New technique detects earliest signs of genetic mutations

Mmutations are changes in the molecular “letters” that make up the DNA code, the blueprint for all living cells. Some of these changes may have little effect, but others can lead to disease, including cancer. Now, a new study introduces an original technique, HiDEF-seq, that can accurately detect early molecular changes in the DNA code that precede mutations.

The study authors say their technique – Hairpin Duplex Enhanced Fidelity Sequencing – could advance our understanding of the underlying causes of mutations in both healthy and cancer cells, and how genetic changes naturally accumulate in human cells as people age.

Led by a team of researchers at NYU Langone Health, with collaborators across North America and in Denmark, the work helps to unravel the earliest steps in how mutations occur in DNA.

The new study is based on the understanding that DNA is made up of two strands of molecular letters, or bases. Each strand consists of four types of letters: adenine (A), thymine (T), guanine (G), and cytosine (C). The bases of each strand pair with the bases on the other strand in a specific pattern, with As pairing with T’s and G’s pairing with C’s. This allows the DNA code to be replicated and passed on accurately from one generation of cells to the next. Importantly, mutations are changes in the DNA code that are present on both strands of DNA. For example, a base pair of G and C, with a G on one strand paired with a C on the other strand, can change to a base pair of A and T.

However, the researchers say, most mutations originate in DNA changes that are present on only one of the two strands of DNA, and these single-stranded changes, such as a G and T base pair mismatches cannot be accurately identified using previous testing techniques. . These changes can occur when one strand of DNA is not copied correctly during replication, such as when a cell divides into two cells, or when one of the two strands of DNA is damaged by heat or other chemicals in the body. If these single-stranded DNA changes are not repaired by the cell, then the changes risk becoming permanent double-stranded mutations.

Published in the magazine Nature online June 12, the study showed that the HiDEF-seq technique detected double mutations with extremely high accuracy, with an estimate of one registration error per 100 trillion base pairs analyzed. Furthermore, HiDEF-seq detected changes in the DNA letter code while they were present in only one of the two DNA strands, before permanent double-stranded mutations were made.

“Our new HiDEF-seq sequencing technique allows us to see the earliest fingerprints of molecular changes in DNA when the changes are only in single strands of DNA,” said senior study author Gilad D. Evrony, MD, PhD, a core member of the Center for Human Genetics and Genomics at NYU’s Grossman School of Medicine.

Because people with genetic syndromes linked to cancer are known to have higher rates of mutations in their cells than people without a predisposition to cancer, the researchers began their experiments by describing the DNA changes in healthy cells from people with these syndromes. Specifically, the investigators worked with healthy cells from people with polymerase chain reaction-associated polyposis (PPAP), an inherited condition associated with an increased risk for colorectal cancer, and congenital mismatch repair deficiency (CMMRD). another hereditary condition that increases the likelihood of certain cancers. in children.

Using HiDEF-seq, the researchers found a greater number of single-strand DNA changes in their cells, such as a T paired with a C instead of the original G paired with a C , than in the cells of people who had neither. the syndrome. Furthermore, the pattern of these single-strand changes was similar to the pattern observed in double-stranded DNA mutations for people with both syndromes.

Subsequent experiments were performed on human spermatozoa, which are known to have among the lowest double mutation rates of any human cell type. The researchers found that the pattern of chemical damage, called cytosine deamination, observed by HiDEF-seq in single DNA strands in sperm closely matched the damage observed in deliberately heat-damaged blood DNA. This, the researchers say, suggests that the two patterns of chemical DNA damage, one natural and one induced by external forces, occur through a similar process.

“Our study lays the foundation for using the HiDEF-seq technique in future experiments to transform our understanding of how DNA damage and mutations arise,” said Dr. Evrony, who is also an assistant professor in the Department of Pediatrics and the Department of Neuroscience. and Physiology at NYU Grossman School of Medicine. Single-strand changes in DNA occur constantly as cells divide and multiply, and while layers of repair mechanisms regulate most changes, some slip through and become mutations.

“Our long-term goal is to use HiDEF-seq to create a comprehensive catalog of single-strand DNA mismatch and damage patterns that will help explain known patterns of double-strand mutations,” said Dr. Evrony. “In the future, we hope to combine profiling of single-strand DNA lesions, as obtained by HiDEF-seq, with double-stranded mutations resulting from lesions to better understand and monitor the day-to-day effects on DNA from exposures environmental.”

Geneticists estimate that there are approximately 12 billion bases, or individual letters of DNA, that can be damaged or mismatched in each human cell, as there are two copies of the genetic code, with one copy inherited from each parent. Each of these copies consists of a double-stranded DNA spanning 3 billion base pairs. Dr. Evrony says that every base position in the genetic code is likely to be damaged or mutated at some point during an individual’s life in at least some cells.

Funding for the study was provided by National Institutes of Health grants UG3NS132024, R21HD105910, DP5OD028158, T32AG052909, F32AG076287, and P30CA016087. Additional funding support was provided by the Sontag Foundation, the Pew Foundation, and the Jacob Goldfield Foundation.

Dr. Evrony and NYU have a patent application pending for the HiDEF-seq method.

Dr. Evrony owns equity in DNA sequencing companies Illumina, Pacific Biosciences and Oxford Nanopore Technologies, some of whose products were adapted for use in this study. All such agreements are being managed in accordance with NYU Langone Health policies and practices.

In addition to Dr. Evrony, other NYU Langone researchers involved in this study are co-lead authors Mei-Hong Liu and Benjamin Costa and co-authors Emilia Bianchini, Una Choi, Rachel Bandler, Marta Gronska-Peski, Adam Schwing, Zachary Murphy, Caitlin. Loh and Tina Truong.

Other study co-investigators include Emilie Lassen, Daniel Rosenkjaer and Anne-Bine Skytte, at the Cryos International Sperm and Egg Bank in Copenhagen, Denmark; Shany Picciotto and Jonathan Shoag, at Case Western Reserve University in Cleveland; Vanessa Bianchi, Lucie Stengs, Melissa Edwards, Nuno Miguel Nunes and Uri Tabori, at the Hospital for Sick Children in Toronto; Randall Brand, at the University of Pittsburgh; Tomi Pastinen, at Children’s Mercy Kansas City in Missouri; and Richard Wagner, at the Université de Sherbrooke in Canada.

Questions for the media:

David March
Phone: 212-404-3528
David.March@NYULangone.org