Investigation of Somatic Mutations in Human Brains Targeting Genes Associated With Parkinson's Disease

BACKGROUND: Somatic single nucleotide variant (SNV) mutations occur in neurons but their role in synucleinopathies is unknown. AIM: We aimed to identify disease-relevant low-level somatic SNVs in brains from sporadic patients with synucleinopathies and a monozygotic twin carrying LRRK2 G2019S, whose penetrance could be explained by somatic variation. METHODS AND RESULTS: We included different brain regions from 26 Parkinson's disease (PD), one Incidental Lewy body, three multiple system atrophy cases, and 12 controls. The whole SNCA locus and exons of other genes associated with PD and neurodegeneration were deeply sequenced using molecular barcodes to improve accuracy. We selected 21 variants at 0.33–5% allele frequencies for validation using accurate methods for somatic variant detection. CONCLUSIONS: We could not detect disease-relevant somatic SNVs, however we cannot exclude their presence at earlier stages of degeneration. Our results support that coding somatic SNVs in neurodegeneration are rare, but other types of somatic variants may hold pathological consequences in synucleinopathies

Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION

BACKGROUND: Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads. METHODS: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. RESULTS: We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55‐base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed. CONCLUSION: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance

Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains

The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients

Venetoclax-based low intensity therapy in molecular failure of NPM1 mutated AML.

Molecular failure in NPM1 mutated AML inevitably progresses to frank relapse if untreated. Recently published small case series show that venetoclax combined with low dose cytarabine or azacitidine can reduce or eliminate measurable residual disease (MRD). Here we report an international multicentre cohort of 79 patients treated for molecular failure with venetoclax combinations and report an overall molecular response (≥1-log reduction in MRD) in 66/79 (84%) and MRD negativity in 56/79 (71%). 18/79 (23%) patients required hospitalisation and no deaths were reported during treatment. 41 patients were bridged to allogeneic transplant with no further therapy and 25/41 were MRD negative assessed by RT-qPCR before transplant. Overall survival (OS) for the whole cohort at 2 years was 67%, event-free survival (EFS) was 45% and in responding patients there was no difference in survival in those who received a transplant using time-dependent analysis. Presence of FLT3-ITD mutation was associated with a lower response rate (64 vs. 91%, p<0.01), worse OS (HR 2.50, 95% CI 1.06-5.86, p=0.036) and EFS (HR 1.87, 95% CI 1.06-3.28, p=0.03). 18/35 non-transplanted patients became MRD negative and stopped treatment after a median of 10 months, with 2-year molecular relapse free survival of 62% from the end of treatment. Venetoclax based low intensive chemotherapy is a potentially effective treatment for molecular relapse in NPM1 mutated AML, either as a bridge to transplant or as definitive therapy