**3.2. Candidate gene panel results**

A total of 703 non-synonymous variants were detected; 75 of these variants were novel and had not been reported in the dbSNP database. An average of eight non-synonymous variants was detected per patient.

Two individuals with known mutations in *GFI1B, GP1BA* and *GP9* by Sanger sequencing were included as controls. NGS successfully called the first, *GFI1B* c.880-881insC, but failed to detect the second, a patient with a phenotype consistent with the inherited macrothrombocytopenia Bernard-Soulier syndrome (BSS). This patient's genotype had previously been confirmed by Sanger sequencing and included mutations in both the *GPIBA* (*GPIBA* c.2217C>T) and the *GP9* genes (c.1829A>G and c.1859T>G). Failure to detect these mutations may have been caused by sequencing errors introduced by GC-rich motifs in these regions [36, 37].

Pathogenic mutations were detected in 16 individuals (17.4% of the cohort) whilst 36 individ‐ uals (39.1%) had VUS and 40 individuals (43.0%) were without identifiable pathogenic mutations (Table 2, Table 3).


\*Parents heterozygous; child with homozygous mutation giving rise to a Glanzmann thrombasthaenia phenotype.

\*\* These mutations are likely pathogenic.That is, the detected variation is unreported in the literature to date, however, based on the type of variation, it's deleterious effect predicted using bioinformatic tools (see data analysis) and the associated phenotypic data, is of the type to cause the disorder

**Table 2.** Mutations detected in the candidate genes. Genes affecting the platelet cytoskeleton (top, white shading), the platelet granules (light gray shading) and platelet-related transcription factors (dark gray shading).


\* Parents heterozygous. Child with homozygous mutation giving rise to a Glanzmann thrombasthaenia phenotype.

\*\* Mutations are likely pathogenic.

Two individuals with known mutations in *GFI1B, GP1BA* and *GP9* by Sanger sequencing were included as controls. NGS successfully called the first, *GFI1B* c.880-881insC, but failed to detect the second, a patient with a phenotype consistent with the inherited macrothrombocytopenia Bernard-Soulier syndrome (BSS). This patient's genotype had previously been confirmed by Sanger sequencing and included mutations in both the *GPIBA* (*GPIBA* c.2217C>T) and the *GP9* genes (c.1829A>G and c.1859T>G). Failure to detect these mutations may have been caused by

Pathogenic mutations were detected in 16 individuals (17.4% of the cohort) whilst 36 individ‐ uals (39.1%) had VUS and 40 individuals (43.0%) were without identifiable pathogenic

> **Number of mutations detected of uncertain significance**

sequencing errors introduced by GC-rich motifs in these regions [36, 37].

**mutations**

*ACTN1* 0 8 *GP1BA* 1\*\* 2 *GP1BB* 0 2 *GP9* 0 1 *MYH9* 6 3 *TUBB1* 0 3 *NBEAL2* 1 7 *FLI1* 0 1 *GATA1* 0 3 *GFI1B* 3 2 *RUNX1* 2\*\* 0 *CD36* 0 13 *F2R* 0 0 *GP6* 0 5 *ITGA2* 0 4 *ITGA2B* 3\* 6 *ITGB1* 0 0 *ITGB3* 0 0 *P2RY12* 0 0

**Total Number** 16 60 mutations in 36 individuals Number of individuals without pathogenic mutations identified: 40

\*Parents heterozygous; child with homozygous mutation giving rise to a Glanzmann thrombasthaenia phenotype. \*\* These mutations are likely pathogenic.That is, the detected variation is unreported in the literature to date, however, based on the type of variation, it's deleterious effect predicted using bioinformatic tools (see data analysis) and the

**Table 2.** Mutations detected in the candidate genes. Genes affecting the platelet cytoskeleton (top, white shading), the

platelet granules (light gray shading) and platelet-related transcription factors (dark gray shading).

**Genes Number of individuals with pathogenic**

394 Next Generation Sequencing - Advances, Applications and Challenges

associated phenotypic data, is of the type to cause the disorder

mutations (Table 2, Table 3).

**Table 3.** Pathogenic genetic variants detected: nucleotide cDNA changes and corresponding protein alterations.

The candidate array was successful in detecting mutations in genes commonly associated with macrothrombocytopenia and included a total of nine *MYH9* mutations (six of which had previously been reported in the literature as pathogenic and three of which are of uncertain significance) (Figure 2) and a compound heterozygous mutation of *NBEAL2* in keeping with Gray platelet syndrome.

**Figure 2.** MYH9 variants detected in the candidate gene panel. Exons 2–20 encode the head and neck domains of NMMHC IIA (Blue block). Exons 21–41 encode the tail domains. Mutations were detected in exons 2, 17, 31 and 33. Six pathogenic mutations (red text) and three variants of uncertain significance (black text) were detected.

A homozygous mutation of *ITGA2B* was also detected and confirmed a suspected Glanzmann thrombasthenia phenotype. Several transcription factor variants were found, including a *FLI1* mutation of uncertain significance in one patient, three *GATA1* mutations of uncertain significance in three individuals from two families, three pathogenic *GFI1B* mutations in three individuals from two families and two of uncertain significance in two individuals in another two families. *RUNX1* mutations were identified in three individuals from three families; two of these were considered likely pathogenic, whilst the third was shown to represent a false positive result (*RUNX1*, heterozygous, stop/gain, c. 966T>G (p.Tyr322X), exon 6). False positivity was confirmed by Sanger sequencing that showed a wild-type sequence across that region.

Sanger sequencing was also performed in selected samples across regions of low coverage (Q < 30) from those genes in which the clinical significance is widely accepted and included, *GP9, GP1BA, GPIBB, FLI1* exon 3, *FLI1* exon 9, *MYH9* exon 20, *MYH9* exon 37 and *GFI1B* exon 5. This confirmatory step detected a novel mutation in *FLI1* [38], not identified by NGS.
