Letter to the Editor

Novel Missense Mutation (Y231C) in a TurkishPatient With Canavan Disease

To the Editor:

Canavan disease (CD) is an autosomal recessive neu-rodegenerative disorder affecting white matter andleading to spongy degeneration. Macroencephaly ischaracteristic in children with this severe leukodystro-phy. The disease is caused by the deficiency of aspar-toacylase (ASPA) and increased levels of N-acetyl-aspartic acid (NAA) in brain [Matalon et al., 1988].Canavan disease is prevalent among Ashkenazi Jewswith two predominant mutations C693A and A854Cfound in 98% of the Jewish cases [Kaul et al., 1993,1994; Matalon et al., 1993; Matalon, 1997]. The carrierfrequency found in a sample of over 4,000 Ashkenazimwas 1/37 [Matalon, 1997]. Canavan disease occurs lessfrequently among non-Jewish populations, and the car-rier frequency has not been established. The mutationsamong the non-Jewish patients are different and morediverse; the most common mutation is C914A observedin 35.7% [Kaul et al., 1994]. Shaag et al. [1995] alsofound the C914A is the most prevalent mutationamong non-Jewish European individuals. Other muta-tions in non-Jewish patients can randomly reside inexons 1–6, however a higher prevalence of mutationshas been detected in exons 3–6 [Kaul et al., 1994, 1995,1996; Shaag et al., 1995; Kobayashi et al., 1998; Mata-lon and Michals-Matalon, 1998]. We report here anovel A692G homozygous missense mutation, whichresults in a Tyr231Cys (Y231C) amino acid changewithin exon 5 of ASPA gene.

We report on a girl born to first-cousin Turkish par-ents from an otherwise healthy family. The parentsnoted poor head control at age 4 months and no furtherdevelopmental progress. The baby was first seen at age8 months, weight was 8.9 kg (50 centile), length 71 cm(50 centile), and head circumference (OFC) 45 cm (> 75centile). The baby was hypotonic, had no head control,could not sit, roll over, or grasp, and had no social

smile. Laboratory tests showed very high levels of N-acetylaspartate in urine, thus confirming the diagnosesof CD. Cranial sonography showed frontal atrophy.Magnetic resonance imaging of the brain showed pro-nounced brain atrophy with slightly delayed myelina-tion and a demyelinating process most pronounced oc-cipitally and in the cerebellum.

Blood samples were used to prepare DNA from leu-kocytes using DNA extraction kit (QIAamp DNA BloodKit, Qiagen Inc, Hilden, Germany). Gene specific ASPAprimers with minor modifications were used to amplifyexons 1–6 and their boundary sequences as we re-ported earlier [Kaul, 1996]. The base excision sequencescanning (BESS) mutation characterization system(BESS T-Scan Kit, Epicentre Technologies, Madison,WI) was used for screening mutations following themanufacturer’s instructions. The BESS T-Scan posi-tive polymerase chain reaction (PCR) products under-went direct sequencing, using the PCR primers as se-quencing primers. The sequencing reactions of the PCRproducts were performed using the Thermo Sequenaseradiolabeled terminator cycle sequencing system (Am-ersham-Pharmacia Biotech, Inc., Piscataway, NJ) with33P-terminators.

The BESS mutation screening procedure docu-mented absence of a band on the antisense strand ofthe exon 5-specific PCR fragment from the DNA of thepatient. This finding is characteristic of disappearanceof thymine (Fig. 1). The direct sequencing of the senseDNA strand of exon 5-specific PCR fragment supportedthe BESS result and identified a homozygous A to Gmutation (Fig. 2) at base position 692 of ASPA cDNA[Kaul et al., 1993]. This novel mutation resulted in analteration of codon 231 and created an amino acidchange: tyrosine to cysteine. No other sequence alter-ation of ASPA gene was found. This missense mutationoccurred within the highly conserved region of theASPA gene [Kaul et al., 1993], which also supports thatthis novel mutation was responsible for the ASPA en-zyme deficiency and Canavan disease [Cotton andScriver, 1998]. Interestingly this mutation occurredwithin codon 231, which is the site of one of the com-mon mutations (nonsense mutation C693A) amongAshkenazim [Matalon et al., 1993; Kaul et al., 1994;Matalon, 1997]. A polymorphic mutation has been alsodescribed at codon 231 (C693T) among the Askhen-

Contract grant sponsor: the John Sealy Foundation Endow-ment Grant; Contract grant sponsor: the National Institutes ofHealth; Contract grant number: RO1 NS38562-01.

*Correspondence to: Professor Reuben Matalon, M.D., Ph.D.,Department of Pediatrics, Genetics Division, Children’s Hospital,Room 3.350, University of Texas Medical Branch, Galveston, TX77555-0359. E-mail: rmatalon@utmb.edu

Received 22 April 1999; Accepted 22 July 1999

American Journal of Medical Genetics 87:273–275 (1999)

© 1999 Wiley-Liss, Inc.

azim, which can interfere with the DNA-based carriertesting using allele-specific oligonucleotide hybridiza-tion [Alford et al., 1998]. However, this polymorphicmutation does not change the amino acid compositionof ASPA at codon 231.

In conclusion, this novel mutation A692G (Y231C) ofASPA gene can lead to ASPA protein modification withdeficient enzyme function. The molecular description ofthis mutation also establishes a molecular basis to de-termine whether this mutation occurs predominantlyamong individuals with Turkish descent.

ACKNOWLEDGMENTS

This study was supported in part by grants from theJohn Sealy Foundation Endowment Grant and NIH-RO1 NS38562-01.

REFERENCES

Alford RL, DeMarchi JM, Richards CS. 1998. Frequency of a DNA poly-morphism at position Y231 in the aspartoacylase gene and its impacton DNA based carrier testing for Canavan disease in the AshkenaziJewish population. Hum Mutat 1:S161–162.

Cotton RGH, Scriver CR. 1998. Proof of “disease causing” mutations. HumMutat 12:1–3.

Fig. 1. BESS T-Scan mutation detection analysis of exon V-specificPCR fragment of ASPA gene from patient (lane 1) and from normal control(lane 2). Briefly, the BESS T-Scan analysis detects mutations by incorpo-rating dUTP into a PCR product substituting dTTP. Subsequent enzymaticcleavage produces a DNA ladder similar to a “T” sequencing pattern. Amissing T nucleotide was detected and located (lane 1) when the antisensePCR primer was labeled with radioactive isotope. This change suggestedthat the sense strand has a deleted “A” base (at position 692) or a basesubstitution occurred.

Fig. 2. Sequence analysis of exon V-specific PCR fragment of ASPAgene from patient (top) and from normal control (bottom). Nucleotidesequence of the sense strand shows homozygous 692 A→G mutation. Thismutation results in Tyr231→Cys amino acid change. Numbers under basesare given by the automated sequencer and do not correspond to their po-sition in the ASPA cDNA or gene.

274 Rady et al.

Kaul R, Gao GP, Aloya M, Balamurugan K, Petrosky A, Michals K, Mata-lon R. 1994. Canavan disease: mutations among Jewish and non-Jewish patients. Am J Hum Genet 55:34–41.

Kaul R, Gao GP, Balamurugan K, Matalon R. 1993. Cloning of the humanaspartoacylase cDNA and a common missense mutation in Canavandisease. Nat Genet 5:118–123.

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Matalon R, Michals K, Sebesta D, Deanching M, Gashkoff P, Casanova J.1988. Aspartoacylase deficiency and N-acetylaspartic aciduria in pa-tients with Canavan disease. Am J Med Genet 29:463–471.

Matalon R, Michals-Matalon K. 1998. Molecular basis of canavan disease.Eur J Paediatr Neurol 2:69–76.

Shaag A, Anikster Y, Christensen E, Glustein JZ, Fois A, Michelakakis H,Nigro F, Pronicka E, Ribes A, Zabot MT, Elpeleg ON. 1995. The mo-lecular basis of Canavan (aspartoacylase deficiency) disease in Euro-pean non-Jewish patients. Am J Hum Genet 57:572–580.

Peter L. RadyTrini VargasStephen K. TyringReuben Matalon*Department of PediatricsDivision of GeneticsUniversity of Texas Medical Branch,Galveston, Texas

Ulrich LangenbeckInstitute for HumangeneticsUniversity of FrankfurtFrankfurt, Germany

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