Web viewWord count: 2354. ABSTRACT. ... Sklar J. Correlation of loss of heterozygosity at chromosome 9q with histological subtype in medulloblastomas. Am J Pathol. 1995;

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

mailto:[email protected]

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

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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.

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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

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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

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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)


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)


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)



2 (1)

11 c.1525G>C; p.GIy509Arg Probably damaging (Score: 0.999)

Damaging (Score: 0)

Class C65 (GV: 0.00 - GD: 125.13)



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


2 (1)

12 c.1660A>C, p.Ser554Arg Possibly damaging (Score: 0.817)

Damaging (Score: 0)

Class C65(GV: 0.00 - GD: 109.21)



8 (2)

15 c.2284G>A, p.Gly762Arg Probably damaging (Score: 0.958)


Class C65 (GV: 0.00 - GD: 125.13)



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)



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)



2 (1)

*Amino acid change seen once but different nucleotide change

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Documents

Web viewWord count: 2354. ABSTRACT. ... Sklar J. Correlation of loss of heterozygosity at chromosome 9q with histological subtype in medulloblastomas. Am J Pathol. 1995;