The management and clinical outcomes of pregnancies in women with urea cycle disorders: A review of the literature and results of an international survey.

An increasing number of women with urea cycle disorders (UCDs) are reaching child-bearing age and becoming pregnant. Improved diagnostics and increased awareness of inherited metabolic diseases has also led to more previously undetected women being diagnosed with a UCD during or shortly after pregnancy. Pregnancy increases the risk of acute metabolic decompensation with hyperammonemia-which can occur in any trimester, and/or the postpartum period, and may lead to encephalopathy, psychosis, coma, and even death, if not diagnosed promptly and treated appropriately. There are also (theoretical) concerns that a maternal UCD, or its treatment, may cause potential risks for the unborn child. Currently evidence on management and outcome of pregnancies in UCDs is limited to case reports and there are no clear guidelines. In order to inform management and investigate outcomes of pregnancies in women with a UCD, we performed a retrospective review of published cases and analyzed data collected from an international online survey. We conclude that, although risk during the intra- and postpartum period exists, multidisciplinary management by an experienced team and a prospective plan usually result in successful pregnancy, labor, delivery, and postpartum period. No deaths were reported in mothers managed accordingly. With the exception of male neonates with Ornithine Transcarbamylase deficiency, the clinical outcome of children born to mothers with UCDs appears positive, although follow-up is limited. The outcome for women presenting with a first acute metabolic decompensation during pregnancy or postpartum is less favorable. Deaths were associated with diagnostic delay/late management of hyperammonemia in previously undiagnosed women.

Visual functions, ocular characteristics and visual quality of life in patients with homocystinuria.

BACKGROUND: Homocystinuria (HCU) is a rare metabolic disease that affects many organs, including the eyes. Aims: to assess visual functions, ocular characteristics, visual quality of life and time from the onset of ocular manifestations to HCU-diagnosis in patients with HCU. MATERIAL AND METHODS: Eighteen patients underwent ophthalmological examinations and visual quality of life questionnaires. RESULTS: Best corrected decimal visual acuity was median 1.0 (range amaurosis - 1.3) right eye and 1.0 (range amaurosis -1.3) left eye. Five patients presented with severe myopia as first HCU manifestation, duration to HCU diagnosis was mean 13.6 years (range 2-25). Two patients had suffered ectopia lentis as first HCU manifestation, HCU diagnosis was established mean 8.0 years (range 7-9) later. One patient had suffered both from thrombosis and ectopia lentis prior to diagnosis. Another four patients suffered thromboembolic events before diagnosis. Median VFQ-25 composite score was 93 (68-98). CONCLUSIONS: The prevalence of myopia, ectopia lentis and monocular blindness was high in HCU-patients, which was reflected in their visual quality of life. Diagnosis was often delayed after the first ocular manifestation, increasing the risk of other severe non-ocular complications.

Novel findings in a Swedish primary familial brain calcification cohort.

INTRODUCTION: Brain calcifications are frequent findings on imaging. In a small proportion of cases, these calcifications are associated with pathogenic gene variants, hence termed primary familial brain calcification (PFBC). The clinical penetrance is incomplete and phenotypic variability is substantial. This paper aims to characterize a Swedish PFBC cohort including 25 patients: 20 from seven families and five sporadic cases. METHODS: Longitudinal clinical assessment and CT imaging were conducted, abnormalities were assessed using the total calcification score (TCS). Genetic analyses, including a panel of six known PFBC genes, were performed in all index and sporadic cases. Additionally, three patients carrying a novel pathogenic copy number variant in SLC20A2 had their cerebrospinal fluid phosphate (CSF-Pi) levels measured. RESULTS: Among the 25 patients, the majority (76%) displayed varying symptoms during the initial assessment including motor (60%), psychiatric (40%), and/or cognitive abnormalities (24%). Clinical progression was observed in most patients (78.6%), but there was no significant difference in calcification between the first and second scans, with mean scores of 27.3 and 32.8, respectively. In three families and two sporadic cases, pathogenic genetic variants were identified, including a novel finding, in the SLC20A2 gene. In the three tested patients, the CSF-Pi levels were normal. CONCLUSIONS: This report demonstrates the variable expressivity seen in PFBC and includes a novel pathogenic variant in the SLC20A2 gene. In four families and three sporadic cases, no pathogenic variants were found, suggesting that new PFBC genes remain to be discovered.

PARKIN is not required to sustain OXPHOS function in adult mammalian tissues.

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson's disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

Visual outcome, ocular findings, and visual quality of life in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal disease, in which diagnosis is often established several years after onset of symptoms. Ocular manifestations can occur in childhood and be a clue to earlier diagnosis. The aim was to report ocular outcome and visual quality of life (QoL) in patients with FD. MATERIAL AND METHODS: FD-patients recruited from Karolinska University Hospital underwent ophthalmological examinations including best corrected visual acuity (BCVA), refraction, biomicroscopy, optical coherence tomography, keratometry, review of medical records and QoL Inventories. A total severity score (TSS), as estimated via Fabry Stabilization Index, was calculated. RESULTS: Twenty-six FD-patients (16 men) mean age 36.4 years (range 5.0-63.5 years) were included. BCVA was median 1.0 (range 0.5-1.6). Conjunctival blood vessel tortuosity occurred in 15/26 patients, chemosis in 2/26 patients, cornea verticillata in 23/26 patients, lens opacities in 19/26 patients, and tortuous or dilated retinal vessels in 20/25 patients. Group-wise comparisons of adult patients showed no differences regarding age, TSS, or ocular parameters. Overall, TSS was correlated to age (r = 0.53, p = 0.02). A linear regression model showed that age and sex explained 38% of the variance in TSS. Keratometry did not reveal corneal ectasia in any of the 12 patients examined. VFQ 25 in 15 patients showed a high median composite score, 93.6 (range: 78.1-100). CONCLUSIONS: BCVA in FD-patients was good despite corneal and lens pathology. Ocular variables did not show an association with TSS in adult patients. Corneal or lens opacities should also lead to a suspicion of FD in children.

Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

Late onset hyperornithinemia-hyperammonemia-homocitrullinuria syndrome - how web searching by the family solved unexplained unconsciousness: a case report.

BACKGROUND: Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a rare inherited urea cycle disorder, can remain undiagnosed for decades and suddenly turn into an acute life-threatening state. Adult presentation of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome has rarely been described, but is potentially underdiagnosed in the emergency room. In the case of acute hyperammonemia, prompt diagnosis is essential to minimize the risk of brain damage and death. CASE PRESENTATION: We present the diagnostics, clinical course, and treatment of a 48-year-old Caucasian man presenting with unexplained unconsciousness in the emergency room. A web search by a family member led to the suspicion of urea cycle disorder. Subsequent analysis of plasma ammonia and amino acids in plasma and urine demonstrated a pattern typical for hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. The diagnosis was confirmed by genetic analysis which revealed two heterozygous mutations in the SLC25A15 gene. The cause of the hyperammonemia crisis was acute upper gastrointestinal hemorrhage, leading to protein overload and subsequent cerebral edema. Continuous renal replacement therapy, scavenger treatment, and tightly controlled nutrition were useful in preventing hyperammonemia and recurrence of cerebral edema. CONCLUSIONS: The case emphasizes the importance of taking rare metabolic genetic disorders into consideration in patients with prolonged unexplained unconsciousness.

Isolated 46,XY gonadal dysgenesis in two sisters caused by a Xp21.2 interstitial duplication containing the DAX1 gene.

CONTEXT: Testis development is a tightly regulated process that requires an efficient and coordinated spatiotemporal action of many factors, and it has been shown that several genes involved in gonadal development exert a dosage effect. Chromosomal imbalances have been reported in several patients presenting with gonadal dysgenesis as part of severe dysmorphic phenotypes. RESULTS: We screened for submicroscopic DNA copy number variations in two sisters with an apparent normal 46,XY karyotype and female external genitalia due to gonadal dysgenesis, and in which mutations in known candidate genes had been excluded. By high-resolution tiling bacterial artificial chromosome array comparative genome hybridization, a submicroscopic duplication at Xp21.2 containing DAX1 (NR0B1) was identified. Using fluorescence in situ hybridization, multiple ligation probe amplification, and PCR, the rearrangement was further characterized. This revealed a 637-kb tandem duplication that in addition to DAX1 includes the four MAGEB genes, the hypothetical gene CXorf21, GK, and part of the MAP3K7IP3 gene. Sequencing and analysis of the breakpoint boundaries and duplication junction suggest that the duplication originated through a coupled homologous and nonhomologous recombination process. CONCLUSIONS: This represents the first duplication on Xp21.2 identified in patients with isolated gonadal dysgenesis because all previously described XY subjects with Xp21 duplications presented with gonadal dysgenesis as part of a more complex phenotype, including mental retardation and/or malformations. Thus, our data support DAX1 as a dosage sensitive gene responsible for gonadal dysgenesis and highlight the importance of considering DAX1 locus duplications in the evaluation of all cases of 46,XY gonadal dysgenesis.

Identification of endothelin-converting enzyme-2 as an autoantigen in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.

Transcriptional profiling of genes induced in the livers of patients treated with carbamazepine.

BACKGROUND: The antiepileptic drug carbamazepine (CBZ) is a potent inducer of human drug metabolism, resulting in serious interactions with many commonly prescribed drugs. The molecular mechanisms underlying this response are not well understood, however, and the spectrum of CBZ-inducible genes in human liver has not been thoroughly investigated. METHODS: The availability of liver ribonucleic acid from 2 epileptic patients treated with CBZ and from 7 control subjects enabled us to study the global induction response of drug-metabolizing enzymes, drug transporters, and nuclear receptors in vivo. RESULTS: Using expression profiling, we identified 64 significantly up-regulated transcripts but only 1 significantly down-regulated transcript (SLC22A5). We confirmed the induction of several genes that previously have been shown to be inducible by drugs in vitro, including multiple cytochrome P450 (CYP) genes in the CYP1A, CYP2A, CYP2B, CYP2C, and CYP3A subfamilies, as well as glutathione S-transferase A1, uridine diphosphate-glucuronosyltransferase 1As, the drug transporter ABCC2, and the nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor). Moreover, we identified a number of additional genes not previously known to be induced by CBZ, including CYP39A1, sulfotransferase 1A1, glutathione S-transferase Z1, and the drug transporters SLCO1A2, ABCG2, and ABCB7, as well as the glucocorticoid and aldosterone receptors. In transactivation studies in CV-1 cells, we demonstrated that both CBZ and its major metabolite, CBZ-10,11-epoxide, activate the nuclear receptor PXR in a concentration-dependent fashion and at therapeutic concentrations with 50% inhibitory concentration values of approximately 50 micromol/L. CONCLUSIONS: CBZ is a potent inducer of a broad spectrum of drug-metabolizing enzymes and drug transporters in the human liver, and these effects are mediated at least in part by activation of PXR.

[Hereditary metabolic diseases with onset in adulthood. Early and correct treatment of acute symptoms can be life-saving]. / Ärftliga metabola sjukdomar med symtomdebut i vuxen ålder - Tidig och korrekt behandling vid akuta symtom kan vara livräddande.

Inherited metabolic diseases usually present in the neonatal period or before school age. A growing portion of the disorders can be treated successfully, and an increasing number of patients are now treated in adult medicine. Several of the disorders also exist as attenuated variants without distinct symptoms in childhood. They can present as an acute onset event during metabolic stress in adulthood. We describe three patients with acute clinical decompensation in adulthood with severe sequelae and propose investigations to help diagnose such patients. One patient was unconscious from ammonia intoxication and developed severe neurological sequelae because of a defect in the urea cycle. A second patient had seizures caused by a defect in the conversion of homocysteine to methionine, resulting in lack of S-adenosylmethionine. A third patient had a stroke and grossly elevated homocysteine caused by undiagnosed homocystinuria. Recently there have been dramatic improvements in diagnostic methods, and new therapies are continuously being developed. Knowledge of these disorders is therefore of increasing importance also in adult medicine.

A nicotine C-oxidase gene (CYP2A6) polymorphism important for promoter activity.

In humans, several polymorphic variants have been described for the gene encoding the major nicotine C-oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5' flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5'-upstream region of the CYP2A6 gene from eight individuals revealed a c.-1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3'-UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele-specific hybridization (DASH) showed that, in both groups, the variants carrying the c.-1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5'-regulatory sequence showed that the region between c.-1005 and c.-1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.-1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.-1013A as compared to the c.-1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.-1005 and c.-1019 in the CYP2A6 gene and that a c.-1013A>G polymorphism in this region affects CYP2A6 transcription.

Characterization of a novel CYP2A7/CYP2A6 hybrid allele (CYP2A6*12) that causes reduced CYP2A6 activity.

The human CYP2A6 enzyme metabolizes certain drugs and pre-carcinogens and appears to be the most important enzyme for nicotine metabolism. At present, more than 10 different allelic variants are known that cause abolished or decreased enzyme activity. Genetic polymorphism in this gene might be of particular importance for an individual's need for nicotine and for susceptibility to lung and/or liver cancer. We have identified a new CYP2A6 allele (CYP2A6*12) which carries an unequal crossover between the CYP2A6 and CYP2A7 genes in intron 2. This results in a hybrid allele where the 5' regulatory region and exons 1-2 are of CYP2A7 origin and exons 3-9 are of CYP2A6 origin, resulting in 10 amino acid substitutions compared to the CYP2A6(*)1 allele. Phenotyping with the CYP2A6 substrate coumarin indicates that it causes reduced CYP2A6 activity in'vivo. Furthermore, when expressed in mammalian COS-1 cells, the enzyme variant catalyzed 7-hydroxylation of coumarin at a rate approximately 60% of that of the wild-type enzyme. The CYP2A6(*)12 allele was present at an allele frequency of 2.2% among Spaniards, but was absent in Chinese.

Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6).

OBJECTIVES: In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2A6 gene variants are known which contribute to the highly variable expression of this enzyme among individuals. In this study we report a novel polymorphism located in the 5' flanking region (-745A > G) of the CYP2A6 gene disrupting a CCAAT box. METHODS AND RESULTS: Electrophoretic mobility shift assays (EMSA) indicated that NF-YA is part of this nuclear protein complex. Chromatin immunoprecipitation revealed that NF-Y recognizes a region of the CYP2A6 5' flanking region located between -932 and -606. EMSA showed that out of the three CCAAT boxes in the CYP2A6 promoter, with CCAAT core sequences located between -839/-835, -748/-744, and -689/-685, only the one at -748/-744 was able to compete with the nuclear protein complex binding to the -748/-744 CCAAT box. Cotransfection experiments indicated that NF-Y acts as a positive regulatory element on CYP2A6 gene regulation. EMSA demonstrated that an NF-Y consensus oligonucleotide but not the -745A > G oligonucleotide competed efficiently with binding of the protein complex to the -748/-744 CCAAT box. Promoter activity of the -745A > G variant was significantly reduced to 78% relative to the wild-type allele in HepG2 cells transfected with luciferase reporter plasmids. Finally, haplotype analysis was carried out comprising the -745A > G variant in combination with all known CYP2A6 3' and 5' flanking single nucleotide polymorphisms: -1013A > G, -48T > G, and the CYP2A6/CYP2A7 3' flank conversion. CONCLUSION: A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.

CYPalleles: a web page for nomenclature of human cytochrome P450 alleles.

Genetic variations in the genes encoding the cytochrome P450s (CYPs) are important determinants for interindividual differences in sensitivity to drugs and environmental chemicals as well as for the pathogenesis of several human diseases. In order to standardise the nomenclature of the rapidly increasing number of alleles described, a web page was established a few years ago. Here, we describe the present status of the web page and summarise the principles used for CYP allele nomenclature.

Characterization of deletions at 9p affecting the candidate regions for sex reversal and deletion 9p syndrome by MLPA.

The distal region on the short arm of chromosome 9 is of special interest for scientists interested in sex development as well as in the clinical phenotype of patients with the 9p deletion syndrome, characterized by mental retardation, trigonocephaly and other dysmorphic features. Specific genes responsible for different aspects of the phenotype have not been identified. Distal 9p deletions have also been reported in patients with 46,XY sex reversal, with or without 9p deletion syndrome. Within this region the strongest candidates for the gonadal dysgenesis phenotype are the DMRT genes; however, the genetic mechanism is not clear yet. Multiple ligation-dependent probe amplification represents a useful technique to evaluate submicroscopic interstitial or distal deletions that would help the definition of the minimal sex reversal region on 9p and could lead to the identification of gene(s) responsible of the 46,XY gonadal disorders of sex development (DSD). We designed a synthetic probe set that targets genes within the 9p23-9p24.3 region and analyzed a group of XY patients with impaired gonadal development. We characterized a deletion distal to the DMRT genes in a patient with isolated 46,XY gonadal DSD and narrowed down the breakpoint in a patient with a 46,XY del(9)(p23) karyotype with gonadal DSD and mild symptoms of 9p deletion syndrome. The results are compared with other patients described in the literature, and new aspects of sex reversal and the 9p deletion syndrome candidate regions are discussed.

Complete androgen insensitivity without Wolffian duct development: the AR-A form of the androgen receptor is not sufficient for male genital development.

BACKGROUND: The androgen receptor (AR) is essential for the differentiation of male external and internal genitalia. It is normally present in two forms, a full-length form B and an N-terminal truncated form A with still unknown function. Mutations in the AR gene cause androgen insensitivity syndrome (AIS), which is divided into subgroups according to the degree of undermasculinization. Patients with completely female external genitalia are classified as complete AIS (CAIS). However, a recent study has shown that some CAIS patients have signs of internal male genital differentiation due to missense mutations that show some degree of residual function. OBJECTIVE: We aimed to study the expression of the different forms of the AR in two CAIS patients in relation to the development of male internal genital structures. One patient had a mutation (L7fsX33) that affects only the full-length AR-B form of the AR, whereas the other had a nonsense mutation (Q733X) affecting both isoforms. MEASUREMENTS AND RESULTS: We thoroughly analysed internal genitalia at surgery and by histological examination. No signs of Wolffian duct (WD) development were present in any of the patients. Western blotting of proteins from gonadal and genital skin fibroblasts was performed with AR antibodies directed against different AR epitopes. The N-terminally truncated A form was expressed in normal amounts in the patient with the L7fsX33 mutation while no AR was detected in the other patient. CONCLUSION: The presence of the AR-A form does not seem to be sufficient for WD maintenance and differentiation.

Effects of rifampicin on global gene expression in human small intestine.

OBJECTIVES: The small intestinal wall serves as an important barrier for the entry of foreign substances into the organism. Of particular importance are enzymes and transporters that can inactivate or prevent the uptake of many xenobiotics including drugs. Some of the genes encoding these proteins are transcriptionally activated by xenobiotics, a response well studied in liver but less so in the intestine. The effect of the inducer drug rifampicin on intestinal cells was therefore evaluated both in vivo and in vitro. METHODS: Seven healthy volunteers were treated with rifampicin for 9 days and the global gene expression profile was analysed in RNA from duodenal biopsies taken before and after drug treatment. The gene expression profile was also assessed in LS174T cells derived from a human colon adenocarcinoma after exposure to 10 micromol/l rifampicin for 24 h. RESULTS: We identified 32 genes that were upregulated and two genes that were downregulated by rifampicin treatment in vivo. The list of rifampicin regulated transcripts expectedly included drug metabolizing enzymes and drug transporters, but also genes involved in lipid and amino acid metabolism as well as genes not previously recognized to be part of the adaptation of intestinal cells to xenobiotic exposure. Only a limited number of these rifampicin-regulated transcripts were however also regulated by rifampicin in LS174T cells. CONCLUSION: The similarities and differences of changes in gene expression after rifampicin treatment between duodenal biopsies and cell culture provide a new assessment of the extent and diversity of systems affected by drug exposure.

Pituitary autoantibodies in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies (Aabs) toward intracellular enzymes are a hallmark for APS1 and serve as diagnostic markers and predictors for disease manifestations. In this study, we aimed to identify pituitary autoantigens in patients with APS1. A pituitary cDNA expression library was screened with APS1 sera and a tudor domain containing protein 6 (TDRD6) cDNA clone was isolated. Positive immunoreactivity against in vitro translated TDRD6 fragments was shown in 42/86 (49%) APS1 patients but not in patients with other autoimmune diseases or in healthy controls. By using immunohistochemistry, sera from 3/6 APS1 patients with growth hormone (GH) deficiency showed immunostaining of a small number of guinea pig anterior pituitary cells, and 40-50% of these cells were GH-positive. No such immunostaining was seen with sera from healthy controls. The APS1 Aab-positive, GH-negative cells may represent a novel subpopulation of anterior pituitary cells. In addition, 4/6 patient sera showed staining of a fiber-plexus in the pituitary intermediate lobe recognizing enzymes of monoamine and GABA synthesis. Thus, we have identified TDRD6 as a major autoantigen in APS1 patients and shown that several sera from GH-deficient patients stain specific cell populations and nerves in the pituitary gland.