Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

Molecular Characterization of a Deletion in the HPRT1 Gene in a Patient with Lesch–Nyhan Syndrome

Lesch–Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch–Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.     

A large Alu-mediated deletion, identified by PCR, as the molecular basis for glycogen storage disease Type II (GSDII)

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder resulting from inherited deficiency of the enzyme lysosomal acid α-glucosidase. Over 40 different mutations have been described but no large deletions have been previously identified. We now describe a homozygous large (9-kb) deletion extending from IVS15 to 4 kb downstream of the terminal exon (exon 20), detected by polymerase chain reaction (PCR)-based methods. The deletion was initially suspected because of failure to amplify a contiguous group of exons by PCR. We hypothesized an Alu/Alu recombination, based on our prior demonstration by Southern blotting of Alu elements in the regions potentially flanking the deletion. Additional sequence analysis of genomic fragments confirmed the presence of Alu elements and allowed the design of flanking primers for PCR amplification. Amplification resulted in a smaller than normal fragment (0.7 vs 10 kb) in homozygosity in the proband and in heterozygosity in her parents. Cloning and sequencing of the smaller than normal 0.7-kb deletion fragment revealed an Alu/Alu deletion junction. In heterozygosity this deletion would not be detected by currently standard PCR mutation detection methods. Based on other Alu-mediated deletions, this deletion is likely to be recurrent and should be screened for in all non-consanguineous GSDII patients, particularly when only one mutation has been identified and none of the 12 single-nucleotide polymorphisms in the deleted region are heterozygous. These observations also suggest that initial characterization of genes at disease-causing loci should include a search for Alu and other repetitive elements to facilitate subsequent PCR-based mutation analysis. Received: 24 August 1998 / Accepted: 13 November 1998     

Molecular characterization of a deletion in the HPRT1 gene in a patient with Lesch-Nyhan syndrome

Lesch-Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch-Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.     

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.     

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms

Several recurrent common chromosomal deletion and duplication breakpoints have been localized to large, highly homologous, low-copy repeats (LCRs). The mechanism responsible for these rearrangements, viz., non-allelic homologous recombination between LCR copies, has been well established. However, fewer studies have examined the mechanisms responsible for non-recurrent rearrangements with non-homologous breakpoint regions. Here, we have analyzed four uncommon deletions of 17p11.2, involving the Smith–Magenis syndrome region. Using somatic cell hybrid lines created from patient lymphoblasts, we have utilized a strategy based on the polymerase chain reaction to refine the deletion breakpoints and to obtain sequence data at the deletion junction. Our analyses have revealed that two of the four deletions are a product of Alu/Alu recombination, whereas the remaining two deletions result from a non-homologous end-joining mechanism. Of the breakpoints studied, three of eight are located in LCRs, and five of eight are within repetitive elements, including Alu and MER5B sequences. These findings suggest that higher-order genomic architecture, such as LCRs, and smaller repetitive sequences, such as Alu elements, can mediate chromosomal deletions via homologous and non-homologous mechanisms. These data further implicate homologous recombination as the predominant mechanism of deletion formation in this genomic interval.     

Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene

Mouse cartilage matrix deficiency (cmd), an autosomal recessive phenotype caused by absence of aggrecan, maps to Chromosome (Chr) 7 and is caused by a 7-bp deletion in exon 5 generating a premature stop codon (Watanabe et al. 1994). Another spontaneous mutation with the same locus and phenotype, cmd-Bc, has now been defined as the complete loss of exons 2 to 18, resulting in a significantly shortened mRNA (1.2 kb). The upstream breakpoint is in intron 1, 18.8 kb 3′ of exon 1; the downstream breakpoint lies 10.5 kb past the final aggrecan exon 18. The deletion is flanked by sequences homologous to topoisomerase I and II cleavage sites and a 7-bp direct repeat, suggesting the defect resulted from a nonhomologous recombination event. Additionally, the size of the first intron and the intron-exon structure between exons 12 and 14 were determined, establishing the length of the murine aggrecan gene as 68.6 kb. This report completes the structural analysis of the murine aggrecan gene, defines a second null mutation, and reinforces the importance of aggrecan in development. Received: 20 May 1999 / Accepted: 26 July 1999     

Identical 3250-bp deletion between two AluI repeats in the ADA genes of unrelated ADA-SCID patients.

Recently, we investigated a Belgian patient with severe combined immune deficiency caused by a dysfunction of the gene for adenosine deaminase (ADA-SCID), which was found to be due to a 3.2-kb deletion spanning the promoter and the first exon of the ADA gene (Berkvens et al., 1987, Eur. J. Pediatr. 146:329). No ADA-specific RNA could be detected in primary fibroblasts derived from this patient. In the present paper we establish via direct sequencing of in vitro amplified DNA that the 3250-bp deletion is due to a recombination within the left arms of two direct AluI repeats. This mutation is identical to one reported for an unrelated patient in the United States (Markert et al., 1988, J. Clin. Invest. 81:1323-1327).     

Hereditary cystatin C amyloid angiopathy: identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis

Summary Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease characterized by amyloidosis, dementia and fatal cerebral hemorrhage of young adults. A method for rapid and simple diagnosis of HCCAA is described. It is based upon oligonucleotide-directed enzymatic amplification of a 275-bp genomic DNA segment containing exon 2 of the cystatin C gene from a blood sample, followed by digestion of the amplification product with AluI. Loss of an AluI recognition site in the amplified DNA segment from HCCAA patients results in a deviating band-pattern at agarose gel electrophoresis, compared with that obtained from normal subjects or unaffected HCCAA family members. In a population of 9 patients with manifest HCCAA, 14 patients with other causes of brain hemorrhage and 16 healthy individuals, the diagnostic procedure displayed a sensitivity and specificity for HCCAA of 100%. Amplified DNA segments from 4 HCCAA patients of four different families were analyzed by nucleotide sequencing; the HCCAA-causing mutation in all families was found to be a single TA substitution in the codon for amino acid residue 68 of cystatin C.     

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 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.     

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.     

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV–VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.     

Phenotypic variability in two families with novel splice-site and frameshift NF2 mutations

Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients. Received: 4 January 1996 / Revised: 26 March 1996     

Genomic rearrangements at the <Emphasis Type="Italic">IGHMBP2</Emphasis> gene locus in two patients with SMARD1

Autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by mutations in the immunoglobulin -binding protein 2 (IGHMBP2) gene. Patients affected by the infantile form of SMARD1 present with early onset respiratory distress. So far, patients with neither juvenile onset nor with larger deletions/rearrangements in IGHMBP2 have been reported. In this study, we investigated one patient with infantile (4 months) and another with juvenile (4.3 years) onset of respiratory distress. Direct sequencing of all exons and flanking intron sequences in both patients revealed a mutation on only one allele. In both patients, we identified genomic rearrangements of the other allele of IGHMBP2 by means of Southern blotting. Putative breakpoints were confirmed by polymerase chain reaction on genomic and cDNA. The patient with juvenile onset had an Alu/Alu mediated rearrangement, which resulted in the loss of ~18.5 kb genomic DNA. At the mRNA level, this caused an in-frame deletion of exons 3–7. The patient with infantile onset had a complex rearrangement with two deletions and an inversion between intron 10 and 14. This rearrangement led to a frameshift at the mRNA level. Our results show that SMARD1 can be caused by genomic rearrangements at the IGHMBP2 gene locus. This may be missed by mere sequence analysis. Additionally, we demonstrate that juvenile onset SMARD1 may also be caused by mutations of IGHMBP2. The complex nature of the genomic rearrangement in the patient with infantile SMARD1 is discussed and a deletion mechanism is proposed.     

A 15-base pair (bp) palindromic insertion associated with a 3-bp deletion in exon 10 of the gp91-phox gene,detected in two patients with X-linked chronic granulomatous disease

Molecular genetic studies were carried out on two maternal cousins with X-linked chronic granulomatous disease (X-CGD). Sequencing analysis of polymerase chain reaction (PCR)-amplified DNA fragments from both patients revealed a 15-base pair (bp) insertion associated with a 3-bp deletion in exon 10 of the cytochrome b heavy chain (gp91-phox) gene. Results of genomic PCR with primers flanking the insertion/deletion site confirmed the mutation, and also demonstrated that their mothers were carriers for the disease. Palindromic sequences were found in the 15-bp insertion as well as in the flanking 3-bp deletion site, which may play a role in the mechanism of this mutation.     

Tyrosinemia type 1 — complex splicing defects and a missense mutation in the fumarylacetoacetase gene

Two mutations are reported in six tyrosinemia type 1 patients from northern Europe. In four patients, a G to A transition at nucleotide position 1009 (G¹⁰⁰⁹A) of the fumarylacetoacetase (FAH) coding sequence caused aberrant splicing by introducing an acceptor splice site within exon 12, thereby deleting the first 50 nucleotides of this exon. The following exon-intron boundary was frequently missed, and a cryptic donor splice site within intron 12 caused a partial intron 12 retention of 105 bp. This point mutation alternatively gave a glycine 337 to serine substitution in instances of correct splicing. The mutation is rapidly detected by PvuII digestion of polymerase chain reaction (PCR)-amplified genomic DNA. Another mutation, g⁺⁵a in the intron 12 donor splice site consensus sequence (IVS12 g⁺⁵a), was found in five of the patients. This caused alternative splicing with retention of the first 105 nucleotides of intron 12, exon 12 skipping, and a combined deletion of exons 12 and 13. Rapid detection of this mutation is achieved by restriction digestion of PCR-amplified genomic DNA; a mismatch primer combined with the point mutation creates a Tru9I restriction site. One patient who was homozygous for the G¹⁰⁰⁹A mutation had a chronic form of tyrosinemia. Three patients were combined heterozygotes for G¹⁰⁰⁹A and TVS12 g⁺⁵a. Their clinical phenotypes varied from acute to chronic, indicating the impact of background genes and/or external factors on the presentation of typrosinemia type 1.