Low-level Mosaicism Research Articles

Prenatal diagnosis of low-level mosaicism for trisomy 18 associated with a favorable fetal outcome

Prenatal diagnosis of low-level mosaicism for trisomy 18 associated with a favorable fetal outcome

Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.

SummaryGenetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine.

Defining the performance parameters of a rapid screening tool for myotonic dystrophy type 1 based on triplet-primed PCR and melt curve analysis

ABSTRACTBackground: DMPK CTG-repeat expansions that cause myotonic dystrophy type 1 (DM1) can be detected more rapidly, cost-effectively, and simply by combining triplet-primed PCR (TP-PCR) with melting curve analysis (MCA). We undertook a detailed technical validation study to define the optimal operational parameters for performing bidirectional TP-PCR MCA assays.Methods: We determined the assays’ analytic specificity and sensitivity, assessed the effect of reaction volumes, DNA diluents, and common contaminants on melt peak temperature, determined the assays’ sensitivity in detecting low-level mosaicism for repeat expansion, and evaluated their performance on two real-time PCR platforms.Results: Both assays were highly specific and sensitive, and performed optimally under a broad range of parameters. Bidirectional TP-PCR MCA analysis also reduces the risk of generating false-negative results associated with the rare CCG-interruptions that may be present at either end of expanded alleles.Conclusion: The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.

Low level mosaicism: incidence and implications on clinical pregnancies

Low level mosaicism: incidence and implications on clinical pregnancies

Application of array-based comparative genomic hybridization technique in genetic analysis of patients with spontaneous abortion

To investigate the value of array-based comparative genomic hybridization (array-CGH) technique for the detection of chromosomal analysis of miscarried embryo, and to provide genetic counseling for couples with spontaneous abortion. Totally 382 patients who underwent miscarriage were enrolled in this study. All aborted tissues were analyzed with conventional cytogenetic karyotyping and array-CGH, respectively. Through genetic analysis, all of the 382 specimens were successfully analyzed by array-CGH (100.0%, 382/382), and the detection rate of chromosomal aberrations was 46.6% (178/382). However, conventional karyotype analysis was successfully performed in 281 cases (73.6%, 281/382), and 113 (40.2%, 113/281) were found with chromosomal aberrations. Of these 178 samples identified by array-CGH, 163 samples (91.6%, 163/178) were aneuploidy, 15 samples (8.4%, 15/178) were segmental deletion and (or) duplication cases. Four of 10 cases with small segmental deletion and duplication were validated to be transferred from their fathers or mathers who were carriers of submicroscopic reciprocal translocation. Of these 113 abnormal karyotypes founded by conventional karyotyping, 108 cases (95.6%, 108/113) were aneuploidy and 5 cases (4.4%, 5/113) had chromosome structural aberrations. Most array-CGH results were consistent with conventional karyotyping but with 3 cases of discrepancy, which included 2 cases of triploids, 1 case of low-level mosaicism that undetcted by array-CGH. Compared with conventional karyotyping, there is an increased detection rate of chromosomal abnormalities when array-CGH is used to analyse the products of conception, primarilly because of its sucess with nonviable tissues. It could be a first-line method to determine the reason of miscarrage with higher accuracy and sensitivity.

Abstract 3619: Copy number estimation of cancer samples with genome, exome and targeted panel next generation sequencing

Abstract Next-generation sequencing (NGS) is mainly used to obtain sequence variants (SNVs). However, obtaining copy number results from NGS has gained momentum in both research and clinical applications. While microarray has traditionally been the gold standard for copy number analysis, NGS is rapidly gaining momentum as the first line analysis for tumor samples. The genomic content captured may vary from batch to batch, where targeted regions may include the full genome, all exonic regions or may be limited to exonic regions in specific genes that have a clear diagnosis, are actionable with prescription drugs or compounds in clinical trials, or have known impacts on prognosis or outcome. Thus, obtaining copy number information from a range of NGS methods is needed. As a result, we have developed a method called BAM (pooled reference) which only requires the loading of BAM files and the NGS design file to generate copy number results. BAM (pooled reference) can derive copy number results from Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES), and targeted panel NGS data. This method builds a reference file out of a pool of BAM files that are either from normal controls or from unrelated experimental samples, then generates copy number calls for each experimental sample. In combination with the VCF files that contain the sequence mutations, an integrated analysis of both events can be easily carried out. To test this algorithm, five colon adenocarcinoma whole exome sequencing samples were processed through the BAM (pooled reference) algorithm in Nexus Copy Number 8.0. GC correction schemes based on a range of window size and presence or absence of GC probe content were applied to the data and assessed for overall quality. Differences in overall read-depth resulted in variable sample quality across the cohorts, however most sample quality was adequate for copy number estimation and a quality threshold was assessed. Among the samples tested, the best quality after GC correction comes from the 50kb region size with or without the probes. Next, the copy number profiles of the TCGA COAD samples from WES and microarray were compared for accuracy. Using microarray results as a reference for assessing calls greater than 5 MB, no false positive and one false negative call were observed; the single false negative call was attributable to low-level mosaicism in the tumor sample. Results indicate that relative copy number can be estimated and is comparable to the results achieved with microarray for the same targeted regions. This analysis series was then repeated using a secondary cohort of unrelated samples subjected to microarray and targeted panel NGS to validate results. The BAM (pooled reference) method has been tested in a variety of cancer samples. This is an ideal tool for copy number estimation with NGS results in cancer samples because it provides a way for non-pair matched analysis with genome, exome and targeted NGS. Citation Format: Soheil Shams, Andrea O’Hara, Zhiwei Che. Copy number estimation of cancer samples with genome, exome and targeted panel next generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3619.

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism.

Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children's Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

A Recurrent Mosaic Mutation in SMO, Encoding the Hedgehog Signal Transducer Smoothened, Is the Major Cause of Curry-Jones Syndrome

Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), aG-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism.

Prenatal diagnosis and molecular cytogenetic characterization of low-level true mosaicism for trisomy 21 using uncultured amniocytes

ObjectiveWe present a prenatal diagnosis and molecular cytogenetic characterization of low-level true mosaicism for trisomy 21 using uncultured amniocytes. Case reportA 35-year-old woman presented with a borderline-positive result of noninvasive prenatal testing for trisomy 21. She underwent amniocentesis at 18 weeks of gestation, which revealed a karyotype of 47,XY,+21(5)/46,XY(53). Repeat amniocentesis at 22 weeks of gestation revealed a karyotype of 47,XY,+21(6)/46,XY(26). Array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed mosaic levels of 10% to 15% for trisomy 21. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes revealed a mosaic level of 21.7% (28/129 cells) for trisomy 21. Following genetic counseling and detailed ultrasound examination, the parents decided to continue the pregnancy. The pregnancy was carried to term, and a normal 3664-g male baby was delivered. The cord blood lymphocytes had a karyotype of 47,XY,+21(2)/46,XY(38). Postnatal interphase FISH analysis of urine detected no trisomy 21 in all 39/39 urinary cells. The neonate was phenotypically normal at age 7 months. ConclusionLow-level true mosaicism for trisomy 21 can be associated with a favorable fetal outcome. aCGH and interphase FISH analyses on uncultured amniocytes are useful for rapid confirmation of low-level true mosaicism for trisomy 21 at repeated amniocentesis.

Prenatal diagnosis of low-level mosaicism for trisomy 2 associated with a favorable pregnancy outcome

Prenatal diagnosis of low-level mosaicism for trisomy 2 associated with a favorable pregnancy outcome

Detection and Quantification of Mosaic Mutations in Disease Genes by Next-Generation Sequencing

The identification of mosaicism is important in establishing a disease diagnosis, assessing recurrence risk, and genetic counseling. Next-generation sequencing (NGS) with deep sequence coverage enhances sensitivity and allows for accurate quantification of the level of mosaicism. NGS identifies low-level mosaicism that would be undetectable by conventional Sanger sequencing. A customized DNA probe library was used for capturing targeted genes, followed by deep NGS analysis. The mean coverage depth per base was approximately 800×. The NGS sequence data were analyzed for single-nucleotide variants and copy number variations. Mosaic mutations in 10 cases/families were detected and confirmed by NGS analysis. Mosaicism was identified for autosomal dominant (JAG1, COL3A1), autosomal recessive (PYGM), and X-linked (PHKA2, PDHA1, OTC, and SLC6A8) disorders. The mosaicism was identified either in one or more tissues from the probands or in a parent of an affected child. When analyzing data from patients with unusual testing results or inheritance patterns, it is important to further evaluate the possibility of mosaicism. Deep NGS analysis not only provides insights into the spectrum of mosaic mutations but also underlines the importance of the detection of mosaicism as an integral part of clinical molecular diagnosis and genetic counseling.

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis.

FMR1 full mutations (FMs) (CGG expansion >200) in males mosaic for a normal (<45 CGG) or gray-zone (GZ) (45-54 CGG) allele can be missed with the standard 2-step fragile X syndrome (FXS) testing protocols, largely because the first-line PCR tests showing a normal or GZ allele are not reflexed to the second-line test that can detect FM. We used methylation-specific quantitative melt analysis (MS-QMA) to determine the prevalence of cryptic FM alleles in 2 independent cohorts of male patients (994 from Chile and 2392 from Australia) referred for FXS testing from 2006 to 2013. All MS-QMA-positive cases were retested with commercial triplet primed PCR, methylation-sensitive Southern blot, and a methylation-specific EpiTYPER-based test. All 38 FMs detected with the standard 2-step protocol were detected with MS-QMA. However, MS-QMA identified methylation mosaicism in an additional 15% and 11% of patients in the Chilean and Australian cohorts, respectively, suggesting the presence of a cryptic FM. Of these additional patients, 57% were confirmed to carry cryptic expanded alleles in blood, buccal mucosa, or saliva samples. Further confirmation was provided by identifying premutation (CGG 55-199) alleles in mothers of probands with methylation-sensitive Southern blot. Neurocognitive assessments showed that low-level mosaicism for cryptic FM alleles was associated with cognitive impairment or autism. A substantial number of mosaic FM males who have cognitive impairment or autism are not diagnosed with the currently recommended 2-step testing protocol and can be identified with MS-QMA as a first-line test.

Next-generation sequencing discloses a nonsense mutation in the dystrophin gene from long preserved dried umbilical cord and low-level somatic mosaicism in the proband mother

Duchene muscular dystrophy (DMD) is a progressive muscle wasting disease, caused by mutations in the dystrophin (DMD) on the X chromosome. One-third of patients are estimated to have de novo mutations. To provide in-depth genetic counseling, the comprehensive identification of mutations is mandatory. However, many DMD patients did not undergo genetic diagnosis because detailed genetic diagnosis was not available or their mutational types were difficult to identify. Here we report the genetic testing of a sporadic DMD boy, who died >20 years previously. Dried umbilical cord preserved for 38 years was the only available source of genomic DNA. Although the genomic DNA was severely degraded, multiplex ligation-dependent probe amplification analysis was performed but no gross mutations found. Sanger sequencing was attempted but not conclusive. Next-generation sequencing (NGS) was performed by controlling the tagmentation during library preparation. A nonsense mutation in DMD (p.Arg2095*) was clearly identified in the proband. Consequently, the identical mutation was detected as an 11% mosaic mutation from his healthy mother. Finally, the proband's sister was diagnosed as a non-carrier of the mutation. Thus using NGS we have identified a pathogenic DMD mutation from degraded DNA and low-level somatic mosaicism, which would have been overlooked using Sanger sequencing.

Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases

BackgroundIn 30–50% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, or POLE or POLD1, causing polymerase-proofreading-associated...

Characterisation of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian polymicrogyria: a next-generation sequencing study

SUMMARYBackgroundBilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment and epilepsy. BPP is etiologically heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic etiologies of BPP and delineate their frequency in this patient population.MethodsWe performed child-parent (trio)-based whole exome sequencing (WES) on eight children with BPP. Following the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 in a cohort of 118 children with BPP who were ascertained from 1980 until 2015 using two methods. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal-large head size. Second, we performed amplicon sequencing of the recurrent PIK3R2 mutation (p.Gly373Arg) on 80 children with various types of polymicrogyria including BPP. One additional patient underwent clinical WES independently, and was included in this study given the phenotypic similarity to our cohort. All patients included in this study were children (< 18 years of age) with polymicrogyria enrolled in our research program.FindingsUsing WES, we identified a mosaic mutation (p.Gly373Arg) in the regulatory subunit of the PI3K-AKT-MTOR pathway, PIK3R2, in two children with BPP. Of the 38 patients with BPP and normal-large head size who underwent targeted next generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient was found to have the recurrent PIK3R2 mutation by clinical WES. Seven patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH). Nineteen patients had the same mutation (Gly373Arg), and one had a nearby missense mutation (p.Lys376Glu). Across the entire cohort, mutations were constitutional in 12 and mosaic in eight patients. Among mosaic patients, we observed substantial variation in alternate (mutant) allele levels ranging from 2·5% (10/377) to 36·7% (39/106) of reads, equivalent to 5–73·4% of cells analyzed. Levels of mosaicism varied from undetectable to 17·1% (37/216) of reads in blood-derived compared to 29·4% (2030/6889) to 43·3% (275/634) in saliva-derived DNA.InterpretationConstitutional and mosaic mutations in the PIK3R2 gene are associated with a spectrum of developmental brain disorders ranging from BPP with a normal head size to the megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. The phenotypic variability and low-level mosaicism challenging conventional molecular methods have important implications for genetic testing and counseling.

High-sensitivity sequencing reveals multi-organ somatic mosaicism causing DICER1 syndrome

BackgroundSomatic mosaicism is being increasingly recognised as an important cause of non-Mendelian presentations of hereditary syndromes. A previous whole-exome sequencing study using DNA derived from peripheral blood identified mosaic mutations...

Description of a case of late-onset cryopyrin-associated periodic syndrome due to low-level somatic NLRP3 mosaicism

Cryopyrin-associated periodic syndromes (CAPS) usually present in early childhood as an urticaria-like skin rash associated with an increased inflammatory response, with additional manifestations (i.e. arthropathy, AA amyloidosis or deafness) typically restricted to certain phenotypes. CAPS are caused by dominantly inherited or de novogain-of-functionNLRP3 mutations. The introduction of next-generation sequencing (NGS) into clinics has revealed the important role of somatic NLRP3 mosaicism in these syndromes by means of its detection in a high proportion of patients who were apparently mutation-negative by Sanger sequencing. Thus, NGS technologies are becoming essential for routinely identifying the genetic cause of the suspected autoinflammatory disease.

Increased incidence of mosaicism among biopsied trophectoderm cells analyzed by Next Generation Sequencing

Preimplantation genetic screening (PGS) using Array CGH, qPCR or SNP arrays provided very high levels of diagnostic accuracy, however, very little ability to recognize low-level mosaicism. The newest Next Generation Sequencing (NGS) technique provides the same level of diagnostic accuracy and allows for an increased chance to detect chromosomal mosaics, down to as low as 20%. We set out to determine the presence, quantity and the types of genetic mosaicism in trophectoderm cells following embryo biopsy in clinical PGS cases using NGS analysis.

Post-zygotic Point Mutations Are an Underrecognized Source of De Novo Genomic Variation

De novo mutations are recognized both as an important source of genetic variation and as a prominent cause of sporadic disease in humans. Mutations identified as de novo are generally assumed to have occurred during gametogenesis and, consequently, to be present as germline events in an individual. Because Sanger sequencing does not provide the sensitivity to reliably distinguish somatic from germline mutations, the proportion of de novo mutations that occur somatically rather than in the germline remains largely unknown. To determine the contribution of post-zygotic events to de novo mutations, we analyzed a set of 107 de novo mutations in 50 parent-offspring trios. Using four different sequencing techniques, we found that 7 (6.5%) of these presumed germline de novo mutations were in fact present as mosaic mutations in the blood of the offspring and were therefore likely to have occurred post-zygotically. Furthermore, genome-wide analysis of "de novo" variants in the proband led to the identification of 4/4,081 variants that were also detectable in the blood of one of the parents, implying parental mosaicism as the origin of these variants. Thus, our results show that an important fraction of de novo mutations presumed to be germline in fact occurred either post-zygotically in the offspring or were inherited as a consequence of low-level mosaicism in one of the parents.

Low-level mosaicism of a de novo derivative chromosome 9 from a t(5;9)(q35.1;q34.3) has a major phenotypic impact

Microdeletion and microduplication syndromes are well-known causes of developmental delay and/or malformations of differing severity. Although homogeneous abnormalities can now be detected relatively easily using microarray technologies, they are more difficult to detect and interpret in cases of mosaicism. Here, we report on a male infant with a mosaic de novo derivative chromosome 9, featuring a 10.2 Mb 5q35 duplication (including the NSD1 gene) and a 687 kb 9q34 deletion (including EHMT1). The infant presented developmental delay, short stature, brachy/plagiocephaly and hyperactivity. The proportion of abnormal cells was 50% in saliva (in a microarray analysis) and 25% in lymphocytes (in a FISH analysis). Despite the low-level mosaicism in lymphocytes, this imbalance appears to be responsible for a distinctive phenotype (suggesting the presence of variable clinical expression and/or major somatic mosaicism).