Genome-wide Scan of Dental Fear and Anxiety Nominates Novel Genes.

Dental care-related fear and anxiety (DFA) is prevalent, affects oral health care utilization, and is related to poor oral health and decreased quality of life. In addition to learned and cultural factors, genetics is hypothesized to contribute to DFA. Therefore, we performed a genome-wide association study to identify genetic variants contributing to DFA. Adult and adolescent participants were from 4 cohorts (3 from the US-based Center for Oral Health Research in Appalachia, n = 1,144, 1,164, and 535, and the UK-based Avon Longitudinal Study of Parents and Children [ALSPAC], n = 2,078). Two self-report instruments were used to assess DFA: the Dental Fear Survey (US cohorts) and Corah's Dental Anxiety Scale (ALSPAC). Genome-wide scans were performed for the DFA total scores and subscale scores (avoidance, physiological arousal, fear of dental treatment-specific stimuli), adjusting for age, sex, educational attainment, recruitment site, and genetic ancestry. Results across cohorts were combined using meta-analysis. Heritability estimates for DFA total and subscale scores were similar across cohorts and ranged from 23% to 59%. The meta-analysis revealed 3 significant (P < 5E-8) associations between genetic loci and 2 DFA subscales: physiological arousal and avoidance. Nearby genes included NTSR1 (P = 3.05E-8), DMRTA1 (P = 4.40E-8), and FAM84A (P = 7.72E-9). Of these, NTSR1, which was associated with the avoidance subscale, mediates neurotensin function, and its deficiency may lead to altered fear memory in mice. Gene enrichment analyses indicated that loci associated with the DFA total score and physiological arousal subscale score were enriched for genes associated with severe and persistent mental health (e.g., schizophrenia) and neurocognitive (e.g., autism) disorders. Heritability analysis indicated that DFA is partly explained by genetic factors, and our association results suggested shared genetic underpinnings with other psychological conditions.

Blueberry muffin rash, hyperbilirubinemia, and hypoglycemia: a case of hemolytic disease of the fetus and newborn due to anti-Kp(a).

Hemolytic disease of the fetus and newborn occurs when maternal IgG antibodies cross the placenta and cause hemolysis of fetal red blood cells. Kp(a) is a low frequency red blood cell antigen that has rarely been implicated in hemolytic disease of the fetus and newborn. The few reported cases attributed to anti-Kp(a) have typically had minimal clinical consequences. We report a critically ill neonate who presented with purpura, respiratory failure, severe liver dysfunction, hyperbilirubinemia, hypoglycemia and anemia. This case report broadens the spectrum of neonatal disease associated with anti-Kp(a), addresses the evaluation of hemolysis with liver failure in a neonate, and emphasizes the importance of screening for antibodies to low frequency red blood cell antigens in suspected hemolytic disease of the fetus and newborn.

Experimental studies of the effects of abnormal venous valves on fluid flow.

The effects of variations in the venous valve anatomy are studied experimentally using an artificial system that mimics the bicuspid valves normally found in veins in the lower extremities. The artificial valves are constructed from thin-walled, latex tubing and polyurethane film. The experimental variables in the study are the gap width between the leaflet attachments at the vein wall and the ratio of the sinus depth to vein diameter. The results show that the antegrade mass flow rate is not affected to the same degree when compared to retrograde flow by the various valve configurations examined in this study. The results also indicate that increases in the gap width, which serve to increase the degree of imperfect wall attachment, have less effect on retrograde mass flow rate in valves with deeper sinuses.

Autosomal albino chicken mutation (ca/ca) deletes hexanucleotide (-deltaGACTGG817) at a copper-binding site of the tyrosinase gene.

We compared tyrosinase cDNA sequences from a line of autosomal albino and Black Silky chickens isolated from cultured melanocytes by reverse transcription-polymerase chain reaction (RT-PCR). Both sources produce a single DNA fragment of predicted normal tyrosinase size. Direct sequencing of the PCR product showed three mutated sites in the tyrosinase gene of the albino chicken. Two silent point mutations and a deletion of six nucleotides (-deltaGACTGG) at 817 bp in the tyrosinase cDNA sequence were observed when compared with the White Leghorn and Black Silky cDNA sequences. The deduced albino chicken tyrosinase protein lacks two amino acids, aspartic acid and tryptophan. The position of these amino acids is consistent with one of the potential copper-binding sites that should be indispensable for function of the enzyme. We speculate that the six-base deletion is responsible for the inactive tyrosinase in this line of albino chickens.

Multiplex amplification of STR loci with gender alleles using infrared fluorescence detection.

The analysis of short tandem repeat (STR) polymorphisms has proven extremely useful for gene mapping, paternity testing, and forensic analysis. Several commercial products are currently available for performing amplification and analysis of STRs. We have adapted Promega Geneprint Systems for use with a high sensitivity infrared (IR) fluorescent automated DNA sequencer. IR-labeled amplification products are generated by including a small quantity of IR-labeled dATP in the reaction. Several Geneprint STR loci can be multiplexed together with the amelogenin sex identification locus in a single amplification reaction. We have successfully amplified up to five Geneprint STR loci together with the amelogenin locus thus improving the throughput of analysis. Purified genomic DNA as well as simulated forensic samples have been utilized for these multiplex amplifications.

Producing STR locus patterns from bloodstains and other forensic samples using an infrared fluorescent automated DNA sequencer.

Short tandem repeat (STR) analysis is increasingly being used in forensic case analysis because of the large number of STR loci in the human genome and their highly polymorphic nature. An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from simulated forensic samples. The amplification strategy used a 19 base pair extension on the 5' end of one of the PCR primers. This sequence is identical to the sequence of a universal M13 Forward sequencing primer which is included in the amplification reaction. Allelic bands were detected by incorporation of the M13 primer-fluorescent dye conjugate into PCR products thus eliminating the need for direct conjugation of fluorescent dye to individual STR primers. By using an IR-based automated DNA sequencer and Tth DNA polymerase, polymorphic STR alleles were detected on-line rapidly and efficiently from bloodstains using only a high temperature incubation to extract DNA from blood cells. Five STR loci were also amplified using Chelex extracted DNA from simulated forensic samples. Multiplexing of three primer pairs in a single PCR mixture for amplification was accomplished using Taq polymerase. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with silver staining and fluor detection systems. Real-time detection permits immediate visualization of the data and STR alleles are displayed as familiar autoradiogramlike images that can be analyzed by computer. By loading a 64 lane gel twice and multiplexing with three primer pairs, forensic scientists can type at least three loci from 120 samples in one day.

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Tissue-specific expression of mouse tyrosinase gene in cultured chicken cells.

A mouse tyrosinase cDNA has been combined with different promoters and inserted into several replication-competent avian leukosis proviruses and the viruses were transferred into cultured albino chick cells by viral infection. Expression of the tyrosinase gene depended on one of four promoter sequences: the resident constitutive promoter (Rous sarcoma virus long-terminal repeat; RSV-LTR), 471 bp from the mouse tyrosinase gene-associated promoter, 519 bp from the Japanese quail tyrosinase gene associated promoter, or 369 bp from the quail tyrosinase promoter. The infected cells expressed tyrosinase and produced pigment which could be seen with the light microscope. Immunofluorescence microscopy, using an anti mouse tyrosinase T1-specific antibody, also showed the presence of mouse tyrosinase. When infected with the same viral titer, gene expression was highest with the constitutive LTR promoter. The quail tyrosinase promoter, while less efficient than the LTR, was more efficient than the other tyrosinase promoter. Fibroblasts and hepatocytes infected with the construct carrying the constitutive promoter or the truncated quail promoter expressed tyrosinase. The mouse and quail promoters appeared to show tissue-specific expression since fibroblasts and hepatocytes infected with viruses carrying these promoters did not express mouse tyrosinase. Toxicity is associated with constitutive expression of tyrosinase in nonmelanocytes. Therefore the viruses that carry the tissue specific promoters should be useful for in vivo studies.

Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons.

The ability to correctly diagnose the molecular cause of genetic diseases is becoming increasingly important in medicine. This requires an efficient method for the analysis of the DNA sequence of specific genes and the detection of mutations in affected individuals. We report a method to determine the mutations responsible for tyrosinase related albinism (OCA1) using a combination of polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis and direct DNA cycle sequencing using fluorescently labeled oligonucleotides and an automated DNA sequencer based on infrared fluorescence technology. This method allows DNA from several individuals to be sequenced quickly and simultaneously so that the specific location of each mutation and the carrier status of family members can be determined.

The expression of mouse tyrosinase in chick cells in vitro and in vivo when controlled by a constitutive promoter.

Virally introduced mouse tyrosinase expression was checked both in vitro and in vivo in chicken cells and tissues. The results indicate that a constitutive promoter is able to express mouse tyrosinase in a variety of cells and tissues both in vitro and in vivo. Tyrosinase expression is marked by pigment production in situ, which is visible at macroscopic as well as microscopic levels without the use of substrates. It is concluded that tyrosinase can be a valuable marker for tracking gene insertion since it is spontaneously expressed. The expression of tyrosinase in some cells and tissues has a detrimental effect, however, and should be controlled by tissue-specific promoters.

Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus.

A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.

Transcription and sequence studies of a 4.3-kbp fragment from a ds-DNA eukaryotic algal virus.

A 4.3-kbp portion of the genome from the Chlorella virus, PBCV-1, has been cloned and sequenced. Minimally, five open reading frames (ORFs) were identified on this fragment. Transcriptional analysis indicates that each ORF encodes complex patterns of RNA. The total length of transcribed RNA exceeds that of the ORF indicating either post-transcriptional modification or multiple transcriptional start/stop sites. The sequence TTTTTNT, previously described as the transcriptional stop site for the early genes of vaccinia virus, is also found downstream of each of our ORFs. The regions 5' to each ORF were very A + T-rich (approx 80%) but distinct promoter sequences were not unambiguously identified.

Expression of virally-transduced mouse tyrosinase in cultured chick embryo cells.

A cDNA encoding mouse tyrosinase was inserted into a plasmid containing the provirus of a replication competent Avian Leukosis Virus (ALV). A viral stock produced from the plasmid was used to infect cultured tyrosinase-negative (ca/ca) unpigmented chick embryo pigment cells. Five days after infection many cells were producing very dark discrete pigment granules. Cultures of tyrosinase positive, sex linked albino (sal) pigment cells produced no additional pigmentation. White Leghorn pigment cells responded to viral infection like the sal pigment cells.

Expression of virally transduced mouse tyrosinase in tyrosinase-negative chick embryo melanocytes in culture.

A cDNA encoding mouse tyrosinase was inserted into a plasmid containing the provirus of a replication competent avian leukosis virus (ALV). A viral stock produced from the plasmid was used to infect cultured tyrosinase-negative (ca/ca) unpigmented chick embryo melanocytes. Five days after infection many cells were producing very dark discrete melanosomes.

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.

Minimal pigment: a new type of oculocutaneous albinism.

Minimal pigment, a new type of oculocutaneous albinism (OCA), is described. At birth, affected individuals had no skin or eye pigment, and white hair and blue irides, but minimal amounts of pigment developed in the iris during the first decade of life. They had no measurable hairbulb tyrosinase activity. A characteristic and unusual pattern of parental activity was found in each of three families studied, with one parent having normal and the other parent having abnormally low tyrosinase activity. The melanocyte ultrastructure was normal and variations in premelanosomal pigmentation correlated with tyrosinase activity. This clinical and biochemical pattern has not been seen in any of the previously described types of OCA. The biochemical defect in minimal pigment OCA is unknown.

Brown oculocutaneous albinism. Clinical, ophthalmological, and biochemical characterization.

The clinical, ophthalmological, and biochemical characteristics of a 28-year-old black woman with brown oculocutaneous albinism were determined. Hair color was medium brown and skin color was light brown, and a faint tan developed with sun exposure. The irides were light brown in the central one-third, blue-gray in the peripheral two-thirds, and showed punctate and radial translucency. Visual acuity was 20/60 in the right eye and 20/100 in the left eye. There was a moderate pendular nystagmus, and previous surgeries had corrected an exotropia. The foveal reflex was muted, and the retinal pigment was reduced. Hairbulb tyrosinase activity was 1.75 pmoles/120 min/hairbulb, hairbulb glutathione content 0.83 nmoles/hairbulb, and urine excretion of 5-S-cysteinyldopa 174.9 ng/mg creatinine. Electron microscopy of hairbulb and skin melanocytes showed arrested melanosomal development. These findings suggest that there is a partial block in the distal eumelanin pathway in this form of albinism. The ophthalmological characteristics of six additional cases of this form of albinism are also presented.