Conservation of sequence and expression of Xenopus and zebrafish dHAND during cardiac, branchial arch and lateral mesoderm development.

dHAND and eHAND are related basic helix-loop-helix transcription factors that are expressed in the cardiac mesoderm and in numerous neural crest-derived cell types in chick and mouse. To better understand the evolutionary development of overlapping expression and function of the HAND genes during embryogenesis, we cloned the zebrafish and Xenopus orthologues. Comparison of dHAND sequences in zebrafish, Xenopus, chick, mouse and human demonstrated conservation throughout the protein. Expression of dHAND in zebrafish was seen in the earliest precursors of all lateral mesoderm at early gastrulation stages. At neurula and later stages, dHAND expression was observed in lateral precardiac mesoderm, branchial arch neural crest derivatives and posterior lateral mesoderm. At looping heart stages, cardiac dHAND expression remained generalized with no apparent regionalization. Interestingly, no eHAND orthologue was found in zebrafish. In Xenopus, dHAND and eHAND were co-expressed in the cardiac mesoderm without the segmental restriction seen in mice. Xenopus dHAND and eHAND were also expressed bilaterally in the lateral mesoderm without any left-right asymmetry. Within the branchial arches, XdHAND was expressed in a broader domain than XeHAND, similar to their mouse counterparts. Together, these data demonstrate conservation of HAND structure and expression across species.

Heterotaxy: associated conditions and hospital-based prevalence in newborns.

PURPOSE: To provide insight into the possible etiology and prevalence of heterotaxy, we studied conditions associated with heterotaxy in a consecutive hospital population of newborns. METHODS: From 1972 to March, 1999 (except February 16, 1972 to December 31, 1978), 58 cases of heterotaxy were ascertained from a cohort of 201,084 births in the ongoing Active Malformation Surveillance Program at the Brigham and Women's Hospital. This registry includes livebirths, stillbirths, and elective abortions. Prevalence among nontransfers (i.e., patients whose mothers had planned delivery at this hospital) was calculated as approximately 1 per 10,000 total births (20 of 201,084). RESULTS: We analyzed a total of 58 patients consisting of 20 (34%) nontransfers and 38 (66%) transfers. Patients were categorized by spleen status as having asplenia (7 nontransfers, 25 total), polysplenia (8, 20), right spleen (4, 11), normal left (0, 1), and unknown (1, 0). Among the 20 nontransfer and 59 total heterotaxy patients, the following associated medical conditions were present: chromosome abnormality (1 nontransfer, 2 total), suspected Mendelian or chromosome microdeletion disorder (1 nontransfer, 6 total), and maternal insulin-dependent diabetes mellitus (1 nontransfer, 2 total). There were 6 twins (1 member each from 6 twin pairs including 1 dizygous, 4 monozygous, 1 conjoined; 2 were nontransfers). An associated condition occurred in 5 (25%) nontransfer and 16 (28%) total patients, or among 10 of 53 singleton births (19%). CONCLUSIONS: Although most cases of heterotaxy in this series were sporadic events, an associated condition was present in about one-fourth of the cases. Not all of these conditions would be considered causative etiologies. Based on this small series alone, maternal insulin-dependent diabetes cannot be viewed as a risk factor for heterotaxy. However, the specific association of diabetes with polysplenia with/without left atrial isomerism is noteworthy, and adds weight to animal and epidemiologic case-control data.

Extracardiac anomalies in the heterotaxy syndromes with focus on anomalies of midline-associated structures.

The extracardiac defects in patients with heterotaxy have not been examined as extensively as cardiac defects. We found a high incidence of midline-associated defects in 160 autopsied cases of heterotaxy (asplenia, polysplenia, or single right-sided spleen). Fifty-two percent of patients with left-sided polysplenia had a midline-associated defect, as did 45% of those with asplenia. Most common were musculoskeletal or genitourinary anomalies, as well as cleft palate. Fused adrenal glands and anal stenosis or atresia occurred exclusively among patients with asplenia. A midline anomaly was twice as likely to be detected on complete autopsy than from clinical findings alone. Linkage studies should take into account that affected subjects may have isolated subclinical midline defects. The high incidence of midline-associated defects supports the theory that the midline plays a critical role in establishing left-right asymmetry in the body. Comparison of these defects with mouse models of laterality defects suggests that mutations that disrupt the transforming growth factor beta pathway may result in heterotaxy.

Structural and functional brain asymmetries in human situs inversus totalis.

OBJECTIVE: To determine whether individuals with situs inversus totalis (SI), a condition in which there is a mirror-image reversal of asymmetric visceral organs, have alterations in brain asymmetries. BACKGROUND: The human brain is asymmetric in structure and function. Although correlations between anatomic asymmetries and functional lateralization in human brain have been demonstrated, it has been difficult to further analyze them. Characterization of asymmetries of brain structure and function in SI might advance the understanding of these relationships. METHODS: Using anatomic and functional MRI techniques, we analyzed asymmetries in the brains of three individuals with SI. RESULTS: Two major anatomic asymmetries of the cerebral hemispheres, the frontal and occipital petalia, were reversed in individuals with SI. In contrast, SI subjects had left cerebral hemisphere language dominance on functional MRI analysis as well as strong right-handedness. CONCLUSION: These observations suggest that the developmental factors determining anatomic asymmetry of the cerebral petalia and viscera are distinct from those producing the functional lateralization of language.

Utility of direct measurement of low-density lipoprotein cholesterol in dyslipidemic pediatric patients.

BACKGROUND: Low-density lipoprotein cholesterol (LDL-C) levels are the primary basis for treatment guidelines established for hyperlipidemic children and adolescents. Levels of LDL-C are commonly monitored by means of the Friedewald formula, an indirect calculation that requires an overnight fast. A new method has been developed for the direct measurement of LDL-C (DLDL-C) that does not require fasting. We evaluated the clinical utility of this method. DESIGN: We determined LDL-C concentrations simultaneously by the DLDL-C method, Friedewald equation, and beta-quantification (reference procedure). SETTING: Pediatric dyslipidemia clinic at Children's Hospital, Boston, Mass. PATIENTS: Ninety-two fasting hyperlipidemic pediatric patients. RESULTS: At the LDL-C concentration cutoffs commonly used for making therapeutic decisions, the DLDL-C method had a significant negative bias (P< or =.05) and misclassified patients into incorrect treatment groups more often than the Friedewald method. The negative predictive value for the DLDL-C method was lower than that for the Friedewald method (P< or =.05), and the cost of determining LDL-C level with the new method was 3 times greater. CONCLUSIONS: The misclassification potential for LDL-C, and the assay costs, were greater for the DLDL-C method than for the Friedewald calculation. Despite the apparent advantages of the DLDL-C method, we conclude that for hyperlipidemic children the utility of this new method is not advantageous over the conventional Friedewald method. In some conditions, such as in diabetes or marked hypertriglyceridemia, the DLDL-C method may be useful.

Patterning the heart's left-right axis: from zebrafish to man.

Normal left-right asymmetry is highly conserved among vertebrates. Errors in the proper patterning of this axis are believed to lead to congenital anomalies of the heart and abdominal viscera, often with profound clinical consequences. We review briefly the nature of potential signals and signaling sources that lead to the break in left-right symmetry. The evidence suggests that left-right reversal, or homogenization, of these signals may lead to different consequences, and we explain some malpositions and malalignments of the atria, ventricles, and/or outflow tract that are seen in a variety of congenital cardiac diseases. We speculate that there are units of organ assembly responsive to laterality signals, and these units may be driven independently. One crucial source of signals appears to be the notochord and floorplate. In order to examine the clinical relationship of these midline structures to putative disorders of laterality, we review all patients with disturbances of normal laterality seen at the Massachusetts General Hospital over the past 20 years. We find a significant association between laterality defects and anomalies of the spine and other midline structures.

Three zebrafish MEF2 genes delineate somitic and cardiac muscle development in wild-type and mutant embryos.

The zebrafish is an important experimental system for vertebrate embryology, and is well suited to the molecular analysis of muscle development. Transcription factors, such as the MEF2s, regulate skeletal and cardiac muscle-specific genes during development. We report the identification of three zebrafish MEF2 genes which, like their mammalian counterparts, encode factors that function as DNA-binding transcriptional activators of muscle specific promoters. The pattern of MEF2 expression in zebrafish defines discrete cell populations in the developing somites and heart and has mechanistic implications for developmental regulation of the MEF2 genes, when compared with other species. Alteration of MEF2 expression in two mutants affecting somitogenesis provides insight into the control of muscle formation in the embryo.

Lack of detection of enteroviral RNA or bacterial DNA in magnetic resonance imaging-directed muscle biopsies from twenty children with active untreated juvenile dermatomyositis.

OBJECTIVE: To investigate for the presence of increased titers of circulating antibody to putative infectious agents and for detectable viral RNA or bacterial DNA in children with active recent-onset juvenile dermatomyositis (DM). METHODS: Magnetic resonance imaging-directed muscle biopsies were performed in 20 children with active, untreated, recent-onset juvenile DM and in age-matched children with neurologic disease. Sera were tested for complement-fixing antibody to Coxsackievirus B (CVB), influenza A and B, parainfluenza 1 and 3, Mycoplasma pneumoniae, mumps, respiratory syncytial virus, and Reovirus; and by immunofluorescence for IgG antibody to Toxoplasma gondii cytomegalovirus and IgM antibody to Epstein-Barr virus. Muscle from juvenile DM patients and control children, CD-1 Swiss mice with and without CVB1 infection, and viral stock positive for CVB1-6 were tested using reverse-transcriptase polymerase chain reaction with 5 primer sets, 4 probes (1 Coxsackievirus, 3 Enterovirus), and universal primers for DNA. RESULTS: No increased antibody, viral RNA, or bacterial DNA was present in the juvenile DM patients or the control children. CONCLUSION: Juvenile DM may be triggered by unidentified agent(s) in the genetically susceptible host.

Variable location of accessory pathways associated with the permanent form of junctional reciprocating tachycardia and confirmation with radiofrequency ablation.

Permanent junctional reciprocating tachycardia (PJRT) occurs primarily in young patients and causes nearly incessant tachycardia that is frequently refractory to pharmacologic treatment. Previous nonpharmacologic therapy has included surgical or direct-current catheter ablation of either the His bundle or the accessory pathway. The accessory pathway in PJRT has been described as having retrograde and anterograde decremental conduction properties, and is typically identified in the posteroseptal location. This report describes radiofrequency catheter ablation of accessory pathways in 8 patients with PJRT. All ablations were successful and without adverse effects. Accessory pathway potentials were detected just before atrial activation in 6 of 8 patients. A new finding was that 5 of the 8 pathway locations, as identified by the site of successful ablation, were not in the typical posteroseptal region. In 1 patient it was located in the right posteroseptal region, 2 were in the right atrial freewall, 1 was in the right anterior septum and 1 was in the left posterior region just outside of the septal region. In conclusion, radiofrequency catheter ablation can be a highly effective and safe method for treatment of young patients with PJRT. Because the accessory pathways can be located outside of the posteroseptal region, careful mapping of both the right and left atrioventricular groove may be necessary for successful ablation.

The characterization of yeast mitochondrial RNA polymerase. A monomer of 150,000 daltons with a transcription factor of 70,000 daltons.

Transcription in organelles is regulated by both organellar and nuclear mechanisms. In order to study further the control of organellar transcription, we have purified and characterized the RNA polymerase from mitochondria of Saccharomyces cerevisiae and identified a transcription factor required for promoter recognition. The RNA polymerase can be separated into two forms, selective and nonselective. The nonselective form was purified over 11,000-fold and appears to be active as a monomer with a molecular weight of 150,000. The Mr 150,000 polypeptide acts as a core RNA polymerase, and an Mr 70,000 polypeptide appears to confer selectivity for the promoter upon this core. The Mr 70,000 transcription factor binds specifically to the mitochondrial initiation site in the absence of polymerase and decreases nonselective initiation by the polymerase. The Mr 150,000 polymerase is immunologically related to an Mr 145,000 protein purified from yeast as a primase, although it is thought to be a functional unit of mitochondrial RNA polymerase (Kelly, J. L., and Lehman, I. R. (1986) J. Biol. Chem. 261, 10340-10347). Antibodies to the Mr 145,000 protein inhibit transcription by the mitochondrial RNA polymerase purified here.

In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast.

The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.