Louise Stanley Newcastle

1. What is mosaicism? Describe how mosaicism can be detected by both cytogenetic and molecular genetic techniques and discuss the limitations of different methods, with reference to clinically relevant situations. Louise Stanley Newcastle

2. Keywords • Mosaicism - definition • Detection methods of mosaicism by Cytogenetic and Molecular Techniques • Karyotyping, FISH, QF-PCR, Sequencing, Fluorescent PCR, Melt Curve Analysis, MLPA

3. Definition • In genetics, mosaicism is defined as the presence in an individual of two or more cell lines that are karyotypically or genotypically distinct which are derived from a single zygote (post-zygotic) • Because mutations can distributed throughout the body – severity, symptoms and tissue affected can vary from individual to individual • Can be caused by DNA mutations, epigenetic alterations of DNA, chromosomal abnormalities and spontaneous reversion of inherited mutations • For example an individual mosaic for Down’s syndrome [Not to be confused by chimerism which is where two or more genotypes arise from the fusion of more than one fertilised zygote in the early stages of embryonal development in a single organism]

4. Types of mosacism • Somatic mosaicism • Gonadal mosaicism – restricted to the germline therefore difficult to assess recurrence risks and pedigrees often mimic those showing autosomal recessive inheritance • Tissue specific mosaicism – mutation only observed in certain cell lines

5. Mechanism - chromosomal numerical abnormality GAIN LOSS Diagrams taken from - http://www.ds-health.com/mosaic.htm

6. Mechanism – Point Mutation • Can progress from “normal” to “abnormal” or in reverse direction • Many mechanisms possible for spontaneous reversion – including • Homologous recombination • Back mutation • Compensatory mutations (see Youssoufian and Pyeritz, NGR for further details)

7. Disorders where mosaicism is frequently observed • Down’s Syndrome (2-4 % of cases) • Trisomy 8 Mosaic Syndrome (all) • Angleman Syndrome – individuals with an epigenetic imprinting centre defect (i.e. not a micro-deletion) are often mosaic • NF2 (>25-30% of sporadic cases) • FAP (APC - mosaicism in ~ 10-20% sporadic cases) • BWS (paternal isodisomy) • McCune Albright Syndrome (see notes for further info) • Mitochondrial disease (heteroplasmy) • Neoplasia

8. Karyotyping • Non-targeted screening method (metaphase spreads) • Limited by the resolution (~5Mb) and the number of cells that can be feasibly examined (therefore level of mosaicism that can be detected - abnormality will not be present in all cells) • Hook’s table: e.g. examining 10 cells one can be 90% confident that you are excluding 21% mosaicism, 95% confident excluding 26% mosaicism and 99% confident that you are excluding 37% mosaicism. • Clinical referral for query Turner’s Syndrome for older lady – examine increased number of cells, may observe age-related loss of X chromosome. Look at alternative tissue e.g. skin • Duplicate cultures in cases of suspected mosaicism to exclude artefact

9. FISH -Fluorescent In-Situ Hybridisation • Requires fluorescently labeled probes to target specific chromosomal regions • Increased resolution over karyotyping but target specific • Problems with signal overlap – i.e. if chromosomes lie on top of one another can’t differentiate if true loss of signal • Probe may not bind • E.g. Used for detection of DiGeorge Syndrome – loss of signal at 22q11 • Interphase FISH useful for cancer genetics as cells don’t need to be actively dividing – bcr-abl

10. QF-PCR • Useful for rapid aneuploidy screen (Chrs 13, 18, 21) • Unlike Karyotyping and FISH not looking at single cells but a “DNA average” • Quick but trisomy must be present in a sufficient number of cells to be detected above wt background • Detect mosaicism to ~ 15-20% (quote in Newcastle laboratory). • Normal range values can be quite spread therefore makes it difficult to be confident of low level mosaics.

11. Molecular techniques • Many examples: Mutation ScreeningTargeted Analysis Sanger Sequencing Fluorescent PCR Melt Curve Analysis ARMS/AS-PCR dHPLC Pyrosequencing CSCE MALDI-TOF (Sequenom) MLPA Real-time PCR Next-Gen Sequencing • Discuss a few

12. Sanger Sequencing • Applicable to wide variety of clinical situations –versatile and non-targeted approach to mutation detection • Limited in its sensitivity in detecting mosaics – mutant peak needs to be ~ 20% of total DNA. • Will depend on other factors such as sequence context, type of change, quality etc. Confirm by Pyrosequencing assay c.3250G>T in FBN1 Taken from presentation in CMGS members area

13. Next-Generation Sequencing • Relatively new technology – able to produce vast quantities of sequence data • 3 main platforms – FLX-Roche (pyrosequencing), Illumina (Reversible terminators), SoLID ABI (sequencing by ligation – colour space) • Able to investigate samples with very high fold coverage and therefore sensitive to pick up mosaicism down to 1% (lower possible?) – e.g. use for profiling genetic changes tumour samples • Expensive technique for diagnostics, involved practical process requiring specialist training, laboratories limited access to machines at present, data analysis……

14. Fluorescent PCR – AS case example • Targeted Analysis (one inherent limitation of PCR – preferential amplification of smaller sized product) • PWS/AS Paternal Maternal MS-MLPA (partial results shown) Low level maternal contribution PWS +ve Non-mosaic AS case probe signal to 0 AS +ve

15. FRAX • ~1% affected males are mosaic for a full expansion and have a normal sized allele (best practice guidelines) • This would be detected on PCR only as a normal sized allele due to preferential amplification and therefore reported as a false negative (PCR test only)– Southern blotting required to detect expansion

16. Real-time PCR • Quantitative method • Variety of probes available to use • Taqman (quencher and emitter) • SYBR green • FRET probes • Example of use – measuring levels of bcr-abl transcript in cases of CML • Sensitive method but requires more extensive setting up and a limited number of fluorescent dyes available therefore can only report on a few targets of interest simultaneously

17. Techniques to enrich mutant target over WT • Use of restriction endonucleases with nested PCR to selectively digest the wild-type allele • Multiple rounds of nested PCR prone to contamination • Use of a PNA primer • PNAs form hybrids with DNA that are stable and sensitive to internal base mismatches with their DNA complement. PNA primer is designed to be complementary to WT sequence which overlaps one of the PCR primer binding sites and thus prevents amplification of WT allele. Incomplete blockage by PNA primer may lead to false negative results • Cold PCR • Involved optimisation process for each mutation

18. References • Youssoufian, H and Pyeritz, R.E. (2002) Nature Genetic Reviews, 3, 748 – 758 – Mechanisms and consequences of somatic mosaicism in humans. • Rohlin et al (2009) Human Mutation 30 (6), 1012-1020. Parallel Sequencing Used in Detection of Mosaic Mutations: Comparison with Four Diagnostic DNA Screening Techniques • Hook 1977, Am J Human Genet, 29, 94-97 – Confidence levels for mosaicism in Karyotyping – Copy of Table in Handout • Ley et al. (2008) Nature, 456, 66-72 – Next-Gen sequencing of cytogenetically normal AML patient. • Smith’s Recognizable Patterns of Human Malformation 5th Edition 1997 - Jones, Kenneth Lyons, W.B. Saunders Company. • Kalfa et al. (2006) European Journal of Endocrinology, 155, 839-843 – McCune Albright syndrome (MAS) and detection by nested PCR vs PNA primers • Schwindinger et al (1992) PNAS, 89, 5152-5156 – Molecular cause of MAS • Slatter et al (1994) J Med Genetics, 31, 749-753 – Mosaic paternal UPD in BWS • Bullman et al (2008) J Med Genetics, 45, 396-399 – Mosaic maternal UPD in SRS • Chen et al. (2007) Journal of Molecular Diagnostics, 9, 272 – 276 – Detection methods for JAK2 mutation (V617F) • Strachan and Read – Human Molecular Genetics 3