Analysis of 51 proposed hypertrophic cardiomyopathy genes from genome sequencing data in sarcomere negative cases has negligible diagnostic yield.

PURPOSE: Increasing numbers of genes are being implicated in Mendelian disorders and incorporated into clinical test panels. However, lack of evidence supporting the gene-disease relationship can hinder interpretation. We explored the utility of testing 51 additional genes for hypertrophic cardiomyopathy (HCM), one of the most commonly tested Mendelian disorders. METHODS: Using genome sequencing data from 240 sarcomere gene negative HCM cases and 6229 controls, we undertook case-control and individual variant analyses to assess 51 genes that have been proposed for HCM testing. RESULTS: We found no evidence to suggest that rare variants in these genes are prevalent causes of HCM. One variant, in a single case, was categorized as likely to be pathogenic. Over 99% of variants were classified as a variant of uncertain significance (VUS) and 54% of cases had one or more VUS. CONCLUSION: For almost all genes, the gene-disease relationship could not be validated and lack of evidence precluded variant interpretation. Thus, the incremental diagnostic yield of extending testing was negligible, and would, we propose, be outweighed by problems that arise with a high rate of uninterpretable findings. These findings highlight the need for rigorous, evidence-based selection of genes for clinical test panels.

A multicentre study of patients with Timothy syndrome.

Aims: Timothy syndrome (TS) is an extremely rare multisystem disorder characterized by marked QT prolongation, syndactyly, seizures, behavioural abnormalities, immunodeficiency, and hypoglycaemia. The aim of this study was to categorize the phenotypes and examine the outcomes of patients with TS. Methods and results: All patients diagnosed with TS in the United Kingdom over a 24-year period were reviewed. Fifteen centres in the British Congenital Arrhythmia Group network were contacted to partake in the study. Six patients with TS were identified over a 24-year period (4 boys and 2 girls). Five out of the six patients were confirmed to have a CACNA1C mutation (p.Gly406Arg) and the other patient was diagnosed clinically. Early presentation with heart block, due to QT prolongation was frequently seen. Four are still alive, two of these have a pacemaker and two have undergone defibrillator implantation. Five out of six patients have had a documented cardiac arrest with three occurring under general anaesthesia. Two patients suffered a cardiac arrest while in hospital and resuscitation was unsuccessful, despite immediate access to a defibrillator. Surviving patients seem to have mild developmental delay and learning difficulties. Conclusion: Timothy syndrome is a rare disorder with a high attrition rate if undiagnosed. Perioperative cardiac arrests are common and not always amenable to resuscitation. Longer-term survival is possible, however, patients invariably require pacemaker or defibrillator implantation.

Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples.

PURPOSE: The accurate interpretation of variation in Mendelian disease genes has lagged behind data generation as sequencing has become increasingly accessible. Ongoing large sequencing efforts present huge interpretive challenges, but they also provide an invaluable opportunity to characterize the spectrum and importance of rare variation. METHODS: We analyzed sequence data from 7,855 clinical cardiomyopathy cases and 60,706 Exome Aggregation Consortium (ExAC) reference samples to obtain a better understanding of genetic variation in a representative autosomal dominant disorder. RESULTS: We found that in some genes previously reported as important causes of a given cardiomyopathy, rare variation is not clinically informative because there is an unacceptably high likelihood of false-positive interpretation. By contrast, in other genes, we find that diagnostic laboratories may be overly conservative when assessing variant pathogenicity. CONCLUSIONS: We outline improved analytical approaches that evaluate which genes and variant classes are interpretable and propose that these will increase the clinical utility of testing across a range of Mendelian diseases.Genet Med 19 2, 192-203.

Synthesis and anticancer activity of a hydroxytolan series.

This paper describes the development of novel anticancer poly-hydroxylated tolans. Based on structural similarity to resveratrol, a series of hydroxytolans were synthesized and evaluated for their antitumor capability against three tumor cell lines and one fibroblast cell line for selectivity comparisons. The 4,4'-dihydroxytolan (KST-201) exhibited the most significant anticancer activity with increased selectivity when compared to resveratrol and other hydroxytolans. Unlike resveratrol, KST-201 can boost hydrogen peroxide in tumor cells, which are often at high basal level of reactive oxygen species, to cause cell death by overwhelming the cellular tolerance of oxidative stress.

A rare, human prostate oncocyte cell originates from the prostatic carcinoma (DU145) cell line.

DU145 human prostate carcinoma cells are typically poorly differentiated and contain only scantily distributed organelles. However, among numerous tumor cells randomly examined by electron microscopy out of in vitro cultivation, a peculiar, rare oncocyte-like cell type has been observed whose nucleus appears to be of small dimension and with a cytoplasm almost entirely filled with often distorted mitochondria. A few small, dispersed lysosomal bodies, small cisterns of the endoplasmic reticulum and a few glycogen patches can be found among highly osmiophilic contrasted, cytosolic spaces filled by innumerable ribonucleoproteins. The excessive population of mitochondria may have arisen from a more populated tumor cell type wherein the altered mitochondria are found to appear burgeoning into a spherical-like size progeny crowding the tumor cells. Literature cited between 1950 and the present suggests that this rare, oncocytic, benign prostatic tumor cell type is likely appear epigenetically, stemming from an original secretory cell, which is confirmed by the origin of the cell line originally maintained as cell line out of a brain metastatic, adenocarcinoma niche.

The Voice of Mothers Who Continue to Express Milk After Their Infant's Death for Donation to a Milk Bank.

Background: The second stage of lactation with copious milk production occurs after birth regardless of the infant's survival. Previous research indicates that milk donation following a perinatal loss may help some bereaved mothers come to terms with their loss. The purpose of this study was to explore the experience of women choosing to continue to express milk after a perinatal loss specifically for donation to a nonprofit milk bank governed by the Human Milk Banking Association of North America (HMBANA). Materials and Methods: Participants were recruited through HMBANA's milk bank directors' listserv, their bereavement committee, and through their website. Participants were eligible if they donated to an HMBANA milk bank and specifically continued to pump milk for the purpose of milk donation following a perinatal loss. Qualitative interviews were conducted with each participant using a secured web-based platform. Data collection and analysis occurred concurrently using qualitative content analysis until there was acknowledged informational redundancy. Participants' demographic and lactation data were collected and analyzed using descriptive statistics. Results: Over 10 months, 21 participants were interviewed. Donating after perinatal loss has been described as a positive, valuable, and nurturing experience. Diminished grieving, enhanced connection to the infant, establishment of legacy, and creation of a positive from a negative are highlighted in several themes that illuminate the phenomena of bereaved donation. Discussion: The findings of this research are consistent with published literature, but in this study, two subgroups were formed. This enabled the researchers to compare the experience of those with previous breastfeeding experience and those without, which adds to the knowledge about the phenomenon. It provides information for health care professionals (HCPs) to understand the experience of bereaved milk donors and serves as a call to action for HCPs to develop best practices and incorporate lactation management in enhanced, individualized bereavement care for these women.

Provocation Testing and Therapeutic Response in a Newly Described Channelopathy: RyR2 Calcium Release Deficiency Syndrome.

BACKGROUND: A novel familial arrhythmia syndrome, cardiac ryanodine receptor (RyR2) calcium release deficiency syndrome (CRDS), has recently been described. We evaluated a large and well characterized family to assess provocation testing, risk factor stratification and response to therapy in CRDS. METHODS: We present a family with multiple unheralded sudden cardiac deaths and aborted cardiac arrests, primarily in children and young adults, with no clear phenotype on standard clinical testing. RESULTS: Genetic analysis, including whole genome sequencing, firmly established that a missense mutation in RYR2, Ala4142Thr, was the underlying cause of disease in the family. Functional study of the variant in a cell model showed RyR2 loss-of-function, indicating that the family was affected by CRDS. EPS (Electrophysiological Study) was undertaken in 9 subjects known to carry the mutation, including a survivor of aborted sudden cardiac death, and the effects of flecainide alone and in combination with metoprolol were tested. There was a clear gradation in inducibility of nonsustained and sustained ventricular arrhythmia between subjects at EPS, with the survivor of aborted sudden cardiac death being the most inducible subject. Administration of flecainide substantially reduced arrhythmia inducibility in this subject and abolished arrhythmia in all others. Finally, the effects of additional metoprolol were tested; it increased inducibility in 4/9 subjects. CONCLUSIONS: The Ala4142Thr mutation of RYR2 causes the novel heritable arrhythmia syndrome CRDS, which is characterized by familial sudden death in the absence of prior symptoms or a recognizable phenotype on ambulatory monitoring or exercise stress testing. We increase the experience of a specific EPS protocol in human subjects and show that it is helpful in establishing the clinical status of gene carriers, with potential utility for risk stratification. Our data provide evidence that flecainide is protective in human subjects with CRDS, consistent with the effect previously shown in a mouse model.

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy.

Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cells by a strongly absorbing dye present in the extracellular medium. Cells are placed in a shallow compartment, and a nontoxic and cell-impermeant dye, such as acid blue 9, is added to the medium. Transmission images are collected at the wavelength of maximum dye absorption (630 nm). Where the cell body displaces the dye, the thickness of the absorbing layer is reduced; thus, an increase in cell thickness produces brighter images and vice versa. The absolute values for cell thickness and volume can be easily extracted from the image by computing the logarithm of intensity and dividing it by the absorption coefficient. The method is fast, impervious to instability of the light source, and has a high signal-to-noise ratio; it can be realized either on a laser scanning or a conventional microscope equipped with a bandpass filter. For long-term experiments, we use a Bioptechs perfusion chamber fitted with a 0.03-mm spacer and an additional port to enable rapid switching of solutions. To show possible applications of this method, we investigated the kinetics of the cell volume response to a hypotonic buffer and to the apoptotic agents staurosporine and ionomycin.

Reevaluation of the South Asian MYBPC3Δ25bp Intronic Deletion in Hypertrophic Cardiomyopathy.

BACKGROUND: The common intronic deletion, MYBPC3Δ25, detected in 4% to 8% of South Asian populations, is reported to be associated with cardiomyopathy, with ≈7-fold increased risk of disease in variant carriers. Here, we examine the contribution of MYBPC3Δ25 to hypertrophic cardiomyopathy (HCM) in a large patient cohort. METHODS: Sequence data from 2 HCM cohorts (n=5393) was analyzed to determine MYBPC3Δ25 frequency and co-occurrence of pathogenic variants in HCM genes. Case-control and haplotype analyses were performed to compare variant frequencies and assess disease association. Analyses were also undertaken to investigate the pathogenicity of a candidate variant MYBPC3 c.1224-52G>A. RESULTS: Our data suggest that the risk of HCM, previously attributed to MYBPC3Δ25, can be explained by enrichment of a derived haplotype, MYBPC3Δ25/-52, whereby a small subset of individuals bear both MYBPC3Δ25 and a rare pathogenic variant, MYBPC3 c.1224-52G>A. The intronic MYBPC3 c.1224-52G>A variant, which is not routinely evaluated by gene panel or exome sequencing, was detected in ≈1% of our HCM cohort. CONCLUSIONS: The MYBPC3 c.1224-52G>A variant explains the disease risk previously associated with MYBPC3Δ25 in the South Asian population and is one of the most frequent pathogenic variants in HCM in all populations; genotyping services should ensure coverage of this deep intronic mutation. Individuals carrying MYBPC3Δ25 alone are not at increased risk of HCM, and this variant should not be tested in isolation; this is important for the large majority of the 100 million individuals of South Asian ancestry who carry MYBPC3Δ25 and would previously have been declared at increased risk of HCM.

A 12 week, open label, phase I/IIa study using apatone for the treatment of prostate cancer patients who have failed standard therapy.

PURPOSE: To evaluate the safety and efficacy of oral Apatone (Vitamin C and Vitamin K3) administration in the treatment of prostate cancer in patients who failed standard therapy. MATERIALS AND METHODS: Seventeen patients with 2 successive rises in PSA after failure of standard local therapy were treated with (5,000 mg of VC and 50 mg of VK3 each day) for a period of 12 weeks. Prostate Specific Antigen (PSA) levels, PSA velocity (PSAV) and PSA doubling times (PSADT) were calculated before and during treatment at 6 week intervals. Following the initial 12 week trial, 15 of 17 patients opted to continue treatment for an additional period ranging from 6 to 24 months. PSA values were followed for these patients. RESULTS: At the conclusion of the 12 week treatment period, PSAV decreased and PSADT increased in 13 of 17 patients (p < or = 0.05). There were no dose-limiting adverse effects. Of the 15 patients who continued on Apatone after 12 weeks, only 1 death occurred after 14 months of treatment. CONCLUSION: Apatone showed promise in delaying biochemical progression in this group of end stage prostate cancer patients.

The relationship between weight gain and blood pressure in children and adolescents.

BACKGROUND: The aim of this study was to determine the relationship between the trajectories of weight gain and systolic blood pressure (SBP) in youth. METHODS: Annual surveys of anthropometry, fitness, SBP, and its determinants (cardiac output, systemic vascular resistance, and arterial compliance) were conducted in youth (aged 5 to 19 years) in a school-based setting between 2004 and 2006. Children were stratified according to change in body mass index (BMI) over time. RESULTS: Within the entire cohort (n = 2089), mean SBP (121 +/- 16 SD v 112 +/- 15 SD mm Hg; P < .01) and the prevalence of high blood pressure (48% v 18%, P < .01) were significantly higher and fitness levels were lower (P < .01) in obese children, relative to healthy-weight peers. After 2 years of follow-up, despite similar SBP and BMI at baseline, the average change in SBP was approximately 4.5-fold greater in children with the largest increase in BMI, relative to children who experienced minimal weight gain. This group also experienced a significantly greater increase in stroke volume (P < .05), while the change in heart rate, arterial compliance, and systemic vascular resistance was comparable with that of children who experienced minimal weight gain. Multiple linear regression analysis revealed that SBP increased 0.77 mm Hg for every kilogram of weight gain over a period of 2 years (P < .01). CONCLUSIONS: Overweight and disproportionate weight gain in children are associated with elevated SBP. These data support the need for interventions to prevent excessive weight gain and obesity in children and adolescents.

Transforming a care delivery model to increase breastfeeding.

This article describes the process of changing the care delivery model for maternity practice in a New York State Regional Perinatal Center to support exclusive breastfeeding, defined as providing nothing other than human milk feedings. Barriers exist in hospitals that inhibit exclusive breastfeeding of newborns at the time of discharge and fail to meet the recommendations outlined by the World Health Organization and New York State Department of Health. All aspects of mother/baby care were evaluated to meet the recommendations and increase exclusive breastfeeding. Transforming the care delivery model for mothers and babies began in 2010 with an invitation to participate in the New York State Breastfeeding Quality Improvement in Hospitals Learning Collaborative. Twelve hospitals were selected to participate with the following objectives: increase exclusive breastfeeding; improve hospital breastfeeding policies, practices, and systems that are consistent with New York State hospital regulations, laws and recommended best practices; increase staff skills and knowledge of breastfeeding and lactation support through education; empower, educate, and support new mothers to successfully breastfeed and change the culture and social norm relative to breastfeeding. The transformation of the care delivery model resulted in an increase in exclusive breastfeeding from 6% to 44%.

Nucleolar changes and fibrillarin redistribution following apatone treatment of human bladder carcinoma cells.

Ascorbate and menadione (Apatone) in a ratio of 100:1 kills tumor cells by autoschizis. In this study, vitamin-induced changes in nucleolar structure were evaluated as markers of autoschizis. Human bladder carcinoma (T24) cells were overlain with vitamins or with culture medium. Supernatants were removed at 1-hr intervals from 1 to 4 hr, and the cells were washed with PBS and prepared for assay. Apatone produced marked alterations in nucleolar structure including redistribution of nucleolar components, formation of ring-shaped nucleoli, condensation and increase of the proportion of perinucleolar chromatin, and the enlargement of nucleolar fibrillar centers. Immunogold labeling of the nucleolar rRNA revealed a granular localization in treated and sham-treated cells, and immunogold labeling of the rDNA revealed a shift from the fibrillar centers to the condensed perinucleolar chromatin. Fibrillarin staining shifted from the fibrillar centers and adjacent regions to a more homogeneous staining of the entire nucleolus and was consistent with the percentage of autoschizic cells detected by flow cytometry. Because autoschizis entails sequential reactivation of DNase I and DNase II, and because the fibrillarin redistribution following DNase I and Apatone treatment is identical, it appears that the nucleolar and fibrillarin changes are markers of autoschizis.