Next-generation sequencing (NGS) can accurately assess reportable insertion, deletion, and deletion-insertion variants (indels) as long as 68 base pairs (bp), according to recent research (Clin Chem 2023; doi: 10.1093/clinchem/hvad110). The paper also adds that Sanger sequencing confirmation of indels assessed by NGS usually is unnecessary for particular regions or variants, provided the variants meet appropriate coverage and allele frequency thresholds.
The clinical genetics laboratory community lacks consensus on which NGS testing situations require Sanger sequencing prior to reporting. Previous studies have led to variable recommendations. Many clinical laboratories continue to confirm variants with Sanger sequencing, increasing testing costs and turnaround time. Although many studies have demonstrated NGS’s accuracy in detecting single nucleotide variants (SNVs), few have done so for indels.
Thanks to recent advances in NGS, labs are detecting indels more frequently now. Recent recommendations on orthogonal confirmation note that indels are far less prevalent than SNVs, but account for a proportion of pathogenic variants, particularly in genes where loss of function can cause disease. These variants require special consideration for confirmation because indels have been thought to have higher error rates.
To address this situation, the researchers retrospectively analyzed indel results from NGS-targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. The indels came from a variety of sequencing capture reagents, bioinformatics pipelines, and clinical tests.
They found 100% concordance between NGS and Sanger sequencing for 492 indels—including 217 unique ones—ranging in size from 1 to 68 bp. Seventy percent of the indels were deletions, and 90% were 1 to 5 bp long. Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2,562x. NGS accurately detected the 7% of indels located in complex regions of the genome.
Researchers also demonstrated 100% reproducibility of detection of 179 indels during intra-assay validation, according to the paper.
BLOOD TEST MAY PREDICT HEART AND KIDNEY DISEASE RISK IN TYPE 2 DIABETES
A four-biomarker blood test may predict risk of kidney disease progression in patients with type 2 diabetes and albuminaria (Circulation 2023; doi: 10.1161/CIRCULATIONAHA.123.065251).
The four biomarkers are N-terminal pro-B-type natriuretic peptide, high-sensitivity cardiac troponin T, growth differentiation factor-15, and insulin-like growth factor binding protein 7 (IGFBP7). They were identified in the Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation (CREDENCE) trial. The trial involved 2,627 participants and also found that patients treated with the sodium-glucose cotransporter-2 inhibitor (SGLT2) canagliflozin had lower levels of the biomarkers after 1 year than those who took a placebo.
The researchers observed high baseline concentrations of the four individual biomarkers in plasma samples from trial participants, who had diabetic kidney disease and were treated with canagliflozin or placebo. The researchers assessed each biomarker’s prognostic potential for end-stage kidney disease.
Baseline concentrations of all four biomarkers in these samples were generally elevated, compared with samples from healthy reference populations. The median concentrations for study participants were: NT-proBNP, 180 ng/L; high-sensitivity cardiac troponin T, 19 ng/L; growth differentiation factor-15, 2,595 ng/L; and IGFBP7, 121.8 ng/mL. A 50% increase in all four biomarkers was associated with risk of end-stage kidney disease.
Treatment with canagliflozin for 1 year modestly reduced the longitudinal increase in all biomarkers. The biomarkers all rose by 6% to 29% in the placebo arm, compared with 3% to 10% in the canagliflozin arm. The reduction in NT-proBNP with canagliflozin was particularly noteworthy, averaging a decrease of 15% after 1 year, with a drop in the number of individuals above a prognostic NT-proBNP threshold of 125 ng/L.
The researchers noted that these results further strengthen earlier findings that have shown the value of biomarkers for prognosticating major complications and the consistent benefit of SGLT2 inhibitors in reducing event rates across patients with wide ranges of risk.
NEW POSSIBLE BREAST CANCER GENES IDENTIFIED
Recent research has identified multiple new genes associated with breast cancer that may eventually be included in tests for increased risk of the disease (Nature Genetics 2023; doi.org/10.1038/s41588-023-01466-z).
Current tests consider only a few genes, such as BRCA1, BRCA2, and PALB2. Linkage and candidate gene studies have identified several breast cancer susceptibility genes, but their overall contribution to breast cancer is unclear.
To better determine the role of rare coding variants, researchers performed a meta-analysis of three whole exome data sequencing studies, which included a total of 26,368 breast cancer patients and 217,673 control subjects. Subjects were primarily of European ancestry. The researchers performed burden tests for 15,616 protein truncating variants and 18,602 genes.
The researchers found associations between protein-truncating variants and breast cancer in five previously known susceptibility genes at exome-wide significance: ATM, BRCA1, BRCA2, CHEK2, and PALB. They also found a similar association in another gene, MAP3K1, and associations for LZTR1, ATR, and BARD1. Finally, they discovered an association of exome-wide significance between predicted deleterious rare missense or protein-truncating variants and breast cancer identified for CDKN2A.
The researchers concluded that their results demonstrate how large exome sequencing studies, combined with efficient burden analyses, can identify additional breast cancer susceptibility genes. They called for further studies to replicate their findings in large datasets.