Clinical and molecular analysis of human reproductive disorders in Brazilian patients

Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone(LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG. Correspondence


Introduction
Over the past decade, many genes that influence the development and function of the hypothalamic-pituitary-gonadal axis (HPG) have been identified (1).These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG, and regulate the complex developmental, paracrine, and endocrine interactions that are necessary for normal reproduction (1).
We review here the extensive experience of a single Brazilian Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by disorders of the HPG, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function.Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR and LHR (Table 1).The mutations identified in Brazilian patients in these genes were shown in Table 2.

KAL gene
Kallmann syndrome (KS) consists of con- genital hypogonadotropic hypogonadism (HH) associated with anosmia (absent sense of smell).During fetal development, GnRHreleasing neurons originate in the olfactory placode and migrate with olfactory neurons through the cribform plate to the olfactory bulb and into the fetal hypothalamus.Abnormalities in these processes explain the association of HH with anosmia in patients with KS (2).The gene known to be responsible for the X-linked form of KS, KAL, encodes a protein, anosmin, that plays a key role in the migration of GnRH neurons and olfactory nerves to the hypothalamus (1).Anosmin is also expressed in the development of Purkinje cells located in the cerebellum, meso-, and meta-nephros, oculomotor nucleus, and facial mesenchyme, explaining the association of X-linked KS with synkinesia, renal agenesis, visual abnormalities and midline facial defects (3).In addition to X-linked pedigrees, autosomal dominant and recessive kindreds with KS have been reported (4).The frequency of KAL deletions or mutations is currently unknown because these are rare disorders and large series are unusual.We examined 23 patients with KS and determined their modes of inheritance and incidence of deletions and mutations in the coding sequence of the KAL gene.The diagnosis of HH was based on absent or incomplete sexual development after 17 years of age associated with low or normal levels of luteinizing hormone (LH) in both sexes and low levels of testosterone in males and estradiol in females.All patients had anosmia or hyposmia which was confirmed by an olfactory specific test, i.e., "The Smell Identification Test" (5).No associated features were observed in our patients.Of the 23 KS patients, 12 were familial, whereas 11 were sporadic cases.No deletion in the coding sequence of KAL was found and a point mutation, R191X, was identified for the first time in a patient with a familial history that suggested X-linked inheritance without other associated abnor-malities.This variable penetrance of features is common in families with KAL mutations, suggesting that modifier genes or epigenetic phenomena influence phenotypic expression (6).We also confirmed that mutations in the coding sequence of the KAL gene occur in a minority of KS cases, as previously reported by Oliveira et al. ( 4) who demonstrated that only 14% of familial cases and 11% of sporadic cases of KS had mutations identified in the KAL gene.These findings suggest that autosomal genes not yet identified account for the majority of familial cases of KS.

SF1 gene
The orphan nuclear receptor steroidogenic factor 1 (SF1) gene, officially designated NR5A1, was initially identified as a regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases (7,8).Subsequent studies have shown that SF1 is a key regulator of endocrine function within the HPG axis and adrenal cortex (9).Disruption of the mouse Nr5a1 gene encoding SF1 causes adrenal and gonadal agenesis, XY sex reversal, structural abnormalities of the ventromedial hypothalamic nucleus, and altered gonadotropin secretion by the pituitary gonadotrope.
To date, only three mutations in SF1 have been described in humans (10)(11)(12).In each of these patients, the SF1 mutation caused adrenal insufficiency with varying degrees of gonadal dysfunction, supporting the importance of the role of SF1 in human adrenal development.We identified a novel 8-bp microdeletion of the SF1 gene in a 46,XY Brazilian patient with primary amenorrhea and HH and unexpected normal adrenal function.This microdeletion resulted in a premature termination upstream of sequences encoding the activation function 2 domain, suggesting that this defect is the cause of loss of function of the SF1 leading the XY sex reversal in this patient (Correa RV, Domenice S, Bingham NC, Billerbeck AEC, Rainey WE, Parker KL and Mendonca BB, unpublished data).

DAX1 gene
The DAX1 (DSS locus-dosage sensitive sex reversal-adrenal insufficiency, X chromosome gene 1) gene has been mapped to the short arm of the X chromosome (Xp21) and encodes a transcriptional repressor factor that is classified as an orphan nuclear receptor (13,14).DAX1 is expressed in adrenal cortex, gonads, hypothalamus and pituitary and plays a key role in the development of the adrenal gland and HPG axis (15,16).Mutations in this gene in humans cause an X-linked adrenal cytomegalic form of adrenal hypoplasia and HH (17,18).Recently, a unique case extended the clinical spectrum of the disease including mild forms of HH, delayed-onset of adrenal insufficiency and abnormal spermatogenesis (19).The majority of DAX1 mutations reported are frameshift or nonsense mutations.These mutations cause truncation of the functionally important carboxyl-terminal region of the protein and impair DAX1 interaction with other factors and genes (20).
We described a 2-year-old boy with Xlinked adrenal hypoplasia congenita due to a nucleotide G insertion between nucleotides 430 and 431 in exon 1, resulting in a frameshift mutation and premature stop codon at position 71 of DAX1 (21).This boy showed isosexual gonadotropin-independent precocious puberty as the first clinical manifestation (21).He presented pubic hair, enlarged penis and testes, and advanced bone age.His testosterone levels were elevated, whereas basal and GnRH-stimulated LH levels were compatible with a prepubertal pattern.Chronic GnRH agonist therapy did not reduce his testosterone levels, supporting the diagnosis of gonadotropin-independent precocious puberty.In this patient, steroid replacement therapy resulted in a decrease of testicular size and testosterone levels to the prepubertal range indicating an ACTH-dependent precocious puberty of testicular origin (21).These findings strongly suggest that chronic excessive ACTH levels resulting from adrenal insufficiency may stimulate Leydig cells and lead to gonadotropinindependent precocious puberty in boys with DAX1 gene mutations.
More recently, another boy who presented adrenal insufficiency at 2 years of age was referred to us for clinical and molecular analysis.He was successfully treated with glucocorticoid and mineralocorticoid replacement and at the age of 16 years he was re-evaluated due to delayed puberty.Hormonal data confirmed adrenal insufficiency and HH.DNA analysis revealed a C>T transition at nucleotide 1133 in exon 1 of DAX1 which caused an alanine (GCC) to valine (GTC) substitution at codon 300 in this boy.This missense mutation was not detected in his mother.Therefore, the de novo missense mutation A300V in the DAX1 gene, located at the carboxy-terminal end the of DAX1 protein, caused a severe phenotype in this boy characterized by adrenal insufficiency and HH, suggesting that this region is fundamental for DAX1 action.

GnRH gene
GnRH is a peptide that exerts an important regulatory control over the neuroendocrine axis by stimulating the production and release of LH and FSH.In patients with HH, both gonadotropins are low and GnRH replacement restores the reproductive potential in most of them.Based on these data, GnRH deficiency has been considered to be the etiologic defect in isolated HH and the GnRH gene would be an obvious candidate for mutations in patients with isolated HH (22).This idea was also supported by a naturally occurring animal model of human isolated HH, i.e., mice with autosomal recessive HH caused by a GnRH gene deletion (23,24).However, no human GnRH gene deletions or mutations were identified in humans with HH (25)(26)(27).We have investigated the GnRH gene in four Brazilian patients with isolated HH and to date no mutations or deletions were found in this gene.

GnRHR gene
Several investigators have characterized GnRH receptor (GnRHR) gene mutations as the first identifiable cause of autosomal recessive isolated HH (28).de Roux et al. (28) hypothesized that partial-loss-of-function mutations similar to those described for the LH/hCG receptor gene could occur in patients with incomplete isolated HH.They studied a family with an affected brother and sister and found a compound heterozygous mutation, G106R/R262G, in the GnRHR gene.An increasing number of point mutations in the GnRHR gene have been described in congenital isolated HH.It currently appears that up to 20% of isolated HH patients may have mutations in this receptor (28,29).The clinical features of patients with GnRHR gene mutations are highly variable, even in the same kindred.
We investigated 17 patients from 14 different families, with HH and normal olfaction confirmed in all patients by the specific olfactory test (30).A homozygous missense mutation, Arg139His, located in the conserved DRS motif at the junction of the third transmembrane and the second intracellular loop of the GnRH receptor, was identified in one female with complete isolated HH.The Arg139His mutation completely eliminated detectable GnRH binding activity and prevented GnRH-induced stimulation of inositol phosphate accumulation in vitro.In another family, a compound heterozygous mutation (Asn10Lys and Gln106Arg) was identified in four Brazilian siblings (two males and two females) with partial isolated HH.The Gln106Arg mutation, located in the first extracellular loop of the GnRHR gene, was previously described and in vitro analysis indicated that the mutant receptor was able to bind GnRH, but with a reduced affinity.The Asn10Lys mutation in the extracellular amino-terminal domain of the receptor also reduced the affinity for GnRH in vitro.A good correlation between genotype and phenotype was found in the Brazilian patients: the female, who was homozygous for the completely inactivating Arg139His mutation, had complete HH with undetectable basal serum LH and FSH levels which failed to respond to GnRH stimulation.In addition, the affected patients, who were compound heterozygotes for the Asn10Lys/Gln106Arg mutations, had partial HH with low basal serum LH levels which were responsive to GnRH stimulation.No clinical or hormonal differences were found between HH patients with and without mutations in the GnRHR gene, indicating that these data do not contribute to the identification of HH patients with GnRHR mutations (30).

FSHß gene
The gonadotropins are heterodimers consisting of specific ß-subunits that are noncovalently bound to a common α-subunit.Selective FSH deficiency due to FSHß-subunit gene mutations causing hypogonadism and infertility is a very rare condition.To date, three distinct FSHß-subunit gene mutations have been described in four unrelated females and two males with hypogonadism (1).These mutations often affect the seatbelt region of the protein, and interfere with the synthesis and stability of the heterodimer complex.Consequently, patients have been found to have undetectable serum FSH and elevated serum LH.Recently, we identified an FSHß-subunit gene mutation in a woman with delayed puberty, who presented partial breast development and primary amenorrhea.Her basal and GnRH-stimulated LH levels were elevated, whereas her FSH levels were undetectable in both conditions.Direct sequencing revealed a mutation at codon 76 (Tyr76X) in homozygous state in this patient.This nonsense mutation was previously described in another apparently unrelated woman with a partial phenotype of FSH deficiency (31).These findings, taken together, indicate that Tyr76X, the mutation of the FSHß subunit, is associated with a partial phenotype of FSH deficiency, indicating a good phenotype-genotype correlation in this rare condition.

FSHR gene
The FSH receptor (FSHR) gene exerts its action by binding to a specific membrane receptor which belongs to the G-protein coupled receptor superfamily (32).An inactivating mutation (C566T) in exon 7 of the FSHR gene was first described in highly inbred Finnish families with autosomal recessive premature ovarian failure and normal karyotype (33).This mutation was associated with variable pubertal failure and primary or secondary amenorrhea (34).When we investigated the presence of abnormalities in the FSHR gene in 15 women with familial or sporadic premature ovarian failure we did not identify any inactivating mutations in exons 7-10 of these patients (35).Although the number of Brazilian patients evaluated was small, these findings support the hypothesis that C566T mutation is probably restricted to Finland.We demonstrated a high allelic frequency of two different nucleotide substitutions in exon 10 of the FSHR gene, G919A and G2039A, in patients with familial and sporadic premature ovarian failure (35).These polymorphisms also were found at similar frequencies in healthy women, indicating that these substitutions are of no biological significance.

LHR gene
The human LH receptor (LHR) gene is a member of the G protein-coupled receptor family with seven transmembrane helices (36).Distinct activating and inactivating mutations in the LHR have been described in males with gonadotropin-independent precocious puberty and in a rare form of pseudohermaphroditism (Leydig cell hypoplasia), respectively (32,36).
Familial male-limited precocious puberty, also known as testitoxicosis, is an autosomal dominant condition.Affected males develop rapid virilization, growth acceleration, and skeletal advancement between 2-4 years of age with elevated levels of testosterone, despite prepubertal LH levels.We reported the clinical and molecular aspects of 8 boys with testitoxicosis belonging to 7 unrelated families (37).Sequencing of exon 11 of the LHR gene revealed five different mutations, Ala568Val, Leu457Arg, Thr577Ile, Leu368Pro and Met571Leu (37)(38)(39)(40).These mutations lead to high basal cAMP production in transfected COS-7 cells, reflecting an intrinsic ability of the mutant receptors to couple with and stimulate Gs in the absence of the ligand (36).The Ala568Val in the third intracellular loop was the most frequent defect in Brazilian boys with testitoxicosis (37).Interestingly, one boy had this mutation in homozygous state due the maternal isodisomy, which was demonstrated by microsatellite analysis.In addition, the common ancestral origin of the Ala568Val mutation was demonstrated by the high frequency of a polymorphic allele (D2S123) located within the short arm of chromosome 2 in three families studied.Female patients who were carriers of LHR-activating mutations did not have any clinical or hormonal abnormalities, indicating the lack of phenotypic expression of these mutations in females (37).
Leydig cell hypoplasia is characterized by failure of fetal testicular Leydig cell differentiation.Affected 46,XY individuals display female external genitalia or a micropenis associated or not with hypospadias (36).We reported the first occurrence of micrope-nis and of amenorrhea and infertility due to inactivating mutations in LHR in a Puerto Rican boy and in a 46,XX Brazilian woman, respectively (41).This female patient had a homozygous Arg554Stop which truncated the LHR within the third transmembrane domain.A distinct homozygous deletion of two consecutive amino acids (Leu-608 and Val-609) in the seventh transmembrane helix of the LHR was also found in another family (42).In both families, the affected 46,XY patients had female external genitalia, whereas their sisters had irregular menses (oligoamenorrhea) or secondary amenorrhea and infertility.
In conclusion, identification of naturally occurring genetic mutations has led to significant benefits for the patients, such as appropriate and educated counseling, as well as specific treatment.

Table 1 .
Single gene disorders causing impaired gonadal development and function.

Table 2 .
Mutations identified in patients with reproductive disorders.