DAX1 mutations map to putative structural domains in a deduced three-dimensional model.

The DAX1 protein is an orphan nuclear hormone receptor based on sequence similarity in the putative ligand-binding domain (LBD). DAX1 mutations result in X-linked adrenal hypoplasia congenita (AHC). Our objective was to identify DAX1 mutations in a series of families, to determine the types of mutations resulting in AHC and to locate single-amino-acid changes in a DAX1 structural model. The 14 new mutations identified among our 17 families with AHC brought the total number of families with AHC to 48 and the number of reported mutations to 42; 1 family showed gonadal mosaicism. These mutations included 23 frameshift, 12 nonsense, and six missense mutations and one single-codon deletion. We mapped the seven single-amino-acid changes to a homology model constructed by use of the three-dimensional crystal structures of the thyroid-hormone receptor and retinoid X receptor alpha. All single-amino-acid changes mapped to the C-terminal half of the DAX1 protein, in the conserved hydrophobic core of the putative LBD, and none affected residues expected to interact directly with a ligand. We conclude that most genetic alterations in DAX1 are frameshift or nonsense mutations and speculate that the codon deletion and missense mutations give insight into the structure and function of DAX1.

Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3' splice site.

A 12-yr-old hypothyroid girl was diagnosed at birth as athyreotic because her thyroid gland could not be visualized by isotope scanning. Goiter development due to incomplete thyrotropin suppression, a thyroidal radioiodide uptake of < 1%, and a low saliva to plasma ratio of 2.5 suggested iodide (I-) transport defect. mRNA isolated from her thyroid gland and injected into Xenopus oocytes failed to increase I- transport. Sequencing of the entire Na+/I- symporter (NIS) cDNA revealed a C to G transversion of nucleotide (nt) 1146 in exon 6, resulting in a Gln 267 (CAG) to Glu (GAG) substitution. This missense mutation produces an NIS with undetectable I- transport activity when expressed in COS-7 cells. Although only this missense mutation was identified in thyroid and lymphocyte cDNA, genotyping revealed that the proposita and her unaffected brother and father were heterozygous for this mutation. However, amplification of cDNA with a primer specific for the wild-type nt 1146 yielded a sequence lacking 67 nt. Genomic DNA showed a C to G transversion of nt 1940, producing a stop codon as well as a new downstream cryptic 3' splice acceptor site in exon 13, responsible for the 67 nt deletion, frameshift, and premature stop predicting an NIS lacking 129 carboxy-terminal amino acids. This mutation was inherited from the mother and present in the unaffected sister. Thus, although the proposita is a compound heterozygote, because of the very low expression (< 2.5%) of one mutant allele, she is functionally hemizygous for an NIS without detectable bioactivity.

Central hypothyroidism reveals compound heterozygous mutations in the Pit-1 gene.

Mutations in the gene encoding the Pit-1 transcriptional activator interfere with the embryologic determination and ultimate functions of anterior pituitary cells that produce growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH). Central hypothyroidism is often the presenting feature of combined pituitary hormone deficiency (CPHD), but it is not detected in screening programs that rely upon elevation of TSH. We report a child whose hypothyroidism was recognized clinically at age 6 weeks, and subsequently found to have GH and Prl as well as TSH deficiency. With thyroxine and GH replacement he has reached the 70th percentile for height and has normal intelligence. Molecular analysis of genomic DNA for Pit-1 revealed the presence of compound heterozygous recessive mutations: a nonsense mutation in codon 172 and a novel missense mutation substituting glycine for glutamate at codon 174. This case is the first demonstration of CPHD due to compound heterozygous Pit-1 point mutations, as most reported cases of the CPHD phenotype involve either the dominant negative R271W allele or homozygosity for recessive Pit-1 mutations. Therefore, in cases of CPHD, the possibilities of compound heterozygosity for two different Pit-1 mutations, or homozygosity for mutations in the epigenetic gene, Prop-1, should be considered.

Response to challenge with gonadotropin-releasing hormone agonist in a mother and her two sons with a constitutively activating mutation of the luteinizing hormone receptor--a clinical research center study.

The pituitary-gonadal axis was evaluated in a mother after two of her sons with familial male-limited pseudoprecocious puberty were found to have a constitutively activating mutation of the LH receptor (LHR). Genotyping demonstrated that all showed a mutation in one of the two alleles, a substitution of Gly for Asp578 in the sixth transmembrane segment of the LHR. Ovarian function was normal in the 36-yr-old mother as assessed by LH dynamics and FSH and androgen levels throughout the menstrual cycle. Hormonal responses to acute GnRH agonist (nafarelin) challenge, chronic GnRH agonist administration, and dexamethasone were also normal. Studies of the boys upon presentation at 2.4 and 3.5 yr of age revealed that acute LH responses to nafarelin were in the hypogonadotropic range, and the FSH responses were prepubertal despite the presence of late pubertal testosterone blood levels. Upon the inception of true puberty at 11 yr of age in the older brother, gonadotropin responses normalized for the state of development. The data show that this activating LHR mutation does not cause functional ovarian hyperandrogenism and causes only incomplete pubertal activation of Leydig cells. The results are compatible with relatively low constitutive activity associated with this structural abnormality of LHR.

Feminizing Sertoli cell tumors in boys with Peutz-Jeghers syndrome.

We report the pathology findings in two cases of multicentric Sertoli cell testicular tumors in two young boys with probable Peutz-Jeghers syndrome. Four cases of such tumors occurring in boys with Peutz-Jeghers syndrome were previously reported. Each of the two boys reported in this paper had prominent gynecomastia, rapid growth, and advanced bone age. Serum levels of estradiol were markedly elevated. Anti-müllerian hormone was measured in the serum of one of the boys and was in the normal range for age. Bilateral orchiectomy was performed in each case because the neoplastic growth would most likely result in sterility, and curtailment of height potential was threatened from continued elevation of estradiol levels. Microscopically, greatly enlarged seminiferous tubules packed with ovoid Sertoli-like cells were present. Prominent eosinophilic basement membrane surrounded the tubules and intersected between the cells, forming hyalinized ovoid globules and microcalcifications. Ultrastructure revealed lamination of basement membranes surrounding adjacent cells, ovoid cells with abundant cytoplasm, and limited smooth endoplasmic reticulum. Studies of testicular tumor tissue from both cases revealed increased transcription of the aromatase cytochrome P450 gene using promoter II, the promoter directing aromatase expression in the normal ovary and testis. The levels of transcripts were comparable to corpus luteum, thus resulting in increased estrogen synthesis. Transcripts specific for placental-type aromatase promoters (I.1 and I.2) were not detected in significant levels in these tumors.

Ovarian hyperandrogynism as a result of congenital adrenal virilizing disorders: evidence for perinatal masculinization of neuroendocrine function in women.

Women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency often have a polycystic ovary-like syndrome, consisting of hyperandrogynism, infertility, menstrual irregularities, and elevated LH levels. This is generally considered secondary to poor control of the congenital adrenal hyperplasia. However, our experience led us to suspect that ovarian hyperandrogenism occurs even when congenital adrenal hyperplasia is well controlled on glucocorticoid therapy. Therefore, we tested the hypothesis that congenital adrenal virilizing disorders result in ovarian hyperandrogenism. We studied eight women with congenital adrenal virilizing disorders, seven with well controlled classic 21-hydroxylase deficiency and one with congenital virilizing adrenal carcinoma removed at 1.7 yr of age. We also studied six women with late-onset 21-hydroxylase deficiency, without signs of congenital virilization. An ovarian source of androgens was assessed after suppressing adrenal function with dexamethasone and then testing pituitary-ovarian function by a GnRH agonist (nafarelin) test. Five women with congenital adrenal virilizing disorders (four with classic 21-hydroxylase deficiency and one with congenital virilizing adrenal carcinoma) and one women with late-onset 21-hydroxylase deficiency had ovarian hyperandrogenism as determined by subnormal suppression of free testosterone after dexamethasone and/or by increased 17-hydroxyprogesterone response to nafarelin while on dexamethasone. All women with congenital adrenal virilization and ovarian hyperandrogenism had elevated LH levels after dexamethasone or elevated early LH response to nafarelin, which suggests that LH excess is the cause of their ovarian hyperandrogenism. This was not the case for the late-onset 21-hydroxylase-deficient woman. Our data are compatible with the hypothesis that congenital adrenal virilization programs the hypothalamic-pituitary axis for hypersecretion of LH at puberty. This is postulated to frequently cause ovarian hyperandrogenism even when adrenal androgen excess is subsequently controlled by glucocorticoid therapy.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Surgical and genetic aspects of persistent müllerian duct syndrome.

Persistent müllerian duct syndrome (PMDS) is characterized by the presence of a uterus, cervix, and fallopian tubes in an otherwise normally differentiated 46.XY male. During embryogenesis, regression of müllerian structures in normal males is mediated by antimüllerian hormone (AMH), also called müllerian inhibiting substance (MIS), produced by fetal Sertoli's cells. PMDS has been attributed to deficient AMH activity or to abnormalities in the AMH receptor. The authors report on two patients with PMDS in whom the abnormalities were discovered during surgery for inguinal hernia and cryptorchidism. During the initial operations in each case, testicular biopsies were obtained, and the gonads and müllerian elements were replaced in the pelvis. A second operative procedure, performed several months later, included proximal salpingectomies with dissection of the vasa deferentia on pedicles of myometrium. This permitted excision of the vestigial uterine corpus, leaving a tiny remnant of cervix with the vasa deferentia. The testes were further mobilized so that bilateral orchidopexies could be completed. In the first case, a molecular abnormality was present at position 377 of the first exon of the AMH gene. Thymine replaced cytosine, which altered a CGG arginine codon to a TGG tryptophan codon, rendering the AMH molecule unstable. The molecular abnormality in the first case differs from the first abnormality in AMH reported by Knebelmann et al, thus indicating heterogeneity in this condition. The molecular basis for deficient AMH activity in the second patient has not yet been defined. No molecular abnormalities were found in the exons of this patient's AMH gene.

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Transmission of ring 14 chromosome from mother to two sons.

We report on a family with transmission of a ring chromosome 14 from an affected mother to her 2 sons. The mother was mosaic, 46,XX,r(14)/45,XX,t(14q21q). Both of her sons, affected by seizures and mental retardation, have the karyotype 46,XY,r(14). In considering the association of translocation 14:21 in the mother with ring 14, we postulate that either the ring chromosome was formed first and then opened with translocation of the partially deleted chromosome 14 to chromosome 21, or the 14:21 translocation was present first, then the chromosomes 14 and 21 broke apart, and the partially deleted 14 formed the ring. The published literature of cases of ring 14 is reviewed.

Computerized tomography in occlusive infantile arteriopathy.

Use of computerized tomography (CT) in the diagnosis of arterial calcification of infancy, including coronary obstructive disease, has not been previously reported. We were able to demonstrate by CT scan the abnormal arteries present in this disease. The calcified arteries in this infant, who died of bowel infarction at three weeks of age, were easily delineated in CT scans of the chest, abdomen, and extremities. Autopsy sections revealed intact elastic fibers in the abnormal arteries, indicating that the basic pathologic process does not primarily involve these fibers.

The relationship of inflammatory periodontal disease to diabetic status in insulin-dependent diabetes mellitus patients.

This study was designed to evaluate the relationship of inflammatory periodontal disease to the diabetic status of the insulin-dependent diabetes mellitus (IDDM) patient. 52 IDDM patients, ages 11-22 years, were evaluated. These patients were closely monitored at regular intervals in the University of Kentucky pediatric diabetic clinic. A periodontal examination was carried out for each patient. The patients were then assigned to a periodontitis or non-periodontitis group. Moderate to advanced periodontitis was found in 5.8% of the subjects. The gingival index and sulcular bleeding index were significantly higher in the periodontitis group (P less than 0.05). There was no significant difference between groups for plaque index, age of diabetic onset, duration of diabetes, present age, insulin dosage/weight, or serum glucose (P greater than 0.05). There was a greater % of ketoacidosis, retinopathy and neuropathy in the periodontitis group. IDDM patients with neurological complications or a history of chronic infections had a significantly higher gingival index score than those without the complication (P less than 0.05).

Association of double NOR variant with Turner syndrome.

A study was made of the possible association of double nucleolus organizers (dNORs) with Turner syndrome. Occurrence of dNORs was determined using the silver staining method of Goodpasture and Bloom [1975]. In 45 control subjects, the incidence of dNORs was 8.8%. Studies were done on 33 Turner syndrome patients. In 28 cases with 45,X or 45,X/46,XX karyotypes, the incidence of the dNOR variant was 50%. Five cases of Turner syndrome with X rings or X isochromosomes were negative for dNORs. Analysis of the data indicates an association between the dNOR variant and the occurrence of Turner syndrome. It is proposed that the presence of the dNOR variant can increase the rate of nondisjunction of the X chromosome in meiosis or in mitotic divisions during early gestation.

Persistent müllerian duct syndrome in a man with transverse testicular ectopia.

A 33-year-old Pakistani man with transverse testicular ectopia underwent surgery for repair of a left inguinal hernia. At operation a uterine structure with attached vasa deferentia was found in the left inguinal area and it was removed. Transverse testicular ectopia has been reported previously in association with the persistent müllerian duct syndrome. A deficiency of activity of a müllerian inhibiting substance during gestation is believed to be responsible for this syndrome. Most patients usually are sterile. Cloning of the gene for a müllerian inhibiting substance should permit studies of the pathogenesis of the persistent müllerian duct syndrome.

Tooth eruption and craniofacial development in congenital hypothyroidism: report of case.

The long-term effects of severe hypothyroidism on craniofacial growth and dental development are illustrated in this case. It is apparent that given a favorable diet, the primary dentition can persist for a long period (early childhood to at least the age of 19) without the development of dental caries. It is also clear that the dental structures can still respond to the effects of L-thyroxine at a relatively late age, with the exfoliation of primary dentition and eruption of the secondary dentition. Impacted mandibular second molars appear to be rare. The lack of proper growth of the mandible and failure of normal resorption of the internal aspect of the ramus associated with deposition of bone on the external aspect with the development of normal-size teeth, resulted in a lack of space for the eruption of mandibular second molars. The impaction of the mandibular second molars in this patient seems to be caused by a dissociation of ramus growth and dental development, resulting in insufficient space for proper eruption of these teeth.

Psychological findings in early treated cases of female pseudohermaphroditism caused by virilizing congenital adrenal hyperplasia.

Nine female adolescents with female pseudohermaphroditism resulting from virilizing congenital adrenocortical hyperplasia (CAH) were studied in terms of gender identity, sex-role behavior, psychological adjustment, and psychosexual development. A group of adolescents with chronic illness was used as a control. The Draw-A-Person, the Bem Sex-Role Inventory, Rorschach, TAT, and a questionnaire reviewing peer and romantic activities were administered to both groups. The two groups were comparable on measures of general personality adjustment, with the CAH girls showing a trend toward greater bodily concerns. Sex-role identity for both groups was near the adolescent girl norms for both Femininity and Masculinity, with virilized CAH girls showing slightly higher Androgyny scores. Significant differences were found on gender identity as measured by greater differentiation of the drawn male figure as well as a trend toward drawing the male figure first. The CAH females also showed consistent patterns of psychosocial delay in dating and sexual relations as compared to the control group. Gender identity in this group appears to be mediated by body image. The resulting ambivalence may be evidence of feelings of incompetence, leading to resistance to social interactions and goals involving intimacy and nurturance.

Characterization of the testicular abnormality in 5 alpha-reductase deficiency.

The testes of five phenotypic women (from four families) with 5 alpha-reductase deficiency were studied. In one of the patients, the enzyme deficiency was similar in the testis and epididymis and in fibroblasts cultured from the labia majora. In testes from four of the patients, the concentrations of the 5 alpha-reduced steroids dihydrotestosterone and 3 alpha-androstanediol were less than 10% of those in normal subjects. We conclude that the testis is involved in 5 alpha-reductase deficiency. Impaired spermatogenesis was evident in testicular biopsies from all five subjects, and in two, sperm production, as estimated in testicular homogenates, was less than 10% of normal. The extent to which spermatogenic arrest is due to 5 alpha-reductase deficiency or testicular maldescent is not clear.