Decreased bioactivity of the guanine nucleotide-binding protein that stimulates adenylate cyclase in hearts from cardiomyopathic Syrian hamsters.

We investigated regulation of cardiac adenylate cyclase in 29-d-old BIO 14.6 Syrian hamsters, which inherit cardiomyopathy as an autosomal recessive trait. Pharmacologic stimulation of adenylate cyclase in cardiac membranes with isoproterenol, fluoride ion, guanine nucleotide, forskolin, and manganous ion indicated that there was defective coupling of the guanine nucleotide-binding protein that stimulates adenylate cyclase (Gs) to adenylate cyclase. Cyc complementation assays revealed congruent to 50% less Gs activity in cardiac and skeletal muscle from cardiomyopathic hamsters. Despite this decrease in functional Gs, there were no changes in immunologic levels of the alpha-subunit of Gs (alpha Gs) or in levels of mRNA encoding alpha Gs. The defect in Gs bioactivity was limited to cardiac and skeletal muscle, occurred only in animals homozygous for the dystrophic trait, and was demonstrable before any cardiac abnormalities were evident on light microscopy. By contrast, cardiac levels of beta-adrenergic receptors were not different in cardiac membranes from BIO 14.6 hamsters. We conclude that a functional defect in alpha Gs may contribute to a contractile abnormalities in the cardiomyopathic BIO 14.6 hamster. However, the etiology of the alpha Gs defect remains obscure.

Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart.

Human heart failure is associated with a diminished contractile response to beta-adrenergic agonists. We hypothesized that alterations in the activity of a guanine nucleotide-binding regulatory protein (G protein) might be partially responsible for this abnormality. We therefore measured the activity of G proteins in failing human myocardium utilizing bacterial toxin-catalyzed ADP ribosylation. The activity of a 40,000-mol wt pertussis toxin substrate (alpha G40) was increased by 36% in failing human hearts when compared with nonfailing controls. In contrast, there was no change in the level of the stimulatory regulatory subunit (Gs). The increased activity in alpha G40 was associated with a 30% decrease in basal as well as 5'-guanylyl imidodiphosphate-stimulated adenylate cyclase activity. These data suggest that increased alpha G40 activity is a new marker for failing myocardium and may account at least in part for the diminished responsiveness to beta 1-adrenergic agonists in the failing human heart.

An arginine to histidine mutation in codon 311 of the C-erbA beta gene results in a mutant thyroid hormone receptor that does not mediate a dominant negative phenotype.

We have examined the c-erbA beta thyroid hormone receptor gene in a kindred, G.H., with a member, patient G.H., who had a severe form of selective pituitary resistance to thyroid hormones (PRTH). This patient manifested inappropriately normal thyrotropin-stimulating hormone, markedly elevated serum free thyroxine (T4) and total triiodothyronine (T3), and clinical hyperthyroidism. The complete c-erbA beta 1 coding sequence was examined by a combination of genomic and cDNA cloning for patient G.H. and her unaffected father. A single mutation, a guanine to adenine transition at nucleotide 1,232, was found in one allele of both these members, altering codon 311 from arginine to histidine. In addition, a half-sister of patient G.H. also harbored this mutant allele and, like the father, was clinically normal. The G.H. receptor, synthesized with reticulocyte lysate, had significantly defective T3-binding activity with a Ka of approximately 5 x 10(8) M-1. RNA phenotyping using leukocytes and fibroblasts demonstrated an equal level of expression of wild-type and mutant alleles in patient G.H. and her unaffected father. Finally, the G.H. receptor had no detectable dominant negative activity in a transfection assay. Thus, in contrast to the many other beta-receptor mutants responsible for the generalized form of thyroid hormone resistance, the G.H. receptor appeared unable to antagonize normal receptor function. These results suggest that the arginine at codon 311 in c-erbA beta is crucial for the structural integrity required for dominant negative function. The ARG-311-HIS mutation may contribute to PRTH in patient G.H. by inactivating a beta-receptor allele, but it cannot be the sole cause of the disease.

Father to son transmission of decreased Ns activity in pseudohypoparathyroidism type Ia.

We found both renal resistance to endogenous and exogenous PTH and reduced activity of the stimulatory guanine nucleotide-binding regulatory protein (Ns) of adenylate cyclase in a man with clinical signs of pseudohypoparathyroidism type Ia (PHP-Ia). Both of his children also had reduced Ns levels and short stature. The girl, 11 yr old, had evidence of partial resistance to PTH, while the son, age 7 yr, had no apparent abnormalities in calcium metabolism or response to administered PTH. Variable expression of the metabolic abnormalities of PHP during childhood has been previously described. The occurrence of reduced Ns activity in father and son is consistent with autosomal dominant inheritance for the primary biochemical defect of PHP-Ia in this family.

Coupling of HLA-A3,Cw6,Bw47,DR7 and a normal CA21HB steroid 21-hydroxylase gene in the Old Order Amish.

HLA-Bw47, a rare human histocompatibility antigen, occurs in strong linkage disequilibrium with HLA-A3,Cw6,DR7 and salt-losing congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and is associated with a contiguous deletion of the active CA21HB gene and the C4B complement gene. We studied the HLA-A3,Cw6,Bw47,DR7 haplotype in 10 subjects of the Old Order Amish of Lancaster County in Pennsylvania and found that this haplotype, which occurs with a similar frequency in this group as in the general caucasoid population, has C4B and Ca21HB genes. These C4B and CA21HB genes are expressed as assessed by C4 typing and iv ACTH testing, respectively. Serological studies indicate that the HLA-D loci of this Amish haplotype are the same as those in patients with HLA-Bw47 and CAH, but different from HLA-D loci coupled to HLA-B13, which some workers have proposed is the progenitor genotype for HLA-Bw47. Our studies demonstrate that 1) HLA-Bw47 is not an invariant marker for salt-losing CAH due to 21-hydroxylase deficiency, and 2) the HLA-Bw47 phenotype coupled to CAH is not derived from the HLA-B13 genotype by a single mutation.

Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein.

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.

Endocrinologic evaluation of children who grow poorly following renal transplantation.

Some children do not grow well following successful renal transplantation. We reviewed 25 children with renal allografts who receive regular medical care in the renal transplant program at The Children's Hospital, were less than or equal to 18 years of age, and had stable renal function. We compared children who were growing well (n = 14) with those who were growing poorly (n = 11). The children with poor growth more frequently had elevated serum creatinine concentrations (8/11 vs. 3/14). The mean age at transplantation was the same, although the duration of follow-up was shorter for the children growing well (3.3 +/- 0.5 years) than for those growing poorly (6.2 +/- 1.0 years, P less than 0.02). Eight of the children who were growing poorly underwent endocrinologic evaluation. Plasma growth hormone (GH) concentrations were measured during sleep, after arginine and L-DOPA administration, and during a 4-hr oral glucose tolerance test. In 4 patients, the maximum GH concentration was inadequate both following pharmacologic stimulation (4.0 +/- 2.6 ng/ml, n = 4) and during sleep (4.4 +/- 0.2 ng/ml, n = 3). In 2 additional patients, maximal GH concentrations were subnormal during sleep despite adequate responses during pharmacologic stimulation. In the final two patients, GH secretion was adequate both during sleep and after stimulation. All children studied had some degree of renal insufficiency with a mean creatinine clearance of 39 +/- 4 ml/min/1.73 m2. Plasma concentrations of thyroxine, thyrotropin, and IGF-I were normal for age in all eight patients. We conclude that abnormalities in GH secretion occur frequently among patients who grow poorly following successful renal transplantation. Evaluation of GH secretion may be useful in evaluating growth failure in this group of patients, and the usefulness of GH therapy should be investigated.

Differential diagnosis in young women with oligomenorrhea and the pseudo-pseudohypoparathyroidism variant of Albright's hereditary osteodystrophy.

Oligomenorrhea was the reason for consultation in three individuals (two sisters and one unrelated woman) with the pseudo-pseudohypoparathyroidism (PPHP) variant of Albright's hereditary osteodystrophy (AHO). All had short stature, Ullrich-Turner-like signs, acral anomalies typical of AHO/brachydactyly E, and hypogonadism. One of the three individuals also had reduced erythrocyte NS (a membrane nucleotide regulatory protein that is required for functional coupling of stimulatory hormone receptors and catalytic adenylate cyclase) activity as described in the pseudohypoparathyroidism variant of AHO. The differential diagnosis of young women with the PPHP phenotype is discussed with special reference to Ullrich-Turner syndrome, brachydactyly E, the "resistant ovary" syndrome, and acrodysostosis.

Congenital heart disease associated with sporadic Kallmann syndrome.

A 17-year-old boy with Kallmann syndrome had complex congenital heart disease that included double-outlet right ventricle, d-mal-position of the great arteries, right aortic arch, and hypoplastic main pulmonary artery. He had neurosensory hearing loss and mental retardation. The 7 previously reported patients with Kallmann syndrome and cardiac abnormalities were short with height > or = 2 standard deviations below the mean for age (5/7), lacked a family history of Kallmann syndrome (6/6), and were mentally retarded (4/4). Patients presenting with Kallmann syndrome and congenital heart defects appear to represent a distinct subgroup of patients with Kallmann syndrome. The cause of this association is unclear, but may involve either autosomal recessive inheritance, sporadic dominant mutation, or a shared teratogenic event during the first trimester of gestation.

Pseudohypoparathyroidism type Ia from maternal but not paternal transmission of a Gsalpha gene mutation.

While loss-of-function mutations in Gsalpha are invariably associated with the short stature and brachydactyly of Albright hereditary osteodystrophy (AHO), the association with hormone resistance (to parathyroid hormone and thyrotropin) typical of pseudohypoparathyroidism type Ia (PHP-Ia) is much more variable. Observational studies and DNA polymorphism analysis suggest that maternal transmission of the Gsalpha mutation may be required for full expression of clinical hormone resistance. To test this hypothesis, we studied transmission of a frameshift mutation in Gsalpha through three generations of a pedigree affected by AHO and PHP-Ia. While all family members carrying this loss-of-function mutation in one Gsalpha allele had AHO, neither the presence of the mutation nor the degree of reduction of erythrocyte Gsalpha bioactivity allowed prediction of phenotype (AHO alone versus AHO and PHP-Ia). Paternal transmission of the mutation (from the patriarch of the first generation to three members of the second generation) was not associated with concurrent PHP-Ia, but maternal transmission (from two women in the second generation to four children in the third generation) was invariably associated with PHP-Ia. No expansion of an upstream short CCG nucleotide repeat region was detected, nor was there evidence of uniparental disomy by polymorphism analysis. This report, the first to document the effects across three generations of both paternal and maternal transmission of a specific Gsalpha mutation, strongly supports the hypothesis that a maternal factor determines full expression of Gsalpha dysfunction as PHP-Ia.

Syndrome of coronal craniosynostosis with brachydactyly and carpal/tarsal coalition due to Pro250Arg mutation in FGFR3 gene.

Recently, a unique Pro250Arg point mutation in fibroblast growth factor receptor 3 (FGFR3) was reported in 61 individuals with coronal craniosynostosis from 20 unrelated families [Muenke et al. (1997): Am J Hum Genet 60:555-564]. The discovery of this apparently common mutation has resulted in the definition of a recognizable syndrome, through analysis of subtle clinical findings in families who were previously thought to have a variety of other craniosynostosis syndromes. Previous diagnoses in some of these families have included Jackson-Weiss, Saethre-Chotzen, and Pfeiffer syndromes, as well as Adelaide-type craniosynostosis and brachydactyly-craniosynostosis syndrome [Adès et al. (1994): Am J Med Genet 51:121-130; von Gernet et al. (1996): Am J Med Genet 63:177-184; Reardon et al. (1997): J Med Genet 34:632-636; Bellus et al. (1996): Nat Genet 14:174-176; Hollaway et al. (1995): Hum Mol Genet 4:681-683; Glass et al. (1994): Clin Dysmorphol 3:215-223]. There appears to be a need to further delineate the phenotype associated with this common mutation in FGFR3. We compare the clinical characteristics of previously reported cases of this unique Pro250Arg mutation with those of two additional families and suggest that this syndrome with a unique mutational basis be designated coronal craniosynostosis with brachydactyly and carpal/tarsal coalition due to Pro250Arg mutation in FGFR3 gene, to emphasize the distinctive findings which may be present even in the absence of coronal craniosynostosis.

Decreased cortical and increased cancellous bone in two children with primary hyperparathyroidism.

The basis for this study is two children with primary hyperparathyroidism (PHPT) who radiographically manifested both marked subperiosteal resorption and prominent osteosclerosis. We hypothesize that the parathyroid hormone (PTH) elevation not only increased osteoclastic resorption of cortical bone but also simultaneously enhanced cancellous bone formation, giving rise to osteosclerosis. In this report, we describe the changes in trabecular and cortical bone density, as measured by quantitative computed tomography (QCT), in these two young patients with severe PHPT, before and after removal of a parathyroid adenoma. Before surgery, the radiographic findings of subperiosteal resorption and osteosclerosis were associated with low cortical and high cancellous bone density values in both children. Within 1 week of surgery, both cortical and cancellous bone density values increased and serum concentrations of calcium and, to a lesser degree, phosphorus decreased due to the "hungry bone syndrome." Twelve weeks after parathyroidectomy, QCT bone density values and skeletal radiographs were normal in both patients. The findings suggest that in patients with severe PHPT, the catabolic effect of PTH on cortical bone may be associated with a simultaneous anabolic effect on cancellous bone, and PTH may cause a significant redistribution of bone mineral from cortical to cancellous bone.

Growth hormone treatment of growth failure among children with renal transplants.

Eight children with growth failure following renal transplant have been selected for recombinant human growth hormone (rhGH) treatment at Children's Hospital using the following criteria: (1) a functioning allograft for at least one year; (2) height < third percentile; (3) growth velocity < 4 cm/year; (4) growth potential; and (5) low-dose alternate-day glucocorticoid dosing. The children were 7.4 to 17.7 years of age; had received transplants 2.6 to 12.3 years before rhGH treatment; and all had decreased allograft function. The growth velocity of these children increased from 1.7 +/- 0.7 to 7.1 +/- 2.1 cm/year during the first year of rhGH treatment (0.05 mg/kg s.c. daily). The mean height SD score improved -3.9 +/- 1.5 to -3.4 +/- 1.3 in the seven children who completed one year of treatment (P < 0.001). There was no change in glucose tolerance during rhGH treatment. The serum creatinine concentration increased in all patients with a concomitant decrease in creatinine clearance. The mean rate of change in the inverse creatinine (1/Cr) increased from -0.005 +/- 0.004 dl/mg/month in the two years prior to rhGH treatment to -0.023 +/- 0.015 dl/mg/month during rhGH treatment (P < 0.01). The relative risks and benefits of rhGH treatment must be carefully considered for each patient.

Pseudohypoparathyroidism.

Pseudohypoparathyroidism includes a genotypically diverse group of syndromes of primary resistance to hormones whose actions are mediated by cyclic adenosine 3':5'-monophosphate. In most cases, clinical presentation results from resistance to actions of parathyroid hormone, while resistance to other hormones remains subclinical. The largest subgroup of patients with this disorder, characterized by a typical somatic phenotype termed Albright's hereditary osteodystrophy, exhibits generalized deficient activity of a membrane protein that couples hormone receptors to stimulation of adenylate cyclase. Phenotypic expression of the disease varies greatly between families, within families, and even during the clinical course of individual patients. Genetic transmission occurs via several distinct loci, which produce different patterns of inheritance.

McCune-Albright syndrome. Long-term follow-up.

This article describes clinical follow-up of 15 patients--13 females and two males--with McCune-Albright syndrome. Osseous fractures occurred only during childhood, while hearing impairment due to temporal bone involvement occurred in four of six adults. Four females with precocious puberty had final heights that were not different from the mean for normal females; they eventually developed regular menses, and two had children. Persistent hyperthyroidism requiring ablative therapy occurred in three subjects, while hypophosphatemia occurred in three subjects. The protean manifestations of this disorder suggest that it results from a basic defect of cellular regulation. We postulate that its varied endocrine abnormalities result from altered regulation of intracellular cyclic adenosine monophosphate effects.

Developmental changes in mRNA encoding cardiac Na+/H+ exchanger (NHE-1) in rabbit.

The faster recovery of cardiac contractility in newborn rabbit hearts during acute acidosis compared to adult hearts correlates with greater cellular activity of the Na+/H+ exchanger. We quantified mRNA encoding Na+/H+ exchanger-1 (NHE-1) in rabbit ventricles of fetal (27 days gestation), newborn (2-5 days), and adult (> 6 months) New Zealand white rabbits using reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay, with GAPD mRNA as standard. Both RT-PCR and RNase protection assay revealed similar (p > 0.05) cardiac levels of NHE-1 mRNA in fetal and newborn hearts, while NHE-1 mRNA in these hearts was 1.7x and 1.6x greater than in adult hearts by both RT-PCR and RNase protection assay. These developmental changes in NHE-1 mRNA correlate with the developmental changes in Na+/H+ activity in sarcolemmal vesicles purified from rabbit heart.

Pyruvate metabolism in bovine epididymal spermatozoa.

Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.

Mitochondrial metabolism of pyruvate in bovine spermatozoa.

Treatment with the polyene antibiotic, filipin, renders the spermatozoan cell membrane permeable to small molecules, but not to the intracellular enzymes aldolase and lactate dehydrogenase. Pyruvate (10 mM) as the sole substrate was metabolized very slowly. L-Carnitine increased pyruvate metabolism 3- to 4-fold and allowed limited rates of oxidative phosphorylation. When spermatozoa treated with filipin were supplemented with malate, there was a rapid, almost linear rate of pyruvate metabolism which was slightly increased by L-carnitine. In the absence of malate, 20 to 30% of the pyruvate used was reduced to lactate; this increased to 57% in the presence of malate. Without malate, about 90% of the pyruvate metabolized was converted to lactate and acetate or L-acetylcarnitine. Rutamycin or rotenone increased both the rate of pyruvate use and the delta lactate/deltapyruvate ratio. Under all treatments, L-carnitine consistently reduced the percentage of pyruvate converted to lactate by about 10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine rather than lactate. The mitochondrial inhibitors, rotenone or rutamycin, did not change the amount of pyruvate that was converted to metabolites other than lactate, or L-acetylcarnitine, or both. Pyruvate-supported State 3 respiration was linear only if L-carnitine, or malate, or both, were added to the incubation medium. Added malate was necessary to produce a rapid State 3 respiratory rate and was also required for significant respiratory activity in the presence of rotenone or rutamycin. From cells metabolizing [2-14C]pyruvate (1.4 mM), 14C-labeled acid-extractable metabolites were separated by ion exchange column chromatography. All of the [2-14C]pyruvate (+/-5%) used was recovered in 14C-labeled metabolites and 14CO2. In the presence of malate, citrate accumulation was significant, and was always large in comparison to flux through the citric acid cycle. Glutamate, beta-hydroxybutyrate, acetoacetate, fumarate, aspartate, and alpha-ketoglutarate did not accumulate in significant amounts. Some 14C-labeled succinate was produced but only in the presence of malate. Alkaline hydrolysis of a fraction containing carnitine esters yielded acetate and a compound tentatively identified as beta-hydroxybutyrate or lactate. As in intact cells, intramitochondrial lactate dehydrogenase competes successfully with the electron transport system for the NADH generated by pyruvate metabolism. The role of lactate and L-carnitine, and conclusions suggested by the accumulation of certain metabolites are discussed in relation to control of citric acid cycle activity.