Transcript Managing people in sport organisations: A strategic human resource

Chapter 28
PART V: Practice of Molecular Medicine
Molecular Diagnosis of Human Disease
Companion site for Molecular Pathology
Author: William B. Coleman and Gregory J. Tsongalis
FIGURE 28.1
Schematic representation of the CGG 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.
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FIGURE 28.2
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 28.1). Eag1 is a methylation-sensitive restriction endonuclease that will not cleave
the recognition sequence if the cytosine in the sequence is methylated. 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 28.1). 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 banding 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 banding 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 a normal allele and a CGG-repeat number of 29 and a larger gray
zone allele with a CGG-repeat number of 54. Lane 7 is the banding 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 full expansion mutation allele that is completely methylated and transcriptionally silenced on one X chromosome
but with a second normal allele with a CGG-repeat number of 33.
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FIGURE 28.3
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.
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FIGURE 28.4
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 4 "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.
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