web viewword count: 2354. abstract. ... sklar j. correlation of loss of heterozygosity at chromosome...
TRANSCRIPT
First evidence of genotype-phenotype correlations in Gorlin syndrome
D. Gareth Evans,1,2,* Deemesh Oudit,3 Miriam J. Smith,1,2 David Rutkowski,1,4 Ernest
Allan,3 William G. Newman,1,2 John Lear4
1. Division of Evolution and Genomic Science, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
2. Manchester Centre for Genomic Medicine, St Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK, Manchester Academic Health Science Centre, Manchester, UK
3. Departments of Plastic Surgery, and Oncology Christie Hospital, Manchester M20 4BX UK
4. Department of Dermatology, MAHSC, Salford Royal Foundation Trust, Salford M6 8HD UK
* Correspondence:
Prof DG Evans
Manchester Centre for Genomic MedicineManchester Academic Health Sciences Centre (MAHSC),St Mary’s HospitalUniversity of ManchesterManchester M13 9WLTel: +44 (0)161 276 6506Fax: +44 (0)161 276 6145Email: [email protected]
Word count: 2354
1
ABSTRACT
Gorlin syndrome (GS) is an autosomal dominant syndrome characterised by multiple
basal cell carcinomas and an increased risk of jaw cysts and medulloblastoma in early
life. Heterozygous germline variants in Patch1 (PTCH1) and SUFU encoding
components of the Sonic Hedgehog pathway (SHH) explain the majority of cases. Here
we have undertaken a genotype-phenotype correlation of 182 individuals Median age
47.1 (IQR: 31.1-61.1) meeting diagnostic criteria for GS. A total of 126 patients had a
heterozygous pathogenic PTCH1 variant, 9 had SUFU pathogenic variants and 46 had
no identified mutation. Patients with PTCH1 variants were more likely to be diagnosed
earlier (p=0.02), have jaw cysts (p=0.002) and have bifid ribs (p=0.003) or any skeletal
abnormality (p=0.003) than patients with no identified mutation. Patients with a missense
variant in PTCH1 were diagnosed later (p=0.03) and were less likely to develop at least
10 BCCs and jaw cysts than those with other pathogenic PTCH1 variants (p=0.03).
Patients with SUFU pathogenic variants were significantly more likely than those with
PTCH1 pathogenic variants to develop a medulloblastoma (p=0.009), a meningioma
(p=0.02) or an ovarian fibroma (p=0.015), but were less likely to develop a jaw cyst
(p=0.0004). In summary, we propose that the clinical heterogeneity of GS can in part be
explained by the underlying PTCH1 or SUFU variant.
Key words: PTCH1, SUFU, Gorlin syndrome, medulloblastoma
INTRODUCTION
2
Gorlin syndrome (GS, MIM #109400), also known as nevoid basal cell carcinoma
syndrome (NBCCS) or basal cell nevus syndrome (BCNS), is a dominantly inherited
cancer-predisposition syndrome. Gorlin and Goltz described a syndrome that included
multiple basal cell carcinomas, jaw cysts and bifid ribs in 1960.[1] The birth incidence of
Gorlin syndrome is approximately 1 in 15,000 births with a prevalence of nearer 1 in
30,000.[2] Affected individuals may show multiple phenotypic abnormalities, with
characteristic facial features in over 50% of individuals that can include coarse facial
features, macrocephaly with frontal bossing, and hypertelorism.[3, 4] Diagnostic criteria
for GS have been previously proposed by several groups.[3, 5, 6, 7] Approximately 70 to
80% of individuals with GS have a first degree relative with the syndrome, and in 20 to
30% no family history is observed.[5] Full diagnostic criteria are shown in Table 1.[8]
GS patients are at risk of developing benign and malignant neoplasms. Multiple basal
cell (BCC) skin carcinomas are the hallmark feature most frequently occurring on sun
exposed areas such as the face, back and neck.[8] Men and women are equally
affected, and as of yet there has not been any clear PTCH1 genotype-phenotype
correlation for the timing or number of basal cell carcinomas that develop.[8] Cardiac
fibromas may develop in infants[5] and ovarian fibromas in adolescent girls and women.
[5] Importantly, approximately 5% of individuals with GS develop medulloblastoma.[5, 9]
Cases tend to present before 3 years of age, significantly younger than in sporadic
cases, predominantly of the desmoplastic subtype[10, 11], and are often the first
manifestation of GS.[9, 11, 12] In one review of 36 cases, 24 occurred aged ≤2 years of
age, with all but one (97%) of the remaining cases occurring in less than 6 years.[11] In
addition, patients who are survivors of medulloblastoma treated with therapeutic
radiation have a high risk of developing a large number of basal cell carcinomas (>1000)
in the radiation field.[13, 14]
3
Germline pathogenic variants in genes of the sonic hedgehog (SHH) signaling pathway,
including PTCH1, SUFU, and in two case reports, PTCH2, have been found in
individuals with GS[15, 16, 17, 18, 19, 20, 21] with PTCH1 variants being more common.
Despite variants in PTCH1 having been known as the cause of GS for more than 20
years no clear genotype-phenotype correlations have been described.
METHODS
GS patients fulfilling syndrome criteria (table 1) have been identified by the Manchester
Centre for Genomic Medicine since the early 1980s. Syndromic features including those
identified from a skeletal x-ray survey have been entered onto a bespoke Filemaker
database. Numbers of BCCs including those previously removed were assessed through
cutaneous examination and questionnaire. The majority of women had undergone a
single ovarian ultrasound to detect ovarian fibroma. Jaw cysts were ascertained by
orthopantogram screening, but the majority had presented symptomatically.
Affected individuals with GS (one from each family) were initially screened for germline
PTCH1 pathogenic variants in lymphocyte DNA by Sanger sequencing and multiple
ligation dependent probe amplification (MLPA). Variant negative families were also
screened for deep intronic pathogenic variants using RNA derived from cell lines.[22] All
negative families with available DNA then underwent Sanger sequencing and MLPA of
SUFU.[18]
Tests for significance were assessed by Chi square two sided tests with Fisher’s exact
correction.
4
RESULTS
Clinical details on 230 individuals from 94 families with GS were available. No DNA
sample was available for testing in 22 families. Of 182 patients who were genotyped (or
where it was concluded from family testing) ages ranged from 0.5-90 years (Median 47.1
IQR 31.1-61.1). PTCH1 pathogenic variants were identified in 43/72 families (60%)
containing 126 affected individuals. SUFU pathogenic variants were found in 9
individuals from 3 (4%) families and no pathogenic variant was identified in 26 families
(36%) containing 47 affected individuals. As such a causative variant was identified in
46/72 families (64%) and 135/182 (74%) individuals. In isolated, apparently de novo,
cases a pathogenic variant was found in 23/40 (57.5%) individuals. In contrast, a
pathogenic variant was identified in 23/32 (72%) of second generation familial cases.
Overall, a PTCH1 or SUFU pathogenic variant was more likely to be found in an
inherited than a de novo case (p=0.02). There were 50 people with truncating PTCH1
pathogenic variants, 26 with splicing variants (including one we have previously
described with a deep intronic pathogenic variant (c.2561-2057A>G)),[22] 16 with exonic
copy number variants detected on MLPA and 34 with missense PTCH1 pathogenic
variants. The SUFU pathogenic variants previously described include a large multiexonic
deletion.[18] The proportion with a number of clinical features including age at diagnosis
and age at last follow up are shown in Table 2. There was no significant difference in
age at last follow up, although those with missense variants had an older median age of
47.6 years. There were a number of clinical features that predicted the presence of a
PTCH1 pathogenic variant (table 1). Patients with identified PTCH1 variants were more
likely to be diagnosed earlier (median age 19 vs 36 years;p=0.0008), have developed
jaw cysts (62.7% vs 34.0%;p=0.002) and have bifid ribs (55.5% vs 34.2%;p=0.003) or
any skeletal abnormality (74.3% vs 51.2%;p=0.003) than patients with no identified
variant. Patients with missense variants in PTCH1 were diagnosed later (median 26
5
years; p=0.03) and less likely to have developed at least 10 BCCs and jaw cysts than
those with other PTCH1 variants (p=0.03) or to have developed at least 20 BCCs
(p=0.05). Those with missense variants were also less likely to have all other GS
features, including bifid ribs and jaw cysts, although this only reached statistical
significance for the presence of any congenital skeletal anomaly (including vertebral
defects) at 56.5% versus 76.5% for those with other PTCH1 gene variants (p=0.03).
There was no other identifiable difference between the phenotypes of individuals with
other PTCH1 variant types. All of the missense mutations were predicted to have some
effect on the protein and were shown to segregate with disease when multiple affected
family members were present. Three were also shown to have arisen de novo (Table 3).
None of the missense variants were reported in the ExAC database of around 121,200
alleles (http://exac.broadinstitute.org/gene/ENSG00000185920).
Patients with SUFU pathogenic variants were significantly more likely than those with
PTCH1 mutations to develop a medulloblastoma (33% vs 2.4%;p=0.009) (as previously
described)[18], a meningioma (22.2% vs 1.6%;p=0.02) or an ovarian fibroma (42.9% vs
5.9%;p=0.015), but were less likely to develop a jaw cyst (0% vs 62.7%;p=0.0004).
DISCUSSION
The present study has found a number of genotype-phenotype correlations in GS. A
recent review identified no such correlations[23] and we were not able to identify any
from a PubMed review-December 2016. There are a number of key clinical features of
GS that predict the presence of a PTCH1 pathogenic variant. These include the
presence of skeletal anomalies (especially bifid ribs) and jaw cysts. The number of
BCCs were not a predictor of the presence of a PTCH1 pathogenic variant as we have
previously shown.[8] It is of note nonetheless that a higher proportion of GS patients
6
without a pathogenic variant were sporadic (without a positive family history) and thus
some may be mosaic for the underlying mutation. Although this is an extremely common
mechanism in some other tumour prone syndromes such as neurofibromatosis type 2
(NF2),[24, 25] it has only reported once in GS.[26] In theory, mosaicism should be
relatively easy to prove with biopsy material potentially available from more than one
BCC, although until recently mutational analysis was difficult on formalin fixed material
and required fresh tissue. With Next Generation Sequencing mosaic mutations may be
found more frequently. As such mosaicism may still explain at least part of the difference
between those with and without PTCH1 pathogenic variants. It is possible that some
other sporadic patients may have fulfilled GS criteria by chance due to excess sun
exposure, although the need for at least two major criteria make this unlikely. It is
possible that our techniques have failed to identify a few patients with PTCH1
inactivation although this is unlikely given that RNA analysis was also performed. It is
therefore likely that further as yet unidentified gene(s), likely within the hedgehog
signaling pathway, account for most of the remaining unexplained cases.[22]
Although we were only able to identify nine patients with SUFU pathogenic variants, the
rates of medulloblastoma and meningioma were significantly higher than those for
individuals with PTCH1 pathogenic variants. A recent study of somatic mutations in
meningiomas found that 5 of 775 contained a somatic SUFU mutation, but none
contained a pathogenic PTCH1 mutation.[27]
A study of 131 childhood medulloblastoma cases identified germline SUFU variants in
eight cases.[10] Variants were identified in all three individuals with medulloblastoma
with extensive nodularity, 4 of 20 with desmoplastic/nodular medulloblastomas, and one
of 108 with other subtypes. The study had already excluded four previously reported
familial SUFU patients. The authors concluded that germline SUFU mutations (12 of
142; 8.5%) were more common than PTCH1 mutations (3 of 142; 2%) as a cause of
7
childhood medulloblastoma, although they had only assessed PTCH1 through features
of GS. A more recent study of 133 childhood medulloblastoma cases found germline
SUFU mutations in 6 of 133 (4.5%) compared to 2 of 133 (1.5%) with a PTCH1
mutation.[28] In contrast, somatic PTCH1 mutations are a more common cause of
childhood medulloblastoma than SUFU [28,29]. Indeed in their study of 133 Sonic
Hedgehog-related medulloblastomas, Kool et al found more PTCH1 mutations (60
cases), than in SMO (19 cases), or SUFU (10 cases) [28}. Definite disease-causing
truncating variants in SUFU are exceptionally rare: none are present on the ExAC
database (http://exac.broadinstitute.org/) of over 60,000 individuals. A frameshift variant
(p.Trp465LeufsTer6) was seen in 41 alleles; however, it was seen in homozygous form
once and occurs in the last exon, meaning that it is likely to escape nonsense mediated
decay and is unlikely to be pathogenic. In contrast, eight PTCH1 truncating variants
were present on the ExAC database. If one considers the 3-4 fold higher frequency of
germline SUFU mutations in a series of medulloblastoma and a possible 8-fold higher
frequency of germline PTCH1 in the general population, then one would expect a 24-32-
fold higher incidence of medulloblastoma in SUFU mutation carriers. Even taking into
account the 1 in 15,000 birth incidence estimate for GS, this would mean a 12-16-fold
higher risk of medulloblastoma, which is consistent with the difference between a 2%
risk in individuals with PTCH1 variants and 33% in individuals with SUFU variants in the
present report. Therefore, whilst there is still some doubt over the true risk of
medulloblastoma in SUFU-associated GS,[18] it is likely to be many times higher than
the risk for PTCH1-associated GS. Knowledge of a germline SUFU or PTCH1 mutation
in a child with medulloblastoma is extremely important as SUFU patients are resistant to
SMO inhibition.[28]
Although other reports have queried whether SUFU mutation carriers show clear
features of GS,[10] we have shown that all nine individuals in the present report met
8
diagnostic criteria with the presence of key features such as skeletal anomalies (57%)
ovarian fibromas (43%) and falx calcification (100%). It is also not clear whether the
carrier parents of a SUFU mutation found in the French report had full assessment
including a skeletal survey for GS.[29] Although the risk of BCC from a SUFU mutation
may be lower than that from a PTCH1 mutation, seven of nine (78%) patients with SUFU
variants in the current study had developed BCCs,[18] and two (22%) had developed
more than 20 BCCs, including one individual (n=45 BCCs) who had not undergone
radiotherapy. There are also reports of patients SUFU pathogenic variants with GS,[19,
30] and an individual with hereditary infundibulocystic BCC has also been reported with
a splicing mutation in SUFU.[31] Meningiomas also appear more commonly in
individuals with SUFU variants, although both in the current series had undergone
radiotherapy for medulloblastoma. Nonetheless, a germline SUFU mutation has been
shown to be the causative mutation in a family with familial meningioma,[32] as well as
one of the previously reported GS patients with a meningioma.[30] To date, jaw
keratocysts do not appear to be a feature of SUFU-associated GS with a highly
significant absence compared to PTCH1-associated GS (62.7%) in the present study
(p=0.0004).
In addition to the phenotypic differences between PTCH1- and SUFU-associated GS, we
have shown there are also phenotypic correlations between different PTCH1 germline
mutation types, with missense variants causing an aparently milder phenotype than
truncating variants, with fewer BCCs, later age at diagnosis, and fewer skeletal
anomalies. Indeed, whilst not significantly reduced, all other GS features were less
frequent. A number of other inherited tumour syndromes show correlations with
missense mutations, including von Hippel Lindau disease,[33] SMARCB1-associated
schwannomatosis[34] and NF2.[35, 36] In von Hippel Lindau, missense variants are
9
associated with later onset and lower risk of retinal angiomas and renal cell carcinoma,
but an increased risk of phaeochromocytoma.[33] In schwannomatosis and NF2,
missense variants cause a milder phenotype with later onset, and longer life expectancy
for NF2.[36] In addition, SMARCB1 missense variants are not asociated with
development of rhabdoid tumours unlike the majority of truncating mutations and large
rearrangements.[34] It is likely that many missense mutations although deleterious may
retain some function in the protein product and thus represent hypomorphic variants.[37]
There are some limitations to the current study. Not all patients underwent a skeletal
survey and if more had done so, further correlations could have been identified. We
cannot be certain that all the missense variants are disease-causing, although they all
segregated with disease in ascertained familes or were shown to have occurred de
novo. Although a missense change could have been a chance association, the
frequencies of these variants in sporadic and inherited GS identified a number of clear
cases that were identical to those with other PTCH1 mutations and quite different to the
ratio in cases with no identified mutation. We have not adjusted for multiple testing within
the same family statistically. Nonetheless, even though the missense variants did not
have significance below an adjusted p value of 0.01, the fact that frequencies were
below other PTCH1 mutations for all features means it is unlikely that these are chance
findings. It is also consistent with our clinical impression that these patients have a
milder phenotype. Finally, we did not screen PTCH2 in our cohort, so it is possible that
PTCH2 mutations may account for a small subset of patients in whom no pathogenic
variant was identified and may have their own phenotypic characteristics.
In summary, the present report has identified a number of clear genotype-phenotype
correlations that predict the presence of a germline PTCH1 or SUFU pathogenic variant.
10
The relatively small numbers of patients with each of the different classes of PTCH1
pathogenic variant means that more of these correlations may emerge in the future with
as they have for larger cohorts of patients with von Hippel Lindau syndrome[33] and
NF2.[36]
Contributorship statement
DGE planned the study, drafted the manuscript and is responsible for the overall content
of the study, MJS carried out in silico analysis and created table 3, DO, MJS, DR, EA,
WGN, and JL collated and analysed the data. All authors reviewed, edited and approved
the final manuscript.
Competing interests
All authors declare no competing interests.
11
REFERENCES
1 Gorlin RJ, Goltz RW. Multiple nevoid basal-cell epithelioma, jaw cysts and bifid rib. A syndrome. N Engl J Med 1960;262:908-12.
2 Evans DG, Howard E, Giblin C, Clancy T, Spencer H, Huson SM, Lalloo F. Birth incidence and prevalence of tumor-prone syndromes: estimates from a UK family genetic register service. Am J Med Genet A 2010;152A(2):327-32.
3 Kimonis VE, Goldstein AM, Pastakia B, Yang ML, Kase R, DiGiovanna JJ, Bale AE, Bale SJ. Clinical manifestations in 105 persons with nevoid basal cell carcinoma syndrome. Am J Med Genet 1997;69(3):299-308.
4 Shanley S, Ratcliffe J, Hockey A, Haan E, Oley C, Ravine D, Martin N, Wicking C, Chenevix-Trench G. Nevoid basal cell carcinoma syndrome: review of 118 affected individuals. Am J Med Genet 1994;50(3):282-90.
5 Evans DG, Ladusans EJ, Rimmer S, Burnell LD, Thakker N, Farndon PA. Complications of the naevoid basal cell carcinoma syndrome: results of a population based study. J Med Genet 1993;30(6):460-4.
6 Bree AF, Shah MR, Group BC. Consensus statement from the first international colloquium on basal cell nevus syndrome (BCNS). Am J Med Genet A 2011;155A(9):2091-7.
7 Kimonis VE, Mehta SG, Digiovanna JJ, Bale SJ, Pastakia B. Radiological features in 82 patients with nevoid basal cell carcinoma (NBCC or Gorlin) syndrome. Genet Med 2004;6(6):495-502.
8 Jones EA, Sajid MI, Shenton A, Evans DG. Basal cell carcinomas in gorlin syndrome: a review of 202 patients. J Skin Cancer 2011;2011:217378.
9 Evans DG, Farndon PA, Burnell LD, Gattamaneni HR, Birch JM. The incidence of Gorlin syndrome in 173 consecutive cases of medulloblastoma. Br J Cancer 1991;64(5):959-61.
10 Brugieres L, Remenieras A, Pierron G, Varlet P, Forget S, Byrde V, Bombled J, Puget S, Caron O, Dufour C, Delattre O, Bressac-de Paillerets B, Grill J. High frequency of germline SUFU mutations in children with desmoplastic/nodular medulloblastoma younger than 3 years of age. J Clin Oncol 2012;30(17):2087-93.
11 Amlashi SF, Riffaud L, Brassier G, Morandi X. Nevoid basal cell carcinoma syndrome: relation with desmoplastic medulloblastoma in infancy. A population-based study and review of the literature. Cancer 2003;98(3):618-24.
12 Schofield D, West DC, Anthony DC, Marshal R, Sklar J. Correlation of loss of heterozygosity at chromosome 9q with histological subtype in medulloblastomas. Am J Pathol 1995;146(2):472-80.
13 Strong LC. Genetic and environmental interactions. Cancer 1977;40(4 Suppl):1861-6.
14 Evans DG, Birch JM, Orton CI. Brain tumours and the occurrence of severe invasive basal cell carcinoma in first degree relatives with Gorlin syndrome. Br J Neurosurg 1991;5(6):643-6.
15 Farndon PA, Del Mastro RG, Evans DG, Kilpatrick MW. Location of gene for Gorlin syndrome. Lancet 1992;339(8793):581-2.
12
16 Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G, Wainwright B, Bale AE. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 1996;85(6):841-51.
17 Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein EH, Jr., Scott MP. Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 1996;272(5268):1668-71.
18 Smith MJ, Beetz C, Williams SG, Bhaskar SS, O'Sullivan J, Anderson B, Daly SB, Urquhart JE, Bholah Z, Oudit D, Cheesman E, Kelsey A, McCabe MG, Newman WG, Evans DG. Germline mutations in SUFU cause Gorlin syndrome-associated childhood medulloblastoma and redefine the risk associated with PTCH1 mutations. J Clin Oncol 2014;32(36):4155-61.
19 Pastorino L, Ghiorzo P, Nasti S, Battistuzzi L, Cusano R, Marzocchi C, Garre ML, Clementi M, Scarra GB. Identification of a SUFU germline mutation in a family with Gorlin syndrome. Am J Med Genet A 2009;149A(7):1539-43.
20 Fan Z, Li J, Du J, Zhang H, Shen Y, Wang CY, Wang S. A missense mutation in PTCH2 underlies dominantly inherited NBCCS in a Chinese family. J Med Genet 2008;45(5):303-8.
21 Fujii K, Ohashi H, Suzuki M, Hatsuse H, Shiohama T, Uchikawa H, Miyashita T. Frameshift mutation in the PTCH2 gene can cause nevoid basal cell carcinoma syndrome. Fam Cancer 2013;12(4):611-4.
22 Bholah Z, Smith MJ, Byers HJ, Miles EK, Evans DG, Newman WG. Intronic splicing mutations in PTCH1 cause Gorlin syndrome. Fam Cancer 2014;13(3):477-80.
23 Fujii K, Miyashita T. Gorlin syndrome (nevoid basal cell carcinoma syndrome): update and literature review. Pediatr Int 2014;56(5):667-74.
24 Evans DG, Ramsden RT, Shenton A, Gokhale C, Bowers NL, Huson SM, Pichert G, Wallace A. Mosaicism in neurofibromatosis type 2: an update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification. J Med Genet 2007;44(7):424-8.
25 Evans DG, Bowers N, Huson SM, Wallace A. Mutation type and position varies between mosaic and inherited NF2 and correlates with disease severity. Clin Genet 2013;83(6):594-5.
26 Torrelo A, Hernandez-Martin A, Bueno E, Colmenero I, Rivera I, Requena L, Happle R, Gonzalez-Sarmiento R. Molecular evidence of type 2 mosaicism in Gorlin syndrome. Br J Dermatol 2013;169(6):1342-5.
27 Clark VE, Harmanci AS, Bai H, Youngblood MW, Lee TI, Baranoski JF, Ercan-Sencicek AG, Abraham BJ, Weintraub AS, Hnisz D, Simon M, Krischek B, Erson-Omay EZ, Henegariu O, Carrion-Grant G, Mishra-Gorur K, Duran D, Goldmann JE, Schramm J, Goldbrunner R, Piepmeier JM,
13
Vortmeyer AO, Gunel JM, Bilguvar K, Yasuno K, Young RA, Gunel M. Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas. Nat Genet 2016;48(10):1253-9.
28 Kool M, Jones DT, Jager N, Northcott PA, Pugh TJ, Hovestadt V, Piro RM, Esparza LA, Markant SL, Remke M, Milde T, Bourdeaut F, Ryzhova M, Sturm D, Pfaff E, Stark S, Hutter S, Seker-Cin H, Johann P, Bender S, Schmidt C, Rausch T, Shih D, Reimand J, Sieber L, Wittmann A, Linke L, Witt H, Weber UD, Zapatka M, Konig R, Beroukhim R, Bergthold G, van Sluis P, Volckmann R, Koster J, Versteeg R, Schmidt S, Wolf S, Lawerenz C, Bartholomae CC, von Kalle C, Unterberg A, Herold-Mende C, Hofer S, Kulozik AE, von Deimling A, Scheurlen W, Felsberg J, Reifenberger G, Hasselblatt M, Crawford JR, Grant GA, Jabado N, Perry A, Cowdrey C, Croul S, Zadeh G, Korbel JO, Doz F, Delattre O, Bader GD, McCabe MG, Collins VP, Kieran MW, Cho YJ, Pomeroy SL, Witt O, Brors B, Taylor MD, Schuller U, Korshunov A, Eils R, Wechsler-Reya RJ, Lichter P, Pfister SM, Project IPT. Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell 2014;25(3):393-405.
29 Pugh TJ, Weeraratne SD, Archer TC, Pomeranz Krummel DA, Auclair D, Bochicchio J, Carneiro MO, Carter SL, Cibulskis K, Erlich RL, Greulich H, Lawrence MS, Lennon NJ, McKenna A, Meldrim J, Ramos AH, Ross MG, Russ C, Shefler E, Sivachenko A, Sogoloff B, Stojanov P, Tamayo P, Mesirov JP, Amani V, Teider N, Sengupta S, Francois JP, Northcott PA, Taylor MD, Yu F, Crabtree GR, Kautzman AG, Gabriel SB, Getz G, Jäger N, Jones DT, Lichter P, Pfister SM, Roberts TM, Meyerson M, Pomeroy SL, Cho YJ. Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations. Nature. 2012 Aug 2;488(7409):106-10
30. Bougeard G, Renaux-Petel M, Flaman JM, Charbonnier C, Fermey P, Belotti M, Gauthier-Villars M, Stoppa-Lyonnet D, Consolino E, Brugieres L, Caron O, Benusiglio PR, Bressac-de Paillerets B, Bonadona V, Bonaiti-Pellie C, Tinat J, Baert-Desurmont S, Frebourg T. Revisiting Li-Fraumeni Syndrome From TP53 Mutation Carriers. J Clin Oncol 2015;33(21):2345-52.
30 Kijima C, Miyashita T, Suzuki M, Oka H, Fujii K. Two cases of nevoid basal cell carcinoma syndrome associated with meningioma caused by a PTCH1 or SUFU germline mutation. Fam Cancer 2012;11(4):565-70.
31 Schulman JM, Oh DH, Sanborn JZ, Pincus L, McCalmont TH, Cho RJ. Multiple Hereditary Infundibulocystic Basal Cell Carcinoma Syndrome Associated With a Germline SUFU Mutation. JAMA Dermatol 2016;152(3):323-7.
32 Aavikko M, Li SP, Saarinen S, Alhopuro P, Kaasinen E, Morgunova E, Li Y, Vesanen K, Smith MJ, Evans DG, Poyhonen M, Kiuru A, Auvinen A, Aaltonen LA, Taipale J, Vahteristo P. Loss of SUFU function in familial multiple meningioma. Am J Hum Genet 2012;91(3):520-6.
14
33 Ong KR, Woodward ER, Killick P, Lim C, Macdonald F, Maher ER. Genotype-phenotype correlations in von Hippel-Lindau disease. Hum Mutat 2007;28(2):143-9.
34 Smith MJ, Wallace AJ, Bowers NL, Eaton H, Evans DG. SMARCB1 mutations in schwannomatosis and genotype correlations with rhabdoid tumors. Cancer Genet 2014;207(9):373-8.
35 Evans DG, Trueman L, Wallace A, Collins S, Strachan T. Genotype/phenotype correlations in type 2 neurofibromatosis (NF2): evidence for more severe disease associated with truncating mutations. J Med Genet 1998;35(6):450-5.
36 Hexter A, Jones A, Joe H, Heap L, Smith MJ, Wallace AJ, Halliday D, Parry A, Taylor A, Raymond L, Shaw A, Afridi S, Obholzer R, Axon P, King AT, English Specialist NFRG, Friedman JM, Evans DG. Clinical and molecular predictors of mortality in neurofibromatosis 2: a UK national analysis of 1192 patients. J Med Genet 2015.
37 Smith MJ, Walker JA, Shen Y, Stemmer-Rachamimov A, Gusella JF, Plotkin SR. Expression of SMARCB1 (INI1) mutations in familial schwannomatosis. Hum Mol Genet 2012;21(24):5239-45.
15
Table 1: Diagnostic criteria* as outlined in Jones et al.
MAJOR CRITERIALamellar or early (prior to age 20) calcification of the falxJaw keratocyst2 or more palmar or plantar pitsMultiple basal cell carcinomas (>5 in a lifetime) or one prior to age 30First degree relative with Gorlin syndromeMINOR CRITERIAMedulloblastoma in childhoodLymphomesenteric or pleural cystsMacrocephaly (head circumference >97th percentile)Cleft lip/palateVertebral/rib anomalies (e.g. bifid/splayed/extra ribs or bifid vertebra)Preaxial or postaxial polydactylyOvarian/cardiac fibromasOcular anomalies (cataract, developmental defects, pigment changes of retinal epithelium)
* Diagnosis made with 2 major or 1 major and 2 minor criteria fulfilled.
16
Table 2: Frequency of GS features in those with and without identified PTCH1 or SUFU mutations
Pathogenic variant
Pathogenic variant
not found
SUFU PTCH1 Missense PTCH1 variant
other PTCH1 variant
not tested Total PTCH1 vs no Pathogenic
variant
SUFU Vs PTCH1
Pathogenic variant
SUFU vs no mutation
missense vs other PTCH1
Number 47 9 126 34 92 50 232Female (%) 27
(57.4%)7
(77.8%) 68 (54%) 18 (53%) 50 (54.3%) 19 (38%)NS NS NS NS
Median age 36 34 19 26 16 27Mean age diagnosis 37.17 31.33 22.49 28.18 20.12
0.0008 NS NS 0.03
Range 2-81 3-76 0.3-63 1-61 0.3-63 0.2-88Median age last
follow up 47.2 42.4 45.1 47.6 42.4 42.1NS NS NS NS
Range 14.8-86 30-81 0.5-90 3.2-82 0.5-90 5.4-91Familial cases 24 7 88 24 64 35 154Single cases 23 2 38 10 28 15 78
% Single cases 48.94% 22.22% 30.16% 29.41% 30.43% 30.00% 33.62% 0.02 NS 0.14 NSJaw cysts 16 0 79 18 61 22 117
No jaw cyst (JC) 31 9 47 16 31 28 115% JC 34.04% 0.00% 62.70% 52.94% 66.30% 44.00% 50.43% 0.002 0.0004 0.05 NS
>10 BCC 21 4 61 12 49 20 106% 44.68% 44.44% 48.41% 35.29% 53.26% 40.00% 45.69% NS 0.1 NS NS
>10 BCC+JC 10 0 47 9 38 9 66% 21.28% 0.00% 37.30% 26.47% 41.30% 18.00% 28.45% 0.05 0.03 NS NS
≥20 BCC 12 2 35 5 30 9 51% 25.53% 22.22% 27.78% 14.71% 32.61% 18.00% 21.98% NS NS NS 0.05
Age >30 years >50 BCC 6 1 16 2 14 5 28
Age >30 years 38 6 92 23 69 31 167% 15.79% 16.67% 17.39% 8.70% 20.29% 16.13% 16.77% NS NS NS NS
Presence of palmar pits 32 5 83 19 64 26 146
All assessed for pits 55 9 109 26 83 38 211
% Pits 58.18% 55.56% 76.15% 73.08% 77.11% 68.42% 69.19% 0.05 NS NS NS
17
Meningioma 0 2 2 0 2 0 4% 0.00% 22.22% 1.59% 0.00% 2.17% 0.00% 1.72% NS 0.02 0.02 NS
Falx calcification 28 9 71 11 60 19 127No falx 14 0 22 7 15 9 45
% 66.67% 100.00% 76.34% 61.11% 80.00% 67.86% 73.84% 0.29 NS NS NSBifid ribs 14 2 56 10 46 15 87
No bifid ribs 27 7 45 13 32 10 89% 34.15% 22.22% 55.45% 43.48% 58.97% 60.00% 49.43% 0.003 NS NS NS
Any skeletal abnormality 21 4 75 13 62 19 119No skeletal abnormality 20 3 26 10 16 6 55
% 51.22% 57.14% 74.26% 56.52% 79.49% 76.00% 68.39% 0.003 NS NS 0.03Medulloblastoma 0 3 3 0 3 1 7
% 0.00% 33.33% 2.38% 0.00% 3.26% 2.00% 3.02% NS 0.009 0.007 NSOvarian fibroma 4 3 4 0 4 4 15
No ovarian fibroma 23 4 64 18 46 19 110
% 14.81% 42.86% 5.88% 0.00% 8.00% 17.39% 12.00% NS 0.015 NS NSCardiac fibroma 0 0 2 0 2 1 3
% Cardiac fibroma 0.00% 0.00% 1.59% 0.00% 2.17% 2.00% 1.29% ns Ns ns nsCleft lip/palate 1 0 6 0 6 0 7
% 2.13% 0.00% 4.76% 0.00% 6.52% 0.00% 3.02% ns Ns ns ns
NS-not significant
18
Table 3: In silico predictions for missense variants
Exon DNA and protein change PP2 SIFT AGVGD Mutation TasterFrequency in >60,000 people in ExAc
Classification of Pathogenicity
Segregation(no families)
2 c.242C>A, p.Ala81Glu Possibly damaging (Score: 0.951)
Tolerated (score: 0.14)
Class C65(GV: 0.00 - GD: 106.71)
Disease causing (p-value: 1) 0
Class 3- Unknownpathogenicity
3 affected (1)
2 c.296G>A, p.Gly99Asp Probably damaging (Score: 1.000)
Tolerated (score: 0.16)
Class C65(GV: 0.00 - GD: 93.77)
Disease causing (p-value: 1) 0*
Class 4- Likely pathogenic 1 (1)
2 c.296G>T, p.Gly99Asp Probably damaging (Score: 1.000)
Tolerated (score: 0.16)
Class C65(GV: 0.00 - GD: 93.77)
Disease causing (p-value: 1) 0*
Class 4- Likely pathogenic
1 de novo (1)
8 c.1195T>C, pTrp399Arg Probably Damaging (Score: 1.000)
Damaging (Score: 0)
Class C65(GV: 0.00 - GD: 101.29)
Disease causing (p-value: 1) 0
Class 3- Unknownpathogenicity
2 (1)
11 c.1525G>C; p.GIy509Arg Probably damaging (Score: 0.999)
Damaging (Score: 0)
Class C65 (GV: 0.00 - GD: 125.13)
Disease causing (p-value: 1) 0
Class 3- Unknownpathogenicity
12 (3)
11 c.1612G>A, p.Gly538Arg Probably Damaging(Score: 1.000)
Damaging(score: 0.04)
Class C65(GV: 0.00 - GD: 125.13)
Disease causing(p-value: 1) 0
Class 3- Unknownpathogenicity
2 (1)
12 c.1660A>C, p.Ser554Arg Possibly damaging (Score: 0.817)
Damaging (Score: 0)
Class C65(GV: 0.00 - GD: 109.21)
Disease causing (p-value: 1) 0
Class 3- Unknownpathogenicity
8 (2)
15 c.2284G>A, p.Gly762Arg Probably damaging (Score: 0.958)
Tolerated (score: 0.34)
Class C65 (GV: 0.00 - GD: 125.13)
Disease causing (p-value: 1) 0
Class 4- Likely pathogenic
1 de novo (1)
18 c.2963T>G, p.Val988Gly Probably damaging (Score: 0.996)
Damaging (score: 0.04)
Class C65(GV: 0.00 - GD: 109.55)
Disease causing (p-value: 1) 0
Class 4- Likely pathogenic
2 (1) first de novo
19 c.3236G>A, p.Gly1079Glu Probably damaging (Score: 1.000)
Damaging (Score: 0)
Class C65(GV: 0.00 - GD: 141.80)
Disease causing (p-value: 1) 0
Class 3- Unknownpathogenicity
2 (1)
*Amino acid change seen once but different nucleotide change
19