Novel inactivating mutation of the calcium-sensing receptor in a young woman with mild hypercalcaemia.

A young woman with mild hypercalcaemia and an inappropriately normal serum parathyroid hormone had parathyroid scintigraphy suggestive of an active ectopic parathyroid tissue in the superior mediastinum. Urinary calcium to creatinine clearance ratio was low, and a subsequent genetic analysis confirmed a novel mutation (Q164K) in the calcium sensing receptor gene, consistent with familial hypocalciuric hypercalcaemia. We propose that this mutation accounts for her clinical and investigational findings, although a double pathology of Q164K and an ectopic parathyroid adenoma is also conceivable.

DNA hypermethylation/boundary control loss identified in retinoblastomas associated with genetic and epigenetic inactivation of the RB1 gene promoter.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB-/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr-/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr-/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB-/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr-/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr-/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.

Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilms' tumor cells.

Children with associated Wilms' tumor, aniridia, genitourinary malformations, and mental retardation (WAGR syndrome) frequently have a cytogenetically visible germ line deletion of chromosomal band 11p13. In accordance with the Knudson hypothesis of two-hit carcinogenesis, the absence of this chromosomal band suggests that loss of both alleles of a gene at 11p13 causes Wilms' tumor. Consistent with this model, chromosomes from sporadically occurring Wilms' tumor cells frequently show loss of allelic heterozygosity at polymorphic 11p15 loci, and therefore it has been assumed that allelic loss extends proximally to include 11p13. We report here that in samples from five sporadic Wilms' tumors, allelic loss occurred distal to the WAGR locus on 11p13. In cells from one tumor, mitotic recombination occurred distal to the gamma-globin gene on 11p15.5. Thus, allelic loss in sporadic Wilms' tumor cells may involve a second locus on 11p.

The characterization of ovine genes for atrial, brain, and C-type natriuretic peptides.

The natriuretic peptides (NPs) play an important role in the homeostasis of blood pressure and sodium balance in all mammals studied to date. Their combined actions on the vasculature, kidneys, and adrenals reduce blood pressure and intravascular volume. In order to provide sequence information about the ovine NP genes for our physiological studies in sheep, we have determined the genomic DNA sequence of each of the NPs; atrial NP, brain NP (BNP), and C-type NP using an ovine genomic library. Strong homology with other species was found for ovine peptide and genomic sequences of atrial NP and for C-type NP. Further, despite previous reports of poor conservation of BNP across species, the peptide sequence for ovine BNP was found to be identical to both the 26 amino acid-residue porcine BNP, and the 35 amino acid peptide known as bovine aldosterone secretion-inhibitory factor. This data also revealed strong homology of BNP mature forms in dog, cow, pig, and sheep, thus permitting the use of porcine antisera to study BNP-level changes in sheep models of cardiac failure. This conservation of the BNP gene sequence suggests that BNP, like the other NPs, plays an important role in mammalian physiology.

Effect of nonsense mutations on PTEN mRNA stability.

Cowden disease is an autosomal dominant disorder associated with an elevated risk of breast, thyroid and skin cancers, in which germline mutations of a tumour suppressor gene (PTEN) have been identified. PTEN has a dual-specificity tyrosine phosphatase domain thought to be essential for tumour suppression. Previous genotype/phenotype correlations have identified several potential associations, for example that truncating mutations result in increased breast cancer risk. Such associations are useful in evaluating the phenotypic functions of PTEN sub-domains. However, genotype/phenotype correlations are likely to be complicated by nonsense mediated mRNA degradation. We report here that three out of four mutations do not significantly affect PTEN transcript stability. Furthermore, we show that manual sequencing methods are better than current dye-based sequencing technologies for detecting heterozygous mutations in PTEN transcripts.

Structural analysis of the human insulin-like growth factor-II P3 promoter.

The expression of insulin-like growth factor-II (IGF-II) has been observed previously in many human cancers. The human IGF-II P3 promoter has been shown by others to give rise to abundant 6.0 kb and 2.2 kb fetal transcripts which are expressed in a variety of both paediatric and adult tumours. In order to determine the mechanism by which the P3 promoter is controlled, the promoter was analysed in cell lines using chloramphenicol acetyltransferase (CAT) assay and DNAase I footprinting techniques. The data indicated that P3 is a complex promoter involving at least nine transcription factor binding sites. Furthermore, high levels of 5-methylcytosine detected in the P3 promoter of HeLa genomic DNA suggest that IGF-II gene expression may also be influenced by DNA methylation.

A bisulfite method of 5-methylcytosine mapping that minimizes template degradation.

The bisulfite method is a highly sensitive approach to 5-methylcytosine mapping that utilizes the capability of the polymerase chain reaction to exponentially amplify DNA. We have observed that the bisulfite reaction results in a significant level of template degradation due to DNA depurination. Furthermore, our data suggest that the DNA fragmentation which occurs limits the sensitivity of the method. We describe a simple solution to limit degradation of the DNA template.

A mitotic recombination in Wilms tumor occurs between the parathyroid hormone locus and 11p13.

Wilms tumor is believed to occur as the result of two mutations affecting both alleles of a critical gene located within the p13 band of chromosome 11 (Knudson and Strong 1972; Riccardi et al. 1978). Several mechanisms by which these mutations occur have already been determined in retinoblastoma (Cavenee et al. 1983) and Wilms tumor (Koufos et al. 1984; Orkin et al. 1984; Reeve et al. 1984; Fearon et al. 1984a; Eccles et al. 1984). Of the various mechanisms, however, no example of a mitotic recombination was demonstrated in Wilms tumor. An example is presented here which has been detected by the use of restriction fragment length polymorphisms (RFLPs) mapping to chromosome 11p. In addition the data presented are consistent with the mapping location of parathyroid hormone (PTH) being proximal to 11p13.

Identification of a novel PTEN mutation (L139X) in a patient with Cowden disease and Sjögren's syndrome.

Cowden disease is an autosomal dominant disorder associated with an increased risk of breast, thyroid, and skin cancer in which germline mutations in a candidate tumour suppressor gene (PTEN) have been identified previously. Sjögren's syndrome is a chronic inflammatory and autoimmune disorder of exocrine glands for which the genetic basis is unknown. This report describes a novel PTEN mutation (L139X) in a patient with Cowden disease and Sjögren's syndrome. This observation raises the possibility of a link between mutations in the PTEN gene and Sjögren's syndrome.