Low-level Mosaicism Research Articles

Abstract P4-12-09: Low-level constitutional mosaicism of a de novo BRCA1 gene mutation

Abstract Background: Germline mutations in the BRCA1 and BRCA2 genes detected in some high-risk breast/ovarian families are used to estimate cancer risk, plan cancer early detection schemes, and make decisions about risk reducing surgeries. The gold standard for detecting BRCA sequence changes has long been Sanger sequencing, but recently next-generation sequencing (NGS) technologies have emerged as an accurate and efficient alternative, with improved sensitivity for detection of mosaic events. Here we report the detection of low-level constitutional germline mosaicism (∼5%) for a de novo pathogenic BRCA1 mutation detected using deep sequencing of three different non-cancerous tissues; and a corresponding high-level detection (∼50%) in cancerous breast tissue. This is the first reported case of multiple tissue constitutional mosaicism in BRCA1 at this level of detection. Patient and Methods: The patient is a woman of mixed Jewish Ashkenazi - Bulgarian heritage, diagnosed with a large (8x10 cm), triple negative, high-grade invasive breast cancer in the right breast at age 43 years. Her cancer family history includes a daughter with acute lymphatic leukemia at age 18 months, a brother with a Central Nervous System (CNS) tumor at age 45 years, a father with a malignant CNS tumor at age 58 years, a maternal grandfather with a malignant tumor in his 70's, and two of this maternal grandfather's sisters who were diagnosed with breast cancer. The patient underwent neoadjuvant chemotherapy followed by bilateral mastectomy (therapeutic mastectomy for the right breast and a contralateral risk-reducing mastectomy). The patient underwent germline testing using NGS, sequencing of 29 hereditary cancer genes in DNA extracted from blood using Invitae’s Hereditary Cancer Panel. This testing was followed up with additional NGS testing of buccal and contralateral healthy breast tissue. The patient also underwent somatic mutation testing on her breast tumor. Results: NGS of DNA extracted from blood identified a pathogenic BRCA1 mutation, c.1953dupG (p.Lys652GlufX21), in 5% of reads (X450 coverage). No other pathogenic mutations were detected in other genotyped cancer susceptibility genes. Analysis of buccal tissue and normal breast tissue removed at initial surgery also identified this mutation in ∼5% of reads, and confirm that this individual is a constitutional mosaic for this mutation. Genetic analyses were subsequently performed on the breast cancer tissue using Genome Health platform (Foundation One) and the same mutation, c.1943dupG was detected in 47% of molecules. Sanger sequencing of BRCA1 and BRCA2 in DNA extracted from peripheral blood had not detected this mutation. Analysis of the maternal DNA did not reveal this mutation, and analysis of the father was not possible. Conclusion: This is the first reported case of a de novo constitutional mosaicism in BRCA1 at this level of detection and confirmed across multiple tissue types, and highlights the need to perform deep sequencing in individuals clinically suspected of having cancer predisposition, prior to considering risk-reducing surgery. Citation Format: Eitan Friedman, Noa Efrat, Lior Soussan-Gutman, Addie Dvir, Yulia Kaplan, Tali Ekstein, Keith Nykamp, Martin Powers, Marina Rabideau, Scott Topper. Low-level constitutional mosaicism of a de novo BRCA1 gene mutation [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-09.

Somatic NOD2 mosaicism in Blau syndrome

Somatic NOD2 mosaicism in Blau syndrome

Next generation sequencing in sporadic retinoblastoma patients reveals somatic mosaicism.

In about 50% of sporadic cases of retinoblastoma, no constitutive RB1 mutations are detected by conventional methods. However, recent research suggests that, at least in some of these cases, there is somatic mosaicism with respect to RB1 normal and mutant alleles. The increased availability of next generation sequencing improves our ability to detect the exact percentage of patients with mosaicism. Using this technology, we re-tested a series of 40 patients with sporadic retinoblastoma: 10 of them had been previously classified as constitutional heterozygotes, whereas in 30 no RB1 mutations had been found in lymphocytes. In 3 of these 30 patients, we have now identified low-level mosaic variants, varying in frequency between 8 and 24%. In 7 out of the 10 cases previously classified as heterozygous from testing blood cells, we were able to test additional tissues (ocular tissues, urine and/or oral mucosa): in three of them, next generation sequencing has revealed mosaicism. Present results thus confirm that a significant fraction (6/40; 15%) of sporadic retinoblastoma cases are due to postzygotic events and that deep sequencing is an efficient method to unambiguously distinguish mosaics. Re-testing of retinoblastoma patients through next generation sequencing can thus provide new information that may have important implications with respect to genetic counseling and family care.

Lymphatic and Other Vascular Malformative/Overgrowth Disorders Are Caused by Somatic Mutations in PIK3CA

To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children's Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Children's Hospital. Most individuals from Boston Children's Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Children's Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

FEMALE PATIENT WITH A VARIANT OF TURNER SYNDROME

AbstractThe X chromosome contains determinants necessary to assure normal structural growth. Deletions of proportions of the X chromosome have been reported in a huge number of patients. We report on a mosaic short arm deletion of X chromosome (Xp-) in 14-years-old female patient with short stature, low set ears and no secondary sexual characteristics, a rare Turner syndrome variant. Chromosomal analysis using GTG-banding showed in 85% of cells and 46, X, del(Xp) in 15% of cells. The Fluorescence in situ hybridization technique of the cultured cells helped to perceive low grade cell line. Hence, molecular cytogenetics methods are essential to detect low level mosaicism which is important in better counseling.

New BAC probe set to narrow down chromosomal breakpoints in small and large derivative chromosomes, especially suited for mosaic conditions.

Fluorescence in situ hybridization (FISH) and/or array-comparative genomic hybridization (aCGH) performed after initial banding cytogenetics is still the gold standard for detection of chromosomal rearrangements. Although aCGH provides a higher resolution, FISH has two main advantages over the array-based approaches: (1) it can be applied to characterize balanced as well as unbalanced rearrangements, whereas aCGH is restricted to unbalanced ones, and (2) chromosomal aberrations present in low level or complex mosaics can be characterized by FISH without any problems, while aCGH requires presence of over 50 % of aberrant cells in the sample for detection. Recently, a new FISH-based probe set was presented: the so-called pericentric-ladder-FISH (PCL-FISH) that enables characterization of chromosomal breakpoints especially in mosaic small supernumerary marker chromosomes (sSMC). It can also be applied on large inborn or acquired derivative chromosomes. The main feature of this set is that the probes are applied in a chromosome-specific manner and they align along the chromosome in average intervals of ten megabasepairs. Hence PCL-FISH provides denser coverage and a more precise anchorage on the human DNA-sequence than most other FISH-banding approaches.

Intent to treat analysis of preimplantation genetic screening (PGS) and in vitro fertilization (IVF) versus expectant management (EM) in patients with recurrent pregnancy loss (RPL)

PGS protocol in the prospective study and 52 blastocysts in the retrospective evaluation. Sequencing was performed using MiSeq instrument. CNV analysis was accomplished by BlueFuse software. RESULTS: Reconstitution experiments results demonstrated that chromosomal mosaicism can be consistently identified by NGS, with the lower representation of detectable aneuploid cells being at 16% level. In addition, NGS was able to detect 86/86(100%) embryos with mosaicism, accounting for a total of 225 chromosomes involved by mosaicism. Paired comparison between NGS and aCGH showed 100% concordant results. CONCLUSION: This study demonstrates that NGS technique has a substantially increased sensitivity for detection low level mosaicism, three times higher compared to the resolution limits of the conventional methods, usually ranging from 35 to 50%. As a consequence, NGS may represent a more robust method for aneuploidy screening of embryos at blastocyst stage.

Next generation sequencing-based aneuploidy screening improves detection of low-level mosaicism in human embryos

Aneuploidy screening of embryos at blastocyst stage can be jeopardized by the presence of chromosomal mosaicism, which is a phenomenon characterized by the presence a mixture of diploid and aneuploid cell lines in embryos. Although its significance for implantation and the developmental potential of embryos is still unclear, it is reasonable to assume that mosaicism is likely to influence IVF success rates. In this study we investigated whether next generation sequencing (NGS) technologies applied for preimplatation genetic screening (PGS) purposes have the potential to improve mosaicism detection in human embryos over conventional methods.

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.

Methylation analysis and diagnostics of Beckwith-Wiedemann syndrome in 1,000 subjects.

BackgroundBeckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing.ResultsOut of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system (sensitivity 75.9%, and specificity 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area under the curve 0.85, 95% CI 0.83 to 0.87) compared to previous criteria.ConclusionsWe suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.

The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.

Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS) has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH) to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.

SNP array detects chromosome aberrations that we thought do not exist: the first case of an isochromosome Xp (i(X)(p10))

What's already known about this topic? Single nucleotide polymorphism (SNP) array detects submicroscopic chromosome aberrations that can be missed by karyotyping. Isochromosome i(X)(p10) is supposed to be lethal if it does not contain the X-inactivation center and has never been detected. What does this study add? Low-level mosaicism is an underestimated cause of congenital anomalies: A low-level mosaicism of an isochromosome Xp was found in a fetus with hygroma colli. SNP array can reveal the real percentage of abnormal cells in uncultured material in contrast to karyotyping that is (highly) influenced by the culture selection, and therefore, SNP array on uncultured material should be the preferred technique for prenatal diagnosis.

Variable levels of tissue mosaicism can confound the interpretation of chromosomal microarray results from peripheral blood

Chromosomal microarray analysis (CMA) has significantly increased the ability to diagnose medical conditions caused by copy-number variation in the human genome. Given that the regions involved in copy-number abnormalities often encompass multiple genes, it has been common practice in recent years to compare the phenotypes of individuals with specific copy-number alterations identified by CMA, with the goal of identifying the critical regions for particular elements of a disease phenotype. It is rarely mentioned that this practice relies heavily on the assumption that the absence of mosaicism on CMA from a peripheral blood sample (the most common source of DNA in current clinical practice) reflects the absence of mosaicism in other tissues. We report here a case that violates that assumption. A 28-year-old male with Charcot–Marie–Tooth disease was found by CMA to have a duplication of 17p12 along with two other abnormalities: A duplication of 12p13.33 translocated to the long arm of chromosome 3 and an interstitial duplication of 12p11.23. The patient did not have any clinical features suggestive of 12p duplication syndrome. Chromosomal microarray analysis on skin fibroblasts revealed the duplications at 17p12 and 12p11.23, but not the terminal duplication of 12p13.33. FISH analysis on skin fibroblasts confirmed the presence of very low levels of mosaicism for the terminal 12p duplication. The case illustrates how the absence of mosaicism in blood is not always indicative of the absence of mosaicism in other tissues. Even in an era of high-throughput, highly accurate DNA-based tests, it is important to remember the limitations of testing before drawing conclusions about the relationship between a test results and a clinical phenotype.

Interphase FISH on uncultured amniocytes at repeat amniocentesis for rapid confirmation of low-level mosaicism for tetrasomy 18p

Interphase FISH on uncultured amniocytes at repeat amniocentesis for rapid confirmation of low-level mosaicism for tetrasomy 18p

Chromosomal microarray analysis allows prenatal detection of low level mosaic autosomal aneuploidy

What's already known about this topic? Mosaic autosomal aneuploidies detected prenatally complicate genetic counselling, despite the presence of relevant ultrasound scan anomalies. Chromosomal microarray analysis is currently reported to detect mosaicism of between 10% and 30% prenatally. What does this study add? Chromosomal microarray analysis is effective in detecting low level mosaic autosomal aneuploidy in a prenatal setting, which traditional methods may fail to detect. This detection of low level mosaicism supports the use of chromosomal microarray analysis in prenatal diagnosis.

Enhanced Sensitivity for Detection of Low-Level Germline MosaicRB1Mutations in Sporadic Retinoblastoma Cases Using Deep Semiconductor Sequencing

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.

Development of a Nationwide, Low-Level Wind Shear Mosaic in France

Abstract An algorithm for the detection of horizontal wind shear at low levels was developed. The algorithm makes use of data collected by all radars from the Application Radar à la Météorologie Infra-Synoptique (ARAMIS) operational network, in order to build a complete mosaic of wind shear over metropolitan France. The product provides an estimation of the maximum horizontal wind shear detected in the low levels, between 0 and 2 km AGL. Examination of the wind shear mosaic for different cases shows that the product is able to retrieve small-scale wind shear signatures that can be linked to either convergence lines ahead of convective cells, which are indicative of gust fronts, or strong convergence areas inside intense cells. A statistical evaluation of the wind shear mosaic was performed, by comparing horizontal wind shear observed inside the area defined by convective objects with wind gusts recorded along their trajectory by weather stations. A link between those different observations was clearly established. Therefore, the use of wind shear for wind gust prediction was tested in combination with other parameters: an estimation of the energetic potential of density currents, the cell surface with reflectivity over 51 dBZ, relative helicity, and cell propagation speed. Different wind gust warning rules were tested on 468 convection nowcasting objects (CONOs). The results clearly highlighted the benefits of using wind shear for wind gust estimation, and also demonstrated the improvement in forecasting skill when combining different parameters. The wind shear mosaic will be produced operationally before the end of 2013 and will be used to improve wind gust warnings provided to end users.

Low-frequency Mosaicism of Trisomy 14, Missed by Array CGH

before and after birth, mental retardation, developmental delay, cardiac anomalies, minor anomalies of genitalia in boys, abnormal skin pigmentation and dysmorphic features. Array comparative genomic hybridization (CGH) analysis has recently become a widely used method for detecting DNA copy number changes, in place of traditional karyotype analysis. In particular, an international consensus statement has now recommended array CGH as a first-line test in patients with unexplained developmental delay, intellectual disability and dysmorphisms. However, the ability of array CGH is limited in case of low-level mosaicism. Here, we report the detailed clinical and cytogenetic findings of the first case in Korea to show lowfrequency mosaicism of trisomy 14, diagnosed by array CGH analysis supplemented with karyotyping. Low-frequency Mosaicism of Trisomy 14, Missed by Array CGH

Sensitive and specific detection of mosaic chromosomal abnormalities using the Parent-of-Origin-based Detection (POD) method

BackgroundMosaic somatic alterations are present in all multi-cellular organisms, but the physiological effects of low-level mosaicism are largely unknown. Most mosaic alterations remain undetectable with current analytical approaches, although the presence of such alterations is increasingly implicated as causative for disease.ResultsHere, we present the Parent-of-Origin-based Detection (POD) method for chromosomal abnormality detection in trio-based SNP microarray data. Our software implementation, triPOD, was benchmarked using a simulated dataset, outperformed comparable software for sensitivity of abnormality detection, and displayed substantial improvement in the detection of low-level mosaicism while maintaining comparable specificity. Examples of low-level mosaic abnormalities from a large autism dataset demonstrate the benefits of the increased sensitivity provided by triPOD. The triPOD analyses showed robustness across multiple types of Illumina microarray chips. Two large, clinically-relevant datasets were characterized and compared.ConclusionsOur method and software provide a significant advancement in the ability to detect low-level mosaic abnormalities, thereby opening new avenues for research into the implications of mosaicism in pathogenic and non-pathogenic processes.

Deficits in human trisomy 21 iPSCs and neurons

Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.