BACKGROUND AND AIM: Gene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis. PATIENTS AND METHODS: Both paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families. RESULTS: WES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations. CONCLUSION: Our findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.
- MeSH
- adaptorové proteiny signální transdukční genetika MeSH
- cholestáza * genetika MeSH
- cytoskeletální proteiny genetika MeSH
- flavoproteiny genetika MeSH
- genotyp MeSH
- intrahepatální cholestáza * genetika diagnóza MeSH
- lidé MeSH
- mitochondriální proteiny genetika MeSH
- mutace MeSH
- protoporfyrinogenoxidasa genetika MeSH
- sekvenování exomu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC-MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe-4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.
- MeSH
- bakteriální proteiny biosyntéza genetika MeSH
- flavoproteiny genetika metabolismus MeSH
- mutace * MeSH
- oxidační stres účinky léků genetika MeSH
- Paracoccus denitrificans genetika metabolismus MeSH
- paraquat farmakologie MeSH
- proteomika MeSH
- regulace genové exprese u bakterií účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.
- MeSH
- aminokyseliny chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biokatalýza MeSH
- difrakce rentgenového záření MeSH
- flavinmononukleotid chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- flavoproteiny chemie genetika metabolismus MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- mutageneze cílená MeSH
- NADH, NADPH oxidoreduktasy chemie klasifikace metabolismus MeSH
- NADP chemie metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů * MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O(2) absent) or > 2 (O(2) present), the apparent K (M) value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chromany metabolismus MeSH
- flavinadenindinukleotid analogy a deriváty metabolismus MeSH
- flavoproteiny genetika metabolismus MeSH
- FMN-reduktasa genetika metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- NAD metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- oxidoreduktasy genetika metabolismus MeSH
- Paracoccus denitrificans enzymologie genetika MeSH
- spotřeba kyslíku MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underlying genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria. However, we could not detect a mutation in the coding or the promotor region of the protoporphyrinogen oxidase gene that is mutated in variegate porphyria. Our results indicate that Chester porphyria is neither a dual porphyria, nor a separate type of porphyria, but rather a variant of acute intermittent porphyria. Further, our findings largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.
- MeSH
- akutní intermitentní porfyrie klasifikace genetika MeSH
- ferredoxiny genetika MeSH
- flavoproteiny genetika MeSH
- hydroxymethylbilansynthasa genetika MeSH
- lidé MeSH
- mitochondriální proteiny genetika MeSH
- molekulární sekvence - údaje MeSH
- mutační analýza DNA MeSH
- nesmyslný kodon * MeSH
- protoporfyrinogenoxidasa genetika MeSH
- sekvence nukleotidů MeSH
- sukcinátdehydrogenasa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The molecular mechanism underlying a generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) is based on interactive negative and positive feedback loops that drive the rhythmic transcription of clock genes and translation of their protein products. In adults, the molecular mechanism is affected by seasonal changes in day length, i.e., photoperiod. The photoperiod modulates phase, waveform, and amplitude of the rhythmic clock genes expression as well as the phase relationship between their profiles. To ascertain when and how the photoperiod affects the circadian core clock mechanism during ontogenesis, the rhythmic expression of clock genes, namely of Per1, Per2, Cry1 and Bmal1 was determined in 3-, 10- and 20-day-old rat pups maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The daily profiles in the level of clock genes mRNA were studied in constant darkness. The photoperiod affected the profile of Per1 and Per2 mRNA in 20- and 10- but not yet in 3-day-old pups. Expression of Cry1 was affected only in 20-day-old pups, whereas expression of Bmal1 was not yet affected even in 20-day-old rats. The results demonstrate no effect of the photoperiod on 3-day-old pups, only partial entrainment of the molecular core clockwork in 10-day-old pups and a more mature, though not yet fully complete, entrainment in 20-day-old pups as compared with adult animals. The developmental interval when the photoperiod begins to entrain the core clock mechanism completely might thus occur around the time of weaning.
- MeSH
- analýza rozptylu MeSH
- biologické hodiny genetika MeSH
- cirkadiánní rytmus genetika MeSH
- financování organizované MeSH
- flavoproteiny genetika metabolismus MeSH
- fotoperioda MeSH
- jaderné proteiny genetika metabolismus MeSH
- krysa rodu rattus MeSH
- messenger RNA analýza MeSH
- nucleus suprachiasmaticus metabolismus účinky záření MeSH
- potkani Wistar MeSH
- proteiny buněčného cyklu MeSH
- proteiny nervové tkáně genetika metabolismus MeSH
- stárnutí genetika MeSH
- světlo MeSH
- trans-aktivátory genetika metabolismus MeSH
- transkripční faktory bHLH genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- vývojová regulace genové exprese fyziologie genetika účinky záření MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
