Table 2 Examples of diagnostic gene sequencing panel considerations and recommendations
Issue | Example | Genetic aberration | Methodology | Recommendations |
|---|---|---|---|---|
a. First-tier non-NGS testing often performed first due to disease mechanism; testing for common disease mechanism, which does not need to be repeated, is not included in the panel | Fragile X | Trinucleotide repeat expansion | Triplet-primed PCR, methylation studies, trinucleotide repeat analysis, Southern blotting | Laboratory must highlight the common disease-causing mechanism in the recommendation and in the limitation; orderable stand-alone test, with option to reflex to or combine with sequencing panel |
Spinocerebellar ataxias | ||||
Huntington disease | ||||
Spinal muscular atrophy | Deletion of SMN1 | CNV assay to differentiate SMN1 and SMN2 copy number (e.g., MLPA) | ||
Charcot–Marie–Tooth | 1.5-Mb duplication at 17p11.2 | CNV assay | ||
Krabbe disease | 30-kb deletion | Allele-specific PCR or CNV assay | ||
Sandhoff disease | 16-kb deletion | |||
Hemophilia A | F8 intron 22 inversion | Allele-specific PCR | ||
b. Certain pathogenic variants are common | Hearing loss | Common GJB2 pathogenic variants | Targeted testing for c.35delG or Sanger sequencing of the single GJB2 coding exon | Laboratory must highlight the common disease-causing mechanism in the recommendation and in the limitation; orderable stand-alone test, with option to reflex to or combine with sequencing panel |
Cystic fibrosis | Common variants in CFTR | Targeted testing for common pathogenic variants | ||
c. Differential diagnosis | Hypertrophic cardiomyopathy | Most commonly sarcomere genes, but mimicked by other syndromic genes (e.g., GLA, PRKAG2, LAMP2) | Detected in routine sequence analysis if gene is analyzed | |
d. Reduced penetrance alleles | Breast cancer | CHEK2 (NM_007194.4): c.1100del | Detected in routine sequence analysis; however, risk allele could be reference allele | Weigh the pros and cons of including genes with low penetrance on a NGS panel; prepare appropriate interpretations |
Hereditary prion disease | PRNP (NM:000311.4):c.532G>A (p.D178N) | |||
Familial Mediterranean fever | MEFV (NM_000243 .2): c.442G>C (p.E148Q) | |||
e. Pseudogenes and gene families | Lynch syndrome | High homology between PMS2, PMS2CL, and other pseudogenes; gene conversion between PMS2 and PMSCL | Long-range PCR prior to sequencing | If PMS2 orGBA genes are included as part of a gene panel, auxiliary methodology must be implemented to address homology issue |
Gaucher disease | High homology between GBA and GBAP; gene conversion between GBA and GBAP | |||
f. Mosaicism | Proteus syndrome | AKT1 somatic variants | Whenever possible, directly test affected tissues | Delineate sample acceptance criteria and limitations of testing easily accessible tissues (e.g., blood/saliva), and threshold for mosaicism detection |
g. Transcript | Hereditary breast ovarian cancer | BRCA1 pathogenic variants are transcript-specific: NM_007294.3:c.2603C>G(p.Ser868Ter) NM_007299.3:c.787+1816C>G | Depending on the transcript used the same variant may appear to either be a truncating variant or to fall in a deep intronic region | During test design ensure that to the extent possible pathogenic variants are captured and annotated, or listed in the test limitations |
