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Autosomal Recessive Idiopathic Hypogonadotropic Hypogonadism: Genetic Analysis Excludes Mutations in the Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Genes

Faculty of Medicine (Y.B.-A.), Kuwait University, Al-Jabriyah, 13110 Kuwait; and Reproductive Endocrine Unit (J.S.A., J.K.S., W.F.C., S.B.S.), Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Stephanie Seminara, M.D., Reproductive Endocrine Unit, Bartlett Hall Extension 505, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: seminara.stephanie{at}mgh.harvard.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Discussion References

 
Failure of the normal pattern of episodic secretion of GnRH from the hypothalamus results in the clinical syndrome of idiopathic hypogonadotropic hypogonadism (IHH), with failure of pubertal development and infertility. The only gene that has been implicated in normosmic IHH is the GnRH receptor gene (GNRHR), which accounts for 10% of cases. This report presents four families with autosomal recessive IHH, including a consanguineous pedigree from the Middle East. Defects within the genomic coding sequence of the GNRHR, and the GnRH gene itself, GNRH1, were excluded by temperature gradient gel electrophoresis, direct sequencing, and haplotypes created from simple sequence polymorphisms flanking the GNRH1 and GNRHR loci. We concluded that: 1) genetic analysis has excluded sequence variations in GNRH1 and GNRHR in four families with recessive IHH, suggesting the existence of a novel, as-yet-undiscovered gene for this condition, and 2) because mutation analysis of genomic coding sequence will fail to detect mutations deep within introns or regulatory regions, haplotype analysis is the preferred genetic methodology to eliminate the role of specific candidate genes.

	Introduction

Top Abstract Introduction Subjects and Methods Discussion References

 
USING GENETIC TOOLS to elucidate the pathophysiology of human reproductive disorders is difficult. The reproductive lethality inherent in many of these conditions results in small pedigrees, limiting options for genetic studies. Candidate gene approaches have been tried in numerous disorders, but they are limited by investigators’ knowledge of the disease pathophysiology and are not, by definition, a means to novel gene discovery. Even patient recruitment can be difficult because affected individuals often shoulder heavy emotional burdens caused by a history of abnormal sexual development.

Progress in the search for genes underlying idiopathic hypogonadotropic hypogonadism (IHH) has been slowed by all of the above challenges. In IHH, gonadotropin levels are low in the setting of low levels of sex steroids, and there are no other abnormalities of the hypothalamus and/or pituitary by functional and anatomic testing. Typically, the diagnosis of IHH is made at adolescence when failure of pubertal development occurs. This delay in the ability to diagnose IHH until the late teen years creates another formidable obstacle to genetic studies.

IHH can occur as an isolated clinical entity or can be part of more complex syndromes, such as Kallmann syndrome, which is characterized by IHH and anosmia (1). In the X-linked form of Kallmann syndrome, somatic abnormalities (renal agenesis and synkinesia) are also characteristic phenotypic features (2). In addition to this clinical complexity, IHH is genetically heterogeneous with autosomal dominant, recessive, and X-linked forms (3). Although most patients appear to be deficient in GnRH as attested to by their generally normal gonadotropin responses to a physiologically designed regimen of exogenous GnRH (4), no genetic defects within the gene encoding GnRH (GNRH1) have been identified (5, 6, 7).

The only gene implicated in isolated IHH to date is the GnRH receptor gene (GNRHR) (8, 9). In a screening study of familial IHH cases, mutations in the coding sequence of GNRHR were identified in only two of five (40%) of families whose inheritance pattern was compatible with an autosomal recessive mode of transmission (10). The current report reexamines these families in greater detail and adds a large, consanguineous Middle Eastern pedigree with six affected individuals. Although mutation analysis failed to identify any GNRHR genomic coding sequence abnormalities in the probands from these families, the possibility of an intronic or regulatory mutation in GNRHR could not be excluded. Therefore, haplotype analysis using microsatellite markers flanking GNRHR was performed to analyze the segregation pattern of alleles with segregation of the disease phenotype. Mutation and haplotype analyses were also performed for GNRH1, another obvious candidate gene for IHH. This layering of genetic analyses may be a helpful approach for investigators looking to combine relatively small pedigrees for further genetic studies.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Discussion References

 
Case presentations

Four families are presented (families 005, 105, 242, and 686) with recessive IHH (see Figs. 1–4). Propositi from families 005 and 105 were referred to the Reproductive Endocrine Unit at Massachusetts General Hospital (MGH) after their diagnoses had been established for further detailed evaluations. Relevant patient history is described below, and Table 1 provides further details for family 686. The hormone concentrations were determined by multiple assay methods and are not standardized.

View larger version (31K): [in this window] [in a new window]   Figure 1. Pedigree 005 and haplotype data for GNRHR. Markers surround GNRHR gene. Chromosomes are depicted by different shading.

View larger version (11K): [in this window] [in a new window]   Figure 2. Pedigree 105. Blood samples were not sufficient to perform haplotype analysis.

View larger version (22K): [in this window] [in a new window]   Figure 3. Pedigree 242 and haplotype data for GNRHR. Markers surround GNRHR gene. Chromosomes are depicted by different shading. Thin bars represent regions of uninformativeness.

View larger version (26K): [in this window] [in a new window]   Figure 4. Pedigree 686 and haplotype data for GNRHR. Markers surround GNRHR gene. Chromosomes are depicted by different shading. Thin bars represent regions of uninformativeness.

Family 005 is an Ashkenazi Jewish family and a member of the Hassidic community (Fig. 1). At age 18 yr, the proband presented with right cryptorchidism and absence of secondary sexual characteristics. Family history was notable for two nephews with IHH and one brother with cryptorchidism. Initial laboratory values revealed an LH level of 1.25 mIU/ml, FSH of 2.46 mIU/ml, and testosterone (T) of 11 ng/dl (0.37 nmol/liter). During a single bolus GnRH (100 µg) challenge performed at an outside hospital, LH rose from a baseline of 1.27 ng/ml to a peak of 2.6 ng/ml, and FSH rose from a baseline of 1.08 ng/ml to a peak of 1.31 ng/ml. Cranial imaging revealed a partially empty sella. Karyotype was 46,XY.

At age 20 yr, IV:4 underwent testicular biopsy revealing primary germinal cell arrest of both testes with atrophic tubules and a predominance of Sertoli cells. No interstitial cells were seen between the tubules. The patient received T therapy with good development of secondary sexual characteristics. When he desired fertility, he was switched to human chorionic gonadotropin and Pergonal but achieved a semen analysis of only 220,000 million/ml sperm. During a period of 12 h of blood sampling every 10 min at MGH, an absence of gonadotropin pulsations, pooled T values between 44 and 51 ng/dl (1.5 and 1.8 nmol/liter) and undetectable estradiol levels were documented. He began therapy with exogenous pulsatile GnRH, requiring large doses (500 ng/kg) because of obesity. His gonadotropin and sex steroid levels achieved normal adult male levels, his semen analysis improved (4.8 million/ml), and his descended testicle increased to normal size (20 cc).

Family 105

The female proband II:1 presented at age 17 yr with absence of menarche and breast development (Fig. 2). Family history was notable for a brother (II:4) with markedly delayed puberty with testicular growth occurring at age 17 yr. Initial laboratory evaluation revealed low gonadotropin levels and frequent blood sampling (every 10 min) revealed an absence of gonadotropin pulsations. A head computed tomography scan revealed no abnormalities. The proband elected treatment with exogenous pulsatile GnRH for ovulation induction. In her first three GnRH cycles, she received 75 ng/kg per bolus with frequency changes to mimic the normal female menstrual cycle (11). Her gonadotropin levels increased, and she developed dominant follicles. However, she failed to mount an LH surge during two cycles, and her endometrial lining remained thin, ranging between only 2 and 4 mm. For the fourth cycle, II:1 was pretreated with 2 mg Estrace orally daily for 7 d to increase the thickness of her uterine lining, and her GnRH dose was reduced to 50 ng/kg per bolus. She developed two dominant follicles, mounted an endogenous LH surge, and conceived.

Family 242

Family 3 are Arab Muslims living in Israel referred by their local physician (Fig. 3). The proband (VI:1), his brother, and his cousin met the diagnostic criteria for IHH. An additional cousin, not under the care of the referring physician, was noted to have absent facial hair and was treated for infertility.

Family 686

Family 4 is from Saudi Arabia but currently reside in Kuwait (Fig. 4). The family was brought to the attention of the Reproductive Endocrine Unit by a physician evaluating the wife of IV:2 for infertility. On further questioning, the possibility was raised that the proband might have congenital hypogonadotropism. Further investigation revealed that multiple other family members (all cared for by different physicians) carried the diagnosis of IHH. Because of the size and complexity of this family, additional clinical data are presented in Table 1.

Mutation analysis

GNRH1. Genomic DNA was isolated from peripheral blood leukocytes. Primers were designed to amplify each of the exons of GNRH1, including acceptor-donor splice sites. Two probands were screened using temperature gradient gel electrophoresis, a mutation detection method that can separate two DNA fragments that differ in sequence by as little as a single base substitution (see Ref. 10 for details of temperature gradient gel electrophoresis methodology). A 40-mer GC clamp (n = CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCC) was attached to the 5' end of either the forward or reverse primer to prevent strand melting. Primers used for exon amplification were: 1F, 5'-ATAGTCCATTTGCAGTATAAT; 1R, 5'-ATGCCCTGTAGACATACAATA; 2F, 5'-GGGTTAGTGATACAGATGCTAGCTT; 2R, 5'-GGCTATCCTGAATGTTTAATATAGTTA; 3F, 5'-AGTCCCTATGCTAATCCTGCAACTT; 3R, 5'-AGGTAGCTGTCCTAAGTGATCCCCA; 4F, 5'-CTCCCTAGCACTAACTAGAGCACAA; and 4R, 5'-CTCCCTTTGGTGGGTTTACAGTGATC. The remaining two families were screened using direct sequencing. Primers were designed to amplify each of the three exons: 1F, 5'-CTCTGACTTCCATCTTCTGC-3'; 1R, 5'-GCCTTATCTCACCTGGAGC-3'; 2F, 5'-CTGCAACTTTCCCAATCTCC-3'; 2R, 5'-GAGGAGTCAGGAATGTAAGC-3'; 3F, 5'-CCTAGCACTAACTAGAGC-3'; and 3R, 5'-GTGCAACTTGGTGTAAGG-3'. PCR was performed using a total volume of 15 µl with approximately 100 ng genomic DNA; 20 pmol of each primer; 0.5 U Taq polymerase; 1.5 mM MgCl₂; 50 mM KCl; 200 µM dATP, dCTP, and dTTP; 20 µM dGTP; and 0.1 µCi [³²P]-dGTP. PCR conditions included initial denaturation at 94 C for 2 min, followed by 30 cycles of 94 C for 20 sec, 53–60 C for 20 sec, and 72 C for 20 sec, concluding with a final extension step of 72 C for 2 min. Annealing temperatures were optimized for each set of primers, and PCR products were electrophoresed on a 6% acrylamide gel (90 W for 2–3 h at constant power) and autoradiographed by exposing of the gels to XAR film (Kodak, Rochester, NY) for 2–4 h. GNRHR. The genomic coding sequence of GNRHR was amplified with primers for each exon, including acceptor-donor splice sites, as described previously (12). Approximately 1.75 kb of sequence in 5' untranslated region of the GNRHR gene was also examined using the following primers: UAF, 5'-CCTAGATAGAGAAGACAAAGAGC-3'; UAR, 5'-GCTGTCTCCTGAATCTTGAGC-3'; UBF, 5'-GTCACAAATCTCAGGTGTGAG-3'; UBR, 5'-GCCAACAAGTTTGCTTCTGC-3'; UCF, 5'-CTAACCTTCTGTGGTAACAAGC-3'; UCR, 5'-ATCTTACTGATCCAATCCATGC-3'; UDF, 5'-CAATAACTAGTTTCCTTATGC-3'; UDR, 5'-CTCAGCAGATGGTCTGGC-3'; UEF, 5'-GACAGAACACTAACACTAAATAG-3'; and UER, 5'-CCCTGCATCAGTGGGATGC-3'. All patient DNA samples were analyzed by PCR (described above) using an ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) and an ABI model 377 DNA sequencing system (PE Applied Biosystems).

Haplotype analysis Polymorphic markers were chosen that flank GNRH1 and GNRHR to perform haplotype analysis and exclude the possibility of mutations in other noncoding sequence. Markers [D4S2432 (66.48 Mb), D4S1568 (67.52 Mb), D4S409 (68.24 Mb), GNRHR (68.52–68.53 Mb), D4S3018 (68.54 Mb), D4S392 (70.01 Mb), D8S360 (23.60 Mb), D8S1739 (24.71 Mb), D8S1725 (24.27 Mb), GNRH1 (25.049–25.054 Mb), D8S1771 (25.21 Mb), D8S451 (26.11 Mb)] were determined from the Human Genome Browser [http://genome uscs.edu/goldenPath/hgTracks, html (June 2002 Assembly)] and chosen sufficiently close to GNRHR and GNRH1 to preclude recombination. The markers were also selected to overlap with other adjacent genes to ensure that promoter and regulatory regions of GNRHR and GNRH1 were captured. Haplotypes were constructed manually by visually inspecting the allele patterns across the region. Haplotype analysis could not be performed on family 105 because of a limited number of blood samples. Haplotype analysis was also performed on two families with known coding sequence GNRHR mutations (families 014 and 476) for illustrative purposes.

These genetic studies were approved by the subcommittee on human studies of MGH; all subjects gave their informed consent.

Results

Pedigree analysis For the pedigrees selected for this report, inheritance of IHH is consistent with autosomal recessive transmission. Family 005 is Ashkenazi Jewish and Hassidic with multiple consanguinity loops. In family 105, the affected subjects included a female proband with IHH and her brother with markedly delayed puberty. Although the incidence of delayed puberty in the general population is less than 1% (13), in our previous series of 106 patients with IHH, 12% had relatives with a history of delayed puberty (3). Because this observation has been noted by other investigators (14), delayed puberty appears to represent the mildest end of the phenotypic spectrum of IHH. Family 242 is from the Middle East and also has multiple consanguinity loops. Family 686 is comprised of three intermarriages between two first-cousin sibling groups. The intermarriages give rise to 19 children (10 males and 9 females) including six affected individuals (both males and females) with IHH. The presence of affected individuals (both male and female) across a single generation within three highly related marriage loops is strongly consistent with autosomal recessive inheritance.

Endocrine profiles Because family 686 is large and contains six individuals with IHH, Table 1 shows the gonadotropin and sex steroid data from the affected family members. All affected individuals had low sex steroid levels and inappropriately low gonadotropin levels consistent with a diagnosis of IHH.

Mutation analysis No genomic coding sequence or consensus splice site mutations were identified in GNRH1 and GNRHR.

Haplotype analysis Individuals from families 005, 242, and 686 were genotyped using microsatellite markers flanking the GNRHR locus (D4S2432, D4S1568, D4S409, D4S3018, D4S392) and the GNRH1 locus (D8S360, D8S1739, D8S1725, D8S1771, D8S451). The segregation pattern of informative alleles at each locus was compared with the segregation of the IHH phenotype. Pedigree and genetic data are summarized in Table 2.

Family 005.
The two affected siblings (V:2 and V:3) inherited distinct parental alleles. If the GNRHR locus were segregating with IHH, the haplotypes should be identical between affected individuals. This observed pattern is discordant with involvement of GNRHR in this family (Fig. 1).

Family 242.
Although the markers D4S2432 and D4S1568 are uninformative, the remaining markers demonstrate that the affected cousins VI:1 and VI:6 do not share alleles, excluding involvement of GNRHR (Fig. 3).

Family 686.
Although affected individuals IV:2 and IV:5 inherited identical parental alleles at this locus, affected subjects IV:4 and IV:6 did not, discordant with involvement of GNRHR (Fig. 4).

For comparative purposes, haplotype data are presented on two families in which GNRHR mutations have previously been described. In family 476, individual II:2 inherited identical maternal and paternal haplotypes. Each unaffected sibling carries only one of these alleles. A mutation involving GNRHR cannot be excluded in this family. A coding sequence abnormality was identified in the proband and, as predicted by the haplotype, was homozygous (Gln106Arg) (Fig. 5; Ref. 10).

View larger version (28K): [in this window] [in a new window]   Figure 5. Pedigree 476 and haplotype data for GNRHR. Markers surround GNRHR gene. Chromosomes are depicted by different shading.

View larger version (29K): [in this window] [in a new window]   Figure 6. Pedigree 014 and haplotype data for GNRHR. Markers surround GNRHR gene. Chromosomes are depicted by different shading.

The segregation pattern of alleles ruled out the possibility of linkage for families 242 and 686 to the GNRH1 locus (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Discussion References

 
As a disease model for genetic studies, isolated IHH poses several challenges to investigators. It is rare, typically interferes with fertility leading to small families, is delayed in its expression until puberty limiting the ability to phenotype individuals, presents a wide range of phenotypic expression, and exhibits several modes of inheritance. So far, only one gene has been implicated in patients with IHH, normal olfaction, and normal adrenal function, GNRHR (8, 9). The majority of mutations discovered to date within this G protein-coupled receptor disrupt or completely eliminate ligand binding, which then prevents GnRH-induced stimulation of inositol phosphate accumulation (8, 10, 16, 17, 18, 19, 20). Some GNRHR mutations alter activation of phospholipase C directly (8, 9) or reduce membrane expression (9, 21).

To date, our group has conducted the largest screening study for coding sequence GNRHR mutations in an IHH population (10). Patients with IHH were classified according to both phenotype and mode of inheritance. Coding sequence mutations in the GNRHR were identified in only two of five probands (40%) within the autosomal recessive subgroup.

Predicting which patients carry GNRHR sequence abnormality mutations on purely clinical criteria remains challenging because the issues of variable expressivity and incomplete penetrance are still being charted. Some but not all individuals with IHH and GNRHR sequence variants have a partial form of IHH (i.e. fertile eunuch syndrome) (22). Some patients have abnormal dose responsiveness to long-term exogenous GnRH administration, but this therapy is available only at research centers. The probands presented in this report have congenital complete IHH and those that were treated with pulsatile GnRH responded appropriately. However, in our experience, these observations do not exclude the possibility that these patients can harbor GNRHR mutations. Even the absence of sequence abnormalities in genomic exon-by-exon analysis does not eliminate a role for the candidate genes GNRHR and GNRH1. In fact, the Human Genome Project has reinforced the concept that amino acid sequence is just one of the building blocks of protein complexity. Sequencing of the human genome has revealed a much smaller number of genes than anticipated (32,000) but a much higher number of expressed sequence forms, suggesting a major role for alternative splicing in the production of protein variation (23). Mutations deep within introns can affect the splicing/expression of consensus or alternatively spliced proteins, and mutations within promoters can affect transcriptional regulation but neither are sought after in conventional mutation analysis approaches. Although pedigree analysis by our group and others demonstrates that IHH is a genetically heterogenous disease, the small families in this condition make it essential to maximize genetic approaches to fully eliminate important candidate genes, allowing samples to be redirected to target novel, undiscovered genetic loci.

It is with this backdrop that haplotype analysis was performed on the pedigrees in this report. Segregation of alleles convincingly eliminated a role for GNRHR in three families and GNRH1 in two. Moreover, the heterozygote patterns observed (as opposed to homozygous) eliminated the possibility of a gene deletion. In retrospect, haplotype analysis should have been performed as a first-pass approach, and only when a candidate gene could not be eliminated, should mutation analysis have been performed. In planning future studies for IHH, or any disease model with small families, such a step-wise, two-tiered approach can lead to the creation of a genetically purified population that, albeit small, could potentially be pooled for either parametric or nonparametric (model-free) linkage studies. In fact, a nonpurified study population that includes undetected candidate gene mutations could lead to the masking of positive linkage signals for other potentially novel genes.

This report includes not only three small pedigrees with IHH but also a relatively large inbred family from Saudi Arabia. In such highly inbred families, consanguineous marriage loops can provide substantial statistical power for linkage, even if the total number of affected individuals is modest. As such, this family may have the potential to lead to the identification of a novel autosomal chromosomal locus for IHH. Such an undertaking will provide important new insights into the mechanisms that govern the synthesis and secretion of GnRH.

	Footnotes

 
Abbreviations: GNRHR, GnRH receptor gene; IHH, idiopathic hypogonadotropic hypogonadism; T, testosterone.

Received December 13, 2002.

Accepted March 7, 2003.

	References

Top Abstract Introduction Subjects and Methods Discussion References

 

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