Table 2 Examples of diagnostic gene sequencing panel considerations and recommendations

From: Diagnostic gene sequencing panels: from design to report—a technical standard of the American College of Medical Genetics and Genomics (ACMG)

IssueExampleGenetic aberrationMethodologyRecommendations
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 panelFragile XTrinucleotide repeat expansionTriplet-primed PCR, methylation studies, trinucleotide repeat analysis, Southern blottingLaboratory 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 atrophyDeletion of SMN1CNV assay to differentiate SMN1 and SMN2 copy number (e.g., MLPA)
Charcot–Marie–Tooth1.5-Mb duplication at 17p11.2CNV assay
Krabbe disease30-kb deletionAllele-specific PCR or CNV assay
Sandhoff disease16-kb deletion 
Hemophilia AF8 intron 22 inversionAllele-specific PCR
b. Certain pathogenic variants are commonHearing lossCommon GJB2 pathogenic variantsTargeted testing for c.35delG or Sanger sequencing of the single GJB2 coding exonLaboratory 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 fibrosisCommon variants in CFTRTargeted testing for common pathogenic variants
c. Differential diagnosisHypertrophic cardiomyopathyMost 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 allelesBreast cancerCHEK2 (NM_007194.4): c.1100delDetected in routine sequence analysis; however, risk allele could be reference alleleWeigh the pros and cons of including genes with low penetrance on a NGS panel; prepare appropriate interpretations
Hereditary prion diseasePRNP (NM:000311.4):c.532G>A (p.D178N)
Familial Mediterranean feverMEFV (NM_000243 .2): c.442G>C (p.E148Q)
e. Pseudogenes and gene familiesLynch syndromeHigh homology between PMS2, PMS2CL, and other pseudogenes; gene conversion between PMS2 and PMSCLLong-range PCR prior to sequencingIf PMS2 orGBA genes are included as part of a gene panel, auxiliary methodology must be implemented to address homology issue
Gaucher diseaseHigh homology between GBA and GBAP; gene conversion between GBA and GBAP
f. MosaicismProteus syndromeAKT1 somatic variantsWhenever possible, directly test affected tissuesDelineate sample acceptance criteria and limitations of testing easily accessible tissues (e.g., blood/saliva), and threshold for mosaicism detection
g. TranscriptHereditary breast ovarian cancerBRCA1 pathogenic variants are transcript-specific: NM_007294.3:c.2603C>G(p.Ser868Ter) NM_007299.3:c.787+1816C>GDepending on the transcript used the same variant may appear to either be a truncating variant or to fall in a deep intronic regionDuring test design ensure that to the extent possible pathogenic variants are captured and annotated, or listed in the test limitations
  1. aCGH array comparative genomic hybridization, CNV copy-number variant, MLPA multiplex ligation probe amplification, NGS next-generation sequencing, PCR polymerase chain reaction.