Characterization of the full fragile X syndrome mutation in fetal gametes.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

A point mutation in the FMR-1 gene associated with fragile X mental retardation.

The vast majority of patients with fragile X syndrome show a folate-sensitive fragile site at Xq27.3 (FRAXA) at the cytogenetic level, and both amplification of the (CGG)n repeat and hypermethylation of the CpG island in the 5' fragile X gene (FMR-1) at the molecular level. We have studied the FMR-1 gene of a patient with the fragile X phenotype but without cytogenetic expression of FRAXA, a (CGG)n repeat of normal length and an unmethylated CpG island. We find a single point mutation in FMR-1 resulting in an lle367Asn substitution. This de novo mutation is absent in the patient's family and in 130 control X chromosomes, suggesting that the mutation causes the clinical abnormalities. Our results suggest that mutations in FMR-1 are directly responsible for fragile X syndrome, irrespective of possible secondary effects caused by FRAXA.

The full mutation in the FMR-1 gene of male fragile X patients is absent in their sperm.

Fragile X syndrome is characterized at the molecular level by amplification of a (CGG)n repeat and hypermethylation of a CpG island preceeding the open reading frame of the fragile X gene (FMR-1) located in Xq27.3. Anticipation in this syndrome is associated with progressive amplification of the (CGG)n repeat from a premutation to a full mutation through consecutive generations. Remarkably, expansion of the premutation to the full mutation is strictly maternal. To clarify this parental influence we studied FMR-1 in sperm of four male fragile X patients. This showed that only the premutation was present in their sperm, although they had a full mutation in peripheral lymphocytes. This might suggest that expansion of the premutation to the full mutation in FMR-1 does not occur in meiosis but in a postzygotic stage.

Deletion of 1 amino acid in Indian hedgehog leads to brachydactylyA1.

Brachydactyly type A1 is a limb malformation characterized by a uniform shortening of the middle phalanges in all digits. Mutations in the Indian hedgehog (IHH) gene were shown to be the cause of this autosomal dominant disorder. The IHH protein is known to be an important signaling molecule involved in chondrocyte formation. So far, only missense mutations in IHH have been reported to cause BrachydactylyA1. We report here on the first deletion in IHH, p.delE95, causing mild BrachydactylyA1 in a small Dutch family. This brings the total number of different mutations found to cause BDA1 to 7.

Risk factors for stillbirth in a socio-economically disadvantaged urban Australian population.

INTRODUCTION: Several risk factors for stillbirth have been extensively investigated. Some risk factors are more common in socio-economically disadvantaged regions. The aim of this study was to identify risk factors for stillbirth in the Northern suburbs of Adelaide, one of the most socio-economically disadvantaged urban areas in Australia. MATERIAL AND METHODS: A retrospective case control study (two controls per case) of all women with a singleton pregnancy resulting in a stillbirth during the decade 2002-2012. RESULTS: One hundred and thirty stillbirths were registered over these 10 years. Using univariate analysis, the following risk factors were identified: obesity ≥40 body mass index (BMI) (OR 4.75), non-Caucasian ethnicity (odds ratio [OR] 2.737), pre-existing diabetes (p <0.000), polycystic ovary syndrome (PCOS) (OR 5.250), in vitro fertilisation (IVF) (OR 4.000), booking systolic blood pressure (SBP) ≥ 140 (OR 5.000) and booking diastolic blood pressure (DBP) ≥ 80 (OR 3.111). Many of these factors have complex interrelationships. Multivariate analysis identified the following independent risk factors: BMI ≥40 (OR 3.940), ethnic minorities (mainly indigenous Australians) (OR 2.255) and social issues (OR 3.079). PCOS had an independent effect to some extent, but this was clearly confounded by BMI. CONCLUSION: These Australian data confirm the presence of several potentially modifiable risk factors for stillbirth, within this socio-economically disadvantaged region. Modifying these risk factors, in particular obesity, is a big challenge not only for maternity and primary care providers, but for overall society.

FMR1 premutation allele (CGG)81 is stable in mice.

Fragile X syndrome is caused by an expansion of the CGG repeat present in the 5' UTR of the FMR1 gene. A lot has been elucidated about the genetics of the disease, but not much is known about the mechanisms involved in repeat instability. Transgenic animals with a premutation allele [(CGG)11AGG(CGG)60CAG(CGG)8] in the human FMR1 promoter were generated to study the inheritance of this repeat in mice. Three independent lines, B6, B7 and B29, in total 263 transgenic animals, were tested for repeat instability. In all meiosis and mitosis tested, the repeat inherited stably. This suggests that other factors might be important in repeat (in)stability.

Mental status and fragile X expression in relation to FMR-1 gene mutation.

The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat in the FMR-1 gene. Clinical expression is associated with a large expansion of the CGG repeat. The mutation in the FMR-1 gene and the cytogenetic expression of the fragile site at Xq27.3 have been studied in 52 fragile X male patients. The percentage of the cytogenetic expression of the fragile site at Xq27.3 positively correlates with the mean size of the full mutation in the FMR-1 gene (p < 0.0001) irrespective of the presence of additional premutation alleles. We noted a less frequent occurrence of additional premutation alleles in adult patients compared with juveniles, suggesting a continued mitotic instability in life. Additionally, the level of mental retardation has been ascertained in 35 patients using the Stanford-Binet or Terman-Merrill test of general intelligence. The presence of a full mutation in the FMR-1 gene seemed decisive for the occurrence of mental impairment in the patient. No correlation is observed between the degree of mental retardation and the size of the full mutation. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in part of the cells in addition to a full mutation. One patient is described with the 'Prader-Willi-like' subphenotype of the fragile X syndrome, showing a deletion in the FMR-1 gene in a part of his cells in addition to a full mutation.

The fragile X phenotype in a mosaic male with a deletion showing expression of the FMR1 protein in 28% of the cells.

The instability of the CGG repeat region of FMR1 is not restricted to the CGG repeat but expands to flanking sequences as well. A mosaic fragile X male is reported with a deletion of part of the CGG repeat and 30 bp immediately 3' of the repeat, thus confirming the presence of a hotspot for deletions in the CGG region of FMR1. The deletion, detected in 28% of his lymphocytes, did not impair the transcription and translation of FMR1, suggesting that regulatory elements are not present in the deleted region. The patient has the characteristic fragile X phenotype and assuming that the mosaic pattern detected in the lymphocytes reflects the mosaic pattern in brain, 28% expression of FMRP may not be sufficient for normal cognitive functioning.

No apparent involvement of the FMR1 gene in five patients with phenotypic manifestations of the fragile X syndrome.

Most fragile X patients have a significant increase in the number of CGG repeats in the FMR1 gene. Two patients were described with a deletion and one patient with a point mutation in the FMR1 gene. We describe 5 patients with a fragile X or Martin-Bell phenotype. Two brothers were discordant for the region containing the FMR1 gene; if there is a common cause for the mental retardation this is not located in the FMR1 gene. In the other 3 patients the expression of the FMR1 gene was found to be normal and no abnormalities were noted in the FMR1 mRNA. No amplification was found in the GCC repeat which is associated with the fragile site FRAXE. We conclude that the Martin-Bell phenotype can also be caused by mutations outside the FMR1 gene.

DNA diagnosis of the fragile X syndrome in a series of 236 mentally retarded subjects and evidence for a reversal of mutation in the FMR-1 gene.

The cloning of the FMR-1 gene and the identification of an expanded CGG repeat in DNA of fragile X patients has made reliable DNA diagnosis feasible. Southern blotting and PCR assays of the CGG repeat in an unselected series of 236 mentally retarded subjects resulted in the identification of 10 new fragile X families. Reevaluation of previously assessed fragile X families resulted in the first observation of the presence of a reversal of mutation in the FMR-1 gene.

Responses of experimental rat tumours and a mouse colon tumour to flavone acetic acid.

The toxicity in rats and mice and the responses of 5 rat lung tumours, a rat rhabdomyosarcoma and a mouse colon tumour to flavone acetic acid, FAA, were studied. The LD50 values of FAA in WAG/Rij and BN rats were about 350 and 525 mg/kg respectively, independent of whether the animals were tumour bearing or not. In contrast, the LD50 value of tumour bearing BCBA mice was 50 percent of that of non-tumour bearing mice. Neither tumour volume regression nor growth delay were observed in the rat tumours growing in syngeneic rats or in nude mice, in contrast to the response of colon 38 tumours in mice. Light microscopy revealed massive tumour necrosis in the mouse tumour while no significant changes were observed in the rat tumours.

Molecular and cellular mechanisms underlying anti-neuronal antibody mediated disorders of the central nervous system.

Over the last decade multiple autoantigens located on the plasma membrane of neurons have been identified. Neuronal surface antigens include molecules directly involved in neurotransmission and excitability. Binding of the antibody to the antigen may directly alter the target protein's function, resulting in neurological disorders. The often striking reversibility of symptoms following early aggressive immunotherapy supports a pathogenic role for autoantibodies to neuronal surface antigens. In order to better understand and treat these neurologic disorders it is important to gain insight in the underlying mechanisms of antibody pathogenicity. In this review we discuss the clinical, circumstantial, in vitro and in vivo evidence for neuronal surface antibody pathogenicity and the possible underlying cellular and molecular mechanisms. This review shows that antibodies to neuronal surface antigens are often directed at conformational epitopes located in the extracellular domain of the antigen. The conformation of the epitope can be affected by specific posttranslational modifications. This may explain the distinct clinical phenotypes that are seen in patients with antibodies to antigens that are expressed throughout the brain. Furthermore, it is likely that there is a heterogeneous antibody population, consisting of different IgG subtypes and directed at multiple epitopes located in an immunogenic region. Binding of these antibodies may result in different pathophysiological mechanisms occurring in the same patient, together contributing to the clinical syndrome. Unraveling the predominant mechanism in each distinct antigen could provide clues for therapeutic interventions.

FBXO7 mutations cause autosomal recessive, early-onset parkinsonian-pyramidal syndrome.

BACKGROUND: The combination of early-onset, progressive parkinsonism with pyramidal tract signs has been known as pallido-pyramidal or parkinsonian-pyramidal syndrome since the first description by Davison in 1954. Very recently, a locus was mapped in a single family with an overlapping phenotype, and an FBXO7 gene mutation was nominated as the likely disease cause. METHODS: We performed clinical and genetic studies in two families with early-onset, progressive parkinsonism and pyramidal tract signs. RESULTS: An FBXO7 homozygous truncating mutation (Arg498Stop) was found in an Italian family, while compound heterozygous mutations (a splice-site IVS7 + 1G/T mutation and a missense Thr22Met mutation) were present in a Dutch family. We also found evidence of expression of novel normal splice-variants of FBXO7. The phenotype associated with FBXO7 mutations consisted of early-onset, progressive parkinsonism and pyramidal tract signs, thereby matching clinically the pallido-pyramidal syndrome of Davison. The parkinsonism exhibits varying degrees of levodopa responsiveness in different patients. CONCLUSIONS: We conclusively show that recessive FBXO7 mutations cause progressive neurodegeneration with extrapyramidal and pyramidal system involvement, delineating a novel genetically defined entity that we propose to designate as PARK15. Understanding how FBXO7 mutations cause disease will shed further light on the molecular mechanisms of neurodegeneration, with potential implications also for more common forms of parkinsonism, such as Parkinson disease and multiple system atrophy.

A test of medical problem-solving scored by nurses and doctors: the handicap of expertise.

There is evidence that nurses fulfil the requirements of objective judgement better than doctors. Simulation of Initial Medical Problem-Solving (SIMP), a paper-and-pencil test for the assessment of medical problem-solving, consists of case histories, followed by an open-ended question. The scoring of open-ended questions is time-consuming and adds subjective bias to measurement error. In order to reduce scoring error, answers on SIMP are scored by means of scoring models in the form of check-lists with descriptions of elements of a correct answer. The reliability of the scoring was analysed in a study, with six nurses rating 500 answers. The overall interrater reliability was high, expressed by an intra-class correlation of 0.83. Selection of raters, and improvement of the scoring models, could increase the interrater reliability even further. In addition to the scoring by the nurses part of the material was scored again by two experienced doctors. The reliability of the scoring method on the whole was confirmed. Nevertheless, some evidence was found of misinterpretation of the scoring models by the nurses. Analysis at the item level revealed several instances in which both doctors agreed on a score for an element in an answer and all the nurses agreed on the opposite score. On the other hand, however, the two doctors were less consistent between themselves than the nurses. The disagreement between the doctors seems to be a consequence of differences in their own medical judgement of the case in question. The impact of the mistakes that are made by the nurses is much smaller than the loss of reliability caused by the inconsistency among the doctors.

Antiplatelet medications and their indications in preventing and treating coronary thrombosis.

Platelets play a pivotal role in the pathophysiology of unstable angina, acute myocardial infarction, and complications following percutaneous coronary intervention. Three classes of platelet-inhibiting drugs, aspirin, thienopyridines and platelet glycoprotein IIb/ IIIa inhibitors, are now commonly used for the prevention and treatment of disorders of coronary artery thrombosis. For the last several decades aspirin has been the sole option for antiplatelet therapy in the treatment and prevention of the manifestations of cardiovascular disease. However, a wider selection of antiplatelet agents, including the thienopyridines (ticlopidine and clopidogrel) and the platelet glycoprotein (GP)IIb/IIIa receptor antagonists, are now available and provide clinicians with the opportunity to potentially improve upon the previous gold standard of aspirin. This review summarizes these drugs and the scientific data that have led to their use in primary and secondary prevention, unstable angina, myocardial infarction, and percutaneous coronary intervention.

Validation of a new measure of clinical problem-solving.

A review of the literature on the assessment of medical problem-solving by means of written tests reveals serious short-comings. Most important is the low correlation repeatedly found among cases, which suggests the inability of the measures to assess a general problem-solving ability. The literature further suggests that instruments should focus on the brief period of time after the first encounter of a clinical problem and warns against the effects of cueing. Based on these considerations a new measure for the assessment of medical problem-solving was developed. This test, called Simulation of Initial Medical Problem-Solving (SIMP), consists of a number of short case histories, followed by an open-ended question. Reliability analysed by means of generalizability theory proved satisfactory and concurrent validity was established by a significant correlation with a global judgement of performance in a simulated patient encounter. The moderate correlation between cases is interpreted as an acceptable correlation among test items and leads to the conclusion that a reliable and valid test of clinical problem-solving should consist of a substantial number of different cases.

Complications of oral antiplatelet medications.

One by-product of the flurry of large-scale clinical trials accompanying the emergence of drugs that inhibit platelet function is volumes of information chronicling the adverse effects of this class of medications. One aspect all antiplatelet drugs share is a propensity toward bleeding. Beyond that similarity, however, the different pharmacologic agents in this broad collection have few attributes in common. Aspirin, by virtue of its long history, has been studied most extensively, and has proven to be an exceptionally valuable therapy. However, the complicated adverse profile of this seemingly simple drug is commonly overlooked by practitioners and deserves clinical review. The thienopyridine class (including ticlopidine and clopidogrel) share certain peculiarities that continue to be clarified, including life-threatening thrombotic thrombocytopenia purpura. Dipyridamole is a veteran drug that is enjoying renewed attention as a prophylactic aid in preventing cerebrovascular events. One class, the oral platelet glycoprotein IIb/IIIa receptor inhibitors, has failed to find its way into clinical implementation due to an unfavorable balance between efficacy and adverse effect. This review summarizes the adverse profiles of each of these drug classes and draws on data gathered in large clinical studies.

Pitfalls in the pursuit of objectivity: issues of reliability.

Objectivity has been one of the hallmarks in the assessment of clinical competence in recent decades. A consistent shift can be noticed in which subjective measures are being replaced by objective measurement methods. In the transition from subjective to objective methods trade-offs are involved, both in the effort expended and in the range of behaviours assessed. The issue of the presumed superiority of objective measures is addressed in two successive papers. In this paper a distinction is made between objectivity as a goal of measurement, marked by freedom of subjective influences in general, and objectivity as a set of strategies designed to reduce measurement error. The latter has been termed objectification. The central claim of this paper is that these two approaches to assessment do not necessarily coincide. By reviewing a number of studies comparing subjective and objectified measurement methods, the claim of the supremacy of the latter with respect to reliability is discussed. The results of these studies indicate that objectified methods do not inherently provide more reliable scores. Objectified methods may even provide unwanted outcomes, such as negative effects on study behaviour and triviality of the content being measured. The latter issues, related to validity, efficiency and acceptability, are discussed in a second paper.