Figure 1: Blood smear
Figure 1: Shows reduced density of red blood cells with severe anisopoikilocytosis and the presence of nucleated RBC’s.
Figure 2: HPLC pattern of the index case with thalassemia major
HPLC of index case with elevated HbA2 and abundant HbF at 95% without HbA.
Figure 3: HPLC pattern of the mother of index case with beta thalassemia trait.
Note the presence of HbA in the mother with isolated elevated HbA2 indicative of thalassemia trait.
Figure 4: Molecular studies
Genomic DNA from peripheral blood leucocytes was extracted using Qiagen QIAamp DNA Blood Midi kit and tested for the beta globin gene mutations.
- Amplification Refractory Amplification System (ARMS) PCR was performed for the Common 5 Indian mutations. ARMS PCR showed positivity for Cd 8/9 (+G) (HGVS c.27_28insG) and Cd 41/42 (-TTCT) (HGVS c.126_129 del TTCT) compound heterozygosity.
No Image is attached in this Figure.
Figure 5: Sanger sequencing
Exon 1 sequencing showed the frameshift mutation Cd 8/9 (+G) in Index case and the mother.
Figure 6: Sanger sequencing
Exon 2 sequencing the frameshift mutation Cd 41/42 (-TTCT) ) in Index case and the father.
Figure 7: Prenatal diagnosis with chorionic villus sampling for next pregnancy (NOT INDEX case)
Prenatal diagnosis was performed for the next pregnancy after a chorionic villous biopsy (CVS) at 11 weeks gestation.
Lane 1, Marker (left most lane);
Lane 2, negative control for Cd 8/9 (+G) mutant;
Lane 3, positive control for Cd 8/9 (+G) mutant;
Lane 4, CVS negative for Cd 8/9 (+G) mutant;
Lane 5, CVS positive for Cd 41/42 (-TTCT);
Lane 6, positive control for Cd 41/42 (-TTCT) mutant;
Lane 7, negative control for Cd 41/42 (-TTCT);
Lane 8, All PCR reagents except DNA to exclude DNA contamination in any of the reagents (right most lane).
Final Interpretation: Fetal sample is positive for the father’s mutation Cd 41/42 (-TTCT) and negative for the mother’s mutation.
Fetus is a beta thalassemia trait.
