Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in thirteen Spanish families

We have determined the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT; HPRT1) deficiency in eight Lesch-Nyhan patients and in five partially HPRT deficient patients with mild to severe neurologic symptoms. Eight of these thirteen mutations have not been previously described. HPRT Zaragoza II (a GG insertion in exon 2), HPRT Murcia (an AG deletion in exon 4), HPRT Asturias (a A deletion in exon 4) and HPRT Cartagena (a A insertion in exon 6) cause a frame-shift resulting in a premature stop codon. HPRT Sevilla is a splice-site mutation resulting in exon 8 skipping in the HPRT mRNA. HPRT Huelva, Madrid II and Zaragoza I are point mutations that result in single amino-acid changes in the mutated HPRT protein (118G-->A, G40R; 143G-->A, R 48 H; 397G-->A, V133 M, respectively). Three mutations have been previously described in unrelated families, and two mutations have been already published. All mutations that resulted in truncated proteins corresponded to patients with the Lesch-Nyhan phenotype. Characterization of the HPRT mutation allowed us to make carrier detection in 33 women and prenatal diagnosis in two fetuses. Hum Mutat 15:383, 2000.     

Molecular analysis of hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in five Korean families with Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome is caused by the complete deficiency of hypoxanthine guanine phosphoribosyl-transferase (HPRT). By the analysis of genomic DNA and mRNA using the polymerase chain reaction (PCR) technique coupled with direct sequencing, five independent mutations in HPRT genes have been identified in Korean Lesch-Nyhan families. Two novel mutations and three previously reported mutations have been found in five independent families. Heterozygous carriers were detected in all the families, and prenatal diagnosis was carried out in two families.     

Normal HPRT coding region in complete and partial HPRT deficiency

Lesch-Nyhan syndrome is an X-linked recessive inborn error of metabolism due to a virtually complete lack of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity (OMIM 300322). Partial deficiency of HPRT (OMIM 300323) is characterized by the effects of excess uric acid synthesis and a continuum spectrum of neurological manifestations, without the manifestations of full-blown Lesch-Nyhan syndrome. Both diseases have been associated with mutations in the HPRT gene. These mutations are heterogeneous and disperse throughout the entire HPRT gene. In 2005 Dawson et al. described, for the first time, an individual with gout in whom HPRT deficiency appeared to be due to a defect in gene regulation. In the present study we present four patients with partial HPRT deficiency and one patient with Lesch-Nyhan syndrome who showed a normal HPRT coding sequence and markedly decreased HPRT mRNA expression. This is the first report of a patient with Lesch-Nyhan syndrome due to a defect in HPRT gene expression regulation.     

Structural and functional analysis of mutations at the human hypoxanthine phosphoribosyl transferase (HPRT1) locus

Hypoxanthine phosphoribosyl transferase (HPRT, also known as HGPRT) is an often-used genetic marker in eukaryotic cells. The gene is conserved from bacteria to human, with retained catalytic activity, although substrate specificity may have changed, and the enzyme is essential in malaria-causing protozoans. Inherited mutations in the human HPRT1 gene result in three different phenotypes: Lesch-Nyhan syndrome (LNS or LND), LND variants, and HPRT-related hyperuricemia (HRH). In cultured cells, loss of HPRT activity gives rise to 6-thioguanine (6-TG) resistance. In general, cells from LND patients are also 6-TG resistant, whereas cells from HRH patients are not, with some interesting exceptions. Using modeling methods, we have studied the correlation between the mutable and nonmutated amino acid residues on one hand, and sequence conservation and predicted phenotypic effects on the other hand. Our results demonstrate that most of the mutations are explainable by the predicted effect on protein structure and function. They are also consistent with sequence conservation. Moreover, the mutational profiles of TG-resistant cells and LND overlap to a great extent, while most of the mutations in HRH are unique to that condition. We have also noticed a strong correlation between mutations in the tetramer interfaces and observed phenotypes, suggesting a functional role for a tetramer transition during catalysis.     

Molecular genetic study of a Japanese family with Lesch-Nyhan syndrome: A point mutation at the consensus region of RNA splicing (HPRTKEIO)

Summary Complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) causes Lesch-Nyhan syndrome. A single nucleotide substitution of G to T at the 3-end of intron 3 in the splicing consensus region has been identified in one allele of the HPRT gene from a mother predicted to be a heterozygous Lesch-Nyhan carrier. Utilizing aBfaI restriction site which was lost in the mutation as an indicator, family study showed that the mother and her only daughter were heterozygotes but the mother's sister did not have the mutant allele. The mutation generated splicing error and resulted in two types of abnormal mRNA. The major altered mRNA, named Type I, skipped the exon 4 and is predicted to produce a protein deleted of 22 amino acid residues. The other, Type II, having a 9-bp deletion at the 5-end of exon 4, can result in a protein lacking 3 amino acids, from codon 107 to 109.     

Different somatic and germline HPRT1 mutations promote use of a common,cryptic intron 1 splice site

Aberrant hypoxanthine phosphoribosyltransferase (HUGO‐approved gene symbol HPRT1; MIM# 308000) mRNA splicing, promoted by splice site mutation or loss, is a common mechanism for loss of the purine salvage enzyme HPRT1 from human cells. We report here two in vivo somatic HPRT1 mutations in human kidney tubular epithelial cells that disrupt HPRT1 intron 1 splicing and lead to the inclusion of intron 1 sequence. We propose an explanation for the use of a common, cryptic intron 1 splice donor site by these two mutations, and by 14 additional human HPRT1 mutations that lead to aberrant splicing with the incorporation of intron 1 sequence into mRNA. Hum Mutat 13:504–505, 1999. © 1999 Wiley‐Liss, Inc.     

Three novel deletions in the alanine:glyoxylate aminotransferase gene of three patients with type 1 hyperoxaluria.

We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.     

Lesch-Nyhan syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome (LNS) or Lesch-Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA.     

A novel missense mutation, c.584A > C (Y195S), in two unrelated Argentine patients with hypoxanthine-guanine phosphoribosyl-transferase deficiency, neurological variant

The hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is an inborn error of purine metabolism, responsible for classic Lesch-Nyhan disease and its neurological and hyperuricemic variants. We report a novel mutation in the HPRT gene, c.584A > C (Y195S), in two unrelated Argentine patients affected with the neurological variant with no HPRT activity in lysed erythrocytes. Using PCR plus DNA sequencing and/or restriction enzyme digestion we were able to confirm the diagnosis and identify new cases and potential carriers.     

Three novel small deletion mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia.

The low-density lipoprotein (LDL) receptor gene from 80 unrelated Korean patients with familial hypercholesterolemia (FH) was analyzed to screen for small structural rearrangements that could not be detected by Southern blot hybridization. Three different small deletions were detected in exon 11 of 3 FH patients and were characterized by DNA sequence analysis. Of them two mutations are in-frame 36-bp (FH 2) and 9-bp (FH 34) deletions that result in the loss of twelve amino acids (from Met510 to Ile521) and three amino acids (Thr513, Asp514 and Trp515), respectively. Both mutations are located in the third of the five YWTD motifs of the LDL receptor gene. The third mutation (FH 400) is a 2-bp deletion that shifts the translational reading frame and results in a prematurely terminated receptor protein. The generation of a 36-bp deletion can be explained by the formation of a hairpin-loop structure mediated by inverted repeat sequences. On the other hand, the mechanism responsible for the 9- and the 2-bp deletions is probably strand-slippage mispairing mediated by short direct repeats. All of these three deletions are novel mutations. Each of the three deletions was detected only in a single pedigree out of 80 FH families analyzed.     

Nine novel mutations in the protoporphyrinogen oxidase gene in Swedish families with variegate porphyria

Variegate porphyria (VP) is an autosomal-dominant disorder that is caused by inheritance of a partial deficiency of the enzyme protoporphyrinogen oxidase (EC 1.3.3.4). It is characterized by cutaneous photosensitivity and/or various neurological manifestations. Protoporphyrinogen oxidase catalyses the penultimate step of haem biosynthesis, and mutations in the PPOX gene have been coupled to VP. In the present study, sequencing analysis revealed 10 different mutations in the PPOX gene in 14 out of 17 apparently unrelated Swedish VP families. Six of the identified mutations, 3G > A (exon 2), 454C > T (exon 5), 472G > C (exon 6), 614C > T (exon 6), 988G > C (exon 10) and IVS12 + 2T > G (intron 12), are single nucleotide substitutions, while 604delC (exon 6), 916-17delCT (exon 9) and 1330-31delCT (exon 13) are small deletions, and IVS12 + 2-3insT (intron 12) is a small insertion. Only one of these 10 mutations has been reported previously. Three of the mutations were each identified in two or more families, while the remaining mutations were specific for an individual family. In addition to the 10 mutations, one previously unreported single nucleotide polymorphism was identified. Mutation analysis of family members revealed two adults and four children who were silent carriers of the VP trait. Genetic analysis can now be added to the conventional biochemical analyses and used in investigation of putative carriers of a VP trait in these families.     

Spectrum of CLN6 mutations in variant late infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative diseases of childhood. CLN6, the gene mutated in variant late infantile NCL (vLINCL), was recently cloned. We report the identification of eight further mutations in CLN6 making a total of 18 reported mutations. These mutations include missense, nonsense, small deletions or insertions, and two splice-site mutations. Ten mutations affect single amino acids, all of which are conserved across vertebrate species. Minor differences in the pattern of disease symptom evolution can be identified. One patient with a more protracted disease progression was a compound heterozygote for a missense mutation and an unidentified mutation. Fifteen CLN6 mutations occur in one or two families only, and families from the same country do not all share the same mutation. Unlike NCLs caused by mutations in CLN1, CLN3, CLN5, and CLN8, there is no major founder mutation in CLN6. However, one mutation (E72X) is significantly more common in patients from Costa Rica than two other mutations present in that same population. In addition, a 1-bp insertion (c.316insC) is associated with families from Pakistan and I154del may be common in Portugal. A group of Roma Gypsy families from the Czech Republic share two disease-associated haplotypes, one of which is also present in a Pakistani family, consistent with the proposed migration of the Roma from the Indian subcontinent 1,000 years ago. All mutations are recorded in the NCL Mutation Database together with their country of origin for use in the development of rapid screening assays to confirm diagnosis and to facilitate carrier testing appropriate to a population.     

Identification of three novel mutations of the palmitoyl-protein thioesterase-1 (PPT1) gene in children with neuronal ceroid-lipofuscinosis

Eight unrelated children with progressive neurological deterioration and granular osmiophilic deposits (GROD) due to an underlying palmitoyl-protein thioesterase deficiency were analyzed for mutations in the PPT1 gene. Three novel mutations (G118D, Q291X and F84del) were identified. The novel Q291X mutation was observed in an African-American child. The G118D and Q291X mutations occurred in infantile-onset subjects. These two mutations would be predicted to have severe effects on enzyme activity. The novel F84del mutation involves an invariant phenylalanine residue. A missense mutation, Q177E, occurred in three subjects from two families with late-infantile NCL, confirming an association of the Q177E mutation with a late-infantile phenotype. Other previously described mutations were R151X (5/16 alleles), T75P (3/16 alleles), R164X (1/16 alleles), and V181M (1/16 alleles). The current study expands the spectrum of mutations in PPT1 deficiency and further confirms the broad range of age of onset of symptoms resulting from an enzyme deficiency previously associated only with infantile NCL.     

Ethyl methane sulfonate- and bleomycin-generated deletion mutations atHPRT locus in xeroderma pigmentosum complementation group D fibroblasts

Immortalized fibroblasts from a male patient with xeroderma pigmentosum from complementation group D (XP-D) were treated with either ethyl methane sulfonate (EMS) or bleomycin (BLM) to obtain mutations in hypoxanthine phosphoribosyltransferase (HPRT) activity. The aneuploid parental cell line, MH3-XPD, was found to have a single copy of the HPRT gene, indicating that this cell line remained physically hemizygous for this locus during the transformation process. Subcloning of 6-thioguanine-resistant (6TG^r) isolates resulted in clones without detectable HPRT activity. Continued maintenance in elevated concentrations of 6TG (30–60 M) produced cell populations with negligible growth in counterselection medium. No HPRT-deficient clones arose from unmutagenized cell cultures. Molecular analysis of the HPRT mutations in five clones with undetectable HPRT activity showed that four had large deletions. Two bleomycin-generated isolates were both found to have an approximately 28-kb intragenic deletion beginning within the first intron near exon 1 and ending within the fourth intron near exon 4. Messenger RNA from these clones was truncated by approximately 370 nucleotides. Our findings indicate that these two clones originated from the same mutational event within a founder cell. The three EMS-induced mutants fell into two classes: a putative point mutation or small deletion and two complete gene deletions.     

Guanine nucleotide depletion induces differentiation and aberrant neurite outgrowth in human dopaminergic neuroblastoma lines: a model for basal ganglia dysfunction in Lesch-Nyhan disease

Lesch-Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch-Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.     

Identification of 13 novel mutations including a retrotransposal insertion in SLC25A13 gene and frequency of 30 mutations found in patients with citrin deficiency

Deficiency of citrin, liver-type mitochondrial aspartate-glutamate carrier, is an autosomal recessive disorder caused by mutations of the SLC25A13 gene on chromosome 7q21.3 and has two phenotypes: neonatal intrahepatic cholestatic hepatitis (NICCD) and adult-onset type II citrullinemia (CTLN2). So far, we have described 19 SLC25A13 mutations. Here, we report 13 novel SLC25A13 mutations (one insertion, two deletion, three splice site, two nonsense, and five missense) in patients with citrin deficiency from Japan, Israel, UK, and Czech Republic. Only R360X was detected in both Japanese and Caucasian. IVS16ins3kb identified in a Japanese CTLN2 family seems to be a retrotransposal insertion, as the inserted sequence (2,667-nt) showed an antisense strand of processed complementary DNA (cDNA) from a gene on chromosome 6 (C6orf68), and the repetitive sequence (17-nt) derived from SLC25A13 was found at both ends of the insert. All together, 30 different mutations found in 334 Japanese, 47 Chinese, 11 Korean, four Vietnamese and seven non-East Asian families have been summarized. In Japan, IVS16ins3kb was relatively frequent in 22 families, in addition to known mutations IVS11 + 1G > A, 851del4, IVS13 + 1G > A, and S225X in 189, 173, 48 and 30 families, respectively; 851del4 and IVS16ins3kb were found in all East Asian patients tested, suggesting that these mutations may have occurred very early in some area of East Asia.