Supplemental Material

Supplemental Methods

The human random control gDNA samples (n=96) used in this study were obtained

from the European Collection of Cell Cultures (Health Protection Agency, Porton Down,

UK). For sporadic pituitary adenoma mRNA expression analysis and cDNA sequencing, we

used surgical tumour samples from 48 consecutive patients with sporadic pituitary adenomas

classified histologically and clinically (1) as somatotroph (n=16), corticotroph (n=6),

lactotroph (n=8) and non-functioning pituitary adenomas (NFPAs) (n=18), plus 10 normal

pituitaries obtained at autopsy.

Leukocyte gDNA was extracted with the Qiagen DNA kit (Qiagen, Crawley, UK).

Tumour tissue RNA was extracted and reverse transcribed to cDNA as described (2).

Leukocyte RNA was collected in PaxGene Blood RNA tubes and extracted with the PaxGene

Blood RNA extraction kit (Preanalytix, Qiagen). Leukocyte gDNA was sequenced with 5

pairs of AIP (GenBank No U78521) primers (3) covering the coding regions and the exon-

intron junctions of the gene. Additionally, we sequenced up to -1200 base-pairs of the

promoter area using two sets of primers. Leukocyte and adenoma tissue cDNA was

sequenced with 4 pairs of exon-intron junction-spanning primers (sequences are available on

request). Sequencing was performed as previously described (4).

RT-PCR

Real-time RT-PCR of 48 human pituitary adenoma cDNA was performed using a

commercial primer and probe set (ABI, Warrington, UK; Assay ID: Hs00610222_m1) and the

ABI PRISM 7900 Sequence Detector System. All the reactions were performed in a duplex

PCR reaction with β-actin (β-ACTB) as the control (VIC®

MGB Probe, Cat. Number:

4326315E, ABI) at these conditions: 1μl cDNA, 5μl TaqMan®

Universal Master Mix, 0.5μl

20X AIP Assay Mix, 0.35μl 20X β-actin Assay Mix and 3μl TE (Tris-EDTA buffer). The

reaction conditions were 50°C for 2min, 95°C for 10min and 40 cycles at 95°C for 15sec and

60°C for 1min. Data were analyzed using the standard curve method (2). The relative

quantities of target transcripts were calculated from duplicate samples after normalization of

the data using the housekeeping gene β-actin.

Immunoblotting

Tissue samples from pituitary tissue were homogenized in protein lysis buffer (5) with

Precellys 24 homogenizer (Stretton Scientific Ltd, Stretton, UK) using ceramic beads. 10-

30μg of tissue- or cell culture-lysates were separated by electrophoresis and transferred to

polyvinylidene difluoride (PVDF) membrane (Amersham, Little Chalfont, UK). Membranes

were incubated with mouse monoclonal AIP antibody (ARA9 [35-2] mouse monoclonal

antibody, Novus Biologicals, Littleton, CO, USA) at 1:2000 or with rabbit polyclonal anti-

Xap2 antibody (Santa Cruz, USA) at 1:2000 and β-actin expression was used to determine

relative protein loading. Infrared fluorescent-labelled anti-rabbit or anti-mouse secondary

antibodies (IRDye 680) were used at a 1:10,000 dilution to quantify immunoreactivity using

the Odyssey infrared-imaging system (Li-Cor, Lincoln, Nebraska). Densitometric readings of

the resulting bands were evaluated using Li-Cor software (Li-Cor).

Immunostaining

For pituitary immunostaining we used 10 adenomas from patients with familial

pituitary adenoma and samples from 2 patients with sporadic gigantism and 47 sporadic

pituitary adenomas (somatotroph (n=14), corticotroph (n=9), lactotroph adenomas (n=10) and

NFPAs (n=14)), and 9 normal controls (autopsy pituitary tissue or part of resection specimens

removed at transsphenoidal surgery where normal pituitary tissue was identified on the basis

of immunostaining (all pituitary hormones expressed) and on a normal reticulin pattern (1)).

Immunostaining was performed with the Avidin Biotin Complex immunoperoxidase system

(Vectastain Elite, Vector Labs, Peterborough, UK) using 1:1000 monoclonal AIP antibody

(ARA9, Novus) (6,7) or pituitary hormone antibodies (polyclonal rabbit GH or mouse

monoclonal (prolactin (PRL), adrenocorticotrophic hormone (ACTH), thyroid stimulating

hormone (TSH), luteinising hormone (LH) and follicle stimulating hormone (FSH); Dako

Cambridge, UK) antibodies following heat-mediated antigen retrieval according to the

protocols used by our clinical service (1). Controls were performed with omission of the first

antibody. For semi-quantitative assessment of cytoplasmic AIP staining in the pituitary,

slides were scored blinded to the diagnosis for pattern (diffuse [score 2] or patchy [score 1])

and for intensity (strong [score 3], moderate [score 2] and weak [score 1]), and the final score

was calculated by multiplying the two (pattern and intensity) scores (1). Photographs of slides

were taken using a Leica DMR microscope and a Leica DC 200 digital camera (Leica GmBH,

Germany).

Immunofluorescent confocal microscopy

Immunofluorescent confocal microscopy was performed as described previously (8).

Autofluorescence was reduced by 0.1% sodium borohydride in Tris-buffered saline (TBS) for

30 min at room temperature. Antigen retrieval was accomplished by microwaving the

sections four times for 2.5min each at 800W in TBS containing 5% urea (w/v). The sections

were double labelled by incubation with mouse monoclonal AIP antibody (Novus, 1:500) and

each of the rabbit polyclonal anti-hormone antibodies against TSH, LH and FSH from

BioGenex (San Ramon, CA), and against GH, PRL and anti-ACTH from Dako (Cambridge,

UK)) overnight at 4°C. Anti-GH was used at a titre of 1:2000. The remaining anti-hormone

antibodies were used as supplied. Secondary antibody incubations were performed with Cy3-

conjugated donkey anti-mouse (1:100) and Cy2-conjugated donkey anti-rabbit (1:100)

antibodies (Molecular Probes, Eugene, OR) for 45 min at room temperature. The pituitary

sections were visualized with a Zeiss LSM510 laser scanning confocal microscope.

Immunogold electron microscopy

Immunogold electron microscopy was used to analyse the subcellular distribution of

AIP. Normal post-mortem pituitary, and available tissue from consecutive sporadic GH-,

ACTH- and non-functioning adenomas (at least three samples for each type) were processed

for immunogold labelling. Tumour and autopsy specimens were fixed in freshly prepared 4%

paraformaldehyde for 24h at 4°C, and then washed briefly in PBS. The tissue was transferred

to a solution of 2.3 M sucrose in PBS overnight and sectioned (400 µm) using a Vibratome

(Camden Instruments, Sileby, UK). The cryoprotected sections were subsequently slam-

frozen (Reichert MM80E; Leica, Milton Keynes, UK), freeze-substituted at -80°C in

methanol for 48h, and embedded at -20°C in LR Gold acrylic resin (London Resin Company

Ltd, Basingstoke, UK) in a Reichert freeze-substitution system (Reichert, Vienna, Austria).

Ultrathin sections were prepared by use of a Reichert Ultracut S ultratome and mounted

onto

formvar-coated nickel grids (Agar Scientific Limited, Reading, UK). For immunogold

labelling the sections were incubated for 2h with anti-AIP monoclonal antibody (1:300;

Novus) and for 1 h with anti-mouse IgG 15nm gold complex (British Biocell, Cardiff, UK),

then lightly counterstained with uranyl acetate and lead citrate. In some cases sections were

double labelled by similar methods for GH using rabbit anti-human GH polyclonal antibody

(NHPP, Torrance, California) and anti-rabbit IgG 5nm gold complex (British Biocell,

Cardiff, UK). All antibodies were diluted in 0.1 M phosphate buffer containing 0.1% w/v egg

albumin. For control sections, the primary AIP antibody was omitted and replaced with non-

immune mouse serum diluted 1:200 in 0.1 M phosphate buffer containing 0.1% w/v egg

albumin; immunogold labelling was absent in these conditions. Sections were examined with

a JEOL 1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA).

Cell culture, transient transfections and Cell proliferation studies

HEK293 and GH3 cells were maintained in Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml) and streptomycin

(100 mg/ml) in a humidified atmosphere, at 37°C with 10% CO2. Cells were plated at a

density of 1x106 cells/well in 6-well plates for Western blotting, and 5x10

5 cells/well in 96-

well plates for MTS assay. Transient transfections of HEK293 cells were carried out using

Lipofectamine Plus (Gibco-BRL, Paisley, UK) or Novafactor (Venna Nova, Pampano Beach,

FL) according to the manufacturer’s instructions. Expression efficiency was followed by co-

transfection of green fluorescent protein plasmid at a ratio of 1:10. There was no difference in

transfection efficiency between AIP wild-type and mutant plasmids. Cell proliferation was

assessed by cell counting and the MTS assay (Promega, Southampton, UK) at 24h, 48h and

72h.

TIG3 human fibroblasts were cultured in DMEM containing 10% fetal calf serum and

passaged at a 1:2 or 1:4 split ratio. To permit uptake of ecotropic retroviruses, cells were

infected with an amphotropic retrovirus (pWXLNeo-Eco) encoding the mouse basic amino-

acid transporter, which serves as the cell surface receptor for mouse ecotropic retroviruses

and selected in 200μg/ml G418 (9). Pools of G418-resistant TIG-3 cells were infected with

ecotropic retroviruses prepared by transient transfection of BOSC23 cells with pBABE-puro

vector containing wild-type (WT) and mutant AIP and selected 24h post-infection in medium

containing 2.5μg/ml puromycin. A virus containing the cell cycle inhibitor p16 was used as a

positive control. At 6 days post-infection, cells were plated at 104 cells per well in 24-well

plates in four replicates and cell proliferation was monitored over the following 8 days by

staining with crystal violet (9). For crystal violet assay cells were fixed and stained with 10%

formalin and 0.1% crystal violet. The intracellular stain was eluted with 10% acetic acid and

relative cell numbers determined by measuring the optical density at 590 nm (9). The results

shown were derived from 2-3 separate experiments.

Statistical analysis

Data is shown as mean and standard deviation unless otherwise stated. Student’s t test was

used for population data and the Kruskal-Wallis test was used for multigroup comparison for

RT-PCR and immunostaining data.

Supplemental Results

Details of the identified sequence changes in the AIP gene

In Family II we identified an early stop codon (E24X, c.70G>T, GenBank accession

number EF643644) (Supplemental Fig. 1B). This mutation was detected in all the affected

subjects, in the father who is an obligate carrier and in one of the siblings not affected at the

time of the study. The mutation in Family V consisted of a stop codon (R81X, c.241C>T,

GenBank accession number EF643647) (Supplemental Fig. 1B). In Family I we identified

an amino-acid change, (C238Y, c.713G>A, EF643648) in the 3 affected siblings and in the

father but not in the unaffected sister (Supplemental Fig. 1B). In Family X we identified an

in-frame insertion mutation: c.794_823dup (EF643650) (Supplemental Fig. 1B); a 30 base-

pair segmental duplication of 794-823 was found. This results in a duplication of a 10 amino-

acid sequence at the beginning of exon 6. This change was found in all 3 affected members of

this family but also in 3 siblings who currently do not have clinical symptoms: these subjects

currently have normal random GH and IGF-I levels and normal GH suppressibility to an oral

glucose tolerance test. There is a ‘hotspot’ in the AIP gene at amino-acid 304. We identified a

stop codon (R304X, c.910C>T) in Family XII and Family XXIV and this mutation was also

identified by 3 other studies (3,10,11) in families unlikely to be related to each other and no

known connection to Italy (where 2 previous families were described) or any other single

country. In Family XXV we identified a mutation also affecting the arginine at 304, but at a

different base resulting in an amino-acid change (c.911G>A). This mutation has been

described in a Polish family with Cushing’s disease (12). In Family XII all subjects with the

detected mutation had a pituitary adenoma (Supplemental Fig. 1B). In Family XVI we

identified a synonymous mutation at the beginning of exon 6 (c.807C>T, F269F, EF643649)

that, according to three different prediction programs (ESEfinder (13), RESCUE-ESE (14)

and PESE (15)), may disrupt five different splicing enhancer sites. This disruption is

predicted to cause the loss of exon 6. A dibasic change (c.-270-269CG>AA) and a single

base-pair change (c.-220G>A, EF643645) were found in the promoter area of AIP in Family

VI (Japanese origin) but in none of the Caucasian (n=96) or Japanese controls (n=78). The -

220 change disrupts a C/EBPbeta binding site and the -270-269 change disrupts WT1, NF-1,

ETF, C/EBPbeta and Oct-1 transcription factor binding sites as judged by a transcription site

searching program (http://wwwiti.cs.uni-magdeburg.de/~grabe/alibaba2). Family I, II, V and

X the genotyping data correspond with the predictions based on the earlier haplotype analysis

{Gadelha, 2000 1687 /id;Soares, 2005 4179 /id}. The complete lack of these changes in a

large number of controls and the segregation with the disease suggest a disease-related role

for this change.

We observed additional AIP sequence changes in the families with acromegaly: a novel 3’

UTR change (c.1053G>C) was found in both affected members of Family VI (from Japan)

and 2/96 of the Caucasian controls; this mutation was also found in a Japanese control

population (19) suggesting that this is a rare SNP.

Sequencing of sporadic acromegalic patient gDNA disclosed a previously described

(12) intronic change (c.IVS1-18C>T) in one of our sporadic giants but also in 4 control

subjects (all heterozygous). A novel change resulting in a synonymous amino-acid change

(c.36G>A, G12G) was found in a sporadic acromegalic patient. Another heterozygous 3’UTR

change (c.1070C>A) was found in one sporadic patient’s leukocyte DNA. This change was

not detected in any of the controls, or in other sporadic or familial cases and family members.

In the promoter region, we identified a SNP at c.-262G>A in a sporadic patient and in one

control.

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in normal corticotroph cells, corticotroph

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A, De Herder W, Penfornis A, Ciccarelli E, Estour B, Lecomte P, Gatta B, Chabre O, Sabate MI, Bertagna X, Garcia BN, Stalldecker G, Colao A, Ferolla P, Wemeau JL, Caron P, Sadoul JL, Oneto A, Archambeaud F, Calender A, Sinilnikova O, Montanana CF, Cavagnini F, Hana V, Solano A, Delettieres D, Luccio-Camelo DC, Basso A, Rohmer V, Brue T, Bours V, Teh BT, Beckers A. 2007 Aryl hydrocarbon

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14. Fairbrother WG, Yeh RF, Sharp PA, Burge CB. 2002 Predictive identification of

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Supplemental Figure 1: A) Schematic structure of the AIP gene and protein with the

identified mutations. Numbers in boxes represent exons and numbers underneath the protein

sequence represent amino-acids. Boxes represent location of peptidyl-prolyl cis-trans

isomerase (PPIase) and tetratricopeptide repeat motif (TPR) domains on the gene sequence.

B) Pedigree of the nine families with AIP mutations. The numbers shown below the symbols

indicate the age at diagnosis in affected individuals and age at the time of study in unaffected

individuals.

Supplemental Figure 2: RT-PCR of AIP in HEK293 cells showing low level of AIP

expression in cells transfected with empty vector and equal AIP transfection in all the cells

transfected with an AIP plasmid; β-actin is shown as control.

Supplemental Figure 3: Proliferation of TIG-3 human fibroblast cells infected with

retroviruses encoding wild-type AIP and p16INK4a

(positive control) compared to the empty

vector control (mean±SEM, P<0.001). Lower panel : immunoblot showing expression of AIP.

Supplemental Figure 4: AIP mRNA expression in normal pituitary and sporadic pituitary

adenomas (mean±SEM, Kruskal-Wallis test, overall P<0.001, ***, P<0.001 NFPA vs.

normal pituitary). NP, normal pituitary, GH, somatotroph, PRL, lactotroph, ACTH,

corticotroph adenoma and NFPA, non-functioning pituitary adenoma.

Supplemental Figure 5: Western analysis of AIP expression in sporadic pituitary adenomas

using monoclonal AIP antibody (1:2000) and beta-actin control.

Supplemental Figure 1

A

B

a helix

PPIase-like domain TPR1 TPR2 TPR3

1 12 90 182 215 234 267 301 330

AIPprotein

Chr 11q13AIP gene

1 2 3 4 5 6

R81X

C238Y

Ins274

R304X, R304Q

F269F splice

E24Xc.-270-269CG>AAc.-220G>A

a helix

PPIase-like domain TPR1 TPR2 TPR3

1 12 90 182 215 234 267 301 330

AIPprotein

Chr 11q13AIP gene

1 2 3 4 5 6

R81X

C238Y

Ins274

R304X, R304Q

F269F splice

E24Xc.-270-269CG>AAc.-220G>A

1 2 3 4 5 6

R81X

C238Y

Ins274

R304X, R304Q

F269F splice

E24Xc.-270-269CG>AAc.-220G>A

Family X

3 2

50 15 52 42

15 27

Family I

23 21 19

Gigantism

Acromegaly

Carrier

Prolactinoma

Not affected

3 Number of individuals

Deceased individual

Family V

32 32

64

36 34

Family XVI

58 47

Family XII

24

42 24

27 25

Family VI

14 10

1617

18

24 15 17 13

Family II

62

20

Family XXIV

31 24

Family XXV

17

30

Family X

3 2

50 15 52 42

15 27

Family X

3 2

50 15 52 42

15 27

Family I

23 21 19

Gigantism

Acromegaly

Carrier

Prolactinoma

Not affected

3 Number of individuals

Deceased individual

Family V

32 32

64

36 34

Family XVI

58 47

Family XII

24

42 24

27 25

Family VI

14 10

1617

18

24 15 17 13

Family II

62

20

Family XXIV

31 24

Family XXV

17

30

Family IFamily I

23 21 19

Gigantism

Acromegaly

Carrier

Prolactinoma

Not affected

3 Number of individuals

Deceased individual

Gigantism

Acromegaly

Carrier

Prolactinoma

Not affected

3 Number of individuals

Deceased individual

Family V

32 32

64

36 34

Family XVI

58 47

Family XVI

58 47

Family XII

24

42 24

27 25

Family XII

24

42 24

27 25

Family VI

14 10

Family VI

14 10

1617

18

24 15 17 13

Family II

62

201617

18

24 15 17 13

Family II

62

20

Family XXIV

31 24

Family XXIV

31 24

Family XXV

17

30

Day

0 1 2 3 4 5 6 7 80.00

0.25

0.50

0.75

1.00pBABE empty vector

p16

AIP wild-type

crys

tal v

iole

tab

sorb

ance

P<0.001

37 kDa

Empty ve

ctor

p16 AIP w

ild-ty

pe

Supplemental Figure 3

Supplemental Figure 2

AIP transfection

AIP

β-actin

Empty vector wild-type R304X C238Y

NP GH NFPA NFPA ACTH GH ACTH PRL

AIP37 kDa

Beta-actin46 kDa

NP GH NFPA NFPA ACTH GH ACTH PRLNP GH NFPA NFPA ACTH GH ACTH PRL

AIP37 kDa

Beta-actin46 kDa

Supplemental Figure 4

Supplemental Figure 5

AIP

/bet

a-ac

tinm

RN

A r

atio

NP ACTHGH NFPAPRL0

100

200

300

400

500

600

AIP

/bet

a-ac

tinm

RN

A r

atio

NP ACTHGH NFPAPRL0

100

200

300

400

500

600***