A novel homozygous mutation IVS6+5G>T in ...

GENE-40459; No. of pages: 4; 4C: Gene xxx (2015) xxx–xxx

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A novel homozygous mutation IVS6+5GNT in CYP11B1 gene in a Vietnamese patient with 11β-hydroxylase deficiency Thi Phuong Mai Nguyen a,b, Thu Hien Nguyen a, Diem Ngoc Ngo b, Chi Dung Vu b, Thi Kim Lien Nguyen a, Van Hai Nong a, Huy Hoang Nguyen a,⁎ a b

Institute of Genome Research, Vietnam Academy of Science and Technology, Vietnam Human Genetics Department, National Hospital of Pediatrics, Vietnam

a r t i c l e

i n f o

Article history: Received 22 January 2015 Received in revised form 11 April 2015 Accepted 20 April 2015 Available online xxxx Keywords: IVS6+5GNT mutant CYP11B1 gene Congenital adrenal hyperplasia (CAH) Splice site 11β-Hydroxylase

a b s t r a c t Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is characterized by a deficiency of one of the enzymes involved in the synthesis of cortisol from cholesterol by the adrenal cortex. CAH cases arising from impaired 11β-hydroxylase are the second most common form. Mutations in the CYP11B1 gene are the cause of 11β-hydroxylase deficiency. This study was performed on a patient with congenital adrenal hyperplasia and with premature development such as enlarged penis, muscle development, high blood pressure, and bone age equivalent of 5 years old at 2 years of chronological age. Biochemical tests for steroids confirmed the diagnosis of CAH. We used PCR and sequencing to screen for mutations in CYP11B1 gene. Results showed that the patient has a novel homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5GNT). The analysis of this mutation by MaxEntScan boundary software indicated that this mutant could affect the gene splicing during transcription. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders resulting from the deficiency of one of the five enzymes: 21-hydroxylase (21-OH), 11β-hydroxylase (11-OH), 17-hydroxylase (17-OH), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20/22 desmolase, which are required for the synthesis of cortisol in the adrenal cortex. The most frequent is 21-hydroxylase (21-OHD) deficiency accounting about 90% of CAH cases and about 5–8% of CAH cases are caused by 11β-hydroxylase deficiency (11β-OHD, OMIM #202010), the rest is the other rare enzyme deficiencies. The incidence reported for 11βhydroxylase deficiency is about 1 in 200,000 live births (Belkina et al., 2001; Cerame et al., 1999). CAH due to 11β-hydroxylase deficiency is frequently associated with hypertension. The conversion of 11deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone is interrupted. Glucocorticoid decline will reduce its reverse process of inactivation on the brain, hypothalamus and anterior pituitary, leading to an increase of ACTH. This will stimulate the stronger adrenocortical activity leading to an excess phase precursor substance such as 17-OHAbbreviations: 11-OH, 11β-hydroxylase; 17-OH, 17α-hydroxylase/17,20-lyase; 21-OH, 21-hydroxylase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; ACTH, adrenocorticotropic hormone; CAH, congenital adrenal hyperplasia; MaxENT, Maximum Entropy Model; MDD, Maximum Dependence Decomposition Model; PWM, Position Weight Matrix. ⁎ Corresponding author at: Functional Genomics Laboratory, Institute of Genome Research, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet Str., Caugiay, Hanoi, Vietnam. E-mail address: [email protected] (H.H. Nguyen).

progesterone. 11β-Hydroxylase deficiency leads to increased adrenal androgen excess and virilization in female. The enzyme 11-OH deficiency resulting in the overproduction of androgen response phenotype results in male, external genitalia in female infants and in both sexes the phenomenon of early puberty associated with early mature and accelerated bone age. Girls with 11-OH enzyme deficiency are generally not born with normal genitalia and signs of androgen increased in adulthood can be manifestations of hirsutism and menstrual irregularities (Belkina et al., 2001; Bhangoo et al., 2006; Chabre et al., 2000). In humans, the CYP11B family includes CYP11B1 gene and CYP11B2 gene. Two genes have homologous sequences to 95% in the coding region and to 90% in the intron. CYP11B1 gene is located on the long arm of chromosome 8 (8q21), about 7 kb including 9 exons and 8 introns (Cerame et al., 1999). 11β-Hydroxylase deficiency can be diagnosed by an increase of ACTH, deoxycorticosterone, 17-OHP and 11-deoxycortisol in serum, or an increase of tetrahydro metabolic process in the urine excretion in 24 h (Cerame et al., 1999). In this study, we describe a 2 year-old-boy with 11β-hydroxylase deficiency. Molecular analysis of CYP11B1 gene identified the homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5GNT). 2. Patient and methods 2.1. Patient The patient is the first child of the healthy non-consanguineous Vietnamese parents. He was admitted to the National Hospital of

http://dx.doi.org/10.1016/j.gene.2015.04.052 0378-1119/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Nguyen, T.P.M., et al., A novel homozygous mutation IVS6+5GNT in CYP11B1 gene in a Vietnamese patient with 11βhydroxylase deficiency, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.052

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T.P.M. Nguyen et al. / Gene xxx (2015) xxx–xxx

Pediatrics at 2 years of age. At that time, he was presented with peripheral precocious puberty symptoms including an enlarged penis (6 cm of length and 8 cm of circumference), normal bilateral testis volume (1.5 mL), muscle development, acne, height of 96 cm (+ 2.8 SD compared with WHO growth chart), high blood pressure (110/60 mm Hg or N 90th percentile for age, gender and height) and bone age in advance of chronologic age (5 years). Biochemical investigations showed that plasma sodium level was 136 mmol/L (normal range, 135–155 mmol/L), potassium level 3.7 mmol/L (normal range, 3.5–5.5 mmol/L), chloride level 105 mmol/L (normal range, 96–108 mmol/L), LH b 0.1 UI/L, FSH 0.13 UI/L, 17hydroxyprogesterone (17-OHP) 1160 ng/dL (normal ≤ 169 ng/dL), testosterone 10.11 nmol/L (normal range, 0.35–2.60 nmol/L), and progesterone 15.93 ng/mL (normal b 0.59 ng/mL). Urinary 17-ketosteroid level was 9.1 mg/24 h (normal b8.75 mg/24 h). No adrenal mass was found in abdominal ultrasound. Growth and blood pressure initially normalized with hydrocortisone 15 mg/m2/day, but at 14 years of age, his high blood pressure was increased 150/100 mm Hg. Recently, he was treated with hydrocortisone and antihypertensive drug. He was diagnosed with CAH due to 11β-hydroxylase deficiency. 2.2. Direct sequencing of the CYP11B1 gene Total DNA was extracted from peripheral blood using the DNA extraction kit − QiaAmp DNA blood mini kit (Qiagen, Germany). The CYP11B1 gene was amplified by PCR. Three pairs of primers were used to amplify exon 1–2, 3–5, and 6–9 of CYP11B1 gene without amplifying CYP11B2 gene as previously described (Nguyen et al., 2012; Riedl et al., 2008). All PCR assays were performed in a 50 μL volume containing 200 ng of genomic DNA, 1.75 mM MgCl2, 0.3 mM of each deoxy-NTP, 50 pmol of each primer and 1 U of DNA Polymerase (Invitrogen) in the appropriate buffer. After a denaturation step at 94 °C for 2 min, the amplification was performed for 30 cycles at 94 °C for 45 s, at 60 °C for 30 s, and at 72 °C for 1 min 30 s, with a final extension at 72 °C for 10 min in the AG. 22331 DNA Cycler (Eppendorf, Germany). Amplicons were separated on 1% agarose gel and purified by column filtration QIAamp PCR purification kit (Qiagen, Germany). All exons and intron/exon boundaries were sequenced on 3130 Genetic Analyzer (Applied Biosystems, USA) using internal primers as described previously (Nguyen et al., 2012). Sequencing data obtained in this study were compared with CYP11B1 gene sequence in ENSEMBL (ENSG00000160882) by using the program CLUSTALW 1.8. 2.3. Splice site scores In order to estimate the strengths of 5′ and 3′ splice junctions in the CYP11B1 gene, splice site sequence motifs were scored using the splice site models introduced by Yeo and Burge (2004) and the software available at: http://genes.mit.edu/burgelab/maxent/5ss. These splice site models assign a log-odd ratio (MaxENT score) to a 9 bases long (3 bases in exon and 6 bases in intron) (5′ splice site). The higher the score, the higher the probability that the sequence is a true splice site. 3. Results Clinical manifestations and biochemical profiles of the patient were consistent with congenital adrenal hyperplasia causing 11βhydroxylase deficiency. Genetic analysis of the patient and his family was performed. The CYP11B1 gene of patient and his family was specifically amplified from the genomic DNA, and all nine exons and the exon/ intron boundaries were sequenced. The patient was found to be homozygous for a novel point mutation from guanine to thymine at position bp 4140 in intron 6 (IVS6+5GNT) of CYP11B1 gene (ENSEMBL: ENSG00000160882) (Fig. 1A and B). Sequencing of the CYP11B1 genes of parents indicated that the mutation (IVS6+5GNT) in intron 6 was

inherited from the mother and father. Both parents bore the mutation on one of alleles and were heterozygous (Fig. 1B and C). To analyze if the mutations are located on the boundary (Splicing) between the exon and intron, we used sequence analysis programs MaxEntScan boundary (Splicing site sequence) which was designed by Yeo and Burge (2004) (http://genes.mit.edu/burgelab/maxent/5ss). This program will make a comparison between the standard (gcgGTGGGT) and mutant sequences (gcgGTGGTT) (last three nucleotides of exon 6 and 5 nucleotides of intron 6) through the score of MaxENT (Maximum Entropy Model), the score of MDD (Maximum dependence Decomposition Model) and the score of PWM (Position Weight Matrix) (Yeo and Burge, 2004). A larger score generally indicates a larger ‘strength’ of the corresponding splice site. Test results on normal (wild type) and mutant sequences are shown in Table 2. IVS6+5GNT mutation at nucleotide position 5 in intron 6 MaxENT value is − 2.03 (MaxENT index of the normal sequence is 6.60); MDD index is 1.88 (MDD index of process normal order is 10.38); PWM index was 1.87 (normal PWM sequence index is 5.77). It can be hypothesized that guanine is replaced by thymine at nucleotide number 5 on the intron 6 located at the exon–intron boundaries, and potentially alters mRNA transcription. 4. Discussion In our study, we describe the clinical and novel CYP11B1 mutation (IVS6+5GNT) from a patient diagnosed with 11-OHD in one Vietnamese family. The patient (46,XY) presented with early development such as a large phallus, muscle development, high bone age and especially high blood pressure. High blood pressure, which is one of the clinical distinguished features between 21-hydroxylase deficiency and 11β-hydroxylase deficiency, causes deoxycorticosterone to accumulate, a steroid with mineralocorticoid (sodium retaining) activity. Furthermore, 21-hydroxylase deficiency leads to the deficiency of both cortisol and aldosterone which results in a patient with “salt wasting” form including hyponatremia, hyperkalemia, and hypovolemic shock. Our patient biochemical data did not show hypokalemia. However, hypokalemia was found in 4 of 6 Vietnamese patients with classic type of 11-OHD before, and just 2 of 6 cases was reported without hypokalemia (not published, for detailed information, please contact to Dr. Vu Chi Dung, National Hospital of Pediatrics, Vietnam). The phenotype of the patient was due to the deficiency of adrenal 11β-hydroxylase activity, because 11-deoxycortisol and 11-deoxycorticosterone are not efficiently converted to cortisol and corticosterone, respectively. Decreased cortisol production causes an elevated plasma ACTH level that lead to the overproduction and accumulation of adrenal androgens and their precursors, and subsequent hyperandrogenism. 11-Hydroxylase deficiency also accumulates the mineralocorticoid precursor 11-deoxycorticosterone (DOC) in the zona fasciculata and leads to hypertension in about two thirds of the 11-OHD patient (White and Speiser, 1994). From the early 1950s, the world has studied a few cases of patients with congenital adrenal hyperplasia with manifest hypertension. So far, more than 55 different mutations (mainly missense mutations) have been detected in CYP11B1 in laboratories in the world such as Germany, Austria, Australia, Japan,… (Bhangoo et al., 2006; Nguyen et al., 2012; Andrew et al., 2007; Hampf et al., 2001; Wu et al., 2011). Nine mutations of splice site on the CYP11B1 gene have been published recently (Table 1) (Chabre et al., 2000; Andrew et al., 2007; Hampf et al., 2001; Matsubara et al., 2014; Merke et al., 1998; Skinner et al., 1996; Soardi et al., 2009; Wu et al., 2011). Splice site nucleotide substitutions may result in exon skipping, activation of cryptic splice sites, creation of a pseudo-exon within an intron, or intron retention of mRNA. These changes lead to the production of a nonfunctional protein of CYP11B1. The phenotype of these patients with splice site mutations was presented congenital adrenal hyperplasia. We used MaxENT modeling method based on short sequences of RNA pairing process, and



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Fig. 1. Mutant analysis by direct DNA sequencing. A) Location of point mutation in the CYP11B1 gene. B) The father and the patient are represented by squares, and the mother is presented by circle. The patient carried a novel homozygous mutation IVS6+5GNT. The parents were heterozygous. C) The base change from guanine (G) to thymine (T) at position bp 4140 in intron 6 (IVS6+5GNT).

calculated the nucleotide positions adjacent to each other or not (Yeo and Burge, 2004). MDD and PWM values are independent assessment of the nucleotide. Score analysis of MaxENT, MDD and PWM indicated that the mutant scores were lower than the wild type scores (Table 2). Some mutations of splice sites had been studied in in vitro such as: One female patient was identified with 11-hydroxylase deficiency with virilization symptoms and persistent hypertension. The patient had a mutation at nucleotide number 4 in intron 8, an adenine to guanine (IVS8+4ANG). Adrenal cell line studies and mitochondrial extract showed that the activity of 11-hydroxylase was decreased. The analysis of messenger RNA (mRNA) by RT-PCR and gene sequencing showed that the expression of the shorter mRNA chain resulted in a decline of normal activity of 11-hydroxylase (Chabre et al., 2000). Soardi (Soardi et al., 2009) described a severely virilized female with homozygous for the g.2791GNA transition in the last position of exon 4. This nucleotide was a part of 5′ intron 4 donor splice site consensus sequence. They used a splice site prediction program in Internet (http://www.fruitfly. org/seq_tools/splice.html) to hypothesize the mutation score. The result showed that the mutation caused a reduction from 0.98 (normal) to 0.42 (mutant); this score suggested that the transition could affect normal splicing process. In the present study, analysis of mutation IVS6+5GNT showed that the PWM, MDD and MaxENT values were lower than the normal Table 1 Mutants of slice site have detected in the CYP11B1 gene. Genotype

Phenotype

Reference

IVS3+16GNT IVS4+1GNA IVS4+5GNC IVS5−1GNA IVS5−1GNC IVS5+1GNA IVS5+1GNC IVS7+1GNA IVS8+4ANG

CAH 11-Hydroxylase deficiency 11-Hydroxylase deficiency CAH 11-Hydroxylase deficiency 11-Hydroxylase deficiency CAH CAH 11-Hydroxylase deficiency

Hampf et al., 2001 Soardi et al., 2009 Andrew et al., 2007 Skinner et al., 1996 Chabre et al., 2000 Merke et al., 1998 Bhangoo et al., 2006 Matsubara et al., 2014 Chabre et al., 2000

value (Table 2), which can predict that the likelihood of splicing site may be located at a different location so mutation affects transcription (mRNA) and leads to nonfunctional enzyme. In conclusion, a novel homozygous mutation g.4140GNT (IVS6+5GNT) was detected in the Vietnamese patient with congenital adrenal hyperplasia. Mutation IVS6+5GNT could affect the slice site

Table 2 Scores of the splice site were detected in the CYP11B1 gene. Genotype

Sequence

MaxENT

MDD

IVS4+5GNC Wild type Mutant

PWM

GCGgtgggt GCGgtggct

9.20 4.75

12.88 5.78

IVS5−1GNA Wild type Mutant

ggatagGCG ggataaGCG

8.06 −0.69

6.34 −2.41

IVS5−1GNC Wild type Mutant

ggatagGCG ggatacGCG

8.06 0.00

6.34 −1.73

IVS5+1GNA Wild type Mutant

ACGgtcagg ACGatcagg

4.63 −3.56

8.88 0.70

4.91 −3.27

IVS5+1GNC Wild type Mutant

ACGgtcagg ACGctcagg

4.63 −3.65

8.88 0.60

4.91 −3.36

IVS6+5GNT Wild type Mutant

GCGgtgggt GCGgtggtt

6.60 −2.03

10.38 1.88

5.77 1.87

IVS7+1GNA Wild type Mutant

GGGgtgagt GGGatgagt

7.93 −0.25

12.38 4.20

8.21 0.03

IVS8+4ANG Wild type Mutant

CATgtgagc CATgtgggc

5.03 0.71

9.78 4.78

6.35 3.98

5.77 1.93

The bold emphasis in table 2 is our results.


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of CYP11B1 gene transcription. Our result might be used to support genetic counseling and prenatal diagnosis. Acknowledgment This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106YS.02-2013.20. References Andrew, M., et al., 2007. Congenital adrenal hyperplasia in a Nigerian child with a novel compound heterozygote mutation in CYP11B1. Clin. Endocrinol. (Oxf.) 66 (4), 602–603. Belkina, N.V., et al., 2001. Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2. J. Inorg. Biochem. 87 (4), 197–207. Bhangoo, A., et al., 2006. Donor splice mutation in the 11beta-hydroxylase (CypllB1) gene resulting in sex reversal: a case report and review of the literature. J. Pediatr. Endocrinol. Metab. 19 (10), 1267–1282. Cerame, B.I., et al., 1999. Prenatal diagnosis and treatment of 11beta-hydroxylase deficiency congenital adrenal hyperplasia resulting in normal female genitalia. J. Clin. Endocrinol. Metab. 84 (9), 3129–3134. Chabre, O., et al., 2000. Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1. J. Clin. Endocrinol. Metab. 85 (11), 4060–4068.

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