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Mol. Hum. Reprod.
Advance Access published January 17, 2011
Title page Identification of novel SRY mutations and SF1 (NR5A1) changes in patients with pure gonadal dysgenesis and 46, XY karyotype
Downloaded from molehr.oxfordjournals.org at All India Institute of Medical Sciences, New Delhi on February 18, 2011
Authors Preeti Paliwal1* M Sc Anshul Sharma1* M Sc Shweta Birla1 M Sc Alka Kriplani2 MD, FICOG Rajesh Khadgawat3 DM
* Arundhati Sharma1 PhD
Affiliations 1 Laboratory of Cyto-Molecular Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India 2 Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India 3 Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India
The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
2 Corresponding author Dr. Arundhati Sharma Mailing address: Laboratory of Cyto-Molecular Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India
e-mail address : arundhati_sharma@yahoo.com, arundhatisharma1@gmail.com Phone number: +91-11-26593489, +91-9868397547 Fax number: +91-11-26588641, +91-11-26588663
3 Abstract
Primary amenorrhea due to 46, XY disorders of sexual development (DSD) is complex with the involvement of several genes. Karyotyping of such patients is important as they may develop dysgerminoma and molecular analysis is important to identify the underlying mechanism and
explore the cascade of events occurring during sexual development. The present study was undertaken for the genetic analysis in seven patients from five families presenting with primary amenorrhea and diagnosed with pure gonadal dysgenesis. Karyotyping was done and the patients were screened for underlying changes in SRY, DHH, DAX1 (NR0B1) and SF1 (NR5A1) genes, mutations in which are implicated in (DSD). All the patients had 46, XY karyotype and two novel SRY mutations were found. In Family 1 (patient S1.1) a missense mutation c.294G>A was seen, which results in a stop codon at the corresponding amino acid (Trp98X) and in Family 2 (patients S2.1, S2.2, S2.3), a missense mutation c.334G>A (Glu112Leu) was identified in all affected sisters. Both mutations were seen to occur in the conserved high mobility group box (HMG) of SRY gene. One heterozygous change c.427G>A resulting in Glu143Lys in DHH gene in one patient and two heterozygous changes in the intronic region of SF1 (NR5A1) gene (c.244+80G>A+ c.1068-20C>T) in another patient were noted. One individual did not show changes in any of the genes analyzed. These results reiterate the importance of SRY and others like SF1 (NR5A1) and DHH that are involved in the cascade of events leading to sex determination and also their role in sex reversal.
Key Words Swyer syndrome, pure gonadal dysgenesis, SRY, DHH, DAX1 (NR0B1), SF1 (NR5A1), mutation
4 Introduction Sex determination in humans is a complex process involving interplay of several genes, and malfunctioning of any one of them leads to disorders of sex development (DSD). DSD is a term that characterizes incomplete or disordered genital or gonadal development leading to discordance between genetic, gonadal and phenotypic sex (Hughes et al., 2006; Nabhan and
Lee, 2007). Gonadal development is bipotential during the first 7-8 weeks of mammalian embryogenesis and differentiation occurs subsequently to give rise to testes or ovaries in individuals with a 46, XY or 46,XX karyotype, respectively. DSD is a heterogeneous disorder which occurs either as pure or partial gonadal dysgenesis with a frequency of 1 in 3000 births (Camerino et al., 2006). Pure gonadal dysgenesis is characterized by fully developed femaletype external genitalia and normal Mllerian structures. Testes are not seen and only streak gonads are present. Partial gonadal dysgenesis is distinguished by genital ambiguity and dysgenetic and/or streak gonads. Several genes encode for proteins required for gonad development including SRY, SF1 (NR5A1), DAX1 (NR0B1), DHH, WT1, WNT4 and SOX9, mutations in which can lead to gonadal disorders of sex development.
The SRY gene (MIM#480000) located on chromosome Yp11.3 plays a pivotal role in testis
determination. SRY consists of two open reading frames, its key sequence involves a high
mobility group (HMG) box encompassing codons 1382 which shares characteristics with other
DNA binding sequences. Occurrence of majority of mutations in the HMG domain proves the
role of SRY in testes differentiation by DNA bending (Giese et al. 1994) and juxtaposing more
than one testicular determining genes and, hence, facilitating transcription or interaction
between certain gene products. Perturbations of SRY are seen in 1015% of XY gonadal
dysgenesis cases (Berta et al., 1990) associated with both pure (Cameron and Sinclair, 1997;
Margarit et al., 1998; Assumpcao et al., 2002) and partial gonadal dysgenesis (Domenice et
al.,1998). Loss of function SRY mutations are shown to result in the female phenotype in 10-
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15% of cases (Cameron and Sinclair., 1997; Margarit et al., 1998; Assumpcao et al., 2002).
Most of the reported mutations are de novo but cases documenting inheritance of mutations
from the unaffected father have also been reported (Isidor et al., 2009).
Apart from SRY, DSD also result from mutations in genes which are involved in the gonadal
development such as SF1 (NR5A1), DAX1 (NR0B1), DHH, WT1, WNT4, and SOX9. An important gene involved in early gonadal development is SF1 (Steroidogenic factor 1) (MIM #184757), located at 9q33, a member of the nuclear hormone receptor family, also known as NR5A1 and is expressed in the developing urogenital ridge, hypothalamus, anterior pituitary and the adrenal glands (Parker et al., 2002; Ferraz-de-Souza et al., 2006). It encodes a nuclear transcription factor regulating the expression of a number of genes that participate in sexual development. SF1 knockout mice fail to develop adrenal glands and gonads and die at birth (Lala et al. 1999). In humans, heterozygous SF1 mutations in XY individuals lead to adrenal and gonadal failure, (Khler B and Achermann JC, 2010), cryptorchidism (Wada et al., 2005), micropenis (Wada et al., 2006) and male infertility (Bashamboo et al., 2010). DAX1 (MIM#300473, dosage sensitive sex reversal adrenal hypoplasia congenital critical region of the X chromosome gene1), located on chromosome Xp21.3-p21.2, is a member of the nuclear receptor superfamily (NR0B1) and encodes a 470 amino acid nuclear receptor. It has a triple repeat motif in the DNA-binding domain (DBD) at the N terminus and a C terminus ligandbinding domain (LBD) that resembles several nuclear receptors. It is considered to play a role of a global negative transcriptional regulator of steroid hormone production by repressing the expression of multiple genes involved in the steroidogenic pathway. DAX1 mutations are associated with X-linked primary adrenal insufficiency and hypogonadotropic hypogonadism (Achermann et al., 1999). It antagonizes the action of SRY resulting in a sex-reversed phenotype (Domenice et al., 2004 and Swain et al., 1996).
6 Another gene involved is the desert hedgehog (DHH) (MIM# 233420) located on 12q13, is a member of the hedgehog family of signalling proteins which encodes a 396 amino acids protein. DHH is expressed in the sertoli cells and is a positive regulator of the differentiation of steroidproducing leydig cells in the fetal testis. Studies using murine homologue (Dhh) have demonstrated that the differentiation of peritubular myoid cells and the consequent formation of
testis cords are regulated by Dhh (Clark et al., 2000; Pierucci-Alves et al., 2001) substantiating its role in gonadal differentiation. Mutations in DHH have been reported in cases of partial and pure gonadal dysgenesis (PGD) with 46, XY karyotype (Canto et al., 2004). WT1 (MIM#67102), located on chromosome 11p13 is essential for the development of the urogenital tract, where it regulates expression of SRY, and later it plays a pivotal role together with SF1 in the production of Anti Mullerian Hormone in Sertoli cells (Nachtigal et al., 1998; Hossain and Saunders, 2001). Heterozygous missense and splice site WT1 mutations are associated with Denys- Drash syndrome (DDS; MIM#194080) and Frasier syndrome (FS; MIM#136680). WNT4 (MIM#603490) located on chromosome 1p36.23-p35 belongs to a family encoding cystein-rich glycoproteins, which act as extracellular signalling proteins. Female patients carrying heterozygous WNT4 mutation present with mullerian duct abnormalities along with clinical and biological evidence of hyperandrogenism (Philibert et al. 2008). WNT4 increases the expression of DAX1 in Sertoli and Leydig cells and duplication of WNT4 in a chromosomal male has been associated with XY gonadal dysgenesis (Jordan et al., 2001). The transcription factor SOX9 (MIM#114290) located at 17q23 is necessary for cartilage formation and testis differentiation, mutations of which lead to campomelic dysplasia (CD), in which about two-thirds of 46,XY individuals also have a partial or complete form of XY gonadal dysgenesis (Wagner et al., 1994). Recently, compound heterozygosity for two missense mutations in the polycomb gene chromobox homologue 2 (CBX2) (MIM 602770) at 17q25, has been documented as being causative for XY gonadal dysgenesis (Biason-Lauber et al., 2009).
7 Despite the knowledge, of the role of these genes in the cascade of events during sexual development, the cause of gonadal dysgenesis is still unclear and needs thorough investigation. In view of the importance of the SRY, SF1 (NR5A1), DAX1 (NR0B1) and DHH genes in normal gonadal development, the present study was undertaken with an aim to systematically identify the underlying changes in these genes in 7 patients from 5 families who were clinically
diagnosed with pure gonadal dysgenesis and a 46, XY karyotype.
Materials and methods
The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee. Informed consent was taken from all the patients and their family members before being enrolled for the study. Clinical examinations and laboratory investigations were carried, detailed family history was collected and pedigree charts were drawn (Figure 1).
Patients
All the 7 patients presented with the chief complaint of primary amenorrhea and were subjected
to clinical and genetic evaluations. Physical examinations revealed well developed secondary
sexual characters with normal breast development; pelvic ultrasound showed small infantile
uterus and hormone levels were noted to be irregular in all the seven patients. A total of 5 mL of
peripheral blood samples were collected in heparin from all the patients and their unaffected
relatives for cytogenetic and molecular investigations. Karyotyping was done using conventional
GTG banding on cultured blood lymphocytes.
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes using standard protocols. The
SRY, DHH, DAX1 (NR0B1) and SF1 (NR5A1) genes were amplified using custom-synthesized
oligonucleotide primers. PCR amplification of the entire coding region of SRY gene was done
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using the primers as described previously (Rajender et al., 2006) using 70-100 ng DNA, 1.5 mM
MgCl2, 0.25 mM of each dNTPs, 10pM of each primer, and 1.0 units of Taq polymerase
(Invitrogen, Carlsbad, CA, USA) in a 25 L volume mixture in a thermocycler (ABI 9700, [ABI],
Foster City, CA). A positive (normal male 46, XY) and negative (normal XX female) controls
were included with the amplification of SRY gene.
The entire coding region of DHH gene was amplified using four primer pairs as described previously (Umehara et al. 2000) to identify coding region and splice site changes. Amplification was done using 10 ng DNA, 1.5 mM MgCl2, 0.25 mM of each dNTP, 10pM of each primer, and 0.5 units of Taq polymerase (Invitrogen) in a total reaction volume of 25 L.
PCR amplification of the entire coding region and splice sites of DAX1 (NR0B1) gene was done using primer pairs and conditions as described previously (Achermann et al. 1999). Amplification was done using 10 ng DNA, 1.5 mM MgCl2, 0.25 mM of each dNTP, 10pM of each primer and 0.5 units of Taq polymerase (Invitrogen) in a total 25 L reaction volume.
The entire coding region of SF1 (NR5A1) gene and the flanking region of all the exons were amplified using primer pairs as described previously (Loureno et al., 2009). The 25 L reaction for PCR consisted of 1X PyrostartTM Fast PCR Master Mix (2X, Fermentas, Life Sciences), 10 pM of each primer and 40-80 ng of DNA template.
Sequencing All the amplified products were subjected to gel purification using QIAmp gel extraction kits (Qiagen, GmBH, Hilden) and the purified PCR products were sequenced bidirectionally using BigDye Terminator Mix version 3.1 (Applied Biosystems [ABI], Foster City, CA) and were analyzed on an ABI-3100 Genetic Analyzer (ABI). Nucleotide sequences were compared with the published cDNA sequences of SRY (GenBank accession number ENSG00000184895), SF1
9 (NR5A1) (GenBank accession number ENSG00000136931) DAX1 (NR0B1) (GenBank accession number ENSG00000169297) and DHH gene (GenBank accession number ENSG00000139549).
Polyphen 2 and SIFT analysis

In silico analysis using Polyphen 2 and SIFT (Sorting Intolerant From Tolerant) tools was carried out for the novel mutations, to look for the pathogenicity of the identified changes. The SIFT tool (http://blocks.fhcrc.org/sift/SIFT.html) generates multiple sequence alignments of a gene over different species and assess the degree of conservation of the substituted positions over the course of evolution. It gives a value as a score and a score whose value of <0.05 is considered potentially damaging.
Results
Cytogenetic analysis
All the affected patients showed the male karyotype (46, XY) on chromosome analysis using
conventional cytogenetics. Biochemical parameters showed irregular levels of LH, FSH, and
testosterone. (Table 1)
Mutation analysis
SRY gene: Two novel pathogenic mutations were identified in the SRY gene.
Family 1: In patient S1.1 a novel mutation c.294G>A was identified which generated a
premature stop codon at amino acid position 98 (Trp98X).
Family 2: Three affected patients (S2.1, S2.2 and S2.3) showed the mutation c.334G>A
resulting in amino acid substitution Glu112Leu. The father and the brother were not available for
any examination. In silico analysis revealed that the amino acid at position 112 is conserved
among orthologs with a score less than 0.05 and the substitution was not tolerated.
DHH gene: Family 3: Sequencing of DHH gene revealed a heterozygous mutation C.427G>A
resulting in amino acid substitution Glu143Lys in an individual (patient S3.1). SIFT and
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Polyphen2 analysis revealed that the amino acid position was conserved among orthologs
during evolution.
SF1 (NR5A1) gene: Sequence analysis of SF1 (NR5A1) gene did not reveal any changes in the
coding region. One patient revealed two heterozygous changes in two different introns. The
heterozygous changes c.244+80G>A and c.1068-20C>T were seen in intron 3 and intron 4
respectively in the individual from family 5 (S5.1). No other changes were identified in this
patient in the other genes that were screened.
DAX1 gene: Two previously reported polymorphisms (rs2269345 and rs6150) were seen in the
DAX1 gene in two patients (patient S1.1, S3.1). No other pathogenic changes were noted in this
gene in any of the affected patients. The individual from family 4 (S4.1) did not show any
sequence changes in the genes analyzed.
All these sequence changes were seen to occur only in the patients and not in any of the 50
healthy and fertile controls who were also studied.
Discussion
Several studies have shown the importance of SRY in testicular development. Mutations in SRY
are reported in about 10 to 15 percent of 46, XY females with pure or partial gonadal
dysgenesis (Hersmus et al., 2009). It has been reported that the mutations in the open reading
frame of SRY in XY female patients leads to dysgenesis of gonads (Cameron and Sinclair,
1997; Margarit et al., 1998; Assumpcao et al., 2002). In the present study, we identified two
novel mutations in SRY gene both leading to pure gonadal dysgenesis. One mutation occurred
de novo while the other mutation was identified in a familial case with three affected individuals.
Both mutations were identified at highly conserved positions within the HMG box.
The binding activity of SRY lies in the HMG domain (Hersmus et al., 2009) which is composed
of three -helices forming an L-shaped structure. The glutamic acid-to-leucine change at
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position 112 introduces a small non-polar residue for an acidic residue. The substitution may not
alter the structure of the protein but can significantly alter its binding properties and therefore
glutamic acid residue at position 112 is conserved among the orthologs throughout the course
of evolution. In silico analysis showed that substitution of glutamic acid with leucine in not
tolerated and is therefore considered pathogenic. This mutation occurs in the third alpha helix of
HMG domain and thus might disrupt the binding activity of HMG box consequentially affecting
the transcription of other testies determining genes.
The second mutation Trp98X leads to the generation of a premature stop codon and results in a
truncated protein. Tryptophan at position 98 lies in the second alpha helix of HMG box and the
truncated protein so formed may not be able to bind to the DNA resulting in loss of function
mutation. All the affected individuals of family 2 had the Trp98X mutation. The presence of this
change in the other family members, especially the father could not be studied, as they were not
available for analysis.
The activation of DHH transcription occurs immediately after the initiation of SRY expression
and is one of the first indications of male-specific development (Bitgood and McMahon, 1998).
The phenotypic presentation of patients with DHH gene mutations may be pure or mixed
gonadal dysgenesis. Most of the cases with pure gonadal dysgenesis have been reported with
homozygous mutations, while a heterozygous exon 3 mutation, 1086delG was seen in a patient
with mixed gonadal dysgenesis (Canto et al., 2009). In the present study a heterozygous
mutation c.427G>A was identified in one patient with pure gonadal dysgenesis. The
corresponding amino acid glutamic acid at position 143 is evolutionarily conserved and thus
might be important for the functioning of the protein. Based on previous studies it is known that
DHH is a key gene in mammalian gonadal differentiation (Bitgood et al., 1998; Clark et al.,
2000; Canto et al., 2009). The absence of nucleotide changes in this patient in any of the other
genes indicates that DHH mutation may be pathogenic even in the heterozygous state, although
the presence of mutations in other genes involved in sexual development cannot be ruled out.
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Loureno et al. (2009) described novel SF1 (NR5A1) mutations associated with 46,XY DSD and
ovarian insufficiency. All of them were heterozygous, except one identified in 2 Brazilian sibs
who were homozygous for a Asp293Asn mutation: one with 46, XY DSD and the other with
46,XX primary ovarian insufficiency. Recently SF1 (NR5A1) mutations have been shown to
cause male infertility and the authors believe that the mutations may be associated with altered
sex hormone levels and mild abnormalities in cellular structure of the testes (Bashamboo et al.,
2010).
In the present study we did not find any SF1 (NR5A1) coding region changes but a compound
heterozygous change (c.244+80G>A and c.1068-20C>T) was identified in two consecutive
introns. The effect of these changes was not assessed by functional studies but it is believed
that the changes may affect pre-mRNA splicing leading to altered protein structure. This may in
turn affect its crucial role of effective action of male sex hormone on testicular tissue and thus
can lead to the phenotype as seen in the patient. We also identified two synonymous polymorphisms c.114C>T (rs6150) and c.498G>A (rs2269345) in DAX1 gene in two patients. Both are located within the first exon, and neither changes the predicted amino acid. Although these DNA changes have not been examined experimentally for their effect on mRNA or protein expression, repeated observations by different groups that these nucleotide changes do not co-segregate with the disease phenotype confirms that these are true polymorphisms (Phelan et al., 2001).
One individual from family 4 (S4.1) did not show sequence changes in any of the genes
analyzed. This individual may have an underlying molecular defect in the other genes like WT1
WNT4 or SOX9, mutations in which have also been implicated in gonadal dysgenesis (de Santa
Barbara et al., 2000).
In conclusion, we report novel mutations in the SRY gene in two families, a heterozygous
change in DHH gene in one patient and compound heterozygous changes in the introns of SF1
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(NR5A1) in one patient, all of whom were phenotypic females with 46, XY karyotype. Primary
amenorrhea due to 46, XY DSD is complex with several factors involved in the sex
determination pathway including SF1 (NR5A1), DAX1 (NR0B1), DHH, WT1, WNT4, and SOX9.
These genes should be systematically analysed in individuals with primary amenorrhea due to
46, XY DSD as early diagnosis of this condition is essential due to the high risk of developing
dysgerminoma by such patients.
Acknowledgements: The Authors thank the patients and their family members for their participation in the study Authors roles
P.P., S.B. and A.S. carried out the cytogenetic and molecular analysis. P.P drafted the manuscript. A.K and R.K. were instrumental in patient recruitment and detailed clinical workup of the patients and their family members. A.S* planned and supervised the experiments, analyzed the results and wrote the paper.
Funding Statement nil
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37) Rajender S, Rajani V, Gupta NJ, Chakravarty B, Singh L, Thangaraj K. (2006) SRYnegative 46,XX male with normal genitals, complete masculinization and infertility. Mol Hum Reprod, 12,341-346. 38) Umehara F, Tate G, Itoh K, Yamaguchi N, Douchi T, Mitsuya T, Osame M. (2000) A novel mutation of desert hedgehog in a patient with 46,XY partial gonadal dysgenesis accompanied by minifascicular neuropathy. Am J Hum Genet, 67,13021305. 39) Wada Y, Okada M, Fukami M, Sasagawa I, Ogata T 2006 Association of cryptorchidism with Gly146Ala polymorphism in the gene for steroidogenic factor-1. Fertil Steril, 85, 787-790. 40) Wada Y, Okada M, Hasegawa T, Ogata T (2005) Association of severe micropenis with Gly146Ala polymorphism in the gene for steroidogenic factor-1. Endocr J, 52, 445-448. 41) Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E, et al. (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell, 79, 1111-1120.
19 Figure legends
Figure 1: Pedigree of the patients with pure gonadal dysgenesis. Filled boxes represent affected individuals. Open boxes represent unaffected individuals. Arrowhead indicates the proband.
20 Table 1: Details of clinical features, hormone levels and mutation status of the individuals diagnosed with pure gonadal dysgenesis
S. N o.
Pati ent no.
Age /Se x
Physical examination
Endocrine studies LH FSH Testo (mIU/mL) (ng/mL)
Ultrasonography reports
1
2
3
S1.1
S2.1
S2.2
18/ F 22/ F 26/ F
Breast tanner stage III Breast tanner stage IV Breast tanner stage IV
Axillary &pubic hair- Tanner stage IV Breast tanner stage III Axillary &pubic hair- Tanner stage III NA
20.4
6.35
ND
275.7
22.70
ND
2.10
1.75
ND
small infantile uterus with small ovaries small uterus and ovaries Hypogonadism with small uterus and ovaries
Gene with underlyi ng mutation SRY

SRY
SRY
cDNA position of Pathogenic change identified c.294G>A

c.334G>A
c.334G>A
Amino acid position of Pathogenic change identified Trp98X

Glu112Leu
Glu112Leu
S2.3
16/ F
ND
ND
ND
Hypogonadism with small uterus and ovaries
SRY
c.334G>A
Glu112Leu
S3.1
14/ F
14.19
11.19
5.74
6
7
S4.1
S5.1
18/ F 24/ F
Breast tanner stage II Breast tanner stage III
23.7
14.90
50.1
56.7
1.2
0.83
Uterine agenesis with small uterus and two ovoid structures seen in the superficial inguinal region small uterus and ovaries rudimentary uterus and small sized ovaries
DHH
c.427G>A
Glu142Lys
No change in any of the genes tested

NR5A1 c.244+80G> A +c.106820C>T (Changes in introns 3 and 4) -
F-Female, LH-Luteinizing Hormone, FSH-Follicle Stimulating Hormone, Testo- Testosterone, ND-Not Done, NA- details not available Reference range for hormones FSH (mIU/mL) Females Follicular Phase- 3.03 - 8.08 Mid-cycle peak2.55 - 16.69 Leuteal Phase1.38 5.47 Post menopausal- 26.72 133.41 Males0.95-11.95 Testosterone (ng/mL) Females- 0.05 - 0.73, Males- 1.95 - 11.3 LH (mIU/mL) Females Follicular Phase- 2.39 - 6.60 Mid-cycle peak- 9.06 - 74.24 Leuteal Phase- 0.90 - 9.33 Post menopausal -10.39 - 64.75 Males1.14-8.75

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Mol. Hum. Reprod.

Advance Access published January 17, 2011

* Arundhati Sharma1 PhD

diagnosed with pure gonadal dysgenesis and a 46, XY karyotype.

Materials and methods

Polyphen 2 and SIFT analysis

dysgerminoma by such patients.

Funding Statement nil

Pubertal Delay. J Clin Endocrinol Metab, 84, 4497-500.

Pati ent no.

Endocrine studies LH FSH Testo (mIU/mL) (ng/mL)

18/ F 22/ F 26/ F

Gene with underlyi ng mutation SRY

cDNA position of Pathogenic change identified c.294G>A

Amino acid position of Pathogenic change identified Trp98X

Hypogonadism with small uterus and ovaries

Breast tanner stage II Breast tanner stage III

No change in any of the genes tested

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