Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.     

The pathology of the heart in progressive muscular dystrophy: epimyocardial fibrosis

The gross and microscopic appearance of the hearts from eight patients with Duchenne's progressive muscular dystrophy are described. Seven hearts had gross evidence of myocardial fibrosis, five of these demonstrating distinctive fibrosis of the epimyocardial portion of the free wall of the left ventricle, often with a striking band-like appearance. On the basis of mapping studies of the myocardial fibrosis, a theory regarding the progression of myocardial fibrosis in Duchenne's progressive muscular dystrophy is presented. Correlation of the pathologic anatomy, electrocardiograms, and vectorcardiograms in these patients and the family studies of others suggests that Duchenne's progressive muscular dystrophy represents a generalized cardiomyopathy that has its gravest and most distinctive effect on the epimyocardial portion of the free wall of the left ventricle.     

Neurologic diseases and chromosome 17

On the short arm of the 17th chromosome is a peripheral myelin protein (PMP22) the duplication or point mutation of which causes the development of some congenital autosomal dominant hereditary demyelinization neuropathies: the most frequent variants of Charcot-Marie-Tooth disease (CMT1A), some cases of Déjérine-Sottas disease and microdeletion of PMP22 and hereditary pressure neuropathies. The pericentric section of the long arm of chromosome 17 comprises a locus conditioning the development of the most frequent phacomatosis-neurofibromatosis 1. As to rarer neuromuscular diseases, genome mutations of chromosome 17 condition the development of some cases of autosomal recessive forms of severe muscular dystrophy (SCARMD), a clinical analogue of Duchenne's form of muscular dystrophy, metabolic storage myopathy of Pompe's type and some muscle diseases associated with impaired function of the ion channels (hyperkalaemic periodic paralysis, congenital paramyotonia, some cases of malignant hyperthermia). Aspartoacylase deficiency, conditioning Canavan's leucodystrophy was also located in the area of the short arm of chromosome 17.     

Duchenne's muscular dystrophy in a girl with 45,X/46,XX chromosomal mosaicism

The paper is concerned with a case history of a girl with a rare combination of 2 rare anomalies (only 4 cases in the world literature); monosomia by X chromosome and Duchenne's muscular dystrophy. Mosaicism 45,X/46,XX in a 5 year old girl with a mild picture of Duchenne's muscular dystrophy was confirmed by a study of the kariotype in lymphocytes of the peripheral blood and skin fibroblasts. The authors indicate to a necessity of a thorough cytogenetical study in girls, if there is a clinical picture of Duchennes muscular dystrophy.     

Laminin alpha 2-chain gene mutations in two siblings presenting with limb-girdle muscular dystrophy

We report two siblings, an 11-year-old boy and his 7-year-old sister, referred to us with a diagnosis of muscular dystrophy. The boy presented at 22 months with delay in walking. A very high serum creatine kinase (CK) level and a dystrophic muscle biopsy lead to a diagnosis of Duchenne muscular dystrophy prior to the identification of the dystrophin gene. Two years later his sister presented with similar problems. A diagnosis of limb-girdle muscular dystrophy was made when they were shown to have inherited different X-chromosomes and normal expression of dystrophin and all sarcoglycans. Their conditions remained static. Recently a slowing of the peripheral motor nerve conduction velocities and T2-weighted brain magnetic resonance imaging showed increased signal of the white matter, both of which are features of merosin-deficient congenital muscular dystrophy. Immunolabelling using a C-terminal laminin alpha 2 chain antibody showed a reduction in expression, while labelling with another antibody that recognises a 300-kDa fragment showed a very significant reduction. Mutational analysis of the LAMA2 gene showed two mutations: one was a G-->C point mutation at position -1 of intron 28 acceptor splicing site. This mutation induced activation of a cryptic splice at nucleotide 4429 of exon 29 and partial skipping of this exon, with conservation of the open reading frame. The other was a nonsense mutation due to a C_T transition at position 5525 of the cDNA sequence (exon 37), resulting in a stop codon. These data confirm that mutations of the LAMA2 gene that do not completely disrupt the production of the protein can give rise to phenotypes considerably milder than classical merosin-deficient congenital muscular dystrophy. Partial laminin alpha 2 deficiency should be considered in the differential diagnosis of limb-girdle muscular dystrophy.     

Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.     

Genetic epidemiology of muscular dystrophies resulting from sarcoglycan gene mutations

BACKGROUND: The autosomal recessive limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous muscle diseases characterised by progressive proximal limb muscle weakness. Six different loci have been mapped and pathogenetic mutations in the genes encoding the sarcoglycan complex components (alpha-, beta-, gamma-, and delta-sarcoglycan) have been documented. LGMD patients affected with primary "sarcoglycanopathies" are classified as LGMD2D, 2E, 2C, and 2F, respectively. METHODS: A geographical area in north east Italy (2,319,147 inhabitants) was selected for a genetic epidemiological study on primary sarcoglycanopathies. Within the period 1982 to 1996, all patients living in this region and diagnosed with muscular dystrophy were seen at our centre. Immunohistochemical and immunoblot screening for alpha-sarcoglycan protein deficiency was performed on all muscle biopsies from patients with a progressive muscular dystrophy of unknown aetiology and normal dystrophin. Sarcoglycan mutation analyses were conducted on all patient muscle biopsies shown to have complete or partial absence of alpha-sarcoglycan immunostaining or a decreased quantity of alpha-sarcoglycan protein on immunoblotting. RESULTS: Two hundred and four patient muscle biopsies were screened for alpha-sarcoglycan protein deficiency and 18 biopsies showed a deficiency. Pathogenetic mutations involving one gene for sarcoglycan complex components were identified in 13 patients: alpha-sarcoglycan in seven, beta-sarcoglycan in two, gamma-sarcoglycan in four, and none in the delta-sarcoglycan gene. The overall prevalence of primary sarcoglycanopathies, as of 31 December 1996, was estimated to be 5.6 x 10(-6) inhabitants. CONCLUSION: The prevalence rate estimated in this study is the first to be obtained after biochemical and molecular genetic screening for sarcoglycan defects.     

Systolic time interval characteristics in children with Duchenne's progressive muscular dystrophy

Systolic time interval (STI) characteristics of 17 boys with Duchenne's muscular dystrophy (DMD) were compared with those of 80 normal boys who served as control subjects. The heart rate decreased linearly with age in normal control subjects (r = -.47, P less than .01). By contrast, heart rate was significantly higher in patients with DMD (P less than .001) and tended to increase further with age. Each STI variable for normal control subjects increased significantly with age (P less than or equal to .01); QII, left ventricular ejection time (LVET), and pre-ejection period (PEP), in addition, decreased with increasing heart rate (P less than or equal to .05). In dystrophic patients QII and LVET decreased with increasing heart rate (P less than .001) but were not influenced by age. None of the other STI values in dystrophic patients was significantly influenced by either age or heart rate. Mean QII, LVET, and QI were shorter and PEP, isometric contraction time (ICT), and PEP/LVET ratio were longer (P less than .001) for DMD patients than for normal control subjects. In 13/17 patients, QII and LVET were below the 95% confidence interval of the normal mean, whereas PEP, ICT, and PEP/LVET exceeded the upper limits of normal in 8, 9 and 11 patients, respectively. For dystrophic patients, the difference (delta) between the observed values and those predicted from regression equations for normal control subjects was lower for QII, LVET, and QI (P less than .01) but higher for PEP (P less than .04), ICT, and PEP/LVET ratio (P less than .001). delta QII and delta LVET increased with age (P = .001 and .032, respectively). Duchenne's muscular dystrophy is thus documented to be associated with substantial alterations in STI characteristics that suggest a compromise of global left ventricular performance. Some of these abnormalities increase with age, probably reflecting the progressive cardiomyopathy characteristics of this disease.     

The sarcoglycan complex in limb-girdle muscular dystrophy

The involvement of the sarcoglycan complex in the pathogenesis of muscular dystrophy is becoming increasingly clear. Sarcoglycan gene mutations lead to four forms of autosomal recessive limb-girdle muscular dystrophy. Recent progress has been made with the identification of novel mutations and their correlations with disease. Through this research, a better understanding the molecular pathogenesis of limb-girdle muscular dystrophy has been gained. Finally, animal models are now being used to study viral-mediated gene transfer for the future treatment of this disease.     

Inherited forms of mineralocorticoid hypertension

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal convoluted tubules and cortical collecting ducts. Excess secretion of aldosterone or other mineralocorticoids or abnormal sensitivity to mineralocorticoids may result in hypertension, suppressed plasma renin activity, and hypokalemia. Such conditions often have a genetic basis, and studies of these conditions have provided valuable insights into the normal and abnormal physiology of mineralocorticoid action. Deficiencies of steroid 11 beta-hydroxylase or 17 alpha-hydroxylase are types of congenital adrenal hyperplasia, the autosomal recessive inability to synthesize cortisol. These two defects often cause hypertension because of overproduction of cortisol precursors that are, or are metabolized to, mineralocorticoid agonists. These disorders result from mutations in the CYP11B1 and CYP17 genes encoding the corresponding enzymes. Glucocorticoid-suppressible hyperaldosteronism is an autosomal dominant form of hypertension in which aldosterone secretion is abnormally regulated by corticotropin. It is caused by recombinations between linked genes encoding closely related isozymes, 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), generating a dysregulated chimeric gene with aldosterone synthase activity. Apparent mineralocorticoid excess is a loss of functional ligand specificity of the mineralocorticoid receptor caused by a deficiency of the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase, an enzyme that normally metabolizes cortisol to cortisone to prevent cortisol from occupying the receptor. This autosomal recessive form of severe hypertension results from mutations in the HSD11K (HSD11B2) gene.     

CYP11B1 mutations causing non-classic adrenal hyperplasia due to 11 beta-hydroxylase deficiency

Steroid 11 beta-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol. Severely affected patients carry mutations in the CYB11B1 gene that destroy enzymatic activity. Such patients have signs of androgen excess and usually have hypertension. Mild or non-classic 11 beta-hydroxylase deficiency has been reported previously but not studied genetically. In this study we report analysis of the CYP11B1 genes of three patients thought to suffer from non-classic 11 beta-hydroxylase deficiency. Mutations were detected in the CYP11B1 genes of two patients. One was a compound heterozygote for missense mutations N133H and T319M, whereas the other carried a nonsense mutation (Y423X) on one allele and a missense mutation (P42S) on the other. All three missense mutations affected enzymatic activity when expressed in vitro. No mutations were detected in the coding regions or intron-exon boundaries of the CYP11B1 genes of the other putative non-classic patient. In addition, we were unable to detect CYP11B1 mutations in two hirsute women with mildly elevated levels of 11 beta-hydroxylase precursors who had previously been identified in a screening study of patients in a reproductive endocrinology clinic. We conclude that nonclassic 11 beta-hydroxylase deficiency is a rare disorder. It is not a significant cause of hyperandrogenism in women and relatively stringent criteria should be used to prevent its misdiagnosis.     

Absence of a calmitine-specific protease inhibitor in skeletal muscle mitochondria of patients with Duchenne's muscular dystrophy

We studied the effect of mitochondrial extracts from skeletal muscle of patients with Duchenne's muscular dystrophy (DMD) on calmitine from the skeletal muscle of normal mice and control subjects. Our results clearly show the existence of an abnormal proteolytic activity of mitochondria from patients with DMD on calmitine from the normal mouse. This proteolytic activity was not found on calmitine from the control subject. Overall, our observations suggest that calmitine concentration in the muscle of the control subject remains elevated because of the presence of a calmitine-specific protease and an inhibitor of this protease which regulates and/or suppresses the activity of the enzyme according to the requirements of the muscle cell. Conversely, the calmitine deficiency observed in the muscle of patients with DMD would be due to the absence of this inhibitor. This would account for the continual activity of the enzyme in degrading calmitine as soon as it is synthesized. The identification of this inhibitor is currently being investigated in our laboratory.     

Characterization of steroid 21-hydroxylase gene mutations in a oligosymptomatic form of congenital adrenal hyperplasia: family study

BACKGROUND: 21-hydroxilase deficiency accounts for over 90% of all cases of congenital adrenal hyperplasia (CAH). There is a non-negligible incidence of both severe and nonclassical forms of this genetic disorder. Enzyme deficiency is due to mutations in the gene encoding adrenal 21-hydroxylase (named CYP 21B) and is inherited in an autosomical recesive way. Complete or partial impairment of enzyme activity has been correlated with the different clinical forms of the disease. PATIENTS AND METHODS: In the present paper CYP 21B gene analysis results obtained in a family with two kindred affected by a nonclassical form of the disease are shown. Clinical assessment of these two kindred showed a very mild form of the disease, whereas biochemical results suggested a late-onset partial 21-hydroxylase deficiency. Genotyping for deletions and 10 point mutations in the CYP 21B gene was performed by Southern blot analysis and polymerase chain reaction (PCR) allele-specific oligonucleotide (ASO) hybridation technique. RESULTS: Molecular genetic analysis performed in the two affected patients and two further relatives allowed us to detect the presence of different mutations in the two alleles of the CYP 21B gene. One of these mutations was severe (655G) and came from maternal line, whereas the other was mild (Val281Leu) and originated in paternal line. CONCLUSION: Molecular genetic analysis allows the possibility of finding severe (and non-expected) mutations in patients with clinically mild and late-onset forms of the 21-hydroxylase deficiency.     

Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the alpha-sarcoglycan gene. To determine how alpha-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed alpha-sarcoglycan- deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of alpha-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of alpha-dystroglycan association with membranes. In contrast, no change in the expression of epsilon-sarcoglycan (alpha-sarcoglycan homologue) was observed. Recombinant alpha-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan-sarcospan complex is requisite for stable association of alpha-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.     

Clinical and molecular characterization of a Brazilian patient with Pit-1 deficiency

We studied a 14 year-old girl with extreme short stature (-9.5 SDS), normal psychomotor development and signs of progressive hypothyroidism. Basal IGF-I and T4 were low. Serum GH was low after insulin-induced hypoglycemia and GH-releasing hormone administration. Both TSH and prolactin were low and did not rise after TRH administration. Gonadotropins were normal and cortisol levels were elevated. In contrast, DHEA-S levels were low and she did not develop pubic hair until 26 years of age, compatible with deficiency of a putative pituitary adrenal androgen stimulating hormone. Pituitary size was reduced on magnetic resonance imaging. Sequencing of the Pit-1 gene revealed a heterozygous C to T transition in codon 271 resulting in substitution of tryptophane for a highly conserved arginine. Her parents were homozygous normal for this locus indicating a de novo mutation with dominant expression. Genetic and phenotypic heterogeneity of patients with Pit-1 gene mutations, particularly the R271W mutation, may reveal further information about the nature of genetic silencing, imprinting, and epigenetic inheritance. The relationship of Pit-1 deficiency to abnormal adrenal secretion remains to be elucidated.     

Molecular diagnosis of salt wasting congenital adrenal hyperplasia, caused by deficit of 21-hydroxylase, in the Chilean population

BACKGROUND: The most frequent cause of congenital adrenal hyperplasia, manifested as virilization and salt wasting, is the deficit of 21-hydroxylase. This disease is originated by mutations of the gene CYP21 that codifies this enzyme, mostly recombination between this gene and its inactive pseudogene called CYP21P. AIM: To study the molecular origin of this enzyme deficiency in Chilean patients. PATIENTS AND METHODS: Twenty five patients with salt wasting congenital adrenal hyperplasia, that had 17-hydroxyprogesterone levels above 30 ng/ml, were studied. In all patients, a polymerase chain reaction (PCR) with selective primers was done with extracted genomic DNA, to amplify the active gene and specific primers for normal or mutated alleles (Allele-specific PCR). RESULTS: The affected allele was identified in 39 (78%) of the 50 chromosomes of the 25 patients. The higher frequency affected the ASIn2 in 26% of cases, followed by mutations Arg357Trp in 22% of cases and Gln319Stop in 12% and deletion in 12%. The identification of two affected alleles in a same patient was achieved in 17 cases (68%). The most frequent genotypes were homozygosity for ASIn2 (16%), homozygosity for Arg357Trp (12%) and the homozygote deletion of the gene in 12%. CONCLUSION: The most frequent mechanisms of genetic damage in this population of patients with salt wasting congenital adrenal hyperplasia due to deficiency of 21-hydroxylase were the mutations ASIn2 and Arg357Trp. This type of studies allows prenatal diagnosis and genetic counseling.