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.

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.

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.

The clinical phenotype of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria): case reports of 23 new patients.

OBJECTIVES: To further define the clinical spectrum of the disease for pediatric and metabolic specialists, and to suggest that the general pediatrician and pediatric neurologist consider succinic semialdehyde dehydrogenase (SSADH) deficiency in the differential diagnosis of patients with (idiopathic) mental retardation and emphasize the need for accurate, quantitative organic acid analysis in such patients. PATIENTS: The clinical features of 23 patients (20 families) with SSADH deficiency (4-hydroxybutyric acid-uria) are presented. The age at diagnosis ranged from 3 months to 25 years in the 11 male and 12 female patients; consanguinity was noted in 39% of families. OUTCOME MEASUREMENTS: The following abnormalities were observed (frequency in 23 patients): motor delay, including fine-motor skills, 78%; language delay, 78%; hypotonia, 74%; mental delay, 74%; seizures, 48%; decreased or absent reflexes, 39%; ataxia, 30%; behavioral problems, 30%; hyperkinesis, 30%; neonatal problems, 26%; and electroencephalographic abnormalities, 26%. Associated findings included psychoses, cranial magnetic resonance or computed tomographic abnormalities, and ocular problems in 22% or less of patients. Therapy with vigabatrin proved beneficial to varying degrees in 35% of the patients. Normal early development was noted in 30% of patients. CONCLUSIONS: Our data imply that two groups of patients with SSADH deficiency exist, differentiated by the course of early development. Our recommendation would be that accurate, quantitative organic acid analysis in an appropriate specialist laboratory be requested for any patients presenting with two or more features of mental, motor, or language delay and hypotonia of unknown cause. Such analyses are the only definitive way to diagnose SSADH deficiency; the diagnosis can be confirmed by determination of enzyme activity in white cells from whole blood. We think that increased use of organic acid determination will lead to increased diagnosis of SSADH deficiency and a more accurate representation of disease frequency. As additional patients are identified, we should have a better understanding of both the metabolic and clinical profiles of SSADH deficiency.

Enzymatic and immunologic identification of succinic semialdehyde dehydrogenase in rat and human neural and nonneural tissues.

We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.

Ontogeny of glycoprotein gB-specific antibody and neutralizing activity during natural cytomegalovirus infection.

The envelope glycoprotein gB (gpUL55) is a candidate for inclusion in subunit cytomegalovirus (CMV) vaccines, although data on gB antibody responses after natural infection are limited. [35S]-labeled gB was partially purified from cells infected with an adenovirus recombinant expressing gB and used in radioimmunoprecipitation assays to characterize responses in solid organ transplant recipients with primary (n = 11) or secondary (n = 8) CMV infection. Seropositive transplant patients without evidence of infection (n = 5) and healthy seroconverters (n = 7) were also studied. gB antibody developed concurrently with CMV-specific IgG, IgM, and neutralizing activity in transplant patients with primary infection. Sustained boosts in gB antibody were seen in patients with secondary infection, and healthy seroconverters developed early gB responses. These data imply that gB antibody is an integral part of the humoral response to CMV infection, and, in view of experimental data regarding immunogenicity, support a role for gB in subunit vaccines.

Pre- and postnatal diagnosis of succinic semialdehyde dehydrogenase deficiency using enzyme and metabolite assays.

We report our cumulative experience for the prenatal diagnosis of succinic semialdehyde dehydrogenase (SSADH) deficiency in seven 'at-risk' pregnancies from four unrelated families. Prenatal diagnosis was performed by determination of 4-hydroxybutyric acid (4-HBA) concentration in amniotic fluid using isotope-dilution gas chromatography-mass spectrometry in conjunction with assay of SSADH activity in biopsied chorionic villus and/or cultured amniocytes. In three of four pregnancies predicted as affected, confirmation was obtained by demonstration of deficient SSADH activity in fetal tissues. Our results suggest that determination of 4-HBA concentration in amniotic fluid combined with enzyme determination in cultured or biopsied tissue represents a reliable method for the prenatal diagnosis of SSADH deficiency.,

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.

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.

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 immunofluorescence and enzyme immunoassay for detection of measles-specific immunoglobulin M antibody.

During a measles outbreak, 283 serum specimens from 221 suspected cases of measles were tested by immunofluorescence and enzyme immunoassay for the presence of measles-specific immunoglobulin M (IgM) antibodies by using commercially available reagents. There was 97% agreement between the two assays; thus, the choice of the method for diagnostic testing is a matter of convenience and experience. In all 62 cases of measles from which a single blood sample was available, measles IgM-specific antibodies were detectable by both methods. Fifty percent of the 62 cases were positive within 3 days after onset of the rash. This increased to 91% 10 days after onset of the rash.

Prospective study of cytomegalovirus antigenemia in allograft recipients.

A recently published technique for direct detection of cytomegalovirus (CMV) antigenemia was tested prospectively in 27 transplant recipients. Eighteen patients developed active CMV infections and 10 of the 18 experienced CMV syndrome. All of the infections were detected by classical virus isolation and/or serologic techniques. No antigenemia was demonstrated.

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.