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| 2. |
- Barbaro, Michela, et al.
(författare)
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Complete androgen insensitivity without Wolffian duct development : the AR-A form of the androgen receptor is not sufficient for male genital development
- 2007
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Ingår i: Clinical Endocrinology. - : Wiley. - 0300-0664 .- 1365-2265. ; 66:6, s. 822-826
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Barbaro, Michela, et al.
(författare)
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In vitro functional studies of rare CYP21A2 mutations and establishment of an activity gradient for nonclassic mutations improve phenotype predictions in congenital adrenal hyperplasia
- 2015
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Ingår i: Clinical Endocrinology. - : Wiley. - 0300-0664 .- 1365-2265. ; 82:1, s. 37-44
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundA detailed genotype-phenotype evaluation is presented by studying the enzyme activities of five rare amino acid substitutions (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile) identified in the CYP21A2 gene in patients investigated for Congenital adrenal hyperplasia (CAH). ObjectiveTo investigate whether the mutations identified in the CYP21A2 gene are disease causing and to establish a gradient for the degree of enzyme impairment to improve prediction of patient phenotype. Design and patientsThe CYP21A2 genes of seven patients investigated for CAH were sequenced and five mutations were identified. The mutant proteins were expressed in vitro in COS-1 cells, and the enzyme activities towards the two natural substrates were determined to verify the disease-causing state of the mutations. The in vitro activities of these rare mutations were also compared with the activities of four mutations known to cause nonclassic CAH (Pro30Leu, Val281Leu, Pro453Ser and Pro482Ser) in addition to an in silico structural evaluation of the novel mutants. Main outcome measureTo verify the disease-causing state of novel mutations. ResultsFive CYP21A2 mutations were identified (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile). All mutant proteins exhibited enzyme activities above 5%, and four mutations were classified as nonclassic and one as a normal variant. By comparing the investigated protein changes with four common mutations causing nonclassic CAH, a gradient for the degree of enzyme impairment could be established. Studying rare mutations in CAH increases our knowledge regarding the molecular mechanisms that render a mutation pathogenic. It also improves phenotype predictions and genetic counselling for future generations.
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| 5. |
- Barbaro, Michela, et al.
(författare)
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Isolated 46,XY gonadal dysgenesis in two sisters caused by a Xp21.2 interstitial duplication containing the DAX1 gene.
- 2007
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Ingår i: Journal of Clinical Endocrinology and Metabolism. - : The Endocrine Society. - 1945-7197 .- 0021-972X. ; 92:8, s. 3305-3313
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Tidskriftsartikel (refereegranskat)abstract
- 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 MA-GEB 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.
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| 6. |
- Barbaro, Michela
(författare)
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Mechanisms in disorders of sex development
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- “Is it a boy or a girl?” This is usually the first question that parents have when their baby is born. Sometimes it is not possible to give an immediate answer. This is the case when the newborn presents ambiguous external genitalia and an immediate sex assignment is not possible. This situation represents the most typical and dramatic presentation of a disorder of sex development (DSD). Other DSDs can manifest later in life, for example in a girl with primary amenorrhea, who finds out that she has a 46,XY karyotype and will never be fertile. The overall aim of this thesis was to identify mechanisms in DSD, in order to better understand normal and atypical sex development, and furthermore to offer better diagnostics and genetic counselling to patients with DSD and their families. Congenital adrenal hyperplasia (CAH) due to CYP21A2 deficiency is the single most common cause of ambiguous external genitalia in the newborn. The wide spectrum of clinical manifestations ranges from prenatal virilisation in XX girls and salt-wasting in the neonatal period to precocious pubarche and late-onset hyperandrogenic symptoms during adulthood, depending on the CYP21A2 genotype. By in vitro expression of CYP21A2 we have evaluated the residual enzyme activities of four mutant enzymes that carry novel or rare missense mutations identified in patients with CAH. All mutants had a residual activity below 1%, and are thus associated with severe enzyme deficiency. Therefore, these mutations are predicted to cause classic CAH if found in trans with other severe mutations (Paper I). Mutations in the androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS). Patients with completely female external genitalia are classified as having complete AIS (CAIS). However, some of these patients have signs of internal male genital differentiation due to missense mutations that show a low degree of residual function. We studied the expression of two isoforms 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, as shown by Western blot analysis of proteins from gonadal and genital skin fibroblasts. No signs of Wolffian duct development were present in any of the patients, indicating that the AR-A form is not sufficient for Wolffian duct maintenance and differentiation (Paper II). A genome wide investigation by high resolution BAC array CGH (Comparative Genomic Hybridization) was used to identify gene dosage imbalances in 10 patients with female external genitalia due to XY gonadal dysgenesis (GD). We identified and characterised a 637 kb duplication at Xp21 containing DAX1 in a girl with isolated 46,XY GD (Paper III). We also identified another XY patient with isolated partial GD and ambiguous external genitalia, by MLPA (Multiplex Ligation Probe-dependent Amplification) analysis using a synthetic probe set that we designed to identify gene dosage imbalances for known genes involved in DSD (Paper IV). These reports describe the first duplications on Xp21.2 identified in patients with isolated GD because all previously described XY subjects with Xp21 duplications presented with GD as part of a more complex phenotype, including mental retardation and/or malformations. These data support DAX1 as a dosage sensitive gene responsible for GD and highlight the importance of considering DAX1 locus duplications in the evaluation of all cases of 46,XY GD. More recently, we identified an additional family with several members affected with XY GD, where a small DAX1 duplication is segregating through the female line. These data suggest that DAX1 duplications might be as common as SRY mutations causing 46,XY GD. A terminal 9p deletion, of a region already involved in DSD, was also identified by array-CGH, and confirmed by a MLPA probe set, designed to enable screening of loss of candidate DSD genes at 9p23.4. The identification of submicroscopic deletions at 9p24 is of help to understand the mechanisms that lead to GD in some patients with 9p deletions, and to narrow down the monosomy 9p syndrome candidate region (Paper V). By array-CGH we have also identified two novel chromosomal imbalances that are candidate regions for XY GD: a duplication of 3.7 Mb at chromosome band 12q21.31 and a duplication on chromosome 6 that extends from exon 5 to exon 12 of the SUPT3H gene. These regions will be the subjects of further investigations in order to identify new genes involved in gonadal development (Paper VI). The work of this thesis has led to the establishment of the genetic diagnosis in several patients with DSD, thus allowing not only a better genetic counselling but also, at least in some cases, a better patient management. Furthermore, our genetic diagnostic arsenal has been expanded, as we can offer sequencing of more genes and gene dosage investigations by MLPA.
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| 7. |
- Filograna, Roberta, et al.
(författare)
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PARKIN is not required to sustain OXPHOS function in adult mammalian tissues
- 2024
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Ingår i: npj Parkinson's Disease. - : Springer Nature. - 2373-8057. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Göngrich, Christina, et al.
(författare)
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First Year of TREC-Based National SCID Screening in Sweden.
- 2021
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Ingår i: International journal of neonatal screening. - : MDPI AG. - 2409-515X. ; 7:3
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Tidskriftsartikel (refereegranskat)abstract
- Screening for severe combined immunodeficiency (SCID) was introduced into the Swedish newborn screening program in August 2019 and here we report the results of the first year. T cell receptor excision circles (TRECs), kappa-deleting element excision circles (KRECs), and actin beta (ACTB) levels were quantitated by multiplex qPCR from dried blood spots (DBS) of 115,786 newborns and children up to two years of age, as an approximation of the number of recently formed T and B cells and sample quality, respectively. Based on low TREC levels, 73 children were referred for clinical assessment which led to the diagnosis of T cell lymphopenia in 21 children. Of these, three were diagnosed with SCID. The screening performance for SCID as the outcome was sensitivity 100%, specificity 99.94%, positive predictive value (PPV) 4.11%, and negative predictive value (NPV) 100%. For the outcome T cell lymphopenia, PPV was 28.77%, and specificity was 99.95%. Based on the first year of screening, the incidence of SCID in the Swedish population was estimated to be 1:38,500 newborns.
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| 9. |
- Jensen, Kristian Vestergaard, et al.
(författare)
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Diagnostic pitfalls in vitamin B6-dependent epilepsy caused by mutations in the PLPBP gene.
- 2019
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Ingår i: JIMD reports. - : Wiley. - 2192-8304 .- 2192-8312. ; 50:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Vitamin B6-responsive epilepsies are a group of genetic disorders including ALDH7A1 deficiency, PNPO deficiency, and others, usually causing neonatal onset seizures resistant to treatment with common antiepileptic drugs. Recently, biallelic mutations in PLPBP were shown to be a novel cause of vitamin B6-dependent epilepsy with a variable phenotype. The different vitamin B6-responsive epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. Unfortunately, metabolic biomarkers for early detection and prognosis of PLPBP deficiency are currently still lacking. Here, we present data from two further patients with vitamin B6-dependent seizures caused by variants in PLPBP, including a novel missense variant, and compare their genotype and phenotypic presentation to previously described cases. Hyperglycinemia and hyperlactatemia are the most consistently observed biochemical abnormalities in pyridoxal phosphate homeostasis protein (PLPHP) deficient patients and were present in both patients in this report within the first days of life. Lactic acidemia, the neuroradiological, and clinical presentation led to misdiagnosis of a mitochondrial encephalopathy in two previously published cases with an early fatal course. Similarly, on the background of glycine elevation in plasma, glycine encephalopathy was wrongly adopted as diagnosis for a patient in our report. In this regard, lactic acidemia as well as hyperglycinemia appear to be diagnostic pitfalls in patients with vitamin B6-responsive epilepsies, including PLPHP deficiency.In vitamin B6-responsive epilepsies, including PLPHP deficiency, there are several diagnostic pitfalls, including lactic acidemia as well as hyperglycinemia, highlighting the importance of a pyridoxine trial, and genetic testing.
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| 10. |
- Karlsson, Leif, et al.
(författare)
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Novel non-classic CYP21A2 variants, including combined alleles, identified in patients with congenital adrenal hyperplasia
- 2019
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Ingår i: Clinical Biochemistry. - : Elsevier BV. - 0009-9120 .- 1873-2933. ; 73, s. 50-56
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH.Methods: All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed.Results: The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40–82%). An additive effect on the reduction of enzymatic activity (1–17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro.Conclusions: Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.
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