Generation and isolation of single cells from mouse brain with mosaic analysis with double markers-induced uniparental chromosome disomy.

Mosaic analysis with double markers (MADM) technology enables concomitant fluorescent cell labeling and induction of uniparental chromosome disomy (UPD) with single-cell resolution. In UPD, imprinted genes are either overexpressed 2-fold or are not expressed. Here, the MADM platform is utilized to probe imprinting phenotypes at the transcriptional level. This protocol highlights major steps for the generation and isolation of projection neurons and astrocytes with MADM-induced UPD from mouse cerebral cortex for downstream single-cell and low-input sample RNA-sequencing experiments. For complete details on the use and execution of this protocol, please refer to Laukoter et al. (2020b).

[Detection of mosaic trisomy 9 missed by conventional cytogenetics using SNP-array and fluorescence in situ hybridization].

OBJECTIVE: To detect mosaic trisomy 9 missed by conventional cytogenetics. METHODS: Peripheral blood genomic DNA from a girl with mental retardation was analyzed using Affymetrix CytoScan (TM) HD array. Fluorescence in situ hybridization (FISH) was also performed on samples from two patients. RESULTS: The SNP-array analysis has revealed multiple duplications along chromosome 9. FISH analysis showed that, for the peripheral blood sample from one patient, 40 of 100 interphase cells and 15 of 100 metaphase cells carried trisomy 9. For the cord blood sample from another patient, 35 of 100 interphase cells and 10 of 100 cultured cells carried trisomy 9. CONCLUSION: SNP-array is useful for detecting low-level mosaicism which may be missed by conventional cytogenetics. Combined with karyotype and microarray analyses, FISH is a focused and targeted approach for diagnosing mosaic trisomy. They may provide a useful tool for differentiating pseudomosaicisms from true mosaicisms.

Environmental exposure to pyrethroids and sperm sex chromosome disomy: a cross-sectional study.

BACKGROUND: The role of environmental pesticide exposures, such as pyrethroids, and their relationship to sperm abnormalities are not well understood. This study investigated whether environmental exposure to pyrethroids was associated with altered frequency of sperm sex chromosome disomy in adult men. METHODS: A sample of 75 subjects recruited through a Massachusetts infertility clinic provided urine and semen samples. Individual exposures were measured as urinary concentrations of three pyrethroid metabolites ((3-phenoxybenzoic acid (3PBA), cis- and trans- 3-(2,2-Dichlorovinyl)-1-methylcyclopropane-1,2-dicarboxylic acid (CDCCA and TDCCA)). Multiprobe fluorescence in situ hybridization for chromosomes X, Y, and 18 was used to determine XX, YY, XY, 1818, and total sex chromosome disomy in sperm nuclei. Poisson regression analysis was used to examine the association between aneuploidy rates and pyrethroid metabolites while adjusting for covariates. RESULTS: Between 25-56% of the sample were above the limit of detection (LOD) for the pyrethroid metabolites. All sex chromosome disomies were increased by 7-30% when comparing men with CDCCA and TDCCA levels above the LOD to those below the LOD. For 3PBA, compared to those below the LOD, those above the LOD had YY18 disomy rates 1.28 times higher (95% CI: 1.15, 1.42) whereas a reduced rate was seen for XY18 and total disomy (IRR = 0.82; 95% CI: 0.77, 0.87; IRR = 0.93; 95% CI: 0.87-0.97), and no association was seen for XX18 and 1818. CONCLUSIONS: Our findings suggest that urinary concentrations of CDCCA and TDCCA above the LOD were associated with increased rates of aneuploidy. However the findings for 3BPA were not consistent. This is the first study to examine these relationships, and replication of our findings is needed before the association between pyrethroid metabolites and aneuploidy can be fully defined.

Semi-automated scoring of triple-probe FISH in human sperm: methods and further validation.

Although the frequency and consequence of sperm chromosomal abnormalities are considerable, few epidemiologic studies in large samples have been conducted to investigate etiologic risk factors. This is, in part, attributable to the labor intensive demands of manual sperm fluorescence in situ hybridization (FISH) scoring. As part of an epidemiologic study investigating environmental risk factors for aneuploidy among men attending a hospital-based fertility clinic, a semi-automated method of slide scoring was further validated and used to estimate sex chromosome sperm disomy frequency in a large number of samples. Multiprobe FISH for chromosomes X, Y, and 18 was used to determine sex chromosome disomy in sperm nuclei. Semi-automated scoring methods were used to quantify X disomy (sperm FISH genotype XX18), Y disomy (YY18), and XY disomy (XY18). The semi-automated results were compared with the results from manual scoring in 10 slides. The semi-automated method was then used to estimate sex chromosome disomy frequency in 60 men. Of 10 slides scored, significant differences between the manual and semi-automated results were seen primarily in one slide that was of poor quality because of over swollen nuclei. Among 60 men analyzed using the semi-automated method, median total sex chromosome disomy frequency was 1.65%, which is higher than seen among normal men but within range with reports from fertility clinic populations. These results further validate that semi-automated methods can be used to score sperm disomy with results comparable to manual methods. This is the largest study to date to provide estimates of sex chromosome disomy among men attending fertility clinics. These methods should be replicated in larger clinic populations to arrive at stable estimates of aneuploidy frequency in men who are members of subfertile couples. © 2011 International Society for Advancement of Cytometry.

Beckwith-Wiedemann syndrome and uniparental disomy 11p: fine mapping of the recombination breakpoints and evaluation of several techniques.

Beckwith-Wiedemann syndrome (BWS) is a phenotypically and genotypically heterogeneous overgrowth syndrome characterized by somatic overgrowth, macroglossia and abdominal wall defects. Other usual findings are hemihyperplasia, embryonal tumours, adrenocortical cytomegaly, ear anomalies, visceromegaly, renal abnormalities, neonatal hypoglycaemia, cleft palate, polydactyly and a positive family history. BWS is a complex, multigenic disorder associated, in up to 90% of patients, with alteration in the expression or function of one or more genes in the 11p15.5 imprinted gene cluster. There are several molecular anomalies associated with BWS and the large proportion of cases, about 85%, is sporadic and karyotypically normal. One of the major categories of BWS molecular alteration (10-20% of cases) is represented by mosaic paternal uniparental disomy (pUPD), namely patients with two paternally derived copies of chromosome 11p15 and no maternal contribution for that. In these patients, in addition to the effects of IGF2 overexpression, a decreased level of the maternally expressed gene CDKN1C may contribute to the BWS phenotype. In this paper, we reviewed a series of nine patients with BWS because of pUPD using several methods with the aim to evaluate the percentage of mosaicism, the methylation status at both loci, the extension of the pUPD at the short arm and the breakpoints of recombination. Fine mapping of mitotic recombination breakpoints by single-nucleotide polymorphism-array in individuals with UPD and fine estimation of epigenetic defects will provide a basis for understanding the aetiology of BWS, allowing more accurate prognostic predictions and facilitating management and surveillance of individuals with this disorder.