Scientists identify over 5,000 genetic variants that enable certain cancers to thrive

In a recent study published in Nature Genetics, researchers performed saturation genome editing (SGE) of the BRCA1-associated protein-1 gene (BAP1), whose dysfunction is related to cancer and impaired neurodevelopment.

Study: Saturation genome editing of BAP1 functionally classifies somatic and germline variants. Image Credit: Billion Photos/
Study: Saturation genome editing of BAP1 functionally classifies somatic and germline variants. Image Credit: Billion Photos/


Heritable loss-of-function (LOF) mutations in the BAP1 gene lead to tumor susceptibility, with some connected to neurodevelopmental problems. Variants of unknown importance create autosomal dominant tumor predisposition syndrome, which presents complications during patient treatment.

Recent screening criteria for individuals with pathogenic germline mutations in BAP1 are critical for identifying at-risk groups and improving monitoring recommendations. Identifying disruptive-type somatic BAP1 mutations in malignancies may help with tailored cancer therapy.

About the study

In the present study, researchers used SGE to characterize single-nucleotide variations in the BAP1 gene, enhancing precision medicine efforts.

The researchers concentrated on addressing variations of unknown significance (VUS) in the BAP1 gene, which are critical for patient treatment. They investigated the function of germline and somatic BAP1 polymorphisms in tumor propensity and targeted oncology therapies. They investigated BAP1 variations' correlations with cancers such as cutaneous melanoma, uveal melanoma, cholangiocarcinoma, mesothelioma, meningioma, and renal cancer.

The researchers conducted experiments on 18,108 distinct variations, of which 6,196 showed aberrant functionalities. They then utilized these findings to assess phenotypic relationships in the United Kingdom Biobank, a population-based tumor collection, cancer pedigrees, and ClinVar.

Researchers developed a HAP1 DNA ligase 4 (LIG4) knockout (KO) strain that included genomic integration of a clonally generated Cas9 (HAP1-A5). They refined plasmids and transfection methods, screening all BAP1-coding exons across five periods. In addition, they conducted two different studies to target each SGE area.

Cell fitness was used as a biological readout for BAP1 function, showing its importance and SGE effectiveness in HAP1 cells. A focused clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) screen was carried out, with 193 sgRNAs and variant libraries distributed over all 22 BAP1 target areas.

The researchers conducted functional analyses of gene architecture and conservation, classifying variations into functional categories. Variants with a false discovery rate (FDR) ≥0.01 were classified as unchanged, while those with an FDR ≤0.01 and a negative functional score were classified as depleted. Variants with an FDR ≤0.01 and a positive functional score were classified as enriched. The researchers compared this measure to SGE results and tested the reliability of functional scores and classifications for each variant by comparing log2 fold changes (LFCs) from different genome editing studies.

Researchers compared the BAP1 SGE test to the American College of Medical Genetics and Genomics (ACMG) evidence framework to establish its usefulness for variant interpretation. They investigated BAP1 variants in cancer and neurodevelopment and classified them as highly deficient or slightly depleted. They used a phenome-wide association study (PheWAS) to identify deficient variants and developed cancer-type phenotypic characteristics and rare variant burden test masks.

They also evaluated the link between UK Biobank BAP1 mutations and quantitative aspects of uveal melanomas using ribonucleic acid sequencing (RNA-seq) data from the Cancer Genome Atlas. They also examined tumor sequencing data from 394,756 patient samples in the Foundation Medicine database to identify new BAP1 variants.


Disruptive, non-synonymous-type germline BAP1 mutations were associated with cancer diagnoses and increased insulin-like growth factor-1 (IGF-1) levels, indicating a possible pathogenic mechanism and therapeutic target. The functional score accurately classified the variants, with >99% sensitivity and >98% specificity and an area under the curve (AUC) value of >0.999. 

The revised SGE method increased experiment quality by establishing BAP1 essentiality in the HAP1 LIG4 KO cell line, increasing Cas9 activity, and maintaining robust haploidy. The refined SGE procedure improved transfection efficiency among HAP1 cells to above 60% from below 5.0%. BAP1 essentiality enabled mutational consequence separation, with stop-gained and frameshift variants having primarily negative functional scores.

Missense variations and codon deletions produced scores with a bimodal distribution, allowing researchers to pinpoint critical BAP1 protein residues and domains. Of the 18,108 BAP1 variations, 11,912 were constant, 5,665 depleted, and 531 enriched. Unchanged variations received no functional evaluations, but enriched variants obtained better scores. The team observed a few synonymous variants depleted on the screen. Synonymous-type depleted variants showed significantly higher SpliceAI scores than unchanged synonymous variants. Depleted versions have a broader scoring range.

The conserved N-terminal UCH domain of BAP1 was more resistant to missense mutations and codon deletions. Functional classifications computed with library A or B LFCs and FDRs yielded a 90% variant classification agreement. The researchers found that 99.8% of variations in the pathogenicity truth set showed anticipated depletion, whereas 97% were unchanged or enriched. The functional classification of tests and SGE is quite similar, allowing for the simultaneous identification of a probable hypomorph.


The study showed that extensive functional evaluation of loci using SGE can improve patient diagnosis, biological understanding of disease causes, and the basic understanding of gene and protein function.

Journal reference:
Pooja Toshniwal Paharia

Written by

Pooja Toshniwal Paharia

Pooja Toshniwal Paharia is an oral and maxillofacial physician and radiologist based in Pune, India. Her academic background is in Oral Medicine and Radiology. She has extensive experience in research and evidence-based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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