Does a glucose-based hydrogen and methane breath test detect bacterial overgrowth in the jejunum?

BACKGROUND: Direct diagnosis of small intestinal bacterial overgrowth (SIBO) requires the collection and culture of fluid from the jejunal lumen, with a finding of over 105 viable bacteria per mL. More often, SIBO is diagnosed indirectly, using a non-invasive test of the exhaled hydrogen and methane generated by microbial fermentation when ingested glucose reaches the jejunum. Our objective was to determine how well this breath test detects chronic overgrowth of jejunal bacteria that is unrelated to gastrointestinal surgery. METHODS: Eighteen patients reporting symptoms consistent with SIBO received a glucose breath test. On a later day, the jejunal lumen was sampled via aspiration during enteroscopy. Jejunal aspirates were cultured on aerobic and anaerobic media. DNA was extracted from the same samples and analyzed by quantitative pan-bacterial PCR amplification of 16S ribosomal rRNA genes, which provided a culture-independent bacterial cell count. KEY RESULTS: Combined bacterial colony counts ranged from 5.7 x 103 to 7.9 x 106 CFU/mL. DNA-based yields ranged from 1.5 x 105 to 3.1 x 107 bacterial genomes per mL. Microbial viability ranged from 0.3% to near 100%. We found no significant correlation of glucose breath test results with either the number of bacterial colonies or with the DNA-based bacterial cell counts. Instead, higher signals in the hydrogen-methane breath test were significantly correlated with a lower viability of jejunal bacteria, at a P-value of .014. CONCLUSIONS & INFERENCES: The glucose-based hydrogen and methane breath test is not sensitive to the overgrowth of jejunal bacteria. However, a positive breath test may indicate altered jejunal function and microbial dysbiosis.

Redefining papillorenal syndrome: an underdiagnosed cause of ocular and renal morbidity.

PURPOSE: To report ocular and renal findings specific to the inheritable entity called papillorenal (also known as renal-coloboma) syndrome and relate these to a common cause. DESIGN: Observational case series and genetic study. PARTICIPANTS: Two unrelated probands presenting with absent central retinal vessels and 11 available family members. TESTING: Doppler ultrasonographic imaging of the optic nerves and kidneys, fluorescein angiography, and genetic testing for PAX2 mutations were performed. In selected cases, indocyanine green angiography, scanning laser ophthalmoscope perimetry, Retinal Thickness Analyzer measurements, visual evoked potentials, and magnetic resonance imaging were also performed. MAIN OUTCOME MEASURES: Better defined characteristics of the papillorenal syndrome. RESULTS: Numerous cilioretinal vessels were present with rudimentary or absent central retinal vessels. Superonasal visual field defects, typical for papillorenal syndrome, corresponded to inferotemporal areas of anomalous retinal and choroidal perfusion and hypoplastic retina. Renal hypoplasia was discovered in two affected members of one family (with previously unsuspected renal failure in one case), and recurrent pyelonephritis was discovered in four affected members of the other family. No PAX2 mutations were detected. CONCLUSIONS: In the papillorenal syndrome, the hereditary absence of central retinal vessels may be missed, leading to confusion with isolated coloboma, low-tension glaucoma, and morning glory anomaly. Greater awareness of this syndrome will avoid unneeded glaucoma therapy, allow earlier recognition of renal diseases, and allow genetic counseling. We propose that the papillorenal syndrome is a primary dysgenesis that causes vascular abnormalities predominantly affecting the eye, kidney, and urinary tract, leading to hypoplasia of these structures. The absence of defects in the PAX2 gene in these families suggests that mutations in other genes may also be responsible for this syndrome.

A CRX null mutation is associated with both Leber congenital amaurosis and a normal ocular phenotype.

PURPOSE: To identify and characterize new cone rod homeobox (CRX) mutations associated with the Leber congenital amaurosis phenotype. METHODS: The human CRX gene was sequenced in 74 consecutive patients carrying the diagnosis of Leber congenital amaurosis. RESULTS: Two mutations were identified in CRX that cause frameshifts and predict severe truncations of the encoded protein. One of these, a 1-bp insertion, spares only nine N-terminal amino acids, removing the homeodomain, WSP motif, and conserved OTX domain at the C terminus. Of the CRX mutations described in the literature, this is the first that convincingly represents a null allele of the gene. Although the patient heterozygous for this null allele is affected with Leber congenital amaurosis, it was surprising that her father, who had normal vision, was heterozygous for the same mutation. CONCLUSIONS: These results strongly suggest that haploinsufficiency of CRX is not sufficient to cause a retinal disorder. Loss of function alleles of CRX appear to cause Leber congenital amaurosis through a recessive or multigenic mechanism.

Genetic basis of total colourblindness among the Pingelapese islanders.

Complete achromatopsia is a rare, autosomal recessive disorder characterized by photophobia, low visual acuity, nystagmus and a total inability to distinguish colours. In this disease, cone photoreceptors, the retinal sensory neurons mediating colour vision, seem viable but fail to generate an electrical response to light. Achromatopsia, or rod monochromatism, was first mapped to 2p11-2q12 (MIM 216900; ref. 3), where it is associated with missense mutations in CNGA3 (ref. 4). CNGA3 encodes the alpha-subunit of the cone cyclic nucleotide-gated cation channel, which generates the light-evoked electrical responses of cone photoreceptors. A second locus at 8q21-q22 has been identified among the Pingelapese islanders of Micronesia, who have a high incidence of recessive achromatopsia (MIM 262300). Here we narrow the achromatopsia locus to 1.4 cM and show that Pingelapese achromatopsia segregates with a missense mutation at a highly conserved site in CNGB3, a new gene that encodes the beta-subunit of the cone cyclic nucleotide-gated cation channel. Two independent frameshift deletions establish that achromatopsia is the null phenotype of CNGB3. Combined with earlier findings, our results demonstrate that both alpha- and beta-subunits of the cGMP-gated channel are essential for phototransduction in all three classes of cones.

Sequence and location of SIX3, a homeobox gene expressed in the human eye.

Mouse Six3 is a homeobox gene expressed almost exclusively in the developing retina, lens, hypothalamus, and pituitary. It belongs to the same family as sine oculis, a Drosophila regulatory gene that encodes a transcription factor essential for eye development. The optix gene is its closest known Drosophila homologue, with a homeodomain that is 95% identical in sequence to the Six3 protein. We have isolated the homologous human gene, SIX3, which is expressed in the adult retina and encodes a 332 amino acid protein that is 98% identical to its mouse counterpart. The SIX3 protein coding region is interrupted by a single intron located just downstream of the homeobox. A surprising feature of the SIX3 gene is a 533 nucleotide 5' untranslated region that contains long polypyrimidine tracts with 96% identity to mouse Six3. We have used in-situ hybridization to map SIX3 to 2p21-p22, a site that is syntenic with the Six3 region of mouse chromosome 17. Large heterozygous deletions associated with human holoprosencephaly type 2 have been previously mapped to 2p21, opening the possibility that SIX3 could be involved in the development of midline structures of the head. Alternatively, the expression pattern of mouse Six3 suggests that human SIX3 could be involved in disorders of eye and pituitary development.

Expression of the optx2 homeobox gene during mouse development.

Optx2, a member of the sine oculis-Six family of homeobox genes, is first expressed in the anterior neural plate of the mouse embryo, and subsequently in the optic vesicle and ventral forebrain. During later development, expression is further restricted to precursors of the neural retina, optic chiasm, adenohypophysis and neurohypophysis. In the adult mouse retina, Optx2 mRNA is found in cells within the ganglion cell layer and inner nuclear layer.

The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes.

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

The Pax-6 homeobox gene is expressed throughout the corneal and conjunctival epithelia.

PURPOSE: Heterozygous defects in the highly conserved PAX6 homeobox gene are associated with aniridia, an inherited human disorder affecting several ocular structures, including the adult cornea. This work establishes the pattern of Pax-6 gene expression in the surface epithelia of the late embryonic and adult eye. METHODS: Chick embryo sections and wholemounts, as well as adult mouse and monkey tissues, were analyzed by in situ hybridization and immunohistochemistry with probes specific to Pax-6. Western immunoblots were used to detect Pax-6 protein, and mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: In days 5 and 6 chick embryos, Pax-6 protein is found in the nuclei of all cells within the corneal epithelium and in the future conjunctiva. Although not detected in the cornea by in situ hybridization, Pax-6 mRNA is, in fact, present at levels comparable to those observed in the retina. In the mature mouse, Pax-6 protein was expressed in all cells of the corneal epithelium, the limbus, and the entire conjunctiva. Similar results were obtained for the monkey cornea. CONCLUSIONS: These data indicate that in addition to its role in the embryo, Pax-6 is expressed strongly in surface epithelia of the adult cornea and conjunctiva. In cells of these tissues, the gene may function by regulating structural or secretory specializations. Pax-6 might play a direct role in the maintenance and proliferation of corneal stem cells, a vital process that appears to be defective in aniridia.

A homeo domain protein reveals the metameric nature of the developing chick hindbrain.

The segmented embryonic hindbrain of vertebrates develops by sequential constriction of the neural tube into eight metameric units known as rhombomeres. The cellular and molecular basis of this segmentation process is largely unknown. Using an antibody, we analyzed the expression pattern of the chick homeo domain-containing protein Ghox-lab in the developing chick hindbrain. At the neural plate stage, prior to the appearance of rhombomeres, Ghox-lab is expressed within a single domain that extends anteriorly up to the site where rhombomere 4 will later form. After rhombomere 4 has appeared and as hindbrain segmentation progresses, the level of Ghox-lab protein increases significantly within the fourth rhombomere. This intensification, accompanied by the elimination of Ghox-lab protein in rhombomeres 5 and 6, eventually results in the formation of a distinct island of expression in rhombomere 4. All cells in the newly formed rhombomere 4 express Ghox-lab, except for the cells of the floor plate. In addition, neural crest cells migrating from the fourth rhombomere are also Ghox-lab-positive. These data raise the possibility that Ghox-lab protein might be one of the factors involved in the specification of the metameric pattern of the vertebrate hindbrain.

Region-specific expression in early chick and mouse embryos of Ghox-lab and Hox 1.6, vertebrate homeobox-containing genes related to Drosophila labial.

A chick gene homologous to the Drosophila homeobox gene labial has been cloned and sequenced. Regions of additional sequence identity outside of the homeobox reveal a close relationship to the mouse gene Hox 1.6. Northern blot analysis demonstrates that Ghox-lab and Hox 1.6 transcripts are both present at high levels during early stages of chick and mouse development, with a subsequent decline in abundance to very low levels by the time limb mesenchyme begins to differentiate. In situ hybridization analysis of chick embryos shows intense expression of Ghox-lab mRNA by Hamburger and Hamilton stage 4 (avian 'mid gastrula') and by stage 6 (pre-somitic neural plate) with expression decreasing shortly thereafter. The pattern of Ghox-lab RNA expression in these early embryos divides the embryo into an anterior and a posterior compartment. At stage 6, considerable signal is observed in the posterior two thirds of the embryo, while none is detected in the anterior third which is fated to become the head. This pattern is purely regional in nature, and does not follow boundaries defined by known tissue types. In situ hybridization of Hox 1.6 probes to mouse embryos of day 7.5 or 8.0 indicate that the Hox 1.6 transcript has a temporal and spatial distribution very similar to that of Ghox-lab in the chick embryo.

SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases.

Isolated SV40 minichromosomes [1-3] were treated with different single-cut restriction endonucleases to probe the arrangement of nucleosomes in relation to the SV70 DNA sequence. While Eco RI and Bam HI each cut 22-27% of the SV40 minichromosomes under limit-digest conditions, Bgl I, which cuts SV40 DNA at or very near the origin of replication [4,5], cleaves 90-95% of the minichromosomes in a preparation. Similar results were obtained with minichromosomes which had been fixed with formaldehyde before endonuclease treatment. One possible interpretation of these findings is that the arrangement of nucleosomes in the compact SV40 minichromosomes is nonrandom at least with regard to sequences near the origin of DNA replication.