Mosaic Variants Research Articles

Genetic and clinical findings in a Chinese cohort of patients with collagen VI-related myopathies.

Collagen VI-related myopathy, caused by pathogenic variants in the genes encoding collagen VI, represents a clinical continuum from Ullrich congenital muscular dystrophy (UCMD) to Bethlem myopathy (BM). Clinical data of 60 probands and their family members were collected and muscle biopsies of 26 patients were analyzed. COL6A1, COL6A2 and COL6A3 exons were analyzed by direct sequencing or next generation sequencing (NGS). Sixty patients were characterized by delayed motor milestones, muscle weakness, skin and joint changes with 40 UCMD and 20 BM. Muscle with biopsies revealed dystrophic changes and showed completely deficiency of collagen VI or sarcolemma specific collagen VI deficiency. We identified 62 different pathogenic variants in these 60 patients, with 34 were first reported while 28 were previously known; 72 allelic pathogenic variants in COL6A1 (25/72, 34.7%), COL6A2 (33/72, 45.8%) and COL6A3 (14/72, 19.4%). We also found somatic mosaic variant in the parent of 1 proband by personal genome machine amplicon deep sequencing for mosaicism. Here we provide clinical, histological and genetic evidence of collagen VI-related myopathy in 60 Chinese patients. NGS is a valuable approach for diagnosis and accurate diagnosis provides useful information for genetic counseling of related families.

Mosaicism of de novo pathogenic SCN1A variants in epilepsy is a frequent phenomenon that correlates with variable phenotypes.

Phenotypes caused by de novo SCN1A pathogenic variants are very variable, ranging from severely affected patients with Dravet syndrome to much milder genetic epilepsy febrile seizures plus cases. The most important determinant of disease severity is the type of variant, with variants that cause a complete loss of function of the SCN1A protein (α-subunit of the neuronal sodium channel Nav1.1) being detected almost exclusively in Dravet syndrome patients. However, even within Dravet syndrome disease severity ranges greatly, and consequently other disease modifiers must exist. A better prediction of disease severity is very much needed in daily practice to improve counseling, stressing the importance of identifying modifying factors in this patient group. We evaluated 128 participants with de novo, pathogenic SCN1A variants to investigate whether mosaicism, caused by postzygotic mutation, is a major modifier in SCN1A-related epilepsy. Mosaicism was investigated by reanalysis of the pathogenic SCN1A variants using single molecule molecular inversion probes and next generation sequencing with high coverage. Allelic ratios of pathogenic variants were used to determine whether mosaicism was likely. Selected mosaic variants were confirmed by droplet digital polymerase chain reaction and sequencing of different tissues. Developmental outcome was classified based on available data on intelligence quotient and school functioning/education. Mosaicism was present for 7.5% of de novo pathogenic SCN1A variants in symptomatic patients. Mosaic participants were less severely affected than nonmosaic participants if only participants with truncating variants are considered (distribution of developmental outcome scores, Mann-Whitney U, P=.023). Postzygotic mutation is a common phenomenon in SCN1A-related epilepsies. Participants with mosaicism have on average milder phenotypes, suggesting that mosaicism can be a major modifier of SCN1A-related diseases. Detection of mosaicism has important implications for genetic counseling and can be achieved by deep sequencing of unique reads.

Abstract PD1-06: Mosaic TP53 variants in women with breast cancer

Abstract Background: Germline TP53 pathogenic variants are indicative of Li-Fraumeni syndrome (LFS), a dominantly inherited hereditary cancer syndrome with high lifetime risks for female breast and other cancers. Genetic testing for LFS may reveal mosaicism, indicating a variant is present in some, but not all, of the cells tested. While these findings may represent a true constitutional event, mosaicism for TP53 variants also has been reported as a somatic event in lymphoblastoid cells from individuals with hematologic malignancy, previous chemotherapy exposure, or due to age-related clonal hematopoietic expansion. Understanding if a mosaic variant is constitutional can influence the patient's management and impact familial risk assessment. We present clinical history and follow-up testing from a series of female breast cancer patients with blood or oral rinse testing revealing a mosaic TP53 pathogenic or likely pathogenic variant (collectively, PV). Methods: We retrospectively reviewed clinical history and genetic testing results to identify women with a personal history of breast cancer and mosaicism for one or more TP53 PV identified on multi-gene hereditary cancer testing at our clinical diagnostic laboratory. Descriptive statistics were employed. Results: Forty-eight women with breast cancer were identified as having a mosaic TP53 PV, defined as an allelic fraction of <35%. Mean age at first breast cancer diagnosis was 49.2 years. Twenty children of 13 women with mosaic TP53 PV pursued targeted testing; none were positive for their mother's TP53 PV. Six of the 48 women (16.7%) pursued cultured fibroblast testing for the mosaic TP53 PV. In five, the PV was not found in fibroblasts. All five were diagnosed with breast cancer >40 years of age and three had other primary cancer diagnoses, including one with sarcoma. In one patient with early-onset and HER2-positive breast cancer, testing of fibroblasts also identified mosaicism for the TP53 PV, indicating constitutional mosaicism. Conclusions: For individuals with a mosaic TP53 PV, identification of the variant in a second tissue is necessary to confirm constitutional mosaicism and heightened risk for other LFS-associated cancers. In this series, additional testing confirmed one of six patients with mosaic TP53 PV pursuing fibroblast testing to have constitutional mosaicism. This individual's breast cancer was HER2-positive and her age at diagnosis was younger than those whose mosaic PV was not identified in fibroblasts. Additional follow-up testing data are needed to understand whether confirmatory testing in a second tissue is indicated for any breast cancer patient with a mosaic TP53 PV, or would be most likely to reveal constitutional mosaicism in individuals whose breast cancers are HER2-positive or early-onset. Citation Format: Mester JL, Postula K, Bissonnette J, Klein RT, Hruska K. Mosaic TP53 variants in women with breast cancer [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 PD1-06.

Robust identification of mosaic variants in congenital heart disease.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n=715) and a cohort of healthy individuals (n=416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

The use of droplet digital PCR and high resolution melt for detection of low level mosaicism

Diagnostic testing and screening for a number of genetic conditions can be complicated by presence of low level mosaicism (LLM) that arises post-zygotically, termed somatic mosaicism. While improvements in genetic testing technologies have significantly increased diagnostic yield in children with intellectual disability (ID), more than half of these children still remain undiagnosed. Many pathogenic variants contributing to ID may go unnoticed if present in a small proportion of cells, below the analytical sensitivity of standard methods in wide use. Importantly, mosaic variants may be present in the brain without being readily detectable in the tissues commonly sampled for genetic analysis, including blood and saliva. Some of these may be copy number and/or epigenetic changes such as DNA methylation involved in regulation of gene expression. This presentation highlights these issues in the context of syndromic forms of ID, including examples of well-characterised cohorts of children affected with fragile X syndrome (FXS), Rett syndrome and chromosome 15 imprinting disorders, as well as in developmental delay referrals. Potential solutions are described that use novel methodologies that utilise droplet digital PCR and high resolution melt to detect LLM in blood, buccal, saliva, and dried blood spots, significantly increasing diagnostic yield.

AutismKB 2.0: a knowledgebase for the genetic evidence of autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with strong genetic contributions. To provide a comprehensive resource for the genetic evidence of ASD, we have updated the Autism KnowledgeBase (AutismKB) to version 2.0. AutismKB 2.0 integrates multiscale genetic data on 1379 genes, 5420 copy number variations and structural variations, 11 669 single-nucleotide variations or small insertions/deletions (SNVs/indels) and 172 linkage regions. In particular, AutismKB 2.0 highlights 5669 de novo SNVs/indels due to their significant contribution to ASD genetics and includes 789 mosaic variants due to their recently discovered contributions to ASD pathogenesis. The genes and variants are annotated extensively with genetic evidence and clinical evidence. To help users fully understand the functional consequences of SNVs and small indels, we provided comprehensive predictions of pathogenicity with iFish, SIFT, Polyphen etc. To improve user experiences, the new version incorporates multiple query methods, including simple query, advanced query and batch query. It also functionally integrates two analytical tools to help users perform downstream analyses, including a gene ranking tool and an enrichment analysis tool, KOBAS. AutismKB 2.0 is freely available and can be a valuable resource for researchers.

Diversity of Mosaic pbp2x Families in Penicillin-Resistant Streptococcus pneumoniae from Iran and Romania.

ABSTRACTPenicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for β-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

Novel mosaic variants in two patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder caused by mutations in genes that encode for either structural (SMC1A, SMC3, RAD21) or regulatory (NIPBL, HDAC8) subunits of the cohesin complex. NIPBL represents the major gene of the syndrome and heterozygous mutations can be identified in more than 65% of patients. Interestingly, large portions of these variants were described as somatic mosaicism and often escape standard molecular diagnostics using lymphocyte DNA.Here we discuss the role of somatic mosaicism in CdLS and describe two additional patients with NIPBL mosaicism detected by targeted gene panel or exome sequencing. In order to verify the next generation sequencing data, Sanger sequencing or pyrosequencing on DNA extracted from different tissues were applied. None of the pathogenic variants was originally detected by Sanger sequencing on blood DNA.Patient 1 displays an unusual combination of clinical features: he is cognitively only mildly affected, but shows severe limb reduction defects. Patient 2 presents with a moderate phenotype. Interestingly, Sanger sequencing analysis on fibroblast DNA of this patient did not detect the disease-causing variant previously observed on the same DNA sample by exome sequencing. Subsequent analyses could confirm the variants by Sanger sequencing on buccal mucosa DNA. Notably, this is the first report of a higher mutational load in buccal mucosa than in fibroblast cells of a CdLS patient.Detection of low-level mosaicism is of utmost importance for an accurate molecular diagnosis and a proper genetic counseling of patients with a clinical diagnosis of CdLS. Next-generation sequencing technologies greatly facilitate the detection of low-level mosaicism, which might otherwise remain undetected by conventional sequencing approaches.

High frequency of mosaic pathogenic variants in genes causing epilepsy-related neurodevelopmental disorders

PurposeMosaicism probably represents an underreported cause of genetic disorders due to detection challenges during routine molecular diagnostics. The purpose of this study was to evaluate the frequency of mosaicism detected by next-generation sequencing in genes associated with epilepsy-related neurodevelopmental disorders.MethodsWe conducted a retrospective analysis of 893 probands with epilepsy who had a multigene epilepsy panel or whole-exome sequencing performed in a clinical diagnostic laboratory and were positive for a pathogenic or likely pathogenic variant in one of nine genes (CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A, or SCN2A). Parental results were available for 395 of these probands.ResultsMosaicism was most common in the CDKL5, PCDH19, SCN2A, and SCN1A genes. Mosaicism was observed in GABRA1, GABRG2, and GRIN2B, which previously have not been reported to have mosaicism, and also in KCNQ2 and MECP2. Parental mosaicism was observed for pathogenic variants in multiple genes including KCNQ2, MECP2, SCN1A, and SCN2A.ConclusionMosaic pathogenic variants were identified frequently in nine genes associated with various neurological conditions. Given the potential clinical ramifications, our findings suggest that next-generation sequencing diagnostic methods may be utilized when testing these genes in a diagnostic laboratory.

Mosaic mutations in blood DNA sequence are associated with solid tumor cancers

Recent understanding of the causal role of blood-detectable somatic protein-truncating DNA variants in leukemia prompts questions about the generalizability of such observations across cancer types. We used the cancer genome atlas exome sequencing (~8000 samples) to compare 22 different cancer phenotypes with more than 6000 controls using a case–control study design and demonstrate that mosaic protein truncating variants in these genes are also associated with solid-tumor cancers. The absence of these cancer-associated mosaic variants from the tumors themselves suggest these are not themselves tumor drivers. Through analysis of different cancer phenotypes we observe gene-specificity for mosaic mutations. We confirm a specific link between PPM1D and ovarian cancer, consistent with previous reports linking PPM1D to breast and ovarian cancer. Additionally, glioblastoma, melanoma and lung cancers show gene specific burdens of mosaic protein truncating mutations. Taken together, these results extend existing observations and broadly link solid-tumor cancers to somatic blood DNA changes.

Abstract P2-02-02: Clinical significance of TP53 mosaic mutations uncovered in constitutional genetic testing

Abstract Background: Mosaic findings (mutations outside of the expected 50:50 allele ratio) are often detected in the course of routine constitutional genetic testing due to the high sensitivity of next-generation sequencing (NGS) technologies. The significance of these findings is however often unclear. They can result from constitutional mosaicism secondary to an early embryological event, an acquired somatic mutation in hematopoietic cells, a technical artifact, or (less likely) a reversion event. Studies have shown that clonal hematopoiesis increases with age, seen in up to 10% and 25% of individuals 65 and 90 years of age, respectively. Additional studies have demonstrated expansion of TP53 mutations in peripheral blood cells following chemotherapy administration. We therefore wanted to investigate the potential clinical significance of mosaic mutations found in the peripheral blood in samples submitted for hereditary cancer testing with a focus on TP53. Methods: We examined high-depth NGS data from blood cell-derived DNA for mutations with pathogenic potential suggestive of mosaicism (5-30% allele frequency) in known hereditary cancer genes. For each gene, we determined the average ages of patients tested and compared these distributions to the distribution of ages of patients found to have mosaic mutations. For the mosaic TP53 mutations, we further characterized the mutations found as well as described the clinical scenarios in which the mosaic findings occurred. Results: To date, 101 patients were found to have possibly mosaic mutations with pathogenic potential, including 26 cases with TP53 mutations (0.07% of patients tested), 16 with CHEK2 mutations (0.05%), and 16 with ATM mutations (0.05%). Significant bias towards older ages is seen in patients with ATM (p= 0.0026) and CHEK2 (p=4.2x10-6) mosaic findings. Mosaic TP53 and NF1 mutations were also enriched in older patients , although not as significantly as CHEK2 and ATM mosaic patients. 24/25 patients with TP53 mosaic mutations with a known history were cancer affected. In the COSMIC database, 20/20 of the TP53 mosaic mutations are associated with neoplastic conditions and 13/20 are associated with hematolymphoid neoplasms. 15/20 of the pathogenic mosaic TP53 findings were seen previously in our lab at a ratio corresponding to germline, thereby significantly lessening the possibility of technical artifact. Conclusion: The statistically significant older ages of patients with ATM and CHEK2 mosaic variants suggests that many of these are acquired changes in hematopoietic stem cells rather than constitutional mosaicism per se. Although NF1 is known to be associated with constitutional mosaicism, the bias towards older patients of the NF1 mosaics we saw similarly suggests that most are acquired changes in the hematopoietic stem cells. The less significant age bias in the TP53 events suggests that chemotherapy administration may be leading to enrichment of TP53 mutated clones, although an increased rate of constitutional mosaicism cannot be ruled out. Citation Format: Deucher A, Lincoln S, Sorenson J. Clinical significance of TP53 mosaic mutations uncovered in constitutional genetic testing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-02-02.

Detection and differentiation of botulinum neurotoxin serotypes C, D, and their mosaic variants by highly specific immunoassays and mass spectrometry

Detection and differentiation of botulinum neurotoxin serotypes C, D, and their mosaic variants by highly specific immunoassays and mass spectrometry

Distinct Patterns of Somatic Mosaicism in the APC Gene in Neoplasms From Patients With Unexplained Adenomatous Polyposis

We investigated the presence and patterns of mosaicism in the APC gene in patients with colon neoplasms not associated with any other genetic variants; we performed deep sequence analysis of APC in at least 2 adenomas or carcinomas per patient. We identified mosaic variants in APC in adenomas from 9 of the 18 patients with 21 to approximately 100 adenomas. Mosaic variants of APC were variably detected in leukocyte DNA and/or non-neoplastic intestinal mucosa of these patients. In a comprehensive sequence analysis of 1 patient, we found no evidence for mosaicism in APC in non-neoplastic intestinal mucosa. One patient was found to carry a mosaic c.4666dupA APC variant in only 10 of 16 adenomas, indicating the importance of screening 2 or more adenomas for genetic variants.

The Contribution of Mosaic Variants to Autism Spectrum Disorder.

De novo mutation is highly implicated in autism spectrum disorder (ASD). However, the contribution of post-zygotic mutation to ASD is poorly characterized. We performed both exome sequencing of paired samples and analysis of de novo variants from whole-exome sequencing of 2,388 families. While we find little evidence for tissue-specific mosaic mutation, multi-tissue post-zygotic mutation (i.e. mosaicism) is frequent, with detectable mosaic variation comprising 5.4% of all de novo mutations. We identify three mosaic missense and likely-gene disrupting mutations in genes previously implicated in ASD (KMT2C, NCKAP1, and MYH10) in probands but none in siblings. We find a strong ascertainment bias for mosaic mutations in probands relative to their unaffected siblings (p = 0.003). We build a model of de novo variation incorporating mosaic variants and errors in classification of mosaic status and from this model we estimate that 33% of mosaic mutations in probands contribute to 5.1% of simplex ASD diagnoses (95% credible interval 1.3% to 8.9%). Our results indicate a contributory role for multi-tissue mosaic mutation in some individuals with an ASD diagnosis.

Analysis of phenotypic heterogeneity of male patients with X-linked Alport syndrome from one family

Objective In this study, the phenotype heterogeneity of 2 male patients with X-linked Alport syndrome from one family was analyzed and the likely reasons were discussed by reviewing the literature. Methods The clinical data at the time of diagnosis and during 5 years follow-up of 2-male patients with X-linked Alport syndrome from one family were collected.The α5(IV) chain expression in the epidermal basement membrane was detected by indirect immunofluorescence method.COL4A5 gene mutations in skin fibroblasts and genomic DNA were detected by using reverse transcription polymerase chain reaction and direct sequencing and PCR sequencing methods from skin fibroblasts and genomic DNA, respectively. Results The diagnostic age of patient Ⅲ1 was 14 years old.He had only microscopic hematuria, and proteinuria was negative.A negative α5(Ⅳ) chain staining pattern was detected in his epidermal basement membrane.After 5 years follow-up without drug treatment, he was 19 years old, had persistent microscopic hematuria and normal renal function.The urinary microalbumin was 19.2-31.8 mg/L.The diagnosis age of patient Ⅱ 4 was 29 years old.The hematuria and proteinuria were found at 22 years old.He was treated with tripterygium wilfordii for 1 year.His disease progressed to an end stage of renal disease and he received hemodialysis therapy at 24 years old.He had the renal transplantation surgery at 29 years old, just 2 months before he came to hospital.And his renal function was restored.After 5 years follow-up, his urine examination and renal function were normal.Both patients had a missense mutation c. 3650G>A(p.G1217D)in exon 41 in COL4A5 gene. Conclusions The different phenotypes of 2 male patients from one family with X-linked Alport syndrome were reported.The most possible reason for this is somatic mosaic variants in COL4A5 gene based on literature review.Physicians should be alert to phenotype heterogeneity in male X-linked Alport syndrome despite having the same gene mutation. Key words: Alport syndrome; Hematuria; Proteinuria; Nephritis, hereditary

Abstract 851: Analysis of cancer-initiating mosaic mutation in germline samples of pediatric cancer patients by next generation sequencing

Abstract A cancer predisposition mutation can occur during post-zygotic stages resulting in a mosaic mutation pattern across tissues. Mosaic germline mutations such as those affecting RB1, TP53 and AKT1 are important for clinical testing. However, it is challenging to detect mosaic mutation by genome-wide sequencing approaches as a mutant allele present at a low fraction in a germline sample can be caused by either tumor-in-normal contamination or mosaicism. Here we propose a two-step analytical approach to distinguish between these two possibilities. The first involves assessing the level of tumor-in-normal contamination using all clonal somatic mutations identified through the analysis of tumor purity and chromosome ploidy. The second step determines the probability of mosaicism for a candidate variant by calculating the binomial probability of its mutant allele fraction (MAF) under the contamination hypothesis. We applied this process to 1,172 paired tumor-normal pediatric cancer samples analyzed by whole-genome or whole-exome sequencing. Six mosaic variants were identified, including three pathogenic TP53 mutations with a mutant allele fractions (MAF) ranging from 0.2 to 0.3, one RB1 mutation in a retinoblastoma case with a MAF of 0.08, one IDH1 p.R132H mutation in an acute myeloid leukemiaý case with a MAF of 0.21, and one mosaic RYR2 p.E1690D mutation in an osteosarcoma case with a MAF of 0.11. At the same time, we were able to reject three candidate TP53 variants caused by contamination with MAFs ranging from 0.06 to 0.33. All predicted mosaic mutations were verified by ultra deep sequencing at 10,000X coverage. This experience demonstrates the feasibility of detecting germline mosaicism from whole-genome or whole-exome sequencing of paired tumor-normal samples and would caution accurate analysis of tumor-in-normal contamination when evaluating candidate germline mosaicism. Citation Format: Xiaotu Ma, Donald Yergeau, Bhavin Vadodaria, Joy Nakitandwe, John Easton, Sheila Shurtleff, Jinghui Zhang. Analysis of cancer-initiating mosaic mutation in germline samples of pediatric cancer patients by 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 851.

Somatically acquired structural genetic differences: a longitudinal study of elderly Danish twins.

Structural genetic variants like copy number variants (CNVs) comprise a large part of human genetic variation and may be inherited as well as somatically acquired. Recent studies have reported the presence of somatically acquired structural variants in the human genome and it has been suggested that they may accumulate in elderly individuals. To further explore the presence and the age-related acquisition of somatic structural variants in the human genome, we investigated CNVs acquired over a period of 10 years in 86 elderly Danish twins as well as CNV discordances between co-twins of 18 monozygotic twin pairs. Furthermore, the presence of mosaic structural variants was explored. We identified four mosaic acquired uniparental disomy events on chromosome 4q and 14q in the follow-up samples from four individuals, and our study thereby supports the increasing prevalence of somatic mosaic variants with age.

Mosaic CREBBP mutation causes overlapping clinical features of Rubinstein–Taybi and Filippi syndromes

Rubinstein-Taybi syndrome (RTS, OMIM 180849) and Filippi syndrome (FLPIS, OMIM 272440) are both rare syndromes, with multiple congenital anomalies and intellectual deficit (MCA/ID). We present a patient with intellectual deficit, short stature, bilateral syndactyly of hands and feet, broad thumbs, ocular abnormalities, and dysmorphic facial features. These clinical features suggest both RTS and FLPIS. Initial DNA analysis of DNA isolated from blood did not identify variants to confirm either of these syndrome diagnoses. Whole-exome sequencing identified a homozygous variant in C9orf173, which was novel at the time of analysis. Further Sanger sequencing analysis of FLPIS cases tested negative for CKAP2L variants did not, however, reveal any further variants. Subsequent analysis using DNA isolated from buccal mucosa revealed a mosaic variant in CREBBP. This report highlights the importance of excluding mosaic variants in patients with a strong but atypical clinical presentation of a MCA/ID syndrome if no disease-causing variants can be detected in DNA isolated from blood samples. As the striking syndactyly observed in the present case is typical for FLPIS, we suggest CREBBP analysis in saliva samples for FLPIS syndrome cases in which no causal CKAP2L variant is detected.

A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome.

X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although men with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and develop end-stage renal disease later in life. However, the molecular mechanisms associated with this milder phenotype have not been fully identified. We genetically diagnosed 186 patients with suspected XLAS between January 2006 and August 2014. Genetic examination involved: (1) extraction and analysis of genomic DNA using PCR and direct sequencing using Sanger's method and (2) next-generation sequencing to detect variant allele frequencies. We identified somatic mosaic variants in the type VI collagen, α5 gene (COL4A5) in four patients. Interestingly, two of these four patients with variant frequencies in kidney biopsies or urinary sediment cells of ≥50% showed hematuria and moderate proteinuria, whereas the other two with variant frequencies of <50% were asymptomatic or only had hematuria. De novo variants can occur even in asymptomatic male cases of XLAS resulting in mosaicism, with important implications for genetic counseling. This is the first study to show a tendency between the variant allele frequency and disease severity in male XLAS patients with somatic mosaic variants in COL4A5. Although this is a very rare status of somatic mosaicism, further analysis is needed to show this correlation in a larger population.