Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency

Round 1

Reviewer 1 Report

Cifuentes R and co-authors describe their experience with MLPA analysis of SERPINC1 in the diagnosis of antithrombin deficiency (ATD). They explored the usefulness and limitations of this genetic method by investigating n=341 samples. According their diagnostic protocol, first they perform Sanger sequencing of the coding regions of SERPINC1, by which the causative mutations are found in most of the cases. Next, Sanger sequencing of the entire SERPINC1 is carried out, by which some intronic or promoter mutations also can be found. MLPA is the third step in ATD genetic diagnosis, here n=54 cases were investigated by this method. They have found 22 SVs. In the remaining 32 cases, whole gene analysis by long-range PCR and NGS sequencing and long-read whole genome sequencing were performed. By these n=4 cases were clarified. After analysis of SVs by alternative methods n=6 cases were found, where the MLPA analysis gave false-positive or false-negative results. They have found a so-called hidden SV (heterozygous deletion of exon 7) in a patient previously diagnosed as type I ATD by Sanger sequencing. Moreover, they have found MPLA results as false-positive in three cases (one small INDEL, and two SNVs). By this study they draw the attention to the misdiagnosis of causative SVs not only in ATD but also in all genetic disorders.

The paper is interesting and well-written, and very useful for those working with ATD and genetic diagnostics. There are only some minor points which should be addressed.

The abbreviation “SVs” should be clarified also in the abstract. Allele frequency “< 10-3 “and “< 10-4”, -3 and -4 should be written in superscript.

Author Response

We would like to acknowledge the kind comments and suggestions by reviewer 1 on our manuscript.

 We totally agree with his/her suggestions which have already been adressed in the revised manuscript

Reviewer 2 Report

The frequency of thrombophilia-related pathology is steadily increasing justifying a constant preoccupation with the detection of its etiopathogenic background. Congenital Antithrombin III deficiency (ATD) is the most severe cause of thrombophilia, and it is important to be detected taking into account the current efficacious modalities of its therapeutical approach.

This article, of great professional expertise, succeeded to give valuable insight into the exploratory field of this thrombophilia-connected factor. On a large ( N=341 ) cohort of unrelated patients with ATD, besides sequencing methods for detection of SNVs (Single nucleotide variant ) and INDELs ( small insertions or deletions), it was used also a Multiplex ligation-dependent probe amplification (MLPA). This targeted analysis, a multiplex PCR technique, aimed at detecting Structural Variants (SVs), and genomic alterations that involve DNA segments larger than 1kb. Although they are less frequent than SNVs or INDELs, involving a larger number of base pairs, they can have a greater pathological impact. The authors using MLPA could identify 22SVs cause of ATD, confirming the utility of this technique; at the same time, the authors underlined also some limitations, determined by false positive or negative results. They recommend the validation of MLPA results by other methods

The article reflects a valuable   activity in the field of exploration, with decisive value  in the present evidence based medicine.

Comments/correction

-p3/r50-to put 3,5  between  brackets

-9 authors of the present article  are included  also in the list  of 19 references

Author Response

We would like to acknowledge the kind comments and suggestions by reviewer 1 on our manuscript.

 We totally agree with his/her suggestions which have already been adressed in the revised manuscript