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A Recurrent Missense Mutation in the KAL Gene in Patients with X-Linked Kallmann’s Syndrome¹

Research Unit in Developmental Biology, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguo Social (G.M.-N., J.P.M.); the Department of Genetics, Hospital General de México, Secretaria de Salud; Faculty of Medicine, Universidad Nacional Autonoma de Mexico (J.C.Z., S.K.-A.); and the Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán (A.U.-A.), Mexico City, Mexico

Address all correspondence and requests for reprints to: Juan Pablo Méndez, M.D., Coordinación de Investigación Médica, Unidad de Investigación Médica en Biología del Desarrollo, Avenida Cuauhtémoc 330, Apartado Postal 73–032, Colonia Doctores, C.P. 06725, Mexico DF, Mexico. E-mail: jpmb{at}servidor.unam.mx

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Kallmann’s syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located on the Xp22.3 region, explaining the X-linked form of the disease. We report molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS.

PCR of the 14 exons of the KAL gene was performed on genomic DNA. PCR products of all exons were purified and sequenced. Genetic defects in the KAL gene were found in 7 patients. One exhibits a deletion from exon 3 to exon 5. Six individuals present a previously unidentified missense mutation in exon 11, consisting of a G to A substitution at codon 514 (GAA to AAA). In the remaining 5 individuals, no mutations were observed. We also found three different polymorphic changes. The first one, in exon 2, had not been reported previously. The other two were located at exons 11 and 12.

The deletion described, comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region that corresponds to the whey acidic protein motif. The same missense mutation was found in 6 of the 12 patients, indicating the possibility that it derived from a common ancestor or suggesting the presence of a hot spot in this region of the gene.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
KALLMANN’S syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. It is due to a neuronal migration arrest within the meninges above the cribriform plate, involving the GnRH as well as the olfactory-producing neurons (1, 2).

Segregation analysis in familial cases has demonstrated autosomal dominant, autosomal recessive, and X-linked recessive inheritance patterns, suggesting the existence of several genes regulating GnRH secretion (3, 4, 5). Due to the 5- to 7-fold excess of affected males vs. females, it was initially proposed that the X-linked form was the most frequent (6, 7); however, recently, it has been suggested that most affected patients have mutations in autosomal genes (8, 9).

A gene (KAL) in Xp22.3 was shown to encode a protein sharing homology with molecules involved in neuronal migration and axonal pathfinding. KAL has 14 coding exons, escapes X inactivation, and has a nonfunctional homolog at Yq11.2 (10, 11). The finding of KAL mutations in patients with KS has demonstrated that this gene is responsible for the X-linked form of the disease (1, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). However, in several of these reports no mutations were found in a high percentage of the patients studied, suggesting that in these cases the abnormality could be either in the nonsequenced regions of the KAL gene or in autosomal genes (1, 13, 19, 22).

Herein we report the molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS.

	Subjects and Methods

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We included a total of 12 unrelated males with a diagnosis of X-linked recessive KS who were selected from a group of 28 patients with KS. X-linked recessive pedigrees were identified according to the following criteria: the presence of at least two affected males, the absence of affected females, the absence of male to male transmission, and the absence of consanguinity. All patients were of Mexican mestizo ethnic origin and from different geographic locations. The families were not related. In all cases, another affected individual besides the propositus from each family was also molecularly studied. Informed consent was obtained from all subjects participating in the study.

The 12 propositi ranged in age from 18–54 yr. All were products of uneventful pregnancies, and anosmia or hyposmia had been present since early childhood. In adolescence there was an absence of or subnormal pubertal development. On physical examination certain clinical characteristics were found in all the individuals: normal stature, infantile genitalia, and scant pubic hair. Hyposmia or anosmia was always detected by performing the olfactory test described by Rosen et al. (24). The high resolution G-banded karyotype was 46,XY, and computed axial tomography of the hypothalamic-pituitary region did not demonstrate any disorder in any of the patients. Intravenous pyelograms showed diverse findings in 4 patients (Table 1).

Baseline plasma levels of LH, FSH, and testosterone were measured as previously described (23). Plasma concentrations of LH and FSH are expressed as international units per L according to the Second International Reference Preparation of human menopausal gonadotropin.

Genomic DNA was prepared from peripheral blood leukocytes using standard techniques (25). For each PCR amplification, genomic DNA (0.5–1.0 µg) in the presence of 0.1 mmol/L deoxy-NTP, 2 U Taq DNA polymerase (AmpliTaq, Perkin-Elmer Corp., Branchburg, NJ), and 250 nmol/L of each specific set of KAL primers were used. The sequences of the KAL primers and splice site junctions, the sizes of the amplified products, as well as the PCR conditions were previously described by Hardelin et al. (1); dimethylsulfoxide concentrations were slightly modified (23). Thirty cycles of PCR amplifications were performed in a thermal cycler with denaturation at 94 C for 1 min, annealing at 55–63 C for 1 min, and extension at 72 C for 1 min.

PCR products of the 14 exons of the KAL gene were purified by GeneClean (BIO-101, Vista, CA). These products were then sequenced (300 nmol DNA template/reaction) on an ABI 373 automated DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA) using the dye terminator cycle sequencing core kit (Perkin-Elmer, Foster City, CA). PCR conditions for cycle sequencing were identical to those described above. For all exons, both strands were sequenced and compared. Each mutation or sequence variation was confirmed in two independent PCR amplifications and sequencings.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In all patients hypogonadotropic hypogonadism was documented. Basal testosterone concentrations were consistently in the prepubertal range (<1.2 ng/mL), and gonadotropin levels were always below the normal adult male range (LH, 3–12 IU/L; FSH, 0.5–5.0 IU/L).

Genetic defects in the KAL gene were found in 7 of the 12 propositi. Patient 10 exhibited a deletion from exon 3 to exon 5; the remaining exons of the gene amplified in a normal fashion (Fig. 1).

View larger version (66K): [in this window] [in a new window]   Figure 1. PCR amplification of exons 2–6 of the KAL gene in DNA from patient 10 (P) and a normal control (C). Deletion of exons 3–5 is observed in the patient’s DNA.

View larger version (30K): [in this window] [in a new window]   Figure 2. Missense mutation detected in 6 of the 12 patients studied. Part of the corresponding exon 11 sequence is shown together with the normal sequence. The point mutation is indicated with a star. The change in the corresponding codon and amino acid is indicated at the right.

In all cases, we observed the same molecular findings in the affected relative studied as in the propositi.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
A search for mutations in the KAL gene has been performed in various groups of patients with X-linked KS. Defects have been found widely distributed throughout this gene in approximately 50% of the patients (1, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22). The absence of KAL abnormalities in five of our propositi extends and confirms previous reports in which no mutations were found in various X-linked KS patients studied (1, 19, 22). Defects present in the regulatory regions of the KAL gene promoter, in the untranslated regions of exons 1 and 14, and within introns creating a new splice site or the existence of another gene that participates in X-linked KS should be considered as possible explanations for the presence of KS in these individuals.

The finding of 7 mutations in 12 X-linked KS patients (58%) is slightly higher than that reported previously (52%) (9). We demonstrated an X-linked inheritance pattern in 13 [12 in the present study and 1 previously reported (23)] of 28 individuals (46%) we have diagnosed with KS in the last few years. This agrees with the observation that the X-linked form accounts for the minority of patients with KS, confirming the genetic heterogeneity of the disease.

Interestingly, we found the same missense mutation in 6 of the 12 patients studied. All individuals were from different geographic locations, and although there is no known relationship between them, the mutation could derive from a common ancestor, by a founder gene effect. An alternative explanation is that the identical transition observed in 6 of our patients might indicate the presence of a hot spot in this region of the KAL gene. Furthermore, the recurrence of this mutation could be the result of both a founder gene effect and a repetitive mutation at this site, as has been demonstrated in hemophilia B (26, 27).

This missense mutation located at codon 514 in exon 11 had not been identified previously, providing support for the concept that mutations in the KAL gene are widely distributed. This mutation occurs within the region encoding the third fibronectin type III-like repeat of the KAL protein, a domain involved in processes of neuronal migration and axonal targeting (28).

A deletion of exons 3–5 of the KAL gene had not been reported previously. This deletion comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region, which corresponds to the whey acidic protein motif. In this region only two mutations have been reported to date (22, 23). The patient who presented this deletion exhibited a severe phenotype that included facial asymmetry, abnormal eye movements, bimanual synkinesis, as well as the absence of the right kidney. Interestingly, this phenotype is strikingly similar to that observed in three patients with KS previously described (29).

We also found three different polymorphic changes that were determined to be polymorphisms, as they were also found in genomic DNA from normal controls. The first one, located at codon 78 in exon 2, had not been described previously. This change, found in patients who had the missense mutation (no. 1 and 11) as well as in patients 2, 6, 9, and 12 and in 54% of the normal population, did not alter the encoded amino acid (glutamine). The second polymorphism was found at codon 534 in exon 11, substituting valine for isoleucine. Patients 4 and 8 who exhibited the missense mutation; patients 2, 5, 6, 9, and 12; as well as 48% of the controls presented this base variation, which was previously described (1, 9). The last polymorphic change found in exon 12 at codon 611 in patients 4, 8, and 11 (who also presented the missense mutation) as well as in patients 5 and 12 and in 24% of the controls did not alter the encoded amino acid (isoleucine). This change was also described previously (1). An interesting observation in our study, although the sample size was small, is that 8 of 12 patients exhibited more than 1 polymorphism, whereas only 26% of the controls had 2 polymorphic changes. It is particularly noteworthy that all KS individuals in whom mutations were not found had more than 1 polymorphism, and they all shared the 1 previously described in exon 11.

Although it is generally accepted that silent polymorphisms do not predispose to diseases, it has been shown that they may affect the type and severity of some monogenic conditions (30, 31). At present, we cannot establish a correlation between the location and number of polymorphisms and the occurrence of the mutation or the severity of the disease in our patients.

	Acknowledgments

 
We thank Arcelia Grajales, Dolores Utrera, and Elsa de la Chesnaye for their help.

	Footnotes

 
¹ This work was supported by the Consejo Nacional de Ciencia y Tecnología, Mexico (Grants F527-M9308 and 4043-M9403).

² Present address: Oregon Regional Primate Research Center, Beaverton, Oregon 97006-3499.

Received December 4, 1997.

Revised January 21, 1998.

Accepted February 5, 1998.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maya-Nuñez, G. Articles by Mendez, J. P. Search for Related Content PubMed PubMed Citation Articles by Maya-Nuñez, G. Articles by Mendez, J. P.