Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity - PubMed (original) (raw)

Comparative Study

doi: 10.3324/haematol.2014.113456. Epub 2014 Sep 19.

Siobán B Keel 2, Tom Walsh 3, Ming K Lee 3, Suleyman Gulsuner 3, Amanda C Watts 3, Colin C Pritchard 4, Stephen J Salipante 4, Michael R Jeng 5, Inga Hofmann 6, David A Williams 7, Mark D Fleming 8, Janis L Abkowitz 2, Mary-Claire King 3, Akiko Shimamura 9

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Comparative Study

Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity

Michael Y Zhang et al. Haematologica. 2015 Jan.

Abstract

Accurate and timely diagnosis of inherited bone marrow failure and inherited myelodysplastic syndromes is essential to guide clinical management. Distinguishing inherited from acquired bone marrow failure/myelodysplastic syndrome poses a significant clinical challenge. At present, diagnostic genetic testing for inherited bone marrow failure/myelodysplastic syndrome is performed gene-by-gene, guided by clinical and laboratory evaluation. We hypothesized that standard clinically-directed genetic testing misses patients with cryptic or atypical presentations of inherited bone marrow failure/myelodysplastic syndrome. In order to screen simultaneously for mutations of all classes in bone marrow failure/myelodysplastic syndrome genes, we developed and validated a panel of 85 genes for targeted capture and multiplexed massively parallel sequencing. In patients with clinical diagnoses of Fanconi anemia, genomic analysis resolved subtype assignment, including those of patients with inconclusive complementation test results. Eight out of 71 patients with idiopathic bone marrow failure or myelodysplastic syndrome were found to harbor damaging germline mutations in GATA2, RUNX1, DKC1, or LIG4. All 8 of these patients lacked classical clinical stigmata or laboratory findings of these syndromes and only 4 had a family history suggestive of inherited disease. These results reflect the extensive genetic heterogeneity and phenotypic complexity of bone marrow failure/myelodysplastic syndrome phenotypes. This study supports the integration of broad unbiased genetic screening into the diagnostic workup of children and young adults with bone marrow failure and myelodysplastic syndromes.

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Figures

Figure 1.

Figure 1.

Detection of genomic copy number variants. Ratios of sample to median corrected depth of coverage within a flow cell lane are plotted across targeted genomic regions of the indicated gene. Diploid bases are shown in black. Deletions and duplications are shown in red and blue, respectively. Genomic positions of exons (vertical bars) and untranslated regions (light blue rectangles) are shown above ratio plots. (A) Whole gene deletion of RUNX1. No diploid bases were present in this region. (B) Deletion of FANCA exons 9–22. (C) Amplification of FANCA exons 15–22. (D) Deletion of FANCA exon 29.

Figure 2.

Figure 2.

Targeted gene capture correction of Fanconi anemia subtype assignment. (A) Biallelic FANCA mutations identified by MarrowSeq in 5 patients. FA patient FH-3 (highlighted in red) was non-ACG subtype by clinical complementation testing. (B) Protein extracts of bone marrow fibroblasts isolated from healthy controls or Fanconi anemia patient FH-42 (FANCD2, p.[Leu683Pro];[Glu906Ilefs*4]) were immunoblotted for FANCD2 with or without 24-h mitomycin C treatment. Fibroblasts from FH-42 exhibit low FANCD2 protein expression (lanes 3 and 4) in comparison to cells from controls (lanes 1 and 2). α-tubulin was used to ascertain equivalent protein loading. (C) Functional validation of Fanconi anemia subtype A in FA patient FH-3 (FANCA p.[Cys218Arg];[Val265Leufs*8]). Protein extracts of bone marrow fibroblasts isolated from healthy control or FH-3 were immunoblotted for FANCD2 with or without 24-h mitomycin C treatment. Fibroblasts from healthy control show both non-ubiquitinated (FANCD2-S) and monoubiquitinated (FANCD2-L) FANCD2 forms (lane 1), with an increased ratio of monoubiquitinated FANCD2 relative to non-ubiquitinated FANCD2 upon mitomycin C treatment (lane 2). Fibroblasts from FH-3 show only the non-ubiquitinated FANCD2-S form with and without mitomycin C (lanes 3 and 4). FANCD2 monoubiquitination is restored upon infection with a pMMP retroviral vector encoding the wild-type FANCA cDNA (lanes 5 and 6).

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