Investigating Genetic Variants: Whole Genome Sequencing and Molecular Modeling of POLA2

Genetic Investigations

Whole genome sequencing (WGS) was done on DNA from peripheral blood of individuals AII:3 and BII:1. The analysis used a bioinformatic pipeline from the Department of Clinical Genetics and Genomics at Karolinska University Hospital. The reference for the human genome was hg19. Sanger sequencing validated WGS findings in the probands and for carrier testing of siblings and parents. Breakpoint PCR confirmed a rearrangement in the gene POLA2 for individuals in family B, which was further validated by Sanger sequencing. Variants in POLA2 were named according to transcript NM_002689.4 and genome hg19. Relative telomere length was estimated with quantitative polymerase chain reaction (qPCR) from blood leukocytes.

In family A, telomere length was also measured through flow cytometry with in situ hybridization at Repeat Diagnostics, Inc. Clinical and laboratory findings were gathered from medical charts.

Molecular Visualization and Modeling of POLA2 Missense Variants

The domains of POLA2 were annotated according to the Uniprot entry Q14181. Its structure was downloaded from the Protein Data Bank (PDB), entry 8B9D, reflecting the human replisome assembly. Due to a disordered region in the N-terminal domain, the available structure does not cover some residues. To model the variant p.Ile96Thr, AlphaFold 2.0 was used. Both p.Ile96Thr and p.Pro424Ala variants were generated via de novo sequence analysis with ColabFold. The analysis and visualization were done using UCSF Chimera v. 1.17.3.

Clash analysis was performed for the benign substitution of p.Pro424Ala alongside p.Pro424Leu. The AlphaFold-generated sequences for wildtype and variants were aligned. The electrostatic potential at residue Ile96, suspected as part of a protein-protein interaction site, was visualized. The distances of alpha-helix spans in relevant residues were also calculated.

Terminal Restriction Fragment Analysis of POLA2 Genetic Modifications in HEK293T Cells

HEK293T cells were acquired from ATCC and maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum in a humidified environment. Human POLA2 p.Ile96Thr variant knock-in clones were created using CRISPR RNA (crRNA) and homology-directed repair (HDR) templates. Silent mutations were included to prevent re-cutting.

For creating POLA2 knock-out clones, CRISPR/Cas9 was utilized. CRISPR gene targeting involved complexing Alt-R S.p. Cas9 Nuclease and crRNA or sgRNA, which were then electroporated into cells. Validation of CRISPR targeting was conducted by Sanger sequencing of genomic DNA.

For Southern blot analysis, genomic DNA from 293T cells was isolated and digested with specific enzymes before loading onto an agarose gel. The blot was probed using the kit reagents. Images were obtained using a Chemidoc Touch Imaging System. Quantification utilized the WALTER web tool, and statistical analysis was performed using GraphPad Prism version 10.2.0.