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Targeted sequencing with the Ion Torrent System is able to identify single nucleotide variants, small insertions and small deletions. Variants in repeat sequences, large homopolymers and large insertions/deletions are not or difficult to identify.

The RBC Hemoglobinopathie AmpliSeq Panel (IAD127644_241) exist of 141 primer pairs, which amplify 151 different amplicons. Five primer pairs were designed based on HBA1 gene coordinates and are also 100% specific for the HBA2 gene and five primer pairs were designed based on HBG1 gene coordinates and are also 100% specific for the HBG2 gene. The panel design is covering 35.28 Kbase, and includes almost all coding regions (exons), flanking intronic regions, untranslated regions and known promotor regions of the genes of interest as depicted in Table 1. Also genomic regions that are involved in the expression of the globins and in fetal γ- to adult β-globin switching, are included in the design, as listed in Table 1.

Table 1: List of genes and genomic regions and the coverage of these regions

 

 

 

 

 

 

 

 

For ZBTB7A gene, 31 bases are missed in exon 1 and 2, divided over 4 different amplicons. Also 2 bases and 1 bases of the regions MYB- HBS1L and BCL11A-enhancer3 are missed in the design. There are no disease causing variants published in these missing areas as there are no consequences reported for copy number variants (HGMD version 2017.2). See Table 2.

Table 2: The panel design does not cover the following bases

In Table 3 is listed the published disease causing variants HGMD version 2017.2) which are not covered in the designed panel. Including these regions in the design failed caused by high sequence homology between HBB and HBD gene.

Table 3: List of missed HGMD disease causing variants in the designed Hb panel

For accepting a sequence run with the Hemoglobinopathie AmpliSeq panel, the %Base20x of all amplicons must be at least 99.2%. With this acceptance criteria, the first 60 bases of exon 1 and the complete 5’UTR of the HBA2 gene will be less covered than 20 times per base. This may result in low coverage for 35 DM and 3 DM? variants located in this region (see Table 4a&b). Also small regions in HBG1, GATA1 and BCL11A have a  low coverage, but without missing published DM variants, as listed in Table 4a.
Preferably, a %Base20x coverage of at least 99.7% must be reached to insure that also exon 1 of gene HBA2 is completely covered. Under these condition, ≥99.7% coverage, only 3DM and 1DM? variant located in the 5’UTR region will have a low coverage.

Table 4: Failed amplicons and their missing published mutations at %Base20x coverage of 99.2%

Table 5: HGMD DM and DM? variants of gene HBA2 missed at %Base20x coverage of 99.2% and 99.7%

Reporting

Only clinical relevant variants will be reported. Variants with classification Certainly Pathogenic (class 5) and Likely Pathogenic (class 4) are always reported. Variants with category Unknown significance (class 3) will only be reported if the variant is expected to be involved in the phenotype of the patient. Category Certainly Benign (class 1) and Likely Benign (class 2) variants will not be reported. (see: Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics).  
Besides reporting the clinical relevant variants we report whether a patients is heterozygous, homozygous, expected compound heterozygous or hemizygous for a mutation and  how this may relate to disease phenotype.
All the variants are annotated and reported as designated by the Human Genome Variation Society (HGVS) nomenclature, as described at their website.