Overcoming a diagnostic blindspot: Identifying balanced translocations in Mendelian rare disease cohorts

Background Short-read genome sequencing generates the signal necessary to detect balanced reciprocal translocations at single-nucleotide resolution, but standard structural variant callers represent them as breakend pairs. Structural variant callers produce thousands of breakend records per genome, the majority of which are false positives arising from segmental duplications, repetitive elements, and reference assembly artifacts. This specificity problem has prevented balanced translocation detection from entering routine genome sequencing workflows. We developed a highly specific pipeline to resolve balanced reciprocal translocations and applied it across five clinically distinct cohorts to characterize the prevalence and diagnostic contribution of balanced reciprocal translocations. Methods Short-read genome sequencing data from 16,131 individuals across five cohorts were analyzed: healthy participants from the 1000 Genomes Project (n=3,202), probands with structural birth defects and their family members (CHOP BDB cohort; n=2,875, including 1017 probands), probands enrolled in the GREGoR consortium (GREGoR cohort; n=3,574 probands), probands with undiagnosed disease and their family members enrolled in the Undiagnosed Diseases Network (UDN cohort; n=6,071 including 2,027 probands) and neonates who underwent rapid genome sequencing (CHOP Baby Eagle cohort; n=409). A computational pipeline was developed to characterize breakend events from standard structural variant callers to high-confidence balanced translocation calls by exploiting well-characterized properties of true balanced translocation signatures in short-read data. Translocation breakpoints were resolved to single-nucleotide precision and interrogated for disruption of known disease genes or potential regulatory consequence otherwise. A subset of the identified breakpoints was confirmed by Sanger sequencing. Results Balanced reciprocal translocations were identified in 0.196% of structural birth defect probands (2/1,017), 0.25% of the GREGoR probands (9/3,574), 0.39% of the UDN probands (8/2,027) and 0.24% of neonates who underwent rapid genome sequencing (1/409). Parental carrier frequency in the birth defects cohort was 0.269% (5/1,858), consistent with previously reported estimates in similarly ascertained families, and 0.098% (3/3,040) in the UDN cohort. All six translocations identified in the birth defects cohort were orthogonally validated using Sanger sequencing, confirming pipeline specificity. Approximately 31% of translocations across the three affected cohorts disrupted a known disease gene at the breakpoint, establishing or refining a molecular diagnosis in five cases previously unresolved. The single translocation identified in the newborn cohort disrupted no disease gene and was classified as a variant of uncertain significance.