Application of DNA-Based Diagnosis to Patient Care: The Example of Hemophilia A  STEVE S. SOMMER, M.D., Ph.D.  Mayo Clinic Proceedings  Volume 62, Issue 5, Pages 387-404 (May 1987) DOI: 10.1016/S0025-6196(12)65443-3 Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 1 Diagram of restriction fragment length polymorphism in factor VIII gene recognized by restriction enzyme Bcl I. A, The human factor VIII gene that contains 26 exons is shown to scale. The 26 exons are shaded, whereas the 25 introns and the sequences flanking the first and last exons are unshaded. B, Expanded portion of gene covering exons 17 and 18 is shown, and positions of Bcl I sites (B) are indicated. The presence or absence of the middle site, denoted by B*, is due to variation of sequence at a single base pair (bp), which renders that site susceptible to cleavage with Bcl I in one case (+) and renders it resistant to cleavage in another case (-). If cleavage occurs, an 879-bp and a 286-bp fragment are produced. If cleavage does not occur, a 1,165-bp fusion fragment is produced. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 2 Southern blot analysis of Bcl I restriction fragment length polymorphism in three females. DNA is cleaved with restriction enzyme Bcl I. Cleaved DNA is separated by size by means of agarose gel electrophoresis. Gel is then placed in contact with a piece of nitrocellulose, and by capillary action, buffer flows through gel onto nitrocellulose filter. This procedure causes the DNA fragments to flow out of the gel and bind to the filter. A replica of the DNA fragments in the gel is created on the filter. Hybridization is performed with a labeled probe complementary to exons 17 and 18. Autoradiography reveals specific DNA fragments with which the probe hybridizes. Female 1 has two copies of the 1,165-bp (1.2-kb) fusion fragment. Female 2 has two copies of the 879-bp (0.9-kb) fragment and of the 286-bp (0.3-kb) fragment produced by the presence of the Bcl I site, which is labeled B* in Figure 1. Because the probe used in this example was complementary only to exons 17 and 18, the 286-bp fragments, although present, are not detectable by autoradiography (see Fig. 1). Female 3 is heterozygous for each allele; consequently, both a 1,165-bp and an 879-bp fragment are seen at 50% of the intensity of homozygotes. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 3 Direct diagnosis of causative mutation in two patients (Pt I and Pt II), illustrating that a deletion will produce an abnormal pattern with all restriction endonucleases but that a point mutation will produce an abnormal pattern only if it occurs in the code sequence of a restriction endonuclease. A, Diagram of a region of a gene, showing location of a deletion in patient I and a nonsense mutation in patient II detected by the restriction enzyme Taq I. B, DNA is extracted from patients I and II, digested with the restriction endonuclease Bcl I, and submitted to Southern blot analysis with use of a probe to the two exons (shaded region in A). Restriction pattern of patient I differs from normal (N) because the 3.5-kb deletion eliminates the middle Bcl I site and generates a single 2.6-6kb fragment that is 3.5 kb shorter than the combined length of the two normal fragments. C, Taq I recognizes the code sequence of TCGA. The dinucleotide CG is frequently methylated at the 5 position of C, and that methylated C is a “hot spot” for mutation to T. Thus, Taq I can be used to detect nonsense mutations at these sites. As illustrated, the nonsense mutation at the site C results in loss of a Taq I site and generation of a 5.2-kb fusion segment. If such a change occurs at a Taq I site in the right coding phase in an amino acid coding region, the result would be replacement of arginine with a nonsense codon. Taq I is particularly useful at detecting nonsense mutations in the factor VIII gene because five of the seven Taq I sites in exons are in the appropriate phase. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 4 Diagnosis by linkage analysis for three types of mendelian diseases. A fully shaded symbol represents an affected person, a half-shaded symbol represents an unaffected carrier, and an unshaded symbol denotes a noncarrier. Circles = females; squares = males. A, Simple pedigree in an X-linked disease such as hemophilia A. An intragenic restriction fragment length polymorphism (RFLP) has two alleles, A and B. These alleles can be determined by Southern blot analysis, as described in the text. The RFLP is used to mark the chromosome carrying the defective gene. Analysis of the DNA of the affected person (II-4) indicates that allele A is linked to the mutation. Because the single X chromosome from the affected person came from his mother who is an obligate heterozygote (assuming that the presence of other cases in the extended family establishes that II-2 is not a sporadic mutation), her chromosome with allele A must be linked to the defective gene, whereas allele B must be linked to a functional gene because she is asymptomatic. Her mate (I-2) has allele A on his single X chromosome, but because he is unaffected, allele A is linked in this case to a functional gene. The older brother (II-3) received allele B from the mother and, as expected, is unaffected. The younger daughter (II-2) is homozygous for allele A, an indication that she is a carrier because she has inherited from her mother the A allele that is linked to the mutated gene and from her father the A allele that is linked to a functional gene. The older daughter (II-1) is heterozygous for alleles A and B. Because her father has only one X chromosome to donate and that contains allele A, this daughter's allele A must be linked to a functional gene. Thus, allele B was obtained from the mother, and she is not a carrier. B, Simple pedigree in an autosomal recessive disease such as β-thalassemia. Affected person (II-3) is homozygous for allele A of an intragenic RFLP, an implication that both alleles are linked to mutated genes. Her parents are both heterozygous for alleles A and B. The older daughter (II-2) is heterozygous for alleles A and B and therefore is a carrier. The son (II-1) is homozygous for allele B, an indication of a noncarrier state. C, Simple pedigree in an autosomal dominant disease. Affected father (I-2) is heterozygous for an intragenic RFLP. Because the mother (I-1) is homozygous for allele A, the affected son (II-2) must have received allele A from his mother and allele B from his father; thus, allele B is linked to the mutation. The daughter (II-1) is homozygous for allele A, and she is unaffected. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 5 Diagram showing potential for alteration in linkage relationships with meiotic recombination. Before meiotic recombination, restriction fragment length polymorphism (RFLP) allele A is linked to the mutation (M), and RFLP allele B is linked to the functional gene (+). A, Meiotic recombination distal to both polymorphism and mutation has no effect on linkage relationship. Likewise, meiotic recombination proximal to both polymorphism and mutation also has no effect on linkage (not illustrated). B, Recombination between polymorphism and mutation changes their linkage relationship. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 6 Diagram showing that identification of biologic father is crucial to correct diagnosis of carrier status. Pedigree shows the six family members participating in carrier testing: mother (M), father (F), three sisters (S1, S2, and S3), and brother with hemophilia (H). A, The father has the 1.2-kb allele on his single X chromosome. Therefore, S1 must have received the 1.2-kb allele from the father. Thus, the 0.9-kb allele is from the mother. This is the allele linked to the defective factor VIII gene, so S1 is a carrier. Conversely, S2 must have obtained the 1.2-kb allele from the mother and therefore is not a carrier. Because S3 does not have a 1.2-kb allele, F could not be her biologic father. B, All restriction patterns are identical to those shown in A, except the father now has a 0.9-kb allele. In this case, S1 must have inherited the 0.9-kb allele from the father. Therefore, the 1.2-kb allele is from the mother, an indication that S1 is not a carrier. Because S2 does not have a 0.9-kb allele, F could not be her biologic father. Finally, S3 must have inherited the 0.9-kb allele from the mother and therefore is a carrier. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions

Fig. 7 Pedigree of hypothetical family with hemophilia A, as discussed in the text. Fully shaded symbols represent affected persons, half-shaded symbols represent carriers, and unshaded symbols represent noncarriers. A line through a symbol denotes deceased. Circles = females; squares = males. For further details, see text. Mayo Clinic Proceedings 1987 62, 387-404DOI: (10.1016/S0025-6196(12)65443-3) Copyright © 1987 Mayo Foundation for Medical Education and Research Terms and Conditions