The spectrum of clinical, hormonal and molecular findings in 280 individuals with nonclassical congenital adrenal hyperplasia caused by mutations of the CYP21A2 gene.

BACKGROUND: Nonclassical congenital adrenal hyperplasia (NC-CAH) is caused by mutations of the CYP21A2 gene. The clinical manifestations and hormonal derangements of NC-CAH are quite variable. OBJECTIVES: (i) To define the phenotype and its relation to genotype according to gender and age and (ii) to evaluate the validity of currently applied hormonal criteria for establishing the diagnosis of NC-CAH. PATIENTS AND METHODS: The clinical, hormonal and molecular data of 280 subjects (235 female) with NC-CAH and a median age of 17·6 years were analysed. CYP21A2 genotyping was performed in all subjects. RESULTS: The majority of females aged less than 8 years presented with premature pubarche (88·3%), while those older than 8 presented with a polycystic ovary-like phenotype (63·2%). A total of 7·7% of the females and 51·1% of the males were asymptomatic at the time of diagnosis. In the total group, 50·4% of the subjects were compound heterozygotes for one classical (C) and one nonclassical (NC) mutation, while 46% of the alleles studied carried the p.V281L mutation. Basal 17OHP values were below 6 nm (2 ng/ml) in 2·1% of the subjects with NC-CAH, but none had peak 17OHP values post-ACTH lower than 30 nm (10 ng/ml). CONCLUSIONS: NC-CAH has a variable phenotype depending on the age, gender and the presence of a classical mutation. A peak cut-off value of 17OHP post-ACTH lower than 30 nm excludes the diagnosis of NC-CAH, whereas basal 17OHP <6 nm may represent a false-negative result. A significant number of patients harboured a classical mutation, a finding which requires genotyping of the partner for genetic counselling.

HNF1A gene mutations and premature ovarian failure (POF): evidence from a clinical paradigm combining MODY 3 and POF.

Premature ovarian failure (POF) defines the occurrence of ovarian failure prior to the age of 40. It occurs in one out of 100 women but is very rare before age 20 (1:10,000). Maturity-onset diabetes of the young (MODY), caused by mutations in the HNF1A gene, is also a rare disorder; all types of MODY account for 1-2% of adult diabetic cases. These two rare nosologic entities coexisted in an adolescent girl evaluated for delayed puberty. Although this combination could represent a chance association, an interrelation might exist. We examined HNF1A expression in human fetal and adult ovaries by immunohistochemistry using a polyclonal HNF1A antibody. HNF1A protein was expressed in both the fetal and adult human ovaries. Based on these findings, we hypothesize that HNF1A participates in ovarian organogenesis and/or function and that mutations in the HNF1A gene might represent another molecular defect causing POF, possibly in combination with other genetic factors. The study underlines the importance of rare clinical paradigms in leading the way to elucidation of the pathogenetic mechanisms of rare diseases.

Congenital adrenal hyperplasia caused by compound heterozygosity of two novel CYP11B1 gene variants.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by pathogenic variants in seven genes involved in the cortisol and aldosterone biosynthetic pathway. The second most common cause, 11ß-hydroxylase deficiency (11ßOHD), is attributed to pathogenic variants in the CYP11B1 gene encoding for the enzyme 11ß-hydroxylase (11ßOH). CASE PRESENTATION: A 13-year-old girl was referred to the pediatric endocrinologist due to a syncopal episode. She is the third child of non-consanguineous parents. She presented with premature adrenarche at the age of 6 years and menarche at the age of 12 years. On physical examination, her height was 154.5 cm and weight 50 kg, while she presented with acne, hirsutism, clitoromegaly, and normal blood pressure. Laboratory investigation revealed increased androgen levels and poor cortisol response to the ACTH stimulation test. From the family history, the mother was diagnosed with CAH at the age of 10 years and was under treatment with methylprednisolone. Previous molecular investigation of the CYP21A2 gene was negative. Due to the increased androstenedione levels in the index patient, the suspicion of 11ßOH was raised, and she was investigated for 11-deoxycortisol, 11-deoxycorticosterone, and CYP11B1 gene pathogenic variants. The patient and her mother were found to be compound heterozygous for two novel variants of the CYP11B1 gene. CONCLUSION: We present a case of CAH due to compound heterozygosity of two novel pathogenic variants of the CYP11B1 gene, emphasizing the importance of molecular investigation in order to confirm clinical diagnosis and allow proper genetic counseling of the family.

The IVS1-2A>G mutation in the SRD5A2 gene predominates in Cypriot patients with 5α reductase deficiency.

BACKGROUND: 5α steroid reductase deficiency (5αSRD) is an autosomal recessive enzymatic deficiency and mutations in the 5α steroid reductase type 2 gene (SRD5A2) result in male pseudohermaphrodism caused by decreased dihydrotestosterone (DHT) synthesis. AIM: To identify the specific mutations of the SRD5A2 gene in Cypriot patients with 5αSRD. SUBJECTS AND METHODS: Five unrelated patients with 46,XY karyotype were examined. Four of them were born with ambiguous genitalia and 1 patient, who was raised as girl, presented with primary amenorrhea. The hCG test was informative (elevated testosterone/DHT) of 5αSRD in 3 out of 4 subjects. Sequencing of the SRD5A2 gene was completed for all patients. Genomic DNA was also isolated from a total of 204 healthy unrelated Cypriot subjects. Screening for the IVS1-2A>G mutation was performed by using direct sequencing and restriction enzyme analysis. RESULTS: The IVS1-2A>G was identified in homozygosity in 3 patients and in a compound heterozygote state in the other 2 patients, in combination with p.P181L and p.R171S in exon 3, respectively. The carrier frequency in the Cypriot population for the IVS1-2A>G mutation was estimated to be 0.98% or 2 in 204. CONCLUSIONS: The same IVS1-2A>G mutation in the SRD5A2 gene seems to characterize all Cypriot patients with 5αSRD diagnosed so far. Furthermore this relatively rare genetic defect, which has only been reported previously in a single case in the Eastern Mediterranean region, is very likely to be the result of a founder effect.

Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation.

We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

Insufficient adrenarche in patients with combined pituitary hormone deficiency caused by a PROP-1 gene defect.

Adrenarche was evaluated in five patients, aged 17.4 +/- 3 years, with combined pituitary hormone deficiency (CPHD), caused by a PROP-1 gene defect. Adrenocorticotrophic hormone (ACTH), cortisol and dehydroepiandrosterone sulfate (DHEAS) were determined prior to and following the administration of corticotropin-releasing hormone (CRH) in four of the five patients, while only basal values of ACTH, cortisol and DHEAS were determined in the fifth. In the four patients in whom a CRH test was carried out, the mean basal values of cortisol, ACTH and DHEAS were 289 +/- 140 nmol/l, 4.5 +/- 1.7 pmol/l and 0.26 +/- 0.36 micromol/l, respectively. The corresponding post-CRH peak values were 584 +/- 204 nmol/l, 12.7 +/- 3.9 pmol/l and 0.43 +/- 0.41 micromol/l. In the fifth patient, basal ACTH, cortisol and DHEAS values were 4 pmol/l, 411 nmol/l, and 2.33 micromol/l, respectively. Thus the basal and post CRH values of DHEAS (a marker of adrenarche) were low for age, while basal and post-CRH cortisol and ACTH values were within normal limits. For the interpretation of these findings two hypotheses can be proposed: 1) The PROP-1 gene is only expressed in the pituitary, and the role of PROP-1 is related to the maturation of the cells which synthesize the presumed adrenal androgen stimulating hormone (AASH). 2) The PROP-1 gene is also expressed in the adrenal cortex and, when defective, the zona reticularis does not function appropriately. Regardless of the interpretation

Pituitary size fluctuation in long-term MR studies of PROP1 deficient patients: A persistent pathophysiological mechanism?

Inactivating PROP1 gene alterations are responsible for over 50% of familial combined pituitary hormone deficiency cases. Pituitary enlargement followed by regression and subnormal pituitary size has been documented in a number of PROP1 deficient patients. Data derived from PROP1 deficient mice (Ames dwarfs) have revealed some of the underlying cellular mechanisms. Nevertheless, long-term magnetic resonance imaging (MRI) findings in two PROP1 deficient patients suggest the evolution of pituitary pathology as more complex and persistent than previously described. Patient A had enlarged pituitary gland (pituitary height: 9-10 mm), demonstrated by serial MRI carried out from age 5 to 8.5 yr, small pituitary gland (4 mm) at age 10 yr and pituitary enlargement (11 mm) at age 19 yr. Patient B had a pituitary gland of normal size at age 7 yr (5 mm), whereas at age 14.3 and 16.3 yr, an enlarged pituitary gland was disclosed (10 and 11 mm, respectively). Both series of events are suggestive of a persistent pathophysiological mechanism in the pituitary gland of patients with PROP1 gene defects. Therefore, long-term pituitary follow-up by MRI in such patients may be necessary even in the case of a small or normal pituitary gland. It must be noted that current data from the Ames dwarf mouse cannot fully explain the observed pituitary size fluctuation.

CAC--the neglected repeat.

It is becoming increasingly clear that repetitive DNA is of biological significance as well as experimental importance. Here we review the information available about one type of repetitive DNA, the trinucleotide repeat (CAC)n, and briefly compare it with other trinucleotide repeats. Although much work has been done in analysing DNA fingerprinting patterns produced using the synthetic oligonucleotide (CAC)5 as a probe, there is relatively little information about individual (CAC)n-containing sequences and their abundance, organisation and distribution in mammalian DNA. From the data that is available, it is clear that there are at least two areas that should repay further study: (1) the organisation and generation of long sequences that contain (CAC)n motifs as part of a larger repeating unit (minisatellites) and (2) the distribution of small (CAC)n sequences (microsatellites), in particular their relationship to genes.

Oligonucleotide (CAC)5 fingerprinting: validity and reliability in paternity testing.

Following the development of DNA fingerprinting (Jeffreys et al., Nature 1985, 314, 67-73) using the minisatellite probes 33.15 and 33.6, many other fingerprinting probes have been described. The wide type M13 phage, 3' HVR region of alpha globin gene, F10 and various synthetic oligonucleotide probes have been shown to produce multiallelic and individual specific DNA fingerprints. The two probes developed by Jeffreys et al. have been extensively used in forensic and paternity determination in various laboratories, however, for the use of DNA fingerprints produced by synthetic oligonucleotide probes, still need critical evaluation. In this paper the statistical evaluations of the fingerprinting probes (CAC)5 was carried out by comparing the results produced by Jeffreys et al., using probes 33.15 and 33.6. The mutation rate calculated for (CAC)5 as 0.015 is nearly similar to the probe 33.15 (0.011) but higher than the probe 33.6 (0.005). The data obtained using (CAC)5 fingerprint in paternity cases from the northeast of England are presented.

Study of three hypervariable DNA loci (D1S7; D7S22 and D12S11) in three European populations.

To investigate the population genetic characteristics and genetic affinity, DNA profiles of three highly polymorphic VNTR (variable number of tandem repeats) loci (D1S7; D7S22 and D12S11) were studied in 405 individuals from three major European populations (English, Spanish and Basques). Like other studies on VNTRs, a large significant heterozygote deficiency was observed in all three populations. This decrease was ascribed to the limitation, coalescence and non-detectibility of alleles associated with the RFLP (restriction fragment length polymorphism) technique, through which the VNTR loci are genotyped. When the non-detectable alleles were taken into consideration, analyses of fragment sizes at these loci within each sample, as well as their fixed binned analyses, reveal that the assumptions of independence of allelelic occurrences within and between loci are valid for this European data. By comparing genetic variation at three VNTR loci with 17 blood groups, proteins and HLA loci in three well defined European populations, it is shown that the pattern of differentiation at these sets of loci are in general parallel especially for the hypervariable loci HLA and VNTR. Fixed-bin allele frequencies, therefore, are the best descriptions of such a database both for population genetic and forensic calculation studies. The Basques, with regard to VNTR loci, do not show any reduced genetic variability compared to other two European populations (English and Spanish).

Recombination between rhodopsin and locus D3S47 (C17) in rhodopsin retinitis pigmentosa families.

Autosomal dominant retinitis pigmentosa (adRP) has shown linkage to the chromosome 3q marker C17 (D3S47) in two large adRP pedigrees known as TCDM1 and adRP3. On the basis of this evidence the rhodopsin gene, which also maps to 3q, was screened for mutations which segregated with the disease in adRP patients, and several have now been identified. However, we report that, as yet, no rhodopsin mutation has been found in the families first linked to C17. Since no highly informative marker system is available in the rhodopsin gene, it has not been possible to measure the genetic distance between rhodopsin and D3S47 accurately. We now present a linkage analysis between D3S47 and the rhodopsin locus (RHO) in five proven rhodopsin-retinitis pigmentosa (rhodopsin-RP) families, using the causative mutations as highly informative polymorphic markers. The distance, between RHO and D3S47, obtained by this analysis is theta = .12, with a lod score of 4.5. This contrast with peak lod scores between D3S47 and adRP of 6.1 at theta = .05 and 16.5 at theta = 0 in families adRP3 and TCDM1, respectively. These data would be consistent with the hypothesis that TCDM1 and ADRP3 represent a second adRP locus on chromosome 3q, closer to D3S47 than is the rhodopsin locus. This result shows that care must be taken when interpreting adRP exclusion data generated with probe C17 and that it is probably not a suitable marker for predictive genetic testing in all chromosome 3q-linked adRP families.

Insulin dependent diabetes mellitus (IDDM) and autoimmune thyroiditis in a boy with a ring chromosome 18: additional evidence of autoimmunity or IDDM gene(s) on chromosome 18.

A 4 year 3 month old boy with insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, slight mental retardation, facial dysmorphism, and a de novo ring chromosome 18 (deletion 18q22.3-18qter) is described. This unique association of defects could represent a chance association. Alternatively, the clinical features could be the result of the chromosomal aberration. If so, one could speculate that a gene or genes on chromosome 18 might act as a suppressor or activator of the autoimmune process by itself or in concert with other IDDM loci.