How to determine loss of heterozygosity in a patient with Primary Macronodular Adrenal Hyperplasia (PMAH) and ARMC5 (Armadillo Repeat-Containing 5) mutations?

Determining Loss of Heterozygosity in PMAH with ARMC5 Mutations

To determine loss of heterozygosity (LOH) in a patient with PMAH and ARMC5 mutations, perform DNA sequencing of both the germline tissue (peripheral blood) and multiple adrenal nodules to identify the germline mutation plus distinct somatic "second-hit" mutations or allelic losses in each nodule, which confirms biallelic inactivation of ARMC5 as a tumor suppressor gene. 1, 2, 3

Technical Approach to LOH Detection

Primary Method: Comparative Sequencing Analysis

• Extract DNA from peripheral blood to establish the germline ARMC5 mutation baseline 1, 2
• Obtain tissue samples from multiple adrenal nodules (ideally from both adrenal glands if bilateral adrenalectomy is performed) 3
• Perform Sanger sequencing of all ARMC5 coding exons on both germline and tumor tissue to identify the germline mutation present in all samples plus additional somatic mutations unique to individual nodules 1, 2
• Use droplet digital PCR (ddPCR) to detect large deletions that may not be identified by conventional sequencing, as germline deletions spanning exons 1-5 have been documented in familial PMAH 1

Confirming Biallelic Inactivation

LOH is confirmed when each adrenal nodule demonstrates the germline ARMC5 mutation PLUS a distinct second somatic alteration (frameshift, nonsense, missense, or splice site mutation), supporting the two-hit tumor suppressor model 2, 3. In one extensively studied case, 16 of 20 nodules from a single patient harbored unique "private" second-hit mutations in addition to the germline p.Trp476* variant 3.

Alternative Molecular Techniques

• Microsatellite analysis using polymorphic markers can demonstrate concordant losses between tumor and normal tissue, though this approach has limitations as concordant LOH does not definitively prove clonality since certain chromosomal variations are common in adrenal tumors 4
• Next-generation sequencing (NGS) with multi-gene panels provides comprehensive detection of both small mutations and copy number variations simultaneously 4
• Comparative genomic hybridization maps chromosomal gains and deletions genome-wide, though high concordance rates may reflect common alterations rather than true clonality 4

Clinical Context and Interpretation

Understanding the Two-Hit Mechanism

ARMC5 functions as a tumor suppressor gene requiring biallelic inactivation to drive tumorigenesis 2, 3. The germline mutation (first hit) is inherited in an autosomal dominant pattern with 50% transmission risk to offspring, but penetrance is only 10-20%, similar to BMPR2 mutations in pulmonary arterial hypertension 5, 4. The somatic mutation (second hit) occurs independently in each developing nodule, explaining the polyclonal nodularity characteristic of PMAH 3.

Genetic Testing Strategy

• Begin with sequencing all coding exons of ARMC5 in the index patient to identify the germline mutation 5
• Include copy number variation analysis via ddPCR since large deletions account for some cases and are missed by standard sequencing 1
• If the germline mutation is identified, offer cascade screening to all first-degree relatives with pre-test genetic counseling explaining the 50% inheritance risk and incomplete penetrance 5

Critical Pitfalls to Avoid

Do not dismiss the clinical diagnosis of PMAH solely because genetic testing is negative, as ARMC5 mutations account for the majority but not all cases—one of five families in a major study had PMAH without identifiable ARMC5 mutations 6. That particular family demonstrated prominent hyperaldosteronism, suggesting genetic heterogeneity 6.

Do not assume a single tissue sample is sufficient for LOH analysis—the extensive genetic variance requires sampling multiple nodules to fully characterize the somatic mutational landscape 3. Each nodule may harbor a completely different second-hit mutation (frameshift, nonsense, missense, or splice site), and analyzing only one nodule provides incomplete information 3.

Do not delay clinical management waiting for complete genetic characterization—treatment decisions should be based on clinical syndrome and biochemical evidence of cortisol excess, with genetic testing serving to confirm diagnosis and guide family screening rather than dictate immediate management 7, 5.

Family Screening Implications

For mutation-positive relatives, implement surveillance protocols including annual clinical assessment for signs of cortisol excess and periodic imaging to detect adrenal enlargement 5. However, recognize that negative genetic testing in family members does not eliminate risk entirely if the familial mutation has not been identified, and phenotypic screening remains important 7.