Molecular Human Reproduction Vol.13, No.3 pp.

165170*, 2007
Advance Access publication on January 9, 2007

doi:10.1093/molehr/gal108

KAL1 mutations are not a common cause of idiopathic

hypogonadotrophic hypogonadism in humans
Balasubramanian Bhagavath1,2,3, Ning Xu2,3, Metin Ozata8, Robert L.Rosenfield4,
David P.Bick5, Richard J.Sherins6 and Lawrence C.Layman2,3,7,9
1

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern
Medical Center, Dallas, TX, 2Section of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and
Gynecology, The Medical College of Georgia, 3Developmental Neurobiology Program, The Institute of Molecular Medicine and
Genetics, The Medical College of Georgia, Augusta, GA, 4Pediatric Endocrinology, Department of Pediatrics, University of Chicago,
Chicago, IL, 5Division of Medical Genetics, Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of
Wisconsin, Milwaukee, WI, 6Genetics and IVF Institute, Fairfax, VA, 7Reproductive Medicine Program, The Institute of Molecular
Medicine and Genetics, The Medical College of Georgia, Augusta, GA, USA and 8GATA Haydarpasa Training Hospital, Department of
Endocrinology, Istanbul Turkey
9

To whom correspondence should be addressed at: Section of Reproductive Endocrinology, Infertility, and Genetics, Department of
Obstetrics and Gynecology, The Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-3360, USA. E-mail: Llayman@
mcg.edu

Hypogonadotrophic hypogonadism results in the absence of puberty and if left untreated leads to infertility. Mutations in KAL1
are known to account for some of the cases of Kallmann syndrome. The aim of this study was to determine the prevalence of KAL1
mutations in a large number of patients with idiopathic hypogonadotrophic hypogonadism (IHH). One hundred and thirty eight
patients (109 males and 29 females) with IHH were studied for mutations in KAL1. DNA from these patients was subjected to
denaturing gradient gel electrophoresis or single strand conformation polymorphism to identify mutations. Sequencing was performed to confirm mutations detected. Four mutations were found in 109 males (3.7%). All four mutations were in anosmic/hyposmic men making the prevalence 4/63 (6.3%) in this group of patients. No mutations were found in the 29 female patients. KAL1
mutations are an uncommon cause of Kallmann syndrome.
Key words: gene mutation/hypogonadotrophic hypogonadism/idiopathic hypogonadotrophic hypogonadism/KAL1 gene/Kallmann
syndrome

Introduction
Kallmann syndrome consists of the combination of hypogonadotrophic hypogonadism and anosmia. Associated clinical features
may include neurologic abnormalities (synkinesia, oculomotor deficits, deafness, mental retardation and cerebellar defects), unilateral
renal agenesis, midline facial defects and pes cavus. The hypogonadotrophic hypogonadism usually presents as absent puberty in males
with small testes, low serum levels of testosterone, follicle stimulating
hormone (FSH) and luteinizing hormone (LH) and azoospermia/oligospermia. Female patients present with absent puberty and absent
breast development. If untreated, infertility results. Replacement of gonadotrophin releasing hormone (GnRH) or gonadotrophins, improves
fecundity. The hypogonadotrophic hypogonadism appears to be
related to impaired GnRH neuron migration or reduced GnRH secretion

*N.B. An error was made in the initial online pagination of Molecular Human
Reproduction 13/3. The page span of this article was originally shown as 25
30. The publisher wishes to apologise for this error.

or pulsatility, or impaired GnRH action at the pituitary, which then

results in low gonadotrophin secretion (MacColl et al., 2002).
Kallmann syndrome was the first inherited cause of human idiopathic hypogonadotrophic hypogonadism (IHH), for which the molecular basis was characterized (Franco et al., 1991; Legouis et al.,
1991). Kallmann syndrome is caused by mutations in the KAL1
gene localized to chromosome Xp22.3, which encodes anosmin-1, a
secreted protein that has neural cell adhesion molecule properties.
The precise function of anosmin-1 remains unknown, but the protein
contains a signal peptide, a cysteine rich region, a highly conserved
four-disulfide core whey acid protein (WAP) domain, four fibronectin
type III (FNIII) repeats, and a C-terminal histidine-rich region (Franco
et al., 1991; Legouis et al., 1991). The WAP domain is found in protease inhibitors, which have been shown to play a role in axonogenesis
and neuron migration. The FNIII domains are present in extracellular
matrix proteins, cellular adhesion molecules and protein tyrosine kinases
and phosphatases that have been implicated in neuronal migration and
in axon targeting (Franco et al., 1991; Legouis et al., 1991).
Anosmin-1 appears to be involved in the migration of olfactory and
GnRH neurons from the olfactory placode region across the cribriform

# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For
Permissions, please email: journals.permissions@oxfordjournals.org
165

B.Bhagavath et al.
plate to synapse with the mitral cells of the olfactory bulb (GonzalezMartinez et al., 2004). It has been suggested that this neural network
provides a scaffold for GnRH neurons, which then arrive at their
normal position in the arcuate nucleus of the hypothalamus. In vitro
evidence in support of effects on both olfactory and neuroendocrine
development include: (i) anosmin-1 initiates neurite outgrowth from
olfactory bulb neurons in C. elegans (Rugarli et al., 2002) and rat
(Soussi-Yanicostas et al., 2002), as well as in primary human olfactory
neuroblasts (Gonzalez-Martinez et al., 2004), but does so in a cell-type
specific manner (Soussi-Yanicostas et al., 1998); (ii) anosmin-1
directly increases GnRH neuron migration in immortalized mouse
GN11 cells (Cariboni et al. 2004); (iii) disruption of the kal1.1 in
zebrafish also interferes with GnRH neuron migration (Whitlock
et al., 2006). It is possible that anosmin-1 interacts with FGFR1 to
effect GnRH neuronal migration (Gonzalez-Martinez et al., 2004).
A variety of KAL1 mutations in hypogonadotrophic hypogonadal
males have been described (Legouis et al., 1991; Bick et al., 1992;
Hardelin et al., 1992, 1993; Wulfsberg et al., 1992; Bouloux et al.,
1993; Martul et al., 1995; Parenti et al., 1995; Quinton et al., 1996;
Georgopoulos et al., 1997; Gu et al., 1998; Maya-Nunez et al.,
1998; ONeill et al., 1998; Weissortel et al., 1998; Persson et al.,
1999; Jansen et al., 2000; Matsuo et al., 2000; Izumi et al., 2001;
Oliveira et al., 2001; Soderlund et al., 2002; Massin et al., 2003;
Albuisson et al., 2005; Trarbach et al., 2005; Chocholska et al.,
2006). Initially, gene deletions were identified by Southern blot
analysis (Bick et al., 1992), but a variety of point mutations were
later characterized (Hardelin et al., 1993). KAL1 mutations were
initially proposed to be the most common cause of inherited human
hypogonadotrophic hypogonadism. Although common in clearly
recognized X-linked recessive families, the prevalence of KAL1
mutations varied between 3.1% and 27.8% in families where there
is only one affected individual. A small study of 25 males and 3
females with Kallmann syndrome suggested that the prevalence of
KAL1 mutations was 27.8% in sporadic IHH patients (5/18)
(Sato et al., 2004) but a larger study of 98 patients concluded that
the incidence was only 3.1% (2/65 cases) (Albuisson et al., 2005).
Although females may have IHH and anosmia, no KAL1 gene
mutations have been described to date. Even though X-linked diseases
affecting females are uncommon, there has been one report of an IHH
female with an adrenal hypoplasia congenita mutation (Merke et al.,
1999) making it important to include female Kallmann patients in
KAL1 mutation analysis. The purpose of the present study was to
determine the prevalence of KAL1 mutations in a large population
of males and females with IHH. Any identified mutations will
provide valuable information for future in vitro studies to understand
anosmin-1 protein function during development.

Materials and methods

Patients
One hundred and thirty eight (109 males and 29 females) consecutive, unrelated probands with IHH were studied for mutations in the KAL1 gene. IHH
was defined as irreversible pubertal delay at an age 17 years for females
and 18 years for males accompanied by low serum gonadotrophins and
normal prolactin and thyroid stimulating hormone (Bhagavath et al., 2006).
Males had serum testosterone ,100 ng dl21 (normal 3001100 ng dl21),
and females had irreversible hypoestrogenic amenorrhoea, as demonstrated
by an estradiol ,30 pg ml21 or a vaginal smear containing parabasal cells.
No pituitary tumour was demonstrated by sellar X-ray films, CT or MRI.
Since the full spectrum of KAL1 mutations is not known, IHH patients with
and without anosmia were studied. Anosmia was present in 50 males and
4 females, whereas hyposmia was present in an additional 13 males and
3 females.

166

IHH was also sub-classified according to the severity of hormone deficiency

into a completely deficient endocrine state (complete IHH) or a partially
deficient state (incomplete IHH). Complete IHH was defined as the complete
lack of sex steroid manifestations of puberty. In females, absent breast development (Tanner stage 1) and primary amenorrhoea constituted complete
IHH, whereas in males, testicular size 3 ml (the largest size found in
normal prepubertal males) constituted complete IHH (Burris et al., 1988).
Incomplete IHH was defined as the presence of any breast development in
females and as testicular volume 4 ml in males. This study was approved
by the Human Assurance Committee at the Medical College of Georgia.

DNA Analysis
DNA was extracted from each patient using standard methodology and subjected to polymerase chain reaction (PCR) for each of the 14 exons of the
KAL1 gene, along with their splice junctions. Primer sequences have been previously published by Hardelin et al. (1993) and conditions for PCR were 948C
(60 s), 558C (45 s), 728C (60 s) for exon 1 and 948C (60 s), 558C (45 s), 728C
(45 s) for exons 2 14. Each PCR included a negative control, which contained
all reagents except DNA. For denaturing gradient gel electrophoresis (DGGE),
a 40 bp GC-clamp was added to one of the primers of a primer pair to increase
the detection of mutations (Sheffield et al., 1989).
PCR products were first run on ethidium containing 1.2% agarose gels in the
presence of a molecular weight marker and photographed under ultraviolet
light to confirm presence of the fragment of interest. PCR products were
then subjected to DGGE or single strand conformation polymorphism (SSCP).
Denaturing gradient gels were 22 cm vertical 6.5% polyacrylamide gels containing 20 80% gradients (100% 7 M urea and 40% formamide). Gels were
electrophoresed at 400 1200 volt-hours, as determined by test gels, in E buffer
at 608C (Layman et al., 1997, 1998). SSCP analysis was performed as
described by Nishimura et al. (1998). Briefly, PCR products were mixed
with formamide dye, heat-denatured and then were electrophoresed on 6% nondenaturing acrylamide gels (5 ml glycerol, 5 ml 5X TBE, 12.5 ml 37 : 5 : 1
acrylamide/bis and 77.5 ml ddH2O) for 3 4 h at room temperature, silver
stained and photographed.
Variant fragments identified by DGGE/SSCP were subjected to DNA
sequencing using the Big Dye Terminator kit on an ABI 310 Automated
DNA sequencer according to the manufacturers instructions. Each sequencing
reaction was confirmed by repeat analysis in both forward and reverse directions. Putative mutations were confirmed at least six times.
For the patient with exon 1 to exon 13 deletion, PCR for the deleted exons
was repeated multiple times with positive controls to confirm the deletion.
To ensure that the deletion was not due to poor DNA quality, several exons
from additional genes (FGFR1 exon 9; TENR exons 8 and 10; DCC exons
15, 16 and 29) were subjected to PCR.
Analysis of sequence variations for possible polymorphism was performed
using Sorting Intolerant From Tolerant (SIFT) (Ng and Henikoff, 2001;
Raevaara et al., 2005), with a P , 0.05 being considered as intolerant.
Species included in this analysis are: human (gi#4557683), zebrafish
(gi#6708054), jungle fowl (gi# 45382209), chick (gi#50403735), puffer fish
(gi#6708052), Japanese quail (gi#50401099), Chimpanzee (gi#55662354),
cat (74007097), fruitfly (gi#45550809), mosquito (gi#58382167), C. elegans
(gi#25149859), moth (gi#18478825), Japanese rice fish (gi#88595354), freshwater pufferfish (gi#47226788), domestic cow (gi#76659851), red flour beetle
(gi#91076820), Bornean orangutan (gi#55728158), brushtail possum
(gi#14582214), honey bee (gi#66509567), mouse (gi#10946922), purple sea
urchin (gi#72095991), pig (gi#47522724), wild sheep (gi#78126182). Functional domains in the anosmin-1protein were identified using the Human
Protein Reference Database (Peri et al., 2003).

Results
For 136 of the 138 IHH patients, agarose gel electrophoresis demonstrated the appropriate sized bands for each of the 14 exons of
KAL1, documenting the lack of deletions. However, two patients
had intragenic deletions of KAL1. One patient, had a deletion of
exons 1 13, which were present in controls (exon 14 was present).
This patient had complete IHH, anosmia and bilateral cryptorchidism
and a maternal uncle and maternal cousin with Kallmann syndrome

Visual field
Hyposmia
Complete
WAP, Whey Acidic Protein; FN3, Fibronectin Type 3 domains.
Polymorphism.
a

Deletion of IVS12, exon13,

IVS13, exon14
Deletion of IVS12, exon13,
IVS13, exon14
IVS12 to 30 UTR

WAP, All FN3 repeats

Figure 1. Mutation/deletion Map of KAL1. S, Signal peptide; Cys rich,

Cysteine rich region; W, Whey acidic protein domain; FNIII, Fibronectin
type III domain; H, Histidine rich region; O, Mutations described in this manuscript; V, Polymorphisms described in this manuscript; " , Mutations
described in the literature; B, Deletions described in this manuscript.

FN3(4th)

Cryptorchidism
Cryptorchidism

Cryptorchidism
FN3 (part of 1; 2 4 deleted)

C163del
S206Sa
R257X
K666Ma R668Ha
K666Ma R668Ha
Deletion of exons 1 13
487 489delTGT
768G . Aa
769C . T
1997A.T 2003G . A
1997A.T 2003G . A
Deletion of exons 1 13
Exon 4
Exon 5a
Exon 6
Exon 14a
Exon 14a
Exon 1 13

WAP
Amino acid change
Nucleotide change

Mutation
Exon

Table I. Specific KAL1 mutations identified, along with summary of phenotypic features

Protein domain affected

IHH Type

Incomplete
Incomplete
Complete
Not available
Complete
Complete

Anosmia
Anosmia
Anosmia
Anosmia
Anosmia
Anosmia

Sense of smell

Renal agenesis

Other anomalies

Other affected
family members

Maternal uncle and

maternal cousins
One affected brother

KAL1 mutations in IHH

(Table I). This patients pedigree corresponds to family 2 in

Bhagavath et al. (2006).
The other patient with deletions of exons 13 and 14 (confirming the
findings previously described by Bick et al.) had unilateral cryptorchidism, micropenis and anosmia (Bick et al., 1992). His brother also
had Kallmann syndrome. Using DGGE and SSCP, 5 DNA sequence
differences were identified. Subsequent DNA sequencing and analysis
revealed five different mutations in five unrelated probands listed in
Table I and diagrammatically represented in Figure 1. These putative
mutations include: 487489delTGT (C163del), 769C . T (R257X),
1997A . T (K666M)/2003G . A (R668H) and 768G . A (S206S).
Two of the point mutations are highly likely to cause Kallmann syndrome. In one patient with incomplete IHH, anosmia and unilateral
renal agenesis, a C163del was observed. This C163del removes a
Cys residue in the WAP domain. Since codons 163 and 164 are
both Cys of the exact same sequence, the deletion could be of either
codon.
In another unrelated IHH proband with complete IHH, anosmia and
cryptorchidism, a R257X nonsense mutation was identified. This
mutation would be predicted to result in a truncated protein lacking
three of the four FNIII domains. A double mutant (K666M/R668H)
was found in two unrelated IHH patients, both of whom were
anosmic (Table I). The silent mutation (S206S) is a polymorphism.
SIFT has been used for assessing the significance of missense
mutations (Raevaara et al., 2005); however, the deletion of a Cys
residue C163del, is predicted to be deleterious as expected since
Cys residues participate in disulfide bond formation. This cysteine is
conserved 88% among 26 organisms analysed and no substitutions
are tolerated. Amino acids K at position 666 and R at position 668
are less well conserved at 31% and 15%, respectively. These substitutions are predicted to be tolerated with scores of 0.05 and 1.00,
respectively. The median sequence conservation is 3.63 for K666M
and 3.70 for R668H, making it unlikely that K666M is a mutation
even though the tolerated score is 0.05 (a score ,0.05 is considered
not tolerated).

Discussion
Kallmann syndrome is a common X-linked recessive form of human
hypogonadotrophic hypogonadism. KAL1 gene mutations account
for 33 70% of the cases of familial X-linked forms of Kallmann syndrome (Maya-Nunez et al., 1998; Sato et al., 2004; Albuisson et al.,
2005). The prevalence of KAL1 mutations in apparent sporadic
forms of IHH, with or without anosmia, has been variably reported
between 3.1% and 27.8% (Sato et al., 2004; Albuisson et al., 2005).
However, most of these are small studies.
In the present study, we screened 138 IHH patients (109 males and
29 females) for KAL1 mutations. No KAL1 mutations were identified
in the 29 females. Of 109 males, 50 had anosmia, 13 had hyposmia, 30
167

B.Bhagavath et al.
had normal sense of smell and data were not available in 16 patients.
Of 109 males, 4 had at least one other affected family member. Among
the 105 apparently sporadic male patients, 2 (1.9%) had KAL1
mutations. Of the 4 familial cases, 2 (50%) had KAL1 mutations/deletions. The overall prevalence of KAL1 mutations in males with IHH
was 4/109 (3.7%), but for anosmic/hyposmic males, the prevalence
was 4/63 (6.3%). These figures correspond to those of Albuisson
et al. (2005).
Four of the mutations, including three deletions (C163del, del exons
113 and the previously described deletion of exons 13 and 14) and
one point mutation (R257X) are highly likely to be causative. Two
unrelated probands had double variants (K666M/R668H). However,
it is uncertain if these represent true mutations or polymorphisms.
They will be tested in vitro to determine their effects upon cell
secretion and GnRH neuron migration.
In this study, we screened both normosmic and anosmic/hyposmic
IHH patients for mutations in KAL1 since variable expressivity with
KAL1 mutations has been reported. Siblings with the same intragenic
deletion (exons 313) in KAL1 have been reported to have very different clinical presentations including varying degrees of anosmia
(Massin et al., 2003). For these reasons, we studied both normosmic
and anosmic/hyposomic IHH patients, but found mutations only in
those with an abnormal sense of smell (Table I).
Initially DGGE and SSCP were used to detect mutations, which
would be expected to determine 80 90% of the mutations (Sheffield
et al., 1989; Nishimura et al., 1998). Using DGGE with GC-clamped
PCR products for subsequent analysis should permit the detection of
95% of the mutations (Sheffield et al., 1989). This makes it very unlikely that many mutations were missed by these gene screening
methods.
The R257X mutation has been described by others (Hardelin et al.,
1992), and this truncating mutant is predicted to remove three FNIII
domains, which are important in neuron migration (GonzalezMartinez et al., 2004; Hu et al., 2004). Our patient has complete
IHH with bilateral cryptorchidism and anosmia, reflecting the severity
of this mutation. In comparison, the patient reported by Hardelin et al.
(1992) had more severe disease: micropenis and bilateral cryptorchidism at birth; by 11 years age he had bimanual synkinesia and mild
bilateral pes cavus.
The IVS12-exon14 deletion has been reported previously (Bick
et al., 1992) in a male with complete IHH, hyposmia, visual field
abnormalities and an affected brother. The deletion of exons 113
and Cys 163 (C163del) is reported here for the first time. This
patient, with a nearly complete KAL1 gene deletion, presented with
complete IHH, anosmia and cryptorchidism and had an affected
maternal uncle and a maternal cousin. The precise breakpoints of
the deletion are currently being mapped.
Interestingly, the patient with the C163del mutation, which
occurs within the WAP domain known to be important in neuron
migration, had incomplete IHH, anosmia and unilateral renal agenesis.
As the deletion involves the sixth cysteine in the WAP domain, it is
expected to result in protein misfolding by disrupting disulfide bond
formation. This mutation is the only deletion identified in a patient
with some evidence of sex steroid production, as indicated by
partial testicular growth. None of these mutations were identified in
50 controls, thereby reducing the probability that these were
polymorphisms.
The double variants of K666M and R668H are likely to be polymorphisms. SIFT analysis shows low levels of conservation at these
positions across different organisms. Even at these low levels of
conservation, the substitutions K666M and R668H are predicted to
be tolerated with scores of 0.05 and 1.00, respectively. Even though
the score is quite low at 0.05 for K666M, the median sequence
168

conservation is 3.63, making it less likely to be a mutation even if

the score was lower than 0.05 (Ng et al., 2001).
These findings suggest that mutations in KAL1 account for a small
minority of cases of hypogonadotrophic hypogonadism in humans,
indicating that other genes play an important role in the pathogenesis
of IHH. Mutations in the FGFR1, GNRHR, NR0B1 (DAX1), GPR54,
LEP and LEPR genes have been shown to cause IHH (Grumbach,
2005). Anosmia has only been reported in patients with KAL1 and
FGFR1 gene mutations. Three genes (KAL1, FGFR1 and GNRHR)
constitute the most frequent molecular etiologies of IHH (6.3%,
7.1% and 3.6%, respectively), but the molecular basis for most
cases of IHH remains unknown (this article; Sato et al., 2004;
Bhagavath et al., 2005). It is however possible that there might be unidentified mutations in the 50 untranslated region of KAL1.
No mutations were identified in females with IHH, suggesting that
KAL1 rarely, if ever, causes phenotypic effects in females. However, it
is clear that anosmia occurs in females, suggesting that other genes
that affect olfactory development and GnRH function must be
involved. FGFR1 is one such gene that has been shown to cause an
autosomal dominant form of Kallmann syndrome (Dode et al.,
2003). As mentioned earlier, FGFR1 has been postulated to interact
with the anosmin-1 in effecting olfactory neuronal migration since it
has similar patterns of expression during development and utilizes
heparan sulfate proteoglycans (Gonzalez-Martinez et al., 2004). We
are currently screening these patients for mutations in FGFR1.
Another gene that has been implicated in GnRH neuron migration is
NELF (Kramer and Wray, 2000), but definitive mutations have not
yet been described (Miura et al., 2004).
In summary, KAL1 mutations are an uncommon cause for male
Kallmann syndrome patients, occurring in about 3.7% of all IHH
males and 6.3% of anosmic/hyposmic IHH males in our study population. Female Kallmann syndrome patients are unlikely to be affected
by KAL1 mutations, although it is still possible if skewed X inactivation, a coexistant X chromosome deletion or two mutant alleles
are also present. There is considerable phenotypic variation among
patients with mutations in KAL1 gene, although most of the patients
affected have complete IHH (Oliveira et al., 2001), which our data
also supports. How exactly these mutations cause Kallmann syndrome
is the subject for new investigation into the molecular and cellular
actions of the anosmin-1 protein.

Acknowledgements
This work was supported by grants from the U.S. Public Health ServiceNational Institute of Child Health and Human Development HD33004 and
HD040287 (L.C.L).
We appreciate the assistance of a large number of students who have
participated in these studies: Melissa Enriquez, Sewit Amde, Sujal Shah,
Grace Boachie-Ansah, Ejaaz Kalimullah, Johan Lane, Amit Khardori,
Sameer Barkatullah, Janice Gupta, David Yu, Peter Kawada, and Kyle
L. Layman. We also appreciate the technical expertise of Lynn Chorich and
Lili Cheng.

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Submitted on November 14, 2006; resubmitted on November 8, 2006; accepted
on November 12, 2006