Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 47: 343-346, 2001;
This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lam, C.-W.
Right arrow Articles by Shek, C.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lam, C.-W.
Right arrow Articles by Shek, C.-C.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2001;47:343-346.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Novel Mutation and Polymorphisms of the HMBS Gene Detected by Denaturing HPLC,

Ching-Wan Lam1,a, Priscilla Miu-Kuen Poon1, Sui-Fan Tong1, Anthony Wing-Ip Lo2, Chi-Kong Lai2, Kin-Lam Choi3, Sau-Cheung Tiu3, Yan-Wo Chan2 and Chi-Chung Shek4

1 Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China

2 Department of Pathology, Princess Margaret Hospital, Hong Kong, China
Departments of
3 Medicine and
4 Pathology, Queen Elizabeth Hospital, Hong Kong, China
a author for correspondence: fax 852-2636-5090, e-mail ching-wanlam{at}cuhk.edu.hk


   Introduction
Top
 Introduction
References
 
Acute intermittent porphyria (AIP) is an autosomal dominant, inborn error of the metabolism of heme biosynthesis caused by partial deficiency of hydroxymethylbilane synthase (HMBS). This enzyme catalyzes the condensation of four molecules of porphobilinogen to a tetrapyrrole, hydroxymethylbilane. AIP is characterized by acute attacks of a neurological disorder manifesting as abdominal pain, hypertension, tachycardia, peripheral neuropathy, and mental dysfunction. Biochemical diagnosis of AIP relies on increased urinary porphobilinogen and normal fecal porphyrin excretion (1). Identification of presymptomatic AIP carriers in families with affected individuals is of clinical importance because avoidance of precipitating agents (e.g., drugs and alcohol) can prevent the occurrence of the first porphyric attack, which may be life-threatening. However, there is a significant overlap between the enzyme activities of healthy individuals and patients with AIP (2). In addition, there is a variant of AIP, in which the red blood cell (RBC) HMBS activity is normal (3). DNA-based diagnosis of presymptomatic AIP has been more reliable than diagnosis by RBC HMBS activity, and, thus, direct detection of mutations in presymptomatic AIP is now the definitive approach.

The human HMBS gene spans ~10 kb of DNA on chromosome 11q24.1-24.2 and contains 15 exons (4). Extensive allelic heterogeneity has been demonstrated in this gene, and 159 mutations have been identified in the HMBS gene (5). Most of these mutations are found only in individual families, except those found in Dutch (R116W) (6) and Swedish (W198X) (7) AIP families. Although DNA sequencing can identify all the mutations, this approach is both labor-intensive and time-consuming (8). Several methods that accelerate mutational screening before sequencing have been developed, such as single-strand conformation polymorphism analysis (9), heteroduplex gel analysis (10), and denaturing gradient gel electrophoresis (11)(12). Recently, denaturing HPLC (DHPLC) appears to be more sensitive than other methods in mutation detection, as exemplified in the detection of TSC1 (13), TSC2 (14), BRCA1 (15)(16), BRCA2 (15), CFTR (13), EXT1 (17), EXT2 (17), and HPRT (18). Here, we describe the use of DHPLC for mutation scanning in a family with AIP.

The patient was a 40-year-old woman presenting with frequent acute episodes of abdominal pain and convulsions. In one episode of acute attack, urine porphobilinogen was 117 µmol/mmol of creatinine (reference limit <10 µmol/mmol of creatinine). Reversed-phase high-performance thin-layer chromatography showed a normal pattern of fecal porphyrin excretion (19)(20). The patient was diagnosed to have AIP, but the RBC HMBS activity, measured at a time when she was anemic, was within the reference interval (35 nmol/mL of RBC per hour; reference interval, 28–66 nmol/mL of RBC per hour). The RBC HMBS activity of the mother was low (27.5 nmol/mL of RBC per hour), whereas the father had normal activity (52.4 nmol/mL of RBC per hour). On the basis of these data, the mother was an asymptomatic carrier and the patient was a manifesting individual of AIP.

Genomic DNA was extracted from whole-blood samples of the patient and the family members by a commercially available method (QIAamp; Qiagen). The study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects. All of the exons and the flanking introns of the HMBS gene were amplified using previously described primers (21). Exon 15 of the HMBS gene was amplified into two fragments [i.e., exon 15A (nucleotide positions 9051–9469) and exon 15B (nucleotide positions 9373–9700)]. Heteroduplex analysis was performed on a WAVETM DHPLC instrument (Transgenomic Inc.). Analysis on the system was performed at a temperature sufficient to partially denature (melt) the DNA heteroduplexes. The partially denatured heteroduplexes were resolved from the corresponding homoduplexes by ion-pair reversed-phase HPLC (22). The stationary phase consisted of 2-µm nonporous alkylated poly(styrene-divinylbenzene) particles packed into a 50 x 4.6 mm (i.d.) column (DNASep column; Transgenomic Inc.). Crude PCR product (10 µL) was loaded on the column and eluted from the column by an acetonitrile gradient in 0.1 mol/L triethylammonium acetate (TEAA; pH 7.0) buffer at a constant flow rate of 0.9 mL/min. The buffers were prepared from TEAA buffer concentrate to give the following: buffer A, 0.1 mol/L TEAA; buffer B, 0.1 mol/L TEAA containing 250 mL/L acetonitrile. The gradient was created by mixing buffers A and B. The recommended gradient for mutation detection was a slope of 2% increase in buffer B per minute. Eluted DNA fragments were detected with ultraviolet absorbance at a wavelength of 260 nm. The WAVE utility software helped determine the correct temperature for mutation scanning based on the sequence of the wild-type DNA. This approach was modified during the analysis of DNA from individuals carrying two identical mutant alleles (homozygous mutation). The PCR product spanning the homozygous mutation was mixed with equal amounts of wild-type amplified DNA and hybridized. After this treatment, a sample contained a mixture of hetero- and homoduplexes.

PCR products were purified by Microspin S300 columns (Amersham Pharmacia), and both strands were sequenced using the amplification primers as sequencing primers and BigDyeDeoxyTM terminator cycle sequencing reagents according to the manufacturer’s instructions (PE Biosystems). Products of sequencing reactions were purified by Centri-SepTM spin columns (Princeton Separations). Purified sequencing fragments were separated by capillary electrophoresis and detected via laser-induced fluorescence on an ABI PRISM 310 Genetic Analyzer (PE Biosystems). Sequencing results were compared with the established human HMBS sequence (GenBank Accession No. M95623).

For the proband (II2), the DHPLC chromatograms of exons 11 (Fig. 1A ), 14, 15A, and 15B (data not shown) showed the presence of heteroduplex peaks. Direct DNA sequencing of exon 11 showed a double heterozygous pattern (data not shown). The mutation is a one-nucleotide deletion, 8193delC. This mutation alters the reading frame of the encoded protein such that a stop codon, TGA, is generated at codon 254 (i.e., L254X). The predicted mutant HMBS is a truncated protein of 253 amino acids, compared with 361 residues for the wild-type protein. Screening of the family members by DHPLC and subsequent sequencing confirmed that only the mother (I2) had a 8193delC mutation. This 8193delC mutation was not detected in 50 apparently healthy Chinese individuals. This is the first disease-causing mutation identified in the HMBS gene in Chinese individuals. Direct sequencing of both exon 14 and exon 15A and the flanking introns of the proband showed a novel transition in intron 14, at position 9103, changing the base from guanine to adenine, i.e., 9103G->A (Fig. 1B ). The size of intron 14 was only 88 bp, and the two PCR products both contained the nucleotide at 9103. When we analyzed 50 apparently healthy Chinese individuals, we found that 49 were 9103G/G and 1 was 9103G/A. The allele frequency of 9103G thus was 0.99, and that of 9103A was 0.01. Direct sequencing of exon 15B and the flanking introns showed our previously described single nucleotide polymorphism (SNP), 9678G->A (23).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. DHPLC chromatograms of exon 11 and the flanking introns of the HMBS gene (A) and haplotype analysis (B).

(A), top to bottom: 8193delC/wild-type (II2), 8193delC/wild-type (I2), 8164C/wild-type (III1), 8164C/wild-type (III2), 8164C/wild-type (III3).

Interestingly, DHPLC chromatograms of exon 11 of the other family members (III1, III2, and III3) also showed the presence of heteroduplex peaks. However, the elution profiles were different from that of 8193delC (Fig. 1AUp ). Direct sequencing of the PCR products showed a transversion in intron 10, at position 8164, changing the base from cytosine to adenine, i.e., 8164C->A (data not shown). The 8164C->A transversion is not present in the patient or her mother, thus confirming that 8164C->A is a neutral polymorphism. When we analyzed 50 apparently healthy Chinese individuals, we found that 11 were 8164A/A, 28 were 8164A/C, and 11 were 8164C/C. The allele frequency of 8164A thus is 0.50, and that of 8164C is 0.50. Using the three genomic SNPs, we constructed complete haplotypes for all family members. The haplotypes for the chromosome carrying the disease-causing mutation are 8164C, 9103G, and 9678G. Thus, only the mother and the proband are carriers, and the other family members do not have the mutant alleles (Fig. 1BUp ). The results of haplotype analysis are consistent with the results of mutation detection.

DNA-based diagnosis of AIP was particularly useful in the proband as RBC HMBS activity is uninterpretable in the presence of hemolysis. In this study, we used DHPLC to identify a novel HMBS mutation that causes AIP, and together with the three SNPs, we have determined the haplotypes for the mutation. The haplotypes are informative for this family. We can use either direct mutation detection or linkage analysis to determine whether other family members carry the AIP chromosome.

DHPLC has several advantages over other mutation detection methods. DHPLC is fully automated, eliminating the time and labor in gel preparation, loading, analyzing, and photographic documentation. The PCR products are loaded directly from a 96-well plate without any prior purification. Heteroduplex DNA is resolved from homoduplex DNA in a matter of minutes. The technology detects single-base changes as efficiently as short deletions and insertions. Heteroduplex peaks elute earlier than homoduplexes and can be observed as separate peaks or as shoulders on the leading edge of homoduplex peaks (22). Elution characteristics of the heteroduplex peaks are influenced by the specific base mismatch present and the melting characteristics of the surrounding bases. As demonstrated in this study, even mutations and SNPs located in the same PCR fragment can be distinguished by the characteristic elution profiles.

Although single-base mutations have been detected in 1.5-kb fragments by DHPLC (24), PCR product <100 bp containing a mutant allele with the C->G transversion cannot be resolved from the wild-type allele (25). To achieve the highest accuracy, it is better to amplify large exons into 150- to 450-bp fragments. In addition, cases of possible homozygous mutations require premixing of subject DNA with wild-type DNA. This is, however, less of a problem in autosomal dominant diseases. Although an initial capital investment in the HPLC instrument is required, the combination of low running costs and the tremendous reduction in the effort of sequencing make the DHPLC technique a suitable method for mutation detection (26)(27).


   References
Top
 Introduction
 References
 

  1. Kappas A, Sassa S, Galbraith RA, Nordmann Y. The porphyrias. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited diseases, 7th ed 1995:2103-2160 McGraw-Hill New York. .
  2. Wassif WS, Deacon AC, Floderus Y, Thunell S, Peters TJ. Acute intermittent porphyria: diagnostic conundrums. Eur J Clin Chem Clin Biochem 1994;32:915-921.[Web of Science][Medline] [Order article via Infotrieve]
  3. Mustajoki P, Tenhunen R. Variant of acute intermittent porphyria with normal erythrocyte uroporphyrinogen-I-synthase activity. Eur J Clin Investig 1985;15:281-284.[Web of Science][Medline] [Order article via Infotrieve]
  4. Yoo HW, Warner CA, Chen CH, Desnick RJ. Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene. Genomics 1993;15:21-29.[Web of Science][Medline] [Order article via Infotrieve]
  5. Krawczak M, Cooper DN. The human gene mutation database. Trends Genet 1997;13:121-122.[Web of Science][Medline] [Order article via Infotrieve]
  6. Gu XF, de Rooij F, Lee JS, Te Velde K, Deybach JC, Nordmann Y, et al. High prevalence of a point mutation in the porphobilinogen deaminase gene in Dutch patients with acute intermittent porphyria. Hum Genet 1993;91:129-130.
  7. Lee JS, Anvret M. Identification of the most common mutation within the porphobilinogen deaminase gene in Swedish patients with acute intermittent porphyria. Proc Natl Acad Sci U S A 1991;88:10912-10915.[Abstract/Free Full Text]
  8. Whatley SD, Woolf JR, Elder GH. Comparison of complementary and genomic DNA sequencing for the detection of mutations in the HMBS gene in British patients with acute intermittent porphyria: identification of 25 novel mutations. Hum Genet 1999;104:505-510.[Web of Science][Medline] [Order article via Infotrieve]
  9. Kauppinen R. Single-strand conformation polymorphism (SSCP) analysis applied to the diagnosis of acute intermittent porphyria. Mol Cell Probes 1992;6:527-530.[Web of Science][Medline] [Order article via Infotrieve]
  10. Schreiber WE, Fong F, Nassar BA, Jamani A. Heteroduplex analysis detects frameshift and point mutations in patients with acute intermittent porphyria. Hum Genet 1995;96:161-166.[Web of Science][Medline] [Order article via Infotrieve]
  11. Gu XF, de Rooij F, Voortman G, Te Velde K, Deybach JC, Nordmann Y, et al. Detection of eleven mutations causing acute intermittent porphyria using denaturing gradient gel electrophoresis. Hum Genet 1994;93:47-52.[Web of Science][Medline] [Order article via Infotrieve]
  12. Puy H, Deybach JC, Lamoril J, Robreau AM, Da Silva V, Gouya L, et al. Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria. Am J Hum Genet 1997;60:1373-1383.[Web of Science][Medline] [Order article via Infotrieve]
  13. Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, et al. Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin Chem 1999;45:1133-1140.[Abstract/Free Full Text]
  14. Choy YS, Dabora SL, Hall F, Ramesh V, Niida Y, Franz D, et al. Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2. Ann Hum Genet 1999;63:383-391.[Web of Science][Medline] [Order article via Infotrieve]
  15. Wagner T, Stoppa-Lyonnet D, Fleischmann E, Muhr D, Pages S, Sandberg T, et al. Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics 1999;62:369-376.[Web of Science][Medline] [Order article via Infotrieve]
  16. Gross E, Arnold N, Goette J, Schwarz-Boeger U, Kiechle M. A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC. Hum Genet 1999;105:72-78.[Web of Science][Medline] [Order article via Infotrieve]
  17. Dobson-Stone C, Cox RD, Lonie L, Southam L, Fraser M, Wise C, et al. Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses. Eur J Hum Genet 2000;8:24-32.[Web of Science][Medline] [Order article via Infotrieve]
  18. Skopek TR, Glaab WE, Monroe JJ, Kort KL, Schaefer W. Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis. Mutat Res 1999;430:13-21.[Web of Science][Medline] [Order article via Infotrieve]
  19. Lam CW, Lai CK, Chan YW. Simultaneous fluorescence detection of fecal urobilins and porphyrins by reversed-phase high-performance thin-layer chromatography. Clin Chem 1998;44:345-346.[Free Full Text]
  20. Lai CK, Lam CW, Chan YW. High-performance thin-layer chromatography of free porphyrins for diagnosis of porphyria. Clin Chem 1994;40:2026-2029.[Abstract]
  21. Lundin G, Lee JS, Thunell S, Anvret M. Genetic investigation of the porphobilinogen deaminase gene in Swedish acute intermittent porphyria families. Hum Genet 1997;100:63-66.[Web of Science][Medline] [Order article via Infotrieve]
  22. Underhill PA, Jin L, Lin AA, Mehdi SQ, Jenkins T, Vollrath D, et al. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res 1997;7:996-1005.[Abstract/Free Full Text]
  23. Law WK, Choy KW, Lam CW. Novel single nucleotide polymorphism (9678G->A) for linkage analysis of acute intermittent porphyria. Clin Chem 1999;45:308-309.[Free Full Text]
  24. Jin L, Underhill PA, Oefner PJ, Cavalli-Sforza LL. Systemic search for polymorphisms in the human genome using denaturing high-performance liquid chromatography (DHPLC). Am J Hum Genet 1995;57(Suppl):A26.
  25. Oefner PJ. Allelic discrimination by denaturing high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2000;739:345-355.[Medline] [Order article via Infotrieve]
  26. Arnold N, Gross E, Schwarz-Boeger U, Pfisterer J, Jonat W, Kiechle M. A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers. Hum Mutat 1999;14:333-339.[Web of Science][Medline] [Order article via Infotrieve]
  27. O’Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoorn B, Guy C, et al. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998;52:44-9.[Web of Science][Medline] [Order article via Infotrieve]




This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lam, C.-W.
Right arrow Articles by Shek, C.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lam, C.-W.
Right arrow Articles by Shek, C.-C.
Related Collections
Right arrow Molecular Diagnostics and Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS