SNP Array Technology Research Articles

Microduplication and Microdeletion Syndromes Diagnosed Prenatally Using Single Nucleotide Polymorphism Array.

We present a series of microdeletion and microduplication syndromes (MMSs) observed in our clinical practice over a three-year period from 2020 to 2023. Microdeletion and microduplication syndromes, characterized by chromosomal deletions or duplications of less than five megabases, pose challenges in terms of diagnosis, especially prenatal and clinical management. Clinically, MMSs encompass a broad spectrum of manifestations, ranging from intellectual disability and developmental delays to congenital anomalies, facial dysmorphisms, and neurobehavioral abnormalities. Notable examples include well-characterized syndromes such as DiGeorge syndrome (22q11.2 deletion), Prader-Willi syndrome (15q11-q13 deletion), and Williams syndrome (7q11 deletion). Our study focuses on the genetic foundations and prenatal ultrasound findings of these syndromes, with an emphasis on cases associated with intellectual disability. Using SNP array technology, we delve into the evolving landscape of diagnostic methods, providing a nuanced understanding of copy number variations (CNVs) and their implications. Prenatal diagnosis allows for the early detection of MMSs, enabling parents and healthcare providers to make informed decisions about the pregnancy and plan for appropriate medical care and interventions. Beyond theoretical considerations, our article bridges the gap between research and practical application by offering insights derived from clinical cases. Through the presentation of specific cases, we aim to contribute valuable data to the broader discourse on MMSs, fostering knowledge exchange and enhancing the medical community's awareness of these complex genetic conditions.

Analysis of Xp22.31q27.1 Region Variation Detection based on SNP Array Technology.

The aim of the study was to investigate the value of single nucleotide polymorphism array (SNP array) technology in the prenatal diagnosis. The variants in Xp22.31q27.1 region of 13 fetuses were analyzed by SNP array technology. Chromosome karyotype analysis was also performed for these fetuses and their parents. Chromosome karyotype analysis found no obvious chromosomal abnormality at 400 bands resolution. Using SNP array technology, we found that all fetuses had mutations in Xp22.31q27.1 region, which was mainly Xp22.31 lesion (61.5%, 8/13) that contained 2 to 5 OMIM genes. Moreover, these mutations consisted of 7 cases of repetition and 6 cases of deletion. In addition, 9 variants were inherited from their mothers, 3 mutations were inherited from the father, and 1 variant was de novo. Compared to traditional analysis of chromosome karyotype, SNP-array technology can detect more chromosomal microdeletions and microduplications. SNP-array technology can act as a supplementary diagnostic method in clinical cytogenetic diagnosis.

Fetal genetic findings by chromosomal microarray analysis and karyotyping for fetal growth restriction without structural malformations at a territory referral center: 10-year experience

BackgroundPrenatal invasive genetic testing is commonly recommended to pregnancies of early-onset FGR or FGR combined with a structural defect. Our study aimed to explore the genetic findings for FGR without structural malformations according to cytogenetic karyotyping and single nucleotide polymorphism array (SNP array) technology over a 10-year period.MethodsA total of 488 pregnancies diagnosed with FGR without structural malformation were retrospectively reviewed. Cytogenetic karyotyping was performed on all the subjects, and SNP array was available from 272 of them. Based on the gestational age at onset, the cohort was classified into four groups: ≤ 24, 25–28, 29–32, and > 32 weeks of gestation. According to the ultrasound findings, they were grouped into isolated FGR, FGR with soft markers, and FGR with non-structural anomalies. In pregnancies of young maternal age, based on the results of maternal serum screening (MSS), they were categorized into high-risk and low-risk MSS groups.ResultsNineteen (3.9%) cases of chromosomal abnormalities were detected by cytogenetic karyotyping, including 11 cases of numerical abnormalities, 5 cases of structural abnormalities, and 3 cases of mosaicism. Trisomy 21 was the most frequent abnormality. Abnormal karyotypes were more frequently observed in cases diagnosed at ≤ 24 weeks (7.2%) than those in any other group. Among pregnancies with normal karyotype, an incremental yield of 4.2% were revealed by SNP array technology regarding clinically relevant aberrations. The additional detection rates by SNP array in cases diagnosed at ≤ 24 weeks (6.5%), cases with soft markers (9.5%), and cases with high-risk MSS (12.0%) were higher than those in other groups within each classification. All the cases with abnormal karyotypes and 7 out of 11 pregnancies with clinically relevant anomalies revealed by SNP array alone resulted in pregnancy terminations.ConclusionChromosome abnormality is an important etiology for FGR with no associated structural malformations, and plays a crucial role in pregnancies decision-making. SNP array improves the detection of genetic anomalies especially in FGR diagnosed at ≤ 24 weeks, FGR combined with soft makers, and FGR combined with high-risk MSS.

P444: SMA carrier screening utilizing SNP array technology and confirmatory ddPCR reveals an isolated SMN1 exon 8 deletion

P444: SMA carrier screening utilizing SNP array technology and confirmatory ddPCR reveals an isolated SMN1 exon 8 deletion

Spatiotemporal monitoring of the rare northern dragonhead (Dracocephalum ruyschiana, Lamiaceae) - SNP genotyping and environmental niche modeling herbarium specimens.

The species we have studied the spatiotemporal genetic change in the northern dragonhead, a plant species that has experienced a drastic population decline and habitat loss in Europe. We have added a temporal perspective to the monitoring of northern dragonhead in Norway by genotyping herbarium specimens up to 200 years old. We have also assessed whether northern dragonhead has achieved its potential distribution in Norway. To obtain the genotype data from 130 herbarium specimens collected from 1820 to 2008, mainly from Norway (83) but also beyond (47), we applied a microfluidic array consisting of 96 SNP markers. To assess temporal genetic change, we compared our new genotype data with existing data from modern samples. We used sample metadata and observational records to model the species' environmental niche and potential distribution in Norway. Our results show that the SNP array successfully genotyped all included herbarium specimens. Hence, with the appropriate design procedures, the SNP array technology appears highly promising for genotyping old herbarium specimens. The captured genetic diversity correlates negatively with distance from Norway. The historical‐modern comparisons reveal similar genetic structure and diversity across space and limited genetic change through time in Norway, providing no signs of any regional bottleneck (i.e., spatiotemporal stasis). The regional areas in Norway have remained genetically divergent, however, both from each other and more so from populations outside of Norway, rendering continued protection of the species in Norway relevant. The ENM results suggest that northern dragonhead has not fully achieved its potential distribution in Norway and corroborate that the species is anchored in warmer and drier habitats.

P–381 Deciphering the genetic cause of recurrent and sporadic pregnancy loss

Abstract Study question To investigate the prevalence and effect of (mosaic) de novo genomic aberrations in recurrent pregnancy loss (RPL) and sporadic abortion (SA). Summary answer Prevalence of maternal uniparental disomies (UPDs) was high in both cohorts. While chromosomal UPDs were found in both cohorts, genome wide UPDs were RPL specific. What is known already Spontaneous abortion occurs in 10–15% of clinically recognized pregnancies and recurrent pregnancy loss in 1–3%. SA and RPL are associated with reduced quality of life. Multiple factors contribute to SA and RPL, such as uterine malformations and parental/fetal chromosomal abnormalities. However, in ∼60% of SA and RPL the cause remains unknown. UPD is defined as the presence of two homologues chromosomes originating from a single parent. This phenomenon can lead to imprinting disorders that are characterised by clinical features affecting growth, development and metabolism in liveborn offspring. However, it could also be responsible for pregnancy loss. Study design, size, duration We recruited 32 families with pregnancy loss (n = 16 RPL cohort, n = 16 SA cohort) with no known genetic predispositions and normal karyotyping results in both parents and the fetus. Average maternal age was 28.68 years (SD = 5.43), paternal age 30.3 years (SD = 5.53), and the gestational age at pregnancy loss was 8.65 weeks (SD = 2.47). The average number of miscarriages in the RPL group was 3.57 (SD = 0.84). We profiled the genomic landscape of both cohorts using SNP typing. Participants/materials, setting, methods We isolated DNA from blood of both parents and the placental tissues from the miscarried products of conception. The placenta tissues were sampled from two distinct extraembryonic and embryonic germ layers, the extraembryonic mesoderm and the chorionic villi cytotrophoblast. Subsequently, we performed SNP-genotyping using Illumina’s Global-Screening Array–24 v2.0 BeadChips and applied haplarithmisis to delineate allelic architecture of fetal tissues of both cohorts. This allowed us to detect large de novo copy-number and -neutral (>10kb) changes. Main results and the role of chance In this pilot study, we have analyzed 132 DNA samples (n = 32 families), of which 16 families were in the RPL cohort and 16 in the SA cohort. Within the RPL cohort, we found: one family with mosaic genome wide hexaploidy both in the extraembryonic mesoderm and chorionic villi, one family with a non-mosaic genome wide hetero UPD of the chorionic villi tissue, one family with a mosaic UPD of chromosome 14 in both tissues and tetraploidy exclusively in the chorionic villi, one family with a mosaic UPD of chromosome 16 in both tissues, one family with a mosaic UPD of chromosome 6 in both tissues, and another family with a mosaic UPD of chromosome 5 in the extraembryonic mesoderm. Within the SA group, one family showed a UPD of chromosome 7 and another family showed a segmental UPD of chromosome 5 in both tissues. Strikingly, all the UPDs found in this study were maternal in origin. Limitations, reasons for caution The main limitation of this study is the resolution of detecting copy-neutral and copy-number variations, which is an inherent limiting factor of SNP-array technology. In addition, in the sample in which we observed non-mosaic genome wide UPD, maternal contamination is likely that can be investigated by other technologies. Wider implications of the findings: Multiple genome wide UPDs are found in the RPL group but none in the SA group, indicating an association between genome wide mosaic UPD and RPL. These findings could lead to a better understanding of causative factors for SA and RPL and the need for a SNP-based non-invasive prenatal testing. Trial registration number Not applicable

Prenatal diagnosis of rearrangements in the fetal 22q11.2 region

Background22q11.2 deletion syndrome (22q11.2DS) and 22q11.2 duplication syndrome (22q11.2DupS) are the most common copy number variations in humans. The clinical phenotypes of these two syndromes are variable, and there are no large sample data on the prenatal detection rate for these two syndromes in the Chinese population.ResultsWe recruited 411 pregnant women who showed either abnormal prenatal ultrasound findings or positive prenatal BoBs™ results or who had given birth to a child with chromosomal abnormalities. SNP-array analysis and interphase FISH analysis identified five fetuses with 22q11.2 copy number variants (CNVs), three of which were 22q11.2 deletion syndrome (22q11.2DS) (3/411) and two of which were 22q11.2 duplication syndrome (22q11.2DupS). In all 5 cases of diagnosed 22q11.2 abnormalities, inheritance could not be identified because the parents did not undergo further testing.ConclusionOur case reports provide a detection rate of 22q11.2 CNVs for fetuses with prenatal diagnostic indications, and early diagnosis of these two syndromes was essential for prenatal intervention in these cases. SNP-array technology is an effective tool in the prenatal diagnosis of 22q11.2 CNVs. The prenatal diagnosis of these two syndromes is helpful for early intervention, which is of great clinical significance.

Assessing the power of principal components and wright\u2019s fixation index analyzes applied to reveal the genome-wide genetic differences between herds of Holstein cows

BackgroundDue to the advent of SNP array technology, a genome-wide analysis of genetic differences between populations and breeds has become possible at a previously unattainable level. The Wright’s fixation index (Fst) and the principal component analysis (PCA) are widely used methods in animal genetics studies. In paper we compared the power of these methods, their complementing each other and which of them is the most powerful.ResultsComparative analysis of the power Principal Components Analysis (PCA) and Fst were carried out to reveal genetic differences between herds of Holsteinized cows. Totally, 803 BovineSNP50 genotypes of cows from 13 herds were used in current study. Obtained Fst values were in the range of 0.002–0.012 (mean 0.0049) while for rare SNPs with MAF 0.0001–0.005 they were even smaller in the range of 0.001–0.01 (mean 0.0027). Genetic relatedness of the cows in the herds was the cause of such small Fst values. The contribution of rare alleles with MAF 0.0001–0.01 to the Fst values was much less than common alleles and this effect depends on linkage disequilibrium (LD). Despite of substantial change in the MAF spectrum and the number of SNPs we observed small effect size of LD - based pruning on Fst data. PCA analysis confirmed the mutual admixture and small genetic difference between herds. Moreover, PCA analysis of the herds based on the visualization the results of a single eigenvector cannot be used to significantly differentiate herds. Only summed eigenvectors should be used to realize full power of PCA to differentiate small between herds genetic difference. Finally, we presented evidences that the significance of Fst data far exceeds the significance of PCA data when these methods are used to reveal genetic differences between herds.ConclusionsLD - based pruning had a small effect on findings of Fst and PCA analyzes. Therefore, for weakly structured populations the LD - based pruning is not effective. In addition, our results show that the significance of genetic differences between herds obtained by Fst analysis exceeds the values of PCA. Proposed, to differentiate herds or low structured populations we recommend primarily using the Fst approach and only then PCA.

Landscape of copy number aberrations in esophageal squamous cell carcinoma from a high endemic region of South Africa

BackgroundEsophageal squamous cell carcinoma (ESCC) is an aggressive cancer with one of the highest world incidences in the Eastern Cape region of South Africa. Several genome wide studies have been performed on ESCC cohorts from Asian countries, North America, Malawi and other parts of the world but none have been conducted on ESCC tumors from South Africa to date, where the molecular pathology and etiology of this disease remains unclear. We report here tumor associated copy number changes observed in 51 ESCC patients’ samples from the Eastern Cape province of South Africa.MethodsWe extracted tumor DNA from 51 archived ESCC specimens and interrogated tumor associated DNA copy number changes using Affymetrix® 500 K SNP array technology. The Genomic Identification of Significant Targets in Cancer (GISTIC 2.0) algorithm was applied to identify significant focal regions of gains and losses. Gains of the top recurrent cancer genes were validated by fluorescence in situ hybridization and their protein expression assessed by immunohistochemistry.ResultsTwenty-three significant focal gains were identified across samples. Gains involving the CCND1, MYC, EGFR and JAG1 loci recapitulated those described in studies on Asian and Malawian cohorts. The two most significant gains involved the chromosomal sub-bands 3q28, encompassing the TPRG1 gene and 11q13.3 including the CTTN, PPFIA1and SHANK2 genes. There was no significant homozygous loss and the most recurrent hemizygous deletion involved the B3GAT1 gene on chromosome 11q25. Focal gains on 11q13.3 in 37% of cases (19/51), consistently involved CTTN and SHANK2 genes. Twelve of these cases (23,5%), had a broader region of gain that also included the CCND1, FGF19, FGF4 and FGF3 genes. SHANK2 and CTTN are co-amplified in several cancers, these proteins interact functionally together and are involved in cell motility. Immunohistochemistry confirmed both Shank2 (79%) and cortactin (69%) protein overexpression in samples with gains of these genes. In contrast, cyclin D1 (65%) was moderately expressed in samples with CCND1 DNA gain.ConclusionsThis study reports copy number changes in a South African ESCC cohort and highlights similarities and differences with cohorts from Asia and Malawi. Our results strongly suggest a role for CTTN and SHANK2 in the pathogenesis of ESCC in South Africa.

SELECTION OF A MINIMAL NUMBER OF SIGNIFICANT PORCINE SNPs BY AN INFORMATION GAIN AND GENETIC ALGORITHM HYBRID MODEL

A panel of a large number of common Single Nucleotide Polymorphisms (SNPs) distributed across an entire porcine genome has been widely used to represent genetic variability of pigs. With the advent of SNP-array technology, a genome-wide genetic profile of a specimen can be easily observed. Among the large number of such variations, there exists a much smaller subset of the SNP panel that could equally be used to correctly identify the corresponding breed. This work presents a SNP selection heuristic that can still be used effectively in the breed classification. The features were selected by combining a filter method and a wrapper method–information gain method and genetic algorithma“plus a feature frequency selection step, while classification used a support vector machine. We were able to reduce the number of significant SNPs to 0.86 % of the total number of SNPs in a swine dataset with 94.80 % classification accuracy.

Multi-allelic positional Burrows-Wheeler transform

BackgroundRecent advances in whole-genome sequencing and SNP array technology have led to the generation of a large amount of genotype data. Large volumes of genotype data will require faster and more efficient methods for storing and searching the data. Positional Burrows-Wheeler Transform (PBWT) provides an appropriate data structure for bi-allelic data. With the increasing sample sizes, more multi-allelic sites are expected to be observed. Hence, there is a necessity to handle multi-allelic genotype data.ResultsIn this paper, we introduce a multi-allelic version of the Positional Burrows-Wheeler Transform (mPBWT) based on the bi-allelic version for compression and searching. The time-complexity for constructing the data structure and searching within a panel containing t-allelic sites increases by a factor of t.ConclusionConsidering the small value for the possible alleles t, the time increase for the multi-allelic PBWT will be negligible and comparable to the bi-allelic version of PBWT.

Application of next-generation sequencing technique in genetic analysis of spontaneous abortion

Objective To investigate the value of next-generation sequencing (NGS) technique for genetic analysis of spontaneous abortion. Methods From January to June 2017, 154 patients who visited the First Affiliated Hospital of Zhengzhou University for spontaneous abortion were enrolled. All abortion tissue samples were analyzed by both NGS combined with short tandem repeat (STR) and single nucleotide polymorphism array (SNP-array). Results of the two methods were compared by Chi-square or Fisher's exact test. Results (1) Chromosomal abnormalities were detected in 109 of the 154 cases (70.7%), including 52 (47.7%) of numerical chromosomal abnormalities, 49 (45.0%) of structural chromosomal abnormalities, six (5.5%) of mosaicism, and two (1.8%) of uniparental disomy (UPD). In those 52 cases of numerical chromosome abnormalities, there were 45 of chromosome aneuploidy and seven of polyploidy. The top three numerical chromosomal abnormalities were 45,X (27.0%, 14/52), trisomy 22 (9.6%, 5/52) and trisomy 16 (7.7%, 4/52). Forty-nine structural abnormality cases carried 67 copy number variations (CNV), including 13 pathogenic CNV (pCNV, 19.4%), 24 variants of unknown clinical significance (35.8%) and 30 benign CNV (44.8%). In those 13 pCNVs, two were responsible for microdeletion and microduplication syndromes. (2) SNP-array was successful in 152 cases, but failed in two (1.3%) due to genomic DNA <200 ng. However, NGS technology was successful in all 154 cases and identified chromosomal abnormalities in the two cases that SNP-array had failed. No statistically significant difference was shown in the detection rate of chromosomal abnormalities between SNP-array and NGS technology [70.4% (107/152) vs 67.5% (104/154), χ2=0.293, P=0.588]. (3) No significant difference in the detection of chromosome aneuploidy (six cases in each group, 3.9% vs 3.9%) and mosaicism (45 cases in each group, 29.2% vs 29.6%) was found between NGS technology and SNP-array. Three cases of polyploidy (69, XXX) and two of UPD were identified by SNP-array, but not by NGS. When combined with STR, NGS was able to detect all three cases of polyploidy (69, XXX). (4) Forty-seven structural abnormality cases detected by SNP-array carried 53 CNVs, and 49 detected by NGS carried 67 CNVs. (5) NGS detected ten, three and one more CNVs than SNP-array did when the genome lengths were 100-<500, 500-<1 000 and ≥1 000 kb, respectively. Conclusions NGS can be used to detect chromosomal aneuploidy and mosaicism that can be identified by SNP-array with fewer limitations on total amount of genome. Moreover, CNVs that fail to be identified by SNP-array can also be detected by NGS. When combined with STR, NGS can effectively detect chromosomal polyploidy. Therefore, NGS could be a potential genetic analysis method for spontaneous abortion and of importance for genetic counseling. Key words: Abortion, spontaneous; High-throughput nucleotide sequencing; Chromosome aberrations; Polymorphism, single nucleotide

Abstract PD5-05: Genomic instability and poor antiproliferative response to aromatase inhibitor treatment: A POETIC study

Abstract Background: More than 20% of early-stage patients with estrogen positive (ER+) disease relapse. Higher levels of the proliferation marker Ki67, and lack of reduction of Ki67 in response to AI indicate poorer prognosis. Somatic mutations have been the focus of research in treatment resistance. However, recurrent somatic copy number alterations (SCNAs) are more common and affect more genes in primary breast cancer (BC) than somatic mutations. Previous studies have suggested an increased risk of recurrence for patients with high genomic instability and for patients with loss of heterozygosity (LOH) at the TP53 locus, but it is unknown if these SCNA events impact response to AI treatment. In addition, LOH and mutations at the TP53 locus had a higher risk of recurrence than LOH or mutations at TP53 alone. We hypothesised that genomic instability and SCNAs at particular loci would be increased in early BC patients with high baseline Ki67, and particularly in patients with high Ki67 despite pre-operative AI therapy. Methods: In a substudy of POETIC (UK-wide, phase III, randomised trial with 4483 women testing perioperative AI in postmenopausal women with early BC), SNParray technology was used to determine SCNAs in baseline and surgical tumour core-cuts and blood from 76 patients (59 AI-treated, 17 controls). Proliferation rate was estimated as percentage (%) of cancer cells staining for Ki67 by IHC. Poor AI responders (PR, &amp;lt;60% reduction in Ki67 between baseline and surgery, n=31) and good AI responders (GR, &amp;gt; 75% reduction in Ki67, n=28) were selected from POETIC samples. Mutation data from exome sequencing was available for tumours from 75 of the patients. Results: The fraction of the genome with SCNAs correlated with Ki67 expression in both baseline and surgical samples (baseline Spearman rho=0.5, p &amp;lt; 10-5; surgical Spearman rho=0.44, p &amp;lt; 10-3). In paired baseline vs surgical samples, 24% of samples showed discordance in SCNAs that covered &amp;gt; 10% of the genome. The samples showing the highest discordance were from PRs. The fraction of the genome with LOH was greater in PR (median PR 20%, GR 10%, p = .065), and the best SCNA to predict the fraction of the genome altered in a sample were segments with LOH at Chr17p13.3 (adjusted p &amp;lt; .001, logistic regression). There was a higher percentage of patients with LOH at Chr17p13.3 that contains the TP53 gene in the PR compared to GR group (PR 71%, GR 39%, p = .029), and integration of previously generated mutation data with SCNA showed that 9 out of 31 PRs have mutations and LOH at the TP53 locus compared to 3 out of 28 GRs (p = 0.16). Conclusions: There is discordance between the observed SCNAs in paired samples with high genomic instability and multiple biopsies may be needed to confidently assess all SCNAs. However, LOH at Chr17p13.3 is a biomarker for genomic instability and frequency of LOH is significantly greater in patients that show a poor response to AI treatment. Finally, high genomic instability is associated with high proliferation rates at baseline and surgery after 2 weeks of AI treatment suggesting de novo resistance in tumours with high instability that may lead to a higher rate of recurrence seen in these patients. Citation Format: Schuster EF, Gellert P, Segal CV, López-Knowles E, Buus R, Morden J, Robertson J, Bliss J, Smith I, Dowsett M, POETIC Trial Management Group and Trialists P. Genomic instability and poor antiproliferative response to aromatase inhibitor treatment: A POETIC study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-05.

Application of SNP-array technology in the genetic analysis of pediatric patients with growth retardation

To explore the value of single nucleotide polymorphism array (SNP-array) for the analysis of pediatric patients with growth retardation. One hundred eighty one children with growth retardation were enrolled. DNA was extracted from peripheral samples from the patients, and whole genome copy number variations (CNVs) were detected using Illumina Human Cyto SNP-12. All identified CNVs were further analyzed with reference to databases including ClinGen, ClinVar, DECIPHER, OMIM and DGV as well as comprehensive review of literature from PubMed to determine their pathogenicity. Forty seven patients (26%) with abnormal CNVs were detected, which included 12 known microdeletions/microduplications syndrome (26%), 10 pathogenic non-syndromic CNVs (21%), 3 numerical chromosome aberrations (6%), 3 unbalanced translocations (6%), 4 pathogenic mosaicisms (9%) and 15 cases with unknown clinical significance (32%). After excluding obvious numerical and/or structural chromosomal abnormalities, this study has detected 15 pathogenic microdeletions/microduplications sized 5 Mb or less, which may be missed by routine chromosomal karyotyping. In addition, there were 3 cases with loss of heterozygoisty (LOH) containing known or predicted imprinting genes as well as 2 cases with suspected parental consanguinity. SNP-array technology is a powerful tool for the genetic diagnosis of children with growth disorders with advantages of high resolution and improved accuracy.

Validation of Whole Genome SNP Array As the Primary Genetic Test in Myelodysplastic Syndrome, to Replace Chromosome and FISH Copy Number Analysis, in a UK Diagnostic Genetic Laboratory

▪Metaphase cytogenetic (MC) analysis in patients with myelodysplastic syndrome (MDS) remains the only essential recommended genetic test in Europe(Malcovati et al, Blood 2013). The chromosome abnormalities identified provide evidence of clonality and information regarding disease characterisation, risk stratification, treatment strategies and disease monitoring. With the exception of the 3q/MECOM gene rearrangement, observed in 1% MDS, the common, recurrent chromosome abnormalities and all specified abnormalities in the revised International Prognostic Scoring System (IPSS-R) for MDS are copy number changes (deletion, duplication, amplification) (Greenberg et al, Blood 2012). However, the identification of gene mutations is increasingly becoming important to confirm a diagnosis of MDS in problematic cases, refine risk (particularly in those with lower risk IPSS-R scores) and improve patient management (Bejar et al, JCO 2011;Haferlach et al, Leukemia 2014). Currently there is insufficient prognostic evidence to use gene mutation analysis as the sole genetic test, but SNP array technology may be seen as a stepping stone towards providing some gene mutation information as it detects copy number variation (CNV) at single gene, and sometimes intragenic, resolution and copy neutral loss of heterozygosity (CN-LOH), regions which are known to harbor bi-allelic gene mutations. CN-LOH is common in hematological neoplasia, including MDS.We undertook a prospective validation study using the Affymetrix Cytoscan® HD SNP array, with more than 2.6 million copy number markers, to compare SNP array with MC in patient samples at presentation with confirmed or highly suspected MDS. Subsequent monitoring by SNP array was performed in those patients where samples were received routinely for MC. Expectations were that SNP array analysis would be at least comparable to MC, by detecting all copy number changes detected by chromosome analysis; increase the diagnostic yield of MDS, due to the detection of both sub microscopic CNV and chromosome cryptic CN-LOH; and increase the number of prognostically relevant genetic abnormalities identified.Samples from 358 patients at disease presentation were received for MC at the West Midlands Regional Genetics Laboratory, UK and were used in this study. Of the 358 patients, 207 had a diagnosis of MDS confirmed and 29 had a diagnosis of MDS/MPN confirmed. The remaining 122 cases were classified as follows: 26 other hematological neoplasia (including MPN, AML, CLL and MM), 51 reactive, 32 non-neoplastic disorders and 13 non-diagnostic.In the 236 confirmed MDS or MDS/MPN cases, marrow was available in 219 and peripheral blood (PB) only in 17. PB samples were abnormal by SNP array in 11/17 (65%). Of the 219 marrows, 146/219 (67%) had comparable SNP array and MC results. A normal karyotype or failed MC result was observed in 130 samples, of these 35 (27%) had an abnormal SNP array. Cases were abnormal by both SNP array and MC in 84 (38%) samples, of these additional abnormalities were detected by SNP array in 30 (36%), including gene mutations with prognostic significance, such as KMT2A-itd, TP53 deletion and RUNX1 deletion. A normal SNP array was observed in 5 (2%) samples with abnormal MC due to balanced rearrangements (SNP array will not detect balanced rearrangements) and low level (1-3%) abnormal clones. In marrow samples, the diagnostic yield by MC alone was 91/219 (42%) and by SNP array analysis alone 119/219 (54%). Therefore, SNP array increased the diagnostic yield of MDS and MDS/MPN by 12% and identified further abnormalities in an additional 30/219 (14%) samples.In conclusion, this validation study demonstrates that SNP array may be offered as an alternative test to MC in patients with a confirmed or highly suspected diagnosis of MDS. Patients with features suggesting 3q/MECOM involvement should have FISH for MECOM rearrangements. DisclosuresGriffiths:Affymetrix: Research Funding.

Copy number variation analysis reveals additional variants contributing to endometriosis development.

Endometriosis is a gynecological disease influenced by multiple genetic and environmental factors. The aim of the current study was to use SNP-array technology to identify genomic aberrations that may possibly contribute to the development of endometriosis. We performed an SNP-array genotyping of pooled DNA samples from both patients (n = 100) and controls (n = 50). Copy number variation (CNV) calling and association analyses were performed using PennCNV software. MLPA and TaqMan Copy-Number assays were used for validation of CNVs discovered. We detected 49 CNV loci that were present in patients with endometriosis and absent in the control group. After validation procedures, we confirmed six CNV loci in the subtelomeric regions, including 1p36.33, 16p13.3, 19p13.3, and 20p13, representing gains, while 17q25.3 and 20q13.33 showed losses. Among the intrachromosomal regions, our results revealed duplication at 19q13.1 within the FCGBP gene (p = 0.007). We identified CNVs previously associated with endometriosis, together with six suggestive novel loci possibly involved in this disease. The intergenic locus on chromosome 19q13.1 shows strong association with endometriosis and is under further functional investigation.

Genome-Wide Paternal Uniparental Disomy as a Cause of Beckwith-Wiedemann Syndrome Associated with Recurrent Virilizing Adrenocortical Tumors

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by fetal macrosomia, macroglossia, and abdominal wall defects. BWS patients are at risk to develop Wilms tumor, neuroblastoma, hepatoblastoma, and adrenal tumors. A young woman with BWS features, but with inconclusive genetic evidence for the disease, came to clinical observation for signs of virilization at the age of 16 years. An adrenocortical tumor was diagnosed and surgically resected. The tumor underwent 2 local relapses that were also surgically treated. The patient was also operated to remove a breast fibroadenoma. SNP arrays were used to analyze chromosome abnormalities in normal and tumor samples from the patient and her parents. The patient presented genome-wide mosaic paternal uniparental disomy (patUPD) both in the adrenocortical and the breast tumors, with different degrees of loss of heterozygosity (LOH). The more recent relapses of the adrenocortical tumor showed a loss of part of chromosome 17p that was absent in the first tumor. Analysis of a skin biopsy sample also showed mosaic patUPD with partial LOH, while no LOH was detected in leukocyte DNA. This case shows that virilizing adrenocortical tumors may be a clinical feature of patients with BWS. The SNP array technology is useful to diagnose genome-wide patUPD mosaicism in BWS patients with an inconclusive molecular diagnosis and underlines the tumorigenic potential of the absence of the maternal genome combined with an excess of the paternal genome.

Identification of multiple diagnostic SNP loci for differentiation of three salmonid species using SNP-arrays

This paper reports the use of SNP-array technology in a cross-species study for non-ambiguous species identifications. Based on an existing SNP-array for Atlantic salmon (cross)hybridisations with samples of salmon, brown trout and rainbow trout were analyzed to identify species-specific diagnostic markers. In total 566 SNP loci were identified to be highly polymorphic across the three salmonid species providing the molecular basement for various monitoring applications in aquaculture and food industries.

Clinical utility and dilemmas of SNP microarray testing.

The ability to diagnose patients with developmental delay, intellectual disability, congenital anomalies, and dysmorphic features has significantly improved with the introduction of SNP microarray technologies which includes more than 1.8 million markers on a single array. This high-density array will allow for sensitively detecting all known abnormalities with defined loci of interest as well as the discovery of ‘‘new syndromes’’ as a cause of mental retardation or autism. The most common micro deletion disorders include15q11-q13 Prader-Willi/Angelman, 22q11.3 velo-cardiofacial, 17pl3.3 William, 1p36and 16p11.2 microdeletion syndromes. SNP arrays are able to detect segmental uniparental disomy (UPD) as in Prader Willi syndrome and UPD14. SNP arrays identify parental consanguinity which may otherwise go undetected. Long stretches of homozygosity can be analyzed for recessive genes in patients born to consanguineous parents. (http://www.ccs.miami.edu/cgi-bin/ROH/ROH_analysis_tool.cgi) Arrays cannot however identify balanced chromosomal rearrangements, such as translocations or inversions, Marker chromosomes may also be missed, depending on the size, marker composition, and array coverage of the specific chromosomal region present on the marker. Dilemmas arise when ‘‘CNVs of uncertain clinical significance’’ are identified, or if a parent is not available for testing, thus making it difficult to identify if the rearrangement is inherited or arose de-novo. Further dilemmas arise if a parent is identified with the same rearrangement and is apparently asymptomatic, should one then consider the presence of a mutation in a recessive gene in the other allele to explain autism, mental retardation or other disorders. Overall SNP array technology has become the test of choice, permitting a 5.9% detection rate in patients with negative microarray-based CGH and an overall detection rate approaching 29%.

Continuing difficulties in interpreting CNV data: lessons from a genome-wide CNV association study of Australian HNPCC/lynch syndrome patients

BackgroundHereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome (LS) is a cancer syndrome characterised by early-onset epithelial cancers, especially colorectal cancer (CRC) and endometrial cancer. The aim of the current study was to use SNP-array technology to identify genomic aberrations which could contribute to the increased risk of cancer in HNPCC/LS patients.MethodsIndividuals diagnosed with HNPCC/LS (100) and healthy controls (384) were genotyped using the Illumina Human610-Quad SNP-arrays. Copy number variation (CNV) calling and association analyses were performed using Nexus software, with significant results validated using QuantiSNP. TaqMan Copy-Number assays were used for verification of CNVs showing significant association with HNPCC/LS identified by both software programs.ResultsWe detected copy number (CN) gains associated with HNPCC/LS status on chromosome 7q11.21 (28% cases and 0% controls, Nexus; p = 3.60E-20 and QuantiSNP; p < 1.00E-16) and 16p11.2 (46% in cases, while a CN loss was observed in 23% of controls, Nexus; p = 4.93E-21 and QuantiSNP; p = 5.00E-06) via in silico analyses. TaqMan Copy-Number assay was used for validation of CNVs showing significant association with HNPCC/LS. In addition, CNV burden (total CNV length, average CNV length and number of observed CNV events) was significantly greater in cases compared to controls.ConclusionA greater CNV burden was identified in HNPCC/LS cases compared to controls supporting the notion of higher genomic instability in these patients. One intergenic locus on chromosome 7q11.21 is possibly associated with HNPCC/LS and deserves further investigation. The results from this study highlight the complexities of fluorescent based CNV analyses. The inefficiency of both CNV detection methods to reproducibly detect observed CNVs demonstrates the need for sequence data to be considered alongside intensity data to avoid false positive results.