Imprinting centre deletions in two PWS families: implications for diagnostic testing and genetic counseling.

Prader-Willi syndrome (PWS) is a complex genetic syndrome involving imprinted genes on chromosome 15. It is usually sporadic, and very few affected siblings have been described. Here, we report the clinical and molecular findings in two families with a microdeletion affecting the chromosome 15 imprinting centre (IC). Carrier males have a 50% risk of having children with an imprinting defect leading to PWS, and in one of the two families, a father has two affected daughters. In the other family, diagnostic testing was confounded by the presence of a neutral microdeletion close to the IC. The silent transmission of PWS IC deletions through the female germline and the occurrence of neutral microdeletions close to the IC can impose considerable problems on diagnostic testing and genetic counselling in affected families.

A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES.

Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells. These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif. Such loops are important in RNA-RNA and RNA-protein interactions. Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN. Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements. The sequence of individual selected IRES elements was determined. All functional IRES elements had a tetraloop with a 3' terminal A residue. Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus. In contrast, IRES elements containing YCYA tetraloops were severely defective.

Vaccinia virus protein synthesis has a low requirement for the intact translation initiation factor eIF4F, the cap-binding complex, within infected cells.

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both beta-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (beta-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5' noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5' and 3' termini.

Prevalence of cystic fibrosis mutations in pregnancies with fetal echogenic bowel.

OBJECTIVE: To determine the prevalence of the most common cystic fibrosis mutations in pregnancies complicated by fetal echogenic bowel by using DNA testing. METHODS: Forty-five pregnancies with fetal echogenic bowel were studied prospectively for cystic fibrosis mutations. Using polymerase chain reaction, DNA from fetal amniocytes (n = 21), fetal blood (n = 5), or parental blood (n = 19) was amplified and tested for delta F508, G551D, G542X, and 621 + 1G-->T cystic fibrosis mutations, which account for about 85% of the mutations in the British population. In selected cases, further mutations were tested according to the parental ethnic background. RESULTS: Only one of the 26 fetuses screened was heterozygous for cystic fibrosis mutations. Among 38 parental samples screened from the remaining 19 pregnancies, cystic fibrosis mutations were detected in two cases, only one of the parents being a carrier in each case. The prevalence of cystic fibrosis carrier status in fetal and parental samples (1:26 and 1:19, respectively) is within the expected prevalence in the British population (1:25). No fetuses were affected by cystic fibrosis in this series, but five were found to have growth restriction, two trisomy 21, two congenital infection, and two bowel obstruction. CONCLUSION: Our results suggest that ultrasonographic detection of fetal echogenic bowel is not associated with an increased prevalence of cystic fibrosis mutations in pregnancies at low risk for this disease.

Prenatal analysis of the insulin receptor gene in a family with leprechaunism.

We report on the prenatal diagnosis of a fetus at risk of leprechaunism. We had previously determined the nature of the causative mutation in the insulin receptor gene in this family. The mutation removes a restriction site for the enzyme Mbo II. Genomic DNA was extracted from a chorionic villus sample and the 3' half of exon 2 was amplified by the polymerase chain reaction (PCR) followed by restriction digest. Using this method, we correctly predicted an unaffected child.

Absence of Y specific DNA sequences in two siblings with 46XX hermaphroditism.

We report two siblings with 46XX hermaphroditism in whom we were unable to show the presence of Y specific DNA sequences using the DNA probes Y-190, GMGY-7, pHY2.1, pDP34, and 27a. We conclude that an autosomal or X chromosome gene mutation is the most likely mechanism of inheritance in this family with 46XX hermaphroditism.

Cellular and molecular studies on infant null acute lymphoblastic leukemia.

We have studied the cellular and molecular basis of eight cases of infant null acute lymphoblastic leukemia (ALL). All eight patients were under 9 months of age and presented with leukocyte counts in excess of 60 X 10(9)/L, organomegaly, and in two cases CNS infiltration. Although seven cases were morphologically classified as ALL, one patient had both lymphoid and myeloid features. Phenotypic analysis of leukemic blasts from all patients showed a typical null ALL pattern, ie, CD10 (common ALL antigen)-negative, strongly HLA-DR-positive, and CD19 (B4)-positive. The presence of terminal deoxynucleotidyl transferase (TdT) at presentation was positive in six patients' cells and negative in two. Two patients also expressed the myeloid-associated markers CD33 (MY9) and CD15 (TG1), and coexpression of CD19 and CD33 was confirmed in these two by using dual marker flow cytometry (fluorescence-activated cell sorting). Electron microscopic examination of the same two patients' cells showed the presence of monocytoid blasts that labeled with the pan-B cell antibody B4 (CD19). Short-term culture of one of these patients cells in the presence of phorbol ester resulted in the majority of the cells exhibiting myeloid markers, strong nonspecific esterase positivity, and phagocytic properties. Cytogenetic analysis showed the common feature in 7 of 8 cases to be a break in band 11q23. Molecular analysis of DNA from the blast cells of all eight patients showed rearrangement of the immunoglobulin heavy-chain genes in all cases without, however, any evidence of kappa light-chain rearrangement. T cell receptor genes were present in the germline configuration in all cases. Rearrangements of the c-ets 1 oncogene, which maps to band 11q23, were not detected, thus providing no evidence for involvement of this oncogene in the common disease process. Our data indicate that although infant null ALL may present as a heterogeneous disease the similarity of many features between cases suggests a common derivation from a precursor cell sharing phenotypic and genotypic features of both B and myeloid progenitor cells.

Service experience using DNA analysis for genetic prediction in Duchenne muscular dystrophy.

In August 1985 we instituted a carrier and prenatal testing service for Duchenne muscular dystrophy (DMD) using direct DNA analysis. The experience over the first nine months is described. We have analysed samples for RFLPs from 154 people including 53 women at risk of being DMD carriers from 37 families. We used the probes pERT87.8 (BstXI and TaqI polymorphisms), 87-15 (TaqI polymorphism), and pXJ1.1 (TaqI polymorphism). Forty-one of the women have had their risks altered. We found one deletion (pERT87-8) out of 23 DNA samples analysed from affected boys. We used a recombination fraction of 0.05 in risk calculations but did not detect any known crossovers. In nine of the families there is only an isolated case of DMD. In families where we have not been able to alter the risk of the women being a carrier (for example, because all brothers are dead), we have offered prenatal exclusion and have carried out one first trimester prenatal diagnosis on this basis. Lowering the risk of an affected fetus to less than 2.5% appears to be a satisfactory situation for many (most) of the women involved and seems to justify the introduction of genetic prediction based on single intragenic probes despite the 5% recombination frequency.

Prenatal diagnosis of X-linked choroideremia with mental retardation, associated with a cytologically detectable X-chromosome deletion.

We describe a family in which an X-chromosome deletion is segregating with choroideremia, and X-linked recessive condition. The DNA sequences DXYS1 and DXS3, defined by the probes pDP34 and 19.2 respectively, are absent in the affected male (who is also mentally retarded), and hemizygous in his mother and in his carrier sister, who presented early in pregnancy. Analysis of chorionic villus DNA formed the basis of prenatal exclusion of choroideremia in her male fetus. In three female relatives, studied with late-labelling techniques, the deleted X was preferentially inactivated in 86-100% of cells studied. This family confirms the localisation of the choroideremia locus to within Xq13----21, and places the loci for anhidrotic ectodermal dysplasia and the X-linked immunodeficiencies outside this region.

Insulin-like growth factor-II gene expression in Wilms' tumour and embryonic tissues.

Wilms' tumour (nephroblastoma) is an embryonal neoplasm occurring in hereditary and spontaneous forms. Both types show rearrangements of the short arm of chromosome 11. The germ line of children with the rare inherited triad of aniridia, genito-urinary abnormality and mental retardation carry a chromosome 11 that has a deletion in its short arm (band 11p13) and these children are at increased risk of developing Wilms' tumour. Neonates with the Beckwith-Wiedemann syndrome, in which there may be duplication of the 11p13-11p15 region, are similarly predisposed. In the spontaneous form of the tumour a deletion of the 11p14 band in tumour cells, but not in normal cells, has been reported, and the development of homozygosity for recessive mutations in the 11p region is implicated in the aetiology of Wilms' tumour. In view of these chromosomal rearrangements and because Wilms' tumour is histologically indistinguishable from the early stages of kidney development, we have now examined the expression of genes localized to 11p in Wilms' tumour and human embryonic tissue. In 12 sporadic tumours examined, the expression of the gene coding for insulin-like growth factor-II (IGF-II), localized to the 11p15 region, was markedly increased relative to adult tissues, but was comparable to the level of expression in several fetal tissues including kidney, liver, adrenals and striated muscle. This may reflect the stage of tumour differentiation, but could also contribute to the malignant process, as IGF-II is an embryonal mitogen.

The human apolipoprotein AII gene: structural organization and sites of expression.

The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver.

High-density lipoprotein composition is altered by a common DNA polymorphism adjacent to apoprotein AII gene in man.

25 of a group of 87 White men had Msp 1 restriction site polymorphism within an Alu sequence 3' to the human apo AII gene. Homozygosity for the polymorphism in 8 men was associated with a significant increase in serum apo AII levels (35.4 +/- 1.70 mg/dl, mean +/- SEM) and altered HDL composition, compared with heterozygotes (31.7 +/- 1.29; n = 17) and normal subjects (29.4 +/- 0.64; n = 62). This is the first account of a common variant of an HDL apoprotein gene that affects HDL composition. In view of its association with a high apo AII concentration homozygosity may protect against atherosclerosis.

Chromosomal localization of the human apoprotein CI gene and of a polymorphic apoprotein AII gene.

Human apoprotein(apo) CI and apo AII cDNA probes have been used to analyze the segregation of the human genes in panels of human-mouse hybrids. The apo CI (APOCI) gene segregates with chromosome 19 and the apo AII (APOA2) gene with chromosome 1. Somatic cell hybrids containing chromosome translocations were used to map the apo AII gene to the 1p21-1qter region. Human APOA2 is polymorphic for the restriction endonuclease Msp I. Comparison of human and mouse chromosome 1 reveals a conserved group including apo AII, renin and peptidase genes and suggests that APOA2 will be found distal to this group on human chromosome 1. The mouse apo AII gene is closely linked with genes that regulate HDL structure. Similar HDL regulatory genes will probably be found near human APOA2.

Characterisation of mRNAs encoding the precursor for human apolipoprotein CI.

cDNA clones encoding human apolipoprotein CI have been isolated from an adult liver cDNA library. Apo CI mRNA was shown to have two species of approximately 580 and 560 bases by RNA blot hybridisation. The intracellular precursor of apo CI was inferred from the cDNA sequence to be an 83 amino acid polypeptide consisting of the 57 residue mature protein and an additional 26 residue amino terminal signal peptide. The 5' untranslated regions of the messages are 63 and 40 bases as determined by primer extension and the 3' untranslated region 111 bases. A polyadenylation signal is situated 10 bases 3' of the poly(A) tall. The mRNA level of apo CI in human liver was significantly greater than that of apo All and apo E.

Current status of the peritoneovenous shunt for the management of intractable ascites.

Renewed interest in the management of intractable ascites has led to the use of a peritoneovenous shunt for its control. Analysis of Canadian experience with this technique in the last 2 years has demonstrated that there are problems associated with it that have not been reported in the surgical literature. A group of 60 patients who underwent peritoneovenous shunting at several Canadian centres was analysed. The operative mortality was 20% but was related to the underlying disease rather than to the operative procedure. Although the initial response to shunting was excellent in 53%, long-term patency (more than 3 months) was achieved in only 43%, but the procedure greatly improved the quality of life in those patients. The indications for shunting, the complications, results and cumulative patency rates are discussed.

Initial experience with the polytetrafluoroethylene graft for limb salvage. A report on twenty patients.

The early results of the bypass procedures for limb salvage using the new polytetrafluoroethylene (PTFE) graft are analyzed. Of twenty patients presenting with either severe rest pain or gangrene, patency has been maintained in fourteen for a mean period of thirteen months to date. Particularly satisfying results have been achieved when bypassing into single dominant arteries below the knee where limb salvage and graft patency was obtained in all cases.