Aa a a

Figure 40-9. Mutations in the P-globin gene causing P-thalassemia. The P-globin gene is shown in the 5' to 3' orientation. The cross-hatched areas indicate the 5' and 3' nontranslated regions. Reading from the 5' to 3' direction, the shaded areas are exons 1-3 and the clear spaces are introns 1 (I,) and 2 (I2). Mutations that affect transcription control (•) are located in the 5' flanking-region DNA. Examples of nonsense mutations (A), mutations in RNA processing (O), and RNA cleavage mutations (°) have been identified and are indicated. In some regions, many mutations have been found. These are indicated by the brackets.

Table40-6. Structural alterations of the ß-globin gene.

E. Pedigree Analysis

Table40-6. Structural alterations of the ß-globin gene.


Function Affected


Point mutations

Protein folding Transcriptional control Frameshift and nonsense mutations RNA processing

Sickle cell disease





mRNA production

P0-Thalassemia Hemoglobin Lepore


mRNA production

P-Thalassemia type III

tion of P-globin; P-thalassemia is the result of these mutations. (The thalassemias are characterized by defects in the synthesis of hemoglobin subunits, and so P-thalassemia results when there is insufficient production of P-globin.) Figure 40-9 illustrates that point mutations affecting each of the many processes involved in generating a normal mRNA (and therefore a normal protein) have been implicated as a cause of P-thalassemia.

D. Deletions, Insertions, & Rearrangements of DNA

Studies of bacteria, viruses, yeasts, and fruit flies show that pieces of DNA can move from one place to another within a genome. The deletion of a critical piece of DNA, the rearrangement of DNA within a gene, or the insertion of a piece of DNA within a coding or regulatory region can all cause changes in gene expression resulting in disease. Again, a molecular analysis of P-thalassemia produces numerous examples of these processes—particularly deletions—as causes of disease (Figure 40-8). The globin gene clusters seem particularly prone to this lesion. Deletions in the a-globin cluster, located on chromosome 16, cause a-thal-assemia. There is a strong ethnic association for many of these deletions, so that northern Europeans, Filipinos, blacks, and Mediterranean peoples have different lesions all resulting in the absence of hemoglobin A and a-thalassemia.

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure.

Sickle cell disease again provides an excellent example of how recombinant DNA technology can be applied to the study of human disease. The substitution of T for A in the template strand of DNA in the ß-globin gene changes the sequence in the region that corresponds to the sixth codon from to





Coding strand Template strand

Coding strand Template strand and destroys a recognition site for the restriction enzyme MstlI (CCTNAGG; denoted by the small vertical arrows; Table 40-2). Other MstlI sites 5' and 3' from this site (Figure 40-10) are not affected and so will be cut. Therefore, incubation of DNA from normal (AA), heterozygous (AS), and homozygous (SS) individuals results in three different patterns on Southern blot transfer (Figure 40-10). This illustrates how a DNA pedigree can be established using the principles discussed in this chapter. Pedigree analysis has been applied to a number of genetic diseases and is most useful in those caused by deletions and insertions or the rarer instances in which a restriction endonuclease cleavage site is affected, as in the example cited in this paragraph. The analysis is facilitated by the PCR reaction, which can provide sufficient DNA for analysis from just a few nucleated red blood cells.

F. Prenatal Diagnosis

If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from as little as 10 mL of amniotic fluid (or by chorionic villus biopsy) can be analyzed by Southern blot transfer. A fetus with the restriction pattern AA in Figure 40-10 does not have sickle cell disease, nor is it a carrier. A fetus with the SS pattern will develop the disease. Probes are now available for this type of analysis of many genetic diseases.

G. Restriction Fragment Length Polymorphism (RFLP)

The differences in DNA sequence cited above can result in variations of restriction sites and thus in the length of restriction fragments. An inherited difference in the pattern of restriction (eg, a DNA variation occurring in more than 1% of the general population) is known as a restriction fragment length polymorphism,

A. Mstll restriction sites around and in the j-globin gene

Sickle (S) 5'

f 1.35 kb

B. Pedigree analysis

Fragment size

1.35 kb

1.15 kb

AS AS SS AA AS AS Phenotype

Figure 40-10. Pedigree analysis of sickle cell disease. The top part of the figure (A) shows the first part of the j-globin gene and the MstlI restriction enzyme sites in the normal (A) and sickle cell (S) j-globin genes. Digestion with the restriction enzyme Mstll results in DNA fragments 1.15 kb and 0.2 kb long in normal individuals. The T-to-A change in individuals with sickle cell disease abolishes one of the three Mstll sites around the j-globin gene; hence, a single restriction fragment 1.35 kb in length is generated in response to Mstll. This size difference is easily detected on a Southern blot. (The 0.2-kb fragment would run off the gel in this illustration.) (B) Pedigree analysis shows three possibilities: AA = normal (open circle); AS = heterozygous (half-solid circles, half-solid square); SS = homozygous (solid square). This approach allows for prenatal diagnosis of sickle cell disease (dash-sided square).

or RFLP. An extensive RFLP map of the human genome has been constructed. This is proving useful in the human genome sequencing project and is an important component of the effort to understand various single-gene and multigenic diseases. RFLPs result from single-base changes (eg, sickle cell disease) or from deletions or insertions of DNA into a restriction fragment (eg, the thalassemias) and have proved to be useful diagnostic tools. They have been found at known gene loci and in sequences that have no known function; thus, RFLPs may disrupt the function of the gene or may have no biologic consequences.

RFLPs are inherited, and they segregate in a mendelian fashion. A major use of RFLPs (thousands are now known) is in the definition of inherited diseases in which the functional deficit is unknown. RFLPs can be used to establish linkage groups, which in turn, by the process of chromosome walking, will eventually define the disease locus. In chromosome walking (Figure 40-11), a fragment representing one end of a long piece of DNA is used to isolate another that overlaps but extends the first. The direction of extension is determined by restriction mapping, and the procedure is repeated sequentially until the desired sequence is obtained. The X chromosome-linked disorders are particularly amenable to this approach, since only a single allele is expressed. Hence, 20% of the defined RFLPs are on the X chromosome, and a reasonably complete linkage map of this chromosome exists. The gene for the X-linked disorder, Duchenne-type muscular dystrophy, was found using RFLPs. Likewise, the defect in Huntington's disease was localized to the terminal region of the short arm of chromosome 4, and the defect that causes polycystic kidney disease is linked to the a-globin locus on chromosome 16.

H. Microsatellite DNA Polymorphisms

Short (2-6 bp), inherited, tandem repeat units of DNA occur about 50,000-100,000 times in the human genome (Chapter 36). Because they occur more frequently—and in view of the routine application of sensitive PCR methods—they are replacing RFLPs as the marker loci for various genome searches.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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