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A) for Linkage Analysis of Acute Intermittent Porphyria
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China
a Author for correspondence. Fax 852-2636-5090; e-mail ching-wanlam{at}cuhk.edu.hk.
To the Editor:
Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of metabolism caused by a partial deficiency of the third enzyme of the heme biosynthetic pathway, hydroxymethylbilane synthase (EC 4.3.1.8.; HMBS). This enzyme catalyzes the condensation of four molecules of porphobilinogen to a tetrapyrrole hydroxymethylbilane. Clinically, AIP is characterized by acute attacks of neurological disorders manifesting as abdominal pain, hypertension, tachycardia, peripheral neuropathy, and mental dysfunction. Biochemical diagnosis of AIP relies on increased urinary porphobilinogen and fecal porphyrin excretion within the health-related reference interval. The definitive diagnosis requires the assay of red blood cell (RBC) HMBS activity (1).
AIP is a genetic disease characterized by poor penetrance; therefore, identification of presymptomatic AIP carriers in families with manifesting individuals is of utmost clinical importance because avoidance of precipitating agents, e.g., drugs and alcohol, can prevent the occurrence of the first porphyric attacks, which may be life-threatening. However, the identification of AIP carriers by assaying the RBC HMBS activity is problematic because there is substantial overlap between the enzyme activities of healthy individuals and patients with AIP (2). In addition, in one variant of AIP, the decreased enzyme activity is confined to the liver, and the RBC HMBS activity is within health-related values (3).
DNA-based diagnosis of presymptomatic AIP has proven to be more reliable than diagnosis by RBC HMBS activity. Direct detection of mutations in presymptomatic AIP is the definitive approach; however, this requires the prior identification of the mutations in the probands. To date, 117 mutations have been identified in the HMBS gene. Sixty-five are missense/nonsense mutations, 21 are splicing mutations, 18 are small deletions, 11 are small insertions, 1 is a gross deletion, and 1 is a gross insertion and duplication (4). Most of these mutations are found only in individual families, except those found in Dutch (R116W) (5) and Swedish (W198X) (6) AIP families. In most instances, the mutations can only be identified by DNA sequencing, an approach that is both labor-intensive and time-consuming. Although the exons containing the mutations can often be identified by one of the screening methods, such as single strand conformation polymorphism analysis (7), heteroduplex analysis (8), and denaturing gradient gel electrophoresis (9)(10), no methods at present can reliably detect all of the mutations.
In the course of mutation analysis of the HMBS gene in our patients with AIP, we have identified a single nucleotide polymorphism (SNP) by direct DNA sequencing (data not shown). The SNP is located 72 base pairs (bp) downstream of the last exon, flanking the 3' end of the HMBS gene, at nucleotide position 9678 (numbered according to Genbank accession no. M95623), where the presence of G instead of A abolishes a BsrI restriction site. We developed a PCR-restriction analysis method to study this polymorphic marker in 48 unrelated Chinese individuals without symptoms or family histories of AIP.
Genomic DNA was extracted from whole blood samples by the salting out method as described (11). The study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects.
The sequence of the forward primer was 5'-TGCTGTCCAGTGCCTACATC-3'. The sequence of the reverse primer was 5'-GAACTCTGGGCAAAAGTCCC-3'. Genomic DNA (50 ng) was amplified in a 25-µL reaction mixture containing 1x Mg2+-free Taq DNA polymerase buffer, 200 µmol/L of each dNTP, 1.0 mmol/L MgCl2, 15 pmol of each primer, and 1.0 U of Taq DNA polymerase (Life Technologies). A Perkin-Elmer 480 thermal cycler was used, and the reactions were carried out with manual hot-start at 95 °C for 5 min, followed by 35 cycles of denaturation (30 s at 95 °C), annealing (60 s at 70 °C), and extension (40 s at 72 °C), and a final elongation step at 72 °C for 7 min.
PCR products (15 µL) were digested with 10 units of BsrI
(New England Biolabs) at 65 °C overnight as recommended by the
manufacturer. The digested products were then loaded on 6%
acrylamide:bisacrylamide (19:1, by weight) gels and electrophoresed in
Tris-borate-EDTA buffer. Electrophoresis was carried at 40 mA in 1x
Tris-borate-EDTA buffer. When the primers listed above were used, the
PCR product size was 331 bp. Digestion of wild-type DNA with
BsrI generated fragments of 213, 104, and 14 bp for 9678G.
In the presence of 9678A, fragments of 213, 86, 14, and 18 bp were
observed (Fig. 1
).
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When we analyzed 48 apparently healthy Chinese individuals, we found that 4 were 9678A/A, 27 were 9678A/G, and 17 were 9678G/G. The allele frequency of 9678A was 0.36, and that of 9678G was 0.64. The unbiased estimation of heterozygosity was 0.47, and the polymorphism information content was 0.36. Thus, this SNP is informative in Chinese; however, it would be interesting to know whether this dimorphism will also be informative in other ethnic groups.
Linkage analysis using polymorphic DNA markers is suitable for the identification of presymptomatic AIP carriers in families with manifesting individuals. AIP is an autosomal dominant disease; hence, more than one family member is usually affected, facilitating the phasing of the polymorphic markers. In addition, the entire genomic sequence encoding the HMBS gene is only 10 kilobases in size (12). Thus, recombination between the mutation and the polymorphic marker is negligible, increasing the reliability of this approach. Because highly polymorphic microsatellite markers have not been identified in this gene, SNPs (12)(13) remain the only available genetic markers for linkage analysis to identify presymptomatic carriers in AIP families.
Acknowledgments
We thank S.F. Tong for technical support.
References
The following articles in journals at HighWire Press have cited this article:
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  C.-W. Lam, P. M.-K. Poon, S.-F. Tong, A. W.-I. Lo, C.-K. Lai, K.-L. Choi, S.-C. Tiu, Y.-W. Chan, and C.-C. Shek Novel Mutation and Polymorphisms of the HMBS Gene Detected by Denaturing HPLC, Clin. Chem., February 1, 2001; 47(2): 343 - 346. [Full Text] [PDF]
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A by BsrI restriction
analysis.