Elsevier

European Journal of Medical Genetics

Volume 48, Issue 3, July–September 2005, Pages 263-275
European Journal of Medical Genetics

Original article
X chromosome array-CGH for the identification of novel X-linked mental retardation genes

https://doi.org/10.1016/j.ejmg.2005.04.008Get rights and content

Abstract

Array-CGH technology for the detection of submicroscopic copy number changes in the genome has recently been developed for the identification of novel disease-associated genes. It has been estimated that submicroscopic genomic deletions or duplications will be present in 5–7% of patients with idiopathic mental retardation (MR). Since 30% more males than females are diagnosed with MR, we have developed a full coverage X chromosome array-CGH with a theoretical resolution of 82 kb, for the detection of copy number alterations in patients with suspected X-linked mental retardation (XLMR). First, we have validated the genomic location of X-derived clones through male versus female hybridisations. Next, we validated our array for efficient and reproducible detection of known alterations in XLMR patients. In all cases, we were able to detect the deletions and duplications in males as well as females. Due to the high resolution of our X-array, the boundaries of the genomic aberrations could clearly be identified making genotype–phenotype studies more reliable. Here, we describe the production and validation of a full coverage X-array-CGH, which will allow for fast and easy screening of submicroscopic copy number alterations in XLMR patients with the aim to identify novel MR genes or mechanisms involved in a deranged cognitive development.

Introduction

Mental retardation (MR) is one of the most common disorders affecting 2–3% of the human population [10], [20]. It is a non-progressive cognitive impairment mostly affecting normal brain development marked by learning (IQ < 70) and behavioural disabilities. Since MR patients are unable to adapt their behaviour to changing environmental factors, it implicates constant support from their family or from social workers. Therefore, the disorder is a very important medical and socio-economical problem. MR is classified into syndromic, in which MR is associated with other clinical characteristics, and non-syndromic forms, where MR is the only phenotype [24]. Although many environmental factors can play determining roles in the occurrence and severity of the disease, the impact of genetic factors is estimated at 25–35%. Since 30% more males than females suffer from MR, the search for disease-associated genes has, up to now, predominantly been focused on the X chromosome ('X-linked mental retardation' or XLMR) [22], [28].

Cytogenetically detectable chromosomal anomalies are present in only a very small number of MR patients, and the number of MR families amenable to linkage analysis is rather restricted (http://xlmr.interfree.it/home.htm; http://www.ggc.org/xlmr.htm). Therefore, the genetic defect remains unexplained in most MR patients. Subtle genomic copy number changes (100 kb up to several Mb) have been identified in 5–7% of patients with idiopathic MR [18], [27], [30], [37] but they cannot be detected by routine karyotyping because of its limited resolution. Fluorescent in situ hybridisation (FISH) and comparative genomic hybridisation (CGH) are currently routinely applied in the clinic to detect genomic copy number changes [17], [47]. FISH has the advantage of high resolution (50–100 kb) but has an extremely low throughput if an unknown aberration has to be detected. On the contrary, CGH can scan a whole genome at once but has a rather limited resolution (5–10 Mb) [17]. Other PCR-based methods (marker analysis, MAPH, MLPA) have been described but their throughput is rather restricted [3], [19], [46]. The recently developed array-CGH technique combines the property of a complete genome scan of CGH, with the hybridisation on sorted genomic DNA fragments from the microarray technology [26], [38]. In addition, array-CGH is able to detect and quantify segmental aneuploidy with a resolution comparable to that of FISH. Nowadays, array-CGH has become the method of choice for detection of chromosomal copy number anomalies in tumours as well as in genetic diseases providing direct information on the genomic position of that aberration. The applicability of this technique is demonstrated by a rapidly increasing number of reports. Analyses have been reported on subtelomeric regions [42], topic- and chromosome-specific regions [4], [34], [40], [41], whole chromosomes [5], [43], and the whole genome [15]. Additional studies are referred to in reviews on this technology [1], [23], [25].

In this report, we will focus on the applicability of array-CGH for the detection of X chromosomal submicroscopic aberrations in patients with MR. We will describe our efforts in this field with the development of a full coverage X chromosome-specific array.

Section snippets

Patient samples

Informed consent was obtained from all patients or their families. Genomic DNA from patients as well as from healthy controls was isolated from peripheral blood according to standard procedures. DNA was dissolved in TE buffer at a concentration of 0.33 μg/μl and stored at –20 °C.

Development of a full coverage X chromosome array

Arrays were constructed using 2005 genomic (BAC, PAC, cosmid and fosmid) clones from the X chromosome and 96 from autosomal origin. Clones were obtained from the Children's Hospital Oakland Research Institute (CHORI; //bacpac.chori.org/home.htm

Validation of the X clones

In order to validate our X-array, we first checked the correct assignment of the presumed 2005 X chromosomal-derived clones that we obtained from three different sources (see Section 2.2). This clone validation is not only important for subsequent data interpretation but also to assess the final coverage of the X chromosome. For this, we hybridised four different female versus male control sample mixtures onto the array. A female sample was labelled with Cy5 or Cy3 and hybridised together with

Focus on the X chromosome

At present 931 genes have been identified on the X chromosome (Ensembl v30 build 35), which is about 3.75% of the total number of genes on the genome. Since the X chromosome comprises almost 155 Mb it constitutes about 4.8% of the total genome. It is a gene poor chromosome with a high percentage of interspersed repeats [31]. In OMIM, there are 1244 entries for the word 'MR' and 356 (28.6%) are mapped on X indicating a disproportionate distribution of genes involved in cognitive development.

Summary

The molecular basis for cognitive development is still poorly understood. Based on the 30% excess of male patients suffering from MR, the search for disease-associated genes on the X chromosome has lead to the identification of about 24 genes for which mutations result in non-syndromic MR. However, many more gene on the X chromosome seem to exist. We have developed and validated a full coverage high resolution X chromosome array-CGH for screening of subtle copy number changes in presumed XLMR

Acknowledgements

We would like to thank the patients and their families as well as the medical staff for their cooperation. We want to acknowledge Dr. J.P. Fryns, Dr. K. Devriendt and Dr. J. Vermeesch from the Clinical Genetics Department of the University Hospital Gasthuisberg (Leuven, Belgium) for providing us with patient material and information, and Dr. P. Vanhummelen and T. Bogaerts from the Microarray Facility of the VIB (Leuven, Belgium) for their help with the production of the X-array. We also thank

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