Annurev Genom 120121 105450

Annual Review of Genomics and Human Genetics
Mosaicism in Tumor Suppressor

Gene Syndromes: Prevalence,
Diagnostic Strategies, and
Transmission Risk
Jillian L. Chen,1,2 David T. Miller,3 Laura S. Schmidt,4,5
David Malkin,6,7 Bruce R. Korf,8 Charis Eng,9,10,11,12
David J. Kwiatkowski,1 and Krinio Giannikou1,13
1
Cancer Genetics Laboratory, Division of Pulmonary and Critical Care Medicine and Division
of Genetics, Brigham and Women’s Hospital and Harvard Medical School, Boston,
Massachusetts, USA; email: kgiannikou@bwh.harvard.edu
2
Boston University School of Medicine, Boston, Massachusetts, USA
3
Division of Genetics and Genomics, Boston Children’s Hospital and Harvard Medical School,
Boston, Massachusetts, USA
4
Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda,
Maryland, USA
5
Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick,
Maryland, USA
6
Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada
7
Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada
8
Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
9
Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio,
USA
10
Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, USA
11
Department of Genetics and Genome Sciences, Case Western Reserve University School of
Medicine, Cleveland, Ohio, USA
12
Germline High Risk Cancer Focus Group, Case Comprehensive Cancer Center,
Annu. Rev. Genom. Hum. Genet. 2022. 23:331–61 Case Western Reserve University, Cleveland, Ohio, USA
The Annual Review of Genomics and Human Genetics 13
Division of Hematology and Oncology, Cancer and Blood Disease Institute, Children’s
is online at genom.annualreviews.org Hospital Los Angeles, Los Angeles, California, USA; email: kgiannikou@chla.usc.edu
https://doi.org/10.1146/annurev-genom-120121-
105450
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331
Keywords
mosaicism, tumor suppressor, NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, TP53
Abstract
A mosaic state arises when pathogenic variants are acquired in certain cell lineages during postzy-
gotic development, and mosaic individuals may present with a generalized or localized phenotype.
Here, we review the current state of knowledge regarding mosaicism for eight common tumor
suppressor genes—NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, and TP53—and their related
genetic syndromes/entities. We compare and discuss approaches for comprehensive diagnostic
genetic testing, the spectrum of variant allele frequency, and disease severity. We also review
affected individuals who have no mutation identified after conventional genetic analysis, as well
as genotype–phenotype correlations and transmission risk for each tumor suppressor gene in full
heterozygous and mosaic patients. This review provides new insight into similarities as well as
marked differences regarding the appreciation of mosaicism in these tumor suppressor syndromes.
1. INTRODUCTION
Mosaicism is characterized by the presence of two or more genetically distinct cell populations in
a developing embryo due to spontaneous genetic alterations (82). These spontaneous changes can
give rise to cell lineages with different genomes, resulting in mosaic status that demonstrates cell
and tissue heterogeneity (24). Mosaicism is seen in all organisms, including humans (15, 55, 82). It
presents with a variant allele frequency (VAF) at less than heterozygous levels (<50%) (47), and the
vast majority of alterations occur in the noncoding genome and typically have no apparent effect
on fitness or phenotype (15). Spontaneous genetic alterations affecting the coding genome can po-
tentially cause a de novo pathogenic phenotype in individuals with no family history for a disease.
Alfred Blaschko first theorized that the range of clinical features in an individual with mo-
saicism depends on the stage at which the pathogenic variant (PV) occurs during postzygotic
development (55). Mosaicism is characterized as gonadal or germline when only gametes (sperm
or/and egg) contain the PV and as somatic when only somatic cells contain the PV. Additionally,
the terms generalized mosaicism and localized mosaicism (Figure 1) refer to the body distribution
and physical extent of affected tissues (9, 85), with variability in VAF among different tissues and
organs. The mosaic variants may be present in both somatic and germ cells at varying frequencies
(15, 82), likely reflecting the chance distribution of cells carrying de novo mosaic variants among
the progenitor cells for different tissues during embryogenesis.
Although mosaicism has been studied in great detail for numerous genetic disorders (9, 15,
24, 27, 55, 82, 85, 88, 122, 125), it is poorly documented with limited reports for others, in-
cluding tumor suppressor gene (TSG) syndromes such as PTEN hamartoma tumor syndrome
(PHTS) and Li–Fraumeni syndrome (LFS) (6, 96). Here, we review the literature on mosaicism
for eight genes: NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, and TP53, which are among the
most broadly studied TSGs. These genes are implicated in the two canonical growth signaling
pathways: the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)
pathway and the receptor tyrosine kinase (RTK)/Ras/Raf/mitogen-activated protein kinase kinase
(MEK)/extracellular signal-regulated kinase (ERK) pathway (95) (Figure 2). Individuals carrying
inactivating alleles in these different TSGs have unique tumor predisposition or tissue overgrowth
syndromes, collectively referred to in this review as TSG syndromes (88).
332 Chen et al.

Embryonic development
Fertilization Cleavage Birth/adulthood
and differentiation
a
Germline
genetic event
Full
heterozygous
disease
b
Early postzygotic
alteration
Generalized
mosaicism
c
Later somatic
alteration
Localized
mosaicism
Early stages of
Fertilization Zygote Predifferentiation cell differentiation Later stages
Figure 1
Mosaicism in humans. (a) A germline genetic alteration occurs in gametes (sperm and/or egg). The resulting zygote is heterozygous for
the alteration, and the mutation is transmitted to all cells in the body, leading to an offspring with full heterozygous disease. (b) In
generalized mosaicism, a postzygotic alteration may occur very early (e.g., in the blastocyst stage or later), prior to differentiation into
the different germ layers of the future embryo (82). In general, early mutational events result in cells that carry the genetic alteration
giving rise to all cell and tissue types (112). (c) A somatic alteration acquired at a later stage in embryogenesis (e.g., after some
differentiation has occurred) will be found only in the progeny cells derived from that single cell. As a genetic event occurs later in
development, fewer cells and tissues will be affected by the alteration. Such individuals may have a milder phenotype than those with
full heterozygous genetic alterations, since there are fewer cells at risk of second-hit mutations to cause tumor formation. Ultimately,
only cells within the affected lineages and tissue types have the genetic alterations, resulting in localized mosaicism. A special case of
localized mosaicism is that in which only the germ cells of the testes in males or the ovaries in females are affected. This is gonadal
mosaicism, in which the individual may have no phenotype at all because the variant is present only in germ cells, but the variant can be
transmitted to the individual’s offspring at high frequency in some cases (82). Figure adapted from images created with BioRender.com.
In this review, we considered a representative set of original and comprehensive peer-reviewed

studies focusing on large patient cohorts. Our searches in PubMed and Google Scholar in-
cluded keywords for each gene and syndrome and mosaicism (e.g., “NF1” and “mosaic” or “mo-
saicism”). Below, we provide an overview of each TSG syndrome, reporting prevalence, genotype–
phenotype correlations, and risk of disease transmission, as well as current molecular diagnostic
www.annualreviews.org • Mosaicism in Tumor Suppressor Gene Syndromes 333

strategies to detect mosaicism. We used the following classification system: high-level mosaicism
(VAF 20–35%), moderate-level mosaicism (VAF 5–20%), low-level mosaicism (VAF 1–5%), and
very-low-level mosaicism (VAF <1%) (81).
2. TUMOR SUPPRESSOR GENE SYNDROMES DEMONSTRATE

VARYING RATES OF MOSAICISM
The seven TSG syndromes studied here present with an autosomal dominant pattern of inheri-
tance (57, 88), and affected individuals are predisposed to multiple different tumors as well as other,
non-tumor-related phenotypes (Supplemental Material). The clinical manifestations of TSG
syndromes arise from several different mechanisms (Figure 3), with Knudson’s two-hit model
being the primary mechanism of tumor development (43, 60, 117, 139, 147). For example, early-
onset retinoblastoma in children with inherited RB1 alterations first elucidated the role of somatic
second hits in TSG-mediated tumor formation, which occurs due to biallelic loss of RB1 func-
tion (8). Knudson observed that individuals with sporadic retinoblastoma cases typically present
RTK GFR
PIP3 PIP2 GRB2 GTP
PI3K SOS RAS

*
PTEN GDP
PDK1 RAS-GTP
*
TP53
* *
NF2 NF1
FAK2
AKT RAF
AMPK
MEK
*
TBC1D7
TSC1
mTORC2 *
TSC2 ERK1
Cell survival, RHEB

cell cycle,
progression, *
PTEN
cytoskeleton *
remodeling RB1 ERK1
mTORC1
* CDK4/6
VHL HIF-α
Protein Cyclin D1
synthesis,
cell growth
Stimulation Angiogenesis Gene transcription
Translocation
Inhibition Nucleus
* Tumor suppressor genes
(Caption appears on following page)
334 Chen et al.

Figure 2 (Figure appears on preceding page)
The PI3K/AKT/mTOR and RTK/Ras/Raf/MEK/ERK pathways. Amino acid levels, growth factors (e.g.,
EGFR), and insulin stimulate both pathways to control cell growth and metabolism. Growth factors and
nutrients stimulate GFRs, often RTKs, activating GRB2 and SOS, a guanine nucleotide exchange factor.
Following exchange of GDP for GTP, GTP-bound Ras initiates a phosphorylation cascade, ultimately
activating ERK (95). Growth factors also stimulate the PI3K pathway. Activation of the insulin RTK
activates PI3K, which phosphorylates PIP2 to become PIP3. PDK1 is recruited to the membrane and allows
activation of AKT (149). Both ERK and AKT activate mTORC1 through inhibition of the TSC protein
complex (88, 95, 112). PTEN’s phosphatase activity indirectly inhibits signaling through PI3K by
dephosphorylating PIP3 back to PIP2 (146). NF1 encodes neurofibromin, a GTPase-activating protein that
regulates the RAS-cAMP pathway and MAPK pathway (30). NF2 is also known to act within the PI3K
pathway, as evidenced by elevated levels of phospho-ERK, phospho-MEK (146), and phosphorylated AKT
in NF2. TP53 acts within the nucleus to regulate repair mechanisms and apoptosis following DNA damage.
RB1 also influences cell survival and apoptosis through interactions with other nuclear proteins (69). TP53
and RB1 products have additional tumor suppressor functions outside of the two pathways discussed here
(69). VHL plays a role in regulating the hypoxia-inducible factor complex, maintaining normal levels of
blood vessel formation (69). Tumor suppressor genes are marked with asterisks. Abbreviations: AMPK,
AMP-activated protein kinase; cAMP, cyclic AMP; EGFR, epidermal growth factor receptor; ERK,
extracellular signal-regulated kinase; FAK2, focus adhesion kinase 2; GFR, growth factor receptor; GRB2,
growth factor receptor-bound protein 2; HIF-α, hypoxia-inducible factor alpha; MAPK, mitogen-activated
protein kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin;
mTORC1/2, mammalian target of rapamycin complex 1/2; NF2, neurofibromatosis type 2; PDK1,
phosphoinositide-dependent kinase 1; PI3K, phosphoinositide-3-kinase; PIP2, phosphatidylinositol
4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; RHEB, Ras homolog enriched in brain;
RTK, receptor tyrosine kinase; SOS, Son of Sevenless; TSC, tuberous sclerosis complex. Figure adapted
from images created with BioRender.com.
with clinical symptoms at a later age than those with familial retinoblastoma. This led Knudson
to hypothesize that retinoblastoma in sporadic cases required two hits to occur, and thus tumori-
genesis occurs at a later age due to the additional time required for both the first- and second-hit
alterations to occur (52). By contrast, in individuals with inherited or familial retinoblastoma, the
single hit required tends to occur at an earlier age.
Other clinical, non-tumorigenic features of TSG syndromes may arise via haploinsufficiency
(8, 52, 57), in which loss of one copy of the gene is sufficient to cause reduced gene function, leading
to an aberrant phenotype (57). Haploinsufficiency of PTEN has been related to hamartomas, be-
nign polyps, and developmental disorders in PHTS (110). Second hits have been reported in two-
thirds of cases in tuberous sclerosis complex (TSC), including renal angiomyolipomas, subependy-
mal nodes, and subependymal giant cell astrocytomas, but in only 35% of TSC-related cortical
tubers, demonstrating that these lesions may have very low clonal purity (81).
The TSG syndromes discussed here display immense phenotypic variability despite the func-
tional relatedness of the relevant causative genes as well as their critical functional involvement
in the same biological pathways (Figure 2). It is possible that the TSG syndromes with the high-
est risk of malignancy and severe disease depend on the appearance of de novo PVs (32). Severe
clinical features may prevent affected individuals from surviving to reproductive age or may cause
developmental disabilities that make it less likely for the individual to have offspring. Thus, inher-
ited PVs with these effects are not transmitted to the next generation.
Here, we describe mosaicism for each TSG syndrome and present maps of the variant distribu-
tion across each relevant tumor suppressor gene (Figures 4–11, below), including a comprehen-
sive report of known pathogenic/likely pathogenic (P/LP) variants for all TSGs (Supplemental
Table 1). We included substitutions/single-nucleotide variants and small insertions or deletions
(indels) compiled from the Leiden Open Variation Database, as well as P/LP somatic variants from
tumor samples from ClinVar and cBioPortal. PolyPhen, SIFT, and PROVEAN tools were used

a
Copy-loss LOH
TSG
locus
Mosaic or fully Wild-type copy
heterozygous at birth fully or partially deleted
Mosaic or fully Second somatic alteration

heterozygous at birth distinct from first
c
Copy-neutral LOH Tumorigenesis
Mosaic or fully Second hit due to mitotic

heterozygous at birth recombination or gene conversion
Mosaic or fully Epigenetic/transcriptomic

heterozygous at birth events in other genetic loci
Figure 3
Knudson’s two-hit hypothesis and mechanisms of tumor formation in TSG syndromes. Several mechanisms
of two-hit loss occur for TSGs, leading to tumor development. The first three mechanisms (panels a–c) apply
to heterozygous as well as mosaic individuals in all eight TSGs discussed in this review. (a) An individual is
somatic or germline mosaic (or full heterozygous) at birth for a TSG alteration. Copy-loss LOH occurs
when all or part of the second functional allele is deleted. In general, copy-loss LOH means that the entire
copy of the wild-type allele is lost, usually through partial chromosome arm loss, full chromosome arm loss,
or entire chromosome loss. (b) An individual acquires a second inactivating alteration in the wild-type allele
that is distinct from the inherited germline event. (c) Copy-neutral LOH occurs due to a mitotic
homologous recombination event in which a chromosomal region bearing the mutant allele replaces the
same region bearing the wild-type allele (as in panel a, this can involve a focal chromosomal locus, an entire
chromosomal arm, or an entire chromosome) (8, 43, 109). In TSC, 95% of renal angiomyolipomas (147) and
85% of subependymal giant cell astrocytomas demonstrate copy-neutral LOH (147). (d) While somatic
second-hit events are seen in approximately 30% of inherited retinoblastoma cases, hypermethylation as a
second hit has been noted in a small percentage of tumors (117). Epigenetic events that suppress expression
of the wild-type allele are an alternative mechanism for which there is limited evidence currently (147).
Abbreviations: LOH, loss of heterozygosity; TSC, tuberous sclerosis complex; TSG, tumor suppressor gene.
Figure adapted from images created with BioRender.com.
336 Chen et al.

649 Missense variant
189 In-frame variant
2,482 Truncating variant
88 Splice
27 Other
p.Q514/Q514Rfs*43/Q514*
p.G2376_A2419del/G2376fs
p.R1276Q/G/L/P
p.K1640*/K1640Gfs*36
p.K1423E/M/Q/N/R
p.F2215/F2215Hfs*6
p.E2122*/E2122Gfs*27
p.L844F/P/R/H
p.M1149V/I/T
p.K1640*/K1640fs
p.W784R/S/C
p.I679/Dfs*21
Number of NF1 variants
p.S340/Cfs*12
26
p.Q83*/fs
p.R2496*
p.R2616*
0
Ras_GAP CRAL_TRIO_2
0 400 800 1,200 1,600 2,000 2,400 2,839

Amino acid position
Figure 4
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in NF1 (from the Leiden Open Variation Database,
ClinVar, and cBioPortal). Vertical lines represent each genetic variant; single-nucleotide substitutions or small indels are shown. Variant
labels indicate examples of specific amino acid changes at select nucleotide positions (37, 42, 73). Large deletions and duplications/
amplifications are not shown. Abbreviation: indel, insertion or deletion. Figure adapted from images created with cBioPortal.
for the in silico prediction of missense variants to clarify their pathogenicity (17, 37, 42, 73). Mu-
talyzer analysis was performed to confirm the appropriate annotation for each variant according
to the Human Genome Variation database (GRCh17/hg19).
2.1. NF1: Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is caused by inactivating alterations in NF1 (OMIM 162200 and
613113), which encodes neurofibromin, a Ras GTPase-activating protein. Clinically, the NF1
syndrome is distinguished by the neurofibroma, a hallmark feature, which is a nerve sheath tu-
mor associated with spinal, peripheral, or cranial nerves (52, 75; for a full list of clinical features,
see Supplemental Figure 1). NF1 affects approximately 1 in 3,000 births regardless of sex or
ancestry; 70% of affected individuals are diagnosed in their first year of life, and 97% develop fea-
tures that are diagnosed by 8 years of age (36). Malignancies, including malignant peripheral nerve
sheath tumors or other malignant tumors, are the most frequent cause of NF1-related mortality
(99). Isolated malignant peripheral nerve sheath tumors occur with an incidence of 1 in 100,000
per year (40), and although these tumors are seen in NF1, they are characterized as a separate en-
tity and may not be representative of mosaic NF1. Malignant tumors reduce mean life expectancy
in individuals with NF1 by 8–21 years (36, 52, 70).
Approximately 50% of NF1 PVs are de novo. The full spectrum of known P/LP variants for
NF1 is shown in Figure 4 (71) (see also Supplemental Table 1). NF1 is the largest gene among

the TSGs discussed in this review, containing 61 exons and covering 476.74 kb (29, 31, 143),
possibly in part explaining its high frequency of PVs and the relatively high prevalence of NF1 in
the general population (52, 150).
Mosaicism in NF1 is approximately 10–20 times rarer than full heterozygous inherited NF1
in the general population (118, 129). The true rate of mosaicism in NF1 is undetermined and may
be underappreciated, as many individuals with NF1 clinical features do not pursue genetic testing.
Furthermore, some testing methods with less depth of coverage may lack sensitivity, thus missing
a mosaic PV. Notably, segmental NF consists of a localized mosaic NF1 form and is distinguished
by lesions restricted to particular body regions.
2.2. NF2: Neurofibromatosis Type 2

NF2 (OMIM 101000 and 607379) is a syndromic entity that is characterized by variable pheno-
typic severity and presents most frequently with bilateral vestibular schwannoma (i.e., schwanno-
mas on both vestibulocochlear nerves and subcapsular cataracts) (36, 107). Other benign tumors
are often seen in NF2 (Supplemental Figure 2), including schwannoma of peripheral or other
cranial nerves, meningioma, and ependymoma (36). It affects approximately 1 in 33,000 births
(36), with a nearly complete penetrance of 95% (70) and a variable age of onset; the average age
of onset is 35 years, but it can be present as early as birth. The functional NF2 gene product,
Merlin, a 595-amino-acid protein, is a membrane–cytoskeleton scaffolding protein and a regula-
tor of contact-dependent inhibition of proliferation (140).
NF2 has the highest reported rate of mosaicism among the TSG syndromes discussed here
(22). Half of all individuals with NF2 have de novo genetic alterations (36), and 30–60% of these
individuals are mosaic at a VAF between 0.2% and 30% in blood (32). This has consequences for
diagnosis, as 25–30% of de novo affected individuals have no PV detected in blood lymphocytes
due to somatic mosaicism (34). Additionally, between 33% and 60% of individuals with unilateral
vestibular schwannoma are mosaic for NF2 (34). The spectrum of known P/LP variants for NF2
is shown in Figure 5.
Over the last two decades, several NF2 studies have uncovered mosaic cases in different pe-
diatric cohorts and in founder individuals with no clinically affected parents (53, 54, 67). Mosaic
11 Missense variant 18 Splice

10 In-frame variant 1 Other
p.R57*/Afs*28
169 Truncating variant

p.E445Gfs*9/Rfs*50
p.R198*
p.L14Qfs*34
p.R341*
p.R466*
p.R262*
Number of NF2 variants
p.X482_splice
p.X333_splice
6
p.X81_splice
p.V92Tfs*13
p.Y153*
p.X375_splice
p.A399Pfs*24
p.T512Mfs*3
p.L306Yfs*3
p.Q538P
p.N220Y
p.Y144*
p.D354fs
p.S288*
p.L549*
p.E427*
p.E593*
0
FERM_N FERM_M FERM_C ERM
0 100 200 300 400 500 595

Amino acid position
Figure 5
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in NF2 (from the Leiden Open Variation Database,
338 Chen et al.

33 Missense variant
22 In-frame variant
15 Splice
115 Other
p.N891Tfs*40/Kfs*13/Kfs*41
p.L113*/Afs*12/Cfs*5/Ffs*13/Ffs*6
p.E787*/Nfs*19/Rfs*20/Vfs*37
p.L203Cfs*7/*/Ffs*8/Ifs*15
p.Q654*/Dfs*12/Tfs*34
p.Y185*/Sfs*25/Tfs*25
p.E478*/Kfs*53/Rfs*7
p.Y271*/Hfs*48
p.Q527*/Rfs*7
p.Y761*/Sfs*7
p.W347*/Efs*21
Number of TSC1 variants
11
p.R246K
p.G385Efs*55
p.S1141Ffs*27
p.L64*
p.R1093Q
p.F1059L
p.R992G
0
Hamartin
0 200 400 600 800 1,000 1,164

Amino acid position
Figure 6
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in TSC1 (from the Leiden Open Variation Database,
NF2 often presents with only schwannoma, without other typical features of NF2, and patients
with this phenotype share clinical diagnostic features with schwannomatosis. Schwannomatosis
is a schwannoma predisposition syndrome associated with PVs in SMARCB1 or LZTR1 (33, 36,
140), and approximately 50% of apparent schwannomatosis cases without an LZTR1 or SMARCB1
variant are mosaic for NF2 (33).
2.3. TSC1/TSC2: Tuberous Sclerosis Complex

TSC is characterized by seizures, intellectual disability, and autism, as well as by development
of benign tumors (hamartomas) in the skin, heart, brain, kidneys, lungs, and other organs (89,
137) (Supplemental Figure 3). It is fully penetrant, affecting 1 in 6,000–10,000 individuals. TSC
is caused by inactivation of either TSC1, on chromosome 9q34.13, or TSC2, on chromosome
16p13.3 (OMIM 191100 and 191092). TSC1, composed of 23 exons, encodes the protein hamartin,
and TSC2, composed of 42 exons, encodes tuberin. Hamartin and tuberin form a complex that
inhibits mTOR complex 1 (mTORC1) (Figure 2). Variants in TSC1 constitute 26% of all genetic
alterations, while variants in TSC2 are more common in TSC, constituting 74% of all genetic
alterations (112) (Figures 6 and 7). Two-thirds of TSC cases are sporadic (72).
The hallmark features of TSC include neurocognitive delay and a high predilection for
epilepsy, which is seen in more than 90% of patients in many series (47, 112). Additionally,
55–75% of individuals with TSC develop renal angiomyolipoma, a benign kidney tumor com-
posed of abnormal vasculature and distinctive myomelanocytic cells and adipocyte cells (112).

119 In-frame variant
1,643 Truncating variant
18 Splice
105 Other
p.H522fs/Pfs*66/Pfs*67/Qfs*14/Tfs*13/Tfs*67
p.E482_L493delinsV/E482_Q492del/E482*
p.W304*/Gfs*33/Ffs*27/Sfs*60
p.W1610*/Cfs*15/_D1613delins*
p.R1743Q/G/W/L/P
p.I39Lfs*8/Sfs*7/Tfs*5/Yfs*28
p.R1459*/Efs*17/Sfs*65
p.P1675L/Q/R/S
p.Y407*/Ffs*2/_R413del
p.N1651S/H/T
p.Y1033*/Rfs*10
p.L922Cfs*26fs*4
p.C696Y/R/W
p.Y598C/H
p.Q1503P/H/R
p.Q1395*/Gfs*12
p.R751*/Efs*20
p.E1313*/Gfs*14
p.Y1250*/fs
p.Q373H/P
p.L844P/Q/R
p.S1095N
p.C244R
Number of TSC2 variants
9
p.T147K
0
DUF3384 Tuberin Rap_GAP
0 400 800 1,200 1,600 1,807

Amino acid position
Figure 7
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in TSC2 (from the Leiden Open Variation Database,
Mosaicism is seen in 10–15% of all individuals meeting diagnostic criteria for TSC (13, 92,
130, 131). Given the high frequency (15–20%) of TSC-affected individuals in whom a causative
PV in TSC1/TSC2 cannot be identified by standard methods—a category referred to as no mu-
tation identified (NMI)—TSC is one of the first genetic syndromes to be analyzed for mosaicism
in TSC1/TSC2 using massively parallel sequencing (MPS) (98). Although mosaicism in TSC is
associated with milder clinical features in general (131), 10% of infants diagnosed with TSC were
found to have mosaicism (134), with VAFs ranging from 0.7% to 32% (90, 134).
2.4. PTEN: PTEN Hamartoma Tumor Syndrome/PTEN-opathies

PHTS is caused by genetic alterations in the phosphatase and tensin homolog gene, PTEN,
on chromosome 10q23.31. It is characterized by various clinical features, including increased
lifetime risk of cancer of the breast, thyroid, endometrium, kidney, colon, and skin (146).
PTEN encodes a dual-specificity phosphatase with multiple functions, including inhibition of
cell spreading via dephosphorylation of focal adhesion kinase and dephosphorylation of phos-
phatidylinositol 3,4,5-trisphosphate (PIP3) to counterbalance the activity of phosphoinositide 3-
kinase (PI3K) (Figure 2). Between 11% and 48% of all PHTS cases are estimated to be due to
de novo PTEN variants (48, 96). The spectrum of known P/LP variants for PTEN is shown in
Figure 8.
340 Chen et al.

13 In-frame variant
49 Splice
8 Other
p.C136Y/R/F/W
p.G293*/Efs*14/Vfs*13
p.A328Qfs*16/*/Sfs*3
p.X212_splice
p.R15S/I/K/T
p.R335*/Dfs*9
p.M35V/I/K/R/T
p.N117Kfs*9/Sfs*5
Number of PTEN variants
p.H196Lfs*4/Sfs*6
p.R233*/Q
p.D252G/N/V/Y
p.Y155C/H/S
p.L146*/Ffs*34
19
p.X55_splice
p.X85_splice
p.C105Y/G/R
p.E7*/Gfs*17
p.K267Rfs*9
p.N48K/I
p.E373Lfs*42
p.Y188*
0
DSPc PTEN_C2
0 100 200 300 403

Amino acid position
Figure 8
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in PTEN (from the Leiden Open Variation Database,
Four syndromic entities are included under the umbrella of PHTS and are due to different
genetic alterations in PTEN: Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome (BRRS),
Proteus syndrome, and Proteus-like syndrome (94, 144).
Cowden syndrome (OMIM 158350) is a hamartoma syndrome characterized by macrocephaly,
trichilemmoma, and mucocutaneous papules (45, 47) (Supplemental Figure 4) and affects ap-
proximately 1 in 200,000–250,000 individuals (111, 144). Approximately 30–35% of Cowden syn-
drome cases that meet clinical diagnostic criteria have a PTEN PV (94, 126); germline AKT1 and
PIK3CA PVs can also cause Cowden syndrome (96).
BRRS (OMIM 158350) is a congenital syndrome characterized by macrocephaly, Hashimoto
thyroiditis, intestinal hamartomatous polyposis, vascular malformations, lipomas, and genital
freckling (149) (Supplemental Figure 4). Approximately 60% of affected individuals have a
PTEN alteration (94). Cowden syndrome and BRRS have overlapping spectra of PVs and can
be caused by either inherited or de novo PTEN alterations (94).
By contrast, Proteus syndrome and Proteus-like syndrome are nearly always caused by de
novo PTEN alterations (144). Proteus syndrome (OMIM 176920 and 164730) is an overgrowth
syndrome characterized by hemihypertrophy and macrocephaly that arise postnatally (10, 112).
Proteus-like syndrome was suggested to be part of PHTS in 2000 and is suspected when the
patient fails to meet full diagnostic criteria for Proteus syndrome (144, 149).
Approximately 10–40% of individuals with PTEN alterations have de novo alterations and most
frequently present with a generalized distribution of cutaneous hamartomas and internal lesions
resembling inherited Cowden syndrome (88). Mosaicism for PTEN in individuals with PHTS is
not well documented (149), and only four case reports have analyzed mosaic PHTS, identifying
five unique PTEN alterations (41, 96, 111, 149). Specific mosaic VAFs were confirmed in only one
of these cases, whereas deep sequencing detected a PV at a VAF of 1.7% in blood. Multi-tissue
analysis using Sanger sequencing detected the same PV at 25% in normal colonic and endocervical

7 In-frame variant
14 Splice
p.R167W/Q/G/L/P
p.S65/W/L/P/A
9 Other
p.G144*/Dfs*15/Sfs*14
p.Q73*/Pfs*59/Pfs*87
p.L158P/V/Q/R
p.X155_splice
p.I151S/T/F/M/N
p.W117C/R
p.S111N/C/G/R
p.W88C/R/S
p.I206Cfs*49/Nfs*50
p.L188P/Q/R/V
p.Y98C/H/S
Number of VHL variants
p.L178P/Q/R
14
p.F136L/C/S
p.T124I/A
p.R200W/P
p.R82P/L
p.G104fs
p.E70K
p.M54I
p.S33*
0
VHL
0 100 213
Amino acid position
Figure 9
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in VHL (from the Leiden Open Variation Database,
mucosa and at heterozygous levels in skin fibroblasts and dysplastic gangliocytoma tissue (96).
Mosaic second-hit alterations have been seen at a VAF below 10% in individuals with inherited
heterozygous PTEN alterations and vascular malformations that do not meet PHTS diagnostic
criteria (127).
2.5. VHL: Von Hippel–Lindau Syndrome

Genetic alterations in the VHL gene (OMIM 608537) on chromosome 3p25 cause a benign and
malignant tumor predisposition syndrome called von Hippel–Lindau syndrome (VHL) (OMIM
193300). The two protein products of VHL, pVHL19 and pVHL30, act in the oxygen-sensing
pathway (Figure 2), microtubule stability and orientation, cilia formation, aging, cytokine signal-
ing, collagen IV regulation, and extracellular fibronectin matrix assembly (93). VHL is seen in 1
in 36,000–45,000 individuals, has 90% penetrance by age 65, and may be first diagnosed at any
age (93). Approximately 80% of VHL cases are familial, with the rest being sporadic, as a result
of de novo PVs (114). The spectrum of known P/LP variants for VHL is shown in Figure 9.
VHL causes hemangioblastomas in the brain, spinal cord, and retina, and can variably cause
renal cysts, clear cell carcinoma, and pheochromocytoma (135) (Supplemental Figure 5). The
leading cause of mortality from VHL is renal cell carcinoma (135).
VHL is classified into two types: type 1, which carries a low risk of pheochromocytoma, and
type 2, which carries a high risk of pheochromocytoma (Supplemental Figure 5). Type 2 is sub-
divided into type 2A, which carries a low risk of renal cell carcinoma; type 2B, which carries a high
risk of renal cell carcinoma; and type 2C, which carries a risk of pheochromocytoma only (61).
Although the true rate of mosaicism among individuals who meet VHL diagnostic criteria is
not known, mosaic cases have been estimated to constitute approximately 5% of all VHL cases
(23). Analysis of 42 VHL patients revealed 2 cases who had a mosaic parent, giving a 4.8% likeli-
hood of having mosaic parents in sporadic cases with no documented family history (116).
342 Chen et al.

2.6. RB1: Retinoblastoma
Retinoblastoma, a malignant pediatric tumor of the retina, consists of a tumor entity rather than
a typical syndrome and is usually seen in children under 3 years of age (125). Osteosarcoma is
the most common second malignancy among others (e.g., soft tissue sarcomas, melanoma) and is
generally more aggressive and less curable than retinoblastoma (76) (Supplemental Figure 6).
The lifetime risk of secondary malignancies is 15–20%, which increases to 40–50% if radiation
therapy is given. Retinoblastoma affects approximately 1 in 15,000–18,000 live births (46) and
is caused by genetic alterations in RB1 (∼98%) (117), located in chromosome 13q14.2 (OMIM
180200), or, rarely, by amplification of MYCN (∼1.5%) (76). Unilateral retinoblastoma constitutes
60% of cases and has an average age of diagnosis of 24 months (77), while bilateral retinoblastoma
constitutes 40% of cases and is diagnosed at an average age of 12 months (77); 90% of all cases are
de novo (25), and only 10% are familial (18, 61). Trilateral retinoblastoma is a syndromic form of
retinoblastoma and is seen in 0.6–12.7% of patients with either unilateral or bilateral retinoblas-
toma (Supplemental Figure 6). Patients with trilateral retinoblastoma have pineoblastoma in
addition to retinoblastoma (142).
Somatic mosaicism has been reported in 4.5% of retinoblastoma cases (59, 117, 130). The true
incidence of mosaicism may be higher, as older studies had analyzed only individuals with a known
family history of retinoblastoma or with higher VAFs (117). Recently, using MPS, Rodríguez-
Martín et al. (103) detected high-level mosaicism in 14 of 100 individuals with bilateral retinoblas-
toma and in 11 of 29 individuals (38%) with unilateral retinoblastoma. Mosaicism has been de-
scribed as a major cause of low-penetrance retinoblastoma. Germline-only mosaicism is known
to occur in retinoblastoma, as evidenced by four children with constitutive RB1 variants born to
three different unaffected fathers but the same mother, who harbored a gonadal mosaic PV that
was transmitted to her offspring (4, 105). The spectrum of P/LP variants for RB1 is shown in
Figure 10.
47 Missense variant
16 In-frame variant
56 Splice
13 Other
p.L769*/Ffs*26_K928delinsF
p.E748*/Dfs*6/Kfs*8/Sfs*6
p.S318Ffs*2/Lfs*14/Nfs*13
p.E19Pfs*3/Afs*4/Nfs*46
p.E545fs/*/Kfs*2/Nfs*9
p.F162Lfs*9/Pfs*13/Qfs*8
p.E204Kfs*10/*/Gfs*8
p.G617Vfs*6/Rfs*36
p.L389*/Ffs*3/Ffs*6
p.R73fs/Lfs*3/Sfs*36
p.E53*/Kfs*12/Qfs*13
p.R445*/R445fs
p.W99*/Gfs*10
p.X500_splice
p.E137*/Lfs*15
p.E675*/Nfs*2
Number of RB1 variants
p.Q354*/Efs*7
p.X703_splice
10
p.R255*
p.E287D
0
DUF3452 RB_A RB_B RB_C
0 200 400 600 800 928

Amino acid position
Figure 10
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in RB1 (from the Leiden Open Variation Database,

2.7. TP53: Li–Fraumeni Syndrome
TP53 (OMIM 191170), on chromosome 17p13.1 (69), is the most common somatically mutated
gene across various cancers (20). It contains 11 exons and encodes the ubiquitous p53 protein,
which acts in the nucleus and is important in transcriptional regulation, microRNA processing, cell
cycle control, apoptosis, angiogenesis, and aging. Germline pathogenic TP53 variants are the only
known cause of LFS, a genetic disorder predisposing individuals to multiple tumors, including
early-onset sarcomas, brain tumors, adrenocortical carcinoma, premenopausal breast cancer, and
leukemia (20) (Supplemental Figure 7). The frequencies of tumors seen in LFS may vary based
on age. The frequency of adrenocortical carcinoma is very high in the first 5–6 years of life but
then drops dramatically; a similar pattern is seen with choroid plexus carcinoma in LFS. Breast
cancer frequency, on the other hand, peaks between the ages of 30 and 50 (1).
In contrast to TSC, NF1, NF2, and PHTS, LFS has no distinctive hallmark features and is
characterized by tumors often seen in nonsyndromic cancers. LFS affects 1 in 20,000 individu-
als, and the precise rate of TP53 alterations in LFS is undetermined (71, 115). The syndrome is
characterized by early onset and high penetrance. Germline TP53 PVs are seen in up to 75% of
patients with classic LFS (51, 97), whereas de novo alterations may constitute at least 14% of PVs
(12, 74). The spectrum of known P/LP variants for TP53 is shown in Figure 11.
Individuals with germline TP53 alterations have a 42% risk of developing a TP53-related can-
cer before the age of 16 (20). The lifetime likelihood of a male germline carrier developing cancer
is approximately 73% (12, 71), whereas the risk of developing breast cancer is almost 100% in
female carriers (51).
The true rate of TP53 mosaicism is uncertain, and severe mosaic LFS has been reported in
two case studies (7, 97) that demonstrated the effects of timing on postzygotic alterations. Iden-
tical TP53 alterations were present in tissues and tumors derived from two different germ layers,
689 Missense variant 93 Splice

76 In-frame variant 10 Other
p.H179Q/L/N/R/Y/D/P
p.R273C/H/G/L/P/S
p.K132N/E/M/R/Q/T
p.Y205C/D/H/N/F/S
p.A86Cfs*63/Pfs*34/Vfs*55
p.C242S/F/G/Y/R
p.Q317*/Afs*19/Pfs*20
p.W53*/Cfs*4/Mfs*4
p.R337H/C/P/L/S
p.V73Rfs*76/Wfs*50
p.K120E/Sfs*3/Tfs*2
p.G105D/R/C/S/V
p.Y220/C/S/D/H
Number of TP53 variants
19
p.X367_splice
p.X25_splice
p.X10_splice
p.S392Tfs*76
0
TAD DNA binding domain TETD
0 100 200 300 393

Amino acid position
Figure 11
Map (lollipop diagram) of known pathogenic and likely pathogenic variants in TP53 (from the Leiden Open Variation Database,
344 Chen et al.

demonstrating that the genetic events occurred prior to gastrulation in both individuals, as is typ-
ically seen in mosaicism.
3. IMPROVING RATES OF MOSAICISM DETECTION

During the last decade, the development of advanced, increasingly sensitive, robust, and high-
throughput techniques, including MPS and digital PCR, has significantly improved the molecular
diagnosis of mosaicism, thus enabling better detection of genetic alterations with very-low-level
VAFs (27, 52, 66) (Table 1). For some TSG syndromes, MPS has significantly improved the sen-
sitivity of detection for VAFs down to 0.02% (133) and is considered the gold-standard testing
approach for mosaicism (32, 47). Previously, Sanger sequencing was only able to detect alterations
with a VAF higher than 10–15%, whereas today, MPS—which is extensively used in many clinical
and research laboratories—provides a mean depth of coverage between 300× and 1,000× (27).
The broad range of detection rates for each TSG is due partly to the different sampling criteria
used across different studies and cohorts. Some studies have ascertained patients from national
registries who met strict diagnostic criteria, and others specifically referred affected individuals
who did not meet the full diagnostic criteria, but whose phenotypes were suggestive of a particular
syndrome. The sensitivity and specificity of methods and the robustness of bioinformatics tools
applied for genetic analysis also affect the variability seen in detection rate. Older conventional
methods, such as Sanger sequencing, might have been used to analyze only the exons of the genes
(19). Relevant tumor tissues are often difficult to access, and small sample sizes may confound the
true rate of mosaicism in TSG syndromes.
Another limitation is the lower rates of PV detection in individuals who do not meet the diag-
nostic criteria for full heterozygous disease, which is often the case in individuals with mosaicism.
In LFS, for example, TP53 PVs have been identified in fewer than 20% of affected individuals
who meet the more relaxed Chompret diagnostic criteria (7).
The importance of appropriate tissue sampling is also evident in unilateral retinoblastoma, in
which there is a major discrepancy between the rate of detection in blood (as low as 13.5%) (108)
and the rate when analyzing ocular tumor tissue (as high as 94.9%) (91). Two reported cases of
mosaic retinoblastoma had genetic alterations detected in blood at a VAF of 2–4% (130). However,
50% of sporadic retinoblastoma cases do not have a genetic alteration that could be detected in
blood by Sanger sequencing (3). Studies using allele-specific PCR, quantitative multiplex PCR,
and MPS to test blood DNA have identified higher levels of mosaicism in sporadic bilateral (30%)
than in sporadic unilateral (6%) retinoblastoma cohorts (19, 108).
Limited MPS analysis has been conducted on large NF1 mosaic cohorts. To reassess the num-
ber of mosaic NF1 cases previously reported as segmental NF, García-Romero et al. (44) con-
ducted a systematic review of the literature, including probable and confirmed cases of NF1 that
met defined inclusion criteria. They found that only 15 out of 157 mosaic NF1 cases available for
analysis, documented between 1977 and 2012, had undergone genetic testing, only 12 of which
harbored a pathogenic alteration.
NF1 molecular testing of blood genomic DNA can detect more than 95% of NF1 PVs (39).
Given that café-au-lait macules are the only feature commonly seen in mosaic cases of NF1, skin
biopsies from hyperpigmented tissue can be analyzed for NF1 (58). Before 2019, fluorescence in
situ hybridization (FISH) was commonly used to detect low-grade mosaicism (1–5%) for NF1
whole-gene deletions. Legius et al. (75) have proposed diagnostic criteria to distinguish mosaic
NF1 from mosaic and inherited Legius syndrome, a rare genetic skin pigmentation disorder that
is known to be an NF1-like syndrome, as 50% of cases with Legius syndrome share diagnostic
overlap with NF1, according to National Institutes of Health criteria.

Table 1 Summarized data regarding the molecular analysis of the TSGs reported here and their mosaicism rates
Success rate of PV Frequency of Persistent NMI Risk of disease

TSG identification Molecular method mosaicism cases Tissues analyzed transmission VAF
NF1 94–99% (52) MPS, Sanger sequencing, 40% (62, 66) ND Blood, urine, skin 5–50% (segmental ND
PCR, MLPA, FISH fibroblasts, buccal disease-affected
346
cells, neurofibroma parent) (52)
tissue (62)
NF2 68% (ependymoma) MPS, Sanger sequencing, 33–60% (34) 17% (128) Blood, tumors, buccal 8–12% (36) 1.6% (21)
(33) MLPA, SSCP, TGGE 9% (54) 33.2% (56) mucosa, hair follicles 10% (13)
Chen et al.
16.7–24.8% (67) 40.6% (sporadic) (32) 19.7% (119)
25–58% (32, 53) 68% (blood) (67) 0.2–30% (32)
33% (128)
TSC1/TSC2 85–99% (81, 90, Ultra-deep sequencing, 6% (98) 0.3% (81) Skin lesions (ungual 2–3% (in sporadic 0.06–42% (72,
133) Sanger sequencing, 5.8% (105) 10–15% (90, 133) fibroma, Shagreen parents) (105) 90, 137)
targeted MPS, 9.7% (137) 15% (133) patch, hypomelanotic 0.2–10% (47)
amplicon MPS 7.5–58% (133) 94% (98) macules), blood, 0.21–34%
saliva, angiofibroma, (133)
normal skin, urine, 0.7–32% (90)
semen
PTEN (CS) 85% (meeting full MPS, Sanger sequencing ND (only case 15% (meeting full Endocervical mucosa, ND 1.7–25% (case
diagnostic reports) diagnostic criteria) colonic mucosa, study) (96)
criteria) (145) (145) dysplastic
25% (meeting less gangliocytoma,
stringent criteria) cutaneous fibroma,
(145) epidermal nevi,
lipoma
PTEN 60–65% (26, 145) MPS, MLPA, FISH ND ND Blood, epidermal nevi, ND 17% (26)
(BRRS) vascular
malformations,
overgrown muscle
tissue
PTEN (PLS) ND ND ND ND Nevus, lipomatous mass, ND ND
arteriovenous
malformations
(Continued)
Table 1 (Continued)
Success rate of PV Frequency of Persistent NMI Risk of disease

TSG identification Molecular method mosaicism cases Tissues analyzed transmission VAF
VHL 89–95% (22) MPS, Sanger sequencing 5–16.6% (22, 23) 5% (135) Blood, tumors (e.g., ND 1.7–34.6%
pheochromocytoma, (22, 23)
renal cell carcinoma)
RB1 92–97% (29) MPS, quantitative 0.8% (25) 5.1% (130) Blood, urine, ocular 0.7–1.3% (from 2–4% (25, 59,
(bilateral) multiplex PCR, 9.3% (64) 5.2% (108) tumors, oral mucosa clinically unaffected 130)
AS-PCR, array CGH, 5.5% (108) 7.5% (3) parent) (25) 8–24% (3)
Sanger sequencing, 9.6% (29)
FISH, SSCP, DGGE, 10–30% (3, 59,
MLPA, methylation 117)
assay 14% (103)
RB1 (unilat- 92.7–94.9% (tumor) 1.3% (117) 86.5% (108) Blood, urine ocular 0.5% (in unilateral 2–4% (130)
www.annualreviews.org
•
eral) (29) 3.9% (unilateral tumors, oral mucosa probands who are 8–24% (3)
13.5–42.4% (blood) with tumor) NMI in blood)
(29) (108) (108)
3.7% (unilateral
with no tumor)
(108)
7.3% (130)
10–30% (3, 59,
117)
12% (29)
38% (103)
TP53 (LFS) 60–80% (115) MPS, Sanger sequencing 2.4% (102) 14–17% (84, 102) Blood, fibroblasts, ND 3.3–20% (7)
12.4% (84) adenoid cystic
17% (de novo) carcinoma, breast
Mosaicism in Tumor Suppressor Gene Syndromes

(102) sarcoma, breast
carcinoma, saliva
347
Abbreviations: AS-PCR, allele-specific PCR; BRRS, Bannayan–Riley–Ruvalcaba syndrome; CGH, comparative genomic hybridization; CS, Cowden syndrome; DGGE, denaturing gradient gel
electrophoresis; FISH, fluorescence in situ hybridization; LFS, Li–Fraumeni syndrome; MLPA, multiplex ligation-dependent probe amplification; MPS, massively parallel sequencing; ND, no
data; NMI, no mutation identified; PLS, Proteus-like syndrome; PV, pathogenic variant; SSCP, single-stranded conformation polymorphism; TGGE, temperature gradient gel electrophoresis;
TSG, tumor suppressor gene; VAF, variant allele frequency.
Regarding NF2, the rate of NF2 somatic mosaicism was previously determined to be between
16.7% and 24.8% in individuals with bilateral vestibular schwannoma using single-strand con-
formational polymorphism, temperature gradient gel electrophoresis, or Sanger sequencing on
blood and tumor samples (67). Using MPS, Halliday et al. (53) found that the rate of detection
of mosaicism in blood and schwannoma tissue was 58% in a cohort of 142 individuals with either
unilateral or bilateral vestibular schwannoma who met Manchester diagnostic criteria for NF2.
Most recently, in a study by Teranishi et al. (128), the rate of mosaicism detection, imple-
menting Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and/or
targeted MPS, was improved from 23.5% to 37.7% for individuals with de novo NF2 by analysis
of blood, buccal mucosa, hair follicle, and/or tumor. In this analysis, 9 of 53 de novo affected indi-
viduals (17%) were NMI cases. Prior to this study, the detection rate of pathogenic alterations was
17–25% in de novo affected individuals in blood and tumor tissue (32–34, 67, 78). Tumor tissue
samples were limited in these studies, so the true rate of detection of mosaicism could be as high
as 60% among those who had no PV identified in blood and also present with unilateral vestibular
schwannoma (34).
In retinoblastoma, MPS has improved PV detection in blood to 97% in bilateral cases and
18% in unilateral cases (29). Li et al. (76) reported a rapid and sensitive MPS method to detect
point mutations, small indels, and large deletions or duplications across the RB1 gene as well as
amplification of the MYCN gene.
A diagnostic algorithm recently proposed by Treichel et al. (132) to expedite diagnosis and
genetic counseling for mosaic TSC suggested stratification of the affected individuals based on
the clinical features. Specifically, cases with phenotypic features indicative of germline inherited
TSC (e.g., ungual fibroma with onset before 15 years of age or angiofibroma before 5 years of
age) should undergo sequencing of blood DNA. Also, individuals with clinical features common to
mosaicism (e.g., fewer than three mucocutaneous fibromas or absence of tubers and subependymal
nodules) should be assessed using MPS on available TSC tumors as a first line of analysis, followed
by validation with amplicon MPS performed on other lesioned samples (132).
The genetic analysis of multiple tissue types using MPS is limited in PHTS (41, 121), whereas
MPS performed on epidermal nevi, vascular malformations, and overgrown muscle samples has
provided the best rates of detection of mosaicism in Proteus syndrome (88). Larger patient series
are necessary to determine a more representative range of VAFs common in PHTS mosaic dis-
eases. Several cases of mosaic Cowden syndrome have been unraveled using a variety of methods,
including Sanger sequencing and MPS (41, 96, 111, 121), and two cases of BRRS with mosaic
10q23 deletions have been identified in blood using FISH (26) and MLPA (48).
4. MOSAIC GENETIC ALTERATIONS ARE FREQUENTLY UNCOVERED

IN INDIVIDUALS WITH NO MUTATION IDENTIFIED
Individuals who meet standard diagnostic criteria for the TSG syndromes discussed in this re-
view may have no PV identified following conventional genetic analysis. These so-called NMI
cases may be due to misdiagnosis, limitations in the sensitivity and efficacy of genetic testing, or
mosaicism occurrence, thus leading to the lack of variant identification and disease confirmation.
Currently, the advent of MPS has reduced the number of NMI individuals and improved the rate
of detection of low- and very-low-level mosaic VAFs in TSC, VHL, LFS, and retinoblastoma.
In TSC, individuals with NMI status have been analyzed in serial studies, with steady improve-
ments regarding tissue selection, depth of gene coverage, and interpretation of sequencing data,
leading to higher overall detection rates (47, 98, 105, 120, 133). In the initial MPS analysis of
blood, normal skin, saliva, angiofibroma, and other skin lesion biopsies, 45 of 53 individuals with
TSC NMI status (85%) had a causative PV, and 26 of these 45 (58%) had mosaicism. VAF levels
348 Chen et al.

ranged from 0.21% to 34%, with 17 patients having a VAF below 5%, 5 having a VAF of less than
1%, and 2 having variants detectable only in skin tumor biopsies, highlighting the importance of
analyzing lesion tissues in TSC NMI cases (133). Some of those with persistent NMI status af-
ter MPS analysis marginally met the standard TSC diagnostic criteria. Most recently, Klonowska
et al. (65) identified one subject in whom spontaneous distinct two-hit PVs were seen in a facial
angiofibroma and a kidney angiomyolipoma (four PVs in total), consistent with sporadic occur-
rence of each of those lesions, and not an underlying TSC syndrome. Notably, fewer TSC mosaic
cases were observed at VAFs below 3%, in contrast to a predicted increase in mosaic cases at in-
creasingly lower-level VAFs (47). The deviation from this expected trend indicates that cases of
mosaic TSC at very-low-level VAFs may be undetected clinically because the individuals exhibit
few or no profound symptoms (47).
Regarding NF2, MPS analysis of 1,055 individuals with sporadic NF2 revealed that 232 proven
and probable mosaic individuals had VAFs ranging from 0.2% to 30%, whereas 428 de novo
affected individuals (40.6%) were classified as NMI, likely due to the limited availability of tumor
samples. Of the 160 individuals who had at least one tumor analyzed, mosaicism was detected in
134 (83.8%), while the rest remained NMI cases (32). In a study that found 76 NMI cases out
of 1,361 de novo affected individuals who met diagnostic criteria, the PV detection rate for NF2
when using MLPA and MPS was 95% (33).
Out of 84 individuals from the Danish health registry clinically diagnosed with VHL [75 (89%)
of which were familial cases], 7 had a previous negative VHL genetic test. Out of 69 additional
individuals who met Danish diagnostic criteria, 15 also had a negative VHL genetic analysis; 61
(88%) of these cases were nonfamilial. Mosaicism was not identified in any individuals but may
be suspected in the NMI individuals, especially within the cohort not yet diagnosed with VHL,
of which a large proportion (89%) of cases are sporadic. Most of the parents of the sporadically
affected individuals might be carriers of gonadal mosaicism (11). In the largest MPS study to date,
blood analysis was performed on 47 individuals who had phenotypes indicative of VHL, but did
not all meet diagnostic criteria. Out of 47 individuals who met VHL diagnostic criteria, 4 (8.5%)
had mosaic PVs identified, thus enhancing the rate of mosaicism to 16.7% (4/24) among those
meeting diagnostic criteria (23). It is possible that those who did not meet diagnostic criteria had
lower mosaic VAFs that remained undetected. The rate of VHL mosaicism may range from 5%
to 16.6% for those who meet diagnostic criteria with mild phenotypes and are characterized as
NMI (23).
The only large study conducted to specifically assess the role of mosaicism in LFS used
Sanger sequencing and MPS on blood from individuals with phenotypes suggestive of LFS but
without detected TP53 PVs; this group included individuals with adrenocortical carcinoma (97
children and 49 adults), choroid plexus tumors (43 individuals), female breast cancer before
age 31 (31 individuals), or multiple primary tumors (1 individual). The PV detection rate in
individuals with adrenocortical carcinoma was 46% in children and 14% in adults; mosaicism
was identified in 3 children. In the choroid plexus tumor group, 17 individuals were heterozygous
and 2 were mosaic, and 19 individuals remained NMI. In early-onset female breast cancer, the
germline TP53 detection rate is 4–6%. One woman was mosaic, and 30 women remained NMI.
Of 328 individuals who met the Chompret criteria for LFS and were previously classified as
heterozygotes, 2 were mosaic (102).
Reanalysis of 40 retinoblastoma cases using MPS identified 6 mosaic individuals with VAFs
between 8% and 45% (3); 30 of the 40 individuals had previously been characterized as NMI.
The study improved the diagnostic rate of RB1 PVs from 3% to 10% in unilateral cases and from
81% to 92% in bilateral cases (3). Another study analyzed 529 retinoblastoma individuals from
the Dutch National Retinoblastoma Registry (29), including 257 nonfamilial unilateral cases, of

which 222 were NMI, whereas 10 of the 125 nonfamilial bilateral cases were NMI (29). Recently,
using MPS, Rodríguez-Martín et al. (103) detected 3 mosaic VAFs in 16 individuals with unilateral
retinoblastoma who were previously NMI using conventional sequencing methods.
5. GENOTYPE–PHENOTYPE CORRELATIONS
The spectrum of different clinical features in mosaic TSG syndromes depends on the cell and
tissue types affected, thus contributing to broad phenotypic heterogeneity within each syndrome.
While mosaicism is often associated with milder phenotypes compared with the inherited forms
of disease, mosaic cases with severe clinical features have been reported, including cases of LFS,
VHL, BRRS, and Cowden syndrome (8, 23, 50, 74, 96, 97). Also, while the VAFs detected in
blood in these cases may be low, it is possible that VAFs may be much higher in tissue related
to the specific phenotype of a syndrome (e.g., in the brain in TSC) than in blood. In addition,
there is potentially an impact of modifier genes on phenotype and the unsuspected coexistence
or inheritance of other genetic variants that may enhance or diminish the phenotype. Here, we
report known genotype–phenotype correlations, comparing full heterozygous and mosaic disease
status across the different TSG syndromes.
In NF1, the cell types affected result in a variety of clinical manifestations (124). Melanocytes
undergo biallelic NF1 inactivation in café-au-lait macules, and Schwann cells are affected in plex-
iform and cutaneous neurofibromas, while the development of malignant peripheral nerve sheath
tumors requires additional alterations, such as loss of heterozygosity in TP53 (52) or CDKN2A
inactivation by point alterations, deletion, or translocation (79, 91). The most common cate-
gory of genetic alterations is loss-of-function single-nucleotide variants, but no particular single-
nucleotide variant is recurrent at a rate as high as NF1 microdeletion, identified in 4.7–11% of
all NF1 patients (63). Type I microdeletions encompass 1.4 Mb, involve complete deletion of
the SUZ12 (JJAZ1) gene on chromosome 17q21 (OMIM 606245), and constitute 70–80%
of all microdeletions, while type II microdeletions encompass 1.2 Mb, involve partial deletion of
SUZ12, and constitute 10–20% of microdeletions (62, 123). Although whole-gene deletions are
the most common genetic alterations detected in NF1 cases, they are not very common, overall.
SUZ12 is highly expressed in brain structures associated with memory (e.g., the cerebellum, Purk-
inje fibers, and pyriform cortex) (62), and SUZ12 haploinsufficiency is associated with cognitive
impairment in NF1 patients with constitutional type I microdeletions (62). However, in a 2004
study by Kehrer-Sawatzki et al. (62), none of the mosaic individuals with type II microdeletions
displayed cognitive impairments. Approximately 91% of type I microdeletions are maternally in-
herited, and 63% of type II microdeletions account for low-level mosaicism (VAF <15%) (123).
Overall, microdeletions are associated with severe phenotypes, intellectual disability (14, 123), and
a higher risk of developing malignant peripheral nerve sheath tumors (16–26%) (63). Mosaic cases
of type I microdeletions are infrequent (123), and an MLPA analysis of 116 patients with type I
microdeletions found that 3.4% of their deletions were mosaic (83).
Because up to 60% of atypical NF1 deletions likely exhibit mosaicism (123), more detailed
analysis of such deletions is required. Aside from large deletions, the distribution of the different
genetic alterations seen in mosaic NF1 is similar to that seen in inherited NF1. The p.R1809C
missense variant is correlated with Noonan-like features, pulmonic stenosis, and short stature
(104). Genetic events affecting codons 844–848 are correlated with plexiform neurofibromas, optic
gliomas, and spinal neurofibromas (83). Individuals with p.M992del variants lack neurofibromas or
gliomas, while those with p.M1149, p.R1276, or p.K1423 variants have Noonan-like features. The
p.M1149 and p.R1276 variants are also correlated with cardiovascular anomalies, and p.K1423 is
correlated with spinal neurofibroma (68). Finally, genetic alterations near the 5 end of NF1 and in
350 Chen et al.

the cysteine/serine-rich domain have been reported to correlate with an increased risk of autism
spectrum disorder and glioma (86).
While many inherited TSG syndrome cases can be diagnosed without the need for genetic
testing, mosaic NF1 cases may present with fewer or milder features that have a later age of onset
or fail to manifest at all. For these reasons, mosaic NF1 is likely underdiagnosed (30, 39, 44, 58,
66, 148).
Studies of monozygotic twins have provided insight into the occurrence of mosaicism for both
NF1 and NF2 as well as for retinoblastoma (2, 100). In one case, only one monozygotic twin
presented with mosaic NF1, while the other was clinically unaffected. In another case, one twin
was mosaic, while the other had full heterozygous disease, further highlighting the importance
of the particular timing of genetic alterations occurring during postzygotic development (138).
Individuals with a mosaic NF1 PV typically have milder features and fewer clinical manifestations
than those with non-mosaic NF1 (44). Several studies have shown that the presence of only café-
au-lait macules (52), without other features of NF1, can be indicative of mosaicism (58, 74, 136).
Identification of unilateral or bilateral pigmentary changes confined to a particular skin region or
distribution is included in the diagnostic criteria for mosaic NF1 (58, 75).
Genotype–phenotype associations are well established for non-mosaic heterozygous NF2 and
are also stronger than those seen in NF1 (56). Missense alterations are correlated with the least se-
vere phenotypes and lowest mortality rates (54). The most severe phenotypes, including impaired
vision, hearing, and mobility along with lower reproductive fitness, are correlated with nonsense
and frameshift variants that result in gene truncation, followed by large deletions whose pheno-
typic outcomes are more variable (54). Splice-site variants are associated with more severe disease
states when they are in exons 1–5 than when they are in exons 6–15, as reported by studies of two
large-scale cohorts that analyzed partially overlapping data sets (5, 56). Nonsense variants were
identified in 41.8% of mosaic cases (32).
Mosaic cases of NF2 typically present at later ages but have the same spectrum of tumors (67)
as those with inherited disease, and they have a higher rate of unilateral vestibular schwannoma
(35.9%) than those with inherited disease (12.6%) (53). In one study, 54% of people with a mosaic
PV presented with classic bilateral vestibular schwannoma, as compared with 96–100% of inher-
ited cases (53). Clinically, mosaic NF2 may also be suspected based on the occurrence of unilateral
eighth-nerve schwannoma, associated ipsilateral meningioma, or multiple schwannomas localized
to part of the peripheral nervous system (106). Additionally, the growth rate of schwannomas dif-
fers between mosaic and non-mosaic cases based on the age of onset, whereas the growth rate
of meningiomas does not (128). Survival depends on the type of variant, with truncating variants
showing the highest mortality and mosaic individuals showing less than one-third the mortal-
ity seen in non-mosaic, constitutional cohorts (56, 106). Inherited NF2 cases with asymptomatic
presentations have shown the highest rates of survival, and de novo constitutional variants have
worse survival than both de novo mosaic and inherited cases (38).
In TSC, TSC1 variants are associated with a milder phenotype than TSC2 variants, likely due
to the gene’s smaller size and decreased frequency of second-hit events (16, 81). TSC1-associated
cases usually have lower tumor counts in multiple organs and fewer cortical tubers. In addition,
TSC2-associated cases usually have earlier onset of seizures, including infantile spasms, that are
more difficult to treat on average, and they also have higher rates of autism spectrum disorder and
intellectual disability (112).
Nonsense, splice-site, and missense variants make up approximately half of all TSC-causing
variants, while small indels constitute approximately 38%. Individuals with mosaic variants of
either TSC1 or TSC2 at a VAF of less than 10% and individuals classified as NMI are associated
with a milder phenotype, fewer seizures, and fewer cortical tubers (112). Asymmetric facial

angiofibromas and an absence of subependymal nodules or cortical tubers are commonly seen in
TSC mosaicism (131). Mosaicism is more frequently seen in cases with genomic TSC2 deletions
than in those with smaller TSC2 indels and point variants (98). Mosaicism was seen in 27%
of individuals with combined TSC2 and polycystic kidney disease syndrome, which is due to
genomic deletion of parts or all of both TSC2 and the adjacent PKD1 gene (98). TSC diagnosed
in infancy is due to mosaicism in 10% of cases and is associated with a milder phenotype and
improved seizure-free survival in comparison with heterozygous cases (90).
Few genotype–phenotype correlations have been established in PHTS (145). Variants that re-
sult in the accumulation of stable, inactive PTEN protein are predicted to lead to more severe
PHTS phenotypes and the development of malignancies (145). Frameshift and nonsense variants
constitute the majority of the limited PHTS mosaic cases that have been identified (41, 96, 149).
Only two cases of mosaic deletions involving PTEN have been described (20, 37). Mosaic PHTS
is typically mild (41), but several cases presenting with severe features have been documented (48,
96, 111). Mosaic second-hit PVs may also exacerbate heterozygous germline disease. This phe-
nomenon has been reported in two cases: one patient with Proteus-like syndrome who developed
lipomas by the age of 2.5 years and had worsening hemihypertrophy throughout childhood (149)
and another individual presenting with an atypical case of Cowden syndrome who, in addition
to carrying a germline PTEN variant that causes Cowden syndrome, exhibited features of Pro-
teus syndrome (16). However, mosaic second-hit cases should be distinguished from cases of true
germline mosaic disease. Mosaic PTEN variants resulting in features that are not consistent with
or do not meet diagnostic criteria for PHTS have also been documented (49).
Specific genotypes may be responsible for the different phenotypes seen in VHL (types 1,
2A, 2B, and 2C). Truncating and missense variants that significantly affect protein folding are
associated with type 1, which is distinguished by a low risk of pheochromocytoma. Several studies
have reported a reduced risk of renal cell carcinoma in individuals with complete deletions of VHL
that extend in the 5 direction to include BRK1. It is possible that this phenotype is associated with
a subset of type 1 VHL (type 1B) (80, 135). Missense variants are most frequently associated with
the type 2 phenotypes, which carry a greater risk of pheochromocytoma (135).
Implication of other driver events requires further investigation in mosaic VHL cases with
clinically unique presentations, as well as in mosaic cases that present with uncharacteristically
severe phenotypes (22, 72, 141). By contrast, several mosaic VHL cases with milder phenotypes
have been reported (87, 114).
Genotype–phenotype associations for retinoblastoma focus primarily on the correlations be-
tween PV types and the laterality of retinoblastoma tumors. Full heterozygous germline nonsense
variants are associated with an earlier age of diagnosis and higher risk of bilateral retinoblastoma,
while missense variants, large gene rearrangements, and in-frame splice variants are associated
with a lower risk of bilateral retinoblastoma. Somatic alterations, including mosaic PVs, often
manifest in unilateral retinoblastoma (113). Children with mosaic RB1 PVs often have fewer tu-
mors in each eye than those with inherited PVs, and their tumors are more likely to remain unilat-
eral (101). More than 90% of individuals with an inherited heterozygous PV will develop at least
one retinoblastoma (117). Among sporadic cases, 60% are unilateral and 40% are bilateral (77).
Genetic modifiers (e.g., polymorphism of p.P72R) may account for phenotypic variability
within and across LFS families, but there is no predictive diagnostic algorithm to determine age
of onset (51). The 2009 Chompret criteria outline more relaxed criteria for diagnosis of LFS;
however, in those meeting the Chompret criteria, TP53 alterations are identified in fewer than
20% of cases (7, 97). Interestingly, there is a possible founder effect in the Brazilian population for
the germline, non-mosaic TP53 variant p.R337H, which predisposes individuals to adrenocortical
carcinoma (51). Childhood-onset adrenocortical carcinoma associated with this variant has a low
352 Chen et al.

penetrance of 1 in 30–40 carriers (115), but the carrier rate in the Brazilian population may be as
high as 1 in 350 (51).
6. RISK OF DISEASE TRANSMISSION DEPENDS ON THE EXTENT

AND DISTRIBUTION OF MOSAICISM LEVEL
The transmissibility of mosaicism across different syndromes is not well studied, as there are no
reliable predictors of whether a mosaic PV is present in germ cells (82). Little is known about the
risk of transmission to offspring of mosaic affected individuals based on their VAFs and extent of
mosaicism across different tissues and organs (33, 47, 52, 121).
Although it is difficult to unravel how often this occurs, there have been reports of unaffected
individuals transmitting pathogenic alterations to multiple offspring for all the TSG syndromes.
An NF1 study followed a Danish sperm donor who was later determined to be a gonadosomatic
mosaic carrier for NF1 (30). MLPA analysis found that 9 of his 23 offspring carried an inherited
NF1 deletion of exons 15–29. The study did not report whether the donor himself had manifes-
tations of NF1, and other tissues were not tested. Although semen analysis may predict the risk of
transmission of a mosaic TSC variant to offspring, it is still unclear how the VAF in other tissues
correlates with transmission risk (47). Parents with sporadic TSC have an estimated 2–3% risk of
transmission (105).
In some TSG syndromes, differences in transmission rate are correlated with unilateral versus
bilateral disease. The risk of transmission has been estimated to be 1 in 12 for NF2 patients with
unilateral vestibular schwannoma and 1 in 8 in those with bilateral vestibular schwannoma (34).
In individuals presenting with bilateral vestibular schwannoma at less than 20 years of age, the
risk of transmission is predicted to be 29.3%, as compared with 5.5% in those presenting with
asymmetric disease past the age of 40 years, since there is a 99% chance that an individual with
unilateral presentation at a later age is mosaic (35). Parents with mosaic NF1 PVs detected in
blood (VAF range 0.2–30%, median 17.5%) gave birth to eight (10.7%) positive NF2 cases. All
children of presumed mosaic parents with no PV detected in blood had negative presymptomatic
tests for NF2 (32). Overall, the risk of transmission to offspring of individuals with mosaic NF2
is 8–12%, depending on the parent’s extent of mosaic variation (70).
Regarding retinoblastoma, only 7–15% of all individuals affected with unilateral retinoblas-
toma had affected offspring (1, 39), while all individuals with bilateral retinoblastoma had affected
offspring (50). MPS analysis on blood was performed to assess the variant transmission or disease
transmission risk from parents with retinoblastoma germline mosaicism. Dehainault et al. (25)
found that among the parents of 124 affected offspring, only one carried an RB1 variant at 11%
VAF in leukocytes, corresponding to a transmission risk of 0.8% for RB1 mosaicism in a parent
if their first child was fully affected with retinoblastoma. Additionally, if all germline cells of an
affected parent carry the PV, the maximum risk of recurrence in a second child is 0.4% (25). No-
tably, the risk of transmission for sporadic and mosaic retinoblastoma cases is reported from what
has been identified thus far in the literature and reflects current data analysis (28).
7. CLOSING REMARKS AND FUTURE PERSPECTIVES

Mosaicism is known to be one of the mechanisms, alongside intronic alterations and epigenetic
modifiers, that underlie phenotypic heterogeneity seen in TSG syndromes (11, 44, 58, 92). The
advent of high-throughput MPS methods along with multi-tissue sampling has facilitated and
improved the detection of many previously masked and undiagnosed cases of low-level mosaicism,
thus allowing more precise and accurate genetic counseling. MPS has enabled more sensitive

detection and quantitation of mosaicism, and in combination with better recognition of common
mosaic clinical phenotypes, it has improved our understanding of low-level disease across TSG
syndromes for clinicians and geneticists, thus improving the treatments, outcomes, and lives of
patients with these hereditary genetic diseases.
One of the main limitations in study design for TSG syndromes is the limited availability of
tissues other than blood samples for analysis (130). Analysis of tumor biopsies or archival tissue
samples when available would allow better detection of mosaicism. Regular screening may then
be recommended based on mosaic clinical findings. It is important to note that higher rates of
mosaicism have been documented and reported for TSG syndromes with multiple distinctive
features, such as TSC and NF2, which enable clinical recognition and definite diagnosis. By con-
trast, mosaicism is much less well documented for LFS, which has no pathognomonic features
but rather a risk of early onset of several common cancers, which may occur sporadically or be
due to other germline variants (18). In addition, it is possible that the syndromes with the highest
mosaicism rates are the ones where the presence of the genetic alteration in every cell is more
likely to affect early cell viability and/or reproductive fitness. For these TSG syndromes, analysis
of tumor tissues can greatly enhance variant detection rates. Furthermore, once identified in any
sample, genetic alterations may be sought in multiple other tissues to assess the global prevalence
and distribution of the mosaic variant. Further study of germ cell tissue, especially sperm, in in-
dividuals with mosaicism will hopefully illuminate correlations between mosaic PV prevalence
and the risk of disease transmission, at least in males. Overall, this review has summarized the ef-
fect of mosaicism on diagnosis, phenotypic variability, and transmission risk as well as important
methodological advances in the detection of mosaic alterations.
DISCLOSURE STATEMENT
B.R.K. is a member of the medical advisory boards for SpringWorks Therapeutics, Genome Med-
ical, and iNFixion Bioscience. L.S.S. was supported by the Intramural Research Program of the
Center for Cancer Research, National Cancer Institute, National Institutes of Health, and in part
by federal funds from the Frederick National Laboratory for Cancer Research, National Insti-
tutes of Health, under contract HHSN261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or organizations imply endorsement by the
US government.
ACKNOWLEDGMENTS
We acknowledge the contributions of all scientists in each TSG syndrome and regret that we
could not report all relevant well-described studies due to space limitations.
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Mosaicism in Tumor Suppressor

Copyright © 2022 by Annual Reviews. This work is

332 Chen et al.

In this review, we considered a representative set of original and comprehensive peer-reviewed

www.annualreviews.org • Mosaicism in Tumor Suppressor Gene Syndromes 333

2. TUMOR SUPPRESSOR GENE SYNDROMES DEMONSTRATE

PIP3 PIP2 GRB2 GTP

PI3K SOS RAS

Cell survival, RHEB

(Caption appears on following page)

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Mosaic or fully Second somatic alteration

Mosaic or fully Second hit due to mitotic

Mosaic or fully Epigenetic/transcriptomic

336 Chen et al.

0 400 800 1,200 1,600 2,000 2,400 2,839

2.1. NF1: Neurofibromatosis Type 1

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2.2. NF2: Neurofibromatosis Type 2

11 Missense variant 18 Splice

169 Truncating variant

0 100 200 300 400 500 595

338 Chen et al.

0 200 400 600 800 1,000 1,164

2.3. TSC1/TSC2: Tuberous Sclerosis Complex

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0 400 800 1,200 1,600 1,807

2.4. PTEN: PTEN Hamartoma Tumor Syndrome/PTEN-opathies

340 Chen et al.

0 100 200 300 403

www.annualreviews.org • Mosaicism in Tumor Suppressor Gene Syndromes 341

2.5. VHL: Von Hippel–Lindau Syndrome

342 Chen et al.

0 200 400 600 800 928

www.annualreviews.org • Mosaicism in Tumor Suppressor Gene Syndromes 343

689 Missense variant 93 Splice

0 100 200 300 393

344 Chen et al.

3. IMPROVING RATES OF MOSAICISM DETECTION

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Success rate of PV Frequency of Persistent NMI Risk of disease

Success rate of PV Frequency of Persistent NMI Risk of disease

Mosaicism in Tumor Suppressor Gene Syndromes

4. MOSAIC GENETIC ALTERATIONS ARE FREQUENTLY UNCOVERED

348 Chen et al.

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350 Chen et al.

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352 Chen et al.

6. RISK OF DISEASE TRANSMISSION DEPENDS ON THE EXTENT

7. CLOSING REMARKS AND FUTURE PERSPECTIVES

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354 Chen et al.

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