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Experimental Studies |
Reproductive Endocrine Sciences Center and National Center for Infertility Research at Massachusetts General Hospital (N.A.G., F.P.P., W.F.C., M.V.), and the Department of Genetics and Howard Hughes Medical Institute (C.E.S., J.G.S.), Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Mario Vallejo, M.D., Ph.D., Reproductive Endocrine Unit, BHX-516, Massachusetts General Hospital, Boston, MA 02114, Phone: 617-726-5384, Fax: 617-726-5357.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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Whether IHH and Kallmann’s syndrome represent a spectrum of manifestations of GnRH deficiency rather than two distinct syndromes is still a matter of controversy. In some families, GnRH deficiency and anosmia occur dissociated in different individuals that exhibit one but not the other. In these cases, a clear-cut distinction between IHH and Kallmann’s syndrome based exclusively on clinical observations is difficult (6). Segregation analysis in previous familial cases of IHH and/or Kallmann’s syndrome demonstrated several modes of inheritance, suggesting the existence of multiple genes regulating the expression of reproductive potential via their influences on endogenous GnRH secretion. These include X-linked, autosomal recessive, and autosomal dominant patterns of inheritance (7, 8, 9, 10, 11). Because the number of affected males is about 6-fold higher than that of affected females (12), it has been suggested that the X-linked form is the most frequent.
A candidate gene for the X-linked form of Kallmann’s syndrome was mapped by linkage analysis and deletion studies to the Xp22.3 region (13, 14, 15) and was subsequently isolated by positional cloning (16, 17). This gene, known as KAL-1, consists of 14 exons (18, 19) encoding a predicted protein product with characteristic C-terminal fibronectin type III-like repeats that bears homology with neural cell adhesion molecules (20) and may be involved in the process of migration of GnRH neurons from their place of origin in the olfactory placode to the hypothalamus (16, 17, 21). The finding of mutations and genetic deletions in patients with Kallmann’s syndrome has provided formal evidence for the involvement of the KAL-1 gene in the pathogenesis of the X-linked form of this disease (22, 23, 24, 25, 26).
However, previous studies from our group of a series of 106 patients
with isolated GnRH deficiency have documented that most cases of
isolated IHH and Kallmann’s syndrome are sporadic (6, 27, 28). A
positive family history was established in only 19 of the 106 cases.
These findings suggest that the frequency of spontaneous mutations
accounting for GnRH deficiency may be relatively high. Furthermore,
most of the familial cases exhibited a mode of inheritance incompatible
with an X-linked form, suggesting the existence of at least 2 other
genes, inherited on autosomes in a dominant or a recessive pattern,
respectively, involved in the human syndrome of GnRH deficiency (6, 28). In fact, the X-linked mode of inheritance, characterized
clinically by the predominance of males affected, the presence of
unaffected female carriers, and an absence of male to male
transmission, appeared to account for the minority (i.e.
18%) of familial GnRH deficiency (6).
In the present study, we sought to determine the relative proportion of sporadic cases that arise as a consequence of the appearance of de novo mutations in the KAL-1 gene. To this end, we used a PCR-based technology to screen for mutations within the coding region of the KAL-1 gene in patients with sporadic GnRH deficiency. We reasoned that the finding and frequency of de novo mutations within the KAL-1 gene would provide an approximate index for the occurrence of the X-linked mode of inheritance in sporadic cases of isolated GnRH deficiency in the human. In addition, any mutations detected might prove useful for both studying the functional properties of the KAL-1 protein and assessing its role in the clinical expression of Kallmann’s syndrome.
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Subjects and Methods |
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A total of 21 unrelated males with isolated GnRH deficiency were included in this study. GnRH deficiency in these patients was determined to be sporadic by carrying out a detailed family history showing the absence of hypogonadism, anosmia, or delayed puberty in any known relative.
One patient with a positive family history consistent with an X-linked
mode of inheritance was also included. The X-linked mode of
transmission in this familial case was determined according to the
following criteria: presence of asymptomatic female carriers, presence
of another affected male in the maternal family or among male siblings,
absence of affected females, and absence of male to male transmission
(Fig. 1
, DJ family).
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, MS family). Whereas the mode of
transmission in this patient could not be determined due to the absence
of affected paternal or maternal relatives, an X-linked pattern of
transmission could not be ruled out, and thus, we argued that the
possible finding of a defect in the KAL-1 gene in this case would
confirm its X-linked inheritance. Likewise, one remaining patient with
a brother who had anosmia but not GnRH deficiency was also included in
this study (Fig. 1, FF family). Diagnostic criteria for isolated GnRH deficiency were strict and uniformly included age greater than 18 yr, clinical signs and symptoms of hypogonadism, serum testosterone levels in the prepubertal range (<100 ng/dL), gonadotropin levels within or below the normal adult male range (LH, 1.4–4.3 IU/L; FSH, 1.7–3.7 IU/L), absence of a normal adult pattern of pulsatile gonadotropin secretion (29), normal baseline and reserve testing of other anterior pituitary hormones, and normal radiological imaging of the hypothalamic-pituitary region. The diagnosis of delayed puberty in family members was based on criteria established in the U.S. Health Examination Survey (30, 31).
All patients were tested for the presence of anosmia with either multiple odorants or a commercial smell test kit (Olfacto Laboratories, El Cerrito, CA) using a carbinol derivative. Thirteen of 21 sporadic patients (60%) and the three familial cases were found to be anosmic.
Informed consent was obtained from all patients before the extraction of blood.
Methods
Genomic DNA extraction. Genomic DNA was extracted from whole blood (10–20 mL) using a Genomix kit (Washington Biotechnology). The procedure includes an initial cationic lysis of blood, followed by an extraction step with chloroform and a final precipitation with a cationic detergent and 96% ethanol (32). Cycle sequence analysis of all 14 exons spanning the entire coding region of the KAL-1 gene was carried out using genomic DNA from the patients. Genomic DNA from healthy unrelated male volunteers was used for identification of polymorphisms.
PCR amplification. Genomic DNA (100–200 ng) was used as a template for the amplification of each of the 14 exons of the KAL-1 gene. Primers for PCR amplifications were identical to those described by Hardelin et al. (24). For exons 2–13, PCR products correspond to a segment of DNA spanning the coding region and the adjacent splice site junctions. For exons 1 and 14, which contain the 5'- and 3'-untranslated regions respectively, only the coding segment was amplified, using an intronic primer and a second primer annealing to the corresponding untranslated sequence (24).
Reactions were carried out in a Perkin-Elmer 9600 thermocycler (Norwalk, CT) using the following parameters (30 cycles): denaturation, 30 s at 94°C; annealing, 30 s at 55°C; and extension, 30 s at 72°C. For the amplification reactions of exons 1, 2, and 3, annealing was performed at 63°C, and 10% dimethylsulfoxide was included.
DNA sequencing. PCR products were purified and subjected to
cycle sequencing. For this purpose, either of the oligonucleotide
primers used for the PCR amplification was end labeled with
polynucleotide kinase and [
-32P]ATP. Sequencing was
carried out with 20–40 fmol DNA template/reaction. The dideoxy chain
termination procedure (33), was carried out using Taq
polymerase and a dsDNA Cycle Sequencing System kit (Life Technologies,
Gaithersburg, MD). PCR conditions for cycle sequencing were identical
to those described above. Sequencing reaction products were
electrophoretically resolved on denaturing 6% polyacrylamide gels,
followed by overnight autoradiography. For all exons, both strands were
sequenced and compared. Each potential mutation or deletion was
confirmed by a second independent PCR amplification and sequencing.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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A genetic defect in the KAL-1 gene was found in only 1 of the 21
patients with sporadic IHH included in this study (Table 1, CG). The KAL-1 gene of this patient who had anosmia
was found to contain a deletion of 14 bp (TGAAGCGTGTGCCC) encoding
amino acids 464–468 (exon 10). This defect results in a frame shift at
codon 464. No abnormal splicing of exons 10 and 11 is predicted by this
alteration, but as a consequence of the frame shift, a stop codon (TAA)
is introduced at position 487 (exon 11). Thus, the predicted protein
encoded by this defective gene would lack half of the third and the
fourth fibronectin III-like repeats (Fig. 2).
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). These changes were determined to be
polymorphisms, as they were also found in genomic DNA obtained from
normal individuals. The first polymorphism is a G to A substitution at
codon 668 in one nonanosmic patient, resulting in an arginine to
histidine change. An identical change was detected in 1 of 4 normal
individuals tested. A different polymorphism was detected in 5
unrelated patients with sporadic IHH, 4 of whom had anosmia. This
change consists of an A to G substitution at codon 534 (exon 11),
turning isoleucine into valine. A similar polymorphic change was
detected in 3 of 20 normal individuals tested. An identical
polymorphism has been previously reported by Hardelin at al. (24) in
association with a silent polymorphism in exon 12, which was not
identified in our patients. Interestingly, however, this polymorphism
was associated with an additional polymorphism in exon 13 or 14 in 3
patients (Table 1). Finally, G to A substitutions at codon 651 encoding
arginine were found in 2 patients (BA and DS), but this change did not
alter the encoded amino acid (Table 1). Note that in 3 patients (LW,
BA, and DS) the KAL-1 gene was found to have 2 different polymorphic
changes (Table 1). A similar association of the 2 polymorphisms found
in patients BA and DS has previously been reported in a healthy
individual (24); thus, these changes do not appear to be the cause of
the disease. Future studies will be needed to determine the frequency
and associations of polymorphisms in both GnRH-deficient and normal
individuals. Familial cases
The pedigrees for the families corresponding to the three cases of
familial Kallmann’s syndrome included in this study are shown in Fig. 1. These three patients had genetic defects in the KAL-1 gene, strongly
supporting the idea of an X-linked pattern of IHH inheritance. In one
patient with X-linked Kallmann’s syndrome (DJ), a single base mutation
was found in exon 7. This mutation consists of a C to G substitution
turning codon 328 (TAC) encoding tyrosine into a stop codon (TAG; Table 1). The predicted consequence of this mutation is the generation of a
truncated KAL-1 protein (the wild-type protein is 680 amino acids long)
lacking most of the fibronectin III-like repeats (Fig. 2).
In another patient with familial Kallmann’s syndrome (MS), a small
deletion (9 bp) involving the sequence AACAACAGT was detected (Table 1). This deletion is located at the 3'-end of exon 8, corresponding to
amino acids 400–403 and extends 2 bp into the adjacent intron. As a
consequence, the donor site necessary for correct messenger ribonucleic
acid splicing of exons 8 and 9 is altered.
A third familial case for which an X-linked pattern of inheritance
could not be unequivocally determined (Fig. 1, FF) was included in this
study. In this patient, a single base mutation was detected in exon 11.
This mutation consists of a C to G substitution at codon 517 (TTC to
TTG), turning phenylalanine into leucine in the translated sequence
(Table 1). Because this substitution involves two amino acids with
nonpolar (hydrophobic) chains, we considered it possible that it may
represent a polymorphic variant generating a normal KAL-1 protein. The
presence of the identified mutation in the mother and the anosmic
brother, but not in unaffected brother, would unequivocally confirm its
association with the disease. Unfortunately, no DNA samples were
available from those family members. Therefore, to address the
possibility that the observed C to G substitution may constitute a
polymorphic change, we carried out cycle sequencing analysis of exon 11
of the KAL-1 gene using genomic DNA from 53 unrelated normal subjects.
No mutations were found in these normal individuals. This circumstance
together with the observation that Phe517 located in the
third fibronectin type III-like repeat (Fig. 2) is conserved in all
species tested to date (21, 34) strongly suggest that this
phenylalanine to leucine substitution represents a mutation with
functional consequences. In this individual, we also found a
polymorphic A to G substitution at codon 534 (Ile to Val) identical to
that described above for some sporadic patients (Table 1).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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In a series of 106 patients with GnRH deficiency studied at our center, 34% of the cases were familial, and 66% were sporadic (6). Using a strict phenotype of complete GnRH deficiency and anosmia, the incidence of X-linked inheritance among our familial cases was estimated to be 18%. With the addition of delayed puberty as a surrogate marker of the genetic defect, the incidence of X-linked inheritance further decreased to 11%. A search for mutations in the KAL-1 gene has previously been reported in patients with X-linked Kallmann’s syndrome, and defects in this gene were found in 52% of cases (24, 26). If the assumption is made that all cases with an X-linked mode of inheritance are due to defects in the KAL-1 gene (although the existence of an additional gene participating in X-linked Kallmann’s syndrome cannot be ruled out), it follows that 48% of patients with X-linked Kallmann’s syndrome may bear unidentified mutations in other regions of the KAL-1 gene. Therefore, based on our finding of KAL-1 gene mutations in 8% of the patients with sporadic Kallmann’s syndrome, it could be expected that perhaps an additional 7% of these patients may also harbor mutations in this gene. Thus, the overall incidence of KAL-1 gene mutations in patients with sporadic Kallmann’s syndrome would be 15%. This calculated incidence is in good agreement with our estimate, based on clinical genetic studies, that the X-linked form of the disease accounts for the minority of patients and that most cases are presumably due to mutations in autosomal genes (6), thus indicating the genetic heterogeneity of the disease. It is likely that those autosomal genes encode proteins that regulate different aspects of a complex cascade of developmental processes in which the KAL-1 gene is partly involved (differentiation of olfactory and GnRH neurons in the olfactory placode, cell migration into the olfactory bulb, olfactory bulb development, and differentiation, etc.).
The only genetic defect identified among our cases of sporadic GnRH deficiency consists of a 14-base deletion, presumably resulting in the production of a protein that lacks the third and fourth fibronectin type III repeats. These types of repeats are shared by a group of proteins that have been implicated in neuronal migration and axonal growth (20, 36). The fourth repeat shares homology with the neural cell adhesion molecule, which appears to be important for the development of the olfactory system (35, 37, 38).
Three new genetic defects were detected in our patients with X-linked Kallmann’s syndrome: a mutation introducing a stop codon at position 328, a 9-base deletion affecting the ribonucleic acid splice donor site of exon 8, and a missense mutation at codon 517, turning phenylalanine to leucine. Although the latter mutation might appear as a conservative substitution involving 2 hydrophobic amino acids, a similar change was not detected in the DNA of 53 normal individuals tested. In addition, a comparison of the sequences of the human, quail, and chicken proteins indicates that Phe517 is absolutely conserved across species in the least conserved repeat of the protein (34). These observations suggest that this amino acid substitution may well be the causative genetic defect in this patient.
It should be noted that all genetic defects identified to date in patients with sporadic or X-linked GnRH deficiency, with the exception of large chromosomic deletions, occur within the region encoding the four fibronectin type III repeats of the KAL-1 protein. Interestingly, no mutations have yet been described in the conserved cysteine-rich N-terminal region corresponding to the whey acidic protein motif (16, 17). The mutations reported in the present study have not been previously identified, providing further support to the emerging idea that mutations in the KAL-1 gene are not clustered, but are widely distributed throughout a region encoding the C-terminal two thirds of the protein (22, 24, 26). To date, only two patients have been reported with identical mutations (26). This circumstance together with the genetic heterogeneity thought to be responsible for this disease make genetic diagnosis in familial or sporadic cases difficult. However, the increasing identification of genetic defects provides an opportunity to carry out a molecular diagnosis among the siblings of affected individuals. Notably, prenatal diagnosis of Kallmann’s syndrome has been reported in a fetus with a Xp22.3 contiguous gene syndrome (40).
In conclusion, our study shows that the incidence of genetic defects within the coding region of the KAL gene in our series of patients with sporadic GnRH deficiency is low. Thus, the X-linked mode of inheritance seems to be relatively infrequent, indicating the existence of genetic heterogeneity involving yet unidentified genes whose defects would result in the expression of similar phenotypes characterized by GnRH deficiency with or without anosmia.
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Note Added in Proof |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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2 Supported in part by a Fullbright Fellowship and a grant from the
Gerondelis Foundation.
3 Present address: Division d’Endocrinologie, Centre Hospitalier
Universitaire Vaudois, 1011 Lausanne-VD, Switzerland
4 Investigator with the Howard Hughes Medical Institute.
Received June 21, 1996.
Revised August 9, 1996.
Accepted September 4, 1996.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionNote Added in ProofReferences |
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