- Email: [email protected]
A Case Series Of Infants With Increased Vamp7 Gene Dosage At Birth And Virilization Defects.
Journal Pre-proof A Case Series Of Infants With Increased Vamp7 Gene Dosage At Birth And Virilization Defects. Shadai Chávez-López, José de Jesús Lugo-Trampe, Marisol Ibarra-Ramírez, Geovana Calvo-Anguiano, Laura Elia Martínez-de-Villarreal, Luis Daniel CamposAcevedo PII:
S1477-5131(19)30376-6
DOI:
https://doi.org/10.1016/j.jpurol.2019.11.001
Reference:
JPUROL 3317
To appear in:
Journal of Pediatric Urology
Received Date: 18 January 2019 Accepted Date: 4 November 2019
Please cite this article as: Chávez-López S, de Jesús Lugo-Trampe J, Ibarra-Ramírez M, CalvoAnguiano G, Martínez-de-Villarreal LE, Campos-Acevedo LD, A Case Series Of Infants With Increased Vamp7 Gene Dosage At Birth And Virilization Defects., Journal of Pediatric Urology, https:// doi.org/10.1016/j.jpurol.2019.11.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company.
A CASE SERIES OF INFANTS WITH INCREASED VAMP7 GENE DOSAGE AT BIRTH AND VIRILIZATION DEFECTS Shadai Chávez-López, José de Jesús Lugo-Trampe, Marisol Ibarra-Ramírez, Geovana Calvo-Anguiano, Laura Elia Martínez-de-Villarreal, Luis Daniel Campos-Acevedo* Departamento de Genética, Facultad de Medicina y Hospital Universitario José E. González, Universidad Autónoma de Nuevo León (UANL), Av. Gonzalitos s/n cruce con Av. Madero, Col. Mitras Centro, Monterrey, 64460, Nuevo León, Mexico
A CASE SERIES OF INFANTS WITH INCREASED VAMP7 GENE DOSAGE AT BIRTH AND VIRILIZATION DEFECTS SUMMARY Background Genitourinary disorders are the most frequent congenital defects in newborns, however little is known about their etiology. Several studies have been carried out to find genetic risk factors in the development of these malformations. The expression of VAMP7 is found in testes, epididymis, seminal vesicles, prostatic tissues, penis and urethra. Alterations in gene dose of VAMP7 were recently reported in a subset of male patients initially identified clinically by the presence of congenital genitourinary disorders. In 2016, we developed a diagnostic algorithm for early detection of sex chromosome aneuploidies by quantifying the SHOX, VAMP7 and SRY gene dose in newborns by qPCR using dried blood spot (DBS) samples. Objective Correlate the increased gene dose of VAMP7, obtained by qPCR using dried blood spot (DBS), with genitourinary congenital defects attributable to disorders in virilization and verify the increased gene dose by microarrays. Study design Samples that only presented increased VAMP7 gene dosage were selected from a previously analyzed group of 5,088 males in which the early detection of sex chromosomes aneuploidies was performed. Eight males were found with an increased gene dose of VAMP7 (RQ> 1.3) and were called in for a complete clinical evaluation aimed at the identification of genitourinary anomalies, qPCR and microarrays. Results Eight males from 5,088 samples, were identified with increased VAMP7 gene dosage of which six patients were clinically evaluated, of which 50% were identified with
1
alterations in genital development (bilateral cryptorchidism, unilateral cryptorchidism and glandular hypospadias) and speech delay, while the rest presented different types of atopy.
Patient #
Clinical manifestations
VAMP7 gene dosage by qPCR (DBS)
VAMP7 gene dosage by qPCR (Blood)
Genomic Position
Increase in copy number (kb) by aCGH
Involved genes
1
BIC, LDD
1.4
1
-
No increase
None
2
RUIC, GH, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
3
BIC, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
4
ARh/AD
1.79
1.68
ChrX:154931044155185493
254.449
SPRY3, VAMP7
5
AD/AS
1.39
0.99
-
No increase
None
6
ACMP
1.45
1.94
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
Correlation of clinical data with results of qPCR (RQ>1.3) and aCGH. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism.GH: Glandular hypospadias BPC: bilateral prescrotal cryptorchidism. LDD: Language development delay. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma. ACMP: Allergy to cow’s milk protein.
Discussion Tannour-Louet et al. postulated on 2014, that the duplication of the Xq28 region, specifically of VAMP7, plays a role in the human masculinization disorders of the urogenital tract. The study was based on aCGH results performed to 116 males with disorders of sexual differentiation. In the present study, the patients were initially selected due to an increased gene dose of VAMP7 detected by qPCR, then the clinical evaluation and the aCGH were performed, inverse to what was reported previously but with similar percentages between both studies.
2
Conclusion In this work, we report cases of cryptorchidism, hypospadias, language delay and atopy in male preschoolers initially identified because they have an increased gene dose of VAMP7. INTRODUCTION Cryptorchidism, hypospadias and micropenis are the most common alterations of male sexual differentiation, with a frequency of 3/100, 2.6/10,000 and 1.5/10,000 full-term live births in Mexico, respectively [1-6]. Regarding cryptorchidism, the testes can descend spontaneously into the scrotum during the first year of life, decreasing the prevalence from 3% to 0.8 - 1.2% [7,8]. Sexual differentiation of male external genitalia is under the influence of androgens, whose signaling is mediated through the nuclear androgen receptor (AR) [9]. During embryogenesis, androgens are critical for the proper virilization of external genitalia and migration of testicles through the inguinal canal into scrotum [9,10]. The critical time of genital development known as "masculinization programming window" (MPW) corresponds to the embryonic stage during which the genital tubercle becomes sensitive to androgen signals between 8 and 12 weeks of human gestation [7, 9, 11, 12]. Urethral plate canalization and subsequent incorporation of the urethra into the glans occur during the MPW [1,9]. Interruption of AR signaling will cause a developmental disorder, and the time when it occurs will influence its specific phenotype. Differentiation of the male reproductive system depends on the balance between the action of androgens and estrogens rather than on the circulating concentrations of these hormones. Any change in such homeostasis can alter the internal environment needed for the normal
3
development and function of male genital tract and result in a wide range of genitourinary disorders [9,13]. The VAMP7 gene, formerly known as SYBL1, is located in the second pseudoautosomal region (PAR) of sex chromosomes (Xq28, Yq12). This gene encodes a transmembrane protein, member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family
[14, 15], which is involved in several important cellular
functions, including phagocytosis, mitosis, cell migration, as well as in the repair and growth of plasma membrane through the fusion of vesicles derived from the Golgi apparatus, late endosomes and lysosomes. Additionally, the participation of VAMP7 in neurite growth of developing neurons has been identified through a pathway related to secretory lysosomes in non-neuronal cells; and it is also important for the exocytosis of synaptic vesicles in mature neurons [14]. VAMP7 expression is found in testes, epididymis, seminal vesicles, prostatic tissues, penis and urethra. An increase in VAMP7 protein could affect the function of AR, by altering their subcellular localization at the time of ligand-dependent stimulation [16]. This overexpression stimulates the transcriptional activity of estrogen alpha receptor gene (ESR1), increasing the protein content of ESR1 by stimulating the ligand and upregulating the expression estrogen-responsive genes. This study strongly suggests the possible participation of VAMP7 as cause of hypospadias development. [11, 13, 16, 17] Previously, we developed a diagnostic algorithm for early detection of sex chromosome aneuploidies by quantifying the SHOX, VAMP7 and SRY gene dose in newborns (18, unpublished data). This work shows a group of patients that presented only increased
4
VAMP7 gene dosage and their analysis of genitourinary development disorders, correlating the molecular and the clinical findings. MATERIALS AND METHODS Study design The design of the present analysis is observational, descriptive, and transversal. The project was approved by the Research Ethics Committee of the School of Medicine and University Hospital of the Autonomous University of Nuevo Leon (GN17-00001).
Sample Selection Samples that presented only increased VAMP7 gene dosage were selected from a group analyzed previously in which the early detection of sex chromosomes aneuploidies was performed by quantifying the SHOX, VAMP7 and SRY gene dose by qPCR using dried blood spot (DBS) samples. The sample donors were called in for a complete clinical evaluation performed by a certified geneticist aimed at the identification of genitourinary anomalies, as well as for taking blood samples for qPCR and microarrays.
Determination of the gene dose by qPCR The copy number assays of SHOX, VAMP7 and SRY were performed using TaqMan Probes following the manufacturer's instructions (Applied Biosystems, Foster City, CA). The genes were labeled with the 6-FAM fluorophore, while the reference gene (RNaseP) was labeled with VIC (Catalog No. 4403328). Real-time PCR was performed using a StepOneplus thermal cycler (Applied Biosystems, Foster City, CA). PCR was carried out with 4 µL of TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA), 0.5 µl of each
5
TaqMan probe (20x), 0.5 µL of the reference gene probe (20x), and 5 µL to 10 ng/µL of genomic DNA to a final volume of 10 µL in a 96 well MicroAmp Fast Optical reaction plate. The amplification was carried out under the following conditions: 95°C for 10 minutes followed by 45 cycles at 95°C for 20s and 60°C for 1 min. For normalization, the internal RNaseP gene control was amplified in parallel in the same well with the target gene under identical conditions and cycling. Relative quantitation (RQ) values were obtained using the delta-delta CT (∆∆CT) method. Values > 1.3 were considered as gain and < 0.7 as loss. The correlation coefficient between the CT and the template DNA concentration for each TaqMan assay was calculated by regression analysis and amplification efficiency using the following formula: Efficiency = -1 + 10 (-1/slope).
Array Comparative Genomic Hybridization (aCGH) The aCGH was performed using the human CGH microarrays SurePrint G3 4 × 180 K (Agilent Technologies) following the manufacturer's instructions. For this, 250 ng of genomic DNA from each sample and human reference controls corresponding to the sex of the samples were fragmented by enzymatic reaction. Test-sample and reference DNAs were labeled fluorescently with Cy3-dUTP and Cy5-dUTP respectively using the SureTag DNA Complete Labeling kit (Agilent Technologies). Unincorporated nucleotides and dyes were removed with purification columns (Agilent). Labeled DNA mixed with human Cot-1 DNA were added to the matrix slides. Hybridizations were performed in a hybridization oven at 67°C at 20 rpm for 24 hours. The arrays were scanned on the Agilent SureScan Dx Microarray Scanner
(Agilent
6
Technologies).
The
Agilent
CytoGenomics software (Agilent Technologies) was used to visualize, detect, and analyze the chromosomal changes within the X and Y chromosomes. RESULTS In a total of 10,033 DBS samples (4945 females and 5088 males), 8 males were found with an increased gene dose of VAMP7 (RQ> 1.3) (Table 1). Only 6 patients decided to continue in the current study. RQ Patient #
SHOX
VAMP7
SRY
1
1.17
1.4
0.7
2
1.11
1.44
1.04
3
1.07
1.44
0.91
4
1.05
1.79
1.04
5
0.93
1.39
0.99
6
1.09
1.38
1.06
7
0.93
1.45
0.98
8
0.8
1.35
1.07
Table 1. DBS qPCR results. VAMP7 gene dose above RQ 1.3 .
Clinical evaluation Patients were evaluated from June 2017 to February 2018. The clinical evaluation included: clinical history, pedigree, and physical examination with somatometry and specific search for virilization disorders (cryptorchidism and hypospadias). Table 2 summarizes the results. The detail of the cases is as follows: Case 1. A 3 years old male, product of the third pregnancy of apparently healthy not consanguineous parents. Showing a delay in language development, beginning monosyllables and sentences at 2 years; motor development is normal. 7
Physical
measurements were found within normal parameters. Bilateral inguinal cryptorchidism and 4 cm long penis (p10) were identified. Case 2. A 2 years 3 months old male, product of the second pregnancy of apparently healthy not consanguineous parents. Presence of delayed language development, beginning to speak at 7 months. Dentition was delayed, starting at 14 months. Motor development was referred as normal. Physical measurements were found within normal parameters. Right unilateral inguinal cryptorchidism, 3 cm long penis (p10) and glandular hypospadias were identified. Case 3. A 3 years 1-month old male, product of the third pregnancy of apparently healthy not consanguineous parents. He has delayed language development, still without forming short sentences; motor development was normal. Physical examination revealed weight of 11.5 Kg (-2DS), height of 87cm (-2.5DS) and OFC 46cm (-3.7 DS). Hypoplastic scrotal sac, bilateral prescrotal cryptorchidism and 4 cm long penis (p10) were identified. Case 4. A 3 years 5 months old male, product of the second pregnancy of apparently healthy not consanguineous parents. The development milestones were normal. He presents allergic rhinitis and atopic dermatitis. Physical measurements including penile length was found within normal parameters. Case 5. A 3 years old male, product of the sixth pregnancy of apparently healthy not consanguineous parents. At the present time with normal development milestones. He has atopic dermatitis and diagnosis of asthma. Physical examination was found within normal.
8
Case 6. A 3 years 8 months old male, product of the second pregnancy of apparently healthy not consanguineous parents, with normal development milestones. Allergic to cow's milk protein. Physical examination was found within normal.
Case #
Age (y,m)
Weight (kg)
Height (cm)
OFC (cm)
Penis length (cm)
Language development
Cryptorchidism
Hypospadias
Allergic reactions
1
3
16
92
48.7
4
Delayed
BIC
No
No
2
2,3
11
87.5
47
3
Delayed
RUIC
Glandular
No
3
3,1
11.5
87
46
4
Delayed
BPC
No
No
4
3,5
14.5
93
48.5
4.5
NL
No
No
ARh/AD
5
3
14
94
49.5
4
NL
No
No
AD/AS
3,8 15 100 51 4 NL No No ACMP 6 Table 2.Clinical data of the patients. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism. BPC: bilateral prescrotal cryptorchidism. NL: Normal. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma, ACMP: Allergy to cow’s milk proteins.
qPCR After the clinical evaluation, the quantification by PCR of VAMP7 in peripheral blood of the 6 participants was performed. In two cases (Case 1 with bilateral inguinal cryptorchidism and language delay and case 5 with atopic dermatitis and asthma) the qPCR result was normal.
aCGH Table 3 shows the correlation between qPCR analysis, microarray analysis, and clinical data of the patients. Patient #
Clinical manifestations
VAMP7 gene dosage by qPCR (DBS)
VAMP7 gene dosage by qPCR (Blood)
Genomic Position
Increase in copy number (kb) by aCGH
Involved genes
1
BIC, LDD
1.4
1
-
No increase
None
9
2
RUIC, GH, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
3
BIC, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
4
ARh/AD
1.79
1.68
ChrX:154931044155185493
254.449
SPRY3, VAMP7
5
AD/AS
1.39
0.99
-
No increase
None
6
ACMP
1.45
1.94
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
Table 3. Correlation of clinical data with results of qPCR (RQ>1.3) and aCGH. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism.GH: Glandular hypospadias BPC: bilateral prescrotal cryptorchidism. LDD: Language development delay. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma. ACMP: Allergy to cow’s milk protein.
DISCUSSION Six patients with increased VAMP7 gene dosage, identified with DBS qPCR, were clinically evaluated, of which 50% were identified with virilization defects (bilateral cryptorchidism, unilateral cryptorchidism and glandular hypospadias) and speech delay. The etiology of congenital malformations of the genitourinary tract has not yet been elucidated. It is thought it can be due to either monogenic or multifactorial causes. Mutation in genes such as: WT1, SF1, BMP4, BMP7, HOXA4, HOXB6, FGF8, FGFR2, AR, HSD3B2, SRD5A2, ATF3, MAMLD1, MID1 and BNC2 may be responsible for some genitourinary defects. [19] Recent reports have shown an association between the increase of VAMP7 gene dosage and virilization disorders. aCGH analysis of 116 cases of males with disorders of sexual differentiation including alterations in testicular descent and defects in penis morphology identified 2 subjects (1.7%) with an identical gain in the number of copies in the Xq28 region, specifically VAMP7. Based on these observations, it was postulated that the duplication of the Xq28 region, specifically of VAMP7, plays a role 10
in the human masculinization disorders of the urogenital tract [16]. In another cohort, 180 individuals with similar clinical manifestations were evaluated and 2 subjects (1.1%) were reported with gain in the number of copies in VAMP7, this time using the qPCR technique. In both studies, the selection of subjects started from the clinical manifestations, specifically the presence of virilization disorders such as cryptorchidism and hypospadias, to later analyze the VAMP7 gene dosage [16, 20]. Here, unlike the previously mentioned work, the cases were initially due to an increased gene dose of VAMP7 in a study for the early detection of sex chromosome aneuploidies using DBS qPCR. This analysis showed that 8/10,033 individuals had an increased VAMP7 gene dosage. Six patients were clinically evaluated, of which three (50%) were identified with alterations in genital development (2 bilateral cryptorchidism and 1 unilateral cryptorchidism and glandular hypospadias) and speech delay, while the rest presented different types of atopy. Clinical evaluation of the other two cases was not possible because the parents decided not to continue in the study. Considering the previous studies, in which an association of increased gene dosage of VAMP7 with alterations in the genitourinary development in about 1.3% of the analyzed individuals was found; our study, through the search of cases from increased gene dose achieved the identification of this association, supporting what was reported by other authors. [17]. It is interesting that the patients identified with alterations in the expression of VAMP7 are similar to that reported by Tannour-Louet et. al. In relation to the expected incidence for virilization disorders we found 1.97% for cryptorchidism and 1.96% for hypospadias. On the other hand, three of the cases that did not present genitourinary malformations coincided in presenting different types of atopy. 11
After performing a second qPCR in peripheral blood and aCGH, the increase in the VAMP7 gene dose was confirmed in 4 of the 6 cases, excluding two which initially were identified as high-dose gene by qPCR in DBS, considering these results as false positives. It has been suggested that the role of the VAMP7 protein in the neuritic growth of developing neurons could explain the language alterations exhibited in our patients Although there are no studies that clearly demonstrate their relationship with genitourinary development, the results obtained in our study, as well as those previously reported, strongly suggest a role for this gene in the development of these defects [14]. In Xq28-Xqter region identified by the aCGH were also found the IL9R and SPRY3 genes, which could be correlated with the clinical manifestations identified in the patients. IL9R was the first gene identified in the pseudoautosomal region of chromosomes Xq and Yq. It has 11 exons and encodes a ligand-specific alpha chain for the heterodimeric receptor. This receptor is expressed by a variety of hematopoietic cells such as T cells, mast cells, dendritic cells, epithelial cells, eosinophils, neutrophils and macrophages; being able to have biological significance in the development of asthma, as higher IL9R expression in the airways is noted in asthmatic patients. IL9R belongs to the group of genes with a clear potential role in allergic diseases. Also, the overexpression of IL9R in inflammatory bowel disease suggests a functional role in the perpetuation of gut inflammation. [21 - 23] The SPRY3 gene is made up of 2 exons and encodes for a member of the SPROUTY proteins (SPRY) family, which plays a role in the morphogenesis of the neuronal branching and it is suggested that it can inhibit the growth factor signaling in the neuronal development and the branching of the ganglion cells during postnatal development and in the adult life. [20,
12
24]. Future research in this area may shed additional light on the pathogenesis of language developmental delay. One strength found in this study was the approach to select the sample which was from the laboratory data (molecular biology) to the clinical features, inverse to what was reported by Tannour-Louet et. al (clinical features – laboratory), with similar percentages between both studies. CONCLUSION After clinical and molecular evaluation of six infants, we found the increased VAMP7 gene dosage could be responsible for one of many causes of virilization disorders, and probably language delay and atopies.
Conflict of Interest: None
REFERENCES
1. Urh K, Kunej T. Molecular mechanisms of cryptorchidism development: update of the database, disease, comorbidity, and initiative for standardization of reporting in scientific literature. Andrology. 2016, 1-9 2. Diagnóstico y tratamiento del testículo no descendido. México: Secretaría de Salud; 2014. 3. Diagnóstico y Tratamiento de Hipospadias. México. Secretaría de Salud, 2010
13
4. Pantoja BM, Kofi BE, Rio HG, Sánchez RO, Camué MR. Caracterización clinicoepidemiológica y terapéutica de pacientes con hipospadias. MEDISAN 2009; 13 (6) 5. Leung A.K., Robson W.L. Hypospadias: an update. Asian J Androl 2007 Jan; 9: 1622 6. Sagodi L, Kiss A, Kiss-Toth E, Barkai L: Prevalence and possible causes of hypospadias (in Hungarian). Orv Hetil 2014; 155: 978–985 7. Blanco S., Gottlieb S., Grinspon R., Rey R. Criptorquidia: desde la embriología al tratamiento MÉD UIS. 2015;28(3):371-80 8. Tekgül S. ,et. al. Guía Clínica sobre Urología Pediátrica. European Society for Pediatric Urology (2009),14: 991,1002. 9. Matsushita S. et. al. Regulation of masculinization: androgen signalling for external genitalia development. Nat. Rev. Urology (2018) 10. Wilhelm D, Koopman P. The makings of maleness: towards an integrated view of male sexual development. Nat Rev Genet. 2006; 7:620–631. 11. Blaschko SD, Cunha GR, Baskin LS: Molecular mechanisms of external genitalia development. Differentiation 2012; 84: 261–268 12. Suzuki, H., Suzuki, K. & Yamada, G. Systematic analyses of murine masculinization processes based on genital sex differentiation parameters. Dev. Growth Differ. 57, 639–647 (2015). 13. Watanabe M, Yoshida R, Ueoka K, Aoki K, Sasagawa I, et al: Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities. Hum Reprod 2007 22: 1279–1284.
14
14. D’Esposito et. al. A synaptobrevin-like gene in the Xq28 pseudoautosomal region undergoes X inactivation. Nat Genet.1996; 13: 227-229. 15. Matarazzo et. al. Allelic inactivation of the pseudoautosomal gene SYBL1 is controlled by epigenetic mechanisms common to the X and Y chromosomes. Human Molecular Genetics, 2002, Vol. 11, No. 25 3191–3198 16. Tannour-Louet et. al. Increased gene copy number of VAMP7 disrupts human male urogenital development through altered estrogen action. Nat. Med. (2014).20, 715– 724 17. Wang Z, Liu BC, Lin GT, Lin CS, Lue TF, et al: Up-regulation of estrogen responsive genes in hypospadias: microarray analysis. J Urol 2007 177: 1939–1946. 18. Campos-Acevedo et. al. Dosage of Sex Chromosomal Genes in Blood Deposited on Filter Paper for Neonatal Screening of Sex Chromosome Aneuploidy. Genetic Testing and Molecular Biomarkers. 2016; 12:1-5 19. van der Zanden LF, van Rooij IA, Feitz WF, Franke B, Knoers NV, Roeleveld N Aetiology of hypospadias: a systematic review of genes and environment. Hum Reprod Update. 2012 May-Jun;18(3):260-83. 20. De Bonis, M. L., et. al. Maintenance of X- and Y-inactivation of the pseudoautosomal (PAR2) gene SPRY3 is independent from DNA methylation and associated to multiple layers of epigenetic modifications. Hum. Molec. Genet. 15: 1123-1132, 2006. 21. Fatahi F., et. al. Assessment of the Effects of IL9, IL9R, IL17A, and IL17F Gene Polymorphisms on Women with Allergic Rhinitis in Shahrekord, Iran Ann Med Health Sci Res. 2016 Jul-Aug; 6(4): 216–223.
15
22. Kauppi P., Laitinen T. ,Ollikainen V., Mannila H., Laitinen, L.A., Kere J. The IL9R region contribution in asthma is supported by genetic association in an isolated population. European Journal of Human Genetics (2000) 8, 788–792 23. M.F. Neurath, S. Finotto, IL-9 signaling as key driver of chronic inflammation in mucosal immunity, Cytokine Growth Factor Rev (2016) 24. Ning Z. et. al. Regulation of SPRY3 by X chromosome and PAR2-linked promoters in an autism susceptibility region. Hum Mol Genet. 2015 Sep 15;24(18):5126-41
16
S1477-5131(19)30376-6
DOI:
https://doi.org/10.1016/j.jpurol.2019.11.001
Reference:
JPUROL 3317
To appear in:
Journal of Pediatric Urology
Received Date: 18 January 2019 Accepted Date: 4 November 2019
Please cite this article as: Chávez-López S, de Jesús Lugo-Trampe J, Ibarra-Ramírez M, CalvoAnguiano G, Martínez-de-Villarreal LE, Campos-Acevedo LD, A Case Series Of Infants With Increased Vamp7 Gene Dosage At Birth And Virilization Defects., Journal of Pediatric Urology, https:// doi.org/10.1016/j.jpurol.2019.11.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company.
A CASE SERIES OF INFANTS WITH INCREASED VAMP7 GENE DOSAGE AT BIRTH AND VIRILIZATION DEFECTS Shadai Chávez-López, José de Jesús Lugo-Trampe, Marisol Ibarra-Ramírez, Geovana Calvo-Anguiano, Laura Elia Martínez-de-Villarreal, Luis Daniel Campos-Acevedo* Departamento de Genética, Facultad de Medicina y Hospital Universitario José E. González, Universidad Autónoma de Nuevo León (UANL), Av. Gonzalitos s/n cruce con Av. Madero, Col. Mitras Centro, Monterrey, 64460, Nuevo León, Mexico
A CASE SERIES OF INFANTS WITH INCREASED VAMP7 GENE DOSAGE AT BIRTH AND VIRILIZATION DEFECTS SUMMARY Background Genitourinary disorders are the most frequent congenital defects in newborns, however little is known about their etiology. Several studies have been carried out to find genetic risk factors in the development of these malformations. The expression of VAMP7 is found in testes, epididymis, seminal vesicles, prostatic tissues, penis and urethra. Alterations in gene dose of VAMP7 were recently reported in a subset of male patients initially identified clinically by the presence of congenital genitourinary disorders. In 2016, we developed a diagnostic algorithm for early detection of sex chromosome aneuploidies by quantifying the SHOX, VAMP7 and SRY gene dose in newborns by qPCR using dried blood spot (DBS) samples. Objective Correlate the increased gene dose of VAMP7, obtained by qPCR using dried blood spot (DBS), with genitourinary congenital defects attributable to disorders in virilization and verify the increased gene dose by microarrays. Study design Samples that only presented increased VAMP7 gene dosage were selected from a previously analyzed group of 5,088 males in which the early detection of sex chromosomes aneuploidies was performed. Eight males were found with an increased gene dose of VAMP7 (RQ> 1.3) and were called in for a complete clinical evaluation aimed at the identification of genitourinary anomalies, qPCR and microarrays. Results Eight males from 5,088 samples, were identified with increased VAMP7 gene dosage of which six patients were clinically evaluated, of which 50% were identified with
1
alterations in genital development (bilateral cryptorchidism, unilateral cryptorchidism and glandular hypospadias) and speech delay, while the rest presented different types of atopy.
Patient #
Clinical manifestations
VAMP7 gene dosage by qPCR (DBS)
VAMP7 gene dosage by qPCR (Blood)
Genomic Position
Increase in copy number (kb) by aCGH
Involved genes
1
BIC, LDD
1.4
1
-
No increase
None
2
RUIC, GH, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
3
BIC, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
4
ARh/AD
1.79
1.68
ChrX:154931044155185493
254.449
SPRY3, VAMP7
5
AD/AS
1.39
0.99
-
No increase
None
6
ACMP
1.45
1.94
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
Correlation of clinical data with results of qPCR (RQ>1.3) and aCGH. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism.GH: Glandular hypospadias BPC: bilateral prescrotal cryptorchidism. LDD: Language development delay. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma. ACMP: Allergy to cow’s milk protein.
Discussion Tannour-Louet et al. postulated on 2014, that the duplication of the Xq28 region, specifically of VAMP7, plays a role in the human masculinization disorders of the urogenital tract. The study was based on aCGH results performed to 116 males with disorders of sexual differentiation. In the present study, the patients were initially selected due to an increased gene dose of VAMP7 detected by qPCR, then the clinical evaluation and the aCGH were performed, inverse to what was reported previously but with similar percentages between both studies.
2
Conclusion In this work, we report cases of cryptorchidism, hypospadias, language delay and atopy in male preschoolers initially identified because they have an increased gene dose of VAMP7. INTRODUCTION Cryptorchidism, hypospadias and micropenis are the most common alterations of male sexual differentiation, with a frequency of 3/100, 2.6/10,000 and 1.5/10,000 full-term live births in Mexico, respectively [1-6]. Regarding cryptorchidism, the testes can descend spontaneously into the scrotum during the first year of life, decreasing the prevalence from 3% to 0.8 - 1.2% [7,8]. Sexual differentiation of male external genitalia is under the influence of androgens, whose signaling is mediated through the nuclear androgen receptor (AR) [9]. During embryogenesis, androgens are critical for the proper virilization of external genitalia and migration of testicles through the inguinal canal into scrotum [9,10]. The critical time of genital development known as "masculinization programming window" (MPW) corresponds to the embryonic stage during which the genital tubercle becomes sensitive to androgen signals between 8 and 12 weeks of human gestation [7, 9, 11, 12]. Urethral plate canalization and subsequent incorporation of the urethra into the glans occur during the MPW [1,9]. Interruption of AR signaling will cause a developmental disorder, and the time when it occurs will influence its specific phenotype. Differentiation of the male reproductive system depends on the balance between the action of androgens and estrogens rather than on the circulating concentrations of these hormones. Any change in such homeostasis can alter the internal environment needed for the normal
3
development and function of male genital tract and result in a wide range of genitourinary disorders [9,13]. The VAMP7 gene, formerly known as SYBL1, is located in the second pseudoautosomal region (PAR) of sex chromosomes (Xq28, Yq12). This gene encodes a transmembrane protein, member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family
[14, 15], which is involved in several important cellular
functions, including phagocytosis, mitosis, cell migration, as well as in the repair and growth of plasma membrane through the fusion of vesicles derived from the Golgi apparatus, late endosomes and lysosomes. Additionally, the participation of VAMP7 in neurite growth of developing neurons has been identified through a pathway related to secretory lysosomes in non-neuronal cells; and it is also important for the exocytosis of synaptic vesicles in mature neurons [14]. VAMP7 expression is found in testes, epididymis, seminal vesicles, prostatic tissues, penis and urethra. An increase in VAMP7 protein could affect the function of AR, by altering their subcellular localization at the time of ligand-dependent stimulation [16]. This overexpression stimulates the transcriptional activity of estrogen alpha receptor gene (ESR1), increasing the protein content of ESR1 by stimulating the ligand and upregulating the expression estrogen-responsive genes. This study strongly suggests the possible participation of VAMP7 as cause of hypospadias development. [11, 13, 16, 17] Previously, we developed a diagnostic algorithm for early detection of sex chromosome aneuploidies by quantifying the SHOX, VAMP7 and SRY gene dose in newborns (18, unpublished data). This work shows a group of patients that presented only increased
4
VAMP7 gene dosage and their analysis of genitourinary development disorders, correlating the molecular and the clinical findings. MATERIALS AND METHODS Study design The design of the present analysis is observational, descriptive, and transversal. The project was approved by the Research Ethics Committee of the School of Medicine and University Hospital of the Autonomous University of Nuevo Leon (GN17-00001).
Sample Selection Samples that presented only increased VAMP7 gene dosage were selected from a group analyzed previously in which the early detection of sex chromosomes aneuploidies was performed by quantifying the SHOX, VAMP7 and SRY gene dose by qPCR using dried blood spot (DBS) samples. The sample donors were called in for a complete clinical evaluation performed by a certified geneticist aimed at the identification of genitourinary anomalies, as well as for taking blood samples for qPCR and microarrays.
Determination of the gene dose by qPCR The copy number assays of SHOX, VAMP7 and SRY were performed using TaqMan Probes following the manufacturer's instructions (Applied Biosystems, Foster City, CA). The genes were labeled with the 6-FAM fluorophore, while the reference gene (RNaseP) was labeled with VIC (Catalog No. 4403328). Real-time PCR was performed using a StepOneplus thermal cycler (Applied Biosystems, Foster City, CA). PCR was carried out with 4 µL of TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA), 0.5 µl of each
5
TaqMan probe (20x), 0.5 µL of the reference gene probe (20x), and 5 µL to 10 ng/µL of genomic DNA to a final volume of 10 µL in a 96 well MicroAmp Fast Optical reaction plate. The amplification was carried out under the following conditions: 95°C for 10 minutes followed by 45 cycles at 95°C for 20s and 60°C for 1 min. For normalization, the internal RNaseP gene control was amplified in parallel in the same well with the target gene under identical conditions and cycling. Relative quantitation (RQ) values were obtained using the delta-delta CT (∆∆CT) method. Values > 1.3 were considered as gain and < 0.7 as loss. The correlation coefficient between the CT and the template DNA concentration for each TaqMan assay was calculated by regression analysis and amplification efficiency using the following formula: Efficiency = -1 + 10 (-1/slope).
Array Comparative Genomic Hybridization (aCGH) The aCGH was performed using the human CGH microarrays SurePrint G3 4 × 180 K (Agilent Technologies) following the manufacturer's instructions. For this, 250 ng of genomic DNA from each sample and human reference controls corresponding to the sex of the samples were fragmented by enzymatic reaction. Test-sample and reference DNAs were labeled fluorescently with Cy3-dUTP and Cy5-dUTP respectively using the SureTag DNA Complete Labeling kit (Agilent Technologies). Unincorporated nucleotides and dyes were removed with purification columns (Agilent). Labeled DNA mixed with human Cot-1 DNA were added to the matrix slides. Hybridizations were performed in a hybridization oven at 67°C at 20 rpm for 24 hours. The arrays were scanned on the Agilent SureScan Dx Microarray Scanner
(Agilent
6
Technologies).
The
Agilent
CytoGenomics software (Agilent Technologies) was used to visualize, detect, and analyze the chromosomal changes within the X and Y chromosomes. RESULTS In a total of 10,033 DBS samples (4945 females and 5088 males), 8 males were found with an increased gene dose of VAMP7 (RQ> 1.3) (Table 1). Only 6 patients decided to continue in the current study. RQ Patient #
SHOX
VAMP7
SRY
1
1.17
1.4
0.7
2
1.11
1.44
1.04
3
1.07
1.44
0.91
4
1.05
1.79
1.04
5
0.93
1.39
0.99
6
1.09
1.38
1.06
7
0.93
1.45
0.98
8
0.8
1.35
1.07
Table 1. DBS qPCR results. VAMP7 gene dose above RQ 1.3 .
Clinical evaluation Patients were evaluated from June 2017 to February 2018. The clinical evaluation included: clinical history, pedigree, and physical examination with somatometry and specific search for virilization disorders (cryptorchidism and hypospadias). Table 2 summarizes the results. The detail of the cases is as follows: Case 1. A 3 years old male, product of the third pregnancy of apparently healthy not consanguineous parents. Showing a delay in language development, beginning monosyllables and sentences at 2 years; motor development is normal. 7
Physical
measurements were found within normal parameters. Bilateral inguinal cryptorchidism and 4 cm long penis (p10) were identified. Case 2. A 2 years 3 months old male, product of the second pregnancy of apparently healthy not consanguineous parents. Presence of delayed language development, beginning to speak at 7 months. Dentition was delayed, starting at 14 months. Motor development was referred as normal. Physical measurements were found within normal parameters. Right unilateral inguinal cryptorchidism, 3 cm long penis (p10) and glandular hypospadias were identified. Case 3. A 3 years 1-month old male, product of the third pregnancy of apparently healthy not consanguineous parents. He has delayed language development, still without forming short sentences; motor development was normal. Physical examination revealed weight of 11.5 Kg (-2DS), height of 87cm (-2.5DS) and OFC 46cm (-3.7 DS). Hypoplastic scrotal sac, bilateral prescrotal cryptorchidism and 4 cm long penis (p10) were identified. Case 4. A 3 years 5 months old male, product of the second pregnancy of apparently healthy not consanguineous parents. The development milestones were normal. He presents allergic rhinitis and atopic dermatitis. Physical measurements including penile length was found within normal parameters. Case 5. A 3 years old male, product of the sixth pregnancy of apparently healthy not consanguineous parents. At the present time with normal development milestones. He has atopic dermatitis and diagnosis of asthma. Physical examination was found within normal.
8
Case 6. A 3 years 8 months old male, product of the second pregnancy of apparently healthy not consanguineous parents, with normal development milestones. Allergic to cow's milk protein. Physical examination was found within normal.
Case #
Age (y,m)
Weight (kg)
Height (cm)
OFC (cm)
Penis length (cm)
Language development
Cryptorchidism
Hypospadias
Allergic reactions
1
3
16
92
48.7
4
Delayed
BIC
No
No
2
2,3
11
87.5
47
3
Delayed
RUIC
Glandular
No
3
3,1
11.5
87
46
4
Delayed
BPC
No
No
4
3,5
14.5
93
48.5
4.5
NL
No
No
ARh/AD
5
3
14
94
49.5
4
NL
No
No
AD/AS
3,8 15 100 51 4 NL No No ACMP 6 Table 2.Clinical data of the patients. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism. BPC: bilateral prescrotal cryptorchidism. NL: Normal. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma, ACMP: Allergy to cow’s milk proteins.
qPCR After the clinical evaluation, the quantification by PCR of VAMP7 in peripheral blood of the 6 participants was performed. In two cases (Case 1 with bilateral inguinal cryptorchidism and language delay and case 5 with atopic dermatitis and asthma) the qPCR result was normal.
aCGH Table 3 shows the correlation between qPCR analysis, microarray analysis, and clinical data of the patients. Patient #
Clinical manifestations
VAMP7 gene dosage by qPCR (DBS)
VAMP7 gene dosage by qPCR (Blood)
Genomic Position
Increase in copy number (kb) by aCGH
Involved genes
1
BIC, LDD
1.4
1
-
No increase
None
9
2
RUIC, GH, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
3
BIC, LDD
1.44
1.83
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
4
ARh/AD
1.79
1.68
ChrX:154931044155185493
254.449
SPRY3, VAMP7
5
AD/AS
1.39
0.99
-
No increase
None
6
ACMP
1.45
1.94
ChrX:154947945155232877
284.933
SPRY3, VAMP7, IL9R
Table 3. Correlation of clinical data with results of qPCR (RQ>1.3) and aCGH. BIC: bilateral inguinal cryptorchidism. RUIC: Right unilateral inguinal cryptorchidism.GH: Glandular hypospadias BPC: bilateral prescrotal cryptorchidism. LDD: Language development delay. ARh: Allergic rhinitis. AD: Atopic dermatitis. AS: Asthma. ACMP: Allergy to cow’s milk protein.
DISCUSSION Six patients with increased VAMP7 gene dosage, identified with DBS qPCR, were clinically evaluated, of which 50% were identified with virilization defects (bilateral cryptorchidism, unilateral cryptorchidism and glandular hypospadias) and speech delay. The etiology of congenital malformations of the genitourinary tract has not yet been elucidated. It is thought it can be due to either monogenic or multifactorial causes. Mutation in genes such as: WT1, SF1, BMP4, BMP7, HOXA4, HOXB6, FGF8, FGFR2, AR, HSD3B2, SRD5A2, ATF3, MAMLD1, MID1 and BNC2 may be responsible for some genitourinary defects. [19] Recent reports have shown an association between the increase of VAMP7 gene dosage and virilization disorders. aCGH analysis of 116 cases of males with disorders of sexual differentiation including alterations in testicular descent and defects in penis morphology identified 2 subjects (1.7%) with an identical gain in the number of copies in the Xq28 region, specifically VAMP7. Based on these observations, it was postulated that the duplication of the Xq28 region, specifically of VAMP7, plays a role 10
in the human masculinization disorders of the urogenital tract [16]. In another cohort, 180 individuals with similar clinical manifestations were evaluated and 2 subjects (1.1%) were reported with gain in the number of copies in VAMP7, this time using the qPCR technique. In both studies, the selection of subjects started from the clinical manifestations, specifically the presence of virilization disorders such as cryptorchidism and hypospadias, to later analyze the VAMP7 gene dosage [16, 20]. Here, unlike the previously mentioned work, the cases were initially due to an increased gene dose of VAMP7 in a study for the early detection of sex chromosome aneuploidies using DBS qPCR. This analysis showed that 8/10,033 individuals had an increased VAMP7 gene dosage. Six patients were clinically evaluated, of which three (50%) were identified with alterations in genital development (2 bilateral cryptorchidism and 1 unilateral cryptorchidism and glandular hypospadias) and speech delay, while the rest presented different types of atopy. Clinical evaluation of the other two cases was not possible because the parents decided not to continue in the study. Considering the previous studies, in which an association of increased gene dosage of VAMP7 with alterations in the genitourinary development in about 1.3% of the analyzed individuals was found; our study, through the search of cases from increased gene dose achieved the identification of this association, supporting what was reported by other authors. [17]. It is interesting that the patients identified with alterations in the expression of VAMP7 are similar to that reported by Tannour-Louet et. al. In relation to the expected incidence for virilization disorders we found 1.97% for cryptorchidism and 1.96% for hypospadias. On the other hand, three of the cases that did not present genitourinary malformations coincided in presenting different types of atopy. 11
After performing a second qPCR in peripheral blood and aCGH, the increase in the VAMP7 gene dose was confirmed in 4 of the 6 cases, excluding two which initially were identified as high-dose gene by qPCR in DBS, considering these results as false positives. It has been suggested that the role of the VAMP7 protein in the neuritic growth of developing neurons could explain the language alterations exhibited in our patients Although there are no studies that clearly demonstrate their relationship with genitourinary development, the results obtained in our study, as well as those previously reported, strongly suggest a role for this gene in the development of these defects [14]. In Xq28-Xqter region identified by the aCGH were also found the IL9R and SPRY3 genes, which could be correlated with the clinical manifestations identified in the patients. IL9R was the first gene identified in the pseudoautosomal region of chromosomes Xq and Yq. It has 11 exons and encodes a ligand-specific alpha chain for the heterodimeric receptor. This receptor is expressed by a variety of hematopoietic cells such as T cells, mast cells, dendritic cells, epithelial cells, eosinophils, neutrophils and macrophages; being able to have biological significance in the development of asthma, as higher IL9R expression in the airways is noted in asthmatic patients. IL9R belongs to the group of genes with a clear potential role in allergic diseases. Also, the overexpression of IL9R in inflammatory bowel disease suggests a functional role in the perpetuation of gut inflammation. [21 - 23] The SPRY3 gene is made up of 2 exons and encodes for a member of the SPROUTY proteins (SPRY) family, which plays a role in the morphogenesis of the neuronal branching and it is suggested that it can inhibit the growth factor signaling in the neuronal development and the branching of the ganglion cells during postnatal development and in the adult life. [20,
12
24]. Future research in this area may shed additional light on the pathogenesis of language developmental delay. One strength found in this study was the approach to select the sample which was from the laboratory data (molecular biology) to the clinical features, inverse to what was reported by Tannour-Louet et. al (clinical features – laboratory), with similar percentages between both studies. CONCLUSION After clinical and molecular evaluation of six infants, we found the increased VAMP7 gene dosage could be responsible for one of many causes of virilization disorders, and probably language delay and atopies.
Conflict of Interest: None
REFERENCES
1. Urh K, Kunej T. Molecular mechanisms of cryptorchidism development: update of the database, disease, comorbidity, and initiative for standardization of reporting in scientific literature. Andrology. 2016, 1-9 2. Diagnóstico y tratamiento del testículo no descendido. México: Secretaría de Salud; 2014. 3. Diagnóstico y Tratamiento de Hipospadias. México. Secretaría de Salud, 2010
13
4. Pantoja BM, Kofi BE, Rio HG, Sánchez RO, Camué MR. Caracterización clinicoepidemiológica y terapéutica de pacientes con hipospadias. MEDISAN 2009; 13 (6) 5. Leung A.K., Robson W.L. Hypospadias: an update. Asian J Androl 2007 Jan; 9: 1622 6. Sagodi L, Kiss A, Kiss-Toth E, Barkai L: Prevalence and possible causes of hypospadias (in Hungarian). Orv Hetil 2014; 155: 978–985 7. Blanco S., Gottlieb S., Grinspon R., Rey R. Criptorquidia: desde la embriología al tratamiento MÉD UIS. 2015;28(3):371-80 8. Tekgül S. ,et. al. Guía Clínica sobre Urología Pediátrica. European Society for Pediatric Urology (2009),14: 991,1002. 9. Matsushita S. et. al. Regulation of masculinization: androgen signalling for external genitalia development. Nat. Rev. Urology (2018) 10. Wilhelm D, Koopman P. The makings of maleness: towards an integrated view of male sexual development. Nat Rev Genet. 2006; 7:620–631. 11. Blaschko SD, Cunha GR, Baskin LS: Molecular mechanisms of external genitalia development. Differentiation 2012; 84: 261–268 12. Suzuki, H., Suzuki, K. & Yamada, G. Systematic analyses of murine masculinization processes based on genital sex differentiation parameters. Dev. Growth Differ. 57, 639–647 (2015). 13. Watanabe M, Yoshida R, Ueoka K, Aoki K, Sasagawa I, et al: Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities. Hum Reprod 2007 22: 1279–1284.
14
14. D’Esposito et. al. A synaptobrevin-like gene in the Xq28 pseudoautosomal region undergoes X inactivation. Nat Genet.1996; 13: 227-229. 15. Matarazzo et. al. Allelic inactivation of the pseudoautosomal gene SYBL1 is controlled by epigenetic mechanisms common to the X and Y chromosomes. Human Molecular Genetics, 2002, Vol. 11, No. 25 3191–3198 16. Tannour-Louet et. al. Increased gene copy number of VAMP7 disrupts human male urogenital development through altered estrogen action. Nat. Med. (2014).20, 715– 724 17. Wang Z, Liu BC, Lin GT, Lin CS, Lue TF, et al: Up-regulation of estrogen responsive genes in hypospadias: microarray analysis. J Urol 2007 177: 1939–1946. 18. Campos-Acevedo et. al. Dosage of Sex Chromosomal Genes in Blood Deposited on Filter Paper for Neonatal Screening of Sex Chromosome Aneuploidy. Genetic Testing and Molecular Biomarkers. 2016; 12:1-5 19. van der Zanden LF, van Rooij IA, Feitz WF, Franke B, Knoers NV, Roeleveld N Aetiology of hypospadias: a systematic review of genes and environment. Hum Reprod Update. 2012 May-Jun;18(3):260-83. 20. De Bonis, M. L., et. al. Maintenance of X- and Y-inactivation of the pseudoautosomal (PAR2) gene SPRY3 is independent from DNA methylation and associated to multiple layers of epigenetic modifications. Hum. Molec. Genet. 15: 1123-1132, 2006. 21. Fatahi F., et. al. Assessment of the Effects of IL9, IL9R, IL17A, and IL17F Gene Polymorphisms on Women with Allergic Rhinitis in Shahrekord, Iran Ann Med Health Sci Res. 2016 Jul-Aug; 6(4): 216–223.
15
22. Kauppi P., Laitinen T. ,Ollikainen V., Mannila H., Laitinen, L.A., Kere J. The IL9R region contribution in asthma is supported by genetic association in an isolated population. European Journal of Human Genetics (2000) 8, 788–792 23. M.F. Neurath, S. Finotto, IL-9 signaling as key driver of chronic inflammation in mucosal immunity, Cytokine Growth Factor Rev (2016) 24. Ning Z. et. al. Regulation of SPRY3 by X chromosome and PAR2-linked promoters in an autism susceptibility region. Hum Mol Genet. 2015 Sep 15;24(18):5126-41
16