CDKN2A mutations and melanoma risk in the Icelandic population.

BACKGROUND: Germline CDKN2A mutations have been observed in 20-40% of high risk, melanoma prone families; however, little is known about their prevalence in population based series of melanoma cases and controls. METHODS: We resequenced the CDKN2A gene, including the p14ARF variant and promoter regions, in approximately 703 registry ascertained melanoma cases and 691 population based controls from Iceland, a country in which the incidence of melanoma has increased rapidly. RESULTS: We identified a novel germline variant, G89D, that was strongly associated with increased melanoma risk and appeared to be an Icelandic founder mutation. The G89D variant was present in about 2% of Icelandic invasive cutaneous malignant melanoma cases. Relatives of affected G89D carriers were at significantly increased risk of melanoma, head and neck cancers, and pancreatic carcinoma compared to relatives of other melanoma patients. Nineteen other germline variants were identified, but none conferred an unequivocal risk of melanoma. CONCLUSIONS: This population based study of Icelandic melanoma cases and controls showed a frequency of disease related CDKN2A mutant alleles ranging from 0.7% to 1.0%, thus expanding our knowledge about the frequency of CDKN2A mutations in different populations. In contrast to North America and Australia where a broad spectrum of mutations was observed at a similar frequency, in Iceland, functional CDKN2A mutations consist of only one or two different variants. Additional genetic and/or environmental factors are likely critical for explaining the high incidence rates for melanoma in Iceland. This study adds to the geographic regions for which population based estimates of CDKN2A mutation frequencies are available.

Blood pressure and pulse pressure development in a population sample of women with special reference to basal body mass and distribution of body fat and their changes during 24 years.

OBJECTIVE: To study blood pressure and pulse pressure longitudinally and their association with basal and change of body mass index (BMI) and waist to hip ratio (WHR). DESIGN: A prospective population study of 1462 women in Gothenburg, Sweden, aged 38-60 y at baseline, with a longitudinal follow-up of 24 y. OUTCOME MEASURES: Incidence of hypertension, systolic and diastolic blood pressure, and pulse pressure at baseline and after 12 and 24 y of follow-up. RESULTS: Systolic and diastolic blood pressure as well as pulse pressure increased with age and turned down again at high age. BMI and WHR at baseline were each independently associated with baseline systolic and diastolic blood pressure, but only BMI with pulse pressure. However, baseline BMI and WHR were not associated with change of systolic, diastolic or pulse pressure during 12 or 24 y of follow-up. Increase in BMI during the follow-up period was associated with increase in systolic and diastolic blood pressure but not with increase in pulse pressure. There were no such associations with WHR changes which, were either unrelated or in one analysis inversely related with blood pressure changes. When considering incidence of hypertension during the first 12 y of follow-up, BMI and change in BMI were significant predictors, independent of WHR. CONCLUSION: Age, BMI and increments in BMI seem to be strong predictors for hypertension and increased systolic and diastolic blood pressure in women. In contrast, WHR plays a lesser and uncertain role in the development of hypertension in middle-aged women. Changes in BMI seem not to be accompanied by changes in pulse pressure during a long time follow-up.

Allelic frequencies and patterns of single-nucleotide polymorphisms in candidate genes for asthma and atopy in Iceland.

Numerous asthma and atopy loci have been reported in studies demonstrating associations of the asthma-related phenotypes atopy, elevated IgE levels, and bronchial hyperresponsiveness with alleles of microsatellite markers and single-nucleotide polymorphisms (SNPs) within specific cytokine/chemokine and IgE-regulating genes. Although the studies reporting these observations are compelling, most of them lack statistical power. We assessed the nature, pattern, and frequency of SNPs in 24 candidate genes in Iceland and looked for associations with asthma and atopy. We identified 42 SNPs with an average minor allele frequency of 20.3% (asthma) and 20.7% (control). Twenty SNPs (48%) were within coding sequences and 90% of those led to a predicted change in protein sequence. No differences were detected in the allelic frequencies of SNPs in any of these candidate genes between control subjects and the patients with atopic asthma. Moreover, linkage analysis that included 269 patients with atopic asthma uncovered no evidence of linkage to markers associated with these genes. We conclude that this study has failed to produce evidence in support of the notion that variations within these 24 candidate atopy and asthma genes significantly influence the expression of the atopic asthmatic phenotype or contribute to the susceptibility of atopic asthma.

Microproteinuria and long-term prognosis with respect to renal function and survival in normotensive and hypertensive women--a 24-year follow-up of a representative population sample of women in Gothenburg, Sweden.

OBJECTIVE: This study aimed to assess albuminuria and subclinical proteinuria, their association with hypertension and their role as predictors of hypertension, impaired renal function and mortality. MATERIAL AND METHODS: A baseline population study comprising 1462 women in five different age groups in Gothenburg, Sweden, was carried out in 1968-69. Comprehensive clinical examinations and laboratory tests were performed, including blood pressure measurement and an Albustix test. A systematic subsample of women additionally collected a 24 h urine sample for quantitative protein analysis. Values of urinary protein (u-protein) excretion between 80 and 300 mg/24 h were defined as microproteinuria. The results described in this paper are based on a 24-year follow-up. RESULTS: The baseline Albustix test was positive in 6.8% of 1458 women, from whom a urine sample was obtained. Of 741 baseline urine collections for u-protein excretion, 16.9% were in the microproteinuric range (80-300 mg/24 h), 1.1% in the macroproteinuric range (> 300 mg/24 h) and 82.1% in the normoproteinuric range (< 80 mg/24 h). Hypertension was more common in Albustix-positive women than in those with negative Albustix, and hypertension was also more prevalent in women with microproteinuria than in women with normoproteinuria. Neither positive Albustix nor microproteinuria was related to later renal impairment. Hypertension was associated with increased mortality in both Albustix-positive and Albustix-negative women, and in women with both normoproteinuria and microproteinuria at baseline. The mortality ratio during the follow-up period was, however, not significantly influenced by positive Albustix or by microproteinuria at baseline, in either hypertensive or non-hypertensive women. CONCLUSIONS: This study demonstrated that both a positive Albustix test and microproteinuria were associated with hypertension. Hypertension at baseline increased the risk for death during the follow-up period, while neither albuminuria, defined as a positive Albustix test, nor microproteinuria was associated with an impaired long-term prognosis with respect to renal function or survival in this cohort of Swedish middle-aged women during 24 years of follow-up. Microproteinuria in otherwise healthy normotensive or hypertensive women does not appear to impair the long-term prognosis.

Protection of privacy by third-party encryption in genetic research in Iceland.

As the new human genetics continues its dramatic expansion into many laboratories and medical institutions, the concern for the protection of the personal privacy of individuals who participate increases. It seems that even the smallest of laboratories must confront the issue of how to protect the genetic and phenotypic information of participants in their research. Some have promoted the use of anonymity as a way out of this dilemma. But we are reminded by others that the future cannot be predicted, and that future benefits may be lost when the links to these benevolent volunteers are gone forever. More recently, some ethical bodies have suggested, without specific recommendations, that a reversible third-party encryption system may be a solution to this problem. However, they have not provided a route or even examples of how to proceed. We present here the Icelandic approach to this issue by developing a third-party encryption system in direct collaboration with the Data Protection Commission (DPC) of Iceland. We have incorporated the encryption system within our sample collection and storage software, which minimises inconvenience but enhances security. The strategy assures a barrier between the laboratory and the outside world that can only be crossed by the DPC.

[Complete androgen insensitivity in an Icelandic family caused by mutation in the steroid binding region of the androgen receptor.].

INTRODUCTION: Androgen insensitivity syndrome (AIS) is a X-linked rescessive disorder characterized by impairment of the androgen-dependant male sexual differentiation. The cause of AIS is in most cases a mutation in the gene of the androgen receptor on the X chromosome. In this study we describe an Icelandic family with two girls with AIS. A search for mutations in the androgen receptor gene was performed in order to identify the genetical and molecular basis for AIS in this family. MATERIAL AND METHODS: Genomic DNA was isolated from two girls with complete AIS and their close relatives. PCR was used to amplify all eight exons of the androgen receptor gene of the two AIS girls and SSCP used to screen for mutations. DNA fragments showing abnormal SSCP pattern were subjected to nucleotide sequencing. PCR based diagnostic method was developed and used to detect the mutation causing AIS in the family. RESULTS AND CONCLUSIONS: Using SSCP and DNA sequencing a CGA to CAA missense mutation in exon 5 at codon 752 was identified. The mutation causes in an Arg to Gln amino acid substitution (R752Q mutation) in the ligand binding domain of the androgen receptor and a complete androgen insensitivity. Members of the family were genotyped using a PCR based method for identification of the mutant allele. The results strongly indicated a de novo mutation in a germ cell of the maternal grandmother, as the mutation was not found in her blood leucocytes. The diagnostic test provided a basis for genetic counselling for the family.

Mapping of a familial essential tremor gene, FET1, to chromosome 3q13.

Essential tremor (ET), the most common movement disorder in humans, appears to be inherited as an autosomal dominant trait in many families. The familial form is called familial essential tremor (FET), which seems similar to sporadic essential tremor. ET is a cause of substantial disability, particularly in the elderly. The prevalence of Parkinson's disease and dystonia may be increased in families with ET, but other movement disorders are seldom encountered in these families. Here we report the results of a genome-wide scan for FET genes in 16 Icelandic families with 75 affected individuals, in whom FET was apparently inherited as a dominant trait. The scan, which was performed with a 10-cM framework map, revealed one locus on chromosome 3q13 to which FET mapped with a genome-wide significance when the data were analysed either parametrically, assuming an autosomal dominant model (lod score = 3.71), or non-parametrically (NPL Z score = 4.70, p < 6.4 x 10(-6).

Association of BsmI vitamin-D receptor gene polymorphism with combined bone mass in spine and proximal femur in Icelandic women.

OBJECTIVE: To evaluate whether there is an association between BsmI-vitamin-D receptor (VDR) gene polymorphism and combined bone mass in the spine and proximal femur in a group of adult Icelandic women with high and low bone mineral density (BMD). DESIGN: Comparison of distribution of VDR genotypes (BB, Bb and bb) and allele frequency (B and b) in two groups of women: a group with 'strong bones' with high BMD in both the spine and proximal femur (> 1 standard deviation [SD]) above the age-matched mean (n = 35) and a group with 'weak bones' with BMD > 1.5 SD below the age-matched mean at both sites using dual energy X-ray absorptiometry. SETTING: Iceland, a population with a mean calcium intake > 1000 mg day-1. The calcium intake in the study group was however not evaluated. SUBJECTS: Eighty-three Icelandic women, aged 22-65, free of diseases affecting bone and not taking drugs affecting calcium or bone metabolism, recruited from women undergoing bone densitometry at the Reykjavik Hospital. MAIN OUTCOME MEASURES: Frequency of VDR genotypes and alleles in the two groups. RESULTS: The distribution of VDR genotypes was significantly different in the two groups (P < 0.01); the b allele frequency was 70% in the group with high BMD compared to 48.5% in the group with low BMD. CONCLUSIONS: In this selected group of adult Icelandic women the b allele in the vitamin-D receptor gene seems to be associated with high bone mass in the spine and proximal femur.

Cloning, genetic mapping, and expression analysis of a mouse renal sodium-dependent phosphate cotransporter.

Renal tubular reabsorption of phosphate is critical to the maintenance of phosphate homeostasis in mammals, and the brush-border membrane Na-P(i) cotransport systems in proximal tubules play a major role in this process. We have isolated a cDNA encoding a mouse sodium-dependent phosphate transport protein (Npt1), which is expressed primarily in the kidney. This protein is highly similar to its human and rabbit homologues, based on nucleotide and amino acid comparisons. The presence of potential Asn-linked glycosylation and protein kinase C phosphorylation sites that are conserved among all three homologues suggests that these sites may be important in the function and regulation of this protein. The Npt1 gene was mapped to mouse chromosome 13, close to the Tcrg locus. By both in situ hybridization and reverse transcription-polymerase chain reaction, Npt1 mRNA was localized predominantly to the proximal tubule.

[Falling by the wayside? On priorities in the allocation of health-care resources.].

This essay serves a twofold purpose. The first is to offer a brief overview of recent discussions of what constitutes a just model of rationing in health care, i.e. how the problem of scarce health resources is to be correctly analysed and solved. Moreover, the relevance of general philosophical theories of justice to this specific problem is given some consideration. A whole gamut of already-suggested models is examined, with special emphasis on the weaknesses of each particular proposal. The second goal of the essay is to evoke serious doubts about the viability of some of the most prominent and popular models of just rationing; the main target being the ageism inherent in them. Another model is suggested and defended: a utilitarian one, which puts the whole issue of resource allocation in a wider perspective, taking into account, inter alia, the extent to which patients deserve the service they require.

Can social bees be influenced to choose a specific feeding station by adding the scent of the station to the hive air?

The behavioral response of honeybees (Apis mellifera L.) and bumblebees (Bombus terrestris L.) to the flower volatiles 2-ethyl-1-hexanol and myrcene isolated in situ from white clover (Trifolium repens L.) and oil seed rape (Brassica napus oleifera), respectively, were investigated on a rotating arena with 12 visually identical, but differently scented, feeding stations. When locating a feeding station, neutral in both shape and color, foragers used scent as orientation cue. Introduction of 2-ethyl-1-hexanol to the honeybee hives induced significantly more visits to sites containing this compound. In contrast, introduction of myrcene to the hives did not influence the foraging choices of honeybees significantly. No effect of hive scent composition on the choices made by bumblebees could be detected. "Experienced" bumble bees, i.e., bees with more than five visits to the feeding stations, tended to visit a particular position on the arena without discriminating between the two volatiles. In contrast, honeybees showed no positioning behavior on the arena, using primarily odoriferous stimuli. The observed influences of addition of scents to the hives are discussed in relation to the general knowledge on foraging behavior of social bees and the emission of volatiles from leaves and flowers.

Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystallographic structure of the glucocorticoid and oestrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations. DESIGN: Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and functional responses to this hormone. These cells were also used to isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patients was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)2D3. Computer analysis of mutations in the VDR gene was carried out using the glucocorticoid and oestrogen receptors as model systems. PATIENTS: Two patients with HVDRR from unrelated families. MEASUREMENTS: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in control and affected patients, and functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)2D3 following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS: Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of cDNA revealed single mutations, in patient I (Phe44-->IIe) and in patient II (Lys42-->Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR. CONCLUSIONS: Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.

Myophosphorylase deficiency: an unusually severe form with myoglobinuria.

Myophosphorylase deficiency (McArdle disease) is characterized by exercise intolerance that usually starts in childhood. Severe cramps and myoglobinuria are rarely problems in children. We describe an 8-year-old boy with exercise-induced myoglobinuria; he was homozygous for the mutation most commonly encountered in patients with typical McArdle disease.

Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification.

Due to the limited amount of DNA in a single diploid cell, preimplantation genetic diagnosis has relied on single- or dual-locus analyses in biopsied blastomers. We have applied single-cell whole-genome preamplification to PCR-based analysis of multiple disease loci from the same diploid cell. This method allows diagnosis of multiple disease genes, analysis of multiple exons/introns within a gene, or corroborative embryo-sex assignment and specific mutation detection at sex-linked loci. A blinded study of six genetic loci was performed with whole-genome preamplification followed by nested PCR. Amplification was observed in 103 of 105 assays (98%) and a correct diagnosis was made in 98%. All human blastomeres were correctly diagnosed (100%) at loci where the genotype could be confirmed, attesting to the reliability of the technique. Preamplification has now been applied successfully to the analysis of the two major mutations responsible for Tay-Sachs disease and of a common restriction polymorphism in the gene responsible for hemophilia A. The fidelity and length of product derived from this preamplification step make it an appealing technique for preimplantation genetic diagnoses requiring analyses at more than one locus.

Niemann-Pick-like liver disease and reduced cholesterol esterification in fibroblasts of two male infants.

Niemann-Pick disease type C (NPC) presents in about half of the cases in the newborn period with jaundice, hepato-splenomegaly, and a clinical pattern similar to neonatal hepatitis. The definitive diagnosis can in most instances be made by the appropriate biochemical testing of lipoprotein stimulated cholesteryl ester synthesis and cholesterol accumulation in cultured patient fibroblasts. We report two infants who by liver biopsy had classical findings of NPC and a cholesteryl ester synthesis level about 50% of the normal lower limit. On the other hand neither of these patients' fibroblasts showed any evidence of low density lipoprotein-induced cholesterol accumulation, precluding the possibility of a definitive diagnosis. These cases demonstrate the importance of the appropriate biochemical testing before final counseling is carried out. The possibility of our patients representing allelic or non-allelic variants of NPC are discussed.

Molecular analysis of human platelet antigen system 1 antigen on single cells can be applied to preimplantation genetic diagnosis for prevention of alloimmune thrombocytopenia.

OBJECTIVE: Our purpose was to develop a molecular assay to determine the human platelet antigen system 1 status on single nucleated cells, including human blastomeres. STUDY DESIGN: Eighty single cultured lymphoblasts of known human platelet antigen system 1 genotype and 24 media blanks were mixed in blinded fashion. Amplification of a 246 bp deoxyribonucleic acid fragment and subsequent Nci I restriction digestion were performed to distinguish human platelet antigen system 1a from 1b alleles. Specificity and sensitivity of the technique were determined. Eight blastomeres were also tested. RESULTS: Deoxyribonucleic acid amplification at the human platelet antigen system 1 locus was successful in 95% of the reactions. No media blanks showed amplified deoxyribonucleic acid. The diagnosis was correct in all homozygous human platelet antigen system 1a or 1b cells; three of 23 heterozygous cells amplified but failed to digest with Nci I. Overall specificity was 95%. All blastomeres successfully amplified. CONCLUSIONS: The human platelet antigen system 1 status determination is reliable from a single cell and can be used for preimplantation genetic diagnosis for the prevention of alloimmune thrombocytopenia.

Preimplantation single cell analyses of dystrophin gene deletions using whole genome amplification.

Primer extension preamplification (PEP) increases the scope and capacity of single cell genetic diagnosis by generating sufficient template to perform multiple subsequent DNA analyses using the polymerase chain reaction. We report the simultaneous analysis of single cells at five commonly deleted dystrophin exons and at the ZFX/ZFY loci. Ninety three percent of PEP reactions with single amniocytes, chorionic villus cells and blastomeres were successful, and a blinded analysis of single lymphoblasts from affected males resulted in 93% diagnostic accuracy, demonstrating its applicability in preimplantation prevention of Duchenne muscular dystrophy. Transfer of unaffected male embryos and improved diagnostic reliability are achieved with the ability to perform replicate multilocus analyses from the same blastomere.

Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) is characterized by severe rickets and is often accompanied by alopecia. Mutations in the gene encoding the vitamin D receptor have been found in this condition. In a patient with the characteristic phenotype we have investigated the functional defect and sequenced the gene to seek a mutation. DESIGN: Patient and control cell lines prepared from skin fibroblasts and peripheral blood lymphocytes were used to measure binding of 1,25(OH)2D3 and to isolate vitamin D receptor mRNA. VDR cDNA was sequenced and transfected into receptor defective cells. PATIENT: A child with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in patient and control cells, and functional response to 1,25(OH)2D3 assessed by measurement of 24-hydroxylase activity. VDR mRNA was prepared, reverse transcribed, and cDNA sequenced. VDR cDNA was also transfected into VDR-deficient CV-1 cells and functional response to 1,25(OH)2D3 assessed by co-transfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS: VDR from the patient were able to bind 1,25(OH)2D3 but showed no nuclear localization resulting in an absence of functional response to 1,25(OH)2D3. Sequencing revealed that the VDR coding region was normal. Expression studies of the patient's VDR showed functionally normal VDR as evidenced by normal transactivation in the presence of 1,25(OH)2D3. CONCLUSION: These data indicate a new cause of tissue resistance to 1,25(OH)2D3 which occurs in the absence of mutations in the coding region of VDR gene and which is characterized by defective nuclear localization of this receptor.

Molecular cloning of the cDNA encoding a human renal sodium phosphate transport protein and its assignment to chromosome 6p21.3-p23.

Resorption of phosphate by the kidney is an important function in the maintenance of phosphate homeostasis in mammals, and a defect in renal phosphate uptake has been implicated in at least three human genetic disorders. We have isolated a cDNA encoding a human sodium-dependent phosphate transport protein (NPT1). This cDNA hybridizes to a single 2.5-kb RNA transcript from human kidney cortex, its nucleotide sequence shows 80.3% identity to the rabbit NaPi-1 sequence, and it encodes a polypeptide of 467 amino acids. Amino acid sequence comparisons indicate a 69.7% identity between human NPT1 and rabbit NaPi-1 polypeptides; the inclusion of conservative substitutions increases the homology between the two proteins to 81.5%. Alignment of both sequences also reveals several conserved potential N-glycosylation and protein kinase C phosphorylation sites. Polypeptide hydropathy analysis predicts several membrane-spanning domains. This cDNA maps the location of the gene encoding NPT1 to human chromosome 6q21.3-p23.