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.

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.

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.

Differential activities of the RET tyrosine kinase receptor isoforms during mammalian embryogenesis.

The RET receptor tyrosine kinase has a critical role in kidney organogenesis and the development of the enteric nervous system. Two major isoforms, RET9 and RET51, differ in the amino acid sequence of the C-terminal tail as a result of alternative splicing. To determine the roles of these isoforms in vivo, we used targeted mutagenesis to generate mice that express either RET9 or RET51. Monoisoformic RET9 mice, which lack RET51, are viable and appear normal. In contrast, monoisoformic RET51 animals, which lack RET9, have kidney hypodysplasia and lack enteric ganglia from the colon. To study the differential activities of the two RET isoforms further, we generated transgenic mice expressing ligand-dependent and constitutively active forms of RET9 or RET51 under the control of the Hoxb7 regulatory sequences. Such RET9 transgenes are capable of rescuing the kidney agenesis in RET-deficient mice or causing kidney hypodysplasia in wild-type animals. In contrast, similar RET51 transgenes fail to rescue the kidney agenesis or cause hypodysplasia. Our findings show that RET9 and RET51 have different signaling properties in vivo and define specific temporal and spatial requirements of c-Ret function during renal development and histogenesis of the enteric nervous system.

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.

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.

Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), historically termed Pick's disease. Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics. Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon 10. The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10. This causes more frequent usage of the 5' splice site and an increased proportion of tau transcripts that include exon 10. The increase in exon 10+ messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17.

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.

Characterization of FMR1 promoter elements by in vivo-footprinting analysis.

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

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.

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.

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.

Characterization of FMR1 proteins isolated from different tissues.

FMR1 protein expression was studied in different tissues. In human, monkey and murine tissues, high molecular mass FMR1 proteins (67-80 kDa) are found, as shown in lymphoblastoid cells lines. The identity of these proteins was confirmed by their absence in tissues from patients with the fragile X syndrome and a FMR1 knock-out mouse. An Ile367Asn substitution in the FMR1 protein did not alter the translation, processing and localization of FMR1 proteins in lymphoblastoid cells from a patient carrying this mutation. All the high molecular mass FMR1 proteins isolated from normal lymphoblastoid cells and cells from the patient with the Ile367Asn substitution were able to bind RNA. However, the FMR1 proteins of the patient had reduced affinity for RNA binding at high salt concentrations. In some human, monkey and murine tissues low molecular mass FMR1 proteins (39-41 kDa) were found, which had the same N terminus as the 67-90 kDa isoforms, but differ in their C terminus and are therefore most likely the result of carboxy-terminal proteolytic cleavage. These low molecular mass FMR1 proteins did not bind RNA, in contrast with the high molecular mass FMR1 proteins. The significance of these low molecular mass proteins remains to be studied.

Hotspot for deletions in the CGG repeat region of FMR1 in fragile X patients.

The fragile X syndrome is the most frequent cause of inherited mental retardation. The molecular mechanism of the disorder is based on the expansion of a CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeat containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. We describe four unrelated fragile X patients that are mosaic for both a full mutation and a small deletion in the CGG repeat containing region. Sequence analysis of the regions surrounding the deletions showed that both the (CGG)n repeat and some flanking sequences were missing in all four patients. The 5' breakpoints of the deletions were found to be located between 75-53 bp proximal to the CGG repeat. This suggests the presence of a hot spot region for deletions in the CGG repeat region of the FMR1 gene and emphasizes the instability of this region in the presence of an expanded CGG repeat.

The effect of a single base pair deletion (delta T525) and a C1634T missense mutation (pro545leu) on the expression of lysosomal alpha-glucosidase in patients with glycogen storage disease type II.

Glycogen storage disease type II (GSDII, Pompe's disease) is caused by an autosomal recessive inheritance of lysosomal alpha-glucosidase deficiency. By sequence analysis we have identified the mutations in the lysosomal alpha-glucosidase gene (GAA) of two unrelated patients, who have one and two copies, respectively, of the same missense mutation. The milder affected adult patient was found to be homozygous for a C1634T transition resulting in the substitution of pro545 by leu. The more severely affected adolescent patient had this same mutant allele combined with a 1 base pair deletion (delta T525) in the second allele causing premature termination at nucleotide positions 658-660. Both these mutations were introduced in wild-type alpha-glucosidase cDNA and expressed in COS-1 cells to analyse their effect. The delta T525 mutation prohibits the formation of lysosomal alpha-glucosidase completely. The pro545-->leu substitution is compatible with normal synthesis but hampers enzyme maturation and results in a 92% net loss of lysosomal alpha-glucosidase activity. The patient with adult GSDII has, in accordance with the allelic constitution, a 2-fold higher residual activity than the patient with juvenile GSDII. The delta T525 deletion was detected in two other unrelated patients, and also the C1634T transition was encountered in two more Caucasian patients with GSDII.

Segregation of FRAXE in a large family: clinical, psychometric, cytogenetic, and molecular data.

During an ongoing study on X-linked mental retardation, we ascertained a large family in which mild mental retardation was cosegregating with a fragile site at Xq27-28. Clinical, psychometric, cytogenetic, and molecular studies were performed. Apart from mild mental retardation, affected males and females did not show a specific clinical phenotype. Psychometric assessment of four representative affected individuals revealed low academic achievements, with verbal and performance IQs of 61-75 and 70-82, respectively. Cytogenetically the fragile site was always present in affected males and was not always present in affected females. With FISH the fragile site was located within the FRAXE region. The expanded GCC repeat of FRAXE was seen in affected males and females either as a discrete band or as a broad smear. No expansion was seen in unaffected males, whereas three unaffected females did have an enlarged GCC repeat. Maternal transmission of FRAXE may lead to expansion or contraction of the GCC repeat length, whereas in all cases of paternal transmission contraction was seen. In striking contrast to the situation in fragile X syndrome, affected males may have affected daughters. In addition, there appears to be no premutation of the FRAXE GCC repeat, since in the family studied here all males lacking the normal allele were found to be affected.

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.

A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome.

The vast majority of individuals with the fragile X syndrome show expanded stretches of CGG repeats in the 5' non-coding region of FMR1. This expansion coincides with abnormal methylation patterns in that area resulting in the silencing of the FMR1 gene. Evidence is accumulating that this directly causes the fragile X phenotype. Very few other mutations in FMR1, causing the fragile X phenotype have been reported thus far and all concerned isolated cases. We, however, report a family, in which 11 individuals have a deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene. Although fragile X chromosomes were not detected, all 4 affected males and 2 of the carrier females show characteristics of the fragile X phenotype. Using RT-PCR we could demonstrate that FMR1 is not expressed in the affected males, strongly suggesting that the FMR1 promoter sequences 5' to the CGG repeat are missing. The deletion patients have approximately 45 CGG repeats in their FMR1 gene, though not interspersed by AGG triplets that are usually present in both normal and expanded repeats. It is hypothesized that prior to the occurrence of the deletion, an expansion of the repeat occurred, and that the deletion removed the 5' part of the CGG repeat containing the AGG triplets. Transmission of the deletion through the family could be traced back to the deceased grandfather of the affected males, which supports the hypothesis that the FMR1 gene product is not required for spermatogenesis. Finally, the data provide additional evidence that the fragile X syndrome is a single gene disorder.