Christopher L. Bennett

Genotype-phenotype correlation in Costello syndrome: HRAS mutation analysis in 43 cases

Background: Costello syndrome (CS) is a rare multiple congenital abnormality syndrome, associated with failure to thrive and developmental delay. One of the more distinctive features in childhood is the development of facial warts, often nasolabial and in other moist body surfaces. Individuals with CS have an increased risk of malignancy, suggested to be about 17%. Recently, mutations in the HRAS gene on chromosome 11p13.3 have been found to cause CS. Methods: We report here the results of HRAS analysis in 43 individuals with a clinical diagnosis of CS. Results: Mutations were found in 37 (86%) of patients. Analysis of parental DNA samples was possible in 16 cases for both parents and in three cases for one parent, and confirmed the mutations as de novo in all of these cases. Three novel mutations (G12C, G12E, and K117R) were found in five cases. Conclusions: These results confirm that CS is caused, in most cases, by heterozygous missense mutations in the proto-oncogene HRAS. Analysis of the major phenotypic features by mutation suggests a potential correlation between malignancy risk and genotype, which is highest for patients with an uncommon (G12A) substitution. These results confirm that mutation testing for HRAS is a reliable diagnostic test for CS.

Mutations in ISPD cause Walker-Warburg syndrome and defective glycosylation of α-dystroglycan.

Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant α-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated α-dystroglycan. These results implicate ISPD in α-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.

KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies.

The Viral Interferon Regulatory Factors of Kaposi's Sarcoma-Associated Herpesvirus Differ in Their Inhibition of Interferon Activation Mediated by Toll-Like Receptor 3

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) infection is correlated with three human malignancies and can establish lifelong latent infection in multiple cell types within its human host. In order to establish and maintain infection, KSHV utilizes multiple mechanisms to evade the host immune response. One such mechanism is the expression of a family of genes with homology to cellular interferon (IFN) regulatory factors (IRFs), known as viral IRFs (vIRFs). We demonstrate here that KSHV vIRF1, -2, and -3 have a differential ability to block type I interferon signaling mediated by Toll-like receptor 3 (TLR3), a receptor we have previously shown to be activated upon KSHV infection. vIRF1, -2, and -3 inhibited TLR3-driven activation of IFN transcription reporters. However, only vIRF1 and vIRF2 inhibited increases in both IFN-β message and protein levels following TLR3 activation. The expression of vIRF1 and vIRF2 also allowed for increased replication of a virus known to activate TLR3 signaling. Furthermore, vIRF1 and vIRF2 may block TLR3-mediated signaling via different mechanisms. Altogether, this report indicates that vIRFs are able to block IFN mediated by TLRs but that each vIRF has a unique function and mechanism for blocking antiviral IFN responses.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1 Interacts with a Member of the Interferon-Stimulated Gene 15 Pathway

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus known to establish lifelong latency in the human host. We and others have previously shown that three KSHV homologs of cellular interferon regulatory factors (IRFs), known as viral IRFs (vIRFs), participate in evasion of the host interferon (IFN) response. We report that vIRF1 interacts with the cellular interferon-stimulated gene 15 (ISG15) E3 ligase, HERC5, in the context of Toll-like receptor 3 (TLR3) activation and IFN induction. The ISG15 protein is covalently conjugated to target proteins upon activation of the interferon response. Interaction between vIRF1 and HERC5 was confirmed by immunoprecipitation, and the region between amino acids 224 and 349 of vIRF1 was required for interaction with HERC5. We further report that expression of vIRF1 in the context of TLR3 activation results in decreased ISG15 conjugation of proteins. Specifically, TLR3-induced ISG15 conjugation and protein levels of cellular IRF3, a known ISG15 target, were decreased in the presence of vIRF1 compared to the control. vIRF1 itself was also identified as a target of ISG15 conjugation. KSHV-infected cells exhibited increased ISG15 conjugation upon reactivation from latency in coordination with increased IFN. Furthermore, knockdown of ISG15 in latently infected cells resulted in a higher level of KSHV reactivation and an increase in infectious virus. These data suggest that the KSHV vIRF1 protein affects ISG15 conjugation and interferon responses and may contribute to effective KSHV replication. IMPORTANCE The KSHV vIRF1 protein can inhibit interferon activation in response to viral infection. We identified a cellular protein named HERC5, which is the major ligase for ISG15, as a vIRF1 binding partner. vIRF1 association with HERC5 altered ISG15 modification of cellular proteins, and knockdown of ISG15 augmented reactivation of KSHV from latency.

Diversification among coffee smallholders in the highlands of South Sumatra, Indonesia

Over the past decade international organizations and developing countries have been trying to promote agricultural diversification among smallholders under the assumption that smallholders are too dependent upon a narrow range of crops. The International Coffee Organization and the Government of Indonesia have promoted agricultural diversification among coffee farmers to curb production and, thereby, increase prices. In South Sumatra, Indonesia, coffee smallholders diversify on their own initiative and depend upon numerous resource systems. They diversify out of coffee and even agriculture because other investments yield attractive returns and because further diversification within agriculture is often limited by agronomic and financial constraints.

The Quality of Smallholder Cloves in Maluku: The Local Response to Domestic Demand for a High-Quality Product 1

Indonesias cloves meet higher quality Standards than most of her other agricultural commodilies. This paper presents the results of research on Ambon, Maluku, whose smallholder clove farming System is representative of the principal system used by Indonesien clove producers, in which cloves form part of a well-diversified agricultural portfolio. The paper examines the clove marketing system, the factors involved in clove quality, and the system of quality premia and penalties applied in the marketing of cloves. The largest buyers of cloves, the major kretek cigarette companies, demand high-quality produce, because of the highly competitive domestic market and their more recent attempts to penetrate the international market. These buyers transmit their preferences along the marketing chain through the well-defined incentive system, while poorer quality cloves are bought by smaller kretek producers. The result is a system in which high quality standards are maintained. 1 The Centre for Policy and Implement...

Surgical Resident Duty Hours.

To the Editor: As Bilimoria et al. (Feb. 25 issue)1 note in their article on the Flexibility in Duty Hour Requirements for Surgical Trainees (FIRST) Trial, the 2011 duty-hour reforms of the Accreditation Council for Graduate Medical Education (ACGME) created controversy in the medical community.2-5 Several groups have questioned the benefits of these reforms.1-4 Important to the controversy is underreporting of duty hours by residents.2,5 Underreporting may be a significant source of bias in studies of ACGME duty-hour reforms.1,3,4 To examine underreporting, we surveyed general surgery residents in the United States using methods based on previous studies.2,5 Our study was approved by the institutional review board at the University of North Carolina, and informed consent was obtained from all participants. We received 1003 responses (response rate, 31.9%) (see Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org); 71.6% of the respondents had exceeded duty-hour limits without reporting violations. Of this subgroup, 67.9% exceeded duty hours at least monthly and 60.4% worked more than 80 hours during an average week (Table 1). When asked why they exceeded work-hour restrictions, 61.4% of the respondents said they did so to prevent adverse outcomes in patient care, 42.6% thought it was expected of them, 27.7% reported guilt about leaving the hospital, and 24.1% reported external pressure from authority figures (Table 1). Among all respondents, 36.4% reported working from home to avoid duty-hour violations; 50.7% of this subgroup reported doing so at least once a week (Table 1, and Table S2 in the Supplementary Appendix). Although the response rate of 31.9% limits the generalizability of these results, the response rate is higher than that in other studies,2,5 and the age, sex, and geographic distribution of the respondents were similar to the demographic characteristics of U.S. general surgery residents overall.6 The prevalence of underreporting limits the validity of conclusions drawn by other studies,3,4 including the recent findings from the FIRST Trial. Our study ran concurrently with the FIRST Trial; 52 of the hospitals enrolled in that trial train residents from programs in our study. The ACGME is currently considering revising the duty-hour policy on the basis of studies1,3,4 that are biased because of underreporting. Before any such revision in graduate medical education is considered, the phenomenon of dutyhour falsification should be acknowledged and the reasons why residents falsify their duty hours need to be better understood.

Massive hemoptysis in Loeys-Dietz syndrome.

We describe four unrelated individuals with Loeys–Dietz syndrome (LDS) who presented with massive hemoptysis of unknown etiology. LDS is an autosomal dominant connective‐tissue disorder characterized by altered cardiovascular, craniofacial, and skeletal development that is attributed to mutations in the TGFBR1, TGFBR2, SMAD3, or TGFB2 genes. Massive hemoptysis (MH) is a rare and often fatal pulmonary medical emergency. This is the first report of MH in individuals with LDS and establishes it as part of the LDS spectrum. It compels providers to educate their LDS patients on MH, although much investigation needs to be done to determine etiology and appropriate treatment for this newly described LDS feature.

North Carolina Medical Student Views on Abortion

BACKGROUND Abortion is a controversial yet common procedure within the United States and North Carolina. Although much effort has been directed at understanding the views of physicians and the general public, the views of medical students on abortion are understudied. This study examines the views of medical students at different stages in training on abortion at a public institution, the University of North Carolina at Chapel Hill School of Medicine. METHODS We surveyed incoming, second year, fourth year, and leave-of-absence medical students at the University of North Carolina at Chapel Hill about their views on abortion. We utilized an IRB-approved, anonymous online Qualtrics-based, cross-sectional survey during the spring of 2014. RESULTS We received 315 responses (58% response rate) from 98 incoming (54.5%), 126 second year (70.0%), and 91 fourth year and leave-of-absence (49.2%) students. The majority of students, regardless of stage of training, felt abortion was morally acceptable (incoming 64.3%, second year 74.0%, and fourth year and leave-of-absence 70.0%). Of the students who found abortion to be acceptable, second year (80.9%) and fourth year and leave-of-absence (85.5%) students found second-trimester abortion more acceptable than incoming students (57.15%, P = .002); second year students (42.2%) had greater acceptability of third-trimester abortions compared to incoming (26.2%) and fourth year and leave-of-absence students (22.2%; P = .03). Religiosity and Republican political affiliation were associated with more conservative views on abortion (P = .002); however, gender or intention to enter into an obstetrics/gynecology residency were not. LIMITATIONS Our study reflects views from a single institution, the University of North Carolina at Chapel Hill School of Medicine. This limits the generalizability of our findings to the greater population of medical students across the country. CONCLUSION Despite the controversy surrounding abortion, our work suggests that medical students at the University of North Carolina at Chapel Hill School of Medicine have accepting views of abortion.