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.10.1093/brain/awy157_video1awy157media15813519976001.

Another look at follicular lymphoma: immunophenotypic and molecular analyses identify distinct follicular lymphoma subgroups.

AIMS: The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl-2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B-cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype. METHODS AND RESULTS: Two hundred and five follicular lymphomas were investigated with immunohistochemistry and fluorescence in-situ hybridization (FISH). The use of three distinct anti-bcl-2 antibodies together with CD10 expression data and FISH analysis for bcl-2 and bcl-6 rearrangements allowed subclassification of follicular lymphoma into four distinct subgroups: (i) CD10-positive/bcl-2-positive, (ii) CD10-positive/bcl-2-negative, (iii) CD10-negative/bcl-2-positive, and (iv) CD10-negative/bcl-2-negative. All cases were bcl-6-positive. STMN1 (stathmin) was shown to be helpful in diagnosing bcl-2-negative and/or CD10-negative follicular lymphomas, and in their distinction from marginal zone B-cell lymphoma. CONCLUSIONS: Combined immunohistological and molecular analyses reveal that follicular lymphomas showing an atypical immunophenotypic and molecular profile exist, and we demonstrate that STMN1 represents a novel useful diagnostic marker for these.

Alteration of SMRT tumor suppressor function in transformed non-Hodgkin lymphomas.

Indolent non-Hodgkin lymphomas are characterized by a prolonged phase that is typically followed by a clinical progression associated with an accelerated clinical course and short survival time. Previous studies have not identified a consistent cytogenetic or molecular abnormality associated with transformation. The development of a transformed phenotype, evolving from the original low-grade component, most likely depends on multiple genetic events, including the activation of synergistic dominant oncogenes and a loss of tumor suppressor gene functions. Complex karyotypes and relatively bad chromosome morphology are typical of transformed non-Hodgkin lymphomas, rendering complete cytogenetic analysis difficult. Here, we report the use of transformed non-Hodgkin lymphoma cell lines and primary samples to identify the involvement of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) gene that maps at chromosome 12q24 in transformed non-Hodgkin lymphomas. We also show that down-regulation of SMRT in the immortalized "Weinberg's model" cell lines induces transformation of the cells. Assessment of cDNA array profiles should further help us to design a working model for SMRT involvement in non-Hodgkin lymphoma transformation as a novel, nonclassical tumor suppressor.

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.

A 6.4 Mb duplication of the α-synuclein locus causing frontotemporal dementia and Parkinsonism: phenotype-genotype correlations.

IMPORTANCE: α-Synuclein (SNCA) locus duplications are associated with variable clinical features and reduced penetrance but the reasons underlying this variability are unknown. OBJECTIVES: To report a novel family carrying a heterozygous 6.4 Mb duplication of the SNCA locus with an atypical clinical presentation strongly reminiscent of frontotemporal dementia and late-onset pallidopyramidal syndromes and study phenotype-genotype correlations in SNCA locus duplications. DESIGN, SETTING, AND PARTICIPANTS: We report the clinical and neuropathologic features of a family carrying a 6.4 Mb duplication of the SNCA locus. To identify candidate disease modifiers, we completed a genetic analysis of the family and conducted statistical analysis on previously published cases carrying SNCA locus duplications using regression modeling with robust standard errors to account for clustering at the family level. MAIN OUTCOMES AND MEASURES: We assessed whether length of the SNCA locus duplication influences disease penetrance and severity and whether extraduplication factors have a disease-modifying role. RESULTS: We identified a large 6.4 Mb duplication of the SNCA locus in this family. Neuropathological analysis showed extensive α-synuclein pathology with minimal phospho-tau pathology. Genetic analysis showed an increased burden of Parkinson disease-related risk factors and the disease-predisposing H1/H1 microtubule-associated protein tau haplotype. Statistical analysis of previously published cases suggested there is a trend toward increasing disease severity and disease penetrance with increasing duplication size. The corresponding odds ratios from the univariable analyses were 1.17 (95% CI, 0.81-1.68) and 1.34 (95% CI, 0.78-2.31), respectively. Sex was significantly associated with both disease risk and severity; men compared with women had increased disease risk and severity and the corresponding odds ratios from the univariable analyses were 8.36 (95% CI, 1.97-35.42) and 5.55 (95% CI, 1.39-22.22), respectively. CONCLUSIONS AND RELEVANCE: These findings further expand the phenotypic spectrum of SNCA locus duplications. Increased dosage of genes located within the duplicated region probably cannot increase disease risk and disease severity without the contribution of additional risk factors. Identification of disease modifiers accounting for the substantial phenotypic heterogeneity of patients with SNCA locus duplications could provide insight into molecular events involved in α-synuclein aggregation.

Construction and application of a full-coverage, high-resolution, human chromosome 8q genomic microarray for comparative genomic hybridization.

BACKGROUND: Array-based comparative genomic hybridization (aCGH) enables genome-wide quantitative delineation of genomic imbalances. A high-resolution contig array was developed specifically for chromosome 8q because this chromosome arm is frequently altered in many human cancers. METHODS: A minimal tiling path contig of 702 8q-specific bacterial artificial chromosome (BAC) clones was generated with a novel computational tool (BAC Contig Assembler). BAC clones were amplified by degenerative oligonucleotide primer (DOP) polymerase chain reaction and subsequently printed onto glass slides. For validation of the array DNA samples of gastroesophageal and prostate cancer cell lines, and chronic myeloid leukemia specimens were used, which were previously characterized by multicolor fluorescence in situ hybridization and conventional CGH. RESULTS: Single and double copy gains were confidently demonstrated with the 8q array. Single copy loss and high-level amplifications were accurately detected and confirmed by bicolor fluorescence in situ hybridization experiments. The 8q array was further tested with paraffin-embedded prostate cancer specimens. In these archival specimens, the copy number changes were confirmed. In fresh and archival samples, additional alterations were disclosed. In comparison with conventional CGH, the resolution of the detected changes was much improved, which was demonstrated by an amplicon of 0.7 Mb and a deletion of 0.6 Mb, both spanned by only six BAC clones. CONCLUSIONS: A comprehensive array is presented, which provides a high-resolution method for mapping copy number alterations on chromosome 8q.

Genomic imbalances in CML blast crisis: 8q24.12-q24.13 segment identified as a common region of over-representation.

The acute phase of chronic myeloid leukemia (CML) is accompanied by secondary chromosomal changes. The additional changes have a non-random pattern; however, highly abnormal (marker) chromosomes are reported in some 20% of abnormal karyotypes. These marker chromosomes have proved to be beyond the resolution of conventional G-banding analysis. We used molecular cytogenetic techniques to determine the structure of complex chromosome markers in 10 CML-derived cell lines after our investigations of CML patients in blast crisis. Multicolor fluorescence in situ hybridization identified a multitude of structural chromosome aberrations. In addition, genomic gains identified by comparative genomic hybridization (CGH) were mapped to highly complex marker chromosomes in more than one cell line. The most common genomic loss detected by CGH affected chromosome 9, whereas the most common genomic gains affected, in order of frequency, the sequences of 8q, 6, and 13q. The smallest discrete amplification on 8q was identified in cell line MEG-01. This amplicon contains sequences represented by the marker D8S263/RMC08P029 but did not contain the proximal MYC gene or a more distal marker, D8S256/RMC08P025. We determined the size of the amplicon to be less than the chromosome segment 8q24.12-q24.13. The use of region- and locus-specific probes to analyze the organization of highly complex marker structures aided the identification of preferentially amplified genomic regions. The resultant amplifications could harbor gene(s) driving disease progression.

A Polycythemia Vera Updated: Diagnosis, Pathobiology, and Treatment.

This review focuses on polycythemia vera (PV)-its diagnosis, cellular and genetic pathology, and management. In Section I, Dr. Pearson, with Drs. Messinezy and Westwood, reviews the diagnostic challenge of the investigation of patients with a raised hematocrit. The suggested approach divides patients on their red cell mass (RCM) results into those with absolute (raised RCM) and apparent (normal RCM) erythrocytosis. A standardized series of investigations is proposed for those with an absolute erythrocytosis to confirm the presence of a primary (PV) or secondary erythrocytosis, with abnormal and normal erythropoietic compartments respectively, leaving a heterogenous group, idiopathic erythrocytosis, where the cause cannot be established. Since there is no single diagnostic test for PV, its presence is confirmed following the use of updated diagnostic criteria and confirmatory marrow histology. In Section II, Dr. Green with Drs. Bench, Huntly, and Nacheva reviews the evidence from studies of X chromosome inactivation patterns that support the concept that PV results from clonal expansion of a transformed hemopoietic stem cell. Analyses of the pattern of erythroid and myeloid colony growth have demonstrated abnormal responses to several cytokines, raising the possibility of a defect in a signal transduction pathway shared by several growth factors. A number of cytogenetic and molecular approaches are now focused on defining the molecular lesion(s). In the last section, Dr. Barbui with Dr. Finazzi addresses the complications of PV, notably thrombosis, myelofibrosis and acute leukemia. Following an evaluation of published data, a management approach is proposed. All patients should undergo phlebotomy to keep the hematocrit (Hct) below 0.45, which may be all that is required in those at low thrombotic risk and with stable disease. In those at high thrombotic risk or with progressive thrombocytosis or splenomegaly, a myelosuppressive agent should be used. Hydroxyurea has a role at all ages, but (32)P or busulfan may be used in the elderly. In younger patients, interferon-alpha or anagrelide should be considered. Low-dose aspirin should be used in those with thrombotic or ischemic complications.

Imatinib improves but may not fully reverse the poor prognosis of patients with CML with derivative chromosome 9 deletions.

Deletions of the derivative chromosome 9 occur in a subset of patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML) and are associated with a poor prognosis on standard drug therapy. However, it is currently unknown if the presence of deletions influences the response to imatinib, an Abl-specific tyrosine kinase inhibitor, that has recently shown excellent hematologic and cytogenetic responses in patients with CML. We, therefore, compared hematologic and cytogenetic responses with imatinib in 397 patients with CML, and survival and progression in 354 of these patients, according to deletion status and disease phase. We found no difference in survival between patients with and without deletions, contrasting with previous reports in cohorts with a lower proportion of patients treated with imatinib. However, the time to disease progression on imatinib treatment was significantly shorter for patients with deletions, both in chronic phase (P =.02) and advanced phases (P =.02). Moreover, both in chronic phase and more advanced phases of CML, hematologic and cytogenetic responses were uniformly lower in patients with deletions, with significant differences seen for hematologic response (P =.04), for major cytogenetic response (P =.008) in chronic phase, and for hematologic response in advanced phases (P =.007) of CML. This finding suggests that differences in survival may become apparent with longer follow-up.

Derivative chromosome 9 deletions in chronic myeloid leukemia: poor prognosis is not associated with loss of ABL-BCR expression, elevated BCR-ABL levels, or karyotypic instability.

Deletions of the derivative chromosome 9 have recently been reported in chronic myeloid leukemia. These deletions are large, occur at the time of the Philadelphia (Ph) translocation, span the translocation breakpoint, and represent a powerful prognostic indicator. However, the molecular mechanisms responsible for the poor prognosis associated with deletions are obscure, and several possible models are investigated here. First, we demonstrate that all derivative chromosome 9 deletions detected by fluorescence in situ hybridization were associated with an absence of ABL-BCR expression. However, loss of ABL-BCR expression also occurred without an overt deletion, suggesting the existence of other mechanisms by which ABL-BCR transcription can be abolished. Furthermore, analysis of survival in 160 patients demonstrated that loss of ABL-BCR expression, in contrast to deletion status, was not an indicator of poor prognosis. Second, we addressed the possibility that concomitant small deletions of the Ph chromosome modulate BCR-ABL transcription. Real-time reverse-transcription polymerase chain reaction was used to demonstrate that derivative chromosome 9 deletions were not accompanied by altered levels of BCR-ABL transcripts. Third, deletions may represent a consequence of genetic instability within the target cell at the time of the Ph translocation, with the poor prognosis reflecting a predisposition to subsequent additional genetic alterations. However, patients with deletions do not exhibit an increased frequency of secondary cytogenetic changes following disease progression. Taken together, these data support a model in which deletions of the derivative chromosome 9 result in rapid disease progression as a result of the loss of one or more genes within the deleted region.