Genetic and Epigenetic Characterization of a Discordant KMT2A/AFF1-Rearranged Infant Monozygotic Twin Pair

Russo A, Viberti C, Mareschi K, Casalone E, Guarrera S, Birolo G, Cazzaniga G, Corral L, Trentin L, Basso G, Fagioli F, Matullo G.

Int J Mol Sci. 2021 Sep 9;22(18):9740. doi: 10.3390/ijms22189740

Abstract

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment-genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

Keywords: DNA methylation; NRAS; RNA sequencing; acute lymphoblastic leukemia; exome.

Cell‐free DNA screening for fetal aneuploidy using the rolling circle method: A step towards non invasive prenatal testing simplification

Enza Pavanello, Andrea Sciarrone, Varvara Guaraldo, Elisabetta Muccinelli, Valentina Pia Ciuffreda, Pina Sauro, Giulia Bondielli, Sandra Mirante, Giulio Mengozzi, Elsa Viora, Howard Cuckle, Barbara Pasini  

Prenat Diagn. 2021 Dec;41(13):1694-1700. doi: 10.1002/pd.6050. Epub 2021 Oct 7.

Abstract

Objective: To assess the efficacy of cell-free (cf)DNA screening for aneuploidy using the automated system based on rolling circle replication.

Methods: A prospective study among women referred for invasive prenatal diagnosis between July 2018 and December 2019. The plasma fraction was extracted within 5 days from blood collection, stored at -20°C and cfDNA measured between January and December 2019.

Results: A total of 805 women were recruited; 778 with singleton pregnancies and 27 twins. There were 48 Down syndrome, 25 Edwards syndrome and 3 Patau syndrome cases. Overall, the no-call rate was 2.6% (95% confidence interval 1.6%-3.9%) which reduced from 4.7% to 1.1% after relocation of the system (p < 0.002) to ensure a constant ambient temperature below 25°C. In singletons the Down syndrome detection rate (DR) was 100% (93%-100%) and false-positive rate (FPR) 0.14% (0.00%-0.79%). The Edwards syndrome DR was 96% (80%-100%) and FPR 0.78% (0.29%-1.7%). One false-positive had a confined placental trisomy 18 and the remaining five a z-score requiring sample repetition; all the false-positives occurred before system relocation (p < 0.005). Patau syndrome DR and FPR were 67% (9.4%-99%) and 0.26% (0.03%-0.95%).

Conclusion: The cfDNA rolling circle method yields similar results to other methods provided that room temperature is adequately controlled.

KCNK18 Biallelic Variants Associated with Intellectual Disability and Neurodevelopmental Disorders Alter TRESK Channel Activity

Pavinato L., Nematian-Ardestani E., Zonta A., De Rubeis S., Buxbaum J., Mancini C., Bruselles A., Tartaglia M., Pessia M., Tucker S.J., D'Adamo M.C., Brusco A.

Int J Mol Sci. 2021 Jun 4;22(11):6064. doi: 10.3390/ijms22116064.

Abstract

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.

Keywords: ASD; K2P; KCNK18; TRESK; autism spectrum disorder; intellectual disability; potassium channel.

Elovl5 is required for proper action potential conduction along peripheral myelinated fibers

Hoxha E, Balbo I, Parolisi R, Audano M, Montarolo F, Ravera F, Guglielmotto M, Muratori L, Raimondo S, DiGregorio E, Buffo A, Brusco A, Borroni B, Mitro N, Caruso D, Tempia F.

Glia. 2021 Oct;69(10):2419-2428. doi: 10.1002/glia.24048. Epub 2021 Jun 17.

Abstract

Elovl5 elongates fatty acids with 18 carbon atoms and in cooperation with other enzymes guarantees the normal levels of very long-chain fatty acids, which are necessary for a proper membrane structure. Action potential conduction along myelinated axons depends on structural integrity of myelin, which is maintained by a correct amount of fatty acids and a proper interaction between fatty acids and myelin proteins. We hypothesized that in Elovl5-/- mice, the lack of elongation of Elovl5 substrates might cause alterations of myelin structure. The analysis of myelin ultrastructure showed an enlarged periodicity with reduced G-ratio across all axonal diameters. We hypothesized that the structural alteration of myelin might affect the conduction of action potentials. The sciatic nerve conduction velocity was significantly reduced without change in the amplitude of the nerve compound potential, suggesting a myelin defect without a concomitant axonal degeneration. Since Elovl5 is important in attaining normal amounts of polyunsaturated fatty acids, which are the principal component of myelin, we performed a lipidomic analysis of peripheral nerves of Elovl5-deficient mice. The results revealed an unbalance, with reduction of fatty acids longer than 18 carbon atoms relative to shorter ones. In addition, the ratio of saturated to unsaturated fatty acids was strongly increased. These findings point out the essential role of Elovl5 in the peripheral nervous system in supporting the normal structure of myelin, which is the key element for a proper conduction of electrical signals along myelinated nerves.

Keywords: Elovl5; action potential; axon; myelin; polyunsaturated fatty acids.

New DNA Methylation Signals for Malignant Pleural Mesothelioma Risk Assessment

Cugliari G, Allione A, Russo A, Catalano C, Casalone E, Guarrera S, Grosso F, Ferrante D, Sculco M, La Vecchia M, Pirazzini C, Libener R, Mirabelli D, Magnani C, Dianzani I, Matullo G.

Cancers (Basel). 2021 May 27;13(11):2636. doi: 10.3390/cancers13112636.

Abstract

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (>0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = -0.09, 95% CI = -0.12|-0.06, p = 1.2 × 10-7), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10-6). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10-11) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10-8) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells); area under the curve, AUC = 0.75; BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10-7; BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10-8. Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects.

Keywords: DNA methylation; asbestos exposure; epigenome-wide analysis; interaction analysis; malignant pleural mesothelioma.