further extension at 72°C for 5 min. Primers used in the RT-PCR were 5⬘-TCTGGGCATGGTAGCCGACTG-3⬘ (foreward) and 5⬘GGACTGGATGAGTTCTGAAAAATTC-3⬘ (back). For amplification of exons 4, 5, and 8 from genomic DNA, primer sequences were based on the submitted sequence (NCBI Accession No. AL031230)
219
and were as follows: exon 4, 5⬘-GGTTTGTCAATCAGTTGTGC-3⬘ (foreward) and 5⬘-ATAGATACCATTTACAGTAGG-3⬘ (back); exon 5, 5⬘-GTAAATTGTTGGCACATGTTTG-3⬘ (foreward) and 5⬘-TGGTGATCAGGATGAAATAG-3⬘ (back); exon 8, 5⬘-CTGAATCTCTGCAAATGTGGTTCC-3⬘ (foreward) and 5⬘-CTCTTTCAGGGTTTCCTATG-3⬘ (back). The temperature profile was identical to that of the RT-PCR, except for a shorter elongation time (1 min). PCR products were purified using a Qiagen (Valencia, CA) PCR cleanup kit. The purified PCR products were sequenced using BigDye terminators (Perkin Elmer, Boston, MA) and an ABI (Foster City, CA) 370 automated sequencer. To compare conservation of amino acid residues in the SSADH polypeptide, multiple protein alignments were done using the Clustal method. SSADH protein alignment included the following species: Agrobacterium tumefaciens, Rhizobium sp. NGR234, Synechocystis PCC6803, Streptomyces coelicolor, Sphingonomas aromaticivorans, Schizosaccharomyces pombe (two different SSADH proteins), Saccharomyces cerevisiae, Zymomonas mobilis, Mycobacterium leprae, Ralstonia eutropha, Rattus norvegicus, Emericella nidulans, Escherichia coli, Deinococcus radiodurans (two different SSADH proteins), Clostridium kluyveri, Caenorhabditis elegans, Bacillus subtilis, and Arabidopsis thaliana. RESULTS AND DISCUSSION Mutations in patients and parents were detected using a combination of RT-PCR, genomic PCR, and sequencing. All mutations found with RT-PCR and sequencing were verified by genomic PCR and sequencing (Fig. 1; Table 1). The mutations identified were not detected in a total of 140 control chromosomes, suggesting that these mutations were not simply common polymorphisms. The patient from the first family has been described (1); patient IC. Sequencing the RT-PCR product suggested the patient was homozygous for a 803G ⬎ A mutation in exon 5, leading to a glycine to glutamate substitution at position 268 (G268E, numbering according to Chambliss et al. (8)), but sequencing genomic DNA showed that the patient was heterozygous for this mutation and that the mother was a carrier. Further analysis showed that the father and the patient were heterozygous for a 612G ⬎ A substitution in exon 4, leading to the introduction of a stop codon at position 204 (W204X). Analysis of genomic DNA
220
HOGEMA ET AL.
FIG. 1. Schematic diagram of the human SSADH gene showing mutations described in the current report. Solid boxes represent exons, while straight lines represent introns. Nonsense alleles are shown on the top, while missense alleles are depicted on the bottom. The 3⬘-untranslated region of the gene is depicted by the shaded box (gene structure is not drawn to scale).
from amniocytes and chorionic villi showed that the at-risk fetus carried both mutations. Whereas it is highly likely that the introduction of a stop codon will affect the function of the protein, the G268E substitution could be a polymorphism that is not pathogenic. Comparing human SSADH with other SSADH proteins and other human aldehyde dehydrogenases from the GenBank database showed that the glycine at position 268 is conserved in 15 out of 22 (putative) SSADH proteins in different species, and in 13 out of 16 human aldehyde dehydrogenases. Other amino acids at this position have aliphatic side chains (alanine in two cases, valine in three, isoleucine in one) or a hydroxyl group (threonine in three cases, serine in one). The strong conservation of the glycine, even in species as distantly related to humans as bacteria and yeast, suggests that a substitution to glutamate would affect protein
function. Determination of GHB in amniotic fluid and SSADH activity measurement in amniocytes and a chorionic villus sample suggested an affected pregnancy (Table 1). The family elected to terminate the pregnancy and analysis of fetal DNA showed the expected two mutations. For the second family (Table 1), genomic PCR and sequencing showed that the proband (5) was heterozygous for two mutations (612G ⬎ A and 1234C ⬎ T) that introduce stop codons in exons 4 and 8, respectively, at positions 204 and 412 (W204X and R412X). Further analysis showed that the mother was a carrier of the 612G ⬎ A mutation and the father carried the 1234C ⬎ T mutation. Father and fetus also shared an interesting polymorphism (a deletion/insertion of 1 bp approximately 75 bp upstream of exon 8 never encountered in other patient or control samples). DNA from the proband did
TABLE 1 Prenatal Diagnosis of SSADH Deficiency in Three Unrelated Families SSADH activity Family 1 2 3
Proband mutations
GHB AF
G268E W204X W204X R412X G409D G409D
4.01
Mutations in at-risk pregnancy
AC
CV
ND
0 (0.4) —
0.04 (1.1) 0.4
1.12
0.14
0.01
AC Both present — —
CV
Fetal DNA
Both Both present present Neither — present Heterozygote —
Prediction
Outcome
Affected
Terminated
Unaffected
Miscarriage
Unaffected carrier
Healthy newborn
Note. Control GHB levels in amniotic fluid (AF) 0.42–2.2 mol/L (n ⫽ 30). All enzyme activities in units of nmol/min/mg protein. Parenthetic values represent parallel controls of the same tissue type, when available. Additional abbreviations: AC, amniocytes; CV, chorionic villus; GHB, 4-hydroxybutyric acid. The CV sample for Family 3 was delayed 8 days in transit. Marker enzyme activity in this sample (propionyl-CoA carboxylase) was 0.24 (control values 2.2, 5.2 nmol/min/mg protein, n ⫽ 2), suggesting tissue compromise in transit. ND, not determined.
PRENATAL DIAGNOSIS OF SSADH DEFICIENCY
not show this polymorphism. For prenatal diagnosis, DNA was isolated from a chorionic villus sample taken at 9 weeks gestation. PCR and sequence analysis showed no 612G ⬎ A or 1234C ⬎ T mutations; the paternal insertion/deletion polymorphism upstream of exon 8 was present, however, representing a useful internal control verifying that fetus and proband inherited different chromosomes from their father. Determination of the SSADH activity in the chorionic villus sample also suggested an unaffected pregnancy (Table 1). In the third family presenting for prenatal diagnosis (Table 1; Fig. 1), RT-PCR and genomic PCR verified that the proband was homozygous for a 1226G ⬎ A mutation, leading to substitution of aspartic acid for glycine at position 409 (G409D) of the polypeptide sequence. Analysis of SSADH activity in biopsied chorionic villus tissue revealed deficiency, yet the marker enzyme activity was also quite low, suggesting tissue compromise in transit (Table 1). Due to these results, amniocentesis was pursued which revealed a normal level of GHB in amniotic fluid and demonstrable SSADH enzyme activity in amniocytes cultured from the spun amniotic fluid specimen (Table 1). Mutation analysis of the chorionic villus sample using genomic PCR revealed heterozygosity for the G409D mutation. Analysis of position 409 in 13 other aldehyde dehydrogenases from different species revealed conservation of this glycine, suggesting that this residue is critical for proper function of the protein. Our results from the current study verify the importance and utility of having DNA testing available for prenatal diagnosis of SSADH deficiency. This is especially important in those instances in which samples are transported over long distances, as there may be significant delays in transit time. The data also suggest that delays in transit may have an even more pronounced deleterious effect in instances in which the fetal genotype is heterozygous. In the latter case, there is the possibility that deleterious mutations may lead to aberrant protein folding or aggregation. DNA testing will become an increasingly important part of the testing capabilities for SSADH deficiency, especially as the number of affected probands continues to grow, and should enable accurate PND as early as 8 –9 weeks of gestation.
human SSADH gene, and Ms. Wendy Guerand, Danielle Schor, and Virginia Perreira for determination of GHB quantities in amniotic fluid specimens. We are indebted to Dr. Daniel Bluestone and Dr. Seymour Packman who provided clinical evaluation of the proband in Family 1, and Drs. A. Skartoutsou, K. Boudris, and O. Papandreou who provided clinical assessment and follow-up in Family 3.
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ACKNOWLEDGMENTS The authors are indebted to Dr. Patrizia Malaspina and Dr. Andrea Novelletto for supplying genomic sequence data on the
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