Polymorphic Probes Research Articles

Comprehensive Characterisation of the Copy Number Landscape in Acute Lymphoblastic Leukemia Using Digitalmlpa

Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affecting various signaling pathways including cell differentiation and cell cycle control. ALL subtypes are defined by their genetic abnormalities, encompassing gross chromosomal aberrations, ploidy changes, translocations or rearrangements, regional or focal (intragenic) copy number alterations (CNAs), gene fusions and point mutations. Novel biomarkers for risk stratification are still being discovered, further mapping the diverse genetic ALL landscape. Testing for these newly identified genetic biomarkers and characterising the copy number (CN) landscape in ALL samples can provide useful new insights for risk stratification and patient-tailored treatment regimens. Methods: For multiplex detection of CNAs in ALL samples, a new test version of SALSA® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia was developed. Hereafter, this new test version will be referred to as D007 Acute Lymphoblastic Leukemia. This assay contains probes for CNA detection of 70 key genes and regions frequently altered in B-cell ALL and T-cell ALL ( Figure 1). In addition, this probemix contains digital karyotyping probes covering each chromosomal arm for the detection of gross chromosomal aberrations (except the p-arm of acrocentric chromosomes). A selection of these digital karyotyping probes also serve as reference probes. Finally, a set of single nucleotide polymorphism (SNP) probe pairs, targeting both alleles of each of the SNPs, was included for sample identification and guiding the correct CN baseline determination, facilitating optimal data normalisation in samples with complex karyotypes. To test the performance of this new test version of the probemix, DNA samples from 29 ALL cell lines were analysed. digitalMLPA-derived PCR amplicons were sequenced on a NextSeq 1000 Sequencing System (Illumina). FastQ files were analysed using Coffalyser digitalMLPA™ (MRC Holland) and a custom-built algorithm was used for copy number determination using SNP probe pairs. Results: digitalMLPA data of all 29 cell lines was analysed and compared to Cancer Cell Line Encyclopedia (CCLE) copy number data (Depmap, Copy Number Public 23Q2 download). CNAs were detected with D007 Acute Lymphoblastic Leukemia, including clinically relevant biomarkers such as the IKZF1 plus profile, PAX5alt, TCF3 fusions, focal 22q11.22 deletion (incl. VPREB1), intrachromosomal amplification of chromosome 21 (iAMP21), hypo- and hyperdiploidy. Other CNAs of emerging significance in ALL risk stratification included 6q15-q16 deletions, DNMT3B, CDKN1B and KRAS alterations. SNP probe pairs were effectively used to facilitate correct CN baseline determination, and results were confirmed using CCLE data (Depmap, CCLE_ABSOLUTE_combined 2019 download). Correct CN baseline detection is particularly important for samples with complex karyotypes, such as the P12-ICHIKAWA cell line. In this cell line, our data revealed that most chromosomal regions targeted by the SNP probe pairs had a copy number ≥3 ( Figure 2), consistent with the hypotetraploid karyotype previously described for this cell line. Conclusions: Using this new test version of SALSA® digitalMLPA™ D007 Acute Lymphoblastic Leukemia, numerous CNAs can be detected in a single reaction. Results obtained with this probemix showed a high correlation with CN data from the CCLE database in a panel of 29 ALL cell lines. digitalMLPA provides valuable insights into the CN of key target sequences relative to reference sequences in the genome. Moreover, our novel approach using pairs of probes that target both SNP alleles provides an additional layer of information, guiding CN and ploidy status determination in highly complex karyotypes. The SNP probe pair based approach for detection of CN levels in complex samples shows promising results and will be further validated in an independent cohort of 140 ALL patient samples. These results will be presented at the ASH meeting.

Comprehensive genetic analysis in first or second trimester pregnancy loss using chromosomal microarray with single nucleotide polymorphism probes

To explore possible genetic causes associated with early pregnancy loss using chromosomal microarray analysis (CMA) with single nucleotide polymorphism (SNP) probes. A retrospective review was performed by the CMA of samples from 961 patients who spontaneously aborted in our hospital before the 20th week of pregnancy. (1) The total chromosome abnormality rate in miscarriage samples was 54.44% (515/946), including single chromosome abnormality (39.53%), two chromosome abnormality (2.22%), multi-chromosome abnormality (0.42%), triploidy or hypertriploidy (4.86%), copy number variants (CNVs) in 41 cases (4.33%), regions of homozygosity (ROH, 0.74%), mosaic (2.22%) and chimera (0.11%). (2) CNV analysis of 41 cases showed that 85.36% were pathogenic and likely pathogenic, 12.20% were classified as clinical significance unknown and 2.44% were interpreted as likely benign; (3) Among the cases of ROH, 2 cases shown whole-genome homozygosity and 1 case had completely homozygous at chromosome 21. The homozygous regions in 2 cases were located at the end of the short arm of chromosome 16, suggesting the mechanism of ROH in such cases could be the result of isodisomy. Chromosome abnormality is an important genetic factor causing pregnancy loss. The application of CMA with SNP probes can indeed improve the detection rate of chromosome abnormalities and evaluate the risk of reproductive fertility in patients with pregnancy loss.

Detecting regions of homozygosity improves the diagnosis of pathogenic variants and uniparental disomy in pediatric patients.

Chromosomal microarray analysis using single nucleotide polymorphism probes can detect regions of homozygosity (ROH). This confers a potential utility in revealing autosomal recessive (AR) diseases and uniparental disomy (UPD). Results of genetic testing among pediatric patients from 2015 to 2019 were evaluated. Diagnostic findings with detected ROH from large consecutive case series in the literature were reviewed. Of 2050 pediatric patients, 65 (3%) had one or more ROH and 31 (53%) had follow-up whole exome sequencing (WES) and methylation studies. Seven homozygous variants were detected and four of them from three patients (9.6%) were within the detected ROH and classified as pathogenic or likely pathogenic variants for AR diseases. One patient (3%) had segmental UPD15q for a diagnosis of Prader-Willi syndrome. Additive diagnostic yield from ROH reporting was at least 0.2% (4/2050) of pediatric patients. These results were consistent with findings from several large case series reported in the literature. Detecting ROH had an estimated baseline predictive value of 10% for AR diseases and 3% for UPD. Consanguinity revealed by multiple ROH was a strong predictor for AR diseases. These results provide evidence for genetic counseling and recommendation of follow-up WES and methylation studies for pediatric patients reported with ROH.

A near complete genome for goat genetic and genomic research

BackgroundGoat, one of the first domesticated livestock, is a worldwide important species both culturally and economically. The current goat reference genome, known as ARS1, is reported as the first nonhuman genome assembly using 69× PacBio sequencing. However, ARS1 suffers from incomplete X chromosome and highly fragmented Y chromosome scaffolds.ResultsHere, we present a very high-quality de novo genome assembly, Saanen_v1, from a male Saanen dairy goat, with the first goat Y chromosome scaffold based on 117× PacBio long-read sequencing and 118× Hi-C data. Saanen_v1 displays a high level of completeness thanks to the presence of centromeric and telomeric repeats at the proximal and distal ends of two-thirds of the autosomes, and a much reduced number of gaps (169 vs. 773). The completeness and accuracy of the Saanen_v1 genome assembly are also evidenced by more assembled sequences on the chromosomes (2.63 Gb for Saanen_v1 vs. 2.58 Gb for ARS1), a slightly increased mapping ratio for transcriptomic data, and more genes anchored to chromosomes. The eight putative large assembly errors (1 to ~ 7 Mb each) found in ARS1 were amended, and for the first time, the substitution rate of this ruminant Y chromosome was estimated. Furthermore, sequence improvement in Saanen_v1, compared with ARS1, enables us to assign the likely correct positions for 4.4% of the single nucleotide polymorphism (SNP) probes in the widely used GoatSNP50 chip.ConclusionsThe updated goat genome assembly including both sex chromosomes (X and Y) and the autosomes with high-resolution quality will serve as a valuable resource for goat genetic research and applications.

Peripheral Blood Mitochondrial DNA Copy Number Obtained From Genome-Wide Genotype Data Is Associated With Neurocognitive Impairment in Persons With Chronic HIV Infection.

Mitochondrial DNA (mtDNA) copy number varies by cell type and energy demands. Blood mtDNA copy number has been associated with neurocognitive function in persons without HIV. Low mtDNA copy number may indicate disordered mtDNA replication; high copy number may reflect a response to mitochondrial dysfunction. We hypothesized that blood mtDNA copy number estimated from genome-wide genotyping data is related to neurocognitive impairment (NCI) in persons with HIV. In the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study, peripheral blood mtDNA copy number was obtained from genome-wide genotyping data as a ratio of mtDNA single-nucleotide polymorphism probe intensities relative to nuclear DNA single-nucleotide polymorphisms. In a multivariable regression model, associations between mtDNA copy number and demographics, blood cell counts, and HIV disease and treatment characteristics were tested. Associations of mtDNA copy number with the global deficit score (GDS), GDS-defined NCI (GDS ≥ 0.5), and HIV-associated neurocognitive disorder (HAND) diagnosis were tested by logistic regression, adjusting for potential confounders. Among 1010 CHARTER participants, lower mtDNA copy number was associated with longer antiretroviral therapy duration (P < 0.001), but not with d-drug exposure (P = 0.85). mtDNA copy number was also associated with GDS (P = 0.007), GDS-defined NCI (P < 0.001), and HAND (P = 0.002). In all analyses, higher mtDNA copy number was associated with poorer cognitive performance. Higher mtDNA copy number estimated from peripheral blood genotyping was associated with worse neurocognitive performance in adults with HIV. These results suggest a connection between peripheral blood mtDNA and NCI, and may represent increased mtDNA replication in response to mitochondrial dysfunction.

Analytical validation and chromosomal distribution of regions of homozygosity by oligonucleotide array comparative genomic hybridization from normal prenatal and postnatal case series

BackgroundRegions of homozygosity (ROH) are continuous homozygous segments commonly seen in the human genome. The integration of single nucleotide polymorphism (SNP) probes into current array comparative genomic hybridization (aCGH) analysis has enabled the detection of the ROH. However, for detecting and reporting biologically relevant ROH in a clinical setting, it is necessary to assess the analytical validity of SNP calling and the chromosomal distribution of ROH in normal populations.MethodsThe analytical validity was evaluated by correlating the consistency of SNP calling with the quality parameters of aCGH and by accessing the accuracy of SNP calling using PCR based restriction enzyme digestion and Sanger sequencing. The distribution of ROH was evaluated by the numbers, sizes, locations, and frequencies of ROH from the collection of data from parental, postnatal, and prenatal case series that had normal aCGH and chromosome results.ResultsThe SNP calling failure rate was 20–30% with a derivative Log2 ratio (DLR) below 0.2 and increased significantly to 30–40% with DLR of 0.2–0.4. The accuracy of SNP calling is 93%. Of the 958 cases tested, 34% had no ROH, 64% had one to four ROH, and less than 1% had more than five ROH. Of the 1196 ROH detected, 95% were less than 10 Mb. The distribution of numbers and sizes of ROH showed no differences among the parental, pediatric and prenatal case series and test tissues. The chromosomal distribution of ROH was non-random with ROH seen most frequently in chromosome 8, less frequently in chromosomes 2, 6, 10, 12, 11 and 18, and most rarely seen on chromosomes 15, 19, 21 and 22. Recurrent ROH occurring with a frequency greater than 1% were detected in 17 chromosomal loci which locates either in the pericentric or interstitial regions.ConclusionWith a quality control parameter of DLR set at below 0.2, the consistency of SNP calling would be 75%, the accuracy of SNP call could be 93%, and the observed chromosomal distribution of ROH could be used as a reference. This aCGH analysis could be a reliable screening tool to document biologically relevant ROH and recommend further molecular analysis.

Gold-decorated magnetic nanoparticles modified with hairpin-shaped DNA for fluorometric discrimination of single-base mismatch DNA.

The authors describe the use of gold-decorated magnetic nanoparticles (Au/MNPs) in discriminating DNA sequences with a single-base (guanine) mismatch. The Au/MNPs were characterized through dynamic light scattering, X-ray diffraction, superconducting quantum interference device, and UV/visible spectroscopy. They were then conjugated to a probe oligomer consisting of a hairpin-shaped DNA sequence carrying two signalling fluorophores: fluorescein at its 3' end and pyrene in the loop region. When interacting with the target DNA sequences, the hybridized probe-target duplex renders the pyrene signal (at excitation/emission wavelengths of 345/375nm) either quenched or unquenched. Quenching (or nonquenching) of the pyrene fluorescence depends on the presence of a guanine (or a nonguanine) nucleotide at the designated polymorphic site. The linear range of hybridization in these Au/MNPs is from 0.1nM to 1.0μM of ssDNA. Conceivably, this system may serve as a single-nucleotide polymorphism probe. Graphical Abstract Schematic presentation of probe-conjugated Au/MNP preparation (upper panel) and working principle to discriminate DNA with or without single-base (guanine) mismatch sequences at the polymorphic sites (lower panel). Py denotes pyrene-hooked pyrrolocytidine; F denotes fluorescein.

Detection of DNA sequences with a single-base mismatch on a gold-based and pyrene-assisted platform

This paper presents the use of a molecular beacon probe carrying fluorescein and pyrene to produce a combination of integrated on–off signal patterns in response to DNA sequences with the base at a designated polymorphic site varying between guanine and non-guanine nucleotides. Therefore the proposed concept is suitable for single nucleotide polymorphism (SNP) probe development.

Clinical utility of exome sequencing in individuals with large homozygous regions detected by chromosomal microarray analysis

BackgroundChromosomal microarray analysis (CMA) is recommended as the first-tier clinical diagnostic test for individuals with developmental disabilities. In addition to detecting copy number variations, CMA platforms with single nucleotide polymorphism probes can detect large homozygous regions within the genome, which represent potential risk for recessively inherited disorders.MethodsTo determine the frequency in which pathogenic or likely pathogenic variants can be detected in these regions of homozygosity, we performed whole exome sequencing (WES) in 53 individuals where homozygosity was detected by CMA. These patients were referred to our clinical laboratory for a variety of neurodevelopmental conditions including autism spectrum disorder, developmental delay, epilepsy, intellectual disability and microcephaly.ResultsIn 11.3% (6/53) of cases, the analysis of homozygous variants revealed pathogenic or likely pathogenic variants in GJB2, TPP1, SLC25A15, TYR, PCCB, and NDUFV2 which are implicated in a variety of diseases. The evaluation of heterozygous variants with autosomal dominant inheritance, compound heterozygotes and variants with X-linked inheritance revealed pathogenic or likely pathogenic variants in PNPLA4, CADM1, HBB, SOS1, SFTPC, OTC and ASMT in 15.1% (8/53) of cases. Two of these patients harbored both homozygous and heterozygous variants relevant to their phenotypes (TPP1 and OTC; GJB2 and ASMT).ConclusionsOur study highlights the clinical utility of WES in individuals whose CMA uncovers homozygosity. Importantly, we show that when the phenotype is complex and homozygosity levels are high, WES can identify a significant number of relevant variants that explain neurodevelopmental phenotypes, and these mutations may lie outside of the regions of homozygosity, suggesting that the appropriate follow up test is WES rather than targeted sequencing.

Ultrasound findings provide clues to investigate founder mutations expressed as runs of homozygosity in chromosomal microarray studies.

Chromosomal microarray analysis is effectively applied prenatally to detect copy number changes. Single nucleotide polymorphism (SNP) probes included in the microarray platform can detect regions of excessive homozygosity and identical-by-descent genomic stretches. The utility of the latter as part of prenatal diagnosis is not well established. Recessive founder mutations are well recognized within distinct ethnic groups. Combining these data with prenatal sonography provides accurate focused molecular diagnoses quickly. We aimed to evaluate the application of this approach in expectant families presenting to our unit. Three unrelated gravidae presenting with specific fetal sonographic findings: (1) ventriculomegaly with encephalocele; (2) severe polyhydramnion; and (3) enlarged echogenic kidneys, underwent amniocentesis for chromosomal microarray analysis, and genome-wide human SNP array was used to analyze DNA from amniocytes. The Genomic Oligoarray and SNP array evaluation tool v3.0© was used to detect recessive loci associated with the reported clinical findings. Candidate genes were further interrogated using the Israeli National Genetic Database (INGD) and specifically searching and identifying a corresponding founder mutation within the defined ethnic group. Three fetuses from 3 distinct nuclear families in which the parents shared a similar ethnicity (either Ashkenazi or Bukharan Jews) albeit no reported consanguinity were assessed. We found no copy number changes; however, by evaluating regions of homozygosity, we were able to reveal relevant candidate gene for the specific phenotype for each fetus. Using the INGD led to targeted testing of a specific homozygous fetal mutation for which parents were found to be carriers. In the fetus with ventriculomegaly with encephalocele c.1167dupA mutation in the FKTN gene, in the fetus with severe polyhydramnion c.167ins6[TTTCCC] mutation in the BSND gene, and in the fetus with enlarged echogenic kidneys, c.3761_3762delCCinsG in the PKHD1 gene were identified. A tripartite approach integrating sonographic pathology with regions of excessive homozygosity data and INGD-based founder mutation repository yields a comprehensive streamlined approach to provide accurate genetic diagnosis and counselling within the time constraints of an ongoing pregnancy.

Extracting genotype information of Arabidopsis thaliana recombinant inbred lines from transcript profiles established with high-density oligonucleotide arrays.

Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A.thaliana accessions. Individual probes of the GeneChip® that show sequence polymorphisms between different A.thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A.thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A.thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Tailoring high-density oligonucleotide arrays for transcript profiling of different Arabidopsis thaliana accessions using a sequence-based approach.

Excluding polymorphic probes from GeneChip ® transcript profiling experiments via a sequence-based approach results in improved detection of differentially expressed genes in developing seeds of Arabidopsis thaliana accessions Col-0 and C24. GeneChip® arrays represent a powerful tool for transcript profiling experiments. The ATH1 GeneChip® has been designed based on the sequence of the Arabidopsis thaliana reference genome Col-0, hence the features on the array exactly match the sequences of Col-0 transcripts. In contrast, transcripts of other A. thaliana accessions or related species may show nucleotide differences and/or insertions/deletions when compared to the corresponding Col-0 transcripts, therefore, comparisons of transcript abundance involving different A. thaliana accessions or related species may be compromised for a certain number of transcripts. To tackle this limitation, a sequence-based strategy was developed. Only features on the array that were identical in sequence for the specimen to be compared were considered for transcript profiling. The impact of the proposed strategy was evaluated for transcript profiles that were established for developing seeds of A. thaliana accessions Col-0 and C24.

Additional Information Offered By Snpa in Myelodysplastic Syndromes with Excess Blasts (MDS-EB) and Future Perspectives

Introduction: The detection of chromosomal abnormalities in myelodysplastic syndromes (MDS) supports the diagnosis, classification, prognostic stratification, therapy option, treatment monitoring and better understanding of the biology of disease. The main chromosomal abnormalities of MDS are losses and gains of genetic material, and these changes are among the most important prognostic parameters in IPSS-R. As a consequence, major advances have been achieved in the treatment and survival of patients. However, nearly half of the patients present a normal karyotype, which prevents their further characterization. Therefore, there is the desire to increase the abnormalities detection rate by other methods. Single nucleotide polymorphisms array (SNPa), also referred to as chromosomal microarray, is a sensitive technology used to perform high-resolution genome-wide DNA losses, gains and copy-neutral loss of heterozygosity (CN-LOH). The genomic DNA is hybridized to polymorphic probes which are SNP markers and non-polymorphic probes which are copy number markers, providing information about CN-LOH and copy number alteration (CNA), respectively. Previous studies have shown that the addition of the SNPa to karyotype (KT) increases by 28% the detection rate of cytogenetic abnormalities.Objective: To substantiate this idea we describe two cases of MDS to whom SNPa added valued information to karyotype.Methods: DNA was extracted from bone marrow cells for genomic clonal evaluation. The DNA was digested, amplified, fragmented, labeled and hybridized to the chip (Affymetrix - CytoScan HD®) containing the probes. The chip was scanned to detect the signals’ intensity emitted by the hybridized probes which were further analyzed by a software (ChAS) that allows visualization of CNA and CN-LOH. CNA´s analysis relies on the comparison of the obtained signals to a reference diploid DNA signal, and the difference encountered is characterized as loss or gain. CN-LOH analysis is based on two possible nucleotide signals (A or B), which are evaluated to discriminate three genotypes: AA, AB and BB. CN-LOH occurs when one allele is lost and duplicate another, resulting in genotypes AA or BB.Results: Case 1, 64y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II fibrosis. KT: 46,XY,del(5)(q15q33),del(17)(p11.2)[16] / 46,XY[4]. SNPa: 3p21.31p21.2 CN-LOH; 5q21.1q35.3 loss (CN:1.00); -7; +8; 12p13.33p12.3, 12p12.1p11.22, 12q22q23.3 loss (CN:1.00); -16; 17p13.3p11.2 mosaic loss (CN: 1.50) and -Y.Case 2, 74y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II/lll fibrosis. KT: 46,XY[15]. SNPa: 21q21.1q22.3 CN-LOH.Discussion and Conclusion: The SNPa has the advantage of detecting genomic alterations regardless of the cell cycle, even when the cell is quiescent or growth is defective. It also enables the identification of CN-LOH (also known as uniparental disomy, UPD), submicroscopic amplifications and deletions that are not detected by KT. On the other hand, SNPa does not allow the identification of balanced translocations and polyploidy. In case 1, after SNPa analysis, some chromosomal abnormalities (−7, +8, −16 and −Y) were found in sporadic metaphases during the KT reanalysis, but had not initially been described because they did not meet the criterion to be considered as a cytogenetic clone. The risk-stratification (IPSS-R) for this patient was intermediate, but the addition of the SNPa results the risk-stratification could be changed to very poor. Seven months after diagnosis the patient developed acute myeloid leukemia and died. In case 2, CN-LOH detected by SNPa could be responsible for homozygosity of mutations in critical genes located in the 21p region, such as RUNX1 that encodes a protein, which is a transcription factor critical in hematopoiesis. Indeed, sequencing of candidate genes in CN-LOH regions should be considered a priority in the search of driver mutations of MDS. Twenty-four months after diagnosis the patient died due to other non-hematologic causes. In Summary, SNPa analysis may add value to KT non-informative results and occasionally reveal cryptic abnormalities not recognized by karyotyping. However, SNPa analysis should be viewed as a complimentary tool.Acknowledgment: The SNPa test was supported by Grupo Fleury DisclosuresNo relevant conflicts of interest to declare.

Synthesis of a fluorescence resonance energy transfer-based probe containing a tricyclic nucleoside analog for single nucleotide polymorphism typing

Here, we report the synthesis of a fluorescence resonance energy transfer (FRET)-based probe for single nucleotide polymorphism (SNP) typing. The probe contains a fluorescent tricyclic base, 8-amino-3-(2,3-dihydroxypropyl)imidazo[4′,5′:5,6]pyrido[2,3-d]pyrimidine, as a donor molecule and 7-diethylaminocoumarin-3-carboxylic acid as an acceptor molecule. FRET was observed between the donor and acceptor molecules on the probe. The identity of the target bases on DNA and RNA strands could be determined using the probe.

The Mouse Universal Genotyping Array: From Substrains to Subspecies

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.

Mosaic deletion of 20pter due to rescue by somatic recombination

We report on a unique case of a mosaic 20pter-p13 deletion due to a somatic repair event identified by allele differentiating single nucleotide polymorphism (SNP) probes on chromosomal microarray. Small terminal deletions of 20p have been reported in a few individuals and appear to result in a variable phenotype. This patient was a 24-month-old female who presented with failure to thrive and speech delay. Chromosomal microarray analysis (CMA) performed on peripheral blood showed a 1.6 Mb deletion involving the terminus of 20p (20pter-20p13). This deletion appeared mosaic by CMA and this suspicion was confirmed by fluorescence in situ hybridization (FISH) analysis. Additionally, the deletion interval at 20p was directly adjacent to 15 Mb of mosaic copy-neutral loss of heterozygosity (LOH). The pattern of SNP probes was highly suggestive of a somatic repair event that resulted in rescue of the deleted region using the non-deleted homologue as a template. Structural mosaicism is rare and most often believed to be due to a postzygotic mechanism. This case demonstrates the additional utility of allele patterns to help distinguish mechanisms and in this case identified the possibility of either a post-zygotic repair of a germline deletion or a post-zygotic deletion with somatic recombination repair in a single step.

Prognostic significance of acquired copy-neutral loss of heterozygosity in acute myeloid leukemia.

Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Genomic microarray techniques detect recurrent copy-neutral loss of heterozygosity (cnLOH) in addition to copy number aberrations. However, the clinical utility has not been fully established. Therefore, in the current study, the authors examined the prognostic impact of acquired cnLOH in patients with AML, including complete remission (CR) rate, duration of CR, and overall survival (OS). A total of 112 consecutive patients with AML who were undergoing chromosome genomic array testing (CGAT) at the Seattle Cancer Care Alliance were included in the current study. DNA from the bone marrow or blood was analyzed with a microarray platform with both single-nucleotide polymorphism (SNP) probes and non-SNP probes to identify acquired cnLOH. Results were correlated with cytogenetic, molecular, immunophenotypic, and other clinicopathological findings. Patients with cnLOH demonstrated a shorter duration of CR (hazard ratio, 1.87; P =.04) and worse OS (HR, 1.82; P = .03). Multivariate analyses confirmed the independent predictive value of cnLOH for early disease recurrence (P =.02). These results largely reflected those in patients with intermediate and unfavorable cytogenetics. Most strikingly, 13q cnLOH was found to demonstrate a 6.64-fold higher rate of disease recurrence (P =.006) and 3.45-fold worse OS (P = .02) and was enriched with the FLT3-ITD (Fms-related tyrosine kinase 3-internal tandem duplication) mutation. CnLOH has important prognostic significance in patients with AML. CGAT can replace imbalance fluorescence in situ hybridization and the authors recommend the routine use of CGAT to detect cnLOH, particularly among patients with intermediate-risk cytogenetics.

Chromosome Engineering and Physical Mapping of the Thinopyrum ponticum Translocation in Wheat Carrying the Rust Resistance Gene Sr26

ABSTRACTStudies were initiated to induce homoeologous recombination between the 6Ae#1L chromosome segment from Thinopyrum ponticum (Podp.) Barkw. &amp; D. R. Dewey carrying the stem rust resistance gene Sr26 and homoeologous wheat (Triticum aestivum L.) chromosomes. The research was undertaken in an attempt to restore the yielding potential of wheat lines carrying Sr26. DNA probes mapping to chromosome arm 6L of Triticeae species were screened for chromosome 6Ae#1‐specific markers. Thirty out of 32 probes showed useful polymorphisms. A total of 1406 seedlings in a ph1bph1b background were screened for the presence of homoeologous recombinants with 24 of the polymorphic probes, as well as the 6Ae#1 isozyme markers for aconitase and glutamate oxaloacetate transaminase, and stem rust resistance. Eleven genotypes were found to have reduced amounts of 6Ae#1 chromatin and seven of these were resistant to stem rust (+Sr26). Six lines were homoeologous recombinants involving wheat chromosome 6A and four lines involved crossovers with 6D. The physical location of Sr26 was determined to be on the extreme distal segment of the 6Ae#1 chromosome. Rust resistant lines carrying shortened 6Ae#1 segments with improved agronomic performance are now being utilized by wheat breeding programs in several countries.

Comparison of three methods for the genetic diagnosis of spinal muscular atrophy

Objective To evaluate the value of PCR-restriction fragment length polymorphism (PCR-RFLP), real-time PCR and multiplex ligation-dependent probe amplification (MLPA) in the genetic diagnosis of spinal muscular atrophy (SMA) and make laboratory support accessible to clinicians for the molecular diagnosis of SMA. Methods Methodological evaluation. Forty-one suspected SMA cases and 359 control individuals received in Shanghai Children′s Medical Centre from March 2013 to June 2014 were detected for the deletion of exon 7 and 8 in the survival motor neuron gene 1 (SMN 1) by PCR-RFLP, real-time PCR and MLPA, respectively. Then the results of the three methods were compared and the benefits and limitations of the three methods were evaluated. Results The result of real-time PCR was in complete agreement with that of MLPA, showing that 29 suspected cases harbored homozygous deletions of SMN1 and 1 case possessed heterozygous deletion. Among the homozygous deletions, 27 patients demonstrated absence of exon 7 and 8, and 2 cases demonstrated only the absence of exon 7. Meanwhile, both PCR-RFLP and MLPA analysis showed the same results that only 5 out of 395 control cases carried heterozygous deletion. As for cases without heterozygous deletions, PCR-RFLP demonstrated the same result with real-time PCR and MLPA but it missed all the heterozygous ones. Conclusions PCR-RFLP, the conventional SMA gene diagnosis method, was only capable of detecting homozygous deletion of exon 7 and/or 8 of SMN1, but was not as sensitive as to find out the carriers with heterozygous deletions. MLPA could detect the deletion and quantify the copy numbers of exon 7 and 8 of SMN1, efficiently, while the price was relatively high, which brings challenges for its application in the carrier screening of SMA. Compared with these two methods, real-time PCR with high throughput and low input was a rapid, accurate and economic method for the genetic diagnosis of SMA and carrier screening in large populations. (Chin J Lab Med, 2015, 38: 16-20) Key words: Spinal muscular atrophies of childhood; Polymorphism, restriction fragment length; Real-time polymerase chain reaction

An ephemeral sexual population of Phytophthora infestans in the Northeastern United States and Canada.

Phytophthora infestans, the causal agent of late blight disease, has been reported in North America since the mid-nineteenth century. In the United States the lack of or very limited sexual reproduction has resulted in largely clonal populations of P. infestans. In 2010 and 2011, but not in 2012 or 2013, 20 rare and diverse genotypes of P. infestans were detected in a region that centered around central New York State. The ratio of A1 to A2 mating types among these genotypes was close to the 50∶50 ratio expected for sexual recombination. These genotypes were diverse at the glucose-6-phosphate isomerase locus, differed in their microsatellite profiles, showed different banding patterns in a restriction fragment length polymorphism assay using a moderately repetitive and highly polymorphic probe (RG57), were polymorphic for four different nuclear genes and differed in their sensitivity to the systemic fungicide mefenoxam. The null hypothesis of linkage equilibrium was not rejected, which suggests the population could be sexual. These new genotypes were monomorphic in their mitochondrial haplotype that was the same as US-22. Through parentage exclusion testing using microsatellite data and sequences of four nuclear genes, recent dominant lineages US-8, US-11, US-23, and US-24 were excluded as possible parents for these genotypes. Further analyses indicated that US-22 could not be eliminated as a possible parent for 14 of the 20 genotypes. We conclude that US-22 could be a parent of some, but not all, of the new genotypes found in 2010 and 2011. There were at least two other parents for this population and the genotypic characteristics of the other parents were identified.