Low-level Mosaicism Research Articles

Single-cell analysis of human embryos reveals diverse patterns of aneuploidy and mosaicism.

Less than half of human zygotes survive to birth, primarily due to aneuploidies of meiotic or mitotic origin. Mitotic errors generate chromosomal mosaicism, defined by multiple cell lineages with distinct chromosome complements. The incidence and impacts of mosaicism in human embryos remain controversial, with most previous studies based on bulk DNA assays or comparisons of multiple biopsies of few embryonic cells. Single-cell genomic data provide an opportunity to quantify mosaicism on an embryo-wide scale. To this end, we extended an approach to infer aneuploidies based on dosage-associated changes in gene expression by integrating signatures of allelic imbalance. We applied this method to published single-cell RNA sequencing data from 74 human embryos, spanning the morula to blastocyst stages. Our analysis revealed widespread mosaic aneuploidies, with 59 of 74 (80%) embryos harboring at least one putative aneuploid cell (1% FDR). By clustering copy number calls, we reconstructed histories of chromosome segregation, inferring that 55 (74%) embryos possessed mitotic aneuploidies and 23 (31%) embryos possessed meiotic aneuploidies. We found no significant enrichment of aneuploid cells in the trophectoderm compared to the inner cell mass, although we do detect such enrichment in data from later postimplantation stages. Finally, we observed that aneuploid cells up-regulate immune response genes and down-regulate genes involved in proliferation, metabolism, and protein processing, consistent with stress responses documented in other stages and systems. Together, our work provides a high-resolution view of aneuploidy in preimplantation embryos, and supports the conclusion that low-level mosaicism is a common feature of early human development.

Dynamic nature of somatic chromosomal mosaicism, genetic-environmental interactions and therapeutic opportunities in disease and aging

BackgroundSomatic chromosomal mosaicism is the presence of cell populations differing with respect to the chromosome complements (e.g. normal and abnormal) in an individual. Chromosomal mosaicism is associated with a wide spectrum of disease conditions and aging. Studying somatic genome variations has indicated that amounts of chromosomally abnormal cells are likely to be unstable. As a result, dynamic changes of mosaicism rates occur through ontogeny. Additionally, a correlation between disease severity and mosaicism rates appears to exist. High mosaicism rates are usually associated with severe disease phenotypes, whereas low-level mosaicism is generally observed in milder disease phenotypes or in presumably unaffected individuals. Here, we hypothesize that dynamic nature of somatic chromosomal mosaicism may result from genetic-environmental interactions creating therapeutic opportunities in the associated diseases and aging.ConclusionGenetic-environmental interactions seem to contribute to the dynamic nature of somatic mosaicism. Accordingly, an external influence on cellular populations may shift the ratio of karyotypically normal and abnormal cells in favor of an increase in the amount of cells without chromosome rearrangements. Taking into account the role of somatic chromosomal mosaicism in health and disease, we have hypothesized that artificial changing of somatic mosaicism rates may be beneficial in individuals suffering from the associated diseases and/or behavioral or reproductive problems. In addition, such therapeutic procedures might be useful for anti-aging strategies (i.e. possible rejuvenation through a decrease in levels of chromosomal mosaicism) increasing the lifespan. Finally, the hypothesis appears to be applicable to any type of somatic mosacism.

Three Offspring with Cri-du-Chat Syndrome from Phenotypically Normal Parents

Cri-du-chat syndrome is characterized by facial dysmorphism, intellectual disability, and multiple congenital anomalies. Most cases occur de novo. Here, we report 3 siblings with cri-du-chat syndrome born to healthy parents. The proband was admitted to our clinic at the age of 6.5 years due to severe intellectual disability, facial dysmorphism, and heart defect. His karyotype showed a deletion of chromosome 5p. Microarray analysis revealed a 29-Mb deletion in chromosome 5p and a 4.7-Mb duplication in chromosome 19q. FISH analysis indicated an unbalanced translocation between 5p13.3 and 19q13.4. During follow-up, the second and the third child of the family were born with the same chromosome abnormality. Parental peripheral blood and skin fibroblast karyotypes as well as the FISH results using chromosome 5p- and 19q-specific subtelomeric probes were normal. FISH analysis of the father's sperm detected a 5p deletion in 12.8% of 200 cells, and microarray analysis confirmed the same unbalanced chromosome abnormality in a mosaic pattern. Uncultured peripheral blood and buccal smear of the father were also studied by FISH to exclude low-level mosaicism and in vitro culture effect. This is the first study that provides molecular evidence of paternal gonadal mosaicism of an unbalanced translocation detected in 3 siblings with cri-du-chat syndrome.

Quantitative Assessment of Parental Somatic Mosaicism for Copy-Number Variant (CNV) Deletions.

As genome sequencing methodologies have become more sensitive in detecting low-frequency rare-variant events, the link between post-zygotic mutagenesis and somatic mosaicism in the etiology of several human genetic conditions other than cancers has become more clear. Given that current clinical-genomics diagnostic methods have limited detection sensitivity for mosaic events, a copy-number variant (CNV) deletion inherited from a parent with low-level (<10%) mosaicism can be erroneously interpreted in the proband to represent a de novo germline event. Here, we describe three sensitive, precise, and cost-efficient methods that can quantitatively assess the potential degree of parental somatic mosaicism levels for CNV deletions: droplet digital PCR (ddPCR), PCR amplicon-based next-generation sequencing (NGS), and quantitative PCR. ddPCR using the EvaGreen fluorescent dye protocol can specifically quantify the deleted or non-deleted alleles by analyzing the number of droplets positive for a fluorescent signal for each event. PCR amplicon-based NGS assesses the allele frequencies of a heterozygous single-nucleotide polymorphism within a deletion region. The difference in number of reads between the two genotypes indicates the level of somatic mosaicism for the CNV deletion. Quantitative PCR can be applied where the relative quantity of the deletion junction-specific product represents the level of mosaicism. Clinical implementation of these quantitative variant-detection methods enables potentially more accurate assessment of disease recurrence risk in family-based genetic counseling, allowing couples to engage in more informed family planning. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Droplet digital PCR (ddPCR) Alternate Protocol 1: PCR amplicon-based next-generation sequencing Alternate Protocol 2: Quantitative real-time PCR (qPCR).

Возраст-зависимый тканеспецифичный гоносомный мозаицизм у фертильных женщин репродуктивного возраста

Для установления уровня тканеспецифичного гоносомного мозаицизма у здоровых женщин репродуктивного возраста проведено молекулярно-цитогенетическое исследование культивированных лимфоцитов периферической венозной крови (КЛ), некультивированных лимфоцитов периферической венозной крови (НКЛ) и клеток буккального эпителия (БЭ). Установлено, что в КЛ присутствует низкоуровневый гоносомный мозаицизм с тенденцией к повышению частоты клеток с Х-анеуплоидией с увеличением возраста женщины. Средняя частота клеток с моносомией и трисомией по X хромосоме составляет соответственно 1,83% и 0,66% в группе женщин в возрасте 20-29 лет; 2,23% и 0,86% - в возрасте 30-39 лет и 5,88% и 1,95% - в возрасте 40-49 лет. Средняя частота анеуплоидных клеток в НКЛ статистически значимо не отличалась от таковой в КЛ. В БЭ средняя частота мозаичной Х-анеуплоидии во всех возрастных группах составляет 4,01%. Таким образом, нами определён уровень гоносомного мозаицизма в лимфоцитах и БЭ здоровых фертильных женщин и разработан протокол обследования пациентов при обнаружении низкоуровневого мозацизма по хромосоме X. The aim of study was to evaluate X-chromosome mosaicism in health fertile women from different age groups. We found physiological low-level gonosomal mosaicism in peripheral blood lymphocytes with a clear trend in increased proportion of X-aneuploidic cells associated with the increasing age to 1.83%, 2.23% and 5.88% for 20-29, 30-39 and 40-49 years, respectively. The buccal smear also exhibited physiological pattern of a low-level X-chromosome mosaicism, however, the level of gonosomal mosaicism with no statistically significant difference between groups, average level was 4.01%. The obtained data allow to developed diagnostic protocol of low-level X-chromosome mosaicism in fertile women.

Highly Sensitive Blocker Displacement Amplification and Droplet Digital PCR Reveal Low-Level Parental FOXF1 Somatic Mosaicism in Families with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n=14), of unknown parental origin (n=1), or inherited from a parent suspected to be somatic and/or germline mosaic (n=3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n=3) and copy number variant deletion (n=1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low-level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics.

Parental somatogonadal COL2A1 mosaicism contributes to intrafamilial recurrence in a family with type 2 collagenopathy.

The COL2A1 gene encodes the alpha-1 chain of procollagen type 2. Pathogenic variants in the COL2A1 gene are associated with several different types of skeletal dysplasia collectively known as type 2 collagenopathies. Type 2 collagenopathies have an autosomal dominant inheritance. Some germline or somatogonadal mosaicism cases have been reported. We investigated whether somatogonadal mosaicism occurred in a family with two children suspected of type 2 collagenopathies or related diseases. First, we detected a pathogenic variant in the COL2A1 gene in the two affected children by whole exome sequencing (WES). Next, we performed targeted deep sequencing to their parents without the variant by WES. A low level of COL2A1 mosaicism was revealed in the mother's tissues. We concluded that the mother had somatogonadal mosaicism with the COL2A1 mutation arose in the epiblast, and that the intrafamilial recurrence rate of the disease by the somatogonadal mosaicism was higher than by the germline mosaicism. This report suggests that parental low-level mosaicism should be evaluated in those parents with children carrying de novo germline mutations and the targeted deep sequencing is useful to detect them.

RASA1 mosaic mutations in patients with capillary malformation-arteriovenous malformation

BackgroundCapillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around...

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient’s tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Low-level mosaicism in tuberous sclerosis complex: prevalence, clinical features, and risk of disease transmission

PurposeTo examine the prevalence and spectrum of mosaic variant allele frequency (MVAF) in tuberous sclerosis complex (TSC) patients with low-level mosaicism and correlate genetic findings with clinical features and transmission risk. MethodsMassively parallel sequencing was performed on 39 mosaic TSC patients with 170 different tissue samples. ResultsTSC mosaic patients (MVAF: 0–10%, median 1.7% in blood DNA) had a milder and distinct clinical phenotype in comparison with other TSC series, with similar facial angiofibromas (92%) and kidney angiomyolipomas (83%), and fewer seizures, cortical tubers, and multiple other manifestations (p < 0.0001 for six features). MVAF of TSC1/TSC2 pathogenic variants was highly variable in different tissue samples. Remarkably, skin lesions were the most reliable tissue for variant identification, and 6 of 39 (15%) patients showed no evidence of the variant in blood. Semen analysis showed absence of the variant in 3 of 5 mosaic men. The expected distribution of MVAF in comparison with that observed here suggests that there is a considerable number of individuals with low-level mosaicism for a TSC2 pathogenic variant who are not recognized clinically. ConclusionOur findings provide information on variability in MVAF and risk of transmission that has broad implications for other mosaic genetic disorders.

Sequence characterization of RET in 117 Chinese Hirschsprung disease families identifies a large burden of de novo and parental mosaic mutations

BackgroundHirschsprung disease (HSCR) is an inherited congenital disorder characterized by the absence of enteric ganglia in the distal part of the gut. RET is the major causative gene and contains > 80% of all known disease-causing mutations.ResultsTo determine the incidence of RET pathogenic variants, be they Mendelian inherited, mosaic in parents or true de novo variants (DNVs) in 117 Chinese families, we used high-coverage NGS and droplet digital polymerase chain reaction (ddPCR) to identify 15 (12.8%) unique RET coding variants (7 are novel); one was inherited from a heterozygous unaffected mother, 11 were DNVs (73.3%), and 3 full heterozygotes were inherited from parental mosaicism (2 paternal, 1 maternal): two clinically unaffected parents were identified by NGS and confirmed by ddPCR, with mutant allele frequency (13–27%) that was the highest in hair, lowest in urine and similar in blood and saliva. An extremely low-level paternal mosaicism (0.03%) was detected by ddPCR in blood. Six positive-controls were examined to compare the mosaicism detection limit and sensitivity of NGS, amplicon-based deep sequencing and ddPCR.ConclusionOur findings expand the clinical and molecular spectrum of RET variants in HSCR and reveal a high frequency of RET DNVs in the Chinese population.

Uncovering low-level mosaicism in human embryonic stem cells using high throughput single cell shallow sequencing

Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.

85P - Improving detection level of somatic mosaicism in neurofibromatosis type 1

BackgroundNeurofibromatosis type 1 (NF-1) is a tumor predisposition syndrome resulting from mutations in NF1 antioncogene. Differential diagnosis with genetic testing is essential in NF-1 diagnostics due to its clinical variability. Identifying of pathogenic mutations is challenging because of the gene size and structure, especially for low-level somatic mosaicism. Notably, patients with mosaic NF-1 demonstrate no less severe phenotype than those with classic form. Nowadays, NGS and Sanger sequencing are recognized diagnostics methods and they both are not sensitive enough to mosaic genotypes with small fractions of alternative alleles. We believe that this can be resolved with a special approach to sequencing data analysis. MethodsIn order to re-evaluate previously obtained NGS (Ion AmpliSeq technology) results for 275 probands with clinical diagnosis “NF-1” or “NF, unspecified” for whom germline mutations in NF1 and NF2 genes were not identified, we developed an improved data analysis pipeline to search for somatic mutations, which includes: (1) programmatic pool-based division of NGS reads, (2) elimination of out-of-design aligned reads, (3) exclusion of systematic sequencing errors, (4) variant calling with low stringency parameters (alternative allele frequency, AF≥0.05; read depth, DP≥20). Newly detected mutations were verified using Sanger sequencing and heteroduplex analysis. Sanger results were analysed using our in-house SeqBase software, highly sensitive to mosaic variants. ResultsApplication of our NGS data reanalysis algorithm allowed us to detect 12 cases (4.3%) of somatic mosaic mutations among 275 probands otherwise lacking molecular verification of neurofibromatosis diagnosis. The majority of the identified mutations are nonsense or frameshift, with AF ranging from 0.051 to 0.296. Mutations were verified with alternative methods of molecular diagnostics. ConclusionsImproved sequencing data analysis allows to detect NF-1 cases with mosaic genotype in cases unresolved by conventional analysis pipeline. Yet, success rate is strongly dependent upon the fraction of the mutant allele, thus alternative quantitative methods are required for exhaustive NF-1 molecular diagnosis. Legal entity responsible for the studyFederal State Budgetary Institution "Research Centre for Medical Genetics". FundingThe research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation. DisclosureAll authors have declared no conflicts of interest.

Improved Molecular Diagnosis of McCune-Albright Syndrome and Bone Fibrous Dysplasia by Digital PCR.

McCune–Albright syndrome (MAS) is a rare congenital disorder characterized by the association of endocrine and nonendocrine anomalies caused by somatic activating variants of GNAS. The mosaic state of variants makes the clinical presentation extremely heterogeneous depending on involved tissues. Biological samples bearing a low level of mosaicism frequently lead to false-negative results with an underestimation of causative molecular alterations, and the analysis of biopsies is often needed to obtain a molecular diagnosis. To date, no reliable analytical method for the noninvasive testing of blood is available. This study was aimed at validating a novel and highly sensitive technique, the digital PCR (dPCR), to increase the detection rate of GNAS alterations in patients with a clinical suspicion of MAS and, in particular, in blood. We screened different tissues (blood, bone, cutis, ovary, and ovarian cyst) collected from 54 MAS patients by different technical approaches. Considering blood, Sanger was unable to detect mutations, the allele-specific PCR and the co-amplification at lower denaturation temperature had a 9.1% and 18.1% detection rate, respectively, whereas the dPCR reached a 37.8% detection rate. In conclusion, the dPCR resulted in a cost-effective, reliable, and rapid method allowing the selective amplification of low-frequency variants and able to improve GNAS mutant allele detection, especially in the blood.

Postnatal clinical phenotype of five patients with Pallister-Killian Syndrome (tetrasomy 12p): Interest of array CGH for diagnosis and review of the literature.

BackgroundPallister–Killian syndrome (PKS) is a rare sporadic disorder caused by tetrasomy of the short arm of chromosome 12. The main clinical manifestations are global developmental delay, intellectual disability, epilepsy, dysmorphic features, hypopigmented and/or hyperpigmented lesions, and multiple congenital anomalies. PKS is associated with tissue mosaicism, which is difficult to diagnose through peripheral blood sample by conventional cytogenetic methods and fluorescence in situ hybridization.MethodsHere, we report five patients with PKS. We delineate their clinical phenotypes and we compare them with previously published cases. We used array Comparative Genomic Hybridization (aCGH) with DNA extracted from peripheral blood samples. The five patients have also been tested by conventional cytogenetics techniques.ResultsFour out of five patients showed tetrasomy 12p by aCGH. Three of the four patients have typical i(12p) and one of the four demonstrated atypical tetrasomy 12p. The percentage of mosaicism was as low as 20%. Our cohort exhibited the typical PKS phenotypes.ConclusionOur results demonstrate the efficacy of aCGH for the diagnosis of PKS from DNA extracted from lymphocytes. Thus, for patients suspected of PKS, we recommend performing aCGH on lymphocytes at an early age before proceeding to skin biopsy. aCGH on peripheral blood samples is sensitive in detecting low level of mosaicism and it is less invasive method than skin biopsy. We reviewed also the literature concerning the previously published PKS patients diagnosed by aCGH.

Megalencephaly syndromes associated with mutations of core components of the PI3K-AKT-MTOR pathway: PIK3CA, PIK3R2, AKT3, and MTOR.

Megalencephaly (MEG) is a developmental abnormality of brain growth characterized by early onset, often progressive, brain overgrowth. Focal forms of megalencephaly associated with cortical dysplasia, such as hemimegalencephaly and focal cortical dysplasia, are common causes of focal intractable epilepsy in children. The increasing use of high throughput sequencing methods, including high depth sequencing to more accurately detect and quantify mosaic mutations, has allowed us to identify the molecular etiologies of many MEG syndromes, including most notably the PI3K-AKT-MTOR related MEG disorders. Thorough molecular and clinical characterization of affected individuals further allow us to derive preliminary genotype-phenotype correlations depending on the gene, mutation, level of mosaicism, and tissue distribution. Our review of published data on these disorders so far shows that mildly activating variants (that are typically constitutional or germline) are associated with diffuse megalencephaly with intellectual disability and/or autism spectrum disorder; moderately activating variants (that are typically high-level mosaic) are associated with megalencephaly with pigmentary abnormalities of the skin; and strongly activating variants (that are usually very low-level mosaic) are associated with focal brain malformations including hemimegalencephaly and focal cortical dysplasia. Accurate molecular diagnosis of these disorders is undoubtedly crucial to more optimally treat children with these disorders using PI3K-AKT-MTOR pathway inhibitors.

12. WHAT TO DO WHEN THERE IS NO EUPLOID EMBRYOS? TRANSFER MOSAIC EMBRYOS OR PROPOSE ANOTHER PGT-A CYCLE?

Introduction Embryonic mosaicism is defined as the presence of karyotypically distinct cell lines within an embryo and can be detected with Next Generation Sequencing (NGS) at a rate between 20-80%. High incidence of mosaicism has been reported in preimplantation embryos, with the blastocyst mosaicism being between 4-24% (Harton et al., 2017). Although mosaic embryos have a chance to implant (Munne et al., 2017; Spinella et al., 2018), their pregnancy rate is lower, with an increased miscarriage rate, possible risk of intrauterine growth retardation and uniparental disomy. Therefore, low-level mosaics are preferentially selected for transfer if no euploid embryos are found. Material and Methods This retrospective study was based on 1958 preimplantation genetic testing for aneuploidy (PGT-A) cycles initiated between January 2017 and December 2018 in Istanbul Memorial Hospital. At least one euploid embryo was found for 1066 cycles (54.4%) and in 892 PGT-A cycles either full aneuploid or euploid/ mosaic embryos were diagnosed. PGT-A was done by NGS ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific) following trophectoderm biopsy. Among 168 cycles at least one mosaic embryo was found, and 37 mosaic embryo transfer cycles were achieved (mean female age: 36.0). 42 patients refused the transfer of mosaic embryos and went through another PGT-A cycle (mean female age: 37.1). Student's t-test and Mann-Whitney U test were applied for continuous variables and Chi-square test for categorical group comparisons. Results Among the 42 patients who have chosen to go for a second PGT-A cycle, 31 had at least one euploid embryo diagnosed (73.6%) and 28 couples came for a frozen-thawed embryo transfer cycle at the time the abstract was written. The mean mosaicism rate of the transferred embryos was 29% with a range of 20% to 40%. The biochemical pregnancy rate was 64.8% and 78.5% in the mosaic embryo transfer group and in the second PGT-A cycle group, respectively (p=0.23). The biochemical miscarriage rate was similar for both groups (16.7% vs. 13.6%, respectively). The clinical pregnancy rate was 54.1% and 67.9% for mosaic embryos and for the transfer of euploid embryos found in a second PGT-A cycle, respectively (p=0.26). The clinical miscarriage rate was 10% and 15.8% in the mosaic embryo transfer group and in the second PGT-A cycle group, respectively (p=0.71). The ongoing pregnancy rate was 48.6% and 57.1% for mosaics and second cycle's euploids, respectively (p=0.50). Conclusions As no statistically significant differences were found and considering the high rate of at least one euploid embryo in the second PGT-A cycle (73.6%), couples should be encouraged to start a new PGT-A cycle. For advanced maternal age patients (≥42) and/ or patients with a diminished ovarian reserve a second PGT-A cycle may not be a realistic option, thus mosaic embryo transfers may still be feasible. The limitations of our study include the fact that this was a retrospective analysis. Cohort sizes are undeniably small which prevents us to obtain statistically significant results and draw strong conclusions. Prospective studies are warranted.

15. THE INCIDENCE AND ORIGIN OF CHROMOSOME ANEUPLOIDIES IN HIGH QUALITY KARYOMAPPING SNP PROFILES

Introduction Karyomapping serves as the universal PGT-M platform based on Mendelian inheritance of SNP markers which assist in identifying haplotypes linked to a mutated gene. Moreover, karyomapping enables detection of aneuploidies by interrogating genome-wide SNP data in embryo samples and assists in a selection of euploid embryos for a transfer. We have found out that high quality SNP profiles, characterized by overall call rate ≥95%, might be reliably used for the identification of mitotic chromosome errors, aneuploidies in mosaic or segmental chromosome aberrations. Since karyomapping utilizes parental DNA samples, the origin of identified aneuploidies might be tracked in embryo samples. Our study describes the incidence and origin of aneuploidy, aiming at segmental chromosomes errors and mosaic aneuploidies which represents hot topic in respect to their clinical significance. Material & methods SNP data obtained by HumanKaryomap-12 BeadChips, Illumina SNP arrays were analysed in 2085 trophectoderm (TE) samples derived from 319 couples requesting PGT-M. Mean maternal age was 32.4 ± 4.5. Based on the karyomapping SNP data QC metrics, mean and median call rate was 90.4% and 95%, respectivelly. In total, 936 embryos fulfilled criteria of high quality SNP profiles (call rate ≥95%). A subset of twenty TE samples detected with mitotic trisomies, mosaic aneuploidies and segmental chromosome abnormalities by karyomapping were reanalysed using PG-Seq™ kit, which is the fully validated PGT-A platform in our laboratory. We have observed full concordance between both platforms. Results Analysing 936 high quality TE samples, we have observed that 617 (65.9%) of them were euploid. The remaining 319 TE samples were detected with the following aberrations: a) only aneuploid 234 (73.4%) samples; b) only mosaic 43 (13.5%) samples; c) aneuploid + mosaic 42 (13.2%) samples. In other words, overall mosaicism rate in 936 TE samples was 9.1% (85/936) in our study. This number tend to be underestimated, since we did not report low level mosaicism due to a lack of proper sensitivity validation of the karyomapping in last years. We have detected segmental aneuploidies in 12.6% (118/936) of TE samples, 56 (47.4%) of them were present in mosaic form. Regarding the origin of aneuploidies, 87% (220/253) of whole chromosome aneuploidies affected maternal chromosomes, the ratio difference was even more highlighted in whole chromosome trisomies (93%; 91 out of 98). In contrast, segmental chromosome abnormalities affected more frequently paternal chromosomes (61,3%; 38 out of 62). Notably, this trend towards paternally related segmental aneuploidies were also followed in segmental chromosome abnormalities in mosaic (60,7%; 34 out of 56). Conclusions 1. According to our validation, high quality SNP profiles (overall SNP call rate ≥95%) enables detection of mitotic trisomies, mosaicism at 25% sensitivity level and segmental chromosome abnormalities (in 5Mb resolution). 2. Meiotic whole chromosome trisomies prevail over mitotic whole chromosome trisomies (in full or mosaic state) in the ratio 4:1. 3. Segmental chromosome abnormalities (in full or mosaic state) affects more frequently paternal chromosomes (P value 4. Whole chromosome aneuploidies in mosaic affect equally maternal and paternal chromosomes.

Next-generation sequencing with comprehensive bioinformatics analysis facilitates somatic mosaic APC gene mutation detection in patients with familial adenomatous polyposis

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant colorectal tumor characterized by numerous adenomatous colonic polyps that often lead to colon cancer. Although most patients with FAP harbored germline mutations in APC gene, it was recently recognized that patients with clinical FAP, but without detectable pathogenic mutations, could be associated with somatic mosaic APC mutation.MethodsWe reanalyzed the nest-generation sequencing (NGS) gene panel testing results of patients who were diagnosed with FAP, but did not have APC mutations, at Yonsei Cancer Prevention Center between July 2016 and March 2018. We tested several variant calling algorithms to identify low level mosaic variants. In one patient with a low frequency APC mutation, NGS analysis was performed together with endoscopic biopsy. Variant calling tools HaplotypeCaller, MuTect2, VarScan2, and Pindel were used. We also used 3′-Modified Oligonucleotides (MEMO)-PCR or conventional PCR for confirmation.ResultsAmong 28 patients with clinical suspicion of FAP but no detectable pathogenic variants of colonic polyposis associated genes, somatic mosaic pathogenic variants were identified in seven patients. The variant allele frequency ranged from 0.3 to 7.7%. These variants were mostly detected through variant caller MuTect2 and Pindel, and were further confirmed using mutant enrichment with MEMO-PCR.ConclusionsThe NGS with an adequate combination of bioinformatics tools is effective to detect low level somatic variants in a single assay. Because mosaic APC mutations are more frequent than previously thought, the presence of mosaic mutations must be considered when analyzing genetic tests of patients with FAP.