Whole-exome sequencing points to considerable genetic heterogeneity of cerebral palsy.

Cerebral palsy (CP) is a common, clinically heterogeneous group of disorders affecting movement and posture. Its prevalence has changed little in 50 years and the causes remain largely unknown. The genetic contribution to CP causation has been predicted to be ~2%. We performed whole-exome sequencing of 183 cases with CP including both parents (98 cases) or one parent (67 cases) and 18 singleton cases (no parental DNA). We identified and validated 61 de novo protein-altering variants in 43 out of 98 (44%) case-parent trios. Initial prioritization of variants for causality was by mutation type, whether they were known or predicted to be deleterious and whether they occurred in known disease genes whose clinical spectrum overlaps CP. Further, prioritization used two multidimensional frameworks-the Residual Variation Intolerance Score and the Combined Annotation-dependent Depletion score. Ten de novo mutations in three previously identified disease genes (TUBA1A (n=2), SCN8A (n=1) and KDM5C (n=1)) and in six novel candidate CP genes (AGAP1, JHDM1D, MAST1, NAA35, RFX2 and WIPI2) were predicted to be potentially pathogenic for CP. In addition, we identified four predicted pathogenic, hemizygous variants on chromosome X in two known disease genes, L1CAM and PAK3, and in two novel candidate CP genes, CD99L2 and TENM1. In total, 14% of CP cases, by strict criteria, had a potentially disease-causing gene variant. Half were in novel genes. The genetic heterogeneity highlights the complexity of the genetic contribution to CP. Function and pathway studies are required to establish the causative role of these putative pathogenic CP genes.

Quality of life and the role of menopausal hormone therapy.

The quality of life of countless menopausal women world-wide has been significantly diminished following the sensationalist reporting of the Women's Health Initiative (WHI) and the resulting 50% or more decline in the use of hormone replacement therapy (HRT) over the subsequent 10 years. Quality of life is difficult to measure as there are so many contributing factors and a large number of different instruments, some of which assess general health and only a few which specifically include symptoms related to menopause. HRT improves quality of life of symptomatic menopausal women and some studies of the effects of HRT provide reliable evidence on quality of life other than reduction in vasomotor symptoms. Until there is a better understanding of the minimal risks of HRT for the majority of women, too many will continue to suffer a reduced quality of life unnecessarily.

Future long-term trials of postmenopausal hormone replacement therapy - what is possible and what is the optimal protocol and regimen?

The ideal long-term, randomized, placebo-controlled trial of hormone replacement therapy (HRT) from near menopause for up to 30 years to assess major morbidity and mortality is impractical because of high cost, participant retention, therapy compliance, and continuity of research staff and funding. Also the trial regimen may become outdated. It is nihilistic to demand such a long-term trial before endorsing HRT. However, medium-term trials using surrogate measures for long-term morbidity and mortality are possible and two are near completion. If these studies have been able to maintain reasonable participant retention, therapy compliance and minimal breach of protocol, they will set standards for trials of new HRT regimens. This paper discusses lessons learnt from past attempts at long-term trials and suggests the currently optimal protocol and cost of assessing new HRT regimens to optimize potential benefits and minimize adverse effects. A 5-7-year randomized, placebo-controlled trial of a flexible transdermal estrogen regimen ± either a selective estrogen receptor modulator, e.g. bazedoxifene, or micronized progesterone is discussed. Mild to moderately symptomatic women, 1-4 years post menopause, can be recruited via general practice and group meetings. Future trials should be funded by independent agencies and are high priority in women's health.

Evidence-based assessment of the impact of the WHI on women's health.

Following the announcement of the first results of the Women's Health Initiative (WHI) to the media in 2002, prior to their scientific publication, the resulting panic headlines had an immediate and lasting negative effect on use of menopausal hormone replacement therapy (HRT) around the world. Rates of use dropped by 40-80%. Symptomatic women then sought multiple alternative therapies but the majority of these have no greater effect than the effect seen from placebo in well-conducted trials of HRT. Some of these therapies have risks. Although anecdotally most menopause practitioners after 2002 can attest to having to counsel large numbers of women with debilitating menopausal symptoms who were too frightened to consider HRT, it is difficult to document loss of health-related quality of life in large population studies as they were not conducted. Similarly, the positive or negative effects of the marked decline in HRT on long-term morbidities and mortality have yet to be fully assessed. Recent studies have shown an increase in postmenopausal fractures and in some, but not all, populations a small temporary decline in breast cancer. Cardiovascular outcomes may not be apparent for another decade. Short-term, randomized, placebo-controlled trials confirm that HRT is the only therapy that effectively improves health-related quality of life in symptomatic women through a reduction in vasomotor and urogenital symptoms, joint pains and insomnia, while improving sexuality. The results of the re-analyses of the WHI data and new data from other studies do not justify the continuing negative attitude to HRT in symptomatic women who start HRT near menopause.

HRT in difficult circumstances: are there any absolute contraindications?

Many traditional contraindications to hormone replacement therapy (HRT) are based on the theoretical potential for these hormones to worsen a disease process and are rarely based on supporting data. This review addresses the available data and lack of data that make the prescription of HRT difficult in a variety of common morbidities. In each circumstance, it is assumed that conservative evidence-based therapies have been tried and that menopausal symptoms remain debilitating and are reducing quality of life. Tailoring of the product, dose, route and regimen may avoid some of the theoretical risks of HRT in particular women or conditions and guidelines are given for each co-morbidity. Specifically, it is discussed that tailored HRT may be used without strong evidence of a deleterious effect after ovarian cancer, endometrial cancer, most other gynecological cancers, bowel cancer, melanoma, a family history of breast cancer, benign breast disease, in carriers of BRACA mutations, after breast cancer if adjuvant therapy is not being used, past thromboembolism, varicose veins, fibroids and past endometriosis. Relative contraindications are existing cardiovascular and cerebrovascular disease and breast cancer being treated with adjuvant therapies. Consultation with other carers and written consent are recommended in all these difficult circumstances, but no condition is an absolute contraindication to HRT if potential risk is understood, if HRT is effective in symptom control and if quality of remaining life is paramount.

Menopausal hot flushes and night sweats: where are we now?

OBJECTIVE: An overview of the current knowledge on the etiology and treatment of vasomotor symptoms in postmenopausal women. MATERIALS AND METHODS: Acknowledged experts in the field contributed a brief assessment of their areas of interest which were combined and edited into the final manuscript. RESULTS: Women around the world experience vasomotor symptoms as they enter and complete the menopause transition. Vasomotor symptoms, specifically hot flushes, are caused by a narrowing of the thermoneutral zone in the brain. This effect, although related to estrogen withdrawal, is most likely related to changes in central nervous system neurotransmitters. Peripheral vascular reactivity is also altered in symptomatic women. Estrogen replacement therapy is the most effective treatment for hot flushes. Of the other interventions investigated, selective serotonin and selective norepinephrine reuptake inhibitors and gabapentin show efficacy greater than placebo. Objective monitoring of hot flushes indicates a robust improvement with hormone replacement therapy but little to no change with placebo. These data suggest that the subjective assessment of responses to therapy for vasomotor symptom results in inaccurate data. Hot flushes have recently been associated with increased cardiovascular risks and a lower incidence of breast cancer, but these data require confirmation. CONCLUSIONS: Vasomotor symptoms are experienced by women of all ethnic groups. They are caused by changes in the central nervous system associated with estrogen withdrawal and are best treated with estrogen replacement therapy. Objective monitoring of hot flushes indicates that placebo has little to no effect on their improvement. Subjective assessments of hot flushes in clinical trials may be inaccurate based on objective measurement of the frequency of hot flushes. Based on preliminary reports, women experiencing hot flushes have an increased risk of cardiovascular disease and a reduced incidence of breast cancer.

Yield of clinically reportable genetic variants in unselected cerebral palsy by whole genome sequencing.

Cerebral palsy (CP) is the most common cause of childhood physical disability, with incidence between 1/500 and 1/700 births in the developed world. Despite increasing evidence for a major contribution of genetics to CP aetiology, genetic testing is currently not performed systematically. We assessed the diagnostic rate of genome sequencing (GS) in a clinically unselected cohort of 150 singleton CP patients, with CP confirmed at >4 years of age. Clinical grade GS was performed on the proband and variants were filtered, and classified according to American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. Variants classified as pathogenic or likely pathogenic (P/LP) were further assessed for their contribution to CP. In total, 24.7% of individuals carried a P/LP variant(s) causing or increasing risk of CP, with 4.7% resolved by copy number variant analysis and 20% carrying single nucleotide or indel variants. A further 34.7% carried one or more rare, high impact variants of uncertain significance (VUS) in variation intolerant genes. Variants were identified in a heterogeneous group of genes, including genes associated with hereditary spastic paraplegia, clotting and thrombophilic disorders, small vessel disease, and other neurodevelopmental disorders. Approximately 1/2 of individuals were classified as likely to benefit from changed clinical management as a result of genetic findings. In addition, no significant association between genetic findings and clinical factors was detectable in this cohort, suggesting that systematic sequencing of CP will be required to avoid missed diagnoses.

The genomic basis of cerebral palsy: a HuGE systematic literature review.

Cerebral palsy has been associated with a number of candidate genes. To date, no systematic review has been conducted to synthesise genetic polymorphism associations with cerebral palsy. We apply the HuGE NET guidelines to search PubMed and EMBASE databases for publications investigating single nucleotide polymorphisms (SNPs) and cerebral palsy outcome. 22 papers were identified and are discussed in this review. Candidate genes were grouped as (1) thrombophilic, (2) cytokine, (3) apolipoprotein E or (4) other SNPs, largely related to cardiovascular physiology/pathophysiology and the functioning of the immune system. Of the studies identified, cohorts were usually small, without adequate control and ethnically diverse, making direct comparison between studies difficult. The most promising candidate genes include factor V Leiden, methylenetetrahydrofolate reductase, lymphotoxin-alpha, tumour necrosis factor-alpha, eNOS and mannose binding lectin. Large case-control studies are needed to confirm these candidates with attention given to cohort ethnicity, cerebral palsy subtype analysis and possible multiple gene and gene-environment interactions.

Continuing decline in hormone therapy use: population trends over 17 years.

OBJECTIVE: To describe the prevalence of menopausal hormone therapy (HT) in 2008 and trends over the last 17 years in an Australian population. METHODS: Data were obtained from nine representative population face-to-face interview surveys of the South Australian population from 1991 to 2008. The surveys used consistent method and quality control procedures. In 2008, demographic data, HT use and eight dimensions of health, using the SF-36 health survey questionnaire, were measured. Participants Over 3000 South Australian adults were interviewed in their own home by trained health interviewers in each of the surveys; in the 2008 survey, 1555 women participated, of whom 953 were over age 40. RESULTS: After a peak in use in the 2000 survey, HT use fell from 2003 and has continued to decline in 2008. In 2008, current use over age 50 of registered conventional HT products is now 11.8%, with a further 4.0% using non-registered alternative 'hormonal' products. Current HT use is highest between the ages of 50 and 59 years, where 13.4% use conventional HT and 7.7% use unconventional alternative hormones. Use of these unregistered hormonal products was rare in previous surveys. Median and mean length of conventional HT use were 10.0 and 10.5 years, respectively. HT users continued to have a demographic profile similar to those in previous surveys, i.e. they were better educated, employed, partnered, had a higher income and were less inclined to use complementary and alternative medicines. CONCLUSIONS: There has been a continuing decline in both the overall prevalence and length of use of conventional HT from 2003, probably in association with negative media about HT. Of medical concern is that about one-quarter of women using HT around menopause now chooses unregistered hormonal mixtures that are untested for long-term safety and efficacy.

Targeted resequencing identifies genes with recurrent variation in cerebral palsy.

A growing body of evidence points to a considerable and heterogeneous genetic aetiology of cerebral palsy (CP). To identify recurrently variant CP genes, we designed a custom gene panel of 112 candidate genes. We tested 366 clinically unselected singleton cases with CP, including 271 cases not previously examined using next-generation sequencing technologies. Overall, 5.2% of the naïve cases (14/271) harboured a genetic variant of clinical significance in a known disease gene, with a further 4.8% of individuals (13/271) having a variant in a candidate gene classified as intolerant to variation. In the aggregate cohort of individuals from this study and our previous genomic investigations, six recurrently hit genes contributed at least 4% of disease burden to CP: COL4A1, TUBA1A, AGAP1, L1CAM, MAOB and KIF1A. Significance of Rare VAriants (SORVA) burden analysis identified four genes with a genome-wide significant burden of variants, AGAP1, ERLIN1, ZDHHC9 and PROC, of which we functionally assessed AGAP1 using a zebrafish model. Our investigations reinforce that CP is a heterogeneous neurodevelopmental disorder with known as well as novel genetic determinants.

Fetal exposure to herpesviruses may be associated with pregnancy-induced hypertensive disorders and preterm birth in a Caucasian population.

OBJECTIVE: To investigate the role of fetal viral infection in the development of a range of adverse pregnancy outcomes (APOs), including pregnancy-induced hypertensive disorders (PIHD), antepartum haemorrhage (APH), birthweight <10th percentile (small for gestational age, SGA) and preterm birth (PTB). DESIGN: Population-based case-control study. SETTING: Laboratory-based study. POPULATION: The newborn screening cards of 717 adverse pregnancy cases and 609 controls. METHODS: Newborn screening cards were tested for RNA from enteroviruses and DNA from herpesviruses using polymerase chain reaction (PCR). The herpesviruses were detected using two PCRs, one detecting nucleic acids from herpes simplex virus (HSV)-1, HSV-2, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus (HHV)-8, hereafter designated Herpes PCR group A viruses, and the other detecting nucleic acids from varicella-zoster virus (VZV), HHV-6 and HHV-7, hereafter designated Herpes PCR group B viruses. MAIN OUTCOME MEASURE: Odds ratios and 95% CIs for specific APOs. RESULTS: For both term and PTBs, the risk of developing PIHD was increased in the presence of DNA from Herpes PCR group B viruses (OR 3.57, 95% CI 1.10-11.70), CMV (OR 3.89, 95% CI 1.67-9.06), any herpesvirus (OR 5.70, 95% CI 1.85-17.57) and any virus (OR 5.17, 95% CI 1.68-15.94). The presence of CMV was associated with PTB (OR 1.61, 95% CI 1.14-2.27). No significant association was observed between SGA or APH and exposure to viral infection. CONCLUSIONS: Fetal exposure to herpesvirus infection was associated with PIHD for both term and PTBs in this exploratory study. Exposure to CMV may also be associated with PTB. These findings need confirmation in future studies.

The emerging genetic landscape of cerebral palsy.

Cerebral palsy (CP) is a broad clinical descriptor that encompasses a heterogeneous group of nonprogressive neurodevelopmental disabilities affecting movement and posture. While linked by the presence of damage to the developing brain, the etiology of CP is likely varied and the clinical outcomes are diverse. There is now a large body of evidence supporting a significant role for genetics in causation of CP. An increasing number of studies have identified likely causative genetic variants in families with CP, as well as in individual sporadic cases. Next-generation sequencing is now aiding clinicians in making specific molecular diagnoses, providing future opportunities for tailored treatments and for informed reproductive decisions.

On the nature of the active component of haematoporphyrin derivative--Part 2.

It has been suggested recently that neither an ester nor an ether linkage was present in the tumour-localising component of Photofrin II (PFII) (a commercial preparation of DHE). We now report that our chemical and spectroscopic studies, establish the presence of an ester linkage with the possibility that an ether linkage may also be involved.

Nitrogen analogues of haematoporphyrin and haematoporphyrin derivative.

The preparation of a number of amines related to haematoporphyrin (HP) and haematoporphyrin derivative (HPD) have been studied and their composition and structure discussed through examination of their 1H, 13C NMR and mass spectral data and other physical properties. In vitro biological studies have been carried out and have shown these amines to have a similar photodynamic efficiency to that of HPD. One of these showed cytotoxic properties at exceptionally low light energy levels.

Ripening of the human cervix and induction of labor with intracervical purified porcine relaxin.

In a randomized double-blind placebo-controlled trial involving 71 patients, a viscous gel containing distilled water or 1 or 2 mg pure porcine relaxin was instilled in the cervical canal on the evening before the surgical induction of labor. Eleven of 48 patients receiving relaxin labored overnight, whereas only one of 23 patients went into labor. Only the 2-mg dose significantly improved the mean cervical score compared with the placebo treatment; the effect was greatest in primigravid patients with unripe cervixes. Intracervical application appeared to confer no benefit over vaginal application in effecting cervical ripening or inducing labor. Systemic absorption of the porcine relaxin after its intracervical application was confirmed by the measurement of immunoreactive relaxin in a homologous porcine relaxin radioimmunoassay. Thus, the cervical ripening effect of exogenous relaxin may be mediated either systemically or by direct action at the site of local application. This trial confirms the responsiveness of the human term cervix to exogenous relaxin and supports the suggestion that endogenous relaxin may play a similar role at term in facilitating cervical ripening and parturition.

Cervical ripening with combinations of vaginal prostaglandin F2-alpha estradiol, and relaxin.

The first of a 2-part trial consisted of a double-blind randomized pilot study in which 4 groups of 10 patients near term received 1 of the following hormonal combinations in a vaginal gel 15 hours before surgical induction of labor: 1) prostaglandin F2 alpha (PGF2 alpha) and relaxin; 2) relaxin and estradiol; 3) estradiol and PGF2 alpha; and 4) relaxin, estradiol, and PGF2 alpha. In each group the mean cervical score improved after treatment; the relaxin/PGF2 alpha combination was associated with the greatest improvement in cervical score (4.8). The highest incidence of subsequent labor was also seen in the relaxin/PGF2 alpha group (40%). However, with the exception of the latter group, the clinical effects of these hormonal combinations were neither greater nor smaller than the previously published effects of these hormones used individually in similar circumstances. The second part of the study further explored the possibility of an additive effect of relaxin and PGF2 alpha in combination as suggested by the pilot study, and an additional 40 patients were given this combination. Analysis of these larger numbers showed no additive effect when these hormones were used in combination compared with when they were used individually. Thus, in the circumstances described, there is no clinical advantage to the concurrent administration of any combination of relaxin, PGF2 alpha and estradiol with regard to cervical ripening and/or the initiation of parturition.

Inhibin and relaxin concentrations in early singleton, multiple, and failing pregnancy: relationship to gonadotropin and steroid profiles.

OBJECTIVE: To examine the inter-relationships between inhibin, relaxin, steroid concentrations, estradiol (E2), progesterone (P), and gonadotropins in early pregnancy. DESIGN: Hormone concentrations in plasma were measured during the luteal phase of subjects who became pregnant (n = 58) or failed to become pregnant (n = 47) after ovarian hyperstimulation for in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) (group 1). A further group of subjects became pregnant (n = 7) or failed to become pregnant (n = 8) during endocrinology tracking of a natural cycle (group 2). Blood was obtained every 3 days in the luteal phase from day 5 in group I (day 0 was oocyte recovery) and from day 0 (first increase in luteinizing hormone [LH]) in group II. RESULTS: Progesterone and E2 were increased over nonpregnant values by day 11 (P) and day 16 (E2) in group I and by day 11 (E2 and P) in group II. Inhibin and relaxin concentrations were significantly increased by day 16 in group I (often by day 11) and by day 14 in group II pregnancy subjects. A direct relationship existed between inhibin, P, relaxin, and human chorionic gonadotropin (hCG). Subjects who had twin pregnancies demonstrated higher concentrations of all hormones and often exhibited increases earlier (by day 11 in group I) than singleton pregnancy subjects. Pregnancies that ended in miscarriages tended to have lower concentrations of P and inhibin. None of the hormones reliably discriminated between the clinical conditions of blighted ovum and of spontaneous abortion, and the predictive value of any hormone measured for miscarriage was not high. CONCLUSIONS: The trend of inhibin and relaxin concentrations closely parallels rises in P during early pregnancy. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are suppressed very early in pregnancy. The suppression of LH and FSH in hyperstimulated cycles is more governed by E2 than inhibin in stimulated cycles. Some subjects destined to miscarry exhibit abnormal endocrine changes very early in the luteal phase.

Radioimmunoassay of thyroxine and 3,3',5'-triiodothyronine (reverse T3) in human amniotic fluid.

Measurement of amniotic fluid iodothyronine concentrations may enable antenatal detection of fetal thyroid abnormalities; however, the delineation of normal ranges is complicated by methodological problems associated with strong and highly variable protein-binding, and specificity of antisera. Improved radioimmunoassays of thyroxine (T4) and 3,3',5-triiodothyronine (reverse T3, rT3) have been developed to overcome these problems. In normal pregnancy, mean rT3 concentrations at less than 17 weeks, 17-22 weeks and 35-42 weeks gestation were 3.6 nmol/l (n = 21), 6.1 nmol/1 (n = 14) and 0.66 nmol/1 (n = 39) respectively; corresponding mean T4 concentrations were 2.4 nmol/1, 6.5 nmol/1 and 3.6 nmol/1. rT3 concentrations showed a strong positive correlation with T4 concentration in each age range; however, the molar ratio of rT3:T4 decreased progressively with gestational age, from 1.69 at less than 17 weeks to 0.19 at 35-42 weeks. In both mid- and late gestation, rT3 and T4 concentrations were strongly correlated with total amniotic fluid protein concentrations.

Sulfonated phthalimidomethyl aluminum phthalocyanine: the effect of hydrophobic substituents on the in vitro phototoxicity of phthalocyanines.

The photocytotoxicity of sulfonated phthalimidomethyl aluminum phthalocyanine, a more hydrophobic photosensitizer as compared to phthalocyanine substituted with sulfonate groups only, was investigated. Inclusion of 1-2 phthalimidomethyl groups into disulfonated aluminum phthalocyanine, resulted in increased partition coefficients between n-octanol and water, and a six-fold increase in both cellular uptake and photocytotoxicity towards Chinese hamster lung fibroblast cells (line V-79). Reducing the number of phthalimidomethyl groups, or increasing the degree of sulfonation, lead to a decrease in the partition coefficient, cellular uptake, and phototoxicity. The quantum yield of singlet oxygen was comparable for all dyes tested in this series, indicating that no significant change in this photophysical parameter resulted from phthalimidomethylation. These results suggest that the addition of 1-2 phthalimidomethyl groups to disulfonated aluminum phthalocyanine improves cellular uptake, but, as the relative efficiency of cell killing was not effected, the intracellular distribution on photosensitive molecules may not be modified.