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The
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1
original article
Gigantism and Acromegaly Due to Xq26
Microduplications and GPR101 Mutation
G. Trivellin, A.F. Daly, F.R. Faucz, B. Yuan, L. Rostomyan, D.O. Larco,
M.H. Schernthaner-Reiter, E. Szarek, L.F. Leal, J.-H. Caberg, E. Castermans,
C. Villa, A. Dimopoulos, P. Chittiboina, P. Xekouki, N. Shah, D. Metzger,
P.A. Lysy, E. Ferrante, N. Strebkova, N. Mazerkina, M.C. Zatelli, M. Lodish,
A. Horvath, R. Bertollo de Alexandre, A.D. Manning, I. Levy, M.F. Keil,
M. de la Luz Sierra, L. Palmeira, W. Coppieters, M. Georges, L.A. Naves,
M. Jamar, V. Bours, T.J. Wu, C.S. Choong, J. Bertherat, P. Chanson, P. Kamenický,
W.E. Farrell, A. Barlier, M. Quezado, I. Bjelobaba, S.S. Stojilkovic, J. Wess,
S. Costanzi, P. Liu, J.R. Lupski, A. Beckers, and C.A. Stratakis
The author s’ full names, academ ic degrees,
and affiliations are listed in the Appendix.
Address reprint requests to Dr. Stratakis at
10 Center Dr., Bldg. 10, National Institutes
of Health, Clinical Research Center, Rm.
1-3330, MSC1103, Bethesda, MD 20892-
1862, or at stratakc@mail.nih.gov; or to
Dr. Beckers at th e Department of Endocri-
nology, Centre Hospitalier Universitaire
de Liège (B35), Domain e Universitaire du
Sart-Tilman, 4000 Liege, Belgium, or at
albert.beckers@chu.ulg.ac.be.
Drs. Trivellin and Daly, and Drs. Beckers
and Stratakis, contributed equally to this
article.
This arti cle was published on De cember 3,
2014, at NEJM.org.
DOI: 10.1056/NEJMoa1408028
Copyright © 2014 Massachusetts Medical Society.
ABSTRACT
Background
Increased secretion of growth hormone leads to gigantism in children and acro-
megaly in adults; the genetic causes of gigantism and acromegaly are poorly under-
stood.
Methods
We performed clinical and genetic studies of samples obtained from 43 patients
with gigantism and then sequenced an implicated gene in samples from 248 pa-
tients with acromegaly.
Result s
We observed microduplication on chromosome Xq26.3 in samples from 13 patients
with gigantism; of these samples, 4 were obtained from members of two unrelated
kindreds, and 9 were from patients with sporadic cases. All the patients had disease
onset during early childhood. Of the patients with gigantism who did not carry an
Xq26.3 microduplication, none presented before the age of 5 years. Genomic char-
acterization of the Xq26.3 region suggests that the microduplications are generated
during chromosome replication and that they contain four protein-coding genes.
Only one of these genes, GPR101, which encodes a G-protein–coupled receptor, was
overexpressed in patients’ pituitary lesions. We identified a recurrent GPR101 mutation
(p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly
in tumors. When the mutation was transfected into rat GH3 cells, it led to increased
release of growth hormone and proliferation of growth hormone–producing cells.
Conclusions
We describe a pediatric disorder (which we have termed X-linked acrogigantism
[X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by
early-onset gigantism resulting from an excess of growth hormone. Duplication
of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in
some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National
Institute of Child Health and Human Development and others.)
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S
omatic growth is orchestrated by a
complex hormonal crosstalk involving the
hypothalamus, pituitary, and peripheral tis-
sues.
1
Genetic disorders that affect this network
can lead to increased secretion of growth hor-
mone, which results in acromegaly. If the excess
in growth hormone occurs before epiphyseal fu-
sion, the result can be gigantism. Nonsyndromic
gigantism is most frequently caused by pituitary
adenomas occurring as familial isolated pituitary
adenomas or sporadically, usually as a result of
mutations in the gene encoding aryl hydrocarbon
receptor–interacting protein (AIP).
2-4
Other mono-
genic diseases can cause gigantism, but most of
these conditions develop in adulthood in associa-
tion with other tumors.
5
In young children, so-
matic overgrowth that is due to an excess of
growth hormone is rare, and the cause is un-
known. Other syndromic genetic overgrowth con-
ditions in children, such as the Sotos syndrome
and the Simpson–Golabi–Behmel syndrome, are
not associated with pituitary abnormalities.
6,7
We report a striking phenotype of gigantism
that has an onset in early childhood and that is
caused by an excess of growth hormone. The
disorder is associated with heritable microdupli-
cations on chromosome Xq26.3. There are four
genes in the duplicated stretch of DNA; one of
these, GPR101, encodes an orphan G-protein–
coupled receptor and is probably the gene that
drives the phenotype in young children and the
growth of sporadic growth hormone–producing
adenomas in some patients with acromegaly.
Methods
Patients
We analyzed samples obtained from 43 patients
with gigantism who had hypersecretion of
growth hormone, evidence of an anterior pitu-
itary lesion on magnetic resonance imaging, a
height on country-specific growth charts of ei-
ther more than the 97th percentile or more than
2 SD above the mean height for age, and negative
test results for mutations or deletions in genes
associated with pituitary adenomas (Table 1).
Details with respect to one family with this syn-
drome8,9 and two patients with sporadic dis-
ease10,11 have been described previously.
Genetic Analyses
We sequenced the four genes in the duplicated
region on chromosome Xq26.3 in 259 germline
and tumor DNA samples that were obtained
from 248 patients with sporadic acromegaly (Ta-
ble S1 in the Supplementary Appendix, available
with the full text of this article at NEJM.org). We
sequenced GPR101 and performed array compar-
ative genomic hybridization (aCGH) on germline
DNA in samples obtained from 13 families with
familial isolated pituitary adenomas without AIP
mutations. We used quantitative reverse-transcrip-
tase–polymerase-chain-reaction (qRT-PCR) assays
to measure the expression levels of duplicated
genes in both leukocytes and pituitary tumors.
We performed comparative protein-structure
modeling on GPR101 using Modeller software,
version 9.13.12 We determined the level of growth
hormone and cyclic AMP (cAMP) and the rate of
cellular proliferation after transient overexpres-
sion of each of the four implicated genes in GH3
cells obtained from rat pituitary tumors.
Study Oversight
The institutional review board at each of the par-
ticipating institutions approved our studies. We
studied the anonymized samples from interna-
tional acromegaly cohorts with approval from the
National Institutes of Health Intramural Office
for Human Research Protections. Written in-
formed consent was obtained from all adult pa-
tients and parents or guardians of children with
early-onset gigantism.
Results
Clinical Presentation
The clinical and biochemical characteristics of
the 43 patients who had nonsyndromic gigan-
tism without abnormalities in genes associated
with pituitary tumors are presented in Table 1.
Genetic analyses delineated two phenotypes: an
early-childhood form of gigantism with a typical
onset in late infancy (Fig. 1) and a second form
with a typical onset in adolescence.
Identification of Xq26.3 Microduplication
We detected microduplications on chromosome
Xq26.3 in samples obtained from patients with
the early-childhood form of gigantism (Fig. 2,
and Fig. S1, S2, and S3 in the Supplementary Ap-
pendix). Nine of the 13 patients with an Xq26.3
microduplication and the 1 probable carrier (an
affected mother with gigantism) were female
and were of normal size at birth. All the patients
grew rapidly during infancy, attaining a median
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Gigantism and Acromegaly Due to Xq26 abnormalities
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3
height score of +3.8 SD at diagnosis (median age,
36 months). At the time of diagnosis, they
showed marked overall somatic growth, with el-
evated weight and an enlarged head circumfer-
ence (median, 51.2 cm). The onset of accelerated
growth and the onset of accelerated weight gain
usually coincided but were not always synchro-
nous (Fig. 1, and Fig. S4 in the Supplementary
Appendix). As compared with patients who did
not have an Xq26.3 microduplication, those with
the microduplication had an earlier median age
at the onset of abnormal growth (12 months vs.
16 years), an increased acceleration in height,
and elevated levels of insulin-like growth factor 1
and prolactin (
Table 1
). We did not observe pre-
cocious puberty in the microduplication carriers.
Levels of peripheral growth hormone–releasing
hormone did not suggest ectopic secretion of this
hormone, and nuclear imaging scans were nega-
tive for other tumors.
Of the 13 patients who underwent surgery, 10
had pituitary macroadenomas alone (median
maximum diameter, 16 mm), and 3 patients had
pituitary hyperplasia, with or without an identi-
fied adenoma (Fig. 3H). In all the patients, hor-
monal control was not achieved with medical
therapy alone. Such control required either radi-
cal or repeated neurosurgery alone (in 4 pa-
tients) or in combination with the administra-
tion of the
growth hormone receptor antagonist
pegvisomant (in 3 patients) or radiotherapy (in
2 patients). Seven patients had permanent hypo-
pituitarism at the time of this study.
The common duplicated genomic segment
was approximately 500 kb in length, from posi-
tion 135,627,637 to 136,118,269 (GRCh37/hg19)
Table 1. Clinical Characteristics of 43 Patients with Gigantism with and without Xq26.3 Microduplications.
Characteristic Xq26.3 Microduplication
(N = 14) No Xq26.3 Microduplication
(N = 29) P
Value
Female sex — no. (%) 10 (71) 7 (24) 0.007
Median age at onset of rapid growth (range) — yr 1.0 (0.5 to 2.0) 16.0 (5.0 to 18.0) <0.001
Median age at diagnosis (range) — yr 3 (1 to 22) 21 (5 to 34) <0.001
Median height at diagnosis (range) — cm 116 (99 to 175) 187 (171 to 209) <0.001
Median standard-deviation score for height at
diagnosis (range) +3.8 (+1.9 to +7.1) +3.3 (+2.1 to +5.8) 0.45
Elevated levels of growth hormone and insulin-like
growth factor 1 at diagnosis — no. (%) 14 (100) 29 (100) 1.00
No suppression of growth hormone during oral
glucose-tolerance test — no. (%) 14 (100) 29 (100) 1.00
Median factor increase in insulin-like growth factor 1
at diagnosis (range) — multiple of ULN 4.4 (2.4 to 5.2) 2.1 (1.4 to 5.3) 0.005
Elevated prolactin level at diagnosis — no. (%) 13 (93) 6 (21) <0.001
Median maximum tumor diameter (range) — cm 16 (10 to 39) 20 (9 to 41) 0.16
Adenoma or hyperplasia — no. (%)†
Both adenoma and hyperplasia 2 (14) 0
Adenoma only 10 (71) 29 (100) —
Hyperplasia only 1 (7) 0
Type of syndrome — no. (%)
Sporadic 9 (64) 29 (100) —
Familial 5 (36)‡ 0
Siblings with normal growth — no./total no. (%) 9/11 (82) 29/29 (100) —
* ULN denotes upper limit of the normal range.
† The presence of hyperplasia or adenoma could not be determined in one patient who did not undergo surgery.
‡ In one patient with the familial syndrome, pituitary gigantism was diagnosed in the mother and son at the same visit,
when the son was 8 years of age and the mother was 22 years of age. The mother had had tall stature and acromegalic
features since childhood for which she had not been referred for medical attention. The clinical data for the mother, for
whom DNA was not available, are included.
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Birth to 24 months: Boys
Length-for-age and Weight-for-age percentiles
Published by the Centers for Disease Control and Prevention, November 1, 2009
SOURCE: WHO Child Growth Standards (http://www.who.int/childgrowth/en)
98
95
85
75
50
25
10
5
2
98
95
90
75
50
25
10
5
2
Birth to 24 months: Girls
Length-for-age and Weight-for-age percentiles
Published by the Centers for Disease Control and Prevention, November 1, 2009
SOURCE: WHO Child Growth Standards (http://www.who.int/childgrowth/en)
98
95
90
75
50
25
10
5
2
98
95
90
75
50
25
10
5
2
A
B C
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(Fig. 2). One patient had a complex genomic re-
arrangement, with two duplicated segments that
were separated by a short region of normal ge-
nomic sequence. No other patterns of duplica-
tion or deletion or homozygosity were shared
among the affected patients. One family with
familial isolated pituitary adenomas included an
affected mother and two affected sons (who
have been described previously
8
) with the same
Xq26.3 microduplication; the unaffected father
did not have the duplication. In another family
with this condition, the mother had childhood-
onset gigantism and a histologically confirmed
pituitary macroadenoma but had died of com-
plications of hypopituitarism. She had two
children: the son carried the Xq26.3 microdu-
plication and had childhood-onset gigantism
(Patient F2A), and the healthy daughter did not
have the duplication. The most parsimonious ex-
planation is that the son inherited the X-linked
disease from his carrier mother. Hence, Xq26.3
microduplications can be considered to be a
new pathogenic explanation in certain kindreds
with familial isolated pituitary adenomas that
have acrogigantism without AIP mutations.
Chromosomal position
Patient No.
S9
S8
S7
S6
S5
S4
S2
S1
F2A
F1A/F1B/F1C
135,500,000 135,700,000 135,900,000 136,100,000 136,300,000
Familial
Familial
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic
SRO1 SRO2
BRS3
HTATSF1
VGLL1
CD40LG
ARHGEF6 RBMX GPR101
Figure 2. Summary of the Genomic Gains on Chromosome Xq26.3.
Shown are 10 different Xq26.3 microduplications, as seen on array comparative genomic hybridization, that were
found in 12 patients with familial or sporadic gigantism (with the inheritance pattern indicated at right). Duplicated
genomic segments (red) and nonduplicated segments (white) are shown. The genomic coordinates are provided at
base-pair resolution on the x axis. The two smallest regions of overlap (SRO), SRO1 and SRO2, are identified, show-
ing the genomic contents in the corresponding regions. The symbols next to the gene names represent the struc-
ture of the genes, with vertical lines representing exons and horizontal lines (with or without arrows) representing
introns. Adapted from the UCSC Genes track in the UCSC Genome Browser.
Figure 1 (facing page). Familial and Sporadic Cases
of Gigantism and Male and Female Growth Patterns
Due to the Xq26.3 Microduplications.
Panel A shows Patient F1C, who has familial gigantism,
at the age of 3 years. His growth chart up to 24 months
of age shows the rapid acceleration in weight, although
the acceleration in height did not begin until after his
second birthday (Fig. S4 in the Supplementary Appendix).
Panel B shows an unaffected mother and her daughter
(Patient S6), who has sporadic gigantism and whose
height was 120 cm at the age of 3 years. A growth chart
for Patient S4 (Panel C), another girl with sporadic gigan-
tism, illustrates the typical early increase in height and
weight seen in patients with Xq26.3 microduplications,
starting at the age of 6 months in this child.
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ABCD
E G HF
20 µm 20 µm 20 µm 20 µm
Figure 3. Imaging and Histopathological Findings in Patients with Xq26.3 Microduplications.
Panels A through D show progressive changes from normal pituitary tissue (Panel A) to adenoma (Panel D), as indicated by reticulin staining of the pituitary gland in Patient F1C.
In Panel A, normal pituitary gives way to the expanded hyperplastic acini (Panel B), and in Panel C, areas of transformation are evident (circled) with enlarged, hyperplastic, conflu-
ent acini that are caused by breakdown of reticulin fibers and that lead to adenoma (Panel D) with disruption of the reticulin fiber network. Increased GPR101 expression was ob-
served in five tested patients with Xq26 microduplications, whereas there is little if any expression in normal pituitary tissue or growth hormone–producing tumors without Xq26.3
microduplications or GPR101 defects (see also Fig. S7 in the Supplementary Appendix); an example is shown here (Panels E through G) from Patient S3. When the staining of growth
hormone (Panel E) and the staining of GPR101 (Panel F) are merged, GPR101 seems to be expressed in some of the growth hormone–secreting cells (Panel G, arrows) but not in all
such cells. Nuclei (blue) were stained with DAPI. Panel H shows a sagittal view of a macroadenoma on magnetic resonance imaging of Patient S5 with the Xq26.3 microduplication.
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Further Characterization of Xq26.3
Microduplication
Using high-definition analysis of the critical du-
plicated region, we analyzed 10 distinct genomic
duplications in 12 patients, including 4 patients
with the familial form of the disease and 8 pa-
tients with the sporadic form (Fig. S1 and S3 in
the Supplementary Appendix). On genomewide
aCGH, these mutations appeared to be simple
duplications. However, using high-resolution
aCGH, long-range PCR, and Sanger sequencing
of the breakpoints, we found various underlying
genomic complexities (Fig. S3 in the Supplemen-
tary Appendix).
All sporadic Xq26.3 duplications were nonre-
current; the boundaries of the duplicated seg-
ment were unique to each patient. On both
aCGH and breakpoint PCR assays, samples ob-
tained from unaffected parents and siblings of
patients with sporadic disease showed negative
results, documenting the microduplication as a
new mutation (Fig. S3A and S5A in the Supple-
mentary Appendix). The same duplication was
transmitted from an affected mother (Patient F1A)
to her affected offspring, Patients F1B and F1C
(Fig. 2, and Fig. S3 and S5B in the Supplemen-
tary Appendix).
The duplicated genomic regions that were
shared by all affected persons consisted of the
two smallest regions of overlap (SRO), which were
designated as SRO1 and SRO2 (Fig. 2). SRO1
(chromosomal posit ion, 135,627,637 to 135,986,830;
hg19) encompassed three genes in the Online
Mendelian Inheritance in Man (OMIM) data-
base: CD40LG (OMIM number, 300386), ARHGEF6
(OMIM number, 300267), and RBMX (OMIM
number, 300199), whereas SRO2 (chromosomal
position, 136,045,310 to 136,118,269; hg19) in-
cluded GPR101 (OMIM number, 300393) (Fig. 2).
Investigation of Candidate Genes
Sequencing of each of the four genes in the 43
patients with gigantism did not reveal any single-
nucleotide variants of likely pathogenicity. A
quantitative RT-PCR assay of pituitary tumor
RNA from 2 patients with Xq26.3 microduplica-
tions suggested that CD40LG was not expressed in
the pituitary tumors. Neither ARHGEF6 nor RBMX
showed up-regulated expression in the pituitary
tumors of 2 patients with the duplication (Fig. 4).
In contrast, the expression of GPR101 in the pitu-
itaries of the children carrying an Xq26.3 dupli-
cation was increased by a factor as high as 1000,
as compared with unaffected pituitary tissue and
pituitary tumors from persons who tested nega-
tive for microduplications (Fig. 4A). This result
was confirmed at the protein level by increased
immunostaining for GPR101 in pituitary tumors
from patients with Xq26.3 duplications (Fig. 3G,
and Fig. S7 in the Supplementary Appendix). Ex-
perimental overexpression of ARHGEF6, RBMX,
and GPR101 alone in the rat GH3 cell line did not
signif icantly increase either cell proliferation or
the secretion of growth hormone (Fig. 4D and
4E, and Fig. S8 in the Supplementary Appendix).
Nonmutated GPR101 in combination with ARH-
GEF6, RBMX, or both modestly increased cell pro-
liferation but not the secretion of growth hor-
mone (Fig. S8 in the Supplementary Appendix).
The X-chromosome–inactivation pattern was
random in the female patients with sporadic
disease and skewed in Patient F1A, who had fa-
milial disease; CpG islands were identified in
silico only in RBMX and GPR101 (Fig. S9 and S10
in the Supplementary Appendix).
Identification of p.E308D Mutation in GPR101
In a series of 248 patients with sporadic acro-
megaly, none carried a microduplication at
Xq26.3. However, 11 patients had a c.924G→C
substitution (p.E308D) in GPR101, which was not
found in 7600 control samples obtained from
public databases (Tables S1 and S2 in the Supple-
mentary Appendix). Of the 11 mutation carriers,
3 appeared to carry a constitutive mutation,
which was detected in DNA from peripheral-
blood mononuclear cells (PBMCs). We detected
the mutation in the tumor DNA in the remaining
8 patients (Fig. 5A). In one patient, we deter-
mined that the mutation was a de novo somatic
mutation — that is, the GPR101 mutation oc-
curred only in the tumor DNA sequence and not
in the PBMC sequence (Fig. 5B). None of the 13
families with familial isolated pituitary adeno-
mas carried the p.E308D mutation in GPR101.
GPR101 encodes an orphan
G-protein–coupled
receptor
that is highly expressed in rodent hypo-
thalamus (Fig. S11 and S12 in the Supplemen-
tary Appendix) and is predicted to couple to the
stimulatory G protein (G
s
), a potent activator of
adenylyl cyclase.
13,14
A model of human GPR101
in complex with a G
s
heterotrimer shows the phy-
sical relationship between the p.E308D amino
acid change and the activating p.A397K change,
a mutation that has been described previously.
15
The two amino acids, which are predicted to be
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D
Cell Proliferation
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control GPR101-
WT GPR101-
E308D GPR101-
A397K
Relative Units
** *
Normalized mRNA Expression
0NP1 NP2 NP3 NP4 NP5 GH1 GH2 F1C S6
200
400
600
800
1000
1200
1400
A
GPR101
Growth Hormone
E
45
40
35
30
25
20
15
10
5
0
Control GPR101-
WT
GPR101-
E308D
GPR101-
A397K
Relative Units
Vehicle
***
***
***
Forskolin
***
**
***
CRE Activation
F
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control GPR101-
WT GPR101-
E308D GPR101-
A397K
Relative Units
***
**
**
*
0.0
Normalized mRNA Expression
0.5
1.0
1.5
2.0
2.5
NP1 NP2 GH1 GH2 F1C S6
B
ARHGEF6
Normalized mRNA Expression
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0 NP1 NP2 GH1 GH2 F1C S6
C
RBMX
Figure 4. Expression of GPR101 in Pituitary Tissue from Children with Xq26.3 Microduplications.
The expression of GPR101 in pituitary tissue from children carrying Xq26.3 microduplications was increased by a factor as high as 1000, as compared with the expression in unaffected
pituitary tissue (in five samples [NP1 through NP5] obtained on autopsy) and in pituitary tumors from two patients with sporadic acromegaly (GH1 and GH2) who tested negative for
the microduplication (Panel A). These findings, which were obtained on quantitative reverse-transcriptase–polymerase-chain-reaction (qRT-PCR) assay and normalized by a housekeep-
ing gene, contrast with those for two other genes, ARHGEF6 (Panel B) and RBMX (Panel C), in the duplicated stretch of DNA; neither of these two genes showed up-regulated expres-
sion. Also shown are cell proliferation (Panel D), growth hormone secretion (Panel E), and activation of
DNA sequences called cyclic AMP response elements (CRE)
(Panel F) in rat
GH3 cells transfected with mutant (p.E308D and p.A397K) and nonmutant GPR101 constructs. Values for cells transfected with empty (control) vector were set at 1. Also shown are
values for untreated cells (vehicle) and forskolin (which increases CRE activation). Data are expressed as the mean results of three to five independent experiments, each of which was
performed in triplicate. The T bars indicate standard deviations. One asterisk denotes P<0.05, two asterisks P<0.01, and three asterisks P<0.001.
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Gigantism and Acromegaly Due to Xq26 abnormalities
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9
C
D308
IL 3
A397
TM 6
TM 5
AB
G G GA A C
308 309
308 309 308 309 308 309
Germline
DNA
Normal
Tumor
DNA
Mutation
Male
(Hemizygote)
Female
(Heterozygote)
Normal
Tumor DNA
G G G G G G GA A A AAC C C CAS
Figure 5. Effect of the p.E308D Mutation in GPR101 in 11 Patients with Sporadic Acromegaly.
Panel A shows the sequence for GPR101 in growth hormone–producing pituitary tumors obtained from patients with
sporadic acromegaly, as compared with normal tissue. Panel B shows results for a patient with a somatic mutation,
which was determined by the presence of the mutation in the GPR101 sequence of DNA in the tumor sample but
not in the sequence in peripheral-blood mononuclear cells. None of the 13 families with familial isolated pituitary
adenomas carried the p.E308D mutation in GPR101. Panel C shows a structural model of GPR101
bearing the p.E308D
mutation. Residue A397 is located at the cytosolic end of transmembrane (TM) 6 of GPR101. The mutated D308 res-
idue and the nonmutated A397 residue are shown in space-filling representation and colored according to elements,
with carbon atoms in gray, oxygen atoms in red, and nitrogen atoms in blue. The backbone of the receptor and the
G protein heterotrimer is schematically represented as a ribbon, with the receptor shown with a spectrum of colors
that ranges from red at the N-terminal to purple at the C-terminal; the α, β, and γ subunits of the G protein are in gray,
blue, and pink, respectively. The cytosolic ends of TM 5 and TM 6 and intracellular loop (IL) 3, which connects them,
are indicated by labels. The blue arrows show directions of the β-sheet domains of the β subunit of the G protein.
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10
affected by the mutations, are on the cytosolic
side of the receptor (Fig. 5C). The E308 residue
is located in the long intracellular loop 3, which
connects transmembrane domains 5 and 6.
Overexpression of the p.E308D and p.A397K
mutants, but not of nonmutant GPR101, signifi-
cantly increased cell proliferation and secretion
of growth hormone in rat GH3 cells (Fig. 4D and
4E). As in the construct containing the nonmu-
tant receptor, the two mutant constructs result-
ed in increased cAMP signaling in GH3 cells in
an in vitro reporter assay, both at baseline and
in the presence of 10 μM forskolin, a direct
stimulator of adenylyl cyclase (Fig. 4F).
Discussion
Several lines of evidence support the identifica-
tion of a new pituitary gigantism syndrome in
young children carrying microduplications on
chromosome Xq26.3, a disorder that is probably
caused by GPR101 overexpression. We propose that
this syndrome be called X-linked acrogigantism
(X-LAG). First, we did not find disruption of
Xq26.3 in patients with later-onset gigantism
(Table 1). Second, the finding that patients with
other conditions had different duplications with-
in the same region narrowed our focus to the
smallest region of overlap. A duplication encom-
passing CD40LG and ARHGEF6 but not RBMX and
GPR101 occurred in a family with low birth
weight, intellectual disability, and craniofacial
abnormalities,16 which suggests that duplica-
tions with the exclusion of RBMX and GPR101 do
not lead to gigantism. Third, short stature has
been reported in several patients with deletions
in this region, which suggests that the absence
of these genes may lead to the opposite pheno-
type (Table S4 in the Supplementary Appen-
dix).16-18 Other investigators have described at
least 15 additional patients with the same pheno-
type of early-onset growth who may be good
candidates for a diagnosis of X-LAG (Table S3 in
the Supplementary Appendix).
The breakpoint features of Xq26.3 duplica-
tions suggest that they were generated by means
of a replication-based mechanism that under-
lies the genesis of other copy-number variants
(CNVs) and the pathogenesis of other genomic
disorders.
19
The cytogenetic data narrowed the smallest
region of overlap to a segment spanning
CD40LG, ARHGEF6, RBMX, GPR101, one microRNA
(miR-934), and a small nucleolar RNA (SNORD61)
of unknown function. We did not detect CD40LG
expression in the pituitary tissues from our pa-
tients (Fig. 4). Messenger RNA for ARHGEF6 and
RBMX was expressed to a similar degree in af-
fected and unaffected tissues from duplication
carriers. Of all the genes and the noncoding
RNAs in the duplicated segment, only GPR101
had markedly increased expression in the pitu-
itary tumors from the duplication carriers (Fig. 4).
GPR101 is an orphan G-protein–coupled recep-
tor that is strongly expressed in the hypothalamus
in rodents (Fig. S11 and S12 in the Supplemen-
tary Appendix).
13,14,20
It was recently shown that
a fragment of the gonadotropin-releasing hor-
mone could be a ligand for this receptor.
21
The
GPR101 protein may also play a role in hypotha-
lamic control of energy homeostasis.
22
The ef-
fect of a mutation (p.A397K) that is predicted to
activate GPR101 when tested in vitro and in mice
supports such a role.
15
The pituitary-specific over-
expression of GPR101 may be due to a gene-dose
effect (as described in many genomic disorders
23
)
or to an unknown promoter sequence created by
the chromosomal rearrangement, although we did
not identify any putative new promoter, or to per-
turbed chromatin regulation due to the genomic
structural alteration from duplication CNVs.
24,25
On the basis of our data from transfection experi-
ments, we cannot rule out a modest contribution
of RBMX and ARHGEF6 coexpression to cell prolif-
eration. However, unlike GPR101, neither ARHGEF6
nor RBMX was overexpressed in the pituitary
tumors from children with microduplications.
Our studies of sporadic acromegaly provide
further support for a role of GPR101 in X-LAG.
We found a recurrent GPR101 mutation, p.E308D,
in 4.4% of DNA in tumor samples and in 1.9%
of DNA in PBMC samples obtained from patients
with isolated acromegaly. In at least one patient,
the mutation was present only in the tumor DNA.
We did not identify GPR101 mutations in families
with familial isolated pituitary adenomas. A model
of human GPR101 in complex with a G
s
hetero-
trimer showed that both the p.E308D mutation
and the previously described p.A397K mutation
15
are on the cytosolic side of the receptor that
interacts with heterotrimeric G proteins. Residue
E308 is located in a remarkably long intracellu-
lar loop, which connects two transmembrane
domains. But in the absence of a model template
for the GPR101 intracellular loop in which E308
resides, it is difficult to estimate the structural
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Gigantism and Acromegaly Due to Xq26 abnormalities
n engl j med nejm.org
11
effect of the p.E308D substitution. However, trans-
fection of a construct expressing GPR101 contain-
ing the p.E308D mutation increased proliferation
and growth hormone secretion in a rat pituitary
cell line. Moreover, we showed that GPR101 can
strongly activate the cAMP pathway, for which the
mitogenic effects in pituitary somatotropes are
well established.
26
These data further support a
role for variant GPR101 in sporadic acromegaly.
The mechanism by which mutant GPR101
contributes to increased growth hormone secre-
tion is unclear. Some of the patients with early-
onset gigantism whom we evaluated had normal
or mildly elevated levels of circulating growth
hormone–releasing hormone (but below the
threshold required for ectopic tumoral secretion
of this hormone), as was previously noted in
Family F1.
8
The tumor tissue showed strong
expression of the growth hormone–releasing
hormone receptor, in contrast to its expression
of growth hormone–releasing hormone, which
was low or absent (Fig. S15 in the Supplemen-
tary Appendix).
In conclusion, our results suggest that Xq26.3
microduplication is associated with a clinical
syndrome of early-onset gigantism, which we
have termed X-LAG. An increased dose of GPR101
on chromosome Xq26.3 probably causes the dis-
ease, and its activation by mutation occurs in
patients with sporadic acromegaly. Xq26.3 micro-
duplications may explain other historical cases
of gigantism with features that closely resemble
those of X-LAG.
27,28
Our results offer an oppor-
tunity to study a new pathway involved in the
central regulation of human growth.
Supported by a grant from the Intramural Research Program
of the Eunice Kennedy Shriver National Institute of Child Health
and Human Development (NICHD) (Z01-HD008920, to Dr. Strata-
kis), by a grant from the National Institute of Neurological Dis-
orders and Stroke (NINDS) (RO1 NS058529), by a grant from the
National Human Genome Research Institute (U54HG006542, to
Dr. Lupski), by a grant from Fonds d’Investissement de Recherche
Scientifique of Centre Hospitalier Universitaire (CHU) de Liège
(to Dr. Beckers), by an educational grant from Pfizer Belgium (to
Dr. Beckers), and by the Jabbs Foundation (to Dr. Beckers). Com-
puting resources used for the molecular-modeling component
of this work were provided by the American University High
Performance Computing System, which is funded in part by a
grant from the National Science Foundation (BCS-1039497).
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the patients and their families for their participa-
tion in t his study; the nursing and other support sta ff at the NIH
Clinical Research Center (CRC), in particular Dr. Edward Old-
field (now at the University of Virginia, Charlottesville) and Dr.
Russell Lonser (now at Ohio State University, Columbus) who
operated on most of the patients from the NIH who are de-
scribed in this report; Dr. Phillip Gorden of the National Insti-
tute of Diabetes and Digestive and Kidney Diseases for provid-
ing a list of patients with acromegaly who have been seen at the
NIH CRC during the past 30 years; Dr. Timothy Jones (Depart-
ment of Pediatric Endocrinology, Princess Margaret Hospital for
Children and School of Pediatrics and Child Health, University
of Western Australia); Vincent Schram at the Microscopy and
Imaging Core of the NICHD; Dr. Charalampos Lyssikatos and
Dr. Monalisa Azevedo (Section on Endocrinology and Genetics,
NICHD); Ms. Isabelle Besson and Dr. Michèle Bernier (Pathol-
ogy and Cytology Department, Hôpital Foch, Suresnes, France);
Drs. Antonella Forlino, Annalisa Vetro, and Orsetta Zuffardi
(Department of Molecular Medicine, University of Pavia, Pavia,
Italy); Dr. Anna Spada and Dr. Paolo Beck-Peccoz (Endocrinolo-
gy and Diabetology Unit, Fondazione Istituto de Ricovero e Cura
a Carattere Scientif ico Ca’ Granda Ospedale Maggiore Policlinico,
Department of Clinical Sciences, University of Milan); Ms. Carine
Mottard, Ms. Carine Deusings, Mr. Valery Leduc, and Ms. Nathalie
Sacre (Department of Clinical Genetics, CHU de Liege, Liege,
Belgium); Ms. Latifa Karim (GIGA-Genomics, Liège, Belgium);
Dr. Silvia Paoletta (Laboratory of Bioorganic Chemistry,
National
Institute of Diabetes and Digestive and Kidney Diseases
); Ms.
Silke Williams (Laboratory of Pathology, National Cancer Insti-
tute); Dr. Jack A. Yanovski (Section on Growth and Obesity,
NICHD); Dr. S.J. Levine (Cardiovascular and Pulmonary Branch,
National Heart, Lung, and Blood Institute); Dr. Say Viengchareun
(INSERM Unité 693 Le Kremlin-Bicêtre, France); Dr. Paul Hofman
(Liggins Institute, University of Auckland, Auckland, New Zea-
land); Dr. Stephen Butler (Taranaki District Health Board, New
Plymouth, New Zealand); Dr. Yvonne C. Anderson (Liggins In-
stitute, University of Auckland, Auckland, New Zealand, and
Taranaki Base Hospital, New Ply mouth, New Zealand); Dr. Ian
Holdaway (Auckland City Hospital and Greenlane Clinical
Centre, Auckland, New Zealand); and Dr. Karen Carpenter
(Department of Diagnostic Genomics, PathWest Laboratory
Medicine Western Australia, Perth, Australia).
Appendix
The authors are as follows: Giampaolo Trivellin, Ph.D., Adrian F. Daly, M.B., B.Ch., Ph.D., Fabio R. Faucz, Ph.D., Bo Yuan, B.S., Liliya
Rostomyan, M.D., Darwin O. Larco, Ph.D., Marie Helene Schernthaner-Reiter, M.D., Ph.D., Eva Szarek, Ph.D., Letícia F. Leal, Ph.D.,
Jean-Hubert Caberg, Ph.D., Emilie Castermans, Ph.D., Chiara Villa, M.D., Ph.D., Aggeliki Dimopoulos, M.D., Prashant Chittiboina,
M.D., Paraskevi Xekouki, M.D., D.Sc., Nalini Shah, M.D., D.M., Daniel Metzger, M.D., Philippe A. Lysy, M.D., Ph.D., Emanuele Fer-
rante, M.D., Ph.D., Natalia Strebkova, M.D., Ph.D., Nadia Mazerkina, M.D., Ph.D., Maria Chiara Zatelli, M.D., Ph.D., Maya Lodish,
M.D., Anelia Horvath, Ph.D., Rodrigo Bertollo de Alexandre, Ph.D., Allison D. Manning, M.Sc., Isaac Levy, M.D., Margaret F. Keil,
Ph.D., P.N.P., Maria de la Luz Sierra, M.S., Leonor Palmeira, Ph.D., Wouter Coppieters, Ph.D., Michel Georges, M.D., Ph.D., Luciana
A. Naves, M.D., Ph.D., Mauricette Jamar, M.D., Vincent Bours, M.D., Ph.D., T. John Wu, Ph.D., Catherine S. Choong, M.D., M.B., B.S.,
Jerome Bertherat, M.D., Ph.D., Philippe Chanson, M.D., Ph.D., Peter Kamenický, M.D., Ph.D., William E. Farrell, Ph.D., Anne Barlier,
M.D., Ph.D., Martha Quezado, M.D., Ivana Bjelobaba, Ph.D., Stanko S. Stojilkovic, Ph.D., Jurgen Wess, Ph.D., Stefano Costanzi, Ph.D.,
Pengfei Liu, Ph.D., James R. Lupski, M.D., Ph.D., D.Sc., Albert Beckers, M.D., Ph.D., and Constantine A. Stratakis, M.D., D.Sc.
The author’s affiliations are as follows: the Section on Endocrinology and Genetics, Program on Developmental Endocrinology and
Genetics and Pediatric Endocrinology Interinstitute Training Program (G.T., F.R.F., M.H.S.-R., E.S., L.F.L., A.D., P.X., M.L., A.H.,
R.B.A., A.D.M., I.L., M.F.K., M.L.S., C.A.S.), and the Section on Cellular Signaling, Program in Developmental Neuroscience (I.B.,
S.S.S.), Eunice Kennedy Shriver National Institute of Child Health and Human Development, the Surgical Neurology Branch, National
Institute of Neurological Disorders and Stroke (P. Chittiboina), Laboratory of Pathology, National Cancer Institute (M.Q.), and the
The New England Journal of Medicine
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12
Gigantism and Acromegaly Due to X
q26 abnormalities
Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases
(J.W.) — all at the National Institutes of Health, and the Department of Obstetrics and Gynecology, Uniformed Services University of the
Health Sciences (D.O.L., T.J.W.) — both in Bethesda, MD; the Departments of Endocrinology (A.F.D., L.R., C.V., A. Beckers) and
Clinical Genetics (J.-H.C., E.C., M.J., V.B.), Centre Hospitalier Universitaire de Liège, University of Liège, Domaine Universitaire du
Sart-Tilman, and Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-Genomics, Domaine Universitaire Sart-Tilman
(L.P., W.C., M.G.), Liège, and the Pediatric Endocrinology Unit, Department of Pediatrics, Clinique Universitaire Saint-Luc, Université
Catholique de Louvain, Louvain (P.A.L.) — all in Belgium; School of Health and Biosciences, Pontifícia Universidade Católica do Paraná,
Curitiba (F.R.F., R.B.A.), and Department of Endocrinology, Faculty of Medicine, University of Brasilia, Brasilia (L.A.N.) — both in
Brazil; Departments of Molecular and Human Genetics (B.Y., P.L., J.R.L.) and Pediatrics (J.R.L.), Texas Children’s Hospital (J.R.L.),
Baylor College of Medicine, Houston; Department of Anatomical and Cytological Pathology, Hôpital Foch, Suresnes (C.V.), INSERM
Unité 1016, Institut Cochin, Université Paris Descartes, Hôpital Cochin, Service d’Endocrinologie, Paris (C.V., J.B.), Assistance Pu-
blique–Hôpitaux de Paris, Hôpitaux Universitaires Paris-Sud, Service d’Endo crinologie et des Maladies de la Reproduction et Centre
de Référence des Maladies Endocriniennes Rares de la Croissance, and Université Paris Sud 11, Faculté de Médecine, UMR-S693, Le
Kremlin-Bicêtre (P. Chanson, P.K.), and Aix-Marseille Université, Centre National de la Recherche Scientifique, UMR-7286, Centre de
Recherche en Neurobiologie et Neurophysiologie de Marseille, Marseille (A. Barlier) — all in France; Department of Endocrinology,
King Edward Memorial Hospital, Mumbai, India (N. Shah); Endocrinology and Diabetes Unit, BC Children’s Hospital, Vancouver, Brit-
ish Columbia (D.M.), and the Division of Endocrinology, Hospital for Sick Children, Toronto (I.L.) — both in Canada; Endocrinology
and Diabetology Unit, Fondazione Istituto de Ricovero e Cura a Carattere Scientifico Ca’ Granda Ospedale Maggiore Policlinico, Milan
(E.F.), and Section of Endocrinology, Department of Medical Sciences, University of Ferrara, Ferrara (M.C.Z.) — both in Italy; Endocrino-
logical Research Center, Institute of Pediatric Endocrinology (N. Strebkova), and Burdenko Neurosurgery Institute (N.M.) — both in
Moscow; Department of Pharmacology and Physiology, George Washington University (A.H.), and Department of Chemistry and Center
for Behavioral Neuroscience, American University (S.C.) — both in Washington, DC; Department of Paediatric Endocrinology, Princess
Margaret Hospital for Children and School of Pediatrics and Child Health, University of Western Australia, Perth, Australia (C.S.C.); and
Institute for Science and Technology in Medicine, Keele University, Guy Hilton Research Centre, Stoke-on-Trent, United Kingdom (W.E.F.).
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