Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

Abstract

Background: Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization.

Methods: In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)-PCR.

Results: A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes.

Conclusions: The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF.

Figure 1

Summary of the copy number variations (CNV) detected among 42 POF patients. Genomic DNA from peripheral blood lymphocytes from POF patents was hybridized to complete X and Y chromosomes and 1 Mb autosomal BAC tiling path arrays using a normal female DNA sample as a common reference. (A) and (B) present example X chromosome profiles for a CNV amplification (A) and a deletion (B). To be scored as a CNV, a change had to include three or more consecutive BAC clones. The start and end clones are indicated. The inset provides an enlarged view of the clones involved in the CNV. (C) provides a summary of the location on the X chromosome of CNV detected among this cohort of patients after removal of common CNV variants found in the database of genomic variants.

Figure 2

qPCR confirmation of the copy number variations. qPCR analysis was performed on genomic DNA using markers from each CNV interval identified among the cohort of patients. PCR primers were developed for at least one marker locus per interval. (A) illustrates the analysis using primers designed for the BCORL1 genes across a series of patients including a 45, X individual and a normal female normalized to the control gene HPRT1. (B) illustrates the analysis using primers for four genes (ZFX, TSPAN7, VCX and H2BFM) on four selected patients relative to HPRT1 and highlights both sequence amplification (134, 163) and deletion (77, 147) of patients (solid shading) compared with the normal female control (hatched shading).

Figure 3

RT–PCR analysis of selected genes in tissue RNA from a range of 17 human tissues. The figure illustrates the expression pattern of the XPNPEP2 and ZFX genes as determined by semi-quantitative RT–PCR. A, skeletal muscle; B, thyroid; C, spleen; D, heart-fetal; E, adipose tissue; F, brain; G, colon; H, thymus; I, heart-adult; J, Esophagus; K, placenta; L, small intestine; M, bladder; N, lung; O, trachea; P, ovary; Q, blank control.