Genetic testing in infants.

Genetic testing has made diagnosis and treatment possible for many infants. With the addition of many new tests over the past few years, it is important to understand the clinical usefulness of each of these tests. Selecting the correct method of genetic testing assists in obtaining an accurate diagnosis and development of a plan of care for the infant.

Impaired autoproteolytic cleavage of mCLCA6, a murine integral membrane protein expressed in enterocytes, leads to cleavage at the plasma membrane instead of the endoplasmic reticulum.

CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after posttranslational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6(Δ™)E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.

Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia.

CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells.

Persistence of rubella antibodies after 2 doses of measles-mumps-rubella vaccine.

BACKGROUND: Since 1990, most schoolchildren in the United States have received a second dose of measles-mumps-rubella vaccine (MMR2) at kindergarten entry. Elimination of endemic rubella virus circulation in the United States was declared in 2004. The objective of the current study was to evaluate the short- and long-term rubella immunogenicity of MMR2. METHODS: At enrollment in 1994-1995, children (n = 307) in a rural Wisconsin health maintenance organization received MMR2 at age 4-6 years. A comparison group of older children (n = 306) was vaccinated at age 9-11 years. Serum specimens were collected during a 12-year period. Rubella antibody levels were evaluated by plaque-reduction neutralization (lowest detectable titer, 1:10). RESULTS: Before administration of MMR2 in the kindergarten group, 9% of subjects were seronegative, 60% had the lowest detectable titer, and the geometric mean titer (GMT) was 1:13. One month after administration of MMR2, 1% were seronegative, 6% had the lowest detectable titer, and the GMT was 1:42. Four-fold boosts occurred in 62% of subjects, but only 0.3% were immunoglobulin M positive. Twelve years after MMR2 administration, 10% were seronegative, 43% had the lowest detectable titer, and the GMT was 1:17. The middle-school group showed similar patterns. CONCLUSIONS: Rubella antibody response to MMR2 was vigorous, but titers decreased to pre-MMR2 levels after 12 years. Because rubella is a highly epidemic disease, vigilance will be required to assure continued elimination.

Persistence of mumps antibodies after 2 doses of measles-mumps-rubella vaccine.

BACKGROUND: Since 1990, most US schoolchildren have received a second dose of measles-mumps-rubella vaccine (MMR2) at kindergarten entry. The objective of the present study was to evaluate the short- and long-term mumps immunogenicity of MMR2. METHODS: At enrollment in 1994-1995, children (n=308) in a rural Wisconsin health maintenance organization received MMR2 at age 4-6 years. A comparison group of older children (n=308) was vaccinated at age 9-11 years. Serum samples were collected over 12 years. Mumps antibody levels were evaluated by plaque-reduction neutralization (lowest detectable titer, 10). RESULTS: Before MMR2, the geometric mean titer (GMT) for the younger group was 33; no subject was seronegative, but 16% had the lowest detectable titer. In response to MMR2, the GMT tripled to 97, and the proportion with low titers diminished to 3%. Four-fold boosts occurred among 54%, but only 3% were positive for immunoglobulin M. Twelve years after MMR2, the GMT declined to 46, the proportion with titers

Persistence of measles antibodies after 2 doses of measles vaccine in a postelimination environment.

OBJECTIVE: To evaluate the persistence of measles antibodies after 2 doses of measles vaccine in a setting where exposure to wild-type measles was unlikely. Measles was declared eliminated from the United States in 2000, an achievement attributed to effective implementation of a routine 2-dose vaccination policy. Some have questioned whether measles transmission could resume if immunity wanes in the absence of boosting from wild-type measles. DESIGN: Prospective, observational, volunteer cohort study. SETTING: Rural Wisconsin health maintenance organization. PARTICIPANTS: Children who received the second measles vaccine dose at kindergarten (aged 4-6 years) or middle school (aged 10-12 years) in 1994 or 1995. Serum samples were collected periodically during a 10-year period for the kindergarten group and a 5-year period for the middle school group. INTERVENTION: Second dose of measles vaccine. MAIN OUTCOME MEASURE: Measles antibody levels were assessed by plaque-reduction neutralization: titers less than 8 mIU/mL were considered seronegative and suggestive of susceptibility to measles, and titers of 120 mIU/mL or less were considered low and suggestive of potential susceptibility. RESULTS: During the study period, no measles was reported in the study area. Voluntary attrition reduced the study population from 621 at enrollment to 364 (58.6%) by study end. Before the second dose, 3.1% (19/621) had low titers, of whom 74% (14/19) were antibody-negative, with geometric mean titers being significantly higher in kindergarteners (1559 mIU/mL) than in middle schoolers (757 mIU/mL) and rates of negativity significantly lower (1.0% [3/312] vs 3.6% [11/309]). One month after the second dose, 0.2% (1/612) had low titers and none was seronegative, with geometric mean titers being significantly higher in kindergarteners (2814 mIU/mL) than in middle schoolers (1672 mIU/mL). By study end, 4.9% (18/364) had low titers and none was seronegative, with no significant difference in geometric mean titers between kindergarteners (641 mIU/mL) and middle schoolers (737 mIU/mL) when both groups were aged 15 years. Projections suggest that the proportion of persons with low antibody levels may increase over time. CONCLUSIONS: Measles antibody persisted in all vaccinees available for follow-up 10 years after a second dose of vaccine, with no seronegative results detected. Declining titers suggest the need for vigilance in ensuring disease protection for the vaccinated population.

Evaluation of potentially common adverse events associated with the first and second doses of measles-mumps-rubella vaccine.

BACKGROUND/OBJECTIVES: In 1989, the American Academy of Pediatrics and the Advisory Committee on Immunization Practices recommended that school children receive 2 doses of measles-mumps-rubella vaccine. With measles and rubella eliminated from the United States, measles-mumps-rubella vaccine adverse events have come under scrutiny, but no study has compared the reactogenicity of the first (measles-mumps-rubella vaccine dose 1) and second (measles-mumps-rubella vaccine dose 2) doses at the most common ages of administration in the United States. METHODS: From a health maintenance organization, 3 groups of children were recruited: (1) toddlers aged 12 to 24 months receiving measles-mumps-rubella vaccine dose 1; (2) kindergartners aged 4 to 6 years receiving measles-mumps-rubella vaccine dose 2; and (3) middle schoolers aged 10 to 12 years receiving measles-mumps-rubella vaccine dose 2. From 2 weeks before measles-mumps-rubella vaccine administration until 4 weeks afterward, families recorded in diaries the occurrence of potentially common symptoms. Postvaccination symptom rates were compared with the prevaccination baseline, with significance assessed by testing incidence rate ratios estimated by Poisson regression. RESULTS: Of 2173 children enrolled, 373 (17%) were lost to attrition, producing a study population of 1800. Compared with the prevaccination baseline, rates of fever, diarrhea, and rash were significantly elevated postvaccination among 535 toddlers receiving measles-mumps-rubella vaccine dose 1. An estimated net 95 (18%) experienced measles-mumps-rubella vaccine-associated events (median onset 5-10 days postvaccination, duration 2-5 days), with high fever (temperature > or = 39.5 degrees C) occurring in 33 (6%). None required medical attention. For 633 kindergartners and 632 middle schoolers, symptom rates were not significantly elevated after measles-mumps-rubella vaccine dose 2 compared with baseline. CONCLUSIONS: Vaccination-associated adverse events occur in approximately 1 of every 6 toddlers receiving measles-mumps-rubella vaccine dose 1, with high fever occurring in 1 of 20. Adverse events are infrequent for measles-mumps-rubella vaccine dose 2 administered to school-aged children.

Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine.

Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.

Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy.

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.

Atypical antipsychotics in the elderly.

INTRODUCTION: Although their primary purpose is to treat psychosis, antipsychotics are commonly prescribed for the elderly to treat the behavioural disturbances and agitation associated with dementia. Such use is controversial. Atypical antipsychotics cause fewer extrapyramidal sideeffects than the older drugs in younger adults, but the evidence base for their efficacy and tolerability in the elderly is poor. The aims of this study were to determine the prevalence of atypical antipsychotic prescribing for the elderly, the indications for use and documented side-effects. METHOD: The medication cards of all patients from 19 Trusts, occupying a psychiatric bed for the over 65s, were screened during one week in March 2000. Data were collected by pharmacists from the clinical notes. RESULTS: Half of those prescribed an antipsychotic received an atypical, and risperidone was the one most commonly prescribed. Half the sample had a diagnosis of dementia. Documented side-effects from the atypical were uncommon. CONCLUSION: Atypicals are frequently prescribed as first-line antipsychotics for behavioural problems associated with dementia, despite the poor evidence base for their efficacy and safety in this population. Undermonitoring of side-effects may remain a problem.