1. Chapter 29 Essential Concepts in Molecular Pathology Companion site for Molecular Pathology Author: William B. Coleman and Gregory J. Tsongalis

2. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 2 FIGURE 29.1 Schematic representation of the Ce\GG repeat in exon 1 of FMR1 and associated alleles. A CGG-repeat number less than or equal to 45 is normal. A CGG-repeat number of 46 to 54 is in the gray zone and has been reported to expand to a full mutation in some families. A CGG-repeat number of 55 to 200 is considered a premutation allele and is prone to expansion to a full mutation during female meiosis. A CGG-repeat number in excess of 200 is considered a full mutation and is diagnostic of fragile X syndrome.

3. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 3 FIGURE 29.2 Southern blot analysis for the diagnosis of fragile X syndrome. Patient DNA is simultaneously digested with restriction endonucleases EcoR1 and Eag1, blotted to a nylon membrane, and hybridized with a 32P-labeled probe adjacent to exon 1 of FMR1 (see Figure 29.1). Eag1 is a methylation-sensitive restriction endonuclease that will not cleave methylated DNA. Normal male control DNA with a CGG-repeat number of 22 on his single X chromosome (lane 1) generates a band about 2.8 kb in length corresponding to Eag1-EcoR1 fragments (see Figure 29.1). Normal female control DNA with a CGG-repeat number of 20 on one X chromosome and a CGG-repeat number of 25 on her second X chromosome (lane 5) generates two bands, one at about 2.8 kb and a second at 5.2 kb. EcoR1-EcoR1 fragments approximately 5.2 kb in length represent methylated DNA sequences characteristic of the lyonized chromosome in each cell that is not digested with restriction endonuclease Eag1. DNA in lane 2 contains an FMR1 CGG-repeat number of 90 and is characteristic of a normal transmitting male. The pattern observed in lane 3 is representative of a mosaic male with a single X chromosome with a full mutation (>200 repeats). However, the full mutation in some cells is unmethylated; in other cells, the full mutation is fully methylated, hence the term mosaic. In those cells in which the full mutation is unmethylated, digestion by both Eag1 and EcoR1 occurs, and in those cells in which the full mutation is fully methylated, digestion of the DNA by Eag1 is inhibited. The pattern observed in lane 4 is diagnostic of a male with fragile X syndrome, illustrating the typical expanded allele fully methylated in all cells. Lane 6 is characteristic of a female with one normal allele that has 29 CGG-repeats and a larger gray zone allele with a CGG-repeat number of 54. Lane 7 is the pattern observed from a premutation carrier female with one normal allele having a CGG-repeat number of 23 (band at about 2.8 kb) and a second premutation allele with CGG repeats of 120 to about 200 (band at about 3.1 kb). In premutation carrier females, in cells in which the X chromosome with the premutation allele is lyonized, the normal 5.2 kb EcoR1-EcoR1 band is larger because of the increased CGG-repeat number and is about 5.5 kb in length. Lane 8 is diagnostic of a female with fragile X syndrome with one fully expanded mutation allele that is completely methylated and a second normal allele with a CGG-repeat number of 33.

4. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 4 FIGURE 29.3 HCV genotyping assay. Comparison of samples from patients infected with HCV genotypes 1b, 2a/c, 2b, and 3a. Shown are genotype-specific melting transitions for four samples in 2 mM MgCl2. Data were obtained by monitoring the fluorescence of the LCRed640-labeled FRET sensor probe during heating from 40 to 80°C at a temperature transition rate of 0.1°C/s.

5. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 5 FIGURE 29.4 T-cell receptor gamma chain PCR assay for clonality. (A) Polyclonal reactive T-cell proliferation pattern. A polyclonal population of T-cells with randomly rearranged T-cell receptor gamma chain genes produces a normal or Gaussian distribution of fluorescently labeled PCR products from each primer pair in the multiplex reaction. This produces four bell-shaped curves that represent the valid size range for an individual primer pair. Two of the valid size ranges are green and two are blue; G1, B1, G2, B2. The red peaks represent size standards. (B) Clonal T-cell proliferation pattern. A clonal T-cell proliferation results in a relative dominance of a single T-cell receptor gamma chain gene producing a predominant spike of a discrete size on the corresponding electropherogram. Data were obtained using an ABI 3100 capillary electrophoresis system and ABI Prism Software.