HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch–Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch–Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch–Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C→T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT_Sardinia. The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch-Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprt Montreal) by use of T-lymphocyte cultures

Summary The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (TC transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine₅₆. We have designated this new Lesch-Nyhan mutation hprt _Montreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency: analysis of HPRT mutations by direct sequencing and allele-specific amplification

Summary The Lesch-Nyhan syndrome is a severe X chromosome-linked human disease caused by a virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. A partial deficiency in the activity of this enzyme can result in gouty arthritis. To determine the genetic basis for reduction or loss of enzyme activity, we have amplified and sequenced the coding region of HPRT cDNA from four patients: one with LeschNyhan syndrome (HPRT_Perth) and three with partial deficiencies of HPRT activity, which have been designated HPRT_Urangan, HPRT_Swan and HPRT_Toowong. In all four patients, the only mutation identified was a single base substitution in exons 2 or 3 of the coding region, which in each case predicts a single amino acid substitution in the translated protein. Each base change was confirmed by allele-specific amplification of the patient's genomic DNA. It is interesting to note that the mutation found for HPRT_Perth is identical to that reported for HPRT_Flint. It appears that the two mutations are de novo events.     

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.     

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis. Received: 21 April 1998 / Accepted: 16 July 1998     

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland)

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.     

Molecular Analysis of Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Deficiencies: Novel Mutations and the Spectrum of Japanese Mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

DNA restriction fragment length polymorphisms in differential diagnosis of genetic disease: application in neuromuscular diseases

Summary Three mutations causing alpha-1-antitrypsin defiiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) null_cardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In Pi M_malton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.     

An asymptomatic germline missense base substitution in the hypoxanthine phosphoribosyltransferase (HPRT) gene that reduces the amount of enzyme in humans

A 40-year-old normouricemic (5.5 mg/dl) male showed 46% hemolysate and 37% lymphoblast hypoxanthine phosphoribosyltransferase (HPRT) activities but was otherwise completely free of symptoms. His genomic DNA and cDNA had a missense base substitution (CAT-to-CGT in codon 60) leading to the amino-acid substitution His-to-Arg. Western blot analysis revealed that the amount of HPRT protein in lymphoblasts from this individual was 25%–50% of normal cells, suggesting that the decrease in the amount of enzyme protein was responsible for the partial deficiency. This provides the first clear evidence that a genomic missense mutation at the HPRT locus leads to a decrease in the amount of the enzyme protein but that otherwise it has no evident adverse effects in the hemizygote (asymptomatic mutation). Received: 15 May 1996 / Revised: 22 August 1996     

Clinical and biochemical manifestations and molecular characterization of the mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch-Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

Two novel mutations of SURF1 in Leigh syndrome with cytochrome c oxidase deficiency

Cytochrome c oxidase (COX) deficiency is the most common cause of Leigh syndrome (LS). COX consists of ten nuclear-encoded and three mtDNA-encoded structural subunits. Although the nucleotide sequences of all 13 genes are known, no mutation was found in nuclear-encoded subunit genes of COX-deficiency patients. Zhu et al. (1998) and Tiranti et al. (1998) found nine mutations in the surfeit 1 (SURF1) gene in LS families with COX deficiency. The mouse surfeit gene cluster consists of six closely spaced housekeeping genes unrelated by sequence homology. Except for the Surf3 gene, the function is still not known. The juxtaposition of at least five of the surfeit genes is conserved between birds and mammals. We identified two novel mutations of SURF1 in a Japanese LS patient with COX deficiency using direct sequencing analysis. Firstly, a 2-bp deletion at nucleotide position 790 (790delAG) in exon 8 was found, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 264 to the premature stop codon at 290. Secondly, we found a T-to-G transversion at nucleotide 820, resulting in the substitution of tyrosine by aspartic acid at codon 274 (Y274D). We also studied the parents' genes, and found that the Y274D mutation was in his father and the 790delAG mutation was in his mother heterozygously. Therefore, we concluded that the patient was a compound heterozygote with these mutations. These are the first pathogenetic SURF1 mutations identified in a Japanese family.     

A 13 base pair deletion in exon 1 of HPRTIllinois forms a functional GUG initiation codon

More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT₁₁₅₁, results in Lesch-Nyhan syndrome (LNS), and the other, HPRT_Illinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRT_Illinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRT_Illinois. These data provide support for the hypothesis that patient RT, or variant HPRT_Illinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA.

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular basis of beta (0)-thalassemia/HbE disease in Thailand

The molecular basis of beta(0)-thalassemia/HbE disease in 30 Thai patients was investigated using DNA amplification and dot-blot hybridization with a number of allele specific oligonucleotide probes. The mutations identified were 17 cases of 4 base-pair deletion at codons 41-42, 4 cases of amber mutation at codon 17, and one case each of an ochre mutation at codon 35, a single base substitution at position 5 of IVS-1, and a single base substitution at position 654 of IVS-2.     

Molecular analysis of hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: novel mutations and the spectrum of Japanese mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

A missense mutation (Trp86----Arg) in exon 3 of the lipoprotein lipase gene: a cause of familial chylomicronemia.

We have investigated a patient of English ancestry with familial chylomicronemia caused by lipoprotein lipase (LPL) deficiency. DNA sequence analysis of all exons and intron-exon boundaries of the LPL gene identified two single-base mutations, a T----C transition for codon 86 (TGG) at nucleotide 511, resulting in a Trp86----Arg substitution, and a C----T transition at nucleotide 571, involving the codon CAG encoding Gln106 and producing Gln106----Stop, a mutation described by Emi et al. The functional significance of the two mutations was confirmed by in vitro expression and enzyme activity assays of the mutant LPL. Linkage analysis established that the patient is a compound heterozygote for the two mutations. The Trp86----Arg mutation in exon 3 is the first natural mutation identified outside exons 4-6, which encompass the catalytic triad residues.     

Codon substitution mutations at two positions in the L polymerase protein of human parainfluenza virus type 1 yield viruses with a spectrum of attenuation in vivo and increased phenotypic stability in vitro

The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.