Rapid hematologic germline testing using hair follicle-derived DNA for germline predisposition and inherited marrow failure states Free

Germline predisposition states (GPS), including inherited bone marrow failure syndromes (IBMFS) are seen in 5-10% of individuals with myeloid neoplasms (MN). Given overlap with somatic driver genes, blood, saliva, and buccal swabs are suboptimal specimens for GPS testing in MN; the latter two due to contamination with blood cells(Jerez, 2024). Skin biopsy with fibroblast culture-derived DNA remains the gold standard, but the process is expensive, takes 6-8 weeks to complete, and fibroblast culture failure can occur in some patients with telomere biology disorders. Given the need to rapidly screen donors for HSCT and adjust therapy/conditioning in certain IBMFS, we developed a rapid hematologic germline testing algorithm using hair follicle-derived DNA.

The study was conducted after IRB approval through the Mayo Clinic GPS/IBMFS clinic. To perform PCR-based hair follicle testing, we obtained 15-25 hair follicles (bulbs) per patient. The bulbs were placed in a microcentrifuge tube and subjected to a 24-hour digestion and extraction protocol using a modified version of the QIAGEN DNA Purification from Dried Blood Spots protocol. DNA quality was assessed by Nanodrop and Qubit.Testing included either validation of known or suspected germline variants (e.g., RUNX1, DDX41, ETV6), or panel testing (e.g., GPS or IBMFS). Pathogenic variants were validated by repeat testing in a CAP/CLIA certified lab prior to medical decision making. 23 patients underwent whole exome sequencing (WES) in a CAP/CLIA lab, and 13 patients had concurrent germline testing using alternate DNA sources (8 skin biopsies, 1 saliva, 4 buccal swabs).

Ninety-three patients (49 male, 53%) underwent hair follicle-derived germline DNA testing (median age 63 years). Indications for germline testing included the presence of a suspected variant on somatic testing with variant allele frequency (VAF) between 35%-60% ( n= 66, 71%), patient age <50 years (n=26, 28%), antecedent thrombocytopenia (n=15, 16%), presence of syndromic features (n=4, 4%), patient history of multiple malignancies (n=4, 4%), and positive family history (n=15, 16%). Time to reporting variants after receipt of hair follicle-derived DNA was <14 days. There were zero complications associated with the procedure. DNA quality as assessed by Nanodrop revealed a median nucleic acid concentration of 25.64 ng/μL. The median A260/A280 ratio was 1.86 (range 0.701-2.133) and the median A260/A230 ratio was 1.27 (range -8.193-9.223). DNA quality was insufficient for chromosomal microarray (CMA) assessments.

Of 93 patients evaluated, 67 (72%) had ≥1 germline variant, with 3 having >1 germline variant for a total of 72 variants. Pathogenic/likely pathogenic variants identified included: DDX41 (30, 41.7%), TP53 (4, 5.6%), ANKRD26 (4, 5.6%), RUNX1 (4, 5.6%), GATA2 (3, 4.2%), TERT (3, 4.2%), JAK3 (2, 2.8%), SAMD9/SAMD9L (2, 2.8%), ATM (2, 2.8%), and 1 each (1.4%) in BCL2, BCOR, BPGM, CDKN2A, CEBPA, CHEK2, CSF3R, CXCR4, DNMT3A, ETV6, GATA1, MPL, MPO, POT1, PTPN11, RPL5, and SH2B3. In the 13 patients that had concurrent germline testing, results were identical in all cases, except for 1 false negative in a buccal specimen (PTPN11) and 1 case of germline mosaicism, where the TP53 variant was identified in the blood and hair follicle DNA but not in skin fibroblast DNA. After testing, 30 (44.7%) patients had a change in their management plan: 8 (27%) with DDX41-GPS were observed due to known latency of DDX41-mutant MN, 6 (20%) were referred for HSCT based on the germline variant (CEBPA, DDX41, GATA1, GATA2, RPL5, TP53), 6 (20%) were placed on an enhanced surveillance plan for cancer screening (1 ANKRD26, 3 TERT, 2 TP53), 4 (13%) patients undergoing HSCT had related donors removed from consideration as they were carriers (2 DDX41, 1 ANKRD26, and 1 SAMD9), 2 (7%) patients with neutropenia and underlying germline variants (1 ATM, 1 DDX41) had G-CSF therapy discontinued due to concerns for clonal stimulation, and 1 (3%) patient with a germline CDKN2A mutation (hematological diagnosis- CMML) was recommended to avoid radiation therapy for prostate cancer.

In summary, we validate a rapid and simple hair follicle-derived DNA based germline assessment for patients with hematological disorders and demonstrate a positive impact on clinical care in 44% of patients. We are currently optimizing DNA quality so that germline copy number alterations can be better detected using CMA.