Evaluation of a Multiplex PCR Kit Used for Detecting Y Chromosome Microdeletions

Mi Young Park; Hye-Min Kang; Sang-Hyun Hwang; Hyun-Jun Park; Nam-Cheol Park; Kyung Un Choi; Hyung-Hoi Kim; Chulhun L. Chang; Eun-Yup Lee

doi:10.3343/kjlm.2010.30.4.432

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Park, Kang, Hwang, Park, Park, Choi, Kim, Chang, and Lee: Evaluation of a Multiplex PCR Kit Used for Detecting Y Chromosome Microdeletions

Original Article

Korean J Leg Med 2010;30(4):432-439.

Published online: 14 January 2010

DOI: https://doi.org/10.3343/kjlm.2010.30.4.432

Evaluation of a Multiplex PCR Kit Used for Detecting Y Chromosome Microdeletions

Mi Young Park, M.D.¹, Hye-Min Kang², Sang-Hyun Hwang, M.D.^1,⁵, Hyun-Jun Park, M.D.³, Nam-Cheol Park, M.D.³, Kyung Un Choi, M.D.⁴, Hyung-Hoi Kim, M.D.¹, Chulhun L. Chang, M.D.¹, Eun-Yup Lee, M.D.^1,⁵

¹Departments of Laboratory Medicine, Busan, Korea

²Departments of Medicine, Busan, Korea

³Departments of Urology, Busan, Korea

⁴Departments of Pathology, Pusan National University School of Medicine, Busan, Korea

⁵Medical Research Institute, Pusan National University Hospital, Busan, Korea

Corresponding author: Sang-Hyun Hwang, M.D. Department of Laboratory Medicine, Pusan National University, School of Medicine, 205 Gudeok-ro, Seo-gu, Busan 602-739, Korea Tel: +82-51-240-7403, Fax: +82-51-247-6560 E-mail: mindcatch@hanmail.net

^∗ This work was supported by Pusan National University Hospital Research Grant 2010 and supported by Medical Research Institute & Pusan Cancer Center Grant (2010-28), Pusan National University Hospital.

Revised 24 February 201027 April 2010 Accepted 28 April 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

In addition to Klinefelter's syndrome, microdeletion of Yq is the most common genetic cause of male infertility; 15% of azoospermic or 5-10% of oligozoospermic males have Yq deletions. We evaluated a Yq microdeletion kit (LG Life Sciences, Korea) for identifying microdeletions in the azoospermic factor (AZF) regions of the Yq.

Methods:

The kit was designed to amplify 3 regions of the AZF gene (AZFa, AZFb, and AZFc) using 15 sequence-tagged sites. We evaluated the preclinical performance of the kit. For clinical validation, 58 patients including 25 idiopathic azoospermic or oligozoospermic patients were examined.

Results:

We observed clear bands on electrophoresis of DNA, up to a DNA concentration of 3.12 ng/μL; the known microdeletion regions of all 6 reference cell-lines (Coriell, USA) were accurately detected and no false positive/negative results showed with normal female (n=11) and fertile male (n=15) specimens. This kit could identify the same microdeletions in the common regions, similar to another commercial kit. Among the 58 male infertile patients, 7 (12.1%) had microdeletions of the Yq. Among the idiopathic azoospermic (n=22) and oligozoospermic (n=3) patients, 3 (12.0%) had microdeletions. Further, 2 of 21 varicocele patients (9.5%), 1 of 4 patients with testicular failure, and 1 patient with a 45,X/46,XY mosaic had microdeletions.

Conclusions:

The kit was effective for detecting microdeletions of the Yq. We identified microdeletions in 12% of the infertile patients. This Y chromosome microdeletion detection kit is useful for screening Yq microdeletions in infertile patients.

Keywords: Infertility, Y chromosome, Azoospermic factor, Microdeletion

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Fig. 1.

Multiplex PCR products of various concentrations of genomic DNA. The electrophoresis of PCR products revealed clear bands up to a concentration of 3.12 ng/μL of genomic DNA.

Abbreviation: N, No DNA.

Fig. 2.

Seven patients have microdeletions of the Y chromosome. Black squares indicate the sites of deletions. The first choice of STS markers recommended in European Academy of Andrology/European Molecular Genetics Quality Network guideline are marked with an asterix.

Fig. 3.

The boxes on the gel image indicate microdeletions of Y chromosome of patient 4 (A) and patient 7 (B). Patient 4 has deletions of sY254, sY255, SPGY1, sY158, Y152, sY147, sY157, and sY242. Patient 7 has deletions of sY127, sY254, sY134, sY255, SPGY1, sY 158, sY152, sY147, sY157, sY242, sY130, and sY124.

Table 1.

Sets of sequence-tagged sites of Y chromosome microdeletion detection kit

STS set	STS marker	Locus	Size (bp)	STS set	STS marker	Locus	Size (bp)
STS-1	ZFX	X	519	STS-2	ZFX	X	519
	sY14	Yp	472		sY14	Yp	472
	sY254	AZFc	380		sY84	AZFa	326
	sY86	AZFa	318		sY134	AZFb	238
	sY127	AZFb	274		sY255	AZFc	123
STS-3	ZFX	X	519	STS-4	ZFX	X	519
	SPGY1	AZFc	460		sY157	AZFc	286
	sY158	AZFc	215		sY242	AZFc	233
	sY152	AZFc	125		sY130	AZFb	173
	sY147	AZFc	100		sY124	AZFb	109

Abbreviations: STS, sequence-tagged sites; ZFX, zinc finger protein, X-linked; AZF, azoospermic factor.

Table 2.

Clinicopathologic findings of 7 patients with Y chromosome microdeletion determined using Y chromosome microdeletion detection kit

STS set	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6	Patient 7
Age (yr)	36	32	28	34	34	21	37
Sperm count	Azoospermia	Azoospermia	Azoospermia	Oligozoospermia	Azoospermia	Azoospermia	Azoospermia
Clinical impression of infertility	Idiopathic	Idiopathic	Idiopathic	Varicocele	Varicocele	Testicular failure	Chromosomal abnormality
FNAC/biopsy	Hypospermato -genesis	NT	Normal	NT	SCOS	NT	SCOS
Karyotype	46,XY	46,XYqh-	46,XY	46,XY	46,XY	46,XYqh-	mos 45,X[5]/46,X,del(Y)(q12)[25]
AZF deletion	AZFc	AZFb, AZFc	AZFa	AZFc	AZFc	AZF b, AZFc	AZFb, AZFc

Abbreviations: FNAC, fine needle aspiration cytology; NT, not tested; SCOS, sertoli cell-only syndrome; AZF, azoospermic factor.

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