Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, genotype-phenotype correlations and common mechanisms.

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.

Is vaccination a viable method to control Johne's disease caused by Mycobacterium avium subsp. paratuberculosis? Data from 12 million ovine vaccinations and 7.6 million carcass examinations in New South Wales, Australia from 1999-2009.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne's disease is increasing worldwide. In an attempt to control an epidemic of ovine Johne's disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naïve sheep population. METHODS: NSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair®) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology. PRINCIPAL FINDINGS: From 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009, the target commonly espoused for an effective control program based on vaccination. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks. SIGNIFICANCE: We show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of ≤ 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions.

Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johne disease, is a slow growing mycobacterium. Viable MAP detection is difficult, inconstant and time-consuming. The purpose of this study was to compare a rapid phage/qPCR assay performed on peripheral blood mononuclear cells (PBMCs) with three standard methods of MAP detection: fecal MAP PCR; plasma antigen-specific IFN-γ & serum MAP ELISA hypothesizing that, if sensitive and specific, Johne animals would be positive and Control animals negative. We studied a well characterized herd of Holstein cattle that were naturally infected with MAP and their Controls. RESULTS: With phage/qPCR 72% (23/32) of Johne and 35% (6/17) of Controls were MAP positive. With fecal PCR 75% (24/32) of Johne and 0% (0/17) of Controls were MAP positive. With plasma antigen-specific IFN-γ 69% (22/32) of Johne and 12% (2/17) of Controls were MAP positive. With serum MAP ELISA, 31% (10/32) of Johne and 0% (0/17) of Controls were MAP positive. When phage / qPCR and fecal PCR results were combined, 100% (32/32) Johne and 35% (6/17) of Control animals were MAP positive. Younger Control animals (1-3 years) had significantly fewer plaques (25 ± 17 SEM) than older Controls (4-12 years) (309 ± 134 p = 0.04). The same trend was not observed in the Johne animals (p = 0.19). CONCLUSIONS: In contrast to our hypothesis, using the phage/qPCR assay we find that viable circulating MAP can rapidly be detected from the blood of animals infected with, as well as those in the Control group evidently colonized by MAP. These data indicate that the presence of viable MAP in blood does not necessarily signify that an animal must of necessity be demonstrably ill or be MAP positive by standard diagnostic methods.

Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease.

OBJECTIVES: Although controversial, there is increasing concern that Crohn's disease may be a zoonotic infectious disease consequent to a mycobacterial infection. The most plausible candidate is M. avium subspecies paratuberculosis (MAP) that is unequivocally responsible for Johne's disease in ruminants. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Non-identifiable intestine from patients with documented Crohn's disease was assayed according to the manufacturer's instructions and with suggested modifications. Probes were custom designed for MAP and human ß-actin (as the eukaryotic housekeeping gene) from published genomes. RESULTS: Repetitively, false positive signal was observed in our "No-Probe" negative control. Attempts were made to correct this according to the manufacturer's suggestions (by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters). None prevented false positive signal in the "No-Probe" control. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen resected intestine of humans with Crohn's disease.

Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay.

OBJECTIVES: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants. The "gold standard" of MAP detection is by culture, DNA sequencing possibly supplemented by identification of Ziehl-Neelsen positive mycobacteria. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Intestine from a steer with documented Johne's disease was assayed according to the manufacturer's instructions. Probes were custom designed for MAP and bovine ß-actin (as the eukaryotic housekeeping gene) from published genomes. We attempt to prevent false positive signal in the "no-probe" control, by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and "Hope" for Cy-5). RESULTS: Repetitively, false positive signal was observed in our "no probe" negative control. Attempts to correct this according to the manufacturers suggestions, and with multiple derivative techniques have been unsuccessful. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen intestine of cattle with Johne's disease.

Clinical course of sly syndrome (mucopolysaccharidosis type VII).

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is an ultra-rare disease characterised by the deficiency of ß-glucuronidase (GUS). Patients' phenotypes vary from severe forms with hydrops fetalis, skeletal dysplasia and mental retardation to milder forms with fewer manifestations and mild skeletal abnormalities. Accurate assessments on the frequency and clinical characteristics of the disease have been scarce. The aim of this study was to collect such data. METHODS: We have conducted a survey of physicians to document the medical history of patients with MPS VII. The survey included anonymous information on patient demographics, family history, mode of diagnosis, age of onset, signs and symptoms, severity, management, clinical features and natural progression of the disease. RESULTS: We collected information on 56 patients from 11 countries. Patients with MPS VII were classified based on their phenotype into three different groups: (1) neonatal non-immune hydrops fetalis (NIHF) (n=10), (2) Infantile or adolescent form with history of hydrops fetalis (n=13) and (3) Infantile or adolescent form without known hydrops fetalis (n=33). Thirteen patients with MPS VII who had the infantile form with history of hydrops fetalis and survived childhood, had a wide range of clinical manifestations from mild to severe. Five patients underwent bone marrow transplantation and one patient underwent enzyme replacement therapy with recombinant human GUS. CONCLUSIONS: MPS VII is a pan-ethnic inherited lysosomal storage disease with considerable phenotypical heterogeneity. Most patients have short stature, skeletal dysplasia, hepatosplenomegaly, hernias, cardiac involvement, pulmonary insufficiency and cognitive impairment. In these respects it resembles MPS I and MPS II. In MPS VII, however, one unique and distinguishing clinical feature is the unexpectedly high proportion of patients (41%) that had a history of NIHF. Presence of NIHF does not, by itself, predict the eventual severity of the clinical course, if the patient survives infancy.

Rejecting Kentucky Medicaid proposal would reaffirm commitment to full coverage.

Kentucky Gov. Matt Bevin, representing a state where the uninsured rate has plummeted from 14.3% to 6% since adopting the Medicaid expansion under the Affordable Care Act, is proposing sweeping Medicaid changes that would make it harder for Kentuckians to keep their coverage. He threatens to cancel the Medicaid expansion if HHS doesn't approve his plan.

Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications.

BACKGROUND: The development of novel antibiotics to treat multidrug-resistant (MDR) tuberculosis is time-consuming and expensive. Multiple immune modulators, immune suppressants, anti-inflammatories, and growth enhancers, and vitamins A and D, inhibit Mycobacterium avium subspecies paratuberculosis (MAP) in culture. We studied the culture inhibition of Mycobacterium tuberculosis complex by these agents. METHODS: Biosafety level two M. tuberculosis complex (ATCC 19015 and ATCC 25177) was studied in radiometric Bactec or MGIT culture. Agents evaluated included clofazimine, methotrexate, 6-mercaptopurine, cyclosporine A, rapamycin, tacrolimus, monensin, and vitamins A and D. RESULTS: All the agents mentioned above caused dose-dependent inhibition of the M. tuberculosis complex. There was no inhibition by the anti-inflammatory 5-aminosalicylic acid, which causes bacteriostatic inhibition of MAP. CONCLUSIONS: We conclude that, at a minimum, studies with virulent M. tuberculosis are indicated with the agents mentioned above, as well as with the thioamide 5-propothiouricil, which has previously been shown to inhibit the M. tuberculosis complex in culture. Our data additionally emphasize the importance of vitamins A and D in treating mycobacterial diseases.

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture.

BACKGROUND: The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria. METHODS: The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex). PRINCIPAL FINDINGS: Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, ß-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain. SIGNIFICANCE: We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.

A mosaic activating mutation in AKT1 associated with the Proteus syndrome.

BACKGROUND: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

Attitudes toward direct-to-consumer advertisements and online genetic testing among high-risk women participating in a hereditary cancer clinic.

Genetic testing for the breast cancer genes 1/2 (BRCA 1/2) has helped women determine their risk of developing breast and ovarian cancer. As interest in genetic testing has grown, companies have created strategies to disseminate information about testing, including direct-to-consumer advertising (DTCA) and online genetic testing. This study examined attitudes toward DTCA and online testing for BRCA among 84 women at a high-risk clinic as well as additional factors that may be associated with these attitudes, such as personal and familial cancer history, cancer worry and risk perception, and history with genetic testing/counseling. Results showed that the majority of the women held favorable attitudes toward DTCA for BRCA testing but did not support online testing. Factors such as familial ovarian cancer, cancer worry, and satisfaction with genetic counseling/testing were associated with positive attitudes toward DTCA, whereas personal breast cancer history was related to negative attitudes. The findings suggest that women may view DTCA as informational but rely on physicians for help in their decision to undergo testing, and also suggest that cancer history may affect women's acceptance of DTCA and genetic testing.

Growth of M. avium subspecies paratuberculosis in culture is enhanced by nicotinic acid, nicotinamide, and α and ß nicotinamide adenine dinucleotide.

BACKGROUND: Without known mechanisms of action, Crohn's disease is exacerbated, and ulcerative colitis is improved, by the use of tobacco. Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. We hypothesized that tobacco components might alter the growth kinetics of MAP, explaining these divergent clinical observations. METHODS: The effect of nicotine, nicotinic acid, nicotinamide and α and ß nicotinamide adenine dinucleotide (α and ß NAD) were studied on eight strains of three mycobacterial species (MAP, M. avium and M. tb. complex). Data are obtained as "cumulative growth index," (cGI) and presented as "percent increase in cumulative GI" (% + ΔcGI). RESULTS: Nicotinic acid enhances the two human MAP isolates (Dominic; 225% + ΔcGI and UCF-4; 92% + ΔcGI) and M. avium (ATCC 25291; 175% + ΔcGI). Nicotinamide (at 6.4 µg/ml) enhances the human MAP isolates (Dominic; 156% + ΔcGI and UCF-4; 79% + ΔcGI) and M. avium (ATCC 25291; 144% + ΔcGI.) Both α and ß NAD enhance Dominic; (135 and 150 % + ΔcGI) and UCF-4; (81 and 79% + ΔcGI). At the doses tested, nicotine has no effect on any strain studied. CONCLUSIONS: We show enhancement of MAP growth by nicotinic acid, one of ≥4,000 tobacco-related molecules, its amide, nicotinamide and α and ß NAD. Pure nicotine has no enhancing effect at the doses studies.

The thioamides methimazole and thiourea inhibit growth of M. avium Subspecies paratuberculosis in culture.

BACKGROUND: Thyrotoxicosis is conceptualized as an "autoimmune" disease with no accepted infectious etiology. There are increasingly compelling data that another "autoimmune" affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition. METHODS: The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as "cumulative growth index," (cGI) or "percent decrease in cumulative GI" (%-DeltacGI). PRINCIPAL FINDINGS: Methimazole was the most effective thioamide at inhibiting MAP growth. At 128microg/ml: MAP UCF-4; 65%-DeltacGI & MAP ATCC 19698; 90%-DeltacGI. Thiourea inhibited MAP "Ben" maximally; 70%-DeltacGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested. SIGNIFICANCE: We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.