Duplication of 17q11.2 and Features of Albright Hereditary Osteodystrophy Secondary to Methylation Defects within the GNAS Cluster: Coincidence or Causal?

We report a case of Albright hereditary osteodystrophy (AHO) in a three-year-old girl with a microduplication at 17q11.2. The child developed obesity within the first 6 months of life. A diagnosis of Albright was made at age 2 years when biochemical evidence of parathyroid resistance was found. No mutations were identified in guanine nucleotide-binding protein G (s) subunit alpha (GNAS1). Subsequent investigations revealed methylation disturbance at GNAS1A, neuroendocrine secretory protein antisense (NESPAS) and neuroendocrine secretory protein 55 (NESP55) confirming a diagnosis of pseudohypothyroidism type 1B. A deletion of NESP55 and uniparental disomy chromosome 20 were excluded which suggested that the features of AHO arose through a purely epigenetic mechanism. Further investigation revealed a de novo microduplication at 17q11.2 encompassing the neurofibromatosis type 1 (NF1) gene. The combination of two rare de novo events in the same child raises the possibility that duplication of a gene within the 17q11.2 region may have triggered abnormal methylation in the GNAS cluster region on chromosome 20.

Pseudohypoparathyroidism type 1b due to paternal uniparental disomy of chromosome 20q.

CONTEXT: Pseudohypoparathyroidism type 1b (PHP1b) is the result of end-organ resistance to PTH and other hormones such as TSH in the absence of any features of Albright's hereditary osteodystrophy. Patients with PHP1b show imprinting abnormalities at the complex GNAS locus. The molecular cause of autosomal dominant familial PHP1b has been well-defined with identification of microdeletions within the GNAS locus or the nearby STX16, but the molecular mechanism of the GNAS imprinting defects in sporadic PHP1b cases remains elusive. OBJECTIVE: We investigated the underlying molecular mechanism of GNAS imprinting defects in two patients with sporadic PHP1b. RESULTS: We identified paternal uniparental disomy of the long arm of chromosome 20 (patUPD20) in two unrelated patients with sporadic PHP1b. This provides an explanation for the patients' GNAS methylation abnormalities and hormone resistance. Our data and a review of the six published cases of patUPD20 suggest that high birth weight and/or early-onset obesity and macrocephaly may also represent features of patUPD20. CONCLUSION: We suggest that patUPD20 should be considered in the evaluation of patients with sporadic PHP1b.

Isolated imprinting mutation of the DLK1/GTL2 locus associated with a clinical presentation of maternal uniparental disomy of chromosome 14.

The clinical phenotypes of maternal and paternal uniparental disomy of chromosome 14 (UPD14) are attributed to dysregulation of imprinted genes. A large candidate locus exists within 14q32, under the regulation of a paternally methylated intergenic differentially methylated region (IG-DMR). We present a patient with clinical features of maternal UPD14, including growth retardation, hypotonia, scoliosis, small hands and feet, and advanced puberty, who had loss of methylation of the IG-DMR with no evidence of maternal UPD14. This case provides support for the hypothesis that the maternal UPD14 phenotype is due to aberrant gene expression within the imprinted domain at 14q32.

Mosaic maternal uniparental disomy of chromosome 11 in a patient with Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a clinically heterogeneous disorder characterised mainly by intrauterine and postnatal growth retardation. While maternal uniparental disomy of chromosome 7 is found in 5-10% of SRS patients, recently genetic and epigenetic mutations affecting the imprinting centres on chromosome 11p15 have been reported in up to 64% of patients. Chromosome 11p15 abnormalities reported in SRS include methylation defects in the imprinting centre 1 (ICR1) and maternally inherited duplications involving all or part of the imprinted region of 11p15. Here we report the first published case of SRS with mosaic maternal uniparental disomy of chromosome 11.

Isolated imprinting mutation of the DLK1/GTL2 locus associated with a clinical presentation of maternal uniparental disomy of chromosome 14.

The clinical phenotypes of maternal and paternal uniparental disomy of chromosome 14 (UPD14) are attributed to dysregulation of imprinted genes. A large candidate locus exists within 14q32, under the regulation of a paternally methylated intergenic differentially methylated region (IG-DMR). We present a patient with clinical features of maternal UPD14, including growth retardation, hypotonia, scoliosis, small hands and feet, and advanced puberty, who had loss of methylation of the IG-DMR with no evidence of maternal UPD14. This case provides support for the hypothesis that the maternal UPD14 phenotype is due to aberrant gene expression within the imprinted domain at 14q32.

Maternal UPD(14) in the patient with a normal karyotype: clinical report and a systematic search for cases in samples sent for testing for Prader-Willi syndrome.

Maternal uniparental disomy for chromosome 14 causes a recognizable phenotype that has a number of consistent features, irrespective of the underlying chromosome abnormality. To illustrate this, we describe a patient with a 46,XX karyotype whose short stature, hypotonia, feeding problems, truncal obesity, early puberty, learning difficulties, and craniofacial characteristics led to a diagnosis of maternal (mat) UPD(14). No evidence is available to indicate how common mat UPD(14) in patients with a normal karyotype might be. Because of the similarity between Prader-Willi syndrome (PWS) and the mat UPD(14) phenotype in childhood, we systematically tested samples from 35 patients with normal karyotypes and an unexplained PWS-like phenotype referred to the Wessex Genetics Service. We sought to address the practical question should laboratories carry out tests for mat UPD(14) on all samples received for PWS testing when PWS testing gives negative results? None of the samples tested showed evidence of mat UPD(14). Routine screening of DNA from patients with possible PWS cannot be recommended on this basis.

Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome.

Silver-Russell syndrome (SRS) has been associated with maternal uniparental disomy (UPD) of chromosome 7 in approximately 10% of cases, suggesting that at least one imprinted gene on chromosome 7 is involved in the pathogenesis of the disease. We report a proximal 7p interstitial inverted duplication in a mother and daughter both of whom have features of SRS, including marked short stature, low birth weight, facial asymmetry and 5th finger clinodactyly. Fluorescence in situ hybridisation (FISH) with YAC probes enabled delineation of the duplicated region to 7p12.1-p13. This region of proximal chromosome 7 is known to be homologous to an imprinted region in the mouse chromosome 11 and contains the growth-related genes GRB10 (growth factor receptor-bound protein 10), EGFR (epidermal growth factor receptor) and IGFBP1 (insulin-like growth factor binding protein 1), all of which have been suggested as candidate genes for SRS. Molecular analysis showed that the duplication in both mother and daughter spanned a distance of approximately 10 cM and included GRB10 and IGFBP1 but not EGFR. The de novo duplication in the proband's mother was shown to be of paternal origin. In order to test the hypothesis that sub-microscopic duplications of 7p, whether maternal or paternal in origin, are responsible for at least some cases of SRS, we screened a further eight patients referred to our laboratory for SRS. None were found to have duplications of either GRB10 or IGFBP1. The hypothesis that sub-microscopic duplications including GRB10 and IGFBP1 is a cause of SRS remains a possibility and warrants further investigation. Importantly, in contrast to current thinking, our results suggest that imprinted genes may not underlie the SRS phenotype, and we propose an alternative hypothesis to explain the occurrence of maternal UPD 7 seen in some cases of SRS.

Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.

Desmoid tumors are slowly growing fibrous tumors highly resistant to therapy and often fatal. Here, we report hereditary desmoid disease (HDD), a novel autosomal dominant trait with 100% penetrance affecting a three-generation kindred. Desmoid tumors are usually a complication of familial adenomatous polyposis, a predisposition to the early development of premalignant adenomatous polyps in the colorectum due to chain-terminating mutations of the APC gene. In general, one or more members in approximately 10% of the FAP families manifest desmoid tumors. Affected individuals from the HDD kindred are characterized by multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Osteomas, epidermal cysts, and other congenital features were also observed. We show that HDD segregates with an unusual germ-line chain-terminating mutation at the 3' end of the APC gene (codon 1924) with somatic loss of the wild-type allele leading to tumor development.

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy.

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA)n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.

Insert size and flanking haplotype in fragile X and normal populations: possible multiple origins for the fragile X mutation.

A number of recent studies have found non-random association between the fragile X mutation and genotypes for the closest-linked flanking markers, suggesting either a limited number of 'founder' mutations or, alternatively, a predisposing haplotype for the fragile X expansions. Using three microsatellite markers within 150 kb of FRAXA, we have compared haplotypes in a series of fragile X males and in a control population and find a markedly different distribution in the two samples, with apparently greater haplotype diversity in the fragile X sample. In the control sample, various non-random associations of CGG repeat numbers with flanking haplotypes were recorded which provide a clue to the likely origins of the fragile X mutation, suggesting more than one mechanism for the initial expansion event.

Population studies of the fragile X: a molecular approach.

The fragile X mutation can now be recognised by a variety of molecular techniques. We report a pilot screening survey of a population of children with mental impairment in which we used Southern blotting methods to detect the fragile X mutation, augmented by cytogenetic studies on children whose phenotype suggested a possible chromosome abnormality. There were 873 children with special educational needs in our survey and 310 fulfilled our criteria for testing. A sample was obtained from 254, of whom four were found to have a full fra(X) mutation (delta L) and none to have a premutation. The number of CGG repeats in our population of X chromosomes was measured by PCR analysis and the genotype at the closely linked polymorphic locus FRAXAC1 established. The distribution of CGG repeat numbers was very similar to that of the control population reported by Fu et al and the distribution of FRAXAC1 alleles almost identical to that of the control population reported by Richards et al. Among the non-fragile X chromosomes, we found a very significant correlation between the size of the CGG repeat and the FRAXAC1 genotype. There was a dearth of A and D genotypes in subjects with a small number of CGG repeats and an excess of the A genotype in those with a large number of CGG repeats. These observations are considered in the light of the reported disequilibrium between the A (and possibly also the D) genotype and the fra(X) mutation.

Physical characterization of the replication origin of the cryptic plasmid pCB101 isolated from Clostridium butyricum NCIB 7423.

The complete nucleotide sequence of a 3484-bp Sau3A fragment, previously shown to carry the replication origin of the Clostridium butyricum NCIB 7423 plasmid pCB101 (6.05 kb), has been determined. Of the four open reading frames (ORF A-D) identified within this fragment, two (B and C) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that both polypeptides are required for autonomous replication of the plasmid in Bacillus subtilis. ORF C is immediately preceded by a small ORF (C') that encodes a relatively small polypeptide (50 amino acids) that demonstrates significant homology with RepA of plasmid pLS1. Whereas the ORF C polypeptide (27,100 Da) exhibits no homology to any known protein, that encoded by ORF B (RepB, 43,039 Da) exhibits significant homology with the Rep proteins of the pC194/pUB110 subfamily of single-strand (ss) DNA plasmids, which are widely distributed in gram-positive bacteria. Conserved amino acids include the presumed active site of topoisomerase activity and four cysteine residues in the N-terminus of all Rep proteins compared. The repB gene is preceded by a sequence motif exhibiting substantial homology to the "plus" origins of this family of ss DNA plasmids and was shown to act as a "hot spot" for deletion formation in certain plasmid chimaeras. The compelling suggestion that pCB101 replicates via a rolling circle mechanism was substantiated by the demonstration of ss DNA replication intermediates in B. subtilis cells carrying a pCB101-derived plasmid.

Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene.

The complete nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 gene coding for the chemotherapeutic enzyme L-asparaginase has been determined. The structural gene consists of an open reading frame commencing with an ATG start codon of 1044 bp followed by a TGA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid (aa) sequence with that derived by N-terminal aa sequencing of the purified protein. The gene has been shown to code for a 21-aa signal peptide at its N terminus which closely resembles the signal peptides of other secreted proteins. In common with highly expressed Escherichia coli genes, little use is made of modulator codons. The predicted aa sequence of the enzyme exhibits 46% identity with the determined primary sequence of the E. coli L-asparaginase, although the predicted secondary structure of both proteins indicates more extensive homology. Downstream of the TGA stop codon is a G + C-rich region of dyad symmetry (delta G = -25.4 kcal) characteristic of E. coli Rho-independent transcription terminators. Upstream of the structural gene there are no sequences which bear a strong resemblance to the consensus -35 and -10 regions of E. coli promoters. A sequence is present (CTGGCTCTCCTCTTGAT), however, which exhibits strong homology to the nif promoter consensus sequence (CTGGCACN5TTGCA). Upstream of this region is a sequence which strongly resembles the consensus sequence for promoter regions which are subject to catabolite repression.

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora.

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).

Organization and evolution of the class I gene family in the major histocompatibility complex of the C57BL/10 mouse.

The major histocompatibility complex (MHC) encodes several classes of protein vital to the regulation of the immune response. We have isolated 26 class I genes that map to this region in the C57BL/10 mouse and linked these into three gene clusters. The number of genes differs from the number found in the BALB/c strain and comparison of the organization of the class I genes in these two strains shows conserved regions and polymorphic regions which probably result from deletions, insertions and translocations within the MHC.

Expression of murine H-2Kb histocompatibility antigen in cells transformed with cloned H-2 genes.

Cosmids containing H-2 histocompatibility antigen genes of the H-2b haplotype have been isolated. One of these genes expresses a 45,000 molecular weight protein, indistinguishable from H-2Kb when introduced into mouse L cells. These H-2Kb transformed L cells can be killed by allospecific anti-H-2Kb cytotoxic T cells. Moreover, when infected with influenza virus, they can be killed by an H-2Kb-restricted, influenza virus-specific cytotoxic T cell line. These results show that expression of the H-2Kb gene product on the L-cell surface is sufficient to make it a target for specific T-cell killing.

High-pressure liquid chromatography of steroids secreted by human adrenal and testis cells in monolayer culture.

Reversed-phase high-pressure liquid chromatography with gradient elution on Zorbax-ODS columns has been used to separate, identify, and measure, spectrophotometrically, the steroids secreted by both human adrenal and testis cells in primary monolayer culture. Three related systems using exponential concave gradients have been developed with the specific objective of resolving the steroids produced by these two tissues. A methanol-water gradient has been used to separate most adrenal steroids, an acetonitrile-water gradient to separate testis steroids, and a dioxane-water gradient to separate polar steroids, including aldosterone. These three systems together permit the resolution of at least 43 naturally occurring steroids, plus four synthetic steroids with adrenocortical activity, with overall total elution times of 1 h or less for each system. Retention data for these steroids are given and the separation of steroids in the biological samples illustrated.