International variation in physicians' attitudes towards prophylactic mastectomy - comparison between France, Germany, the Netherlands and the United Kingdom.

PURPOSE: Prophylactic mastectomy (PM) has proven to be the most effective method to reduce the risk of breast cancer in high-risk women. The present study aimed to present and compare the attitudes towards PM among physicians in France, Germany, the Netherlands and the United Kingdom (UK). PATIENTS AND METHODS: An international sample of 1196 general practitioners (GPs) and 927 breast surgeons (BS) were surveyed using a mailed questionnaire. RESULTS: Only 30% of the French and 27% of the German GPs were of opinion that PM should be an option for an unaffected female BRCA1/2 mutation carrier, as compared to 85% and 92% of the GPs in the Netherlands and UK, respectively. Similarly, 78% of the French and 66% of the German BS reported a positive attitude towards PM, as compared to 100% and 97% of the BS in the Netherlands and UK, respectively. In the whole sample of GPs, a positive attitude towards PM was associated with country of residence, being female, and having more knowledge of breast/ovarian cancer genetics, while among BS there was a positive association with country of residence and having more knowledge of breast/ovarian cancer genetics as well, and, in addition, with a higher number of newly diagnosed breast cancer patients last year. CONCLUSION: These results demonstrated the international variations in the attitude towards PM among physicians. This might reflect that different policies are adopted to prevent breast cancer in women at-risk.

Evaluation of Clinical Manifestations in Patients with Severe Lymphedema with and without CCBE1 Mutations.

The lymphedema-lymphangiectasia-intellectual disability (Hennekam) syndrome (HS) is characterised by a widespread congenital lymph vessel dysplasia manifesting as congenital lymphedema of the limbs and intestinal lymphangiectasia, accompanied by unusual facial morphology, variable intellectual disabilities and infrequently malformations. The syndrome is heterogeneous as mutations in the gene CCBE1 have been found responsible for the syndrome in only a subset of patients. We investigated whether it would be possible to predict the presence of a CCBE1 mutation based on phenotype by collecting clinical data of patients diagnosed with HS, with or without a CCBE1 mutation. We report here the results of 13 CCBE1 positive patients, 16 CCBE1 negative patients, who were clinically found to have classical HS, and 8 patients in whom the diagnosis was considered possible, but not certain, and in whom no CCBE1 mutation was identified. We found no statistically significant phenotypic differences between the 2 groups with the clinical HS phenotype, although the degree of lymphatic dysplasia tended to be more pronounced in the mutation positive group. We also screened 158 patients with less widespread and less pronounced forms of lymphatic dysplasia for CCBE1 mutations, and no mutation was detected in this group. Our results suggest that (1) CCBE1 mutations are present only in patients with a likely clinical diagnosis of HS, and not in patients with less marked forms of lymphatic dysplasia, and (2) that there are no major phenotypic differences between HS patients with or without CCBE1 mutations.

A simple clinical model to estimate the probability of Marfan syndrome.

BACKGROUND: Marfan syndrome is a heritable connective tissue disease. Definitive diagnosis is complex, and requires sequencing of a large gene, FBN1. AIM: We aimed to develop a simple model to estimate the pre-test probability of Marfan syndrome. DESIGN: Prospective cross-sectional study. METHODS: We applied diagnostic standards for definitive diagnosis or exclusion of Marfan syndrome in 329 consecutive persons. In 208 persons with random assignment to our derivation group, we performed multivariate logistic regression to assess 14 clinical variables for inclusion in a prediction model with derivation of score points from the estimated coefficients. We created cut-offs to classify low, moderate and high probability of Marfan syndrome. For validation, we applied the model to the remaining 121 persons. RESULTS: We identified seven variables for inclusion in the final model, where we assigned four score points to ectopia lentis, two points to a family history of Marfan syndrome, and one point to previous thoracic aortic surgery, to pectus excavatum, to a wrist and thumb sign, to previous pneumothorax, and to skin striae. In the derivation group 12, 42 and 92% of persons with low (≤1 point), moderate (>1-3.5 points) or high pre-test probability (>3.5 points) had Marfan syndrome, compared to 12, 57 and 91%, respectively, in the validation group. Positive likelihood ratios were 13.96 and 8.54 in the high probability group of the derivation and validation group, respectively. CONCLUSION: A simple prediction model provides evidence for Marfan syndrome. This model can be used to identify patients who require definitive diagnostic work-up.

Familial breast cancer: is it time to move from a reactive to a proactive role?

In 2004 the NICE guidelines on familial breast cancer advised Health Care Professionals that they should not actively seek to identify women with a family history of breast cancer. We have carried out a review of the evidence base and a large scale questionnaire survey of health professionals in four European countries. There is overwhelming support amongst GPs and surgeons against the premise that that health care professionals should not be proactive in identifying patients at risk of familial breast cancer. This that suggest the time is right to overturn the NICE decision.

Search for deletion 22q11.2 in interphase nuclei of buccal mucosa of patients ascertained by isolated cleft palate: a new diagnostic approach.

A new approach for the detection of chromosome deletion 22q11.2 in interphase nuclei from buccal mucosa cells obtained by a non-invasive procedure is described. FISH analysis has been performed on samples from a group of 101 patients that presented consecutively for speech therapy and/or surgical correction of cleft palate. A normal result has been obtained in 98 patients; a deletion 22q11.2 was present in three patients (2.8%) with cleft palate.

Estimating the efficacy and efficiency of cascade genetic screening.

Screening for genetic variants that predispose individuals or their offspring to disease may be performed at the general population level or may instead be targeted at the relatives of previously identified carriers. The latter strategy has come to be known as "cascade genetic screening." Since the carrier risk of close relatives of known carriers is generally higher than the population risk, cascade screening is more efficient than population screening, in the sense that fewer individuals have to be genotyped per detected carrier. The efficacy of cascade screening, as measured by the overall proportion of carriers detected in a given population, is, however, lower than that of population-wide screening, and the respective inclusion rates vary according to the population frequency and mode of inheritance of the predisposing variants. For dominant mutations, we have developed equations that allow the inclusion rates of cascade screening to be calculated in an iterative fashion, depending upon screening depth and penetrance. For recessive mutations, we derived only equations for the screening of siblings and the children of patients. Owing to their mathematical complexity, it was necessary to study more extended screening strategies by simulation. Cascade screening turned out to result in low inclusion rates (<1%) when aimed at the identification of heterozygous carriers of rare recessive variants. Considerably higher rates are achievable, however, when screening is performed to detect covert homozygotes for frequent recessive mutations with reduced penetrance. This situation is exemplified by hereditary hemochromatosis, for which up to 40% of at-risk individuals may be identifiable through screening of first- to third-degree relatives of overt carriers (i.e., patients); the efficiency of this screening strategy was found to be approximately 50 times higher than that of population-wide screening. For dominant mutations, inclusion rates of cascade screening were estimated to be higher than for recessive variants. Thus, some 80% of all carriers of the factor V Leiden mutation would be detected if screening were to be targeted specifically at first- to third-degree relatives of patients with venous thrombosis. The relative cost efficiency of cascade as compared with population-wide screening (i.e., the overall savings in the extra managerial cost of the condition) is also likely to be higher for dominant than for recessive mutations. This notwithstanding, once screening has become cost-effective at the population level, it can be expected that cascade screening would only transiently represent an economically viable option.

Familial thrombocytosis as a recessive, possibly X-linked trait in an Arab family.

Familial thrombocytosis (FT) has previously been described as an autosomal-dominant disorder with manifestations similar to those of sporadic essential thrombocythaemia. We studied an Arab family consisting of four brothers, aged 4-8 years, who had either sustained markedly elevated (> 1000 x 109/l) or moderately elevated (> 500 x 109/l) platelet counts, two healthy sisters and their parents who had normal platelet counts. The four brothers with FT had normal plasma thrombopoietin levels and are currently not presenting with any thrombotic or haemorrhagic complications. Mutation analysis at the thrombopoietin gene (THPO) of the affected family members failed to detect the intron 3 G-->C splice mutation that had been described as causing FT. In addition, segregation analysis using a polymorphic CA marker revealed completely discordant THPO alleles among the affected brothers. We postulate the existence of a new locus for FT whereby the disease is transmitted as a recessive, possibly X-linked trait.

Genotypic and phenotypic spectrum in tricho-rhino-phalangeal syndrome types I and III.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities. Three subtypes have been described: TRPS I, caused by mutations in the TRPS1 gene on chromosome 8; TRPS II, a microdeletion syndrome affecting the TRPS1 and EXT1 genes; and TRPS III, a form with severe brachydactyly, due to short metacarpals, and severe short stature, but without exostoses. To investigate whether TRPS III is caused by TRPS1 mutations and to establish a genotype-phenotype correlation in TRPS, we performed extensive mutation analysis and evaluated the height and degree of brachydactyly in patients with TRPS I or TRPS III. We found 35 different mutations in 44 of 51 unrelated patients. The detection rate (86%) indicates that TRPS1 is the major locus for TRPS I and TRPS III. We did not find any mutation in the parents of sporadic patients or in apparently healthy relatives of familial patients, indicating complete penetrance of TRPS1 mutations. Evaluation of skeletal abnormalities of patients with TRPS1 mutations revealed a wide clinical spectrum. The phenotype was variable in unrelated, age- and sex-matched patients with identical mutations, as well as in families. Four of the five missense mutations alter the GATA DNA-binding zinc finger, and six of the seven unrelated patients with these mutations may be classified as having TRPS III. Our data indicate that TRPS III is at the severe end of the TRPS spectrum and that it is most often caused by a specific class of mutations in the TRPS1 gene.

Immortalization of murine male germ cells at a discrete stage of differentiation by a novel directed promoter-based selection strategy.

We developed a novel promoter-based selection strategy that could be used to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.

Renal polyamine excretion, tubular amino acid reabsorption and molecular genetics in cystinuria.

Cystinuria is an autosomal recessive disorder of the tubular and intestinal resorption of cystine, ornithine. lysine and arginine leading to nephrolithiasis. Three cystinuria types can be distinguished by the mode of inheritance (true recessive or intermediate) and by the pattern of the intestinal amino acid transport. In the present study phenotypes were assessed by the urinary excretion of amino acids related to creatinine, the percentage tubular amino acid reabsorption and the urinary excretion of polyamines as a possible indicator of the intestinal transport defect. However, our thorough phenotyping did not reveal more than two cystinuria types. Genotypes were examined in linkage analyses and single-strand conformation polymorphism-based mutation identification. The SLC3A1 mutations M467T and T216M were disease causing in our homozygous patients of type I cystinuria. We can show the association of type I cystinuria with SLC3A1 and of non-type I cystinuria with a yet unidentified gene on chromosome 19q13.1. Our phenotype and genotype analyses provide evidence for only two types of cystinuria in the investigated patient cohort.

Mutation screening for prenatal and presymptomatic diagnosis: cystic fibrosis and haemochromatosis.

UNLABELLED: Hereditary haemochromatosis (HH) and cystic fibrosis (CF) are the most common autosomal recessively inherited disorders in Caucasian populations. In its typical form, CF manifests during the first years of life, while the mean age of onset of organ damage is 54 years in HH. Both disorders can be diagnosed presymptomatically utilising biochemical and/or genetic testing. Since approximately 90% of mid-European HH patients are homozygous for only one specific mutation (C282Y) in the candidate gene for HH, genetic testing is simple and sensitive in HH. In CF, molecular testing is currently hampered by the large number (more than 800) of mutations in the CF transmembrane conductance regulator gene. Several studies have been initiated to investigate the potential benefits and the best time and mode of presymptomatic testing for HH and CF. CONCLUSION: Mutation analysis is widely recommended for presymptomatic diagnosis of cystic fibrosis and hereditary haemochromatosis because of the presumed benefit, although several medical, ethical, social and technical questions warrant further investigations. Prenatal mutation testing is commonly performed for cystic fibrosis but not for hereditary haemochromatosis. Informed consent of tested individuals and the availability of genetic counselling is a prerequisite for any mutation screening approach in either disease.

Identification of 9 novel FBN1 mutations in German patients with Marfan syndrome.

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium-binding (cb) epidermal growth factor-like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo.

Mutation analysis in 46 German families with familial hypercholesterolemia: identification of 8 new mutations. Mutations in brief no. 226. Online.

In order to obtain a survey of the mutations being prevalent in Northern Germany and to enable molecular genetic testing for families with clinically diagnosed familial hypercholesterolemia (FH), we screened 46 unrelated German individuals with elevated LDL levels for mutations in the 18 exons and their flanking intron sequences including the promotor region of the LDL receptor (LDLR) gene. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B-100 (apoB-100). We detected 15 mutations affecting the LDLR gene, 8 of which, designated A29S, 195insAT, 313+1insG, 553insG, 680insGGACAAATCTG, D200N, E267K and L411V have not yet been reported. One patient is heterozygous for the double mutant N543H and 2393del9Bp. Two patients carried the mutation R3500Q (Arg-->Glu) within the apoB-100 gene.

The potential benefits of genetic testing in breast and ovarian cancer.

Full text is available as a scanned copy of the original print version.

Prenatal diagnosis of congenital alveolar proteinosis (surfactant protein B deficiency).

We report on the DNA-based prenatal diagnosis of congenital pulmonary alveolar proteinosis in a family in which alveolar proteinosis was associated with surfactant protein B (SP-B) deficiency. The parents had lost an eight-week-old female child due to this fatal disorder. The affected child was homozygous and both parents were heterozygous for a frame-shift mutation in codon 121 of the surfactant protein B gene (SFTP3-gene). Chorionic villus sampling (CVS) was performed in two subsequent pregnancies. DNA analysis revealed homozygosity for the codon 121 mutation in the first fetus, and the pregnancy was terminated. Homozygosity for the parental wild-type alleles was detected in the following prenatal diagnosis, and a healthy child has been born. DNA-based prenatal diagnosis of congenital alveolar proteinosis is simple, fast and reliable, and can be performed much earlier in pregnancy than any other method, e.g. the direct measurement of SP-B in amniotic fluid. In families with a term infant who dies of unexplained respiratory failure, genetic testing of the parents should be evaluated, since the presence of the codon 121 mutation enables prenatal diagnosis in later pregnancies.

Structure and function of TSPY, the Y-chromosome gene coding for the "testis-specific protein".

The TSPY genes are arranged in clusters on the Y chromosome of many mammalian species. TSPY is believed to function in early spermatogenesis and is a candidate for GBY, the putative gonadoblastoma-inducing gene on the Y. The TSPY family forms part of a superfamily, TTSN, with autosomal representatives, highly conserved in mammals and beyond.

A new missense substitution at a mutational hot spot of the androgen receptor in siblings with complete androgen insensitivity syndrome.

Several mutations have been described in the human androgen receptor gene including constitutional mutations in androgen insensitivity syndrome, somatic mutations in prostate cancer and triplet expansions in Kennedy's disease (Gottlieb et al. 1997). Here we report on two siblings with complete androgen insensitivity and a novel missense mutation, D695V, in their androgen receptor gene. The two XY females are siblings of German descent and presented at the ages of 23 and 19 years, respectively, with typical clinical features of complete androgen insensitivity. We found both siblings to be hemizygous for a new adenine to thymine transversion at the second nucleotide of codon 695 within the fourth exon of the human androgen receptor gene. The resulting missense mutation D695V is located at the amino-terminal border of the ligand-binding domain of the androgen receptor. The aspartic acid residue at this position is highly conserved in the steroid binding domains of other members of the nuclear receptor family and has already been found to be the site of two other missense mutations associated with androgen insensitivity syndrome (Ris Stalpers et al. 1991, Hiort et al. 1996). Three of four reported subjects showed the complete androgen insensitivity phenotype, in accordance with the two siblings in our study. We suggest that the existence of three pathological amino acid substitutions for aspartic acid 695 most likely reflects the essential role of this residue for normal androgen receptor function in male sexual differentiation.

Molecular cloning and characterization of P47, a novel boar sperm-associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins.

P47, a peripherally associated 47-kDa protein of porcine spermatozoa, was identified by affinity chromatography in the fraction of solubilized plasma membrane proteins bound to immobilized porcine zona pellucida glycoproteins. N-terminal and internal amino acid sequences revealed structural similarity between P47 and rat O-acetyl ganglioside synthase, bovine mammary gland protein (MGP)57/53 and mouse milk fat globule protein E8-polypeptides of unknown function secreted by mammary gland epithelial cells in both species. A polyclonal antibody directed against bovine MGP57/53 displayed cross-reactivity with P47. Indirect immunofluorescence analysis located porcine P47 on the acrosomal cap of testicular sperm and on sperm recovered along different sections of the ductus epididymidis, as well as on swim-up and in vitro-capacitated sperm. Porcine P47 was demonstrated on sperm bound to the zona pellucida of a homologous oocyte. Western blot analysis identified P47 (or MGP57/53) homologous proteins in porcine and human milk. Like the sperm-associated protein, porcine milk P47 possesses affinity for isolated, biotinylated sow oocyte zona pellucida glycoproteins. Reverse transcription-polymerase chain reaction was used to isolate P47 homologous cDNAs from porcine testis and mammary gland tissues as well as from bovine, mouse, and human testis. P47 proteins deduced from these cDNA sequences showed 60-100% amino acid sequence identity. These proteins display a mosaic structure organized into two N-terminal, tandemly arranged epidermal growth factor (EGF)-like domains followed by a region with similarity to C1 and C2 domains found in blood clotting factors V and VII. The second EGF-like domain contains an arginine-glycine-aspartic acid sequence, a motif often found in integrin receptor ligands. P47-like proteins are not expressed solely in testicular and mammary gland tissues. Northern blot analysis showed that P47 mRNA is transcribed in several porcine and bovine tissues. These data indicate a potential role for boar sperm-associated P47 in membrane remodeling and/or as a zona pellucida binding protein.

A Commentary on the NIH Consensus Development Statement 'Genetic Testing for Cystic Fibrosis'.

A NIH Consensus Development Statement recommends implementation of a cystic fibrosis carrier screening in the general pregnant and pre-pregnant population. This suggestion is discussed in the light of missing or insufficient data about the reasons for high uptake rates in pilot projects, psychological and social risks, and optimal educational and counselling settings. It is concluded that the recommendation is not defendable and, at best, premature.