Germline Analysis
A Complete Guide to Inherited Variant Testing

A germline variant is a genetic change present in the germ cells, eggs or sperm, and therefore transmitted to every cell in the offspring's body at conception. Germline variants are either inherited from one or both biological parents, or arise as de novo mutations during the formation of germ cells or in the earliest cell divisions of a developing embryo.

Because germline variants are present in every nucleated cell, they can be detected from any tissue sample: peripheral blood, saliva, buccal swabs, or skin. This is a fundamental distinction from somatic variants, which arise in individual cells after birth and are present only in cells descended from the originally mutated cell.

The clinical significance of a germline variant depends on what gene it affects, how it changes gene function, the inheritance pattern of the associated condition, and whether the variant has been seen before in affected individuals.

One of the most important conceptual frameworks in clinical genomics. Increasingly relevant as tumor profiling and germline testing are performed on the same patients.

Before any germline variant can be interpreted, the inheritance pattern of the associated condition must be established. Inheritance determines who in the family is at risk, what allele frequency is expected in affected individuals, and how strong the evidence for pathogenicity must be.

Germline analysis is not a single test with a single purpose. It is a family of clinical applications, each using the same foundational technology to answer a different question for a different patient population.

Germline variants are classified using the 2015 ACMG/AMP framework (Richards et al., Genetics in Medicine), a 5-tier system:

• Pathogenic (P): strong evidence that the variant causes disease.
• Likely Pathogenic (LP): >90% probability the variant is disease-causing.
• Variant of Uncertain Significance (VUS): insufficient evidence to classify definitively.
• Likely Benign (LB): >90% probability the variant is benign.
• Benign (B): strong evidence the variant does not cause disease.

Classification is determined by applying 28 criteria organized into evidence categories: population data, computational/functional evidence, segregation, de novo occurrence, allele data, and others. Each is weighted Very Strong, Strong, Moderate, or Supporting. ClinGen Expert Panels publish gene-specific rule refinements for many clinically important genes, adjusting criteria based on the biology and clinical context of each gene. These refinements improve classification consistency and reduce VUS rates.

For the complete treatment including how the 28 criteria are applied in practice, see the tertiary analysis and genome interpretation guides.

When WES or WGS is performed for any clinical indication, the ACMG recommends labs actively evaluate and report pathogenic and likely pathogenic variants in a defined list of medically actionable genes, even when those genes are unrelated to the primary indication. These are secondary findings (previously "incidental findings").

What is on the list

SF v3.2 (updated 2023) includes 81 genes associated with conditions where (1) a pathogenic variant substantially increases disease risk, (2) effective preventive or clinical interventions exist, and (3) knowledge of the variant enables risk reduction. The list spans hereditary cancer predisposition (BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, APC, RET, others), hereditary cardiovascular conditions (MYBPC3, MYH7, KCNQ1, SCN5A, LDLR, others), and other actionable conditions (RYR1 for malignant hyperthermia, HFE for hereditary hemochromatosis).

What reporting requires

• Pre-test informed consent. Patients must be counseled about secondary findings before testing and given the option to opt in or out. Some patients prefer not to know findings unrelated to their primary indication. That preference must be respected and documented.
• Active analysis. Labs must actually evaluate the SF v3.2 gene list, not simply report secondary findings if they happen to appear during primary analysis. A deliberate analytical step.
• Genetic counseling for positive findings. A pathogenic or likely pathogenic secondary finding triggers referral for specialist genetics counseling and clinical management.

The right test depends on the clinical indication, phenotype specificity, and what prior testing has revealed.

For detailed guidance on WES vs WGS including diagnostic yield comparisons, see the WES vs WGS guide.

As tumor-based NGS expands, germline testing plays an increasingly important role in cancer care, both as a standalone test for hereditary cancer predisposition and as a reflex test triggered by findings from somatic tumor profiling.

When tumor profiling triggers germline testing

4 to 12% of patients undergoing tumor profiling carry pathogenic germline variants in cancer predisposition genes, including patients who would not have met traditional criteria for germline testing based on personal or family history alone. The 2024 ASCO guideline recommends broader germline testing than family-history criteria alone for several cancer types.

When a likely germline variant is identified during tumor profiling (typically by ~50% VAF in tumor tissue, or directly by matched normal sequencing), germline confirmation testing on normal tissue should be performed before clinical action. Tumor-only profiling is not validated as a germline assay, and the analytical approach, variant interpretation framework, and consent process are all different.

Hereditary cancer syndromes and their genes

• HBOC: BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATM, CHEK2. Testing recommended for all women with ovarian cancer, all patients with triple-negative breast cancer diagnosed under 60, all men with breast cancer, and individuals meeting NCCN criteria.
• Lynch syndrome: MLH1, MSH2, MSH6, PMS2, EPCAM. Universal tumor MMR testing (IHC or MSI) in colorectal and endometrial cancers is standard of care, with germline testing when tumor MMR deficiency is identified.
• Hereditary diffuse gastric cancer: CDH1. >80% lifetime risk; prophylactic total gastrectomy is recommended for confirmed carriers.
• Multiple endocrine neoplasia: RET (MEN2A/2B), MEN1. RET guides prophylactic thyroidectomy timing in MEN2 families.
• Hereditary polyposis: APC (FAP, AFAP), MUTYH (MAP). Guides surveillance intensity and surgical timing.

Therapeutic implications

Beyond family counseling and surveillance, germline cancer predisposition variants increasingly have direct therapeutic implications. Germline BRCA1/2 predicts PARP inhibitor eligibility in ovarian, breast, pancreatic, and prostate cancer. Germline MLH1/MSH2 may inform immunotherapy response expectations. Germline ATM/CHEK2/PALB2 have emerging evidence for PARP inhibitor sensitivity and platinum chemotherapy response.

Germline testing is not a standalone laboratory service. It is embedded in a clinical process that requires patient education, informed consent, result interpretation, and post-test planning. Genetic counseling is integral to every stage.