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Research article
The Journal of Clinical Investigation http://www.jci.org
Digenic mutations account
for variable phenotypes in idiopathic
hypogonadotropic hypogonadism
Nelly Pitteloud,1 Richard Quinton,2,3 Simon Pearce,2,4 Taneli Raivio,1 James Acierno,1
Andrew Dwyer,1 Lacey Plummer,1 Virginia Hughes,1 Stephanie Seminara,1 Yu-Zhu Cheng,2,4
Wei-Ping Li,2,4 Gavin Maccoll,5 Anna V. Eliseenkova,6 Shaun K. Olsen,6 Omar A. Ibrahimi,6
Frances J. Hayes,1 Paul Boepple,1 Janet E. Hall,1 Pierre Bouloux,5
Moosa Mohammadi,6 and William Crowley Jr.1
1Reproductive Endocrine Unit of the Department of Medicine and Harvard Reproductive Endocrine Science Centers, Massachusetts General Hospital, Boston,
Massachusetts, USA. 2Department of Endocrinology and 3Royal Victoria Infirmary, School of Clinical Medical Sciences, and 4Institute for Human Genetics,
University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom. 5Department of Endocrinology, Royal Free Hospital, London, United Kingdom.
6Department of Pharmacology, New York University School of Medicine, New York, New York, USA.
Idiopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone
(GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-
gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial
variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the
model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some
families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome
(IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a
heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releas-
ing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and
across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embry-
onic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2
that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize
to produce a more severe phenotype in IHH families than either alone. This genetic model could account for
some phenotypic heterogeneity seen in GnRH deficiency.
Introduction
Genetic analyses of idiopathic hypogonadotropic hypogonad-
ism (IHH), an important human disease model with implications
for the discovery of genes responsible for human puberty, have
provided considerable insight into the genes that control sexual
maturation. IHH is a clinically and genetically heterogenous dis-
order resulting in gonadotropin-releasing hormone (GnRH) defi-
ciency that can be inherited as an X-linked, autosomal recessive,
or autosomal dominant trait. IHH has been considered to be a
monogenic disorder with several loci identified to date: Kallmann
syndrome 1 sequence (KAL1) (1–3), FGF receptor 1 (FGFR1) (4), proki-
neticin 2 (PROK2), and prokineticin receptor 2 (PROKR2) (5) under-
lie cases of Kallmann syndrome (KS), while gonadotropin-releasing
hormone receptor (GNRHR) (6), FGFR1 (7), and G protein–coupled
receptor 54 (GPR54) (8, 9) underlie normosmic IHH (nIHH). Addi-
tionally, nasal embryonic LHRH factor (NELF) has been implicated in
the pathogenesis of KS (10). Despite these advances, conundrums
remain in understanding the genetic basis of IHH. For example,
there is a puzzling clinical heterogeneity of the reproductive and
nonreproductive phenotypes both within and across IHH families
carrying identical single gene mutations (4, 6, 11–14). Therefore,
a given genotype at a single locus cannot reliably predict the phe-
notypic manifestations in any given member of affected families.
Additionally, some mutations in genes accounting for IHH, espe-
cially FGFR1, apparently demonstrate incomplete penetrance (4).
Finally, defects in the identified genetic loci account for only a
small percentage (<30%) of cases. Thus, it is likely that other major
IHH loci remain to be discovered and/or that the remaining (>70%)
cases are caused by the interplay of several contributing genes.
We hypothesized that IHH often involves defects in more than 1
gene. Herein we report evidence of IHH caused by the interaction
of 2-gene defects (FGFR1 and NELF in pedigree 1 and GNRHR and
FGFR1 in pedigree 2). In addition, in vitro biochemical character-
ization of FGFR1 and NELF mutants is provided.
Results
Pedigree 1. Pedigree 1 has several affected members (Figure 1). The
proband (no. 1-03) was referred to an endocrinologist at age 21 for
failure to undergo puberty. His presentation was consistent with a
severe KS phenotype. He was unvirilized, had eunuchoidal propor-
tions (height, 176 cm; span, 185 cm), bilateral gynecomastia, micro-
phallus, prepubertal testes (2 ml; normal, >12 ml), a repaired cleft
lip/palate, and clinodactyly. His serum gonadotropin levels were
undetectable, testosterone (T) was 1 nmol/l, and inhibin B 74 pg/ml;
otherwise, he had normal pituitary function and brain imaging.
Nonstandard abbreviations used: D3, immunoglobulin-like domain 3; FGFR1, FGF
receptor 1; GnRH, gonadotropin-releasing hormone; GNRHR, gonadotropin-releasing
hormone receptor; IHH, idiopathic hypogonadotropic hypogonadism; KAL1, Kall-
mann syndrome 1 sequence; KS, Kallmann syndrome; NELF, nasal embryonic LHRH
factor; nIHH, normosmic IHH; SPR, surface plasmon resonance; T, testosterone.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. doi:10.1172/JCI29884.
research article
The Journal of Clinical Investigation http://www.jci.org
Formal testing revealed hyposmia (score of 29/40, below fifth percen-
tile for his age) (15). Two subsequent years of gonadotropin therapy
induced full virilization and sperm production. His father (no. 1-01)
had a history of delayed puberty (growth spurt and full virilization
after age 17) and congenital anosmia (score of 13/40), and his adult
serum T was 18.1 nmol/l. The proband’s mother (no. 1-02) had clino-
dactyly and Duane ocular retraction syndrome and was menopausal.
The proband’s sister (no. 1-05) exhibited midline defects, including
a bifid nose and high arched palate; the brother (no. 1-04) exhibited
clinodactyly only. The mother and both siblings had normal puberty
and a normal sense of smell as determined by formal testing.
Mutational analysis of the FGFR1 gene. The proband (no. 1-03) car-
ries a unique heterozygous mutation (c.1025 T→C) in exon 7 pre-
dicted to substitute a leucine for serine at position 342 (p.L342S)
in the immunoglobulin-like domain 3 (D3) of FGFR1 (Figure 1,
Figure 2, A and B, and Figure 3G). This change was also found
in the affected father (no. 1-01) and the affected sister (no. 1-05)
(Figure 1) but not in 200 white controls.
Structural and biochemical analysis of the L342S mutation impli-
cate a loss of FGF8b signaling through FGFR1c in the pathology of
KS/IHH. The isoforms FGFR1b and FGFR1c are generated
by alternative splicing of exons 8A and 8B, respectively
(16). To date, FGFR1 mutations causing IHH have only
been identified in exon 8B (17), implicating FGFR1c in the
pathogenesis of IHH. Because L342 is highly conserved
among the “c” splice isoforms of FGFR1–3 across species
(Figure 2B), we used the FGFR2c-FGF8b crystallography
model (18) to study L342S. The corresponding amino acid
in FGFR2c, L343, is a key constituent in the hydrophobic
groove of D3 and is extensively engaged by FGF8b (18) (Figure 3G).
This leucine accounts for the unique binding specificity of FGF8b
for the “c” isoforms of FGFR1–3. Consistent with these structural
data, surface plasmon resonance (SPR) analysis revealed a dramatic
loss (20-fold) in the affinity of the L342S mutant for FGF8b (Figure
3, C and F), with only a small decrease (2-fold) in affinity for FGF1
(Figure 3, A and D) and (3-fold) FGF2 (Figure 3, B and E).
L342S FGFR1c missense mutation is a loss-of-function mutation. L6 myo-
blasts transiently expressing WT FGFR1c were treated with FGF8b,
which induced a 6-fold increase in LUC reporter gene expression (Fig-
ure 3H). In agreement with SPR results, the L342S FGFR1c was silent
when expressed alone. As the KS subject harbored a heterozygous
L342S mutation, we further coexpressed the WT FGFR1 and L342S
in 1:1 and 1:2 ratios. The results are compatible with the hypothesis
that this mutant acts as a dominant-negative mutation.
The variable degree of sexual maturation among family mem-
bers carrying the same FGFR1 mutation led to further candidate
Figure
Identification of FGFR1 (p.L342S)
and NELF (8-bp intronic deletion)
mutations in pedigree 1; identification
of GNRHR [p.Q106R] and [p.R262Q]
and FGFR1 (p.R470L) mutations in
pedigree 2. Only subjects harboring 2
gene defects have IHH. Probands are
identified by arrows; circles denote
females, squares denote males. Del,
NELF intronic deletion.
Figure
Schematic showing location of the 2 FGFR1 mutations and
conservation of L342 and R470 residues across species and
FGFRs. (A) The FGFR1 gene is located on chromosome 8p.
FGFR1 contains 18 exons with intervening introns not drawn
due to scale. SP, signal peptide; TM, transmembrane domain;
TK, tyrosine kinase domain. (B) Comparison of L342 and R470
across species and within the FGFR family.
research article
The Journal of Clinical Investigation http://www.jci.org
gene screening and the identification of an additional deletion in
NELF in the severely affected proband.
Mutational analysis of the NELF gene. A heterozygous 8-bp deletion
ending 14 bp before exon 10 (c.1159-14_-22del) was identified in the
proband (no. 1-03), his mother (no. 1-02), and his brother (no. 1-04)
(Figure 4A). This deletion was not observed in 384 white controls.
RT-PCR. The HEK-293 cells transfected with the WT plasmid
expressed NELF exons 8–11 at the expected size (291 bp). However,
cells transfected with the 8-bp intronic deletion plasmid expressed
an additional 257-bp splice form of NELF mRNA, lacking exon
10 (Figure 4C). The missplicing of mRNA lacking exon 10 results
in a premature stop codon that predicts a truncated NELF pro-
tein product (p.Y376X) rather than the full-length product, which
comprises 528 residues.
Immunohistology of FNC-C4 cells. Colocalization of both NELF and
GnRH1 in FNC-B4 cells was demonstrated by immunohistochemistry
(Figure 4D) and RT-PCR (data not shown).
Genotype-phenotype correlations. Proband no. 1-03 exhibited a
severe KS phenotype with absent puberty, microphallus, hyposmia,
undetectable serum luteinizing hormone and follicle stimulating
hormone levels, hypogonadal T levels, and low inhibin B levels. He
also had clinodactyly and cleft lip and palate. He harbored both a
paternally derived heterozygous FGFR1 mutation (c.1025 T→C,
p.L342S), resulting in weaker binding to FGF8b, and a maternally
derived heterozygous 8-bp intronic deletion of NELF, resulting in
a splicing defect of exon 10 and premature stop codon (Figures
1–4). While a single gene defect was associated with an attenuated
phenotype (i.e., delayed puberty and anosmia in the father carrying
L342S only), 2 mutant genes/gene products (FGFR1 and NELF)
synergized to produce a more severe KS phenotype.
Pedigree 2. All members of the second, previously reported (14)
pedigree were normosmic by formal smell testing, including 2 sis-
ters affected with nIHH (Figure 1). The proband (no. 2-03) present-
ed at 17 with primary amenorrhea, no breast development, short
fourth metacarpals, and osteoporosis. She had undetectable serum
gonadotropin and estradiol (E2) levels but otherwise normal pitu-
itary function and cranial imaging. As previously reported, pulsatile
GnRH induced ovulation in the proband, but she had 5 consecutive
miscarriages (14). Her sister also presented at 18 with primary amen-
orrhea, absent breast development, and scoliosis. She had 2 success-
Figure
The L342S mutation dramatically reduces affinity of FGFR1c for FGF8b, implicating decreased FGF8b/FGFR1c signaling in the etiology of
KS/IHH. (A–F) The L342S mutation reduces the affinity of FGFR1c for FGF8b. Varying concentrations of WT (A–C) or L342S FGFR1c mutant
(D–F) were injected over a CM5 chip onto which FGF1 (A and D), FGF2 (B and E), and FGF8b (C and F) were immobilized. Analyte concentra-
tions are indicated as follows: 31.25 nM in gray, 62.5 nM in violet, 125 nM in green, 250 nM in red, 500 nM in blue. (G) The location of L343 in
FGFR2c, the residue corresponding to L342 in FGFR1c, is mapped onto the FGF8b-FGFR2c structure (18). The L342S mutation should weaken
key hydrophobic contacts between F32, V36, and F93 of FGF8b and D3 of FGFR. Gray: molecular surface of FGFR; orange: FGF8b. The side
chains of selected residues are shown. The molecular surface of the hydrophobic groove of FGFR D3 (yellow) is rendered transparent so that
the side chain of L343 (the residue corresponding to L342 of FGFR1c) is visible. (H) L342S FGFR1 heterozygous mutation is a loss-of-function
mutation. WT and L342S FGFR1c were transiently transfected into L6 myoblasts with an FGFR1-responsive osteocalcin promoter luciferase
construct. FGF8b treatment of WT FGFR1c induced a 6-fold increase in LUC reporter gene expression, while the L342S FGFR1c alone remained
silent. The coexpression of the WT and L342S FGFR1c suggests that this mutation acts as a dominant negative.
research article
The Journal of Clinical Investigation http://www.jci.org
ful pregnancies on gonadotropin therapy. Her daughter (no. 2-06)
underwent normal puberty and has scoliosis, and male twins (nos.
2-07 and 2-08) were born without cryptorchidism or microphallus
and have yet to undergo puberty (10 years old). The proband’s father
has a history of delayed puberty, scoliosis, and bilateral hearing loss.
The brother was born with a cardiac septal defect and 3 fused cervi-
cal bones and went through normal puberty. The mother is meno-
pausal but had normal reproductive function.
GNRHR mutational analysis. A compound heterozygous muta-
tion in GNRHR [Q106R] and [R262Q] was identified in the pro-
band (no. 2-03) and her sister (no. 2-05) (14). Both exhibited severe
nIHH with absent puberty. Their father (no. 2-01) with a history
of delayed puberty was heterozygous for R262Q, while the mother
with no reproductive phenotype (no. 2-02) was heterozygous for
Q106R. In addition, the brother (no. 2-04) and the niece (no. 2-06)
were WT, while the twins (nos. 2-07 and 2-08) were heterozygous for
Q106R and R262Q, respectively (Figure 1). In 1997, de Roux et al.
described 2 siblings with partial nIHH as evidence by spontane-
ous testicular growth and active spermatogenesis in the male and
Tanner IV breast development in the female
carrying the same compound heterozygous
[Q106R] and [R262Q] mutation in the GNRHR
gene. In vitro studies demonstrated that each
mutant, [Q106R] and [R262Q], resulted in loss
of function (6). The variable expressivity rang-
ing from absent puberty to a partial puberty
across families carrying the same compound
heterozygous GNRHR mutation led to further
candidate gene screening, which identified an
additional FGFR1 mutation in our pedigree.
FGFR1 mutational analysis. We identified
a heterozygous mutation in FGFR1 (c.1409
G→T) in exon 10 in both nIHH sisters. This
nucleotide change is predicted to substitute an
arginine for leucine at position 470 (p.R470L)
(Figure 2A). Screening of the entire pedigree
revealed this change only in the father with
delayed puberty (no. 2-01) and the unaffected
niece (no. 2-06) (Figure 1). R470 is conserved
across species (Figure 2B), and R470L was not
detected in 200 white controls.
The R470L mutation reduces the tyrosine kinase
activity of FGFR1. In the FGFR1 kinase struc-
ture, arginine 470 (R470) lies at the junction
between the kinase domain and the juxta-
membrane region (Figure 2A). The side chain
of R470 makes 3 hydrogen bonds with D468;
the latter engages in a hydrogen bond with
K536 from the αC helix (Figure 5A) (19).
Crystal structures of unphosphorylated and
phosphorylated kinase domains show distinct
and reversible movements of the αC helix dur-
ing the kinase activation/inactivation cycle.
R470 both facilitates the conformation of the
juxtamembrane/kinase region and contributes
to proper αC positioning. Therefore, the R470L
mutation should negatively impact the tyrosine
kinase activity of FGFR1. Indeed, comparison
of WT FGFR1 and the R470L mutant revealed a
marked decrease in the tyrosine kinase activity
in the mutant, indicating a loss of function (Figure 5B).
Genotype-phenotype correlations. The sisters severely affected with
nIHH harbor both a compound heterozygous mutation in the
GNRHR gene [Q106R] and [R262Q] and a heterozygous FGFR1
mutation (R470L) — therefore a triallelic pattern of inheritance. The
additional FGFR1 mutation could explain the variable phenotypic
expressivity seen between pedigrees harboring the same compound
heterozygous GNRHR mutation (6). Interestingly, the father (no.
2-01), with a combination of 2 heterozygous mutant alleles (GNRHR
and FGFR1), had delayed puberty, while other family members carry-
ing only 1 mutant allele display a normal reproductive phenotype.
Discussion
IHH appears to follow the pattern of several disorders that were
initially thought to be monogenic but have subsequently proven
to be caused by or modulated by more than 1 gene defect (20, 21).
In contrast to polygenic traits, these oligogenic disorders involve
the synergistic action of mutant alleles at a small number of loci.
This report expands our understanding of the genetics of IHH and
Figure
A 8-bp intronic NELF deletion results in missplicing of exon 10. (A) Direct DNA sequencing of
cloned PCR products of the proband’s genomic DNA revealed a heterozygous 8-bp deletion
in intron 9. This deletion is part of a direct 8-bp repeat (tgtggcct) and occurs 14 bp upstream
of the exon 10 acceptor (5′) splice site. The lower case part of the sequence indicates introns
while the upper case indicates exons. (B) Predicted cDNAs in the exon 8–11 region of the
NELF gene. The proband was found to have an 8-bp deletion in intron 9 (black triangle), 14
bp upstream of exon 10. (C) The result of RT-PCR using HEK-293 cell mRNA from cells
transfected with WT NELF genomic construct containing exons 8–11 and the mutant NELF
construct (8-bp deletion in intron 9). The HEK-293 cells transfected with the WT NELF con-
struct show a normally spliced NELF exon 8–11 RT-PCR product, corresponding to the pre-
dicted size of 291 bp. The cells transfected with the mutant NELF construct, however, show
an additional band of 257 bp corresponding to the expected size of a transcript lacking exon
10. The RNA from cells transfected with either WT or mutant genomic construct show an
additional product of 794 bp (786 bp for the mutant construct), reflecting PCR amplification of
the residual plasmid DNA. (D) Colocalization of NELF and GnRH1 in the olfactory epithelial
cell line FNC-B4 by immunohistochemistry. Original magnification, ×150.
research article
The Journal of Clinical Investigation http://www.jci.org
suggests oligogenicity may underly other monogenic disorders
also characterized by incomplete penetrance and variable pheno-
types within and between families.
IHH is currently described as a monogenic disorder resulting in
defective GnRH secretion or action, with 7 loci implicated in the
pathogenesis of the disease to date. However, the genotype-pheno-
type correlations from specific mutations in different loci have been
imprecise. An illustrative example is FGFR1. Numerous heterozygous
FGFR1 mutations underlie cases of KS (4, 13, 22–25) and nIHH (7,
26), characterized by marked phenotypic variability both within and
between families and apparent incomplete penetrance. Further-
more, although FGFR1 mutations are known as causing an auto-
somal dominant form of IHH, there is a report of 1 KS subject with
a very severe phenotype harboring a homozygous FGFR1 mutation
(4). These data suggest a dosage effect of the FGFR1 mutant alleles
and the existence of modifier genes and/or environmental effects
leading to phenotypic variability across and within families.
Herein, we report a proband who carries heterozygous muta-
tions in both FGFR1 and NELF and exhibits a severe KS pheno-
type with cleft palate (pedigree 1). The FGFR1 mutation, L342S,
alters the spectrum of FGF binding by decreasing FGF8b binding
specifically. These results were corroborated by our reporter gene
assay, which showed the L342S mutation to be a loss-of-function
mutation, potentially having a dominant-negative effect in the
heterozygous state. In addition, these data implicate FGF8b as
a key ligand for FGFR1c in the pathogenesis of KS. These find-
ings are in accordance with studies on mice with a hypomorphic
Fgf 8 allele, which exhibited defected nasal cavity development and
olfactory bulbs dysgenesis, a phenotype similar to that observed
in the Fg fr1 conditional knockout mouse (27–29). Therefore, we
believe that the L342S FGFR1 mutation contributed to both the
KS and the craniofacial phenotype of the proband. However, the
KS phenotype failed to segregate with the L342S mutation, as only
the proband exhibited KS. Interestingly, he carried an additional
heterozygous intronic NELF deletion resulting in missplicing of
exon 10 and a premature stop codon. NELF plays a key role in
GnRH and olfactory neuron outgrowth (30–32) and is colocalized
with GnRH1 in human olfactory stem cells (Figure 4D). NELF has
previously been implicated as a potential locus for IHH in an affect-
ed hypogonadotropic patient with a unique heterozygous muta-
tion (c.1438A→G; p.T480A), though the biology of this mutant
remains unexplored (10). Interestingly, the mother (no. 1-02),
who carried the NELF mutation only, had Duane ocular retraction
syndrome (OMIM 126800), a disorder of neuronal migration that
has been associated with KS (33). Therefore, we believe that the
heterozygous mutations in both the NELF and the FGFR1 gene
synergized to cause severe KS in the proband, while individuals
carrying only 1 of the 2 mutations do not present with KS.
Since 1997, GNRHR mutations have been known to cause a reces-
sive form of nIHH. However, unexplained variable expressivity of
IHH across pedigrees carrying the same compound heterozygous
mutation in GNRHR (6, 14) led us to screen further candidate
genes. As a result, we identified a triallelic mode of inheritance
for IHH in pedigree 2. Subjects carrying only 1 mutant allele
(FGFR1 or GNRHR) had a normal reproductive phenotype. Fur-
ther, the individual (no. 1-01) carrying both heterozygous FGFR1
and GNRHR mutations had only a mild reproductive phenotype
(delayed puberty), while the 2 sisters harboring both a loss-of-func-
tion compound heterozygous mutation in GNRHR and a loss-of-
function heterozygous FGFR1 mutation (R470L) had severe IHH
phenotypes. Although IHH was initially thought to be transmit-
ted as a recessive trait, this pedigree indicates a triallelic digenic
inheritance of IHH, suggesting that in some families, more than
2 mutant alleles might be required to manifest IHH. Similar pat-
terns of mutation lead to Bardet-Biedl syndrome (34).
Only 30% of IHH subjects studied to date harbor a mutation
in 1 of the genes known to cause IHH, and thus it is not possi-
ble to infer the frequency of oligogenicity in IHH. The concept
of digenicity was evoked but not proven in a recent report that
described a KS subject carrying both a KAL1 missense mutation
and a PROKR2 missense mutation (5). However, the functionality
of these mutants was not explored, and no data were presented on
the pedigree of this subject.
The discovery of oligogenic traits in IHH raises both conceptual
and practical issues. Because IHH is no longer considered to be a
monogenic disorder, the transmission of a trait through families is
no longer synonymous with the transmission of 1 specific mutant
allele. Therefore, we must rethink our current approach in study-
ing the genetics of IHH, separating the segregation of a trait from
the segregation of a specific mutant allele (21). The results of this
study indicate that labeling mutant alleles as dominant or recessive
is often an oversimplification. Oligogenicity also has implications
for genetic counseling regarding IHH. Finally, the question remains
Figure
The R470L FGFR1 mutation is loss-of-function. (A) The R→L sub-
stitution abolishes hydrogen bonds that play a role in the positioning
of the C-helix of FGFR1. The unphosphorylated “low-activity” form of
the FGFR1 kinase domain (PDB ID: 1FGK) is represented as a ribbon
diagram. The N-lobe of the FGFR1 kinase is shown in green, with
the exception of the αC helix, which is in blue. Red: C-lobe of FGFR1
kinase; gray: linker connecting the N- and C-lobes; yellow: activa-
tion loop in the C-lobe. Selected residues are shown, and hydrogen
bonds are represented as dashed lines. R470 indirectly contributes to
the proper positioning of αC in the kinase domain and hence kinase
regulation by engaging in 3 hydrogen bonds with D468, which in turn
engages in a hydrogen bond with K536 from αC helix. (B) The R470L
mutation reduces the tyrosine kinase activity of FGFR1.
research article
The Journal of Clinical Investigation http://www.jci.org
as to whether the variability of expressivity in IHH is controlled by a
small number of major loci or a large number of minor loci.
The molecular basis of oligogenicity is poorly understood. To
date, one of the best examples of the molecular basis of digenic
inheritance is the direct interaction of the mutants retinal outer
segment membrane protein 1 and retinal degeneration slow,
which causes retinitis pigmentosa (35). The 2 mutants combine to
prevent the formation of a tetrameric complex that is important
for the integrity of the photoreceptors. Further examples in which
synergistic mutations at different loci cause a disease are found in
diseases characterized by mutations in receptor-ligand pairs, such
as Hirschsprung disease (RET and GDNF) (36). In addition, indi-
rect interactions between 2 mutated proteins have been reported
in severe insulin resistance where PPARG and PPP1R3A mutants
are expressed in 2 different tissues (37).
In contrast, little is known about the molecular pathways where-
by the 2 pairs of mutant proteins (NELF and FGFR1; GnRHR
and FGFR1) contribute to the IHH phenotype. FGFR1 and
NELF are both expressed in GnRH neurons (30, 38, 39). There-
fore, the 2 mutant proteins may act at different levels of the same
intracellular pathway and may quantitatively contribute to its
progressive dysfunction until a critical threshold is reached, thus
producing the disease phenotype. Alternatively, these 2 proteins
may participate in a multiprotein complex that becomes progres-
sively compromised by the additional mutations. In the other
gene pair (FGFR1 and GNRHR), the FGFR1 mutant is anticipated
to reduce the number of GnRH neurons, as evidenced by a mouse
model with targeted dominant-negative FGFR1 in the GnRH neu-
rons (40). These mice, although fertile, display a 30% decrease in
the number of GnRH neurons in the hypothalamus. Conversely,
the GNRHR mutants result in GnRH resistance that would
require a compensatory increase in GnRH to restore reproductive
function. Such a putative compensatory mechanism would be
impaired in the individuals with an additional FGFR1 mutation.
Thus, deficient GnRH migration, secretion, and/or action could
be caused by the interaction of these mutants in the same or dif-
ferent developmental pathways, leading to subtle amplification
effects of each mutant protein.
This report expands our understanding of the genetics of this
disorder by demonstrating that IHH can be caused by the com-
bination of gene defects. The complexity of this genetic model
implies a revision of the genetic terminology of IHH. Furthermore,
these data predict that mutational analysis in multiple genes for
seemingly monogenic disorders will become increasingly frequent
and may lead to greater accuracy in phenotypic predictions. In the
case of IHH, studying the interaction of a small number of key
loci will undoubtedly be key to understanding some of the broader
variability of the timing of normal puberty.
Methods
Mutational analysis. Genetic studies were approved by Partners Health-
care Human Research Committee, and all participants provided written
informed consent.
Whole blood samples were obtained from subjects and genomic DNA
extracted. Exons and exon-intron boundaries were amplified using
standard PCR techniques for GNRHR (Genbank accession number
NM_001012763 [ref. 41]); KAL1 (accession number NM_000216 [ref. 42]);
GPR54 (accession number NM_032551 [ref. 9]); FGFR1 (accession num-
ber BC018128 [ref. 4]); and NELF (accession number ENSG00000165802
[ref. 10]). Nonsense changes resulting in a truncated protein, frameshift,
insertion, or deletion were categorized as definite mutations. Nucleotide
changes, which were (a) absent from the Single Nucleotide Polymorphism
database (http://www.ncbi.nlm.nih.gov/projects/SNP/) and expressed
sequence tags and (b) absent in at least 170 ethnically matched healthy
controls were identified as mutations. All genes and proteins are described
using standard nomenclature (43).
Control population. To determine whether the observed bp changes in the
genes cited above were normal variants, a cohort of adult healthy white
subjects (n ≥ 200) were screened.
Structural analysis of the effects of FGFR1 mutations on FGFR1 function. Crystal
structures of the FGFR1 kinase domain (Research Collaboratory for Struc-
tural Bioinformatics Protein Data Bank identification [PDB ID]: 1FGK)
(19) and the extracellular ligand binding region of FGFR2c in complex
with FGF8b and heparin oligosaccharide (PDB ID: 2FDB) (18) were used
to map the potential effects of the mutations. Crystal structures were visu-
alized using program O (44).
SPR analysis. Real-time biomolecular interactions between WT and L342S
mutant FGFR1c extracellular domains with FGF1, FGF2, and FGF8 were
characterized with a Biacore 3000 instrument as previously described (18).
Plasmids and in vitro mutagenesis. Human WT FGFR1c cDNA (NM_000604)
was subcloned into a pcDNA3.1+ expression vector (Invitrogen). The FGFR1c
L342S mutation was introduced into the human FGFR1c cDNA by site-direct-
ed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Stratagene) and
subcloned into pcDNA3.1+ using HindIII and XhoI restriction sites.
Reporter gene assay. To demonstrate that the heterozygous L342S muta-
tion was a loss-of-function mutation, we used an FGF reporter bioassay. WT
FGFR1c and L342S FGFR1c were transfected along with an osteocalcin FGF
response element (OCFRE) promoter/luciferase reporter (45) into L6 myo-
blasts, a cell line largely devoid of endogenous FGFRs and FGFs (46, 47). L6
myoblasts were maintained in DMEM containing penicillin (100 U/l), strep-
tomycin (100 μg/l), and 10% FCS (Invitrogen). A total of 4 × 104 cells per well
in 24-well plates were seeded and 24 hours later transiently transfected with
a total of 400 ng of DNA consisting of 100 ng of WT FGFR1c cDNA, 30 ng
of OCFRE reporter gene, and 0, 100, and 200 ng of the mutated FGFR1c
(L342S) and empty vector using FuGENE 6 Reagent (Roche Diagnostics).
Twenty-four hours after transfection, the cells were treated with increas-
ing doses of FGF8b (from 0 to 2,000 pM) in culture medium containing
0.1% FCS and 1 μg/ml heparin. Following 16 hours of incubation, the cells
were lysed, and luciferase activities were measured using Promega Luciferase
Assay System (Promega). Since Renilla and β-galactosidase were induced by
FGFs, the luciferase data are reported directly. Experiments were performed
in triplicate and repeated once. The data are reported as mean ± SD of a
representative experiment performed in triplicate.
Kinase assay. The tyrosine autophosphorylation activity of the WT and
R470L mutant FGFR1 kinase domains were quantified using a continuous
spectrophotometric assay as previously described (48).
Generation of the NELF mutant construct. To assess the effect of the intronic
deletion of the NELF gene on exon splicing, WT and mutant genomic NELF
constructs were made in the expression vector pCR3.1 and transfected into
HEK-293 cells (Lipofectamine 2000; Invitrogen). Oligonucleotide primers
(F5′: CAGTGACCTGCAGAGCTC-3′ and R5′: CCAGATCTTGGCTCCCTT-
GTG-3′) spanning the genomic sequence from within exons 8 and 11 were
used to amplify, via PCR, a fragment of WT DNA (794 bp) and mutant DNA
(786 bp in the deleted allele) (Figure 4B). RT-PCR was expected to yield a
291-bp product from a NELF cDNA with correct splicing of exons 9 and 10.
Immunohistochemistry of FNC-B4 cells. FNC-B4 cells, isolated from human
fetal olfactory epithelia, have characteristics of both secretory and migra-
tory GnRH neurons (39, 49). FNC-B4 cells were stained with either GnRH1
or NELF antibodies (30, 49). Secondary antibodies conjugated with either
Texas red or FITC were used to visualize GnRH1 and NELF staining.
research article
The Journal of Clinical Investigation http://www.jci.org
Acknowledgments
We thank David Ornitz for his kind gift of the OCFRE lucifer-
ase reporter. This work was funded by NIH grants DE13686,
HD15788, and HD028138.
Received for publication July 27, 2006, and accepted in revised
form November 27, 2006
Address correspondence to: Nelly Pitteloud, Reproductive Endo-
crine Unit, BHE 5, Massachusetts General Hospital, Boston, 02114
Massachusetts, USA. Phone: (617) 724-1830; Fax: (617) 726-5357;
E-mail: npitteloud@partners.org.
Moosa Mohammadi and William Crowley Jr. contributed equally
to this work.
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