ABSTRACT
Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.
Subject(s)
Epidermolysis Bullosa Simplex/genetics , Myasthenic Syndromes, Congenital/genetics , Plectin/genetics , Receptors, Nicotinic/genetics , Consanguinity , Epidermolysis Bullosa Simplex/complications , Excitatory Postsynaptic Potentials/physiology , Female , HEK293 Cells , Humans , Male , Middle Aged , Miniature Postsynaptic Potentials/physiology , Mutagenesis, Insertional/genetics , Myasthenic Syndromes, Congenital/physiopathology , Neuromuscular Junction/physiopathology , PedigreeABSTRACT
We developed an oblique-incidence reflectivity difference (OI-RD) scanning microscope for label-free imaging of microarrays of biomolecules upon solid substrates. We demonstrate that hybridization reactions in an oligonucleotide microarray fabricated upon a glass slide can be detected by such an OI-RD microscope.
Subject(s)
Biochemistry/methods , Microscopy/methods , Models, Theoretical , Oligonucleotide Array Sequence Analysis , Biochemistry/instrumentation , Equipment Design , Microscopy/instrumentation , Nucleic Acid Hybridization , Scattering, RadiationABSTRACT
Intrauterine growth retardation (IUGR) affects almost 10% of infants born in the United States. It may be responsible for delayed gastrointestinal function and is an important cause of perinatal morbidity and mortality. The New Zealand White rabbit provides an optimal model for the study of naturally occurring IUGR. At term, birth weight is determined by fetal position within the bicornuate uterus. The small intestinal disaccharidase enzymes are indicators of bowel maturity and function. To examine potential differences in disaccharidase development between normal and IUGR fetuses, this rabbit model was investigated. Jejunum was harvested at multiple stages in rabbit development including the third trimester fetus, neonate, and adult. Lactase, maltase, and sucrase enzyme activity, as well as total protein content, was determined. Results were analyzed by the 2-tailed t test and ANOVA. Lactase activity appeared in the mid-third trimester, peaked in the early neonatal period, then declined to adult levels. Maltase activity appeared in the early third trimester and gradually rose to adult levels. Sucrase remained at trace levels until the mid-neonatal period, reaching adult levels by weaning. Both lactase and maltase activity were depressed in IUGR fetuses compared with their normal littermates. This pattern of disaccharidase depression continued into the neonatal period until catch-up growth occurred at 2 wk when levels equalized. This report describes differential small intestinal disaccharidase development between normal and growth-retarded rabbit fetuses in a naturally occurring model of IUGR.
Subject(s)
Disaccharidases/metabolism , Disease Models, Animal , Fetal Growth Retardation/enzymology , Animals , Intestine, Small/embryology , Intestine, Small/enzymology , RabbitsABSTRACT
Mutant alleles of the chemokine receptors CCR5 and CCR2 affect the susceptibility to HIV infection as well as the rate of disease progression. In this article the authors report the results of a survey for presence of the common Delta32ccr5 and ccr2b-V64I mutant alleles in 472 individuals of a multiethnic cohort. Hispanic Americans had the highest observed frequency of the Delta32ccr5 allele (3.57%), whereas African Americans had a lower frequency (1.55%). The mutant allele was absent in Asian Americans and Native Americans. Thus, the Delta32ccr5 allele segregates in populations with a significant white admixture and is rare in genetically distant non-European groups. Native Americans had the highest occurrence of the ccr2b-V64I allele (31.13%), whereas African Americans, Asian Americans, and Hispanic Americans had much lower frequencies (14.36%, 11.94%, and 14.37% respectively). This mutation is probably an ancient one, occurring before the migration of the ancestors of Native Americans across the Bering Straits to the Americas. The twofold greater frequency of ccr2b-V64I in modern Native Americans probably reflects a founder effect. The observed population differences in Delta32ccr5 and ccr2b-V64I frequencies, considered together with their documented effects on sensitivity to HIV infection and rate of disease progression, have implications for HIV transmission patterns in the United States, as well as for AIDS prediction, monitoring, and treatment.
Subject(s)
Ethnicity/genetics , Receptors, CCR5/genetics , Receptors, Chemokine/genetics , Alleles , Asian/genetics , Black People/genetics , DNA/analysis , Gene Frequency , Hispanic or Latino/genetics , Humans , Indians, North American/genetics , Mutation , Polymerase Chain Reaction , Receptors, CCR2ABSTRACT
Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3-30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early-passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms.
Subject(s)
Biomarkers, Tumor/analysis , Brain Neoplasms/genetics , Glioma/genetics , Immunosorbent Techniques , Intercellular Signaling Peptides and Proteins , Viral Proteins/analysis , Viral Proteins/genetics , Animals , DNA, Complementary/analysis , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Gene Library , Humans , Progranulins , RatsABSTRACT
Genomic mismatch scanning (GMS) is a technique that enriches for regions of identity by descent (IBD) between two individuals without the need for genotyping or sequencing. Regions of IBD selected by GMS are mapped by hybridization to a microarray containing ordered clones of genomic DNA from chromosomes of interest. Here we demonstrate the feasibility and efficacy of this form of linkage-mapping, using congenital hyperinsulinism (HI), an autosomal recessive disease, whose relatively high frequency in Ashkenazi Jews suggests a founder effect. The gene responsible (SUR1) encodes the sulfonylurea receptor, which maps to chromosome 11p15.1. We show that the combination of GMS and hybridization of IBD products to a chromosome-11 microarray correctly maps the HI gene to a 2-Mb region, thereby demonstrating linkage-disequilibrium mapping without genotyping.
Subject(s)
ATP-Binding Cassette Transporters , Chromosome Mapping/methods , Genetic Techniques , Hyperinsulinism/genetics , Linkage Disequilibrium , Potassium Channels, Inwardly Rectifying , Child , Chromosomes, Human, Pair 11 , Founder Effect , Humans , Hyperinsulinism/ethnology , In Situ Hybridization/methods , Potassium Channels/genetics , Receptors, Drug/genetics , Sulfonylurea ReceptorsSubject(s)
Genetics, Medical/trends , Molecular Biology/trends , DNA/analysis , Ethics, Medical , Genetic Testing/trends , Heterozygote , HumansABSTRACT
Resistance to activated protein C (APC) is the most common risk factor for venous thromboembolism, a major cause of morbidity and mortality with an incidence of about 1/1,000 per year. The Arg 506 to Gln mutation in exon 10 of the coagulation factor V gene (factor V-Leiden) has been found to be responsible for over 90% of the APC resistance cases and is an autosomal dominant trait. Initial studies have suggested that this mutation is restricted to individuals of European Caucasian extraction with an average allele frequency in European and American Caucasians of 4.4%, making it one of the most common monogenic disorders in the Caucasian population. A limited number of other ethnic populations have been tested and the mutation has been found only rarely. In our multiethnic survey of 602 individuals, Hispanic-Americans had the highest observed frequency of the factor V-Leiden mutant allele, 1.65%, while African-Americans had a somewhat lower frequency, 0.87%. No factor V-Leiden mutations were found in 191 Asian-Americans or 54 Native-Americans tested. These results indicate that the factor V-Leiden mutation segregates in populations with significant Caucasian admixture and is rare in genetically distant non-European groups. This ethnic stratification may be important in developing cost-effective selective screening programs to identify individuals at risk for thromboembolism and offer prophylactic therapy.
Subject(s)
Ethnicity/genetics , Factor V/genetics , Gene Frequency , Mutation , Alleles , Asian/genetics , Black People/genetics , Hispanic or Latino/genetics , Humans , Indians, North American/genetics , Northwestern United States , Southwestern United States , White People/geneticsABSTRACT
Maternal ingestion of alcohol is believed to be one factor that greatly influences the development of intrauterine growth retardation (IUGR) and postnatal growth failure. The present study was undertaken to determine whether maternally ingested alcohol adversely affects fetal growth and intestinal mucosal function. Five time-mated New Zealand white rabbit does were given ethanol intravenously (ETH group) (30% vol/vol; 1.0 g/kg/d) on gestational days (GD) 15 through 29 (term, 31 days). Two other rabbits received the same dose of ethanol. Maternal, fetal, and amniotic fluid alcohol levels were measured on GD 24. Four control rabbits (SH group) received normal saline (25 mL, intravenously). At term, the animals were delivered by cesarean section and killed. Seventeen of the 42 ETH fetuses survived the study period (43%); all 24 SH fetuses survived. On GD 24, within 60 minutes after maternal ethanol infusion, the fetal blood alcohol concentration (BAC) increased to 153 +/- 1.97 mg/dL (v maternal, 179 +/- 1.75 mg/dL); the amniotic ethanol level increased to 46 +/- 1.32 mg/dL. Birth weight was lower in the ETH group (46.88 +/- 2.21 g) than in the SH group (55.78 +/- 1.80 g) (P < .01). Disaccharidase activity, an indicator of intestinal mucosal function, showed that lactase activity (per milligram of protein) was significantly lower in ETH fetuses (2.60 x 10(-2) +/- 0.22 UE/mg) than in SH fetuses (3.50 x 10(-2) +/- 0.25 UE/mg) (P = .01); maltase activity and protein content were not affected significantly. This report provides the first description of the adverse effects of maternal alcohol ingestion on the small intestinal mucosal function of the fetal rabbit.
Subject(s)
Embryonic and Fetal Development/drug effects , Ethanol/adverse effects , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Alcohol Drinking/adverse effects , Amniotic Fluid/chemistry , Animals , Birth Weight/drug effects , Digestive System/drug effects , Digestive System/embryology , Disaccharidases/metabolism , Ethanol/blood , Female , Fetal Blood/chemistry , Fetal Growth Retardation/chemically induced , RabbitsABSTRACT
During the past 25 years, 252 children underwent repair of pectus deformities. There were 195 male and 57 female patients, of whom 227 had pectus excavatum and 25 had pectus carinatum. Of the 252 patients, 113 underwent repair at between 2 and 5 years of age. Exercise limitation was reported by 51%, and 32% had frequent respiratory infections or asthma. Repair was performed through a transverse incision with subperiosteal resection of the lower four or five costal cartilages, from sternum to costochondral junction bilaterally. A transverse wedge osteotomy was made through the anterior table of the sternum, with fracture but no displacement of the posterior table. For children younger than 5 years (n = 108), the periosteal sheath of the fifth rib from each side was sewn together behind the sternal tip. For older patients (n = 136), a thin steel strut was used for sternal support for 6 months. There were no deaths within the first year. Complications included seroma (16), atelectasis (12), pneumothorax (three), and recurrent chest depression (three). More than 98% of patients had improvements in exercise tolerance, endurance, respiratory symptoms, and cosmetic appearance; these improvements were considered excellent results. Operation at an early age with routine use of substernal support with minimal preoperative and postoperative testing has provided excellent results at a low cost.
Subject(s)
Funnel Chest/surgery , Sternum/abnormalities , Sternum/surgery , Adolescent , Adult , Child , Child, Preschool , Congenital Abnormalities/surgery , Female , Humans , Male , Methods , Osteotomy/methods , Postoperative Complications , Prostheses and Implants , Ribs/surgeryABSTRACT
As fetal swallowing is documented in utero, supplementation of the ingested amniotic fluid with nutrients or hormones has been postulated as a potential prenatal treatment for intrauterine growth retardation (IUGR). To study the effect of epidermal growth factor (EGF) on the developing fetal small intestine, 12 pregnant rabbits underwent operation on day 24 of a normal 31-day gestation. Bilateral ovarian end fetuses underwent catheterization of their respective amniotic cavities with attachment to a miniosmotic pump. Study fetuses received recombinant human EGF at approximately 300 micrograms/kg/d for 1 week; controls received carrier solution only at an equivalent rate. On gestational day 31, fetuses were delivered by cesarean section and somatic measurements were recorded. The small intestine was harvested and proximal, middle, and distal regions were analyzed for lactase and maltase enzyme activity. Additionally, the uptake of radiolabeled glucose and proline was measured by a standard everted mucosal sleeve technique for each segment. Results were analyzed by Student's paired t test and reported as mean +/- SEM. Nine fetal pairs survived (75%). Small intestinal (SI) length was increased in EGF fetuses (54.8 +/- 1.9 cm) versus control (50.4 +/- 2.7 cm) (P = .02). Lactase activity, reported as UE/g protein, was significantly increased in the proximal segments in the EGF-infused fetuses; maltase was significantly increased in both the proximal and middle segments (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)