Splicing Analysis of CYP11B1 Mutation in a Family Affected with 11b-hydroxylase Deficiency

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Pages:  5
Wordcount:  1206 Words
Date:  2021-03-19

The article splicing analysis of Cytochrome P450 Family 11 Subfamily B Member 1 (CYP11B1) mutation in a family affected with 11b-hydroxylase deficiency: case report analyzes Congenital Adrenal Hyperplasia (CAH) from caused by a deficiency of 11b-hydroxylase a condition characterized by low renin hypokalemia, hypertension, ambiguous and hyperandrogenemia in females. In their study, the authors emphasized in hormonal, clinical and molecular features of two related children with 11 beta hydroxylase deficiency (11b-OHD) and examining the results of a splicing mutation. 11b-OHD is caused by mutations of a CYP11B1 genetic factor which is responsible for over 5% of the CAH in non-consanguineous inhabitants. It, however, elucidates for about 15% of Jewish Middle Eastern and Muslim populations.

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Hypertension is one of the primary indicators of Congenital Adrenal Hyperplasia (CAH) variant mainly caused by a deficiency of steroid 11b-hydroxylase such as P450C11b. Standard 11 beta hydroxylase deficiency (11b-OHD) because masculinization of the exterior genitalia in the traumatized women accelerated growth, bone maturation and precocious puberty in male and female. Patients suffering from 11b-OHD deficiency can experience more concentrations of 17-hydroxyprogesterone (17OHP) especially when it accumulates in two steps behind enzymatic block such that 17OHP is used to detect 11b-OHD using newborn screening program. CYP11B1 is found at the extended arm of 8q21 DNA that encodes 503 amino acids and had 9 exons. Most of the CYP11B1 alterations are linked with standard 11 beta hydroxylase deficiency (11b-ODH) and scarce cause non-standard 11b-OHD. The authors report describes two siblings with the clinical and endocrinal composition of 11b-OHD but had multiple different alterations in the CYP11B1 genetic factor.

The two came from a non-blood related family and the first individual was 3years old. She was 46 years and had unclear genitals. She had been assessed for abdominal genital growth and experienced first genital operation in Turkey. During her examination, her phallus was 2cm wide and 5cm long and palmar and areola crumbles were pigmented together. Her dehydroepiandrosterone sulfate (DHEAS) was 165.2 ug/dl while androgen levels were 132 ng/dl. Treatment with hydrocortisone helped her improve Blood Pressure (BP) to the normal range while PRA became measurable and testosterone was suppressed. The other patient had spots and masculinization at the age of 2 years and a progressive development of the outer genitals together with a deep voice. An Adrenocorticotropic hormone (ACTH) assessment revealed a rise in ACTH baseline, temperately raised 17OHP later in 60 minutes and non-response to Adrenocorticotropic hormone (ACTH). He was treated with hydrocortisone and his BP was well controlled while plasma renin activity (PRA) increased to 738 ng/dl/h.

During the DNA sequencing the researchers extracted genomic chromosome from outlying blood leucocytes using QIAamp DNA blood mini kit of the parents and individuals. polymerase chain reaction (PCR) outputs were handled with ExoSAP-IT reagent and referred at Macrogen Inc. for straight succession. There was a minigene in a video test done of the CYP11B1 merging alteration implemented and a part of the mutant genomic DNA and wild-type and amplified by polymerase chain reaction (PCR) using the oligonucleotides. PCR responses were performed in a 20ul volume comprising 50ng gDNA, 10uM Deoxynucleoside Triphosphates (dNTPs), and 5U/ul. The polymerase chain reaction (PCR) component was sliced using T4 DNA ligase with Xbal enzymes and BamHI-HF enzyme and duplicated into the corresponding locations of pcDNA 3.1/myc-His B appearance trajectory. There was a culture of the COS-7 cells in penicillin/streptomycin and high aldohexose at 37oC. Cells were grown using Effectene Transfection Reagent on 6-well plates and transfected with the mutant minigene constructs and wild-type.

QIAamp RNA Blood Mini Kit was used in the extraction of total cellular ribonucleic acid (RNA) and treated with DNasel (Qiagen) and then employed as a prototype using Improm-II Reverse Transcription System for complementary DNA (cDNA) fusion. The polymerase chain reaction (PCR) outputs were examined by staining with ethidium bromide and electrophoresis on a 1% agarose gel. The PCR products after subcloning into PCR fragment ligated to a plasmid vector (pGEM-T) were confirmed by Sanger sequencing.

The DNA placement of the whole coding areas and their nearby noncoding DNA of the CYP11B1 gene indicating the family members were multiple genes for a nonsense mutation c.421C. The mutations identified were conveyed before but their pathogenic contrivances have not been explained clearly. The fathers mutation was situated at the merging contributor location of intron 7 and could generate an anomalous merging of the messenger RNA (mRNA). The person c.1200 + 1G>A CYP11B1 minigene became administered to 2 incorrect merged outputs that were less than the WT minigene.

From their study, they found that one mutant avoided the whole exon 7 while comprising all the arrangements of exons 8 and 6 as the additional one missed exons 7 and 8 even though there was a recollection of full arrangements of exons 9 and 6. CYP11B1 mutations were found in the two siblings and they were diagnosed with classic 11b-OHD. Mineralocorticoid signs link off-color with the mark of masculinization in the stricken girls and blood pressure is regular during initial stages of a child. Studies in vitro undertakings below 5% were taken as consistent and severe with nonsense p.R141X and standard 11 beta hydroxylase deficiency (11b-OHD) is projected to focus on an untimely break in exon 3 and produces a shortened catalyst without the vital remains for heme binding area constant that has the children's medical phenotypes. IVS7+1G>A mutation was found to cause an intron retention leading to exon skipping.

The learning outcome of the article included the evaluation of compound heterozygous Cytochrome P450 Family 11 Subfamily B Member 1 (CYP11B1) alteration mainly linked with an abnormality in protein structures. One can also comprehend the severity of classic 11b-hydroxylase and how a mutation analysis is done using PCR and Sanger encoding of the whole coding regions. One also learns the effect of mutations c.421C>T and c.1200 + 1G>An in parents to their children. Finally one acquires knowledge on how CYP11B1 leads to exon skipping.


Charnwichai, Pattaranatcha, Patra Yeetong, Kanya Suphapeetiporn, Vichit Supornsilchai, Taninee Sahakitrungruang, and Vorasuk Shotelersuk. "Splicing analysis of CYP11B1 mutation in a family affected with 11b-hydroxylase deficiency: a case report." BMC Endocrine Disorders 16, no. 1 (2016): 1.

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Splicing Analysis of CYP11B1 Mutation in a Family Affected with 11b-hydroxylase Deficiency. (2021, Mar 19). Retrieved from https://proessays.net/essays/splicing-analysis-of-cyp11b1-mutation-in-a-family-affected-with-11b-hydroxylase-deficiency

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