Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington.

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States.

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

Mixed methods study on elimination of tuberculosis in Hong Kong.

INTRODUCTION: Tuberculosis (TB) commonly affects developing countries. Several developed regions in Asian still have a stagnant intermediate TB burden. Information to adequately inform TB strategies is lacking. We conducted a mixed methods study to fill this information gap in Hong Kong. METHODS: Data from the Hong Kong government were used to analyse trends of TB notification rates compared with World Health Organization (WHO) targets. A review of policy documents and literature was conducted to evaluate TB control and elimination in Hong Kong. RESULTS: Extrapolated trends showed that Hong Kong will be unable to meet the WHO target of a 90% drop in incidence rate by 2030. The policy review showed that the Hong Kong government has not set a clear strategy and timeline for specific goals in TB control and elimination. The literature review found that older adults are largely responsible for the stagnant TB prevalence because of reactivation of latent TB infection (LTBI), while mortality of hospitalised patients with TB is still high because of delayed diagnosis and treatment. CONCLUSION: Tuberculosis incidence is currently under control in Hong Kong, but further actions are needed if the elimination targets are to be achieved. Improved diagnostic tools are required, and policies targeting LTBI in older adults should be implemented to achieve the WHO target by 2030.

The parasite Ichthyophonus sp. in Pacific herring from the coastal NE Pacific.

The protistan parasite Ichthyophonus occurred in populations of Pacific herring Clupea pallasii Valenciennes throughout coastal areas of the NE Pacific, ranging from Puget Sound, WA north to the Gulf of Alaska, AK. Infection prevalence in local Pacific herring stocks varied seasonally and annually, and a general pattern of increasing prevalence with host size and/or age persisted throughout the NE Pacific. An exception to this zoographic pattern occurred among a group of juvenile, age 1+ year Pacific herring from Cordova Harbor, AK in June 2010, which demonstrated an unusually high infection prevalence of 35%. Reasons for this anomaly were hypothesized to involve anthropogenic influences that resulted in locally elevated infection pressures. Interannual declines in infection prevalence from some populations (e.g. Lower Cook Inlet, AK; from 20-32% in 2007 to 0-3% during 2009-13) or from the largest size cohorts of other populations (e.g. Sitka Sound, AK; from 62.5% in 2007 to 19.6% in 2013) were likely a reflection of selective mortality among the infected cohorts. All available information for Ichthyophonus in the NE Pacific, including broad geographic range, low host specificity and presence in archived Pacific herring tissue samples dating to the 1980s, indicate a long-standing host-pathogen relationship.

Case Report: Primary Spinal Lymphoma.

This is the report on the case of a 74 year old male patient who was admitted to hospital emergency because of a distended bladder, which was detected on an MRI. This MRI was performed because of an acute paralysis of the patient's left leg. After various examinations we could conclude that the patient's neurological symptoms were not due to metastases of a solid tumour as we expected, but to a primary spinal diffuse B-cell lymphoma. The central nervous system, and especially the spinal cord, are an extremely rare location for primary B-Cell lymphoma.

Influence of CYP2B6 516G>T polymorphism and interoccasion variability (IOV) on the population pharmacokinetics of efavirenz in HIV-infected South African children.

OBJECTIVE: To investigate the influence of CYP2B6 516G>T polymorphism, as a covariate, and of interoccasion variability (IOV) on the oral clearance (CL/F) of efavirenz (EFV) in treatment-naïve black South African children over a period of 24 months post-antiretroviral therapy (ART) initiation. METHODS: HIV-infected black children (n = 60, aged 3-16 years), with no prior exposure to ART, eligible to commence ART and attending an outpatient clinic were enrolled into this study. Blood samples were taken at mid-dose interval at 1, 3, 6, 12, 18 and 24 months post-ART initiation. EFV plasma samples were determined with an adapted and validated LC/MS/MS method. Genotyping of the CYP2B6 G516T single nucleotide polymorphism (SNP) was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). NONMEM was used for the population pharmacokinetic modelling. RESULTS: EFV concentrations below 1 µg/mL accounted for 18% (116/649), EFV concentrations >4 µg/mL accounted for 29.5% (192/649) and concentrations within the therapeutic range (1-4 µg/mL) represented 52.5% (341/649) of all the samples determined. The covariates age, weight and CYP2B6 G516Tgenotype were included in the final model with population estimates for CL/F determined as 2.46, 4.60 and 7.33 L/h for the T/T, G/T and G/G genotype groups respectively. CONCLUSIONS: The inclusion of both age and weight to predict accurate EFV CL values for the respective genotype groups within this paediatric population was required, whereas the addition of gender and body surface area did not improve the predictions. The importance of introducing IOV in a PK model for a longitudinal study with sparsely collected data was again highlighted by this investigation.

HAART and risk of tuberculosis in HIV-infected South African children: a multi-site retrospective cohort.

SETTING: Four human immunodeficiency virus (HIV) clinics located at South African tertiary hospitals. OBJECTIVE: To assess the effectiveness of highly active antiretroviral therapy (HAART) in reducing incident tuberculosis (TB) in HIV-infected children. DESIGN: Retrospective cohort. RESULTS: A total of 1132 children's records were included in the study. At entry to the cohort, the median (interquartile range [IQR]) age, CD4%, CD4 count and viral load of all children was respectively 6.3 years (4.1-8.8), 15% (9.0-22.2), 576 cells/mm(3) (287-960) and 160 000 copies/ml (54 941.5-449 683); 75.9% were started on HAART. The male:female ratio was 1:1, and median follow-up time was 1.7 years. In children whose follow-up included both pre-HAART and on-HAART periods, the incidence of clinically diagnosed TB was respectively 21.1 per 100 person-years (py; 95%CI 18.2-24.4) and 6.4/100 py (95%CI 4.8-8.1), and when restricted to confirmed cases, respectively 3.1/100 py (95%CI 2.2-4.2) and 0.8/100 py (95%CI 0.5-1.4). Only 23% of all cases of TB were microbiologically confirmed. Multivariate analyses showed that HAART reduced incident TB by approximately 70%, both for confirmed and all TB cases. CONCLUSIONS: In this high TB burden country, the incidence of diagnosis of TB in HIV-infected children is at least as high as that of adults. HAART reduces incident TB, but further prospective TB preventive and diagnostic studies are urgently needed in children.

The utility of fine needle aspiration in HIV positive children.

OBJECTIVE: To determine the spectrum of disease, diagnostic accuracy and adequacy of fine needle aspirates (FNA) in human immunodeficiency virus (HIV) positive children who present with mass lesions. METHODS: Between January 1997 and December 2002, 95 FNAs were performed in 91 children aged 15 years and younger who were known to be infected with the human immunodeficiency virus (HIV). RESULTS: Head and neck masses including salivary gland swellings were the most common presentation (58.9%) followed by axillary masses (25.3%). Groin masses were aspirated in six children, flank and abdominal masses in four children, buttock masses in three children, a chest wall mass in one child and a sonar guided FNA of a lung mass in one child. Eight FNAs (8.4%) proved inadequate. Reactive lymphadenopathy was diagnosed in 42 cases, mycobacterial infection in 22, four children were diagnosed with abscess, one child had a fungal infection and five were found to have non-Hodgkin's lymphoma. There were four cases each of lymphoepithelial lesion and Kaposi sarcoma. There was one case each of nephroblastoma, rhabdomyosarcoma, myeloma, melanotic progonoma and spindle cells, not otherwise specified. CONCLUSION: Fine needle aspiration in HIV positive children is a worthwhile procedure and in most instances allows a rapid diagnosis obviating the need for surgery and enabling swift treatment to be undertaken where necessary. Ancillary studies form an important diagnostic component. Universal safety precautions must be strictly adhered to.

Novel use of accelerator mass spectrometry for the quantification of low levels of systemic therapeutic recombinant protein.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

A limited epizootic of neuroblastoma in coho salmon reared in chlorinated-dechlorinated water.

During the 1976-77 brood year, approximately 12 cases of neuroblastoma were observed in a captive group of 100,000 fingerling coho salmon (Oncorhynchus kisutch) reared in a commercial hatchery. The tumors were large, occurring in the skeletal muscle near the dorsal fin causing conspicuous bulging of the overlying integument. Tumors examined from 3 fish each consisted of neuroblasts in trabecular patterns interspersed by glial fibrillar material and linear cavities resembling central neural canals lined by ependyma-like cells. Ganglion-like cells also were apparent morphologically and by special stain. Cancer of the tumor was characterized by an abundance of mitotic figures with occasional abnormal divisions, local invasion of normal tissues, and potentially metastatic tumor cell aggregates in organ vasculature. The etiology of this tumor may have been related to mutagenic-carcinogenic halogenated compounds possibly formed in the hatchery water supply during continuous chlorination of incoming river water.

Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection.

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

Chemical modification of a variant of human MIP-1alpha; implications for dimer structure.

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.

Full-length genome characterization of HIV type 1 subtype C isolates from two slow-progressing perinatally infected siblings in South Africa.

Isolation and characterization of HIV-1 from asymptomatic, slow-progressing individuals are important in studying viral pathogenesis and facilitate the development of vaccines and antivirals. In this study we identified two slow-progressing HIV-1-infected siblings, isolated viruses, and sequenced the full-length genome, to identify virus attenuations that may contribute to their altered rate of disease progression. Proviral DNA from strains 99ZATM10 and 01ZATM45 was isolated from peripheral blood mononuclear cells (PBMC) coculture.Virtually full-length genomes and long terminal repeat (LTR) regions were polymerase chain reaction (PCR) amplified, sequenced, and assembled to generate the complete genomes. Phylogenetic analysis confirmed that both isolates were subtype C throughout their genome. Predicted amino acid sequence analysis for all the HIV-1 proteins showed that both viruses had open reading frames for all genes, and encoded proteins of the expected length, except for the rev gene. The 3' end of rev exon 2 did not have the 16-amino acid (aa) truncation characteristic of subtype C viruses, and in addition, had a three-aa extension (GlyCysCys). Rev is a necessary regulatory factor for HIV expression, and changes in the protein may affect viral replication. These results suggest that slower HIV disease progression in these children may be attributed, at least in part, to an altered Rev protein.

White matter MR hyperintensities in adult patients with congenital rubella.

PURPOSE: To observe and quantify white matter hyperintensities on MR images in adults with schizophrenialike symptoms who had had congenital rubella, in order to elucidate the neuropathologic sequelae of this perinatal viral infection and to explore the potential relationship of these lesions to schizophrenia. METHODS: Eleven deaf adult patients with documented prenatal rubella virus infection and schizophrenialike symptoms were compared with 19 age-matched patients with early-onset schizophrenia who did not have congenital rubella and with 18 age-matched control subjects. All MR images (obtained at 1.5 T) were evaluated by a neuroradiologist who was blinded to diagnosis and were rated for white matter lesions on a five-point scale: 0 = no lesions; 1 = 1 lesion less than 1 mm in diameter; 2 = 1 to 4 lesions 1 mm or greater; 3 = 5 to 10 lesions; 4 = more than 10 lesions or a single lesion more than 1 cm in diameter. In addition, the white matter hyperintensities were volumed objectively with a manual threshold technique. RESULTS: Ratings of white matter lesions were significantly higher in the rubella patients than in the control subjects: 6 of the 11 patients had ratings greater than 1 compared with 1 of the 18 control subjects and none of the 19 schizophrenic patients. Also, MR images in five rubella patients received ratings at the highest end of the scale of abnormality (3 or 4). The white matter hyperintensities were characterized as bilateral T2 signal hyperintensities in periventricular and subcortical regions, punctate or linear in shape; they were observed predominantly in parietal lobes. CONCLUSION: This quantitative MR study of adult rubella patients disclosed abnormal white matter lesions that may correspond to neurovascular lesions known neuropathologically. They do not appear to be directly related to schizophrenialike symptoms.

Use of an in vivo oxygen electrode to determine the effect of hemorrhagic shock on liver oxygen tension.

Measurement of arterial and liver tissue oxygen tension (PO2) in animals subjected to hemorrhagic shock demonstrates a significant (p less than 0.001) decrease in tissue PO2 while PaO2 remains essentially unchanged. In fact, marked increase in PaO2 fail to increase tissue PO2 to control levels, demonstrating that the act of increasing FIO2 and/or PaO2 is inadequate treatment of decreased tissue oxygenation in marginal or low flow states. Measurement of tissue PO2 in a variety of clinical situations seems warranted to allow alterations of therapy to improve flow when indicated by inadequate tissue oxygenation. The application of this simple but extremely useful technic should result in improved survival rates in critically ill patients.

Recurrent psychogenic coma following tracheal stenosis repair.

Medication, intracranial hemorrhage, infarction, infection, hypoxia, organ failure, and nutritional deficiency may cause unconsciousness following successful emergence from anesthesia. A 39-year-old woman with a history of tracheal stenosis, depression, and anxiety had complete unconsciousness on 3 separate occasions following surgical repair of her tracheal stenosis. In each case, the patient's endotracheal tube had been removed; she was alert and oriented to person, time, and place; and she was admitted to the hospital for observation. Within a few hours after the tube was removed, the patient became abruptly unconscious for periods of 36, 18, and 30 hours. Each time, the results of cardiac, pulmonary, metabolic, and neurologic examinations and radiological studies were normal. We hypothesize that the patient's apparent comas were the result of an underlying conversion disorder precipitated by unresolved psychological conflict surrounding a long history of abuse in which she was repeatedly smothered by a pillow.

Paclitaxel (Taxol) plus doxorubicin plus filgrastim in advanced sarcoma: a phase II study.

The authors evaluated the novel chemotherapeutic regimen of paclitaxel (Taxol, Bristol-Myers Squibb, Princeton, NJ, U.S.A.) plus doxorubicin plus filgrastim--a granulocyte colony-stimulating factor (G-CSF)--in advanced or metastatic sarcoma. Eligible patients must have had histologically confirmed advanced previously untreated soft-tissue sarcoma. All patients must have had bidimensionally measurable metastases. Treatment consisted of doxorubicin, 50 mg/m2 by intravenous push, followed 4 hours later by paclitaxel, 150 mg/m2 by continuous infusion over 24 hours every 3 weeks, plus G-CSF, 5 microg/kg, on days 3 through 12 of each cycle. Cycles were repeated every 21 days. A one-time dose escalation for doxorubicin only (60 mg/m2) was allowed in all patients who experienced no significant toxicity after their first cycle of paclitaxel plus doxorubicin. From November 1993 through May 1996, 29 patients were entered in this study. Grade 3 anemia occurred in three patients. Grade 3--4 neutropenia occurred in 20 patients. Seven patients experienced at least one episode of neutropenic fever, including one death. Grade 3 thrombocytopenia occurred in four patients. There were six partial responses in 27 eligible patients, for a response rate of 22.2% (95% confidence interval, 7%-38%). Median time to progression was 4.5 months, and median overall survival was 10.2 months. The regimen of paclitaxel plus doxorubicin plus filgrastim as used in this study appears to have no more activity than single-agent doxorubicin.

Paclitaxel plus gallium nitrate and filgrastim in patients with refractory malignancies: a phase I trial.

To determine the maximally tolerated dose of paclitaxel with and without filgrastim (G-CSF) when administered as a 24-hour intravenous infusion after a 120-hour infusion of gallium nitrate at a fixed dose of 300 mg/m2/24 hours, 40 patients were entered onto a trial lasting from September 1994 to September 1996. Eligibility included a diagnosis of an advanced malignancy not amenable to curative therapy and up to one previous chemotherapy regimen for metastatic disease. Gallium was administered at a fixed dose of 300 mg/m2/day as a continuous intravenous infusion for 120 hours. Paclitaxel starting at 90 mg/m2 was given concurrently with the last 24 hours of the gallium as a 24-hour intravenous infusion. Cycles were repeated every 21 days. Once the maximum tolerated dose (MTD) of paclitaxel was reached, G-CSF (5 microg/kg/day days 7-16) was added and paclitaxel dose escalation continued. The MTD for paclitaxel without G-CSF was 110 mg/m2 and 225 mg/m2 with G-CSF, with neutropenia being the dose-limiting toxicity. A partial response was noted in a patient who had thymoma and a complete response was achieved in a patient who had colon cancer. The recommended phase II dosage is gallium nitrate at 300 mg/m2/day over 120 hours, with paclitaxel at 110 mg/m2 over 24 hours without G-CSF or 225 mg/m2 over 24 hours with G-CSF and 0.5 mg calcitriol on days 1 through 7. Further trials of this modified regimen for outpatient administration are in progress.

Effect of cyclopropenoid fatty acids on the hepatic microsomal mixed-function-oxidase system and aflatoxin metabolism in rabbits.

Male New Zealand weanling rabbits were fed a diet containing 0.25% cyclopropenoid fatty acids for 28 days. Compared with the controls, the rabbits given cyclopropenoid fatty acids showed retarded growth, some moderate liver histological damage, altered hepatic mixed-function-oxidase activities and minor variations in vitro [14C]aflatoxin B1 metabolism. In in vitro assays the major hepatic metabolite of aflatoxin B1 (AFB1) was aflatoxicol (AFL) and the major AFL metabolite was AFB1. Minor amounts of aflatoxin M1 and a metabolite believed to be AFL-M1 were formed. The similarity of this AFB1 metabolite pattern to that in rainbow trout, taken together with the apparent absence of AFB1 detoxification products is consistent with the sensitivity of both species to the acute effects of AFB1.

Familial Creutzfeldt-Jakob disease: a neuropsychological case study.

The spectrum of neuropsychological features of familial Creutzfeldt-Jakob disease (CJD) have seldom been reported, possibly because of (a) the rarity of this hereditary form of prion disease; (b) frequent delays in diagnosis, and; (c) the typically rapid demise of the patient, which affords little opportunity for comprehensive testing or serial analysis. Here we describe the neurobehavioral characteristics of a 48-year-old right-handed male (JD) who presented with complaints of poor depth perception, unsteady gait, and unusual sensory experiences in his face and neck. JD was followed serially over the final 4 months of his 5-month illness. Immediately following hospital admission, he underwent a neuropsychological evaluation that revealed moderate to severe impairment of delayed (30-minute) verbal memory, tactual performance in his right hand, and word-finding ability. In contrast, other abilities that are commonly classified within the verbal, visuospatial, and memory domains showed minimal or no compromise. Parallel studies of electroencephalographic activity revealed diffuse slowing and, later, 1-Hz rhythmical discharges over the left hemisphere, and mild prominence of the lateral ventricles and cerebral sulci on magnetic resonance imaging. Autopsy revealed spongiform changes and reactive astrocytosis, and genetic testing demonstrated a codon 200 mutation in the prion protein gene. These findings indicate that CJD can result in clinical manifestations compatible with multifocal asymmetric cerebral involvement before more diffuse neurodegeneration ensues, providing a strong impetus for the study of additional cases. This long-term understanding can help to determine whether the multiple loci of clinical involvement are specified by genetic or epigenetic factors, or both.