Compound Heterozygous — The Importance of Phase Over the Number of Variants
Why is this important?
In autosomal recessive disorders, a disease phenotype typically manifests only when both copies of a gene lose sufficient function. Although the identification of two rare variants in the same gene via sequencing may appear conclusive, it is essential to determine whether these variants reside on the same or different chromosomal copies. This positional relationship is defined as phase.
When two variants are located on separate homologous chromosomes, they are described as being in trans; when both are present on the same chromosomal copy, they are in cis. The term compound heterozygous generally refers to a state where two distinct variants are in trans. Therefore, the critical question is not merely the count of variants, but whether each allele has been affected.
Key Concepts
Individuals typically possess two copies of most autosomal genes, one inherited from each parent. In recessive disorders, an individual carrying only one pathogenic variant may have the other copy compensate for its function. If two distinct pathogenic variants are in trans, both copies may be affected, aligning with the disease mechanism. Conversely, if the variants are in cis, the opposite copy is likely unaffected, rendering this explanation inapplicable.
Phase can be determined through parental testing and segregation analysis within families. The presence of one variant in the mother and another in the father generally supports a trans configuration. Long-read sequencing, read-backed phasing, or other molecular assays may also directly link the two loci. However, if the variants are distant from each other or familial samples are unavailable, phase may remain undetermined.
Small Household Example
Suppose a patient has two variants, A and B. If variant A is observed only in the mother and variant B only in the father, the patient can be interpreted as having inherited the two variants on different alleles. Conversely, if both A and B are present together in the mother and absent in the father, it is highly likely that the two variants are in cis.
This example illustrates phasing but does not prove the pathogenicity of each variant. If both A and B are VUSes, confirming trans configuration adds positional information but does not automatically classify the two variants as pathogenic.
How to Interpret PM3 Evidence
PM3 under the ACMG/AMP guidelines is an evidence criterion applied to variants observed in trans with a pathogenic variant in autosomal recessive disorders. ClinGen recommendations adjust the weight of this evidence based on the number of observed cases, the classification of the trans partner variant, and the certainty of phase. Observations with unknown phase (i.e., without parental testing) are considered weaker than those confirmed to be in trans.
It is also important not to count multiple observations from the same patient or members of the same family as independent instances. PM3 is applied within the premise that the gene–disease relationship and the recessive mechanism are already established; it does not constitute a definitive classification on its own.
Remaining Pieces Required for Diagnostic Interpretation
In addition to the phase of the two variants, each population frequency, functional impact, conservation of splice sites, and existing clinical reports are evaluated. The patient’s phenotype is assessed for concordance with the disease, whether the age of onset and severity are explained, and whether other genes or non-genetic causes are more appropriate.
Compound heterozygous states can also be formed by different variant classes, such as when a copy-number variant deletes one allele and a small sequence variant is present on the other allele. If the assay’s scope detects only one type of variant well, the second allele may be missed.
Common Points of Confusion
heterozygousindicates that the two alleles at a single locus are different, whereascompound heterozygousrefers to two distinct variants within the same gene.- The mere fact that two variants reside in the same gene does not imply they are in trans.
- Because trans denotes positional relationship and pathogenicity conveys clinical significance, these must be assessed separately.
- The principles governing recessive disorders cannot be directly applied to dominant or X-linked disorders.
Limitations
Low-level mosaicism in one parent, de novo events, allele dropout, or sample mix-ups can complicate simple pedigree interpretation. Direct phasing is also limited by read length and the positions of informative variants. The report should distinguish between confirmed trans, inferred trans, and phase unknown to convey uncertainty.
The final diagnosis results from a comprehensive review of phase, variant classification, gene–disease validity, inheritance pattern, and patient phenotype.
Reading in Context
How functional evidence of variants is incorporated into classification is covered in the Functional Assays and Variant Interpretation concept, while the pitfalls of mosaicism and allele fraction are addressed in the Germline and Somatic concept.