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Pediatric Endocrinology Unit (G.P., R.M., R.R.), General Pediatric Unit (V.A.), Pediatric Radiology Unit (L.H.-P.), Department of Genetics (J.A., S.L.), Necker-Enfants Malades Hospital, 75015 Paris, France; Department of Radiology (J.B.), Foundation Rothschild, 75019 Paris, France; Department of Pediatric Endocrinology (S.C., I.N.), Armand Trousseau Hospital, A.P.H.P., U.P.M.C., Institut National de la Santé et de la Recherche Médicale U 515, 75012 Paris, France; and Department of Pediatrics (A.M.B.), Besançon Hospital, 25030 Besançon, France
Address all correspondence and requests for reprints to: Irène Netchine, M.D., Ph.D., Pediatric Endocrinology, Armand Trousseau Hospital, A.P.H.P., U.P.M.C. Paris 6, INSERM U 515, 26 Avenue du Dr. Arnold Netter, 75012 Paris, France. E-mail: irene.netchine{at}trs.aphp.fr.
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Abstract |
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Objective: The objective of the study was to study gonadotropic axis function and growth parameters in CHARGE syndrome.
Design: This was a retrospective study.
Patients: The study included 32 children with CHARGE syndrome.
Results: Nineteen of 20 affected boys had micropenis and/or cryptorchidism, consistent with hypogonadotropic hypogonadism during fetal life. None of the boys was of pubertal age. Seven of nine boys tested before the age of 5 months during the neonatal peak period had extremely low testosterone levels. LH response to GnRH stimulation was variable during the first year of life and not correlated with existing clinical abnormalities. None of the girls over the age of 12 yr (n = 7) had begun puberty spontaneously, and a lack of response to GnRH stimulation was documented in five of them. Olfactory evaluation (n = 10) and magnetic resonance imaging (n = 18) of the forebrain revealed defective sense of smell and abnormal olfactory bulbs in all cases. Cardiorespiratory and nutritional problems were corrected, but the mean height of the 25 children who had reached 5 yr of age was –2 ± 0.2 SD score. Height was not correlated with birth length or body mass index. GH deficiency was diagnosed in only three children.
Conclusion: These findings suggest that CHARGE syndrome includes the main features of Kallmann syndrome, which is defined by hypogonadotropic hypogonadism combined with a defective sense of smell and abnormal olfactory bulb development. This forebrain abnormality, if confirmed in a larger group of patients, could serve as a major new criterion for the diagnosis of CHARGE syndrome.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We investigated the cause of genital hypoplasia and growth retardation in CHARGE syndrome by studying gonadotropic axis function, nutritional status, and growth parameters in 32 children with CHARGE syndrome. We carried out magnetic resonance imaging (MRI) of the hypothalamo-pituitary region (n = 16). We also analyzed the patients’ sense of smell (n = 10) by an appropriate olfactory method and performed MRI of the olfactory bulb region (n = 18).
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Thirty-two children, 20 boys (B1-B20) and 12 girls (G1-G12) diagnosed with CHARGE syndrome (five major criteria or four major and three minor criteria) according to criteria by Blake et al. (4) and Amiel et al. (5) were included after informed consent was obtained from their parents, in accordance with national ethics rules. Each patient was examined by a geneticist and a pediatric endocrinologist. Patients were referred to the Pediatric Endocrinology Unit at various ages, depending on their general condition and the age at diagnosis. Some children were referred at an age at which gonadotropic axis evaluation and olfactory evaluation were not possible, and MRI required general anesthesia and therefore could not be performed in some cases. The 20 boys were born between 1991 and 2002, and the 12 girls were born between 1984 and 1998.
Clinical assessment
Micropenis was defined as a penis length of less than –2 SD score (SDS) for a given age (12). Cryptorchidism was defined as testis not palpable in the scrotum after two consecutive examinations by a pediatric endocrinologist. Pre- and perinatal histories were reviewed to determine gestational age at birth. Weights and heights were measured at birth and expressed with respect to Usher and McLean standards (13). Patients were considered to be small for gestational age (SGA) if either of these two measures were less than –2 SDS. Twenty-five patients had reached 5 yr of age. Their heights and weights after birth were plotted on a Sempe growth chart (14) and analyzed according to midparental target heights.
Biochemical assessment
Endocrine status was evaluated in baseline condition or dynamic conditions. Basal plasma testosterone level was determined in seven boys before the age of 5 months, corresponding to the neonatal peak period, and compared with normal values (15, 16). In eight boys, testosterone level was measured after six human chorionic gonadotropin (HCG) injections of 1500 IU and compared with normal values (17). Estradiol levels were determined in five girls over the age of 12 yr, with concentrations less than 30 pmol/liter considered prepubertal. Gonadotropin level was evaluated at baseline (n = 13) and after the infusion of 100 µg/m2 GnRH (n = 13).The normal ranges for baseline LH and FSH levels in boys begin to increase about 2 wk after birth, reaching 0.02–7 IU/liter for LH and 0.16–4.1 IU/liter for FSH within the first 3 months. They then decline to prepubertal values by the end of the first year. Normal baseline LH and FSH levels in prepubertal girls after 2 yr of age were 0.02–0.42 IU/liter and 1–4.2 IU/liter, respectively (18). Eight boys underwent GnRH stimulation in the first year of life, and the results obtained were compared with published values (15, 19) and our own laboratory results. We considered an LH peak exceeding 6 IU/liter in the first 6 months of life to be normal in boys. Five girls over the age of 12 yr also underwent GnRH stimulation. The cutoff point between prepubertal and pubertal LH levels was defined by the 95th percentile of the normal prepubertal population. We considered an LH peak exceeding the cutoff point of 6.9 IU/liter in girls to indicate central activation of the hypothalamo-pituitary-gonadal axis, as suggested by Brito et al. (20).
GH and IGF-I levels were evaluated by RIA. GH secretion was assessed (n = 20) by means of various provocation tests [glucagon test (0.1 mg/kg), arginine test (0.5 g/kg), and ornithine test (14.5 g/m2)], and the response was considered normal if the GH peak after stimulation was more than 20 mU/liter. If the GH peak was below this value, a second provocation test was performed (n = 9). Twenty-five patients who had reached 5 yr of age were included in the growth evaluation part of the study. Patients were divided into two groups. Group I (n = 22) comprised patients with normal GH peaks after stimulation or normal serum IGF-I levels or normal growth parameters (height > –1 SDS). Group II (n = 3) consisted of patients with GH deficiency, defined by peak GH levels of less than 20 mU/liter in response to two stimulation tests and serum IGF-I levels less than –2 SDS for age and pubertal stage. Other pituitary functions were evaluated by determining plasma basal free T4 and TSH (n = 26) and basal plasma cortisol levels at 0800 h (n = 18). The normal limits of these levels in the plasma were 11–28 pmol/liter for free T4, 0.6–5 mIU/liter for TSH, and more than 193 nmol/liter for cortisol.
MRI and olfactory evaluation
MRI of the olfactory bulbs was performed (n = 18) using a 1.5-Tesla unit, in the coronal plane with 2-mm slices and a T2-weighted spin echo sequence. Images were analyzed by two consultant neuroradiologists who were required to meet a consensus. Olfactory bulbs were scored as normal, hypoplastic or aplastic and olfactory sulci as normal, shallow, or absent. MRI of the hypothalamo-pituitary region was carried out (n = 16) in the sagittal and coronal planes, parallel to the pituitary stalk with 3-mm slices and a T1-weighted spin echo sequence, before and after contrast enhancement (0.2 ml/kg gadolinium). The anterior pituitary gland was scored as normal or hypoplastic (21).
Olfactory evaluation was performed for 10 children, with the validated Biolfa method, investigating both quantitative and qualitative olfactory abilities. This method, which was first validated in healthy young adults (22), has since been validated in a control group of children and subsequently used for 10 patients. Children below this age or with intellectual development below the age of 5 yr could not be evaluated in this way. The results of the olfactory evaluation were graded as normal, hyposmic, or anosmic (23).
Statistical analysis
Data are expressed as means ± SE. Data were compared at different ages (1, 2, and 5 yr) by means of Wilcoxon rank tests. Correlations were assessed by Spearman’s rank correlation test.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Clinical and hormonal data for the gonadotropic axis are shown in Table 1
. All but one (19 of 20) of the boys had cryptorchidism and/or micropenis. Cryptorchidism was documented for 16 boys and was bilateral in 12. Fifteen boys had micropenis, associated with cryptorchidism in 12 cases. All of the male patients were of prepubertal age. Seven of nine patients tested before the age of 5 months had extremely low testosterone levels, whereas the remaining two had testosterone levels at the lower end of the normal range. Testosterone levels after HCG injection were variable [normal in two cases (>8.4 nmol/liter), very low in two cases (<3 nmol/liter), and intermediate in three cases] and were not correlated with testosterone levels before HCG injection or with the existing clinical abnormalities. All basal LH and FSH concentrations were in the normal range. LH response to GnRH stimulation was variable during the first year of life (normal in five cases, low in three cases) and was not correlated with testosterone levels before and after HCG or with the existing clinical abnormalities (Table 1). Ten boys with micropenis were treated with testosterone (2 or 4 im injections of 100 mg/m2 testosterone before the age of 2 yr), which restored normal penis length (>3 cm).
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). The other two girls were not evaluated because they had a bone age of less than 11 yr. MRI and olfactory evaluation
MRI of the olfactory bulb region was performed in 18 patients (10 boys, eight girls). Results were abnormal in all patients tested, with olfactory bulbs absent (n = 15), hypoplastic (n = 1), or asymmetric (hypoplastic on one side and absent on the other) (n = 2; Fig. 1 and Table 1). By contrast, MRI of the hypothalamo-pituitary region performed in 16 patients showed hypoplasia of the anterior pituitary in only three patients and no ectopic posterior pituitary.
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). Growth and somatotropic axis
Eleven children (34%) were born SGA. Growth parameters were determined for 25 children (11 girls) to the age of 5 yr and 9 children (5 girls) to the age of 10 yr. Median heights for the patients (SDS) were –1.5 ± 0.2 at 1 yr, –1.5 ± 0.2 at 2 yr, –2 ± 0.2 at 5 yr, and –2.4 ± 0.5 at 10 yr of age. Patients’ heights were lower than their target heights (0.1 ± 0.3, P < 0.001). The deviation of heights from mean scores (SDS) at 5 yr was significantly smaller than that at 1 yr (P < 0.04) and 2 yr (P < 0.02) and was less than –2 SDS in 56% of patients. Feeding difficulties necessitated gastrostomy in 68, 48, and 16% of patients at 1, 2, and 5 yr of age, respectively. Gastrostomy ended at a mean age of 3.5 ± 0.3 yr of age (2 months old to 12 yr old). Body mass index (BMI) did not vary significantly during the first 5 yr: –0.4 ± 0.2 Z-score at 1 yr, –0.2 ± 0.2 Z-score at 2 yr, and –0.4 ± 0.2 Z-score at 5 yr. The heights and BMI of patients born SGA did not differ significantly from those of the other patients at 1, 2, and 5 yr. Cardiorespiratory disease was severe in the first year of life in 17 patients. However, these problems were cured by cardiac surgery and/or transient tracheostomy and therefore ceased to be symptomatic after this age. We therefore did not take them into account in our analysis.
GH secretion and growth were considered normal in 22 of the 25 children who had reached 5 yr of age: normal GH secretion (n = 14) or normal serum IGF-I level for age and pubertal stage (n = 4) or height greater than –1 SDS (n = 4) (group I, data not shown). Heights (SDS) in group I patients were –1.5 ± 0.2 at 1 yr, –1.5 ± 0.2 at 2 yr, and –1.8 ± 0.2 at 5 yr of age. Three children had a documented GH deficiency (group II, Table 2). They all displayed height less than –3 SDS with a growth rate of less than 4 cm/yr, insufficient response to two stimulation tests, and IGF-I levels less than –2 SDS for age and pubertal stage despite good nutritional status.
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Other anterior pituitary functions
Free T4 and TSH (n = 26) and cortisol (n = 18) levels were normal when evaluated even for the three patients with GH deficiency (data not shown).
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Discussion |
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Hypogonadotropic hypogonadism is difficult to diagnose before the age at which puberty is normally reached. The observation of micropenis and/or cryptorchidism in almost all the male CHARGE patients studied (19 of 20) indicates that hypogonadotropic hypogonadism begins in fetal development and strongly suggests abnormal future pubertal development (24, 25). During the first 6 months of life, normal boys have a postnatal surge in FSH, LH, and testosterone levels, attributable to an increase in GnRH pulse amplitude (25). Testosterone levels were extremely low in seven of nine boys before 6 months of age. The increase in testosterone level in response to HCG infusion was variable in the first year of life and not correlated with basal testosterone levels or existing clinical abnormalities. Eight boys underwent GnRH stimulation in the first year of life. In these boys, basal LH and FSH levels did not exceed normal values as they would be in cases of anorchia (26). The LH response after GnRH stimulation was also variable, considered normal in five cases and low in three cases, and did not correlate with the existing clinical abnormalities or testosterone levels. Independently of peak LH concentration after GnRH stimulation, the extremely low testosterone levels documented in seven boys strongly suggest an insufficient spontaneous LH pulsatile secretion as testosterone levels follow the pattern of LH secretion (25, 27). Although most adults with hypogonadotropic hypogonadism do not secrete gonadotropins in response to single-bolus GnRH testing (28), their gonadotropin secretion may normalize after repeated GnRH injection or pulsatile administration of GnRH (29). Infants with hypogonadotropic hypogonadism may respond better than adults to a single bolus of GnRH as the LH receptor is stimulated by HCG during fetal development, even if GnRH secretion is insufficient and the period of hypothalamo- pituitary development with its activation cascade has only recently occurred.
Hypogonadotropic hypogonadism was also strongly suspected in a group of five adolescent CHARGE girls, all of whom displayed no LH or FSH response after GnRH stimulation. In adults, hypogonadotropic hypogonadism is traditionally diagnosed based on the absence of pubertal development by the age of 18 yr, prepubertal sex steroid concentrations, and low/normal gonadotropin levels (30). Long-term follow-up is important for this group of CHARGE patients because most are likely to require the induction of puberty, and the characterization of their gonadotropic function during adulthood would be of value.
Olfactory bulb development was found to be abnormal in all cases in which it was explored (n = 18). If confirmed in a larger group of patients, this abnormality could serve as a major new criterion for the diagnosis of CHARGE syndrome. Arhinencephaly has been reported in postmortem examinations of patients with CHARGE syndrome (3, 31), but the link with hypogenitalism in this syndrome has never before been described. Olfaction is difficult to explore, particularly in infants or children with learning disabilities. Validated olfactory evaluation was performed for 10 patients, according to the Biolfa method (22), and the results obtained were abnormal in all cases, with anosmia diagnosed in seven patients and hyposmia in three (23). We show here that all the patients with CHARGE syndrome who underwent olfactometry and MRI of the olfactory bulb region fulfilled the criteria for Kallmann syndrome, which combines hypogonadotropic hypogonadism with various degrees of deficiency in the sense of smell due to olfactory bulb aplasia or hypoplasia (32). It was recently suggested that cryptorchidism and hypogenitalism should not be considered minor CHARGE criteria because these features are nonspecific (33). However, the combination of hypogenitalism with abnormal olfactory bulb development and other criteria of this syndrome is highly evocative of CHARGE syndrome. Patients with Kallmann syndrome may occasionally display other symptoms: renal agenesis, bimanual synkinesia, cleft lip/palate, dental agenesis, and neurosensory deafness. No mutation has been identified so far in the autosomal recessive form of Kallmann syndrome. Mutations in the X-linked KAL1 gene, encoding the extracellular matrix protein anosmin 1, or in FGFR1(KAL2), encoding fibroblast growth factor (FGF) receptor 1 and involved in an autosomal dominant form of Kallmann syndrome, account for approximately 20% of all cases of Kallmann syndrome. The other, as-yet-undiscovered Kallmann genes may also be involved in FGF signaling (34). A distinct autosomal recessive disorder consisting of hypogonadotropic hypogonadism without abnormalities in the sense of smell is also known (35).
Mutations were recently identified in CHD7, a member of the chromodomain helicase DNA-binding gene family, in 10 of 17 patients with CHARGE syndrome (36). This class of proteins is thought to play a role in embryonic development by affecting chromatin structure and gene expression (37). A de novo mutation in the Semaphorin 3E gene has also been reported in one CHARGE patient (38). Because Kallmann syndrome is thought to result from a deficiency in FGF signaling at the earliest stage of olfactory bulb morphogenesis (34), our study showing that CHARGE syndrome encompasses the main features of Kallmann syndrome suggests there may be a functional connection between CHD7 and FGF signaling in olfactory bulb differentiation. In particular, it is possible that CHD7 controls the transcription of one or several genes encoding proteins involved in FGF signaling. Alternatively, FGF signaling through FGFR1 may control the activity of CHD7 or the rate of transcription of CHD7 in the olfactory bulb primordium.
Hypogonadism in Kallmann syndrome probably results from the failed embryonic migration of GnRH-synthesizing neurons along the olfactory nerve pathway (39). Our findings suggest that hypogonadotropic hypogonadism in CHARGE syndrome may result from the same embryonic defect. Use of the chd7 gene knockout in mice may make it possible to confirm this in the near future.
CHARGE patients have growth impairment that worsens with age, reaching –1.5 SDS at 1 and 2 yr and –2.4 SDS at 10 yr of age. These data are consistent with previous reports (10). Growth retardation may be multifactorial. One in three CHARGE patients is born SGA. However, the postnatal growth retardation of these children is similar to that of children born appropriate for gestational age. Poor nutritional status may contribute to the height deficit but most of the patients have a normal BMI (mean BMI at –0.4 Z-score) because nutritional problems were corrected, when necessary, by enteral feeding (68% of patients at 1 yr of age). Improvements in feeding and respiratory problems with age make it possible for children with this condition to eat normally after the age of 4 or 5 yr. Documented GH deficiency, based on the accepted criteria (40), was detected in only three patients (9%) and was not associated with TSH or ACTH deficiency. These patients had a more pronounced height deficit than the other patients at the age of 5 yr (height –3.1 SDS vs. –1.8 SDS without GH deficiency). However, this difference was not statistically significant, probably due to the small number of patients in this group. Thus, in most cases, growth impairment does not seem to be due to having been born SGA, GH deficiency, or malnutrition. Instead, it seems to be a primary feature of the disease.
In summary, if our data are confirmed in follow-up studies, MRI findings of olfactory bulb abnormalities may become a major criterion of CHARGE syndrome and may facilitate the early diagnosis of prepubertal hypogonadotropic hypogonadism.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; CHARGE, coloboma, heart defect, choanal atresia, retarded growth and development, genital hypoplasia, ear abnormalities, and/or hearing loss defect; FGF, fibroblast growth factor; HCG, human chorionic gonadotropin; MRI, magnetic resonance imaging; SDS, SD score; SGA, small for gestational age.
Received December 16, 2004.
Accepted July 13, 2005.
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