Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations

Abstract

Purpose

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease characterized by impaired adrenal steroidogenesis and most often caused by CYP21A2 gene mutations. For the first time, we reported complete spectrum and frequency of CYP21A2 gene mutations in 61 unrelated patients with classical and non-classical CAH from Serbia.

Methods

Direct DNA sequencing of whole CYP21A2 gene and polymerase chain reaction with sequence-specific primers for detection of CYP21A1P/CYP21A2 chimeras were combined.

Results

We identified 18 different pathogenic alleles—two of them novel. Mutation detection rate was highest in patients with salt-wasting form of CAH (94.7 %). The most prevalent mutation was intron 2 splice site mutation, c.290-13A/C>G (18.5 %). Other mutation frequencies were: CYP21A1P/CYP21A2 chimeras (13 %), p.P30L (13 %), p.R356W (11.1 %), p.G110fs (7.4 %), p.Q318X (4.6 %), p.V281L (4.6 %), p.I172N (2.8 %), p.L307fs (2.8 %), p.P453S (1.9 %), etc. Mainly, frequencies were similar to those in Slavic populations and bordering countries. However, we found 6.5 % of alleles with multiple mutations, frequently including p.P453S. Effects of novel mutations, c.386T>C (p.Leu129Pro) and c.493T>C (p.Ser165Pro), were characterized in silico as deleterious. The effect of well-known mutations on Serbian patients’ phenotype was as expected.

Conclusions

The first comprehensive molecular genetic study of Serbian CAH patients revealed two novel CYP21A2 mutations. This study will enable genetic counseling in our population and contribute to better understanding of molecular landscape of CAH in Europe.

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Fig. 1

Abbreviations

17-OHP:

17-Hydroprogesterone

21-OH:

21-Hydroxylase

Bp:

Base pair

CAH:

Congenital adrenal hyperplasia

CYP21A2:

Cytochrome P450, family 21, subfamily A, polypeptide 2

dNTP:

Deoxyribonucleoside triphosphate

MLPA:

Multiplex ligation-dependent probe amplification

HGMD:

Human gene mutation database

NC-CAH:

Non-classical form of CAH

PCR:

Polymerase chain reaction

PCR-SSP:

Sequence-specific PCR amplification

SV-CAH:

Simple virilizing form of CAH

SW-CAH:

Salt-wasting form of CAH

UTR:

Untranslated region

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Corresponding author

Correspondence to M. Stojiljkovic.

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Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This study was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III41004).

Ethical approval

This study involved human participants and has been approved by the Ethics Committee of the School of Medicine, University of Belgrade in Belgrade, Serbia and the Ethics Committee of the Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic” in Belgrade, Serbia, and has therefore, been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

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Informed consent was obtained from all individual participants included in the study.

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Milacic, I., Barac, M., Milenkovic, T. et al. Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations. J Endocrinol Invest 38, 1199–1210 (2015). https://doi.org/10.1007/s40618-015-0366-8

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Keywords

  • 21-Hydroxylase deficiency
  • Alleles with multiple mutations
  • CYP21A1P/CYP21A2 chimeras
  • Genotype–phenotype correlation
  • Mutation detection
  • Mutations’ effect