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Editorial |
Departments of Human Genetics, and Pediatrics, UCLA School of Medicine, 10833 LeConte Avenue, Los Angeles, CA 90095-1752, Fax 310-206-4584, E-mail evilain{at}ucla.edu
Advances in the Human Genome Project have profoundly influenced not only the search for new genes, but also the molecular diagnosis of genetic disorders. In the early 1980s, the first generation of the human genome map was based on restriction fragment length polymorphisms (RFLPs) identified by Southern analysis (1). Linkage analysis by RFLP became a very powerful tool for molecular diagnosis. The early 1990s saw the rise of the second generation of mapping, based on microsatellite markers and PCR amplification (2). These multiallelic polymorphisms are much more informative than RFLPs and simpler to study. We are now witnessing the third generation of genome mapping, based on single-nucleotide polymorphisms (SNPs), and analyzed by PCR and chip-based microarrays (3).
An SNP involves any nucleotide of the human genome in which two different bases can be found in the population. SNPs are the most common type of human genetic variation. Because they have only two alleles, they are less informative than microsatellites. This drawback is overcome by their high density throughout the genome and the simplicity and possible automation of their analysis. They may become one of the most powerful tools in human genetics for identifying disease genes and mapping complex traits by linkage.
As larger and larger portions of the human genome are sequenced, one might speculate that mapping tools would become obsolete for diagnostic studies. Direct mutation detection is considered the gold standard of molecular diagnosis because all of the methods based on linkage of polymorphic markers may be impeded by a lack of informative markers or by misinterpretations resulting from recombination events, new mutations, or misattribution of paternity. In this issue of Clinical Chemistry, Killeen et al. (4) demonstrate, however, that linkage analysis remains an important diagnostic tool in difficult settings such as 21-hydroxylase deficiency, even when the gene sequence is known.
The accurate molecular diagnosis of congenital adrenal hyperplasia (CAH) is a challenge. This condition is serious, treatable, and common, and molecular diagnosis is essential. Ninety-five percent of all cases of CAH are caused by a defect in the function of steroid 21-hydroxylase (5), producing adrenal insufficiency by impaired synthesis of cortisol and aldosterone. Girls with CAH are exposed to excessive adrenal androgens, which cause virilization of their external genitalia. CAH is treatable by glucocorticoid and mineralocorticoid replacement therapy in addition to plastic surgery for ambiguous genitalia. Prenatally, virilization of genitalia may be prevented by administration of a synthetic glucocorticoid to the patient's mother (6). This classical form of CAH occurs with a frequency of 1 in 15 000 (7). A nonclassical form presenting with hirsutism, dysmenorrhea, and infertility in women has been shown to occur as frequently as 1 in 100 in Caucasian populations (8). A reliable diagnostic approach is crucial not only because of the frequency and the severity of CAH, but also because of the possibility of prenatal diagnosis and treatment. In particular, the long-term effects of steroid treatment of unaffected fetuses have not been evaluated clearly (9), rendering a rapid and accurate molecular diagnostic strategy indispensable.
Identification of mutations in CAH is complicated by the fact that the 21-hydroxylase gene, CYP21, is nearly duplicated in a closely linked gene with 98% similarity but with several deleterious sequence alterations (10). The CYP21 homolog is therefore a nonfunctional pseudogene named CYP21P. The sequence alterations in CYP21P represent mutations that cause CAH when present in CYP21. The pseudogene-derived mutations occur because of gene conversion, a mechanism of genetic transfer between homologous genes [see Ramsden and Sinnott (11) for a review]. In this context, the identification of a disease-causing mutation relies on the specific amplification of CYP21 and not CYP21P. A limited number of CYP21-specific primers are available; however, the high frequency of gene conversion between the two homologs may prevent amplification of the expected gene or may impair the detection of a CYP21 deletion, which occurs in about one-third of CAH cases [see Wedell (12) for a review on the molecular diagnosis of CAH]. Moreover, preferential amplification of one allele of CYP21, or allele dropout, has been described for the most common point mutation of CYP21, a conversion of nucleotide 656 in intron 2, which leads to a premature 3' splice site (13). This allele dropout can cause incorrect typing of asymptomatic heterozygous individuals as homozygous for this mutation.
The presence of SNPs in the second intron of CYP21 can be used to resolve potential ambiguities. Killeen et al. (4) have determined the frequency of three intronic SNPs and have shown that they can be used as polymorphic markers to follow the segregation of alleles in informative families with 21-hydroxylase deficiency. They are analyzed by the cleavase fragment length polymorphism (CFLP) method, a variant of single-strand conformation polymorphism (SSCP) analysis (14), that is based on the ability of a structure-specific endonuclease, cleavase I, to cut the junction between single-stranded and double-stranded DNA. Any difference in conformation caused by DNA sequence variation will be detected as a different banding pattern.
Because the analysis of SNP markers still depends on the successful amplification of the CYP21 alleles, this method should always be used in combination with other molecular diagnostic techniques, including direct sequencing. Availability of the DNA of both biological parents is required. Overall, the biggest advantage of this linkage method is the intragenic localization of the SNPs; use of intragenic polymorphisms reduces considerably the risk of error because of a recombination event.
SNPs will most likely be used widely for the localization of loci for complex traits (15). It remains to be seen whether SNPs will also be major tools for molecular diagnosis. One of their unique characteristics is the possibility of a relatively simple automation of their analysis (3), which may lead in the future to molecular screening of large populations. Because of their potential for high throughput, SNPs will have increasing application for predisposition traits when linkage disequilibrium is demonstrated with disease genes.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Eitan and Y. Kashi Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA) Nucleic Acids Res., June 15, 2002; 30(12): e62 - e62. [Abstract] [Full Text] [PDF]
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 D. H. Oliver, R. E. Thompson, C. A. Griffin, and J. R. Eshleman Use of Single Nucleotide Polymorphisms (SNP) and Real-Time Polymerase Chain Reaction for Bone Marrow Engraftment Analysis J. Mol. Diagn., November 1, 2000; 2(4): 202 - 208. [Abstract] [Full Text]
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