Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome.

Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.

Mutational spectrum of COH1 and clinical heterogeneity in Cohen syndrome.

Cohen syndrome (CS) is an autosomal recessive disorder with variability in the clinical manifestations, characterised by mental retardation, postnatal microcephaly, facial dysmorphism, pigmentary retinopathy, myopia, and intermittent neutropenia. Mutations in the gene COH1 have been found in an ethnically diverse series of patients. Brief clinical descriptions of 24 patients with CS are provided. The patients were from 16 families of different ethnic backgrounds and between 2.5 and 60 years of age at assessment. DNA samples from all patients were analysed for mutations in COH1 by direct sequencing. Splice site mutations were characterised using reverse transcriptase PCR analysis from total RNA samples. In this series, we detected 25 different COH1 mutations; 19 of these were novel, including 9 nonsense mutations, 8 frameshift mutations, 4 verified splice site mutations, 3 larger in frame deletions, and 1 missense mutation. We observed marked variability of developmental and growth parameters. The typical facial gestalt was seen in 23/24 patients. Early onset progressive myopia was present in all the patients older than 5 years. Widespread pigmentary retinopathy was found in 12/14 patients assessed over 5 years of age. We present evidence for extended allelic heterogeneity of CS, with the vast majority of mutations leading to premature termination codons in COH1. Our data confirm the broad clinical spectrum of CS with some patients lacking even the characteristic facial gestalt and pigmentary retinopathy at school age.

New monoclonal antibodies for the detection of immediate early antigens of cytomegalovirus.

Two new monoclonal antibodies, CIE-1 and CIE-2, were developed for the rapid detection of human cytomegalovirus (HCMV) infection. They were found to be reactive with immediate early protein of HCMV in the nuclei of infected fibroblasts, as early as 3 hours post-infection. By radioimmunoprecipitation, CIE-1 was found to react with a protein with an apparent molecular weight of 70,000, whereas CIE-2 precipitated 2 proteins of 70,000 and 72,000 daltons, respectively. Both monoclonal antibodies recognized three prototype strains of HCMV: AD-169, Towne, and Davis, and did not cross-react with other human herpesviruses. CIE-1 and CIE-2 were compared with four commercial anti-HCMV monoclonal antibodies (Clonab, Dupont, Sera-Lab and Syva) by testing 88 clinical isolates. Culture confirmation tests and shell vial assays showed that CIE-1 and CIE-2 were more sensitive than several of these reagents and equally sensitive to the Dupont reagent. Moreover, CIE-1 and CIE-2 produced a bright, sharp staining of the nuclei of infected cells. These monoclonal antibodies should thus be valuable in rapid diagnosis of HCMV.

New case of mosaic tetrasomy 9p with additional neurometabolic findings.

Tetrasomy 9p is a rare chromosomal aberration that was described in 28 previous patients. Here we report on a newborn girl who was referred for genetic evaluation because of developmental delay, hypertonicity, microcephaly, minor anomalies, and neurometabolic findings. She had an isochromosome 9p (pter --> p10 --> pter) in 32% of blood cells. The extra chromosome was not found in amniocytes. Examination of fibroblasts from different skin biopsies also showed mosaicism in this tissue. In a first biopsy from the abdominal wall, the cells (n = 50) had a normal chromosomal complement. Further analysis of fibroblasts from the left forearm showed the isochromosome 9p in 5 out of 8 mitoses. Fluorescence in situ hybridization (FISH), using a whole chromosome 9 probe, confirmed that the extra marker was 9 in origin. Molecular studies showed that the isochromosome was of maternal origin. Meiotic nondisjunction was followed by centromeric misdivision and postzygotic loss of the marker.

Antibodies to human cytomegalovirus structural polypeptides during primary infection.

This study aimed at broadening the understanding of the immunogenic potential of cytomegalovirus (CMV) structural polypeptides during natural infection and to ascertain their possible use in serological diagnosis. Immunoblotting was used to analyse the appearance and development of serum IgG and IgM against human CMV structural polypeptides in sequential sera from renal transplant recipients during the first 1-3 months of primary CMV infection. The results showed that the first IgG to appear is specific for a polypeptide of 66 kDa and these antibodies appear either alone or together with others against another polypeptide of 82 kDa. IgG to the 150 kDa protein appears at least one week later. The first IgM to appear reacts preferentially with a 38 and 66 kDa polypeptide. The early detection of antibody against the major viral antigenic proteins in the diagnosis of CMV primary infection is discussed.

Sensitivity and specificity of enzyme immunofiltration and DNA hybridization for the detection of HCMV-infected cells.

The sensitivity and specificity of enzyme immunofiltration and DNA hybridization were compared in human cytomegalovirus (HCMV) (AD 169)-infected MRC-5 cells. The enzyme immunofiltration was carried out on glass fiber filters in microplates, using an HCMV (AD 169) monoclonal antibody and a peroxidase conjugate. The DNA hybridization was carried out with a microfiltration apparatus, using a 32P-labelled HCMV (AD 169) Eco R1 D fragment probe. The sensitivities of enzyme immunofiltration and DNA hybridization were 1.82 X 10(3) and 1.13 X 10(3) infected cells, respectively. Both methods were highly specific, but enzyme immunofiltration was faster and simpler.

Rapid detection of influenza virus infections in human fetal lung diploid cell cultures.

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.

A preventable case of congenital rubella syndrome and its public health implications.

A failure to provide rubella immunization prior to conception, followed by failure to diagnose typical rubella during the first trimester of pregnancy, resulted in a case of congenital rubella syndrome. The ensuing malpractice suit was settled out of court 11 years later. Several reasons for the failure of prevention and diagnosis are discussed, along with the implications of the case for rubella prevention today.

Comparison of immunological reactivity to two major antigens of homologous and AD169 human cytomegalovirus strains during primary infection in renal transplant patients.

The IgG and IgM immunoblotting patterns for the major HCMV antigens p65 and p55/52 were studied in 5 renal transplant recipients during primary HCMV infections. Sequential sera from the 5 patients were tested in parallel with standardized antigens derived from the reference strain AD169 and from the patient'own isolates. The immunoblotting patterns differed from patient to patient as well as between strain AD169 and the patient's homologous isolates. The differences consisted in a better response to the antigens derived from the reference strain AD169, suggesting a delay of the IgM/IgG switch and of the affinity maturation of IgG antibodies to the homologous p65 and p55/52 antigens. The results suggest caution in the interpretation of immunoblotting data derived from laboratory strains such as AD169.

Supernumerary der(1) marker chromosome derived from a ring chromosome 1 which has retained the original centromere and euchromatin from 1q21.1 --> q21.3 with substantial loss of 1q12 heterochromatin in a female with dysmorphic features and psychomotoric developmental delay.

We report on a 5.5-year-old girl with dysmorphic features and psychomotoric developmental delay with a mitotically stable supernumerary marker chromosome. The origin of the marker was identified by microdissection and reverse painting of marker DNA as the pericentromeric region of chromosome 1. Fine mapping by FISH with selected YAC or BAC clones identified no p-arm material on the marker. The marker has retained its original centromere and euchromatin from 1q21.1-q21.3 but only small remnants of the 1q12 heterochromatin. Furthermore, some FISH clones presented single signals on the marker and others presented double signals indicating a partial duplication within the marker. These observations suggest a multi-step origin of the marker most probably with ring formation as the first step.

Bordetella pertussis in the National Capital Region: prevalent serotype and immunization status of patients.

Over a 2-year period 67 strains of Bordetella pertussis were identified in 231 single specimens of nasopharyngeal secretions submitted from patients suspected to have whooping cough in the National Capital Region; 89.5% of the identifications were made by culture. Serotype 1,3 was predominant. At least 75% of the patients with bacteriologically confirmed whooping cough had not been fully immunized. There was no evidence that adenoviruses or other viruses played any important etiologic role in the 204 cases of whooping cough or whooping cough syndrome studied virologically.

Comparison of immunoblotting with other serological methods and virus isolation for the early detection of primary cytomegalovirus infection in allograft recipients.

Sequential specimens from nine allograft recipients were examined by using a variety of methods to detect primary cytomegalovirus (CMV) infection as rapidly as possible posttransplantation. Sera were examined for immunoglobulin G (IgG) and IgM antibodies by immunoblotting, enzyme immunoassay, and immunofluorescence and also by complement fixation, latex agglutination, and an immunofluorescence test for antibody to CMV early antigen. Urine and occasionally blood, tissue, and other specimens were centrifuged onto cell cultures to enhance CMV infectivity. Eight of the nine patients showed laboratory evidence of primary CMV infection, and CMV was isolated from seven of the eight: in no case was virus isolated before seroconversion had become evident. However, serological tests differed in their abilities to detect antibody response to CMV infection in different patients; while immunoblotting, latex agglutination, and enzyme immunoassay for IgG antibodies generally detected seroconversion before complement fixation, this was not invariably the case. At present, optimal laboratory detection of CMV infections in these patients can be achieved only by a combination of serological methods and virus isolation.

Comparison of five methods of cytomegalovirus antibody screening of blood donors.

A group of 120 sera from blood donors was screened by complement fixation and commercially available immunofluorescence, solid-phase fluorescence immunoassay, enzyme-linked immunosorbent assay, and indirect hemagglutination tests. Twenty-four of the sera were positive by three or more of the five tests and judged to be true positives; 89 were negative by three or more of the tests and considered to be true negatives. The tests were ranked for accuracy, sensitivity, specificity, false-positive rate, and false-negative rate. The indirect hemagglutination test scored best, followed by enzyme-linked immunosorbent assay, solid-phase fluorescence immunoassay, complement fixation, and immunofluorescence, in that order. When the tests were ranked on the basis of technical demands, turnaround time, requirement for special equipment, and subjectivity in reading, the indirect hemagglutination test again scored best, followed by solid-phase fluorescence immunoassay, enzyme-linked immunosorbent assay, immunofluorescence, and complement fixation in that order. Our findings suggest that the indirect hemagglutination test is the most reliable and effective commercially available test for the identification of those donors who are very unlikely to transmit cytomegalovirus to recipients.

Persistence of naturally acquired cell-mediated immunity to rubella virus during the first trimester of pregnancy.

We studied the persistence of naturally acquired cell-mediated immunity to rubella during early pregnancy. We compared lymphocyte transformation responses to phytohemagglutinin and rubella virus in 35 naturally immune women aged 17-37 years, in the first trimester of pregnancy, with 49 naturally immune age-matched nonpregnant controls. A significant lower lymphocyte transformation response to phytohemagglutinin was observed during the first trimester of pregnancy (P = 0.008), but lymphocyte transformation responses to rubella virus were not significantly different (P = 0.901). These data indicate that, in naturally immune women, cell-mediated immunity to rubella virus is not significantly altered by the physiological changes in early pregnancy.