Diagnostic of Large Genetic Defects in Blood Coagulation Genes: NGS vs. MLPA

Background Large deletions/duplications account for 5% in patients with hemophilia A and B. The conventional method used to detect these large gene alterations is MLPA. In majority of cases, this method is good, standardized, and reliable. Nevertheless, there are cases where the MLPA displays pitfalls in routine diagnostic. NGS (next-generation DNA sequencing) is a high-throughput method, which allows not only quick and consistent sequencing results but also overcomes the disadvantages of MLPA in the detection of large deletions/duplications.

Aim Here we report eight cases in which large genetic variants were successfully detected with NGS, where MLPA was not able to obtain reliable results.

Materials and Methods Large genetic variants were analyzed by MLPA and NGS according to the manufacture’s recommendation.

Results Three cases represent the first group, where MLPA gave inconsistent results. In case 1, MLPA diagnosed a false-positive large deletion of exon 1 in the F9 due to small insertion in ligation binding site. In two other cases, large deletions in exon 13 and 18 of the F8 were not detected by MLPA, while the ligation binding site was outside the region of the deletion. All these cases were successfully identified by NGS. The second group summarizes four cases, where Klinefelter and Turner syndromes were accidentally identified in the frame of routine F8 and F9 genetic diagnostic. This event was possible only due to the fact by NGS the number of X-chromosomes can clearly be displayed. The third group comprises two cases. In these female patients, the normal sequencing results reveal missense mutations in the F8 gene, which was enough to confirm the carrier status. As both females showed relative low values of FVIII:C, the samples were subjected to further investigation with NGS for additional defects. Surprisingly, these data reveal additional large heterozygous deletion in one of the females and heterozygous large duplication in the other, which were not diagnosed by standard sequencing method.

Conclusion Putting together all these results and our experience, we can conclude that NGS is a reliable method for routine diagnostic not only of small genetic variants but also for detection of large deletion/duplication in hemi- and heterozygous state. Additionally, this method allows simultaneous detection of aneuploidies. The application of NGS in the routine analyses makes the genetic diagnostic of hemophilia patients safer and quicker.

Publication History

Article published online:
13 November 2020