Print version ISSN 0004-282X
Arq. Neuro-Psiquiatr. vol.57 n.4 São Paulo Dec. 1999
PATRICK O'NEILL*, LUCY TROMBLEY*, MARY GUNDEL*, TIMOTHY HUNTER**, JANICE A. NICKLAS***, MARA LUCIA S. FERREIRA****, MARIA JULIA BUGALLO****, ANTÔNIO CARLOS FARIAS****, ALFREDO LOHR****, MERI DIAMANTOPOULOS****, SALMO RASKIN*****
ABSTRACT - The mutation in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene has been determined in two brothers affected with Lesch-Nyhan syndrome. Female members of the family who are at risk for being heterozygous carriers of the HPRT mutation were also studied to determine whether they carry the mutation. DNA sequencing revealed that the boys' mother is heterozygous for the mutation in her somatic cells, but that three maternal aunts are not heterozygous. Such carrier information is important for the future pregnancy plans of at-risk females. The mutation, an A®T transversion at cDNA base 590 (590A®T), results in an amino acid change of glutamic acid to valine at codon 197, and has not been reported previously in a Lesch-Nyhan syndrome male. This mutation is designated HPRTBrasil.
KEY WORDS: Lesch-Nyhan syndrome, DNA, HPRT.
Identificação de uma nova mutação (HPRTBRASIL) em uma família brasileira com a síndrome de Lesch-Nyhan e análise de mulheres potencialmente heterozigotas
RESUMO - Uma mutação no gene hipoxantina-guanina fosforibosiltransferase (HPRT) foi determinada em dois irmãos afetados pela síndrome de Lesch-Nyhan. O sequenciamento do cDNA do gene HPRT das mulheres desta família, potencialmente portadoras heterozigotas da mutação HPRT, que é uma transissão de A®T na base 590 do cDNA (590A®T), revelou que a mãe dos meninos é heterozigota para esta mutação, mas que três tias maternas não são heterozigotas. Este tipo de informação é importante no planejamento de gestações futuras, nas mulheres em risco. Como esta mutação ainda não havia sido relatada em indivíduos do sexo masculino na síndrome de Lesch-Nyhan, foi, portanto, denominada HPRTBrazil.
PALAVRAS-CHAVE: síndrome de Lesch-Nyhan, DNA, HPRT.
Lesch-Nyhan syndrome, an inborn error of purine metabolism, is caused by loss of the enzyme hypoxanthine-guanine phosphoribosyltransferase due to mutations in the Xchromosome gene HPRT1,2. Because this is an X-chromosome linked disease, only males are generally affected and females can be heterozygous carriers. It has been estimated that one-third of Lesch-Nyhan syndrome males would represent new mutations3. Features of this syndrome are spastic cerebral palsy, choreathetosis, uric acid urinary stones and neurological disfunction including self-destructive biting of fingers and toes.
A method for the diagnosis and the analysis of the HPRT mutations for Lesch-Nyhan syndrome using peripheral blood Tlymphocytes has been developed4. This method is based on the ability of HPRT-deficient T-lymphocytes to proliferate and form colonies in the presence of the cytotoxic purine analog 6-thioguanine4. Applying this T-lymphocyte cloning assay to Lesch-Nyhan syndrome families allows both diagnosis of the disease and analysis of the HPRT mutations responsible for the disease, as well as determination of the carrier status of at-risk females5,6. Here, we report the study of a Lesch-Nyhan syndrome family in which a new HPRT mutation was found, and the determination of the carrier status of at-risk females in the family. This new mutation is termed HPRTBrasil, after the country of origin of the family.
We analyzed two Brazilian caucasoid brothers, aged twelve and fourteen years old, and their mother. The first clinical manifestations on the boys became apparent during the third month of life in both of them, with psychomotor retardation and generalized muscular hypotonia. The younger brother started to present self mutilation (biting of lips and fingers) when he was three years old. The older one, however, presented this manifestation only at fourteen years old, usually biting his hands.
Neurological examination showed spasticity, chorioathetosis and signs of self mutilation. The laboratory tests showed hyperuricemia, urinary uric acid increased, and deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) that belongs to purine metabolism.
T-lymphocyte isolation and culture
Peripheral blood samples were collected in vacutainer tubes containing sodium heparin and shipped by overnight carrier to the University of Vermont. (Alternatively, for genomic DNA analysis, DNA was extracted from peripheral blood samples collected in EDTA and shipped to Vermont). The samples were collected after informed consent was obtained. The mononuclear cell fraction was isolated by centrifugation over Histopaque and the cells plated for the T-lymphocyte cloning assay, as described previously4. Briefly, cells are plated in 96-well microtiter plates in medium RPMI 1640 containing 20% medium HL-1, 5% defined calf bovine serum (Hyclone, Logan, Utah, USA), 10% T-cell growth medium containing interleukin-1 and interleukin-2, 0.25 mg/ml phytohemagglutinin, and 1 x 104 irradiated (90 Gy) HPRT mutant human lymphoblastoid "feeder" cells in the absence or presence of 10 mM 6-thioguanine (TG). Cells from the affected males were plated at 1, 2, 5, 10 or 100 cells/well in the absence or presence of TG. Cells from their mother were plated at 2, 5, 10, 102 and 103 cells/well in the absence of TG and at 102, 103 and 104 cells/well in the presence of TG. Cells from the males were also plated at 1 x 105 cells/ml in 2 ml wells in the same medium containing no addition, hypoxanthine and thymidine (HT), hypoxanthine, aminospterin and thymidine (HAT) or TG. Wild type HPRT+ cells grow in the absence of TG, in the presence of HT or of HAT, but not in the presence of TG. Mutant HPRT - cells grow in the absence of TG and in the presence of either TG or HT, but not in the presence of HAT. Thus, growth in the presence of TG, but not in HAT, is a rapid screen for HPRT mutant cells. HPRT mutant cells from a Lesch-Nyhan syndrome male should clone equally well in the absence and presence of TG and yield a mutant frequency of 1.0. Cells from a non-carrier female will contain a frequency of HPRT mutant cells of 120 x 106 4. Cells from a HPRT heterozygous female will contain HPRT mutant cells at a frequency of 0.15.0%. The frequency in carrier females is less than the 50% expected from random Xchromosome inactivation because of negative selection against mutant cells during hematopoietic stem cell proliferation7. In previous studies, carrier females showed HPRT mutant frequencies in the range of 15%5,6.
HPRT mutation analysis
The HPRT gene contains approximately 42,000 base pairs in genomic DNA, has nine exons and a mRNA of approximately 1,800 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination UAA). The entire gene has been sequenced and primers developed for polymerase chain reaction (PCR) amplification of the nine exons in genomic DNA and the mRNA8,9. Recent review articles have summarized the known HPRT mutations responsible for Lesch-Nyhan syndrome. Approximately 80 different mutations have been reported12,13. This wealth of information allows for rapid analysis of the HPRT gene by cDNA sequencing for point mutations and genomic PCR analysis for deletion mutations.
Cultured Tlymphocytes from the affected males and their mother were used for DNA analysis. Samples of 1 x 104 cells were centrifuged in 0.5 ml microfuge tubes, flash frozen in liquid nitrogen and stored at 70 C. Cell lysates were prepared and first strand cDNA synthesized by reverse transcriptase as described by Yang et al8. Two rounds of PCR amplification were performed with HPRT specific oligonucleotide primers as described previously6. The first round employed 30 cycles with primers 3 and 4b (HPRT base 60 to 41, and 769 to 746, respectively), and the second round 30 cycles with primers B and 4 (HPRT base 36 to 17, and 702 to 721, respectively). The cDNA products were sequenced directly by use of a Taq Dyedeoxy Terminator Cycle Sequencing Kit (Perkin Elmer - ABI) and an automated ABI sequencer Model 373A with HPRT primers B and A (HPRT bases 36 to 17 and 701 to 682, respectively). This is essentially the method originally described by Gibbs et al9.
For genomic DNA sequencing, the primers developed to amplify HPRT exons 7 and 8 were used10. These primers are numbered according to the genomic HPRT gene sequence11. The primers are 38667 to 38691 (sense) and 40199 to 40176 (antisense). The DNA was sequenced with the antisense primer. Exon 8 is genomic bases 40033 to 40109.
The HPRT mutant frequency was determined with cells from the two affected males and their mother. Both males showed similar cell cloning in the absence and presence of TG, consistent with the Lesch-Nyhan syndrome diagnosis. Their mother showed an HPRT mutant frequency of 3.9%, consistent with her being a heterozygous carrier (Table 1). Sequencing of cDNA from males revealed a single base substitution at cDNA base 590 in exon 8. This is the only difference from the published wild type HPRT coding sequence9. This 590A®T transversion changes codon 197 from GAA to GTA, resulting in the amino acid change 197glu®val. Sequencing of cDNA from the mother's cells grown in the absence of TG showed the wild type A at cDNA base 590. Sequencing of cDNA from the mother's TG resistant mutant cells showed the 590A®T transversion mutation. To absolutely confirm the heterozygous nature of the mother and to test the feasibility of direct DNA analysis for testing at risk females in the family, the exon 8 region of genomic DNA was sequenced. The DNA sequence from the males encompassing genomic bases 40151 to 39801 showed only a single change from the normal sequence11. This was an A to T change at base 40090. This 40090A®T mutation is the base designated cDNA base 590. The mother's exon 8 sequence showed both an A and a T at genomic base 40090, confirming that she is heterozygous for the mutation in both her somatic and germinal cells.
An important aspect of Lesch-Nyhan syndrome mutation analysis is the ability to apply these methods to heterozygote detection in order to determine the carrier status of at-risk females in the family. DNA from three maternal aunts of the affected boys was used to determine the sequence of the genomic region containing exon 8. All three showed only an A at 40090, indicating that none is a carrier.
This 590A®T mutation in exon 8 results in a change of codon 197 from GAA to GTA, and an amino acid change of 197 glu®val. This single base substitution reduces the HPRT enzyme activity to very low levels and allows the cells to grow in the presence of 6thioguanine, normally a cytotoxic purine analogue. A mutation at base 590 in codon 197 has not been reported previously in a LeschNyhan syndrome patient12,13. As shown in Table 2, mutations in this region of the HPRT gene (codons 191-202) have been reported both in Lesch-Nyhan syndrome and partial HPRT deficiency (gout). In addition, many mutations have been found in this region of the gene in somatic cell HPRT mutations which were isolated as TG resistant (TGr) mutants. One of these includes the same 197 glu®val change observed in HPRT Brasil14.
Lastly, the 590 A®T mutation creates a TA dinucleotide repeat rich region in exon 8 (TATAATGAATA®TATAATGTATA ), suggesting that the mutation might have been induced by slippage during DNA replication in this region of repeated sequences.
Data generated from this type of studies may have significant implications for diagnosis and prognosis in Lesch-Nyhan syndrome patients and their relatives. The aim of future therapies is to slow or stop the progression of Lesch-Nyhan syndrome in affected persons. Knowledge of the molecular basis of the disease in the population should help families at risk and improve genetic counseling.
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*Genetics Laboratory, University of Vermont, Burlington, VT, USA; **DNA Analysis Facility, University of Vermont, Burlington, VT, USA, ***Molecular Diagnostics Laboratory University of Vermont, Burlington, VT, USA, ****Hospital Infantil Pequeno Príncipe, Curitiba, PR, Brazil, *****Hospital Infantil Pequeno Príncipe e Laboratorio Genetika, Curitiba, PR, Brazil. Aceite: 30-agosto-1999.
Dr. Salmo Raskin - Laboratorio Genetika - Alameda Augusto Stellfeld, 1516 - 80730-150 Curitiba PR - Brasil. Tel: 55-41 2326838 Fax: 55-41 2325206. E-mail: firstname.lastname@example.org