Analysis of cell-free fetal DNA for non-invasive prenatal diagnosis in a family with neonatal diabetes.

AIMS: An early genetic diagnosis of neonatal diabetes guides clinical management and results in improved treatment in ~ 40% of patients. In the offspring of individuals with neonatal diabetes, a prenatal diagnosis allows accurate estimation of the risk of developing diabetes and, eventually, the most appropriate treatment for the baby. In this study, we performed non-invasive prenatal genetic testing for a fetus at risk of inheriting a paternal KCNJ11 p.R201C mutation causing permanent neonatal diabetes. METHODS: A droplet digital polymerase chain reaction assay was used to detect the presence of the mutation in cell-free circulating DNA (cfDNA) extracted from maternal plasma at 12 and 16 weeks' gestation. RESULTS: The mutation was not detected in the cfDNA samples, suggesting that the fetus had not inherited the KCNJ11 mutation. The fetal DNA fraction was estimated at 6.2% and 10.7%, which is above the detection limit of the assay. The result was confirmed by Sanger sequencing after the baby's birth, confirming that the baby's risk of developing neonatal diabetes was reduced to that of the general population. CONCLUSIONS: We report the first case of non-invasive prenatal testing in a family with neonatal diabetes. A prenatal diagnosis in families at high risk of monogenic diabetes informs both prenatal and postnatal management. Although the clinical impact of this novel technology still needs to be assessed, its implementation in clinical practice (including cases at risk of inheriting mutations from the mother) will likely have a positive impact upon the clinical management of families affected by monogenic diabetes.

A CACNA1D mutation in a patient with persistent hyperinsulinaemic hypoglycaemia, heart defects, and severe hypotonia.

Congenital hyperinsulinaemic hypoglycaemia (HH) can occur in isolation or it may present as part of a wider syndrome. For approximately 40%-50% of individuals with this condition, sequence analysis of the known HH genes identifies a causative mutation. Identifying the underlying genetic aetiology in the remaining cases is important as a genetic diagnosis will inform on recurrence risk, may guide medical management and will provide valuable insights into ß-cell physiology. We sequenced the exome of a child with persistent diazoxide-responsive HH, mild aortic insufficiency, severe hypotonia, and developmental delay as well as the unaffected parents. This analysis identified a de novo mutation, p.G403D, in the proband's CACNA1D gene. CACNA1D encodes the main L-type voltage-gated calcium channel in the pancreatic ß-cell, a key component of the insulin secretion pathway. The p.G403D mutation had been reported previously as an activating mutation in an individual with primary hyper-aldosteronism, neuromuscular abnormalities, and transient hypoglycaemia. Sequence analysis of the CACNA1D gene in 60 further cases with HH did not identify a pathogenic mutation. Identification of an activating CACNA1D mutation in a second patient with congenital HH confirms the aetiological role of CACNA1D mutations in this disorder. A genetic diagnosis is important as treatment with a calcium channel blocker may be an option for the medical management of this patient.

Improved genetic testing for monogenic diabetes using targeted next-generation sequencing.

AIMS/HYPOTHESIS: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. METHODS: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested--32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. RESULTS: In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. CONCLUSIONS/INTERPRETATION: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate.

Emerging sexually transmitted viral infections: 2. Review of Zika virus disease.

A sudden increase in the number of newborn infants with microcephaly in Brazil in 2015 brought Zika virus (ZIKV), a less-known infection, to public attention. The rapid increase in the number of cases across the Americas and the devastating complications of infection with ZIKV highlighted the gravity of the situation. Within a relatively short period of time, our knowledge of this infection has significantly increased. This includes the realisation that ZIKV can be sexually transmitted. The aim of the present article is to provide a concise summary on this novel sexually transmitted infection linked to human birth defects and Guillain-Barre Syndrome. According to World Health Organization, individuals living outside areas of ZIKV mosquito transmission where one or both partners have been exposed to ZIKV should abstain from sex or have sex with condoms for at least six months after the last day of possible exposure.

Phenotype of CNTNAP1: a study of patients demonstrating a specific severe congenital hypomyelinating neuropathy with survival beyond infancy.

CHN is genetically heterogeneous and its genetic basis is difficult to determine on features alone. CNTNAP1 encodes CASPR, integral in the paranodal junction high molecular mass complex. Nineteen individuals with biallelic variants have been described in association with severe congenital hypomyelinating neuropathy, respiratory compromise, profound intellectual disability and death within the first year. We report 7 additional patients ascertained through exome sequencing. We identified 9 novel CNTNAP1 variants in 6 families: three missense variants, four nonsense variants, one frameshift variant and one splice site variant. Significant polyhydramnios occurred in 6/7 pregnancies. Severe respiratory compromise was seen in 6/7 (tracheostomy in 5). A complex neurological phenotype was seen in all patients who had marked brain hypomyelination/demyelination and profound developmental delay. Additional neurological findings included cranial nerve compromise: orobulbar dysfunction in 5/7, facial nerve weakness in 4/7 and vocal cord paresis in 5/7. Dystonia occurred in 2/7 patients and limb contractures in 5/7. All had severe gastroesophageal reflux, and a gastrostomy was required in 5/7. In contrast to most previous reports, only one patient died in the first year of life. Protein modelling was performed for all detected CNTNAP1 variants. We propose a genotype-phenotype correlation, whereby hypomorphic missense variants partially ameliorate the phenotype, prolonging survival. This study suggests that biallelic variants in CNTNAP1 cause a distinct recognisable syndrome, which is not caused by other genes associated with CHN. Neonates presenting with this phenotype will benefit from early genetic definition to inform clinical management and enable essential genetic counselling for their families.

Microdosimetry of inhomogeneous radon progeny distributions in bronchial airways.

A Monte Carlo code, initially developed for the calculation of microdosimetric spectra for alpha particles in cylindrical airways, has been extended to allow the computation (i) of additional microdosimetric parameters and (ii) for realistic exposure conditions in human bronchial airways with respect to surface activity distribution and airway geometry. The objective of the present study was to investigate the effects of non-uniform distributions of radon progeny activities in bronchial airways on cellular energy deposition parameters. Significant variations of hit frequencies, doses and microscopic energy deposition patterns were observed for epithelial cell nuclei, depending strongly on the assumed activity distributions. Thus, epithelial cells located at different positions in a given bronchial airway may experience a wide range of biological responses. The results obtained suggest that the hit frequency may be the primary physical parameter for alpha particles, supplemented by microdosimetric single event spectra, to be related to biological effects for chronic low level exposures.

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase.

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.

Referral of patients with pain from pancreatic cancer for neurolytic celiac plexus block.

OBJECTIVE: To assess whether patients with pancreatic cancer-associated pain living near a pain control center were more likely to undergo neurolytic celiac plexus block (NCPB) than those living at a distance and to determine the rationale of physicians at our institution for referring patients for NCPB. DESIGN: We retrospectively reviewed the frequency of use of NCPB in patients with pancreatic cancer and conducted an anonymous physician survey of referral patterns for NCPB for such patients. MATERIAL AND METHODS: A prospective database of medical diagnoses and a clinical database at our institution were used to identify patients with pancreatic cancer within three geographic regions who were assessed during the inclusive years 1980 through 1989: group I ("local") = all patient with pancreatic cancer in Olmsted County, Minnesota; group II ("surrounding") and group III ("distant") = patients referred for pancreatic cancer evaluation who lived within 100 miles of our institution (excluding Olmsted County) or more than 100 miles from our institution, respectively. Medical records were retrospectively reviewed to assess the use of NCPB at any time during the course of pancreatic cancer. For the physician survey component, all medical oncologists, gastroenterologists, and general surgeons at our institution who might be responsible for the care of patients with pancreatic cancer were sent a questionnaire about their referral patterns for NCPB among patients with pancreatic cancer. RESULTS: Overall, approximately 15% of the 292 patients with pancreatic cancer studied underwent NCPB. Distance from our pain control center was not found to be associated with frequency of use of NCPB. Of the 78 physicians surveyed, 59 (76%) responded, and 35 of the responders (59%) had encountered at least 1 patient with pancreatic cancer during the preceding 12 months. In that subset of physicians, perceived barriers for referral for NCPB were limited appointment availability and need for repeating the procedure. CONCLUSION: On the basis of this study, referral patterns for NCPB in patients with pancreatic cancer do not seem to be associated with the geographic distance of a patient's residence from a pain control center. Improving appointment availability for NCPB might increase the number of patients offered this technique for control of pain.

A consistent set of neutron kerma coefficients from thermal to 150 MeV for biologically important materials.

Neutron cross sections for nonelastic and elastic reactions on a range of elements have been evaluated for incident energies up to 150 MeV. These cross sections agree well with experimental cross section data for charged-particle production as well as neutron and photon production. Therefore they can be used to determine kerma coefficients for calculations of energy deposition by neutrons in matter. Methods used to evaluate the neutron cross sections above 20 MeV, using nuclear model calculations and experimental data, are described. Below 20 MeV, the evaluated cross sections from the ENDF/B-VI library are adopted. Comparisons are shown between the evaluated charged-particle production cross sections and measured data. Kerma coefficients are derived from the neutron cross sections, for major isotopes of H, C, N, O, Al, Si, P, Ca, Fe, Cu, W, Pb, and for ICRU-muscle, A-150 tissue-equivalent plastic, and other compounds important for treatment planning and dosimetry. Numerous comparisons are made between our kerma coefficients and experimental kerma coefficient data, to validate our results, and agreement is found to be good. An important quantity in neutron dosimetry is the kerma coefficient ratio of ICRU-muscle to A-150 plastic. When this ratio is calculated from our kerma coefficient data, and averaged over the neutron energy spectra for higher-energy clinical therapy beams [three p (68) + Be beams, and a d (48.5) + Be beam], a value of 0.94 +/- 0.03 is obtained. Kerma ratios for water to A-150 plastic, and carbon to oxygen, are also compared with measurements where available.

Cost analysis of the Ohio nursing home industry.

This study was part of a major review of long-term care policy in the state of Ohio. The authors analyzed 1532 cost reports filed by nursing homes in 1975-1976 with the Ohio Medical Assistance (Medicaid) program. The objective was to guide policy on size (economies of scale), ownership, certification status, and reimbursement. Economies of scale were not found important: skilled nursing facilities (SNFs) offered the only evidence of operation below optimal scale, and the savings attributable to achieving optimal scale (increasing average bed size from 108 to 143) amounted to only $0.20 per patient day. Proprietary facilities were consistently less costly than voluntary or governmental facilities; however, quality measures were not available, and the largest cost differential was in direct cost where quality might be affected. Hypothesized greater efficiency in proprietary facilities could not be rejected--if accurate, the cost savings were very large ($3.92 to $9.14 per patient day for all homes together). As expected, skilled facilities were more costly than intermediate care facilities (ICFs), and the differential ($3.31 per patient day) was large enough to suggest transfer of misplaced patients. High proportional Medicaid utilization of a home tended to reduce cost, possibly because of the very low ceiling rates paid by the Ohio Medicaid program during the period of this study (1975-76 data). High utilization in general reduced average cost, presumably by spreading fixed cost.

Determinants of dental user groups among an elderly, low-income population.

OBJECTIVE: We test whether or not there are differences for selected variables among five dental user groups and one nondental group within an elderly, low-income population. DATA SOURCE: We used ten years of Medicare Part B claims data from the Cincinnati Health Department for all clinic users 62 years of age and older who participated in the Municipal Health Services Program. STUDY DESIGN: A polychotomous logistic regression model determined the ability to differentiate between the groups for each of the selected variables, controlling for race. Next, a polychotomous stepwise logistic regression was used in finding a multivariate model for determining dental user group membership. Logistic regression was used to ascertain which variables were discriminators between any two types of dental users. PRINCIPAL FINDINGS: Mean number of medical visits, mean number of prescriptions filled, and race are determinants of group membership, with the nondental group having more medical visits and more likely to be white. Although year of birth cohort is statistically significant in determining dental user types, the direction of effect is not constant across the comparisons. However, the relative risk for being in the two complete denture groups, compared to both compliant subgroups, increases with each older cohort. CONCLUSIONS: Higher levels of medical use may "crowd out" dental use, even when it is without user cost, either because the medical problems are treated as a higher priority, or because dealing with medical needs leaves too little perceived time or energy to seek dental care. Even in a low-income population seeking dental care, there appears to be a birth cohort effect with a decline in the younger elderly who require two complete dentures.

Analysis of protein-protein interactions during cytomegalovirus infection.

A key area in the study of infection by cytomegalovirus (CMV), or that of any other virus, is to gain an understanding of the manner in which viral proteins interact with those of the host cell. The most widely used method to identify interactions between viral and cellular proteins in the infected cell is that of co-immunoprecipitation; lysates from infected cells are treated with antibody which recognises, say, a viral protein of interest, and the resulting immune complexes are then screened for the presence of a cellular protein of interest: the presence of the second protein in the immune complexes is indicative of an interaction between the two proteins in vivo. However, such interaction need not necessarily be direct, as immunoprecipitation of a viral protein that could interact with a single component of a multiprotein complex might be expected to co-precipitate all the proteins in that complex. Therefore assays might be said to demonstrate protein-protein associations in the cell, rather than direct interactions. The resolving power of co-immunoprecipitations is also limited in other respects. First, the success of the assay relies heavily on the quality of the immunological reagents used; it is also possible that antibody binding will actually disrupt the interaction to be studied.

Predictors of dental use for low-income, urban elderly persons upon removal of financial barriers.

This study examines a low-income, urban elderly population of dental and medical, nondental users. A total of 1,378 medical, nondental users and 2,086 dental users were identified using longitudinal claims data (1983-1992) from a Medicare-waiver program that reimbursed for health care services at cost. Dental users were more likely to be from a younger age cohort (born after 1910, p = .0001) and were more likely to be black (63.3% vs 35.7%, P = .0001) than medical, nondental users. Medical, nondental users had more medical visits (p = .0001), higher medical and pharmacy charges (p = .0001), and more prescriptions (p = .0001) than did the dental users. These findings indicate that among this population of urban elderly, dental users were more likely to be black and have lower medical utilization than nondental users.

Measuring the impact of P.L. 99-252: an economist's view.

This paper examines ways in which standard economic analysis may be used to predict and evaluate the impact of P.L. 99-252, the Comprehensive Smokeless Tobacco Health Education Act of 1986. Simple supply and demand models are used to illustrate the range of possible effects of the act. Analysis indicates that the act should reduce demand, though the magnitude of the impact can only be determined empirically. Given that the act affects only one of the principal determinants of demand, consumer preferences, the impact may be rather limited in magnitude. The impact on supply is more ambiguous, but it is possible that restrictions on advertising might lower the smokeless tobacco producers' costs in ways that would lead to an increase in supply. Several suggestions for empirical measurement of impact are made. Three other issues are identified and discussed: potential negative consequences of the act's required health warnings (as a liability defense for firms); the importance of point of view in dealing with the costs of smokeless tobacco use; and a comparison of the act to alternative strategies to reduce smokeless tobacco use, such as taxation.

Effects of selenium and copper supplementation on the growth of beef steers.

During the grazing seasons of 1978 and 1979, 126 Hereford cross Friesian and 25 Charolais cross Friesian steers were used in controlled trials of the effects of injecting them with copper and, or, selenium. In both seasons the unsupplemented steers had low blood concentrations of copper, selenium and glutathione peroxidase, whereas the supplemented steers maintained their serum copper concentrations within the normal range and had significantly higher whole blood concentrations of selenium and glutathione peroxidase than the unsupplemented animals. Supplementing the steers with 400 mg copper during 1978 increased their growth rate by 0.032 kg/day and supplementing them with 200 mg copper during 1979 increased it by 0.080 kg/day. Supplementing the steers in each year with two doses of selenium, each of 0.15 mg selenium/kg bodyweight, increased their growth rate by 0.041 kg/day in 1978 and by 0.060 kg/day in 1979. There was no interaction between the selenium and copper treatments and the total increases in liveweight gains due to both supplements were around 11 kg in 1978 and 16 kg in 1979.

Modeling energy deposition and cellular radiation effects in human bronchial epithelium by radon progeny alpha particles.

Energy deposition and cellular radiation effects arising from the interaction of single 218Po and 214Po alpha particles with basal and secretory cell nuclei were simulated for different target cell depths in the bronchial epithelium of human airway generations 2, 4, 6, and 10. To relate the random chord lengths of alpha particle tracks through spherical cell nuclei to the resulting biological endpoints, probabilities per unit track length for different cellular radiation effects as functions of LET were derived from in vitro experiments. The radiobiological data employed in the present study were inactivation and mutation (mutant frequency at the HPRT gene) in V79 Chinese hamster cells and inactivation and transformation in C3H 10T1/2 cells. Based on computed LET spectra and relative frequencies of target cells, probabilities for transformation, mutation, and cell killing in basal and secretory cells were computed for a lifetime exposure of 20 WLM. While predicted transformation probabilities were about two orders of magnitude higher than mutation probabilities, they were still about two orders of magnitude lower than inactivation probabilities. Furthermore transformation probabilities for basal cells are generally higher than those for secretory cells, and 214Po alpha particles are primarily responsible for transformations in bronchial target cells.

Energy deposition, cellular radiation effects and lung cancer risk by radon progeny alpha particles.

Slowing down spectra, LET spectra, hit probabilities, and radiation doses were simulated for the interaction of single 218Po and 214Po alpha particles with sensitive basal and secretory cell nuclei in the bronchial epithelium of human and rat lungs for defined exposure conditions. Probabilities per unit track length for transformation, derived from in vitro experiments with C3H 10T1/2 cells, were used to estimate transformation probabilities for randon progeny alpha particles in basal and secretory cells. Different weighting schemes were assumed to relate cellular hit probabilities, doses and transformation probabilities, obtained for different cell depths and airway generations, to lung cancer risk per unit exposure. In vitro transformation and in vivo lung cancer incidence were simulated by a state-vector model which provides a stochastic formulation of dose-rate dependent cellular transitions related to formation of double strand breaks, repair, inactivation, stimulated mitosis and promotion through loss of intercellular communication.