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01.12.2018 | Case report | Ausgabe 1/2018 Open Access

BMC Endocrine Disorders 1/2018

A novel chimeric CYP11B2/CYP11B1 combined with a new p.L340P CYP11B1 mutation in a patient with 11OHD: case report

Zeitschrift:
BMC Endocrine Disorders > Ausgabe 1/2018
Autoren:
Lian Duan, Rufei Shen, Lingyu Song, Yong Liao, Hongting Zheng
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12902-018-0249-z) contains supplementary material, which is available to authorized users.
Abbreviations
11OHD
11β-hydroxylase deficiency
17 KS
17-ketosteroid
17OHP
17-hydroxyprogesterone
ACTH
Adrenocorticotrophic hormone
ALD
Serum aldosterone
CAH
Congenital adrenal hyperplasia
CYP11B1mut
Mutation p.L340P
DOC
11-deoxycorticosterne
SNPs
Single nucleotide polymorphisms

Background

Congenital adrenal hyperplasia (CAH) is one of the most common inheritable metabolic disorders and is characterized by virilization, precocious pseudopuberty and accelerated skeletal maturation, progressing in some cases to severe dehydration, shock, and even death [ 1, 2]. An autosomal recessive disorder, CAH is caused by mutations in genes encoding important enzymes or cofactors in the steroidogenesis pathway [ 3]. One common variant of CAH is 11β-hydroxylase deficiency (11OHD), driven by CYP11B1 inactivating mutations clustered in exons 2, 6, 7 and 8 [ 4], and approximately 148 mutations have been reported in the Human Gene Mutation Database website. However, CYP11B1 mutations may also occur as a result of aberrant incorporation/chimerism of the gene with the highly homologous aldosterone synthase ( CYP11B2) gene sequence. These CYP11B2/CYP11B1 chimeric genes are relatively rare, with breakpoints dispersively distributed, and have been speculated to be pathogenic because of a loss of function in the zona fasciculate/reticularis, despite maintained function in the zona glomerulosa [ 59]. In a few cases, missense mutation and chimera have been found in the same individual [ 6]. However, it is difficult to evaluate the precise contributing mechanism.
In the present paper, we report an 11OHD case who harbored a chimeric CYP11B2/CYP11B1 on one allele with a breakpoint range (g.9559–9742) in the CYP11B2 gene in the junctional zone of exon 6 and intron 6. Interestingly, the other allele held a novel mutation, p.L340P, and bioinformatics and molecular dynamics simulation indicated that this was a disease-causing mutation. We then performed a pedigree analysis, which revealed that both the missense mutation and chimera were synergistically pathogenic in the proband.

Case presentation

A 14-year-old boy (46, XY) was admitted in September 2015 because of precocious puberty and recurrent episodes of periodic paralysis without special intervention. His sexual development, such as pubic hair, laryngeal prominence and spermatorrhea, occurred earlier than that in peers. He was born after an uneventful full-term pregnancy to a non-consanguineous healthy couple of Chinese origin with no family history of congenital adrenal hyperplasia. He was taller than boys of the same age until he was 12 years old. In addition, axillary hair and laryngeal prominence had appeared at 7 years old, and spermatorrhea and pubic hair growth had occurred at age 9. Physical examination showed hypertension (143/106 mmHg), slightly black skin, and external genitalia maturation (pubic hair and axillary hair at stage 5 assessed by Tanner classification and an estimated testicular volume of 25 ml on both sides, as measured by an orchidometer), which confirmed precocious puberty (Fig.  1a). Laboratory data in the absence of medication showed decreased plasma potassium and cortisol but elevated levels of plasma 17 hydroxyprogesterone (17OHP), androstenedione, adrenocorticotrophic hormone (ACTH), and uric 17 ketosteroid (17 KS) (Additional file  1: Table S1). Additionally, serum aldosterone (ALD) was normal with low rennin activity, and renal, liver, metadrenaline, normetadrenaline and thyroid function, were all normal (Additional file 1: Table S1). Radiation imaging showed accelerated bone aging (over 18 years according to the Greulich and Pyle Atlas) (Fig.  1b) and bilateral adrenal hyperplasia (Fig.  1c). The hypokalemic hypertension, elevated 17-OHP and androstenedione were all suggestive of an 11OHD diagnosis. Interestingly, low urine osmotic pressure and specific gravity could not be significantly elevated by water deprivation and a desmopressin test. This phenomenon is consistent with nephrogenic diabetes insipidus that may be due to long-term hypokalemia (Additional file 1: Table S1).
Given this clinical presentation, we then sought to confirm whether it was indeed a case of 11OHD. To find the possible genetic pathogenic mechanism, we conducted sequencing of the nine exons and flanking sequencing of the CYP11B1 gene and identified the novel mutation p.L340P ( CYP11B1mut) (Fig.  1d). Bioinformatics and molecular dynamics simulation indicated that CYP11B1mut altered the free energy, and change the stability and conformation of the protein by driving the uncoiling of a partial helical structure into a loop structure, leading to positive group migration and pocket structure enlargement (Additional file  2: Table S2, Figure  1e, f), suggesting that the mutation could be pathogenic. However, his mother did not carry this pathogenic mutation (Fig.  1g), indicating the proband only carried the point mutation on one allele, with the pathogenicity of the other allele unclear. Since 11OHD is understood to be an autosomal recessive condition, we then investigated the genotype of the patient in greater detail. QPCR of the sequence around the mutation of the proband showed that the copy number was half the normal number, which indicated a fragment deletion, and this was also found in his mother (Additional file 3: Figure S1). Targeted next-generation sequencing of the patient genome then revealed a large fragment deletion that included exons 1 to 6 of CYP11B1 (Additional file  3: Figure S1). To validate the deletion, we conducted PCR in proband with mixed oligonucleotide primers that have been previously reported [ 8] (a forward primer complementary to the CYP11B2 sequence and a reverse primer complementary to the CYP11B1 sequence) and confirmed a chimeric CYP11B2/CYP11B1 gene. Interestingly, extended familial analysis revealed that the chimera was also present in his mother, uncle and grandfather (Fig.  1h), and multiple sequence alignment revealed that the breakpoint was located at g.9559–9742 of CYP11B2, considering the cytosine nucleotide base (C) of rs6391 in this family with 3 single nucleotide polymorphisms (SNPs) (Fig.  1i). Our results suggested that the proband harbored a compound heterozygosity for a chimeric CYP11B2/CYP11B1 gene combined with a novel missense mutation p.L340P (Fig.  1j and k , Additional file  4: Figure S2) as two contributors to pathogenicity.
Since the blood pressure of the patient was poorly controlled by hydrocortisone, the proband was treated with a combination of dexamethasone (0.75 mg/everyday) and levamlodipine (2.5 mg/everyday) for 20 months. After treatment, clinical parameters, such as blood pressure, 17OHP and ACTH decreased, and the volume of the adrenal glands was reduced (Additional file 1: Table S1, Additional file 5: Figure S3). Main methods used in the study are summarizing in Additional file 6. This study was approved by the ethics committees of Xinqiao Hospital, Third Military Medical University, and informed consent was obtained from the patient’s mother (Additional file 7).

Discussion and conclusion

11OHD presents with decreased cortisol and corticosterone synthesis due to impaired conversion of 11-deoxycortisol and 11-deoxycorticosterne (DOC) to cortisol and corticosterone, respectively, and excess androgens due to DOC accumulation [ 10]. Although DOC was not detected, all of the classical 11OHD symptoms, precocious pseudopuberty, accelerated skeletal maturation and hypokalemia hypertension [ 11, 12], were present in our patient. After treatment with the combined medication regimen described above, the clinical symptoms of the patient improved and the adrenal gland volume was reduced.
From our investigation of the genetic contribution to pathogenesis, this patient was found to harbor a chimeric CYP11B2/CYP11B1 gene with a novel breakpoint (g.9559–9742) on one allele, which differs from previous reports. Interestingly, the other allele contained a new disease-causing mutation, p.L340P, which altered the free energy and stability of 11β-hydroxylase. The chimeric mutation leads to deletion of CYP11B1 exons 1 to 6 on one allele, and unfortunately, a pathogenic missense mutation appeared on exon 6 of CYP11B1 in the other allele, which resulted in a seemingly homozygous missense mutation that was predicted by bioinformatics and molecular dynamics simulation to be a disease-causing mutation. At present, only one case of a pathogenic p.G314R mutation combined with a nonfunctional chimeric CYP11B2/CYP11B1 has been reported [ 6]. Our pedigree analysis revealed that the proband carried both the chimera and p.L340P, and their coexistence increased the morbidity of 11OHD in this family. Our results suggest that chimera screening and CYP11B1 mutation screening should be simultaneously conducted and that pedigree study is necessary.

Acknowledgments

The authors would like to thank all the participants in this study, particularly Hongxia Wang, Xu Zhang and Honghe Fang for sequencing and bioinformatics assistance.

Funding

The collection of clinical data, data analysis and interpretation, and writing of the manuscript were supported by grants from the National Key R&D Program of China (No. 2016YFC1101100), National Natural Science Foundation of China (NO. 81471039, No. 81270893, No. 81228023, No. 81401601, and No.81402202), Natural Science Foundation Project of Chongqing (CSTC2014jcyjjq10006, CSTC2012jjB10023, and CSTC2016jcyjA0518), and Clinical Research Project of Xinqiao Hospital of Third Military Medical University (Yclkt-201421).

Availability of data and materials

All data generated or analyzed during this study are included in the article.

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Written informed consent was obtained from the patient’s mother for publication of this case report and any accompanying images.

Competing interests

The authors declare that they have no competing interests.

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