Whole-Exome Sequencing Identifies Germline IDH2 and IDH3 mutations That Predispose to Myeloid Neoplasms

Background: Somatic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2^MT) occur in up to 20% of certain types of myeloid neoplasms, chiefly MDS, MDS/MPN overlap and AML. These mutations induce a metabolic rewiring of cancer cells that affects alpha-ketoglutarate (aKG)-dependent dioxygenases such as TET2 DNA demethylases and Jumanji histone demethylases, which leads to global DNA and chromatin hypermethylation and leukemogenesis. In addition to somatic IDH1/2^MT, germline IDH1/2^MT occur in the context of Ollier and Maffucci syndromes which predispose to cartilaginous tumors and D2HG aciduria, which predisposes to brain tumors. We hypothesized that germline mutations in IDH1/2 or the functionally related IDH3 may predispose to myeloid neoplasms.

Methods: From 409 patients with MDS, MPN or AML, tumor samples from the myeloid compartment and germline samples from CD3⁺ lymphocytes were subjected to whole-exome sequencing. Myeloid cells were adhered to microscopy slides and maximal IDH3, IDH1/2 and, glucose-6-phosphate dehydrogenase (G6PD) glutamate dehydrogenase (GDH) activity was determined using quantitative enzyme cytochemistry.

 

Results: In 409 patients with myeloid diseases, we found 8 patients with germline variants in IDH2 or IDH3. Combined, these variants occur more frequently in these 409 patients with myeloid neoplasms than in the general population (OR = 4.05, P =.0024), suggesting that germline IDH2/3^MT predispose for myeloid diseases (Table 1). Of note, germline IDH2/3^MT were completely mutually exclusive with somatic TET2^MT, suggesting overlapping functions. Notably, all but one of these variants were indicated by two independent software programs to be deleterious for the enzymatic activity. To validate this, we determined maximal IDH3 activity in myeloid cells derived from a 47-year old AML patient with a germline IDH3^MT (c.G626A, p.G209E) (and family history of leukemia) and her sister with IDH3^WT. Maximal IDH3 activity was downregulated in the IDH3^MT sample, while maximal IDH1/2, G6PD and GDH activity were unchanged (Fig. 1).

1 patient had a family history of MDS and 3 patients had a family history of colon, prostrate, gastric, lung and head and neck carcinoma in multiple family members. One patient had 4 family members affected with different cancers. The high age of the affected patients suggest that although germline IDH2/3^MT may predispose to myeloid neoplasms, the development of disease occurs slowly.

Discussion: Whereas somatic IDH2^MT are common in MDS and other myeloid neoplasms, somatic IDH3^MT are not frequently observed, nor in myeloid neoplasms, nor in other types of cancer that are regularly affected by IDH1/2^MT. We show that deleterious germline IDH2/3^MT may predispose to myeloid neoplasms and we postulate that this may occur via decreases in IDH2/3 activity. IDH3^G209E is deleterious for IDH3 enzymatic function and may decrease intracellular aKG levels. This will restrict the function of aKG-dependent dioxygenases such as TET2 and Jumonji and mimic somatic IDH1/2^MT and somatic inactivating TET2^MT. This suggests that IDH1/2/3 are key enzymes and aKG is a central metabolite in maintaining normal function in myeloid cells. Given the family histories of 5/8 of the affected patients, screenings for germline IDH2/3 variants may reveal novel recurring IDH2/3^MT that relate to various types of cancer.

Fig 1. Germline IDH3^G209E mutation decreases maximal IDH3 activity, but not maximal IDH2 or GDH activity.

Representative photomicrographs of primary IDH3^G209E MDS and IDH3^WT myeloid cells after staining IDH3 activity in the presence of various isocitrate concentrations, IDH2 activity (1 mM isocitrate) and GDH activity (5 mM glutamate) using quantitative enzyme cytochemistry. The conversion of colorless tetrazolium salt to the purple formazan directly reflects enzyme activity thus darker cells have a higher maximal activity of the investigated enzyme.

 

Patient ID	Age	Diagnosis	Gender	OS (months)	Variant	SNP nr	Occurrence in general population
1	50	RCMD	F	23	IDH2T495M	rs118053940	0,004848
2	66	CMML-1	M	46	IDH3AR360C	rs116374996	0,004161
3	49	RAEB-1	F	16	IDH3BR359W	rs377682152	0,000154
4	70	RCMD	M	67	IDH2T495M	rs118053940	0,004848
5	76	MDS-U	F	17	IDH3BR334W	rs374613588	0,00008
6	75	sAML	M	5	IDH2R261H	rs118101777	0,00177

Table 1. Clinical characteristics from 6 patients with germline IDH2/3^MT for which clinical information was available.