Accepted Manuscript

Title: A Recurrent Synonymous Mutation in the Human

Androgen Receptor Gene Causing Complete Androgen
Insensitivity Syndrome

Authors: Rafael Loch Batista, Andresa di Santi Rodrigues,

Mirian Yumie Nishi, Nathalia Lisboa Rosa Almeida Gomes,
José Antonio Diniz Faria Junior, Daniela Rodrigues de
Moraes, Luciani Renata Carvalho, Elaine Maria Costa Frade,
Sorahia Domenice, Berenice Bilharinho de Mendonca

PII: S0960-0760(17)30185-1
DOI: http://dx.doi.org/doi:10.1016/j.jsbmb.2017.07.020
Reference: SBMB 4986

To appear in: Journal of Steroid Biochemistry & Molecular Biology

Received date: 30-5-2017

Accepted date: 18-7-2017

Please cite this article as: Rafael Loch Batista, Andresa di Santi Rodrigues,
Mirian Yumie Nishi, Nathalia Lisboa Rosa Almeida Gomes, José Antonio Diniz
Faria, Daniela Rodrigues de Moraes, Luciani Renata Carvalho, Elaine Maria
Costa Frade, Sorahia Domenice, Berenice Bilharinho de Mendonca, A Recurrent
Synonymous Mutation in the Human Androgen Receptor Gene Causing Complete
Androgen Insensitivity Syndrome, Journal of Steroid Biochemistry and Molecular
Biologyhttp://dx.doi.org/10.1016/j.jsbmb.2017.07.020

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A Recurrent Synonymous Mutation in the Human Androgen Receptor Gene Causing
Complete Androgen Insensitivity Syndrome

Rafael Loch Batista, MD

Andresa di Santi Rodrigues, MSc

Mirian Yumie Nishi, PhD

Nathalia Lisboa Rosa Almeida Gomes, MD

José Antonio Diniz Faria Junior, MD

Daniela Rodrigues de Moraes, MD

Luciani Renata Carvalho, MD

Elaine Maria Costa Frade, MD

Sorahia Domenice, MD

Berenice Bilharinho de Mendonca, MD

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética

Molecular/LIM42, Hospital das Clínicas, Disciplina de Endocrinologia e Metabologia, Faculdade
de Medicina da Universidade de São Paulo, São Paulo, Brasil
Abstract

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of
sex development (46,XY DSD). This syndrome is an X-linked inheritance disease and it is caused
by mutations in the human androgen receptor (AR) gene. Non-synonymous point AR
mutations are frequently described in this disease, including in the complete phenotype. We
present a novel synonymous mutation in the human AR gene (c.1530C>T) in four 46,XY
patients from two unrelated families associated with complete androgen insensitivity
syndrome (CAIS). The analysis of mRNA from testis showed that synonymous AR mutation
changed the natural exon 1 donor splice site, with deletion of the last 92 nucleotides of the AR
exon 1 leading to a premature stop codon 12 positions ahead resulting in a truncate AR
protein. Linkage analyses suggested a probable founder effect for this mutation. In conclusion,
we described the first synonymous AR mutation associated with CAIS phenotype, reinforcing
the disease-causing role of synonymous mutations

1. Introduction

Androgen Insensitivity Syndrome (AIS) is a common cause of 46,XY Disorders of Sexual

Development (DSD) caused by AR gene mutations [1-3]. Including prostate cancer, there are
more than 1000 reported mutations in the AR gene, related to different degrees of androgen
activity and phenotype [4]. The clinical spectrum is very broad, ranging from infertility in males
to 46,XY individuals with complete female external genitalia [3]. A substantial number of
patients with Complete Androgen Insensitivity Syndrome (CAIS) with AR mutation has been
described (90 – 95%) [3]. However, in some families with typical CAIS phenotype defects in AR
were not identified.

Synonymous mutations are a specific subset of silent mutations in which the mutation
occurs in the coding region of a gene but does not alter the amino acid sequence. In the past,
synonymous mutations were presumed to exert no functional effect. However, synonymous
mutations has been implicated in the etiology of several human diseases and in cancer [5, 6].
In general, AR mutations in individuals with AIS are more frequently reported in patients with
CAIS phenotype. In most cases, the molecular defect is a single point mutation causing an
amino acid change or a premature stop codon [4, 7]. Synonymous AR mutation causing AIS is
very rare and there are no AR synonymous mutations reported in individuals with CAIS. We
present here one novel recurrent synonymous mutation in AR in two unrelated families with
CAIS. Simultaneously, we described in a PAIS individual an already described AR synonymous
mutation associated with PAIS phenotype

2 - Material
2.1 Patients
The patient’s characteristics are described in the Table 1. The hormonal data in
adult patients 1, 2 and 4# is typical of CAIS, with elevated serum LH and
testosterone levels. In patient #3 the diagnosis of CAIS was realized in early age
due her familial history, after the mini puberty. A PAIS individual No surgical
treatment was performed in this paevaluation in our hospital was at 34 years age.
He was submitted to mastectomy follow by orthophaloplasty and orquidopexy. His
laboratorial data was demonstrated at Table 1. This AR SM was the same
previously described (12).

2.2 - Deoxyribonucleic acid (DNA) sequence analysis

Genomic DNA was extracted from the peripheral blood leukocytes of the patients and
used with specific polymerase chain reaction (PCR) primers to amplify all coding exons (exons 1
to 8) and respective exon-intron boundaries of the AR gene (NC 000023.11 /
ENSG00000169083 / ENS00000374690)Bi-directional sequencing of the PCR products was
performed using the same PCR primers and CEQ DTCS sequencing kit (Beckman Coulter,
Fullerton, CA, USA) and an automated capillary DNA sequencer (GenomeLab TM GeXP, Genetic
Analysis System; Beckman Coulter, Fullerton, CA, USA), following the manufacturer’s
instructions. Sequence variations were described in relation to the AR complementary DNA
(cDNA) reference sequence (NM_000044.3), whereby nucleotide c.1 was the A of the ATG-
translation initiation codon. The Human Splicing Finder 3 bioinformatics tool was used to
predict the functional consequences of the mutation.

2.3 - Reverse Transcription PCR (RT-PCR) analysis

Total RNA was extracted from the testes of patient #1 using TRIzol® Reagent (Thermo
Fischer Scientific, Carlsbad, CA, USA. Reverse Transcription was performed according to the
High-Capacity cDNA Reverse Transcription Kit protocol (Thermo Fischer Scientific, Carlsbad,
CA, USA). The AR cDNA was PCR-amplified and sequenced.
 2.4 - X chromosome Linkage analysis

Six microsatellite markers flanking the AR gene spanning approximately 52.5 Mbp
(DXS993, DXS991 and DXS8029 located upstream and DXS986, DXS990 and DXS8031 located
downstream of the AR gene) and intragene CAG and GGC repeats were choose for patients’
haplotype analysis.

2. -Results

Sequencing analysis of the AR gene identified a synonymous variant in the exon 1 (N-
terminal Domain; c.1530C>T – Figure 1) in four patients from two CAIS families. The Human
Splicing Finder predicted a splicing modification showing that c.1530C>T alters an Exonic
Splicing Enhancer with potential splicing site alteration (Table 1). The sequencing of the c.DNA
showed a deletion involving the last 92 nucleotides of exon 1. The c.DNA sequencing shows
that the splicing position changes from c.1616+1 (wild-type) to c.1525 (mutant). Translation of
this altered transcript produced a human AR protein where a premature stop codon was
created 12 amino acids ahead (Figure 2). The result of the linkage analysis showed the same
haplotype for all X-chromosome markers suggesting a founder effect for this mutation.
Another already known deleterious synonymous AR mutation (c.2667C>T; p.S889S) was found
in a PAIS individual.

3 - Discussion

Synonymous mutations are able to change the sequence of a gene without directly altering
the aminoacid sequence of the encoded protein. Due to the absence of protein change,
synonymous mutations is often assumed to be neutral in mutation’s prediction tools and are
completely ignored in many studies[8]. However, synonymous mutations can have an
important role in many biological processes, such as mRNA splicing, folding, mRNA interaction
and protein translation. There are more than 50 diseases associated with synonymous
mutations [9].
Alterations in the splice site are the most common way that synonymous mutations leads
to an aberrant protein [8]. A specific nucleotide sequence is necessary for a correct splicing
process. A unique nucleotide change can be sufficient to change the donor or acceptor splicing
site causing an incorrect alignment of the spliceosome and changing the exon–intron
boundaries [10]. As consequence, shorter or longer transcript protein results from intron
retention, exon skipping or creation of exonic cryptic splice sites [11].
Currently, more than 300 different AR mutations are related to AIS in human AR gene
worldwide database (http://androgendb.mcgill.ca/). [4]. Synonymous mutations affecting the
human AR gene are very rarely. There are two reports in patients with PAIS phenotype. The
p.S889S mutation leads to a partial deletion of the exon 8 and of the 3’-untranslated region as
demonstrated in the AR cDNA from genital skin fibroblasts [12]. The other deleterious
synonymous mutation (p.G796G) was reported in AR cDNA from labioscrotal skin fibroblast
culture from a PAIS individual [13].
In the present study, we present the novel c.1530C>T synonymous AR mutation in four
individuals from 2 unrelated families with CAIS phenotype. The X-chromosome microsatellite
analysis showed the same extra and intragenic markers in all individuals suggesting a founder
effect of this mutation (Figure 3).This mutation results in an abnormal transcript with deletion
of the last 92 nucleotides in the exon 1 (residues 509 – 539). The posterior inclusion of 36
nucleotides of exon 2 causes a frameshift, with a stop codon 12 positions ahead (Figure 2).

4 - Conclusion

In conclusion, we reported the first synonymous mutation in the AR gene related with CAIS
phenotype. This is a recurrent mutation due to a founder effect. In addition, we found other
already described synonymous mutation in a PAIS individual reinforcing the role of
synonymous mutations in AIS etiology.

Financial Support of this research:

This research was supported by FAPESP (São Paulo Research Foundation): Grants 2013/02162-
8 and 2014/50137-5.
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 Figure 1. Electropherogram of AR c.DNA sequencing of the patient #3. The exon 1 is
transcripted stopped at c.1525 position (first arrow from left), causing a 92 nucleotides
deletion. The subsequent insertion of the exon 2 leads to a premature stop codon (second
arrow) 12 positions ahead (p.V509Ffs*12).

Figure 2. Schematic representation of the AR protein: wild type AR (top) and the
p.V509Ffs*12 mutant (below)
Table 1 – Clinical Characteristics of Individuals with AR synonymous mutation (c.1530C>T)

Age of
Individual/ AR Clinical LH FSH Testosterone
Diagnosis
Family mutation Presentation U/L U/L (ng/dL)
(Yr)
Primary
I/1 p.S510S 29 29 8 800
*amenorrhea
Primary
II/1 p.S510S 23 19 5 679
amenorrhea*

Familial
III/1 p.S510S 2 1 1 793**
*history

Primary
I/2 p.S510S 25 40 22 1443
*amenorrhea
Atypical
I/3 p.S889S 34 18 12 1130
genitalia

*typical female external genitalia

**Testosteron results after beta-HCG stimulation test

Table 2. Haplotype analysis: microsatellite markers flanking the AR gene and the number of
GGC and CAG repetitions in individuals with the p.S510S AR mutation

Chromosomal Individuals
Marker Position Region
#1 #2 #3 #4
41,288,430-
DXS993 41,288,735 p11.4 287 287 287 287
55,492,619-
336 336
DXS991 55,492,898 p11.21 336 336
65,722,200-
264 264
DXS8029 65,722,467 q12 264 264
67,544,032-
AR 67,730,619 f q12
CAG
repeats
(exon 1) 21 21 21 21
GGC
repeats
(exon 1) 15 15 15 15
69,317,205-
DXS8031 69,317,469 q13.1 256 256 256 256
80,125,531-
DXS986 80,125,691 q21.1 165 165 165 165