Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations

Prado, Mayara Jorgens; Singh, Sudhir; Ligabue-Braun, Rodrigo; Meneghetti, Bruna Valandro; Rispoli, Thaiane; Kopacek, Cristiane; Monteiro, Karina Mariante; Zaha, Arnaldo; Rossetti, Maria Lúcia Rosa; Pandey, Amit V.

doi:10.3390/ijms23010296

Cited by 3 publications

(1 citation statement)

References 45 publications

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“…We predicted the impact of the variant P228L using a set of sequence-based in silico tools, PredictSNP2, CADD, DANN, FTHMM, FunSeq2, GWAVA, and Meta-SNP [ 36 , 37 ], using the human POR reference sequence (NCBI: NP_000932.3). Additionally, we used the DynaMut-2.0 (Baker Institute, Melbourne, Australia) [ 38 ] tool to evaluate how the single amino acid change in POR could affect its structure, function, and interactions, based on its crystal structure data (PDB ID # 5FA6 chain A).…”

Section: Methodsmentioning

confidence: 99%

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Velazquez

Noebauer

Pandey

2022

IJMS

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Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

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Section: Methodsmentioning

confidence: 99%

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Velazquez

Noebauer

Pandey

2022

IJMS

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Loss of protein stability and function caused by P228L variation in NADPH-cytochrome P450 reductase linked to lower testosterone levels

Velazquez

Noebauer

Pandey

2022

Preprint

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Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L) identified by genome sequencing has been linked to lower testosterone levels and reduced P450 activities. We expressed POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant reduction of small molecule metabolism was observed with POR P228L while activities of CYP3A4 were reduced by 25%, and activities of CYP3A5, and CYP2C9 were reduced by more than 40% compared to WT POR. The 17,20 lyase activity of CYP17A1 responsible for production of main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in presence of P228L variant of POR. Based on in silico and in vitro studies we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterization of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

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Salt-Wasting Congenital Adrenal Hyperplasia in A 2-Year-Old Patient

Amelia,

Esa,

Kurniawan

et al. 2023

Indonesian J. Clin. Pathol. Med. Lab.

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Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to deficiencies of enzymes involved in steroidogenesis in the adrenal cortex. It is known that 90% of CAH are due to 21-hydroxylase enzyme deficiency caused by mutation of the CYP21A2 gene. A female patient aged two years and one month old was reported with a diagnosis of salt-wasting CAH. The diagnosis was based on complaints of genital enlargement since birth. The patient had developed repeated vomiting and severe dehydration since newborn. The patient was diagnosed with CAH and under treatment with glucocorticoid replacement therapy by a pediatrician since the age of 5 months. Body height/age < -3 Standard Deviation (SD) of the Z-score chart (very short stature) and ambiguous genitalia were reported. Laboratory tests indicated thrombocytosis (795x103/uL), mild hyperkalemia (5.4 mmol/L), and increased testosterone (110.1 ng/dL). Bone age study affirmed an advanced bone age. History of increased 17-hydroxyprogesterone (109.19 ng/mL) and decreased morning serum cortisol levels (1.7 ug/dL). History of gynecological ultrasonography (USG) showed uterus, no bilateral testicular structure, hyperplasia of the left adrenal gland, and suggestive of genital ambiguity. The 21-hydroxylase deficiency causes a decrease in aldosterone and cortisol and an increase in androgens. Hypoaldosteronism causes hypovolemia, hyponatremia and hyperkalemia. Hypocortisolime causes hypoglycemia, decreased response to infection, and stress. Hyperandrogenism causes virilization of the genitalia and short stature in patients. A female patient aged two years and one month old was reported with a salt-wasting congenital adrenal hyperplasia diagnosis. Chromosome analysis was the recommended test in this case.

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Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations

Cited by 3 publications

References 45 publications

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Loss of protein stability and function caused by P228L variation in NADPH-cytochrome P450 reductase linked to lower testosterone levels

Salt-Wasting Congenital Adrenal Hyperplasia in A 2-Year-Old Patient

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