Pseudohypoparathyreoidismus und epigenetische Veränderungen des GNAS-Genlocus

The term pseudohypoparathyroidism (PHP) describes a heterogeneous group of related disorders characterized by end-organ resistance to parathyroid hormone (PTH). PHP is caused by deficiency of the α-subunit of stimulatory G proteins (Gsα), which is crucial for signal transduction of more than 1000 G protein-coupled receptors into the cell. PHP type Ia is caused by heterozygous, maternally inherited inactivating mutations involving those exons of the GNAS locus that encode Gsα. In addition, PHP Ia and Ic patients present with features of Albright hereditary osteodystrophy (AHO), which includes round face, short stature, brachymetacarpia, ectopic ossification, and mental retardation. Paternally inherited GNAS mutations lead to pseudo-PHP and are characterized by only some features of AHO in the absence of hormone resistance. PHP type Ib is caused by heterozygous, maternally inherited deletions up-stream of or within the GNAS locus that are associated with the loss of methylation at one or more maternally methylated regions within GNAS . Typically, these patients lack AHO features. This article provides an overview of the role of epigenetic factors for different PHP subtypes.     

Genetic basis for resistance to parathyroid hormone

Pseudohypoparathyroidism (PHP) is associated with biochemical hypoparathyroidism (i.e. hypocalcemia and hyperphosphatemia) due to parathyroid hormone (PTH) resistance rather than to PTH deficiency. Patients with PHP type 1a have a generalized form of hormone resistance plus a constellation of developmental defects termed Albright hereditary osteodystrophy (AHO). Within PHP type 1a families some individuals will show AHO but have normal hormone responsiveness, a variant phenotype termed pseudo-PHP. By contrast, patients with PHP type 1b manifest only PTH resistance and lack features of AHO. These various forms of PHP are due to defects in the GNAS1 gene that lead to decreased expression or activity of the alpha-subunit of the stimulatory G protein (G(s)alpha). Tissue-specific genomic imprinting of GNAS1 accounts for the variable phenotypes of patients with GNAS1 defects.     

G protein defects in signal transduction

G proteins couple receptors for many hormones to effectors that regulate second messenger metabolism. Several endocrine disorders have been shown to be caused by either loss- or gain-of-function mutations in G proteins or G protein-coupled receptors. In pseudohypoparathyroidism type Ia (PHP Ia), there are generalized hormone resistance (parathyroid hormone [PTH], thyroid-stimulating hormone, gonadotropins) and associated abnormal physical features, Albright hereditary osteodystrophy. Subjects with PHP Ib are normal in appearance and show renal resistance to PTH. In McCune-Albright syndrome (MAS), subjects show autonomous endocrine hyperfunction associated with fibrous dysplasia of bone and skin hyperpigmentation. Germline loss-of-function mutations have been identified in the G(s)-alpha gene in PHP Ia, and recent evidence suggests that the G(s)-alpha gene is paternally imprinted in a tissue-specific manner. Abnormal imprinting of the G(s)-alpha gene may be the cause of PHP Ib. MAS, in contrast, is caused by gain-of-function missense mutations of the G(s)-alpha gene.     

Paternal uniparental isodisomy of chromosome 20q--and the resulting changes in GNAS1 methylation--as a plausible cause of pseudohypoparathyroidism

Heterozygous inactivating mutations in the GNAS1 exons (20q13.3) that encode the alpha-subunit of the stimulatory G protein (Gsalpha) are found in patients with pseudohypoparathyroidism type Ia (PHP-Ia) and in patients with pseudo-pseudohypoparathyroidism (pPHP). However, because of paternal imprinting, resistance to parathyroid hormone (PTH)-and, sometimes, to other hormones that require Gsalpha signaling-develops only if the defect is inherited from a female carrier of the disease gene. An identical mode of inheritance is observed in kindreds with pseudohypoparathyroidism type Ib (PHP-Ib), which is most likely caused by mutations in regulatory regions of the maternal GNAS1 gene that are predicted to interfere with the parent-specific methylation of this gene. We report a patient with PTH-resistant hypocalcemia and hyperphosphatemia but without evidence for Albright hereditary osteodystrophy who has paternal uniparental isodisomy of chromosome 20q and lacks the maternal-specific methylation pattern within GNAS1. Since studies in the patient's fibroblasts did not reveal any evidence of impaired Gsalpha protein or activity, it appears that the loss of the maternal GNAS1 gene and the resulting epigenetic changes alone can lead to PTH resistance in the proximal renal tubules and thus lead to impaired regulation of mineral-ion homeostasis.     

Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine]

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.     

Alteration of mannose transport in fibroblasts from type I carbohydrate deficient glycoprotein syndrome patients

The aim of the present study was to explore how mannose enters fibroblasts derived from a panel of children suffering from different subtypes of type I carbohydrate deficient glycoprotein syndrome: seven carbohydrate deficient glycoprotein syndrome subtype Ia (phosphomannomutase deficiency), two carbohydrate deficient glycoprotein syndrome subtype Ib (phosphomannose isomerase deficiency) and two carbohydrate deficient glycoprotein syndrome subtype Ix (not identified deficiency). We showed that a specific mannose transport system exists in all the cells tested but has different characteristics with respect to carbohydrate deficient glycoprotein syndrome subtypes. Subtype Ia fibroblasts presented a mannose uptake equivalent or higher (maximum 1.6-fold) than control cells with a D-[2-3H]-mannose incorporation in nascent N-glycoproteins decreased up to 7-fold. Compared to control cells, the mannose uptake was greatly stimulated in subtype Ib (4.0-fold), due to lower Kuptake and higher Vmax values. Subtype Ib cells showed an increased incorporation of D-[2-3H]-mannose into nascent N-glycoproteins. Subtype Ix fibroblasts presented an intermediary status with mannose uptake equivalent to the control but with an increased incorporation of D-[2-3H]-mannose in nascent N-glycoproteins. All together, our results demonstrate quantitative and/or qualitative modifications in mannose transport of all carbohydrate deficient glycoprotein syndrome fibroblasts in comparison to control cells, with a relative homogeneity within a considered subtype of carbohydrate deficient glycoprotein syndrome. These results are consistent with the possible use of mannose as a therapeutic agent in carbohydrate deficient glycoprotein syndrome Ib and Ix.     

Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.

We have compared the activities of mouse alpha-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). MersI, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single trans-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.     

Major proteins of the outer cell envelope membrane of Escherichia coli K12: multiple species of protein I differ in primary structure.

Summary Protein I, one of the major outer membrane proteins of E. coli in most K12 strains is represented by two very similar polypeptides Ia and Ib. Sequential mutations (involving selections for phage resistance) can lead to loss of proteins Ia and Ib. Among revertants of such Ia^- Ib^- mutants clones exist that instead of Ia or Ib produce a third species of protein I, polypeptide Ic.Ichihara and Mizushima [J. Biochem. 83, 1095–1100 (1978)] have shown that proteins Ia and Ib exhibit differences in primary structure. Here evidence is presented indicating that protein Ic also is not identical in primary structure with Ia or Ib. Thus, 3 very similar structural genes appear to exist for the protein I species known to date, and that for Ic normally is silent. Introduction of a functional Ic locus into a Ia⁺ Ib⁺ strain caused expression of all three proteins with a reduced rate of synthesis of protein Ia.     

Major proteins of the outer cell envelope membrane of Escherichia coli K-12: multiple species of protein I.

Summary Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia⁺ Ib^-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II^*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.     

Occurrence of the African subgroup (Ia) of BK polyomavirus in younger Japanese children

BK polyomavirus (BKV) is ubiquitous among humans, usually infecting them asymptomatically during childhood. BKV persists in renal tissue of individuals and their progeny are excreted in urine, particularly in immunocompromised patients. JC virus, another human polyomavirus, has been considered to be transmitted from parents to children during prolonged cohabitation. However, BKV has been supposed to be transmitted not only within but also outside the family. In the present study, to clarify this possibility, we analyzed phylogenetically 35 BKV which were excreted in the urine by Japanese children and adults undergoing stem cell transplantation. Subtypes I, III and IV were detected in 15, two and one children and in 15, one and one adults, respectively. Among 15 subtype I isolates from children, three, four and eight belonged to subgroups Ia, Ib-1 and Ic, respectively. All the three children from whom Ia was detected were less than 9 years old. In contrast in the adults, three subtype I belonged to Ib-1 and the other 12 to Ic. These findings may reflect the recent transmission of BKV Ia strains to Japanese children.     

Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver-leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI-MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N-binding sugar chains matches that of a high-mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C-terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3-Cys17, Cys175-Cys191, and Cys300-Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.     

Phylogenetic analysis of the rDNA intergenic spacer subrepeats and its implication for the domestication history of foxtail millet, Setaria italica

We sequenced ribosomal DNA intergenic spacer subrepeats and their flanking regions of foxtail millet landraces from various regions in Europe and Asia and its wild ancestor to elucidate phylogenetic differentiation within each of types I–III found in our previous work and to elucidate relationships among these three types. Type I was classified into seven subtypes designated as Ia–Ig based on subrepeat sequences; C repeats downstream of those subrepeats are also polymorphic. Of these, subtypes Ia–Id and Ig were found in foxtail millet landraces. Subtypes Ia and Ib were distributed broadly throughout Asia and Europe. Subtype Ic was distributed in China, Korea and Japan. Subtype Id has a 20-bp deletion in subrepeat 3 and has a unique C repeat sequence. This subtype was found in a morphologically primitive landrace group from Afghanistan and northwestern Pakistan and differed greatly from other type I subtypes, implying that these landraces were domesticated independently. Subtypes Ig was found in a landrace from Pakistan and Ia and Ie–Ig were in six wild ancestor accessions. Type II was also highly polymorphic and four subtypes were found and designated as subtypes IIa–IId, but sequence analyses indicated type III as monomorphic. The present work indicates that type III should be classified as a subtype of type II (subtype IIe). Sequence polymorphism of subrepeats of types I–III indicated that subrepeats of subtype IIa are greatly divergent from others. Relationships among types I–III are much more complicated than anticipated based on previous RFLP work.     

A gene on chromosome 11q23 coding for a putative glucose- 6-phosphate translocase is mutated in glycogen-storage disease types Ib and Ic.

Glycogen-storage diseases type I (GSD type I) are due to a deficiency in glucose-6-phosphatase, an enzymatic system present in the endoplasmic reticulum that plays a crucial role in blood glucose homeostasis. Unlike GSD type Ia, types Ib and Ic are not due to mutations in the phosphohydrolase gene and are clinically characterized by the presence of associated neutropenia and neutrophil dysfunction. Biochemical evidence indicates the presence of a defect in glucose-6-phosphate (GSD type Ib) or inorganic phosphate (Pi) (GSD type Ic) transport in the microsomes. We have recently cloned a cDNA encoding a putative glucose-6-phosphate translocase. We have now localized the corresponding gene on chromosome 11q23, the region where GSD types Ib and Ic have been mapped. Using SSCP analysis and sequencing, we have screened this gene, for mutations in genomic DNA, from patients from 22 different families who have GSD types Ib and Ic. Of 20 mutations found, 11 result in truncated proteins that are probably nonfunctional. Most other mutations result in substitutions of conserved or semiconserved residues. The two most common mutations (Gly339Cys and 1211-1212 delCT) together constitute approximately 40% of the disease alleles. The fact that the same mutations are found in GSD types Ib and Ic could indicate either that Pi and glucose-6-phosphate are transported in microsomes by the same transporter or that the biochemical assays used to differentiate Pi and glucose-6-phosphate transport defects are not reliable.     

Heterotopic ossifications in a mouse model of albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is characterized by short stature, brachydactyly, and often heterotopic ossifications that are typically subcutaneous. Subcutaneous ossifications (SCO) cause considerable morbidity in AHO with no effective treatment. AHO is caused by heterozygous inactivating mutations in those GNAS exons encoding the α-subunit of the stimulatory G protein (Gα(s)). When inherited maternally, these mutations are associated with obesity, cognitive impairment, and resistance to certain hormones that mediate their actions through G protein-coupled receptors, a condition termed pseudohypoparathyroidism type 1a (PHP1a). When inherited paternally, GNAS mutations cause only AHO but not hormonal resistance, termed pseudopseudohypoparathyroidism (PPHP). Mice with targeted disruption of exon 1 of Gnas (Gnas(E1-/+)) replicate human PHP1a or PPHP phenotypically and hormonally. However, SCO have not yet been reported in Gnas(E1+/-) mice, at least not those that had been analyzed by us up to 3 months of age. Here we now show that Gnas(E1-/+) animals develop SCO over time. The ossified lesions increase in number and size and are uniformly detected in adult mice by one year of age. They are located in both the dermis, often in perifollicular areas, and the subcutis. These lesions are particularly prominent in skin prone to injury or pressure. The SCO comprise mature bone with evidence of mineral deposition and bone marrow elements. Superficial localization was confirmed by radiographic and computerized tomographic imaging. In situ hybridization of SCO lesions were positive for both osteonectin and osteopontin. Notably, the ossifications were much more extensive in males than females. Because Gnas(E1-/+) mice develop SCO features that are similar to those observed in AHO patients, these animals provide a model system suitable for investigating pathogenic mechanisms involved in SCO formation and for developing novel therapeutics for heterotopic bone formation. Moreover, these mice provide a model with which to investigate the regulatory mechanisms of bone formation.     

Clinical heterogeneity of pseudohypoparathyroidism: from hyper- to hypocalcemia

Pseudohypoparathyroidism (PHP) is a rare inherited syndrome characterized by parathyroid hormone (PTH) resistance and is frequently associated with Albright's hereditary osteodystrophy and resistance to other cAMP-mediated hormones. The usual neonatal presentation is mild primary hypothyroidism secondary to resistance to thyroid-stimulating hormone; hypocalcemia usually develops after age 3-5 years. This work describes the diversity in the clinical expression and course of PHP, with emphasis on calcium levels by age and treatment, in 8 children under long-term follow-up at our pediatric tertiary center. The calcium levels at presentation ranged from transient neonatal hypocalcemia to infantile hypercalcemia to childhood/adolescence hypocalcemia. Interestingly, relative hypocalciuria at diagnosis and during therapy, in the presence of renal PTH resistance, was the rule. These findings indicate that transient neonatal hypocalcemia associated with other clinical features or a family history of PHP may be a flag for clinicians to screen for PTH resistance later in life. In addition, PTH resistance may be missed by surveying calcium levels only; thus the PTH levels have to be checked as well. In addition, the recommendation for patients with hypoparathyroidism that strict low-normal calcium levels be maintained during therapy in order to prevent hypercalciuria is probably not applicable in PHP.     

Purification, characterization and substrate specificities of xylanase isoenzymes from Melanocarpus albomyces IIS 68

The ascomycetous fungus Melanocarpus albomyces when grown on wheat straw produced seven extracellular xylanase isoenzymes, designated as la, Ib, Ic, IIa, lib, llc, and lId. All seven xylanases were purified to homogeneity by gel filtration and ion-exchange chromatography. The molecular mass (kDa) of la, lb, Ic, Ila, lIb, lIc, and lId were estimated to be 22.9, 20.7, 18.6, 31.3, 25.4, 38.5, and 34.3, respectively by SDS-PAGE, and 23.7, 20.5, 17.1, 31.7, 25.1, 39.8, and 32.2, respectively by gel filtration. The isoelectric points of Ia, lb, Ic, Ila and IIb were found to be 6.2, 6.3, 6.4, 3.7, and 4.4, respectively. The activity of the isoenzymes was dependent on the type of the xylan substrates; Xylanases Ia, lb, and Ic showed highest specific activity toward larchwood xylan (an arabinoglucuronoxylan), IIa and Ilc toward birchwood xylan (a glucuronoxylan), and llb and IId toward beechwood xylan (a glucuronoxylan). Four isoenzymes la, lb, Ic, and Ila had an arabinose-releasing property on larchwood xylan. Application of specific isoenzymes as prebleaching agents in paper manufacture is proposed.