Fanconi Anemia: Genotype-Phenotype Correlations

National Human Genome Research Institute

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Abstract

Over the years, we have employed NextGen technologies and high-density SNP arrays to identify genetic and genomic variations that cause Fanconi anemia, and influence the various phenotypes associated with the disease. Our custom capture gene sequencing panel consisted of 152 genes that targets the entire length of genes associated with FA and other inherited bone marrow syndromes (59). In addition, ADH/ALDH (27) and 1-Carbon metabolism (47) genes are also included as they encode enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. In addition to sequence variants, the targeted gene panel allowed us to detect deletions/duplications and determine their precise boundaries from the nextgen sequence reads. For identification of large-size genomic changes including isodisomy caused by mitotic recombination in patients displaying somatic mosaicism, we employed high-density (~1.8 million) SNP arrays. For elucidating the nature of cDNA products generated by aberrant splicing, we use PacBio sequencing and other RNAseq technologies. We are also pursuing efforts to develop zebrafish mutants as a model to confirm the role of certain candidate genes in the DNA repair (FA) pathway, and study the FA disease process, particularly hematopoietic disease associated with FA. In recent years, we have reported the molecular diagnosis of individuals with pathogenic variations in FANCA, FANCL, and FANCB. For the FANCB group, we detailed genotype-phenotype correlations. We have now completed similar studies on patients from four rare FA groups, contributing for 1%-4% of affected individuals: FANCE, FANCF, FANCD2 and FANCJ. Determination of the pathogenicity of the identified missense variants is critical, and thus five novel FANCJ missense variants were evaluated by biochemical as well as cell viability assays which found that one of them is hypomorphic while the rest completely inactivate the FANCJ function. Often, the phenotype of an individual is modulated by somatic mosaicism, which occurs when cells revert, or lose, a disease-causing variant, or when cells accrue a novel variant that attenuates the severity of the inherited pathogenic variant. We have identified and defined 32 individuals from 30 families displaying somatic mosaicism. In our FANCB report, we described an individual with a mosaic duplication of exon 3 and we have extended this study to further evaluate the cellular frequency of this event by single cell RNA-seq analysis. We have also integrated the Pacbio Kinnex platform to obtain longer reads of the single cell RNA molecules increasing the likelihood that the duplication event is captured and represented among the single cell population. This data is being evaluated. After reporting studies on zebrafish knockouts of 17 known FA genes, we have now studied null mutants for faap100 and slx4ip genes, demonstrating that the former is indeed a new FA gene, and lack of the latter gene does compromise DNA repair. In order to study the FA disease process, particularly in a hematopoietic lineage, we designed experiments to trace cd41 (hematopoietic stem cell) and gata1 (red cell) lineages in zebrafish lines inactivated for fanca and/or adh5 genes. We have collected single cell RNA-seq data on cells sorted for cd41 and gata1 lineages, with and without treatment with a DNA crosslinking agent. The data is being analyzed to evaluate the consequence from the loss of an FA gene on hematopoiesis, and particularly, any consequences subsequent to the challenge of increased DNA damage. We had described the genomic signature of cancer with FA DNA repair pathway deficiency, which was characterized by the presence of genomic structural variants, and often accompanied by pathogenic sequence variants in TP53. In pursuance of these observations, efforts are underway to develop methodologies to improve early detection of HNSCC. Towards this end, an ongoing study evaluates the genomic and sequence alterations in brushings from multiple sites of buccal cavity from healthy individuals and individuals affected with FA. We aim to develop molecular screening tools for early detection of oral squamous cell carcinoma development in FA patients. We have investigated the biological landscape of FA-deficient cells through comprehensive RNA-seq profiling, focusing on splicing and expression dysregulation. RNA-seq profiles were generated for 189 samples, from two distinct sample sets: One set consisted of five patient-derived fibroblast cell lines (one each of FA-A/C/G/I/J) which were each transfected with either a functional FA gene (WT) or an empty vector (EV), in triplicate, yielding 30 samples. The second sample set consisted of 159 samples, including 147 FA patient-derived cell lines across ten subtypes (FA-A:56, FA-B:16, FA-C:5, FA-D2:25, FA-E:8, FA-F:9, FA-G:2, FA-I:4, FA-J:19, FA-L:4) and 12 samples from four healthy controls, spanning fibroblasts (52) and lymphoblastoid cell lines (107). Differentially expressed genes (DEGs) and differentially alternatively spliced genes (DASGs) between EV and WT were characterized, and these clustered distinctly by FA gene (A/C/G/I/J) and vector type (EV/WT). Conversely, the 147 samples from patient-derived cell lines did not cluster by FA subtype faithfully, reflecting diverse genetic and pathophysiologic backgrounds of the individuals. Supervised clustering improved subtype-relevant grouping, implicating expression and isoform biomarkers specific to each group. Enrichment analysis targeted GO_Biological_Process, BioPlanet, and HallMarks50 gene sets. The DEGs and DASGs shared enrichment in cell cycle, extracellular matrix (ECM), and apoptosis pathways. Notably, DEGs were uniquely enriched in immune pathways (interferon gamma/alpha, inflammation) and DASGs in RNA splicing/processing. FA genes exhibited both shared and gene-specific transcriptomic patterns: FANCJ displayed extensive dysregulation, with strong ECM and EMT enrichment, while FANCC exhibited Hypoxia and Cell Migration focus. These results underscore the multifaceted consequences of a deficient FA pathway.