Principles
The diagnosis of a genetic disease requires a systematic approach that takes clinical and genetic considerations into account. Whereas clinical medicine tends to classify diseases according to organ system, age of onset, gender, or primary method of detection (radiology, imaging techniques), medical genetics, like pathology, is oriented towards the basic cause or lesion, in this case the gene or genes affected by a relevant genetic change. Genetic diagnosis is based on an interdisciplinary analysis of all clinical and laboratory data from a genetic perspective.

• Genetic diagnosis, a multistep procedure
  
  The phenotype, which is the clinical manifestations including individual and family history in medical terms, is the starting point. The first decision the medical geneticist must make is whether a pattern of manifestations can be recognized. Tools that can assist in this decision include training and personal experience; appropriate textbooks and other literature; and online search systems such as OMIM, MEDLINE, PubMed, POSSUM, London Dysmorphology Data Base for congenital malformations, and cytogenetic databases. If a disease pattern can be recognized, the next decision concerns the category of disease. Although difficult to establish in practice, the disease category is important for the next steps to be taken. For this purpose, the McKusick catalog of human genes and diseases, Mendelian Inheritance in Man (MIM) and its online version (OMIM) are indispensable. The possibility of genetic heterogeneity must be considered at this stage. The term genetic heterogeneity refers to a phenotype (disease) that has different causes. A particular phenotype may be caused by mutations at different loci (locus heterogeneity) or by different mutant alleles at the same locus (allele heterogeneity). All genetic diagnostic procedures should be preceded by genetic counseling, which properly includes obtaining the (informed) consent of the persons involved.
  
  
• Genotype analysis by PCR typing of a polymorphic restriction site
  
  Genotype analysis by PCR typing of a polymorphic restriction site is preferred to the more laborious Southern blot hybridization (see p. 62). (Figure adapted from Strachan and Read, 1999).
  
  
• Protein truncation test (PTT)
  
  This is a test for frameshift, splice, or nonsense mutations that leads to a truncated protein due to an early stop codon created downstream of the mutation. The truncated protein is detected in an assay based on an in-vitro translation system. The translation will be interrupted at a premature stop codon resulting from the mutation. The size of the newly translated protein is determined by gel electrophoresis. PTT detects the approximate location of the mutation as reflected by the size of the mutant protein. PTT is useful in studying genes with frequent nonsense mutations, such as the APC, BRCA1, and BRCA2 genes. However, it cannot be applied for genes with frequent missense mutations. The figure shown here is highly schematic. (Adapted from Strachan and Read, 1999; and Beaudet, 1998).
  
  
• References
  
  Aase, J.M.: Diagnostic Dysmorphology. Plenum
  Medical Book Company, New York, 1990.
  Beaudet, A.L.: Genetics and disease, pp. 365–
  395. In: Fauci, A.S., et al., eds., Harrison’s
  Principles of Internal Medicine. 14th ed.
  McGraw-Hill, New York, 1998.
  Jones, K.L.: Smith’s Recognizable Patterns of
  Human Malformation. 5th ed. W.B.
  Saunders, Philadelphia, 1997.
  McKusick, V.A.: Mendelian Inheritance in Man.
  A Catalog of Human Genes and Genetic Disorders.
  12th ed. Johns Hopkins University
  Press, Baltimore, 1998.
  Strachan, T., Read, A.P.: Human Molecular
  Genetics. 2nd ed. Bios Scientific Publishers,
  Oxford, 1999.
  van der Luijit, R., et al.: Rapid detection of translation
  terminating mutations at the adenomatous
  polyposis (APC) gene by direct protein
  truncation test. Genomics 20:1–4,
  1994.

• Detection of a point mutation by oligonucleotides
  
  Short segments of DNA (oligonucleotides) are used to determine whether there is a mutation in a segment of DNA (1, normal DNA; 2, mutation from G to A). An oligonucleotide is a synthetically produced DNA segment about 20 nucleotides long; its sequence is complementary to a corresponding segment of the investigated gene. It hybridizes completely with its complementary segment (3). If a mutation, here from G to A (1), is located in this region, hybridization will not be perfect at this site (mismatch) (4). On the other hand, an oligonucleotide that is complementary to the DNA segment with the mutation will hybridize completely (allele-specific oligonucleotide, ASO) (5). This hybridizes incompletely with the normal DNA (6). By parallel use of both nucleotides, mutant and nonmutant DNA can be differentiated. The test results (7) show the hybridization of mutated DNA and of control DNA with the allele-specific oligonucleotides (ASO 1 for the control, ASO 2 for the mutation). Hybridization is indicated by a signal (dot-blot analysis).
  
  
• Demonstration of a point mutation by ribonuclease A cleavage
  
  The basis for this method is that a normal DNA strand hybridizes completely with mRNA from that region. Completely hybridized DNA and mRNA are protected from the effects of the RNA-splitting enzyme ribonuclease A (ribonuclease protection assay). Hybridization is incomplete in the area of a mutation. In this region, mRNA will be cleaved by ribonuclease A (RNAase A). This can be demonstrated by Southern blot. There will be two fragments formed that together correspond to the size of the completely hybridized fragment (600 base pairs (bp), versus 400 and 200 bp).
  
  
• Denaturing gradient gel electrophoresis
  
  This method exploits differences in the stability of DNA segments with and without mutation. While double-stranded DNA of a control person is completely complementary (homoduplex), a mutation leads to a mismatch at the site of mutation (heteroduplex). This DNA is less stable than completely complementary DNA strands (it has a lower melting point). If normal DNA (control) and DNAwith the mutation are placed in a gel with an increasing concentration gradient of formamide (denaturing gradient gel), the mutant and normal DNA can subsequently be differentiated in a Southern blot. The normal DNA remains stable to higher concentrations of formamide and migrates farther than mutant DNA, which dissociates earlier and therefore does not migrate as far.
  
  
• References
  
  Caskey, C.T.: Disease diagnosis by recombinant
  DNA methods. Science 236:1223–1229,
  1987.
  Dean, M.: Resolving DNA mutations. Nature
  Genet. 9:103–104, 1995.
  Mashal, R.D., Koontz, J., Sklar, J.: Detection of
  mutations by cleavage of DNA heteroduplexes
  with bacteriophage resolvases. Nature
  Genet. 9:177–183, 1995.