Germline mutations and genotype phenotype correlation in Asian … · Conclusion: Asian Indians with PCC/PGL differ from Western cohorts in having preponderance of VHL mutations in

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Published by Bioscientifica Ltd.DOI: 10.1530/EJE-16-0126www.eje-online.org © 2016 European Society of Endocrinology

175:4 311–323R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

175:4

10.1530/EJE-16-0126

Germline mutations and genotype–phenotype correlation in Asian Indian patients with pheochromocytoma and paragangliomaReshma Pandit1, Kranti Khadilkar1, Vijaya Sarathi2, Rajeev Kasaliwal3, Manjunath Goroshi1, Shruti Khare1, Sandhya Nair1, Vijaya Raghavan1, Abhay Dalvi4, Priya Hira5, Gwendolyn Fernandes6, Pragati Sathe6, Amey Rojekar6, Gaurav Malhotra7, Ganesh Bakshi8, Gagan Prakash8, Anil Bhansali9, Rama Walia9, Sadishkumar Kamalanathan10, Jayaprakash Sahoo10, Ankush Desai11, Nikhil Bhagwat12, Prashanth Mappa13, Rajesh Rajput14, Sudha Rao Chandrashekhar15, Vyankatesh Shivane1, Padma Menon1, Anurag Lila1, Tushar Bandgar1 and Nalini Shah1

1Department of Endocrinology, Seth G S Medical College and KEM Hospital, Mumbai, Maharashtra, India, 2Department of Endocrinology, Vydehi Institute of Medical Sciences and Research Centre, Bangalore, Karnataka, India, 3Department of Endocrinology, Mahatma Gandhi Hospital and Medical College, Jaipur, Rajasthan, India, 4Departments of General Surgery, 5Radiology and 6Pathology, Seth G S Medical College and KEM Hospital, Mumbai, Maharashtra, India, 7Radiation Medicine Centre, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India, 8Department of Uro-oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India, 9Department of Endocrinology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, India, 10Department of Endocrinology, Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER), Pondicherry, India, 11Endocrine Unit, Department of Medicine, Goa Medical College, Bambolim, Goa, India, 12Department of Endocrinology, Topiwala National Medical College & BYL Nair Charitable Hospital, Mumbai, Maharashtra, India, 13Department of Medicine, Kannur Medical College and Hospital, Kannur, Kerala, India, 14Department of Endocrinology, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India, and 15Division of Pediatric Endocrinology, Bai Jerbai Wadia Hospital for Children, Mumbai, Maharashtra, India

Abstract

Background: Genetic aetiology of pheochromocytoma (PCC) and paraganglioma (PGL) is increasingly being studied;

however, Asian Indian data on this aspect are scarce.

Objective: To study the prevalence of germline mutations and genotype–phenotype correlation in Asian Indian PCC/

PGL patients.

Design: In this study, 150 index patients (M:F, 73:77) with PCC/PGL were evaluated. Phenotypic data were collected.

Germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested by sequencing and NF1

was diagnosed according to phenotype.

Result: Of the total population, 49 (32.7%) PCC/PGL patients had germline mutations (VHL: 23 (15.3%), RET: 13

(8.7%), SDHB: 9 (6%), SDHD: 2 (1.3%) and NF1: 2 (1.3%)). Amongst the 30 patients with familial and/or syndromic

presentation, all had germline mutations (VHL: 14 (46.7%), RET: 13 (43.3%), SDHB: 1 (3.3%) and NF1: 2 (6.7%)). Out

of 120 patients with apparently sporadic presentation, 19 (15.8%) had a germline mutation (VHL: 9 (7.5%), SDHB:

8 (6.7%) and SDHD: 2 (1.7%)). Mutation carriers were younger (29.9 ± 14.5 years vs 36.8 ± 14.9; P = 0.01) and had a

higher prevalence of bilateral PCC (26.5% vs 2.9%, P < 0.001) and multifocal tumours (12.2% vs 0.96%, P = 0.06). Based

on syndromic features, metastasis, location and number of tumours, around 96% mutations in our cohort could be

detected by appropriately selected single gene testing.

Conclusion: Asian Indians with PCC/PGL differ from Western cohorts in having preponderance of VHL mutations in

multifocal tumours and apparently sporadic unilateral PCC. Syndromic presentation, metastasis, location and number

of PCC/PGL can be effectively used for guiding genetic prioritisation.

Clinical Study

Correspondence should be addressed to R Pandit Email [email protected]

European Journal of Endocrinology (2016) 175, 311–323

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in Asian Indians

Introduction

Pheochromocytoma (PCC) and paraganglioma (PGL) are tumours derived from neural crest cells located in the adrenal medulla and sympathetic/parasympathetic ganglia respectively (1). PCC and sympathetic PGL (sPGL) generally secrete catecholamines responsible for classic symptoms, whereas head and neck PGL (HNPGL) that arise from parasympathetic ganglia are usually non-secretory (1).

Since 1990, germline mutations have been reported in 16 PCC/PGL susceptibility genes: NF1 (2), RET (3), VHL (4), SDHD (5), SDHC (6), SDHB (7), TMEM127 (8), MAX (9), SDHAF2 (10), SDHA (11), FH (12), EGLN1 (13), EGLN2 (14), MDH2 (15), KIF1BB (16) and EPAS1 (17). Germline mutations in genes such as VHL, RET and NF1 have been characterised by typical syndromic components, making their genetic diagnosis easier. The prevalence of germline mutations in PCC/PGL patients with familial and/or syndromic (FS) presentation has been high as expected; however, the genetic yield in patients with apparently sporadic (AS) presentation has always attracted attention. In 2002, Neumann et  al. reported germline mutations (VHL, RET, SDHB and SDHD) in 66 (24%) of 271 PCC/PGL patients with AS presentation, thus ending the 10% rule of hereditary PCC (18). Following this initial report from Germany, analysis of these genes in large PCC/PGL cohorts from other parts of the world also reported a similar yield (19, 20, 21, 22). A recent systematic review reported a germline mutation rate of 11.6% in 5031 PCC/PGL patients with AS presentation (23), ultimately paving the way for the Endocrine Society recommendation of genetic testing of all patients with PCC/PGL (24).

The pursuit of studying genetic aetiology is important for understanding the pathophysiology of the disorder, investigating targeted therapy and genetic counselling. Due to the implications of multiple genes and the cost and labour involved in Sanger sequencing, a clinical feature-driven diagnostic algorithm should be established to guide the prioritisation of genetic testing (24).

Genetic aetiology of PCC/PGL is increasingly being studied; however, Indian data on genotype–phenotype correlation are scarce. Our objective is to study the prevalence of germline mutations in the major susceptibility genes (VHL, RET, SDHB, SDHD, SDHC and NF1) and genotype–phenotype correlation in Asian Indian patients with PCC/PGL.

Subjects and methods

A total of 150 unrelated index patients with PCC/PGL (functional and non-functional) attending the endocrine clinics of eight different centres (120 from Seth G.S. Medical College and K.E.M. Hospital, Mumbai, and 30 from collaborating centres) from India were included in the study. Institutional ethics committee approved the study, and a written informed consent was obtained from all participants. Diagnosis of PCC/PGL was based on surgical histology in 136 patients. In patients for whom surgical histology was not available, the diagnosis was based on plasma free metanephrines, functional imaging (131I-metaiodobenzylguanidine (MIBG)) and anatomical imaging (Computed tomography (CT) in PCC and sPGL, magnetic resonance imaging (MRI) in HNPGL).

A detailed personal and family history was elicited in all patients for familial/syndromic association. Patients were evaluated for catecholamine excess status with 24-h urinary vanillylmandelic acid and/or plasma-free meta-nephrines. Twenty four hour urinary vanillylmandelic acid > 10 mg, plasma-free normetanephrine > 180 pg/mL or plasma-free metanephrine > 90 pg/mL were considered as elevated and tumours were considered as hormonally functional. Tumours with normal biochemistry were considered as non-functional. Tumours with isolated elevation of plasma normetanephrine were considered as normetanephrine secreting, whereas those with elevation of plasma metanephrine with or without elevation of plasma normetanephrine were considered as metanephrine secreting. Patients underwent anatomical imaging (computed tomography of neck to pelvis) and functional imaging (131I-metaiodobenzylguanidine). Head and neck PGL patients were also subjected to MRI of head and neck. Computed tomography of the abdomen was examined for components of VHL (renal cysts and renal cell carcinoma, pancreatic cysts and pancreatic neuroendocrine tumours). Clinical diagnosis of neurofibromatosis 1 (NF1) was based on the established clinical criteria (25).

Familial/syndromic (FS) group was characterised by the presence of stigmata of known syndrome in the index case/family members, and/or history of PCC/PGL in family members. Absence of FS characteristics was considered as apparently sporadic (AS). Tumours were classified into mutually exclusive groups as PCC: unilateral/bilateral, sPGL: single/multiple, HNPGL: unilateral/bilateral and multifocal disease (coexistence of PCC and

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175:4 313Clinical Study R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

PGL). Metastatic disease was defined as the presence of lesions outside the adrenal glands and sympathetic and parasympathetic chain.

Genotype analysis

Genomic DNA was extracted from the peripheral blood using alcohol precipitation method. All patients were screened for pathogenic variations in five genes by Sanger sequencing: VHL (OMIM*608537, ENSG00000134086), SDHB (OMIM*185470, ENSG00000117118), SDHD (OMIM*602690, ENSG00000204370), SDHC (OMIM*602413, ENSG00000143252) and exons 10, 11, 13, 14, 15 and 16 of RET proto-oncogene (OMIM+164761, ENSG00000165731). Patients with wild-type RET, VHL, SDHB, SDHD and SDHC were subsequently reanalysed for genomic rearrangements involving VHL and SDHx genes using multiplex ligation-dependent probe amplification (MLPA) assay. Genotyping for the NF1 gene was not performed, and diagnosis was based on established clinical criteria alone (25). PCR primers were designed using Primer3Plus software (26) to cover exons and intron–exon boundaries. PCR purification and Sanger sequencing were outsourced to a commercial service provider (Applied Biosystems 3730 Genetic Analyser and BigDye Terminator v3.1 cycle sequencing kit) and the results were analysed at our department. Variations were identified by scanning electropherograms using ABI Sequence scanner version 1.0 and evaluated for their pathogenic potential using Mutation Taster software (27), Polymorphism Phenotyping v2 (PolyPhen-2) (28) and

Sorting Intolerant from Tolerant (SIFT) (29). Variations identified as pathogenic were confirmed by resequencing. These mutations were referenced in databases such as Human Gene Mutation Database (HGMD) (30), the TCA Cycle Gene Mutation Database (31), the VHL mutation database (32), Multiple Endocrine Neoplasia type 2 RET proto-oncogene database (33) and literature. Additionally, they were checked in our control population of 150 clinically healthy individuals and Exome Aggregation Consortium (ExAC, consisting of 8256 South Asians; allele frequency <0.01) (URL:http://exac.broadinstitute.org; accessed on December 2015). MLPA was carried out as per the manufacturer’s instructions (SALSA MLPA P016 VHL probemix and SALSA MLPA P226 SDH probemix, MRC-Holland, Amsterdam, The Netherlands).

Statistical analysis

Data are reported as mean ± s.d., actual numbers and percentages. Several statistical methods were used, both parametric (ANOVA, t-tests) and non-parametric (Fisher’s exact t-test and Kruskal–Wallis test). After ANOVA, post hoc comparisons of two groups were performed by the KSD procedure in SPSS version 21.

Results

Phenotype

Overall, 150 unrelated index patients were enrolled in the study (77 females and 73 males). The mean age at presentation was 34.5 ± 14.8 years (age range, 6–75 years).

Table 1 Clinical characteristics of patients based on the mode of presentation.

Characteristic Total: 150Familial and/or syndromic

presentation: 30 (20%)Apparently sporadic

presentation: 120 (80%) P value

Age at presentation (mean ± s.d.) (years) 34.5 ± 14.8 31.1 ± 14.3 35.4 ± 14.9 0.15Range 6–75 6–58 8–75M:F 73:77 11:19 62:58 0.14Tumour size (mean ± s.d.) (cm) 6.0 ± 2.4 5.2 ± 2.4 6.2 ± 2.4 0.013Range (cm) 2.0–22 3–8 2–22Total PCC 103 24 (80%) 79 (65.8%) 0.13Unilateral PCC 87 13 (43.3%) 74 (61.7%) 0.09Bilateral PCC 16 11 (36.7%) 5 (4.2%) <0.001Total sPGL 30 1 (3.3%) 29 (24.2%) 0.001Single sPGL 26 1 (3.3%) 25 (20.8%) 0.028Multiple sPGL 4 0 4 (3.3%) 0.584Total HNPGL 10 0 10 (8.3%) 0.213Unilateral HNPGL 8 0 8 (6.7%) 0.15Bilateral HNPGL 2 0 2 (1.7%) 0.48Multifocal PCC/PGL 7 5 (16.7%) 2 (1.7%) 0.004Malignancy 16 1 (3.3%) 15 (12.5%) 0.196

HNPGL, head and neck paraganglioma; PCC, pheochromocytoma; sPGL, sympathetic paraganglioma.

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Tab

le 2

C

linic

al a

nd

gen

etic

ch

arac

teri

stic

s o

f m

uta

tio

n-p

osi

tive

pat

ien

ts.

Case

Sex/a

ge

FS/A

STu

mo

ur

typ

eG

en

eExo

nN

ucl

eo

tid

e c

han

ge

Am

ino

aci

d c

han

ge

Typ

eM

uta

tio

n

tast

er

Po

lyPh

en

-2SIF

TEX

aC

all

ele

fr

eq

uen

cy

EX

aC

all

ele

fr

eq

uen

cy

in S

ou

th

Asi

an

p

op

ula

tio

n

1F/

27A

SU

nila

tera

l PC

CSD

HB

4c.

292T

>A

p

.(C

ys98

Ser)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Dam

ag-

ing

NR

NR

2F/

38A

SSi

ng

le s

PGL

SDH

B7

c.68

9G>

Ap

.(A

rg23

0His

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 0

.991

Dam

ag-

ing

NR

NR

3F/

19A

SSi

ng

le s

PGL

SDH

B7

c.68

9G>

Tp

.(A

rg23

0Leu

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

.000

Dam

ag-

ing

NR

NR

4F/

27A

SSi

ng

le s

PGL

SDH

B4

c.33

8G>

C (

No

vel)

p.(

Cys

113S

er)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Dam

ag-

ing

NR

NR

5M

/27

AS

Sin

gle

sPG

L (m

alig

nan

t)SD

HB

3c.

251A

>C

(N

ove

l)p

.(A

sp84

Ala

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

.000

Dam

ag-

ing

NR

NR

6M

/19

FSSi

ng

le s

PGL

SDH

B3

c.22

7T>

G (

No

vel)

p.(

Cys

93G

ly)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Dam

ag-

ing

NR

NR

7M

/13

AS

Sin

gle

sPG

LSD

HB

2c.

136C

>T

p.(

Arg

46Te

r)Su

bst

itu

tio

n

(No

nse

nse

)D

isea

se

cau

sin

gN

AD

amag

-in

g d

ue

to s

top

0.00

0016

48

(0.0

001)

0

8M

/23

AS

Mu

ltip

le s

PGL

SDH

B2

c.13

1_13

9del

TCTA

TCG

AT

(No

vel)

p.(

ILe4

4Arg

fs*2

36)

Del

etio

nN

AN

AN

AN

RN

R

9M

/36

AS

Sin

gle

sPG

L (m

alig

nan

t)SD

HB

IVS3

c.28

6+1

G>

ASp

lice

Site

Ch

ang

eSp

lice

Site

ch

ang

eD

isea

se

cau

sin

gN

AN

AN

RN

R

10M

/18

AS

Un

ilate

ral P

CC

SDH

D4

c.38

6_38

6del

T (N

ove

l)p

.(Le

u12

9Trp

fs*6

)D

elet

ion

Dis

ease

ca

usi

ng

NA

NA

NR

NR

11M

/22

AS

B/L

HN

PGL

SDH

D4

c.33

7_34

0del

GA

CT

p.(

Asp

113M

etfs

X21

)D

elet

ion

Dis

ease

ca

usi

ng

NA

NA

NR

NR

12F/

20FS

B/L

PC

Cp

ancr

eati

c cy

sts

VH

L3

c.58

8_58

8du

pA

(N

ove

l)p

.(A

sp19

7Arg

insf

s*?)

Du

plic

atio

nPr

o-

lon

ged

p

rote

in

du

e to

lo

ss o

f o

rig

inal

st

op

co

do

n

NA

NA

NR

NR

13F/

13A

SB

/L P

CC

V

HL

3c.

500G

>A

p.(

Arg

167G

ln)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Dam

ag-

ing

NR

NR

14F/

11A

SB

/L P

CC

+sP

GL

VH

L3

c.50

0G>

Ap

.(A

rg16

7Gln

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

.000

Dam

ag-

ing

NR

NR

15M

/12

FSU

nila

tera

l PC

C+

sPG

L R

enal

an

d p

ancr

e-at

ic c

ysts

Fa

mily

his

tory

+

VH

L1

c.29

3A>

C (

No

vel)

p.(

Tyr9

8Ser

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

Dam

ag-

ing

NR

NR

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175:4 315Clinical Study R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

16F/

20FS

B/L

PC

C

Fam

ily h

isto

ry+

VH

L3

c.48

2G>

Ap

.(A

rg16

1Gln

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

.000

Tole

ran

tN

RN

R

17F/

6FS

Un

ilate

ral P

CC

Fa

mily

his

tory

+V

HL

3c.

482G

>A

p.(

Arg

161G

ln)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Tole

ran

tN

RN

R

18M

/34

AS

B/L

PC

CV

HL

3c.

482G

>A

p.(

Arg

161G

ln)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPr

ob

ably

d

amag

ing

sc

ore

1.0

00

Tole

ran

tN

RN

R

19F/

22FS

B/L

PC

C+

sPG

L

Fam

ily h

isto

ry

PCC

/PG

L

VH

L3

c.59

3T>

Cp

.(Le

u19

8Pro

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

.000

Dam

ag-

ing

NR

NR

20M

/21

FSU

nila

tera

l PC

C

Ret

inal

an

gio

mas

C

NS

hae

man

gio

bla

s-to

mas

, pan

crea

tic

cyst

s Fa

mily

his

tory

+

VH

L3

c.47

9A>

T (N

ove

l)p

.(G

lu16

0Val

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 0

.994

Dam

ag-

ing

NR

NR

21F/

35A

SU

nila

tera

l PC

CV

HL

3c.

548C

>G

(N

ove

l)p

.(Se

r183

Trp

)Su

bst

itu

tio

n

(mis

sen

se)

Dis

ease

ca

usi

ng

Pro

bab

ly

dam

agin

g

sco

re 1

Dam

ag-

ing

NR

NR

22F/

29FS

Un

ilate

ral P

CC

C

NS

hae

man

gio

bla

s-to

ma,

ren

al a

nd

p

ancr

eati

c cy

sts

VH

L1

c.22

4_22

6del

TCT

p.(

Phe7

6del

)D

elet

ion

Dis

ease

ca

usi

ng

NA

NA

NR

NR

23F/

45A

SU

nila

tera

l PC

CV

HL

1c.

224_

226d

elTC

Tp

.(Ph

e76d

el)

Del

etio

nD

isea

se

cau

sin

gN

AN

AN

RN

R

24F/

20FS

Un

ilate

ral P

CC

R

enal

an

d p

ancr

e-at

ic c

ysts

VH

L1

Het

ero

zyg

ou

s d

elet

ion

o

f ex

on

1N

AN

AN

AN

AN

AN

AN

A

25M

/28

AS

Un

ilate

ral P

CC

VH

L3

c.50

9T>

C (

No

vel)

p.(

Val

170A

la)

Sub

stit

uti

on

(m

isse

nse

)D

isea

se

cau

sin

gPo

ssib

ly

Dam

agin

g

sco

re 0

.953

Dam

ag-

ing

NR

NR

26F/

18FS

B/L

PC

C+

sPG

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y175:4 316Clinical Study R Pandit and others Genetic aetiology of PCC/PGL

in Asian Indians

Case

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R

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on

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isse

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)N

R

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isse

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se

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gle

ton

)N

R

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nila

tera

l PC

C

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ys63

4Tyr

)Su

bst

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tio

n

(mis

sen

se)

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ease

ca

usi

ng

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bab

ly

dam

agin

g

sco

re 1

Dam

ag-

ing

NR

NR

47M

/50

FSU

nila

tera

l PC

C

MTC

+

Fam

ily h

isto

ry +

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11c.

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p.(

Cys

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rg)

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stit

uti

on

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isse

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se

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Tab

le 2

(C

on

tin

ued

).

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175:4 317Clinical Study R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

PCC was present in 103 (68.7%) of the 150 patients, of which 16 (15.5%) had bilateral PCC and 87 (84.5%) had unilateral PCC; 30 (20%) had sPGL, of which four had multiple sPGL; and ten (6.7%) had HNPGLs, of which two were bilateral. Multifocal disease was present in seven (4.7%) patients, and all of these had PCC with sPGL. Malignancy was observed in 16 (10.7%) patients.

Out of the 150 patients, 30 (20%) had FS presentation and 120 (80%) had AS presentation. Out of 30 patients with FS, 13 were clinically diagnosed as VHL syndrome, ten as MEN2A, three as MEN2B, two as NF1 and two as familial PCC/PGL syndrome. Amongst the 13 VHL patients, four had syndromic components without family history, four had a family history of syndromic components and the remaining five had both. FS group had a greater proportion of bilateral PCC (36.7% vs 4.2%, P < 0.001) and multifocal disease (16.7% vs 1.7%, P = 0.004), whereas AS group had a greater proportion of sPGL (24.2% vs 3.3 %, P = 0.001). Mean tumour size was smaller (5.2 ± 2.4 cm vs 6.2 ± 2.4 cm, P = 0.013) in FS group compared with AS group. Table 1 summarises the baseline clinical characteristics of patients based on the mode of presentation.

Genotype

Forty-nine (32.7%) PCC/PGL patients had germline mutations (VHL: 23 (15.3%), RET: 13 (8.7%), SDHB: 9 (6%), SDHD: 2 (1.3%), NF1: 2 (1.3%)). None had mutations in SDHC. The mutation yield in FS group was 100% (30/30) with mutations in VHL (14/30, 46.7%), RET (13/30, 43.3%), NF1 (2/30, 6.7%) and SDHB (1/30; 3.3%). The yield for AS group was 15.8% (19/120) with mutations in VHL (9/120; 7.5%), SDHB (8/120; 6.7%) and SDHD (2/120; 1.7%).

VHL mutations

In 23 patients, 15 different VHL mutations (missense mutations (10), splice site mutation (1), deletion (1), duplication (1) and large deletions (2)) were identified, of which five were novel (Table  2). The mean age of presentation was 25.9 ± 14.6 (range: 6–55) years, with 14 patients (60.8%) having FS presentation and nine (39.1%) having AS presentation. Plasma-free metanephrines were available for 14 patients and all had isolated elevation of plasma normetanephrine. Of 23 patients, 18 (78.3%) had PCC (bilateral: 6, unilateral: 12) and five (21.7%) had multifocal disease (PCC with sPGL). The mean tumour

size was 5.3 ± 2.5 (range: 2–8.7) cm. There was no evidence of malignant disease.

RET mutations

There were 13 patients with RET mutations of which ten had mutations in codon 634 (MEN 2A) and three had mutations in codon 918 (MEN 2B). The mean age of presentation was 40.4 ± 14.6 (range: 22–58) years with all patients having past or present evidence of medullary thyroid carcinoma. Cutaneous lichen amyloidosis and primary hyperparathyroidism were observed in three and two patients with MEN2A respectively. All patients with MEN2B had characteristic phenotypic features including Marfanoid habitus, mucosal neuromas and fleshy lips. The mean tumour size was 5.5 ± 2.5 (range: 4–7.2) cm. Plasma-free metanephrines were available for eight patients and all had elevation of both metanephrine and normetanephrine. Twelve (92.3%) patients had PCC, with bilateral PCC in seven. One MEN2A patient had a multifocal (bilateral PCC with an abdominal sPGL) malignant disease, which was unusual for RET phenotype. Apart from RET, we performed PCR for VHL, SDHB, SDHC, SDHD as well as MLPA for VHL and SDHx in this patient and could not find a second mutation.

SDHB mutations

In nine patients, nine different SDHB mutations (missense mutations (7), splice site mutation (1) and deletion (1)) were identified, of which four were novel (Table  2). The mean age of presentation was 25.4 ± 8.1 (range: 13–38) years, with a majority of patients (8/9, 88.9%) having AS presentation. The mean tumour size was 5.9 ± 1.9 (range: 3–9) cm. Plasma-free metanephrines were available for five patients, and all had isolated elevation of plasma normetanephrine. Tumours were mostly sPGL (8/9, 88.9%), with one patient having two abdominal sPGLs. None of the patients had multifocal disease, and only one had unilateral PCC. Of nine patients, two (22.2%) had an evidence of metastatic disease. Amongst the 16 patients with malignant tumours, three (18.8%) harboured a germline mutation of which two were in SDHB.

SDHD mutations

SDHD mutations were identified in two patients; both were deletions (one novel). The mean age of presentation was 20 ± 2.8 years, with both having AS presentation.

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in Asian Indians

Of the two patients, one had a unilateral PCC, whereas the other had bilateral HNPGL. There was no evidence of multifocality or malignancy.

NF1 patients

The diagnosis of NF1 was based only on established clinical criteria without performing genetic analysis of NF1 gene. Both patients with NF1 had classical cutaneous lesions (café au lait pigmented spots and neurofibromas). The average age at presentation of the two patients was 37.5 ± 10.6 years and both had benign unilateral PCC.

Mutation-negative group

Mutation-negative group included 101 patients with all patients having an AS presentation. The mean age at presentation was 36.8 ± 14.8 (range: 8–75) years. Plasma-free metanephrines were available for 74 patients, of which 40 were normetanephrine secreting, 23 were metanephrine secreting and the rest were non-secretory; 69 (68.3%) patients had PCC (bilateral: 3, unilateral: 66), 22 (21.8%) had sPGL (multiple: 3, single: 19), nine (8.9%) had HNPGL (bilateral: 1) and one (1%) had multifocal disease. The mean tumour size was 6.2 ± 2.4 (range: 2.3–22) cm; 13 (12.9%) patients had metastatic disease.

Genotype–phenotype correlation

Patients with VHL and SDHB mutations were younger than those with RET mutations (P = 0.004, P = 0.017 respectively) and those in the mutation-negative group (P < 0.001, P = 0.025 respectively). FS presentation was more common in patients with RET compared with VHL (P < 0.001), SDHB (P < 0.001) and mutation-negative group (P < 0.001). Mutation-negative group had a significantly greater proportion of unilateral PCC compared with RET (P = 0.024) and SDHB (P = 0.002) mutation groups. Patients with mutations had a greater proportion of bilateral PCC (26.5% vs 3%, P < 0.001) compared with no mutation group. Amongst the mutation-positive group, patients with RET mutations had a significantly greater proportion of bilateral PCC than those with VHL mutations (P = 0.004) and SDHB mutations (P < 0.001). Patients with mutations had a greater proportion of multifocal (12.3% vs 1%, P = 0.06) compared with no mutation group. Amongst the mutation-positive group, multifocal tumours were significantly more common in patients with VHL mutations than patients with RET

mutations (P = 0.046) and SDHB mutations (P = 0.007). sPGLs were frequent in patients with SDHB mutations compared with those with VHL mutations (P < 0.001), RET mutations (P < 0.001) and no mutation group (P < 0.001) (Table 3).

Discussion

In this largest Asian Indian PCC/PGL cohort, we report germline mutations in 32.7% of the total patients, which is comparable with the reported cohorts (27.4–39.2%) as shown in Table 4 (18, 19, 20, 22). The genetic yield in patients with FS presentation was high (100%) as reported from previous studies (92.7–100%) (19, 20, 22). VHL and RET constituted 90% of the mutations in the FS group. Similar preponderance of VHL and RET genes (55.4–87.1%) has been reported in FS group in previous large PCC/PGL cohorts (18, 19, 22). The genetic yield in patients with AS presentation (15.8%) was similar to that in previous studies (11.6–17.2%) (18, 19, 20, 22). The most common mutations in AS group were in VHL and SDHB (89.5%). Similar preponderance of VHL and SDHB genes (77.1–90%) has been reported in the literature (18, 19, 20).

Our patients with hereditary disease were younger than those with no mutations. Association of hereditary PCC/PGL with younger age is a universal finding in all previous studies (18, 19, 20, 21, 22). Initial studies rather suggested restriction of genetic testing to younger patients. In our cohort, the genetic yield in patients with AS presentation older than 46 years was 4% (1/25) and nil in ten patients older than 55 years. Hence, we may consider restricting genetic analysis to PCC/PGL patients younger than 46 years.

Apart from FS presentation, bilateral PCC had the strongest association with the presence of a germline mutation. This has been depicted across almost all major studies with 56.3–87.8% patients with bilateral PCC having germline mutations (19, 21, 22). VHL and RET mutations account for most of the mutations (77.7–100%) in bilateral PCC, making the genetic prioritisation easier based on typical syndromic presentation. In addition to bilateral PCC, multifocal tumours were also more common in hereditary disease. Interestingly, our patients with multifocal disease (PCC and sPGL) had mutations in VHL (n = 5) and RET (n = 1) but not in SDHB or SDHD. This is in contrast to reports from previous large cohorts where SDHB and SDHD mutations are equally or more commonly reported in patients with coexisting PCC and sPGL (19, 22).

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175:4 319Clinical Study R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

Tab

le 3

G

eno

typ

e–p

hen

oty

pe

corr

elat

ion

in p

atie

nts

wit

h P

CC

/PG

L.

Ch

ara

cteri

stic

sVHL

RET

SDHB

SDHD

NF1

Here

dit

ary

dis

ease

No

mu

tati

on

P v

alu

e$

P v

alu

e

(her

edit

ary

dis

ease

vs

no

mu

tati

on

)

Tota

l23

139

22

4910

1<

0.00

1<

0.00

1FS

:AS;

30:

120

14:9

13:0

1:8

0:2

2:0

30:1

90:

101

M:F

(73

:77)

9:14

6:7

5:4

2:0

0:2

22:2

751

:50

0.29

10.

422

Mea

n a

ge

at p

rese

nta

tio

n

(34.

5 ±

 14.

8 ye

ars)

25.9

 ± 1

4.6

40.4

 ± 1

4.6

25.4

 ± 8

.120

 ± 2

.837

.5 ±

 10.

629

.9 ±

 14.

536

.8 ±

 14.

80.

006*

0.01

Ran

ge:

(6–

75)

6–55

22–5

813

–38

6–58

8–75

Mea

n s

ize

(6.0

 ± 2

.4 cm

)5.

3 ±

 2.5

5.5 

± 2

.55.

9 ±

 1.9

5.5

5.5 

± 0

.75.

5 ±

 2.4

6.2 

± 2

.40.

602

0.07

3R

ang

e: 2

–22 

cm2–

8.7

4–7.

23–

92–

92.

3–22

Met

anep

hri

nes

(n

 = 1

03)

NM

N/M

N/N

S14

/0/0

0/8/

05/

0/0

1/0/

10/

0/0

20/8

/140

/23/

11

Un

ilate

ral P

CC

(n

 = 8

7)12

(52

.2%

)5

(38.

5%)

1 (1

1.1%

)1

(50%

)2

(100

%)

21 (

42.9

%)

66 (

65.3

%)

0.01

4†0.

014

FS:A

S 13

:74

6:6

5:0

0:1

0:1

2:0

13:8

0:66

Bila

tera

l PC

C (

n =

 16)

6 (2

6.1%

)7

(53.

8%)

00

013

(26

.5%

)3

(3%

)<

0.00

1‡<

0.00

1FS

:AS

11:5

4:2

7:0

11:2

0:3

Sin

gle

sPG

L (n

 = 2

6)0

07

(77.

8%)

00

7 (1

4.3%

)19

(18

.8%

)<

0.00

1§<

0.00

1FS

:AS

1:25

1:6

1:6

0:19

Mu

ltip

le s

PGL

(n =

 4)

00

1 (1

1.1%

)0

01

(2.0

%)

3 (3

%)

0.61

80.

16FS

:AS

0:4

0:1

0:1

0:3

Tota

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Amongst patients with unilateral PCC, 24.1% of patients had mutations, VHL being the most (12/21, 57.1%) affected gene. In previous studies, VHL, RET and NF1 are the commonly affected genes in patients with unilateral PCC. In patients with sPGL, 26.7% of patients had germline mutations and all were in SDHB gene, which is in accordance with the literature (20, 22). Hence, in patients with sPGL, the first gene of choice should be

SDHB. The cases with HNPGL were small in our study with low prevalence of germline mutation (1/10, 10%). However, one of the two patients with bilateral HNPGL had mutation in SDHD gene, which is similar to previous reports favouring prioritisation of SDHD gene testing in bilateral HNPGL (20, 22).

Although the prevalence of germline mutations was less in patients with malignancy (3/16, 18.8%), majority

Table 4 Comparison of genetic yield in various large PCC/PGL cohorts based on the mode of presentation, location and nature

of PCC/PGL.

Centre Total cohort FS AS

Total cohort

Bilateral PCC Multifocal* Unilateral PCC sPGL Malignant

Our cohortIndia 2016† 49/150 30/30 19/120 13/16 6/7 21/87 8/30 3/16n = 150 32.7% 100% 15.8% 81.3% 85.7 24.1 26.7% 18.8%

VHL:23 VHL:14 VHL:9 VHL:6 VHL: 5 VHL:12 VHL:0 VHL:0SDHB:9 SDHB:1 SDHB:8 SDHB:0 SDHB: 0 SDHB:1 SDHB:8 SDHB:2RET:13 RET:13 RET:0 RET:7 RET: 1 RET:5 RET:0 RET:1SDHD:2 SDHD:0 SDHD:2 SDHD:0 SDHD: 0 SDHD:1 SDHD:0 SDHD:0NF1:2 NF1:2 NF1:0 NF1:0 NF1:0 NF1:2 NF1:0 NF1:0SDHC:0 SDHC:0 SDHC:0 SDHC:0 SDHC: 0 SDHC:0 SDHC:0 SDHC:0

Spain 2009† 93/237 64/69 29/168 34‡ 12‡ 20‡ 17‡ 13/17n = 237 39.2% 92.7% 17.2% 76.5%

VHL:20 VHL:13 VHL:7 VHL:12 VHL: 3 VHL:5 VHL:0 VHL:1SDHB:25 SDHB:8 SDHB:17 SDHB:0 SDHB:4 SDHB: 2 SDHB:16 SDHB:9RET:36 RET: 36 RET:0 RET:22 RET: 1 RET:13 RET: 0 RET:0SDHD:11 SDHD:7 SDHD:4 SDHD:0 SDHD:4 SDHD: 0 SDHD:1 SDHD:3NF1:0 NF1:0 NF1:0 NF1:0 NF1:0 NF1:0 NF1:0 NF1:0SDHC:1 SDHC:0 SDHC:1 SDHC:0 SDHC: 0 SDHC:0 SDHC:0 SDHC:0

Italy 2009† 161/501 102/107 59/394 27/48 22/29 58/278 17/40 9/25n = 501 32.1% 95.3% 14.9% 56.3% 75.9% 20.9% 42.5% 36%

VHL:48 VHL:17 VHL: 3 VHL:24 VHL:2 VHL:2SDHB:24 SDHB:0 SDHB: 3 SDHB:5 SDHB:12 SDHB:5RET:27 RET:10 RET: 1 RET:16 RET:0 RET:0SDHD:47 SDHD:0 SDHD: 14 SDHD:4 SDHD:1 SDHD:2NF1:11 NF1:0 NF1:1 NF1:9 NF1:1 NF1:0SDHC:4 SDHC:0 SDHC: 0 SDHC:0 SDHC:1 SDHC:0

France 2005 86/314 56/56 30/258 36/41§ 6/8§ 22/215§ 28/58§ 18/52n = 314 27.4% 100% 11.6% 87.8% 75% 10.2% 48.3% 34.6%

VHL:25 VHL:16 VHL:9 VHL:17 VHL: 2 VHL:4 VHL:4 VHL:2SDHB:21 SDHB: 3 SDHB:18 SDHB:0 SDHB: 3 SDHB:4 SDHB:17 SDHB:15RET:16 RET:15 RET:1 RET:11 RET: 0 RET:5 RET: 0 RET:0SDHD:11 SDHD:9 SDHD:2 SDHD: 3 SDHD: 1 SDHD:1 SDHD:7 SDHD:0NF1:13 NF1:13 NF1:0 NF1:5 NF1:0 NF1:8 NF1:0 NF1:1SDHC:0 SDHC:0 SDHC:0 SDHC:0 SDHC: 0 SDHC:0 SDHC:0 SDHC:0

Germany 2002 66/271 31/31 35/240 21/26|| 45/245|| 8/22n = 271 24.3% 100% 14.6% 80.77% 18.33% 36.36%

VHL:30 VHL:15 VHL:15 VHL:12 VHL:18 VHL:0SDHB:12 SDHB:0 SDHB:12 SDHB:0 SDHB:6 SDHB:8RET:13 RET:12 RET:1 RET:5 RET:8 RET:0SDHD:11 SDHD:4 SDHD:7 SDHD:4 SDHD:7 SDHD:0NF1:0 NF1:0 NF1:0 NF1:0 NF1:0 NF1:0

*Multifocal disease is defined as a coexistence of PCC and PGL in the same patient; †Exclusive HNPGL disease was also included in the cohort; ‡Denominator (total number of patients with each class of PCC/PGL) could not be derived; §The number of patients with multifocal disease and unilateral PCC was derived from the table; ||Bilateral and multifocal tumours were combined and single PCC and sPGL were combined.AS, apparently sporadic presentation; FS, familial and/or syndromic presentation; PCC, pheochromocytoma; sPGL, sympathetic paraganglioma/s.

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175:4 321Clinical Study R Pandit and others Genetic aetiology of PCC/PGL in Asian Indians

(2/3, 66.7%) of these were in SDHB, which is in agreement with previous reports (19, 20, 22). The other patient with malignancy had RET mutation (C634Y). This patient had bilateral PCC along with an abdominal extra-adrenal PGL at diagnosis and a year later presented with multiple abdominal metastases. Unusual presentation made us search for a coexisting mutation in other genes, which was negative. Although malignancy is rarely described in patients with RET mutation (34), rare presence of extra-adrenal tumour in our patient may account for the malignant disease.

Amongst single benign tumours with AS presentation, our genetic yield was 14.6% (PCC: 10.8%, sPGL: 24.1%), which is comparable with that reported in a recent systematic review (11–13%; PCC: 7.1%, sPGL: 13.5%) (23). Amongst the apparently sporadic unilateral PCC with positive mutations, VHL mutations were the most common (6/8, 75%). This is in contrast to a recent systematic review, which reported SDHB as the most common gene involved in apparently sporadic single PCC (23). Observations similar to our study have also been reported in a previous study from India in which none of the 32 patients with apparently sporadic unilateral PCC had SDHB mutations (35). Hence, in Asian Indian patients with unilateral PCC with AS presentation, VHL could be the first gene of choice.

In our cohort, there was a strong genotype–phenotype correlation. The evaluation for syndromic components identified 57.1% (VHL (13), RET (13) and NF1 (2)) of all the mutations. Hence, it is of utmost importance to obtain a detailed personal and family history and relevant evaluation for known syndromic stigmata. The additional prioritisation based on the presence of metastasis, location and number of tumour was useful. Exclusive presence of VHL mutations in non-syndromic bilateral PCC and multifocal PCC/PGL, SDHB mutations in sPGL and metastatic tumours, and SDHD mutation in bilateral HNPGL provided an opportunity to identify 26.5% (VHL (4), SDHB (8) and SDHD (1)) of all the mutations. Further genetic testing for VHL in unilateral PCC identified another six (12.2%) mutations. Overall, an appropriately selected single gene testing could identify around 96% of mutations in the cohort. This is unique to our cohort, perhaps could even be unique to Asian Indians. Based on this genotype–phenotype correlation in our cohort, we propose an algorithm for genetic prioritisation in Asian Indian PCC/PGL patients (Fig. 1). Hence, we suggest that complete screening could be replaced by a genetic triaging. However, confirmation of these findings by larger Asian Indian studies would be required.

The only group in our cohort where there was a need to screen patients for more than one gene was apparently sporadic unilateral PCC. To identify all mutations in this group, patients had to be screened for mutations in three genes (VHL, SDHB and SDHD). Recently, next-generation sequencing for PCC/PGL susceptibility genes is available in India, the cost of which is similar to that of Sanger sequencing of three common genes. Hence, in future, it would be more useful to perform genetic testing by next-generation sequencing in this particular group of patients. Lack of oligogenicity in our study reiterates that identifying a mutation in one causative gene obviates testing of the remaining genes.

The study also had a few limitations. Due to constrained resources, we could study only five genes. However, these five genes are reported to account for majority of the germline mutations in PCC/PGL, whereas the newer genes account for minority (36, 37, 38). Referral bias might have been introduced as proportionately more complex cases get referred to tertiary centres, thereby falsely increasing the genetic yield.

Conclusion

In this largest Asian Indian PCC/PGL cohort, 32.7% of total patients and 15.8% with AS presentation had germline mutations. Asian Indians with PCC/PGL differ from Western cohorts in having preponderance of VHL mutations in multifocal tumours and apparently sporadic unilateral PCC. Around 96% mutations in our cohort could be detected by appropriately selected single gene

Figure 1

Flow chart to triage genetic testing using clinical features.

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testing. Syndromic presentation, metastasis, location and number of PCC/PGL can be effectively used for guiding genetic prioritisation.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis project has been funded by the Scientific and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Grant# SB/SO/HS/041/2013).

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Received 11 February 2016Revised version received 14 June 2016Accepted 15 July 2016

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