Table 1.
Examples of mosaicism identification process in clinical diagnostics and research
| Inquiry/disease/phenotype No. of individuals tested |
Material testeda | Method applied | Results | Remarks | References | |
|---|---|---|---|---|---|---|
| 1. | Choice of material and limitations of Sanger sequencing in molecular diagnostics/Cornelia de Lange syndrome/three unrelated patients (A, B and C) | (a) PBS and BM | (a) Sanger sequencing | (a) No disease-causing mutation in any of the five known CdLS genes | Mosaicism to be considered in CdLS Because of the limited availability of fibroblast cells for most of the patients there is an urgent need of modern and sensitive sequencing technologies in routine molecular diagnostics for CdLS |
Braunholz et al. (2015) |
| (b) PBS (patient B only) | (b) Exome sequencing | (b) No disease-causing mutation | ||||
| (c) PBS (patients B and C only) | (c) CGH array | (c) No disease-causing mutation | ||||
| (d) BM | (d) NGS, high-coverageb | (d) Three mosaic NIPBL mutations | ||||
| (e) PBS | (e) SNaPshot assays | (e) No signal indicating the mutant allele was detected in patients A and B, only a very faint signal detected in patient C | ||||
| (f) BM, fibroblast samples; urine (patients A and C only) | (f) SNaPshot assays | (f) Three mosaic NIPBL mutations | ||||
| (g) Fibroblast samples | (g) Sanger sequencing | (g) Three mosaic NIPBL mutations | ||||
| 2. | Somatic mosaicism in the healthy mother of affected siblings; novel heterozygous mutation in ACTA1/nemaline myopathy/two affected siblings, their both parents, and normal controls | (a) PBS | (a) Sanger sequencing | (a) Mutation in affected siblings only | Possibility of very-low-grade somatic mosaicism in suspected carriers, rather somatic than germline mosaicism HRM could not detect the 8.3 % mosaicism; NGS as the first choice for detecting very-low-grade somatic mosaicism |
Miyatake et al. (2014) |
| (b) PBS (siblings and parents) | (b) NGS, whole-exome sequencing (Illumina HiSeq2000) | (b) Mutation in affected siblings only | ||||
| (c) Saliva, hair, nails, PBS | (c) Sanger sequencing | (c) No mutation in parents | ||||
| (d) PBS (siblings), and saliva, nails, hair and PBS (parents) | (d) NGS, deep targeted resequencing for the mutation in ACTA1 (Illumina MiSeq) | (d) Somatic mosaicism in the mother: 0.4, 1.1, and 8.3 % in saliva, PBS, and nails, respectively | ||||
| (e) PBS (siblings and parents), and saliva, nails, and hair (mother only) | (e) Allele-specific PCR and Sanger sequencing of these amplicons | (e) Mutation in PBS of the siblings and the mother, and in saliva and nails of the mother | ||||
| (f) PBS (normal controls, siblings, father), and nails (mother) | (f) HRM analysis | (f) The melting curves of both affected siblings were aberrant (mutant), those of the parents were called normal | ||||
| 3. | Comparison of HRM analysis, pyrosequencing, allele-specific PCR, NGS and IHC to Sanger sequencing; detection of p.V600E and non-p.V600E BRAF mutations/melanoma/49–82 tumor samples per technique | (a–f) DNA extracted from formalin-fixed paraffin-embedded tissues: 63 samples were melanomas, 11 were lung adenocarcinomas and eight were colorectal carcinomas | (a) HRM analysis | (a) Time and cost saving, 100 % specificity, detection limit of 6.3 % mutated alleles | High sensitivity and multiplexing options of NGS allowing to generate a molecular profile of each tumor sample analyzed A combination of HRM and IHC recommended to increase sensitivity and specificity for routine diagnostic of BRAF mutations |
Ihle et al. (2014) |
| (b) Pyrosequencingc | (b) 90 % specificity, detection limit of 5 % mutated alleles, result interpretation prone to errors | |||||
| (c) Allele-specific PCRd | (c) Limited utilization detecting p.V600E mutations only with 98.3 % specificity and detection limit of 7 % mutated alleles | |||||
| (d) Targeted NGS (Illumina MiSeq) | (d) Should be carefully validated before implementation into routine diagnostics, 100 % specificity, detection limit of 2 % mutated alleles | |||||
| (e) IHCe | (e) Fast and cheap performance, 98 % specificity, 100 % sensitivity, detection limit of 5 % mutated alleles | |||||
| (f) Sanger sequencing | (f) A reliable method, detection 100 % specificity, detection limit of 6.6 % mutated alleles | |||||
| 4. | Clinical utility of CMA using DNA from buccal cells/ASD and other phenotypic features (patient 1), developmental delay and ASD (patient 2), hypergonadotrop hichypogonadis, short stature and mild learning disabilities (patient 3)/three unrelated patients | (a) BC (patient 1 only) | (a) CMA, with both copy number and SNP probesf | (a) A gain of chromosome 21, consistent with 15–20 % mosaicism for trisomy 21 | Buccal samples as a primary sample for certain genetic studies (such as CMA) Additional testing using DNA from buccal samples to be considered in patients for whom a genetic etiology is suspected but a diagnosis has not been achieved using DNA from blood leukocytes |
Sdano et al. (2014) |
| (b) CL (patient 1 only) | (b) FISH (500 interphase nuclei, four probes) | (b) 22/500 interphase nuclei with extra copy of chromosome 21 | ||||
| (c) CL (patient 1 only) | (c) G-banded chromosome analysis (100 metaphase cells) | (c) 4/100 metaphases with extra copy of chromosome 21 | ||||
| (d) PBS (patient 2 only) | (d) CMAg | (d) No abnormalities | ||||
| (e) BC (patient 2 only) | (e) CMA, with both copy number and SNP probesf | (e) 20 % mosaicism for tetrasomy 12p, diagnosis of Pallister–Killian syndrome | ||||
| (f) CL (patient 3 only) | (f) Standard G-banded karyotype ( 20 metaphase cells) | (f) No abnormalities | ||||
| (g) CL (patient 3 only) | (g) Standard G-banded karyotype (46 more metaphase cells) | (g) Four of the 66 total cells were found to be 45,X | ||||
| (h) BC (patient 3 only) | (h) CMA, with both copy number and SNP probesf | (h) Three cell lines suspected: 45,X (in ~45 % of BS and 6 % of CL), 46,XX, and the indeterminate cell line [either (46,X,idic (X)(p11.21) or 46,X,del (X) (p11.21)] in ~15–20 % of BS and not seen in CL Normal chromosome complement in ~40 % of BS and 94 % of CL |
||||
| 5. | Quantitative analysis of the ratio of mutant versus wild-type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues/COL6-RD/four families | (a) PBS, CDF, and saliva (family 1 only) | (a) Real-time PCR | (a) Too weak signal to be detected in PBS and saliva from the mosaic father, 36 % of the mutant allele in the CDF the mosaic father | Somatic mosaicism for dominant collagen 6 mutations suggests that parental mosaicism may be more common than previously suspected in COL6-RD | Donkervoort et al. (2015) |
| (b) PBS, CDF, saliva, and cDNA from CDF (family 2 only) | (b) Real-time PCR | (b) 20, 42, and 16 % of mutant allele ratio in the father, in PBS, CDF and saliva, respectively, while 35 % mutant allele ratio from the cDNA from CDF | ||||
| (c) CDF (family 3 only) | (c) Real-time PCR | (c) 24 % of mutant ratio in CDF of the mosaic father | ||||
| (d) PBS and saliva (family 4 only) | (d) Real-time PCR | (d) 40 % of mutant ratio in PBS and 58 % in the saliva of the mosaic mother | ||||
| (e) Fibroblasts (patients and parents, families 1, 2, and 3 only), PBS (patients and parents, family 4) | (e) Sanger sequencing | (e) Heterozygous mutant alleles identified in the four patients and in one of their parents; peak height of the mutant allele in the carrier parents was significantly smaller | ||||
| 6. | Low-level constitutional mosaicism of a de novo BRCA1 c.1953dupG gene mutation/breast cancer/one case | (a) PBS (blood draw 1) | (a) NGS (Illumina MiSeq, HiSeq 2500) of 29 hereditary cancer genesh | (a) Pathogenic BRCA1 mutation in 5 % of reads | NGS sequencing should be considered in affected individuals whose tumors display a BRCA mutation that cannot be demonstrated in PBS using Sanger sequencing | Friedman et al. (2015) |
| (b) PBS | (b) Sanger sequencing of BRCA1 and BRCA2 | (b) No mutation | ||||
| (c) PBS | (c) Sanger sequencing of c.1953dupG mutation locus | (c) The presence of a small peak of c.1953dupG mutation, with signal almost indistinguishable from background noise | ||||
| (d) PBS (both blood draw 1 again and blood draw 2) and buccal swab | (d) NGS (Illumina MiSeq, HiSeq 2500) of 29 hereditary cancer genesh | (d) Mosaic signal of 4.9–6.8 % was reproducibly detected in all samples | ||||
| (e) Breast tumor tissue | (e) Somatic NGS assayi | (e) Mutation c.1943dupG in 47 % of sequence reads | ||||
| (f) Healthy breast tissue | (f) Somatic NGS assayi | (f) Mutation c.1943dupG was in 5 % of reads | ||||
| (g) Maternal PBS | (g) Deep sequencing | (g) No c.1943dupG mutation |
a BM buccal mucosa, PBS peripheral blood sample (lymphocyte DNA), BC buccal cells, CL cultured lymphocytes, CDF cultured dermal fibroblasts, BT buccal tissue
bIon Torrent PGM with the AmpliSeq Designer Tool
c therascreen ® BRAF Pyro Kit (Qiagen)
dThe cobas® BRAF V600 test (Roche)
eMonoclonal mouse antibody VE1 (Spring Bioscience)
fAffymetrix CytoScanHD®, Affymetrix
gAffymetrix 6.0 array®
hNGS of 29 hereditary cancer genes, performed at Invitae (San Francisco, CA, USA)
iReported as commercially available somatic NGS assay