Untersuchung zur Freissetzung kinetischer Energie bei Dissoziationsreaktionen multifunktionaler Systeme

MALDI erlaubt die Sequenzaufklärung großer Moleküle und ist damit eine wichtige Ionisationsmethode in der Massenspektrometrie. In dieser Arbeit wird das Fragmentierungsverhalten typischer MALDI-Matrices aufgeklärt (Vanillin-, Ferula- und Sinapinsäure). Zu den untersuchten Dissoziationsreaktionen wurden quantenmechanische und kinetische Rechnungen durchgeführt. Schwerpunkt der Arbeit lag auf der Beurteilung der beim unimolekularen Zerfall freigesetzten kinetischen Energie (kinetic energy release). Anhand der experimentellen und theoretischen Daten konnte der Anteil freigesetzter Energie beurteilt werden. Berechnet wurden jeweils die Potentialenergiekurven und Ratenkonstanten des unimolekularern Zerfalls. Neben MALDI-Matrices wurden noch zwei isomere Systeme untersucht, Methoxyacetophenon und Diethylphthalat. Neben der Aufklärung des Fragmentierungsverhalten wurde hierbei der ortho-Effekt mittels Markierungsexperimenten näher untersucht. Im dritten Teil wurden verschiedene Pyrazol-Guanidin-Cluster untersucht. Zu diesen in der Ionenquelle erzeugten Systeme wurden Strukturen und der kinetic energy release bestimmt

Untersuchung zur Freissetzung kinetischer Energie bei Dissoziationsreaktionen multifunktionaler Systeme

MALDI erlaubt die Sequenzaufklärung großer Moleküle und ist damit eine wichtige Ionisationsmethode in der Massenspektrometrie. In dieser Arbeit wird das Fragmentierungsverhalten typischer MALDI-Matrices aufgeklärt (Vanillin-, Ferula- und Sinapinsäure). Zu den untersuchten Dissoziationsreaktionen wurden quantenmechanische und kinetische Rechnungen durchgeführt. Schwerpunkt der Arbeit lag auf der Beurteilung der beim unimolekularen Zerfall freigesetzten kinetischen Energie (kinetic energy release). Anhand der experimentellen und theoretischen Daten konnte der Anteil freigesetzter Energie beurteilt werden. Berechnet wurden jeweils die Potentialenergiekurven und Ratenkonstanten des unimolekularern Zerfalls. Neben MALDI-Matrices wurden noch zwei isomere Systeme untersucht, Methoxyacetophenon und Diethylphthalat. Neben der Aufklärung des Fragmentierungsverhalten wurde hierbei der ortho-Effekt mittels Markierungsexperimenten näher untersucht. Im dritten Teil wurden verschiedene Pyrazol-Guanidin-Cluster untersucht. Zu diesen in der Ionenquelle erzeugten Systeme wurden Strukturen und der kinetic energy release bestimmt

Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

AbstractContext: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia.Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient).Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the α4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein.Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient's phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer

17α-hydroxylase deficiency diagnosed in early infancy caused by a novel mutation of the CYP17A1 Gene

Mutations of the CYP17A1 gene cause 17α-hydroxylase deficiency (17OHD) resulting in 46,XY disorder of sex development, hypertension, hypokalemia and absent pubertal development. It is a rare, autosomal recessive form of congenital adrenal hyperplasia (CAH).We report on a neonate with prenatally determined 46,XY karyotype. At 20 weeks of gestation, lack of development of male external genitalia was noticed. A phenotypically female child was born at 41 weeks of gestation.Postnatal ultrasound revealed testes in both labia majora, an absence of uterus and normal adrenal glands. Steroid hormone analysis in serum revealed low basal levels of cortisol, testosterone and androstenedione in the presence of massively elevated corticosterone at the age of 2 weeks. The urinary steroid profile from spot urine showed excessive excretion of 17-desoxysteroids, decreased glucocorticoid metabolites and absent C19 steroids, thus proving 17OHD. Molecular analysis identified a novel mutation of the CYP17A1 gene: c.896T>A (p.I299N) in exon 5. Substitution with hydrocortisone was started. The child is raised as a girl and is developing well so far.Herein, we report the unusually early diagnosis of a newborn with the rare CAH form of 17OHD allowing an early start of treatment

A recurrent germline mutation in the 5’UTR of the androgen receptor causes complete androgen insensitivity by activating aberrant uORF translation

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11 beta-hydroxylase deficiency (11 beta-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11 beta-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11 beta-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11 beta-OHD showed a nearly complete loss of 11 beta-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11 beta-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11 beta-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP 11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. published online 11 September 201

46,XY disorder of sex development in a sudanese patient caused by a novel mutation in the HSD17B3 gene

In this study, we present a Sudanese 46,XY patient raised as a female and diagnosed at the age of 20 years with having 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency. She presented with primary amenorrhea, undeveloped breasts and a male pattern of secondary sexual characteristics. Examination of her external genitalia showed type IV genital circumcision. Steroid measurements both in urine and serum pointed to 17β-HSD3 deficiency. A novel homozygous splice-site mutation [c.524 + 2T&gt;A] was detected in intron 7 of the &lt;i&gt;HSD17B3&lt;/i&gt; gene. In this patient, steroid concentration clearly supported both the clinical diagnosis of 17β-HSD3 deficiency and the functional relevance of the mutation. Interestingly, despite of the type IV genital circumcision, the patient expressed her interest in reassigning her sex from female to male.</jats:p

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling