|
Figure 1.
Preimplantation genetic
diagnosis (PGD) for B-thalassemia performed by using the
minisequencing technique.
A) Pedigree of a couple carrying B-thalassemia mutations and
examples of different results of the HBB gene mutation
analysis. Informative STR markers are ordered from telomere
(top) to centromere (bottom). The numbers in STR markers
represent the size of PCR products in base pair. STR alleles
linked to the paternal and maternal mutations are
represented in boldface.
B) Examples of minisequencing results obtained for the above
case. The mutations of interest are IVSI-110 G-A and IVSI-6
T/C, analysed in a multiplex reaction format. The y-axis
represents the Relative Fluorescence Units (RFU) of the
detected fragments; the x-axis represents time and is
displayed by data points. Colour is assigned to individual
ddNTPs as follows: green/A, black/C, blue/G, red/T. Mutation
IVSI-110 G-A is shown on the left of the minisequencing
window; the blue peak represents the normal allele (wild
type base G), the green peak (mutant base A) the mutated
allele. Mutation IVSI-6 T/C is shown on the right; the red
peak is the normal allele (wild type base T) and the black
peak (mutant base C) is the mutated allele.
Embryo 1 (upper panel/left) is carrier for IVSI-6 T/C
mutation; Embryo 3 (upper panel/right) is normal; Embryo 2
(lower panel/left) is compound heterozygote for the two
mutations. Embryo 4 (lower panel/right) is also affected,
although minisequencing result shows a heterozygosity for
mutation IVSI-110 G-A. In fact, linked STR markers highlight
an allele drop-out (ADO) of the affected allele (black peak,
mutant base C). |
