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BRIEF REPORT |
Departments of Endocrinology and Metabolism (Y.A., Y.T., K.M., M.A.), Genetics (K.Ku.), and Radiology (K.F., N.A.), Kanagawa Children’s Medical Center, Kanagawa 232-8555, Japan; Department of Genetics (H.K.), Nagano Children’s Hospital, Nagano 399-8288, Japan; and Department of Pediatrics (K.Ko.), Keio University School of Medicine, Tokyo 160-8582, Japan
Address all correspondence and requests for reprints to: Yumi Asakura, M.D., Department of Endocrinology and Metabolism, 2-138-4 Mutsukawa Minamiku Yokohamashi, Kanagawa Children’s Medical Center, Kanagawa 232-8555, Japan. E-mail: yumi79{at}mvb.biglobe.ne.jp.
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Abstract |
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Objective: We performed endocrine and radiological studies to determine whether endocrinological disorders such as hypogonadotropic hypogonadism, GH deficiency, or hypothyroidism are involved and also whether olfactory bulb hypoplasia and semicircular canal aplasia are major signs in patients with molecularly confirmed CHARGE syndrome.
Design: Clinical features, endocrinological assessments, and radiological abnormalities in eight children (five boys and three girls) whose molecular analyses were available were evaluated among 15 children clinically diagnosed with CHARGE syndrome at our institute.
Results: We identified heterozygous CHD7 mutations in all patients screened for mutations. Four boys had micropenis and/or cryptorchidism. One was diagnosed with GH deficiency, and the other was diagnosed with hypothyroidism. Computed tomography findings revealed aplasia of the semicircular canals. Magnetic resonance imaging studies of the olfactory bulb region revealed abnormal olfactory sulci and bulb development in all children.
Conclusion: We suggest that hypogonadism, GH deficiency, and hypothyroidism could be possible endocrinological defects in patients with CHD7 mutations and that olfactory bulb hypoplasia as well as semicircular canal aplasia should be considered as a major sign for CHARGE syndrome and recommend a computed tomography scan of the temporal bone and magnetic resonance imaging study of the olfactory bulb region.
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Introduction |
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Severe hearing loss and semicircular canal hypoplasia were found in almost all patients evaluated (2, 3, 4). Anosmia and archinencephaly have also been reported (5), and magnetic resonance imaging (MRI) enabled us to find anomalies of the olfactory bulbs (6, 7). Sanlaville et al. (8) reported that these two features were constant in fetuses with CHD7 mutations and proposed them as major diagnostic criteria of the syndrome. We carried out computed tomography (CT) scans of the temporal bone to identify semicircular canal abnormalities and MRI studies of the olfactory bulb region in patients with CHD7 mutations.
Hypogonadism is another frequent feature (7, 9, 10). Besides hypogonadism, growth failure with or without GH deficiency (GHD) (7, 11) and hypothyroidism (12) have been reported as endocrinological defects. We evaluated the endocrinological involvement of the children with CHD7 mutations to clarify their endocrinological condition.
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Patients and Methods |
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From 15 patients clinically diagnosed with CHARGE syndrome at the author’s institute, five boys and three girls, whose parents agreed with the molecular analysis, were recruited. A diagnosis of CHARGE syndrome was made according to the diagnostic criteria by Blake et al. (13). Written informed consent was obtained from their parents in accordance with a protocol approved by an institutional review board. Clinical information was collected retrospectively from their medical records.
Molecular studies
The entire CHD7 coding regions (exon 2–38) of the eight patients were screened for mutations. The primer pairs were designed to amplify exon, exon-intron boundaries, and short flanking intronic sequences. PCR amplicons from genomic DNA were scanned for mutations using DNA HPLC (Transgenomic, Omaha, NE). PCR products corresponding to all variant elution profiles of the DNA HPLC were purified using a desalting column and were sequenced bidirectionally using the dideoxy sequencing method (BigDye Dideoxy sequencing kit; Applied Biosystems, Foster City, CA) and an automated sequencer (ABI3100; Applied Biosystems), as reported previously (14).
Endocrinological assessment
Gonadal function was examined by a GnRH test (100 µg/m2), and LH peak levels after GnRH were compared with mean ± 2 SD of the normal Japanese reference calculated through conversion of a logarithmic procedure (15). IGF-I levels were evaluated by immunoradiometric assay and compared with mean ± 1.96 SD of the reference for different ages (16). GH secretion was examined by insulin (0.1 U/kg) and/or arginine (0.6 g/kg) provocation in seven patients, and the response was considered normal if the GH peak was less than 6 ng/ml by immunoradiometric assay calibrated with human GH WHO 98/54. Thyroid function was assessed by baseline TSH and free T4 levels and by TRH provocation (10 µg/kg). The normal limits of these levels were 0.9–2.0 ng/dl (11.6–25.7 pmol/liter) for free T4, and 0.5–5 mIU/liter for TSH as baseline, and the response of TSH after TRH was considered normal if the TSH peak was 10–35 mIU/liter.
Radiological studies
MRIs of the pituitary gland and olfactory bulbs were evaluated in all children using a 1.5-T unit with 2-mm slices and a T2-weighted spin echo sequence and analyzed by two radiologists. Coronal view of the normal olfactory region consists of symmetric distinct sulci and symmetric well defined bulbs. Pituitary height was compared with the mean ± 1 SD of the normal reference for different ages (17). CT scans of temporal bone were analyzed by the same radiologists. Agenesis of semicircular canals was noted when the canals were absent and an isolated vestibule was present.
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Results |
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Six children fulfilled Blake’s diagnostic criteria (13), and two (cases 4 and 7) filled two major and five minor criteria. Their characteristics are shown in Table 1
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We identified heterozygous CHD7 mutations in all children screened: five nonsense mutations (cases 1, 2, 3, 6, and 8), one frameshift mutation predicting a premature stop codon (case 4), and two missense mutations (cases 5 and 7). The frameshift mutation 4480C
T (R1494X) has been reported in a Japanese patient (12), and the other seven have not been reported. The mutation 5050GA (G1684S) of case 7 was proven to be de novo. The mother of case 5 didn’t have the mutation, but a paternal sample was unavailable. Two hundred alleles from 100 control subjects were examined for these two missense mutations of 4529CG and 5050GA, and no mutations were detected.
Endocrinological assessment
Gonadotropin axis Genital hypoplasia was found in four boys, three of whom had micropenis defined as a penile length of less than –2 SD for a given age (18), and four had unilateral or bilateral cryptorchidism. None of the girls had a record describing hypogenitalism. LH responses to GnRH stimulation were less than –2 SD of the reference in six. However, their ages were not the appropriate range that would provide useful information concerning gonadotropin secretion.
GH All patients were born slightly small (average birth height, –1.9 SD) after full-term gestation. Growth impairment became apparent postnatally and resulted in short stature of less than –2 SD in all cases. Cases 2 and 7 were suspected to have GHD due to insufficient GH responses to two provocations, but only case 7 with low-for-age IGF-I levels was diagnosed with GHD.
Thyroid axis Baseline TSH in three patients (cases 4, 5, and 7) was high. Case 4 was accompanied by low free T4 with a high level of serum thyroglobulin (1700 ng/ml) without thyroid autoantibodies. An echogram showed an enlarged thyroid gland with a highly echoic nodule. Cytology by needle biopsy revealed no malignant findings, and the high echoic region was not detectable after the biopsy. The peak TSH was within the normal range, but TSH at 12.8 mIU/liter at 120 min seemed to be prolonged. Case 5 had a high peak TSH with a normal level of free T4. The thyroid gland was normal by echogram despite a high level of thyroglobulin (130 ng/ml). These findings were normalized with L-T4 replacement. In case 7, the thyroid axis was considered normal. In cases 1, 2, and 6, peak levels of TSH were slightly low, but the free T4 levels were normal.
Radiological imaging
MRIs of the hypothalamo-pituitary region showed normal structure of the anterior pituitary gland. In three patients, the central height of the pituitary gland was slightly low. By contrast, MRIs of the olfactory bulb region were abnormal in all patients. Olfactory sulci were shallow or absent and asymmetric. Olfactory bulbs were hypoplastic or aplastic (Fig. 1). CT studies of the temporal bone of six patients showed bilateral agenesis of the semicircular canals and dysplastic vestibules.
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Discussion |
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Since the CHD7 gene was discovered as a major gene involved in this syndrome, wide phenotypic spectrum has been reported (2, 3, 8, 12). Semicircular canal agenesis (2, 8) and archinencephaly (8) have been reported in all patients with CHD7 mutations examined. Recently, heterozygous mutant mice carrying a loss-of function allele were reported to reveal abnormalities similar to the features of CHARGE syndrome (19, 20). Widespread expression of Chd7 in early development of the mouse was recognized in organs affected in CHARGE syndrome including semicircular canals and olfactory epithelium (8, 19, 20). In this study, we confirmed semicircular canal and olfactory bulb dysplasia in patients with CHD7 mutations. We agree with Sanlaville’s proposal that these two features should be considered as major signs for CHARGE syndrome (8).
On the issue of endocrinological status, we suggest that endocrinological evaluation not only for hypogonadism but also for GH secretion and thyroid function are necessary. Hypogonadotropic hypogonadism has been reported as an endocrinological defect in CHARGE syndrome (9). Unfortunately, the ages of the patients in our study were not in the appropriate range that would provide useful information concerning gonadotropin secretion, but the observation of cryptorchidism and/or micropenis in four boys provides circumstantial evidence that CHD7 mutations could cause hypogonadotropic hypogonadism, and genital hypoplasia in boys deserves special attention. In girls, female genital hypoplasia has been reported (2, 8, 12) but was not identified in our children, because hypogonadism in girls is difficult to determine before the age at which puberty is normally started. Only one of eight had a documented GHD. One had marginally low stimulated GH responses, but this was associated with a normal IGF-I level and could be due to hypogonadism. GHD doesn’t explain all of their growth failure.
One case had hypothyroidism, and one had hyperthyrotropinemia. Hypothyroidism and thyroid tumor were described as atypical features of CHD7 mutations by Aramaki et al. (12). Case 4 had primary hypothyroidism with an enlarged thyroid gland. His TSH response after TRH was prolonged and seemed to be stunted as in primary hypothyroidism in comparison with his low free T4. The cause of hypothyroidism has not been clarified yet, because the number of patients was too small. Hypothyroidism is not a constant feature but could be part of the phenotypic spectrum. We could not deny that case 4 might have hypothalamo-pituitary insufficiency as well as primary hypothyroidism.
Bosman et al. (19) reported the identification of mutations in the Chd7 gene in mouse N-ethyl-N-nitrosource (ENU) mutagenesis programs. Hurd et al. (20) generated mice carrying Chd7Gt as a reporter-tagged loss-of function allele and tracked Chd7 mutant cells in CHARGE-relevant organs to clarify developmental mechanism of Chd7 deficiency. CHD7 expression has been reported within the hypothalamus and pituitary gland during the human embryonic period (8) and Chd7 in Rathke’ pouch (19). These suggest that the endocrine deficiencies including GHD and hypothyroidism as well as hypogonadism may occur centrally in the differentiation of the hypothalamic nuclei and/or more peripherally in the differentiation of the tropic cells of the anterior pituitary. Chd7 mutant cells were observed in the developing pituitary, but no morphological abnormalities of the pituitary gland were reported in heterozygous Chd7 mutants (20). Additional studies with hormone-specific markers are necessary in Chd7 mutant mice to clarify whether primary pituitary dysfunction might contribute to the hormonal defects observed in patients with CHD7 mutations.
No apparent genotype-phenotype correlations have been noted so far or observed in the present study, but it is still possible that less severe mutations result in less specific phenotypes. It would be interesting to know whether CHD7 mutations could be identified in patients with semicircular canal agenesis and/or olfactory bulb anomalies with fewer features of the CHARGE phenotype. Another interesting issue is whether CHARGE syndrome could cause a Kallmann-like phenotype. Although we could not confirm hypogonadotropic hypogonadism, the observation of cryptorchidism and micropenis in boys provides circumstantial evidence that CHD7 mutations could cause hypogonadotropic hypogonadism and a Kallmann-like phenotype.
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Footnotes |
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First Published Online December 18, 2007
Abbreviations: CT, Computed tomography; GHD, GH deficiency; MRI, magnetic resonance imaging.
Received June 26, 2007.
Accepted December 11, 2007.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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