The identification of new disease-associated genetic mechanisms in patients with mental retardation

Abstract

Failure of normal brain development or functioning can lead to mental re tardation (MR), now often referred to as intellectual disability, charac terized by limitations in mental capabilities and adaptive skills. MR ha s a prevalence of 2-3% in the general population in developed countries. Though environmental factors significantly contribute to the appearance and severity of the disease, genetic factors are estimated to account f or 25-35%. Gene defects on the X chromosome (X-linked) have been conside red to be important contributors to MR and research has been mainly focu sed on this X-linked form of MR (XLMR). The etiology of XLMR is extremel y heterogeneous and requires the use of several molecular techniques to find them. This research project focuses on the identification of new XL MR-associated genetic factors or disease mechanisms. To this end, the de velopment and implementation of novel technologies were crucial. Mutations altering the coding sequence of a gene or one of its regulator y elements are the classical genetic defects associated with inherited h uman disorders. The standard molecular technique to track such changes i s via sequence analysis. Other, less common disease-associated mutations are chromosomal aberrations, which often are very informative. Whereas classical karyotyping allows rough estimation of the breakpoints of the chromosomal aberration, walking FISH can delineate the breakpoint region to a few tens of kb. Genome browsers are then consulted to check whethe r a gene could be interrupted by the breakpoint. Subsequently, altered e xpression of this candidate MR gene can be analyzed in patient derived c ells, which could explain the patient s phenotype. We characterized the breakpoints of a translocation t(X;9)(q13.2;p24) present in a severely d evelopmentally retarded female patient. The breakpoint on the X chromoso me was found to disrupt the MCT8 gene. Moreover, X-inactivation studi es revealed that the normal X chromosome was always inactivated in her c ells suggesting the lack of MCT8 expression, which was confirmed in t he patient s fibroblasts (part 1). As such, this patient represents t he first female with the Allen-Herndon-Dudley syndrome (AHDS). Up to now, 82 XLMR genes have been described but the mutation frequency of each gene in the XLMR population is very low (0.1-1%). The novel tech nology of array-CGH allowed the identification of submicroscopic genomic gains and losses (>5 Mb to 10 kb), which added another layer of disease -causing mutations in XLMR. The resolution of array-CGH correlates with the type and number of probes spotted onto the array: the initial resolu tion obtained with large-insert BAC/PAC clones spaced every 1 Mb was sig nificantly lower than the currently used oligonucleotides arrays spaced every 100 bp. We developed a full-tiling X chromosome-specific BAC array (X-array) wit h a theoretical resolution of 80 kb (part 2). The X-array was validat ed by hybridisation of DNA from patients with known X-chromosomal imbala nces of different sizes. All clones within the aberration (deletion as w ell as duplication) were easily detected, and the borders of the imbalan ce could be readily delineated demonstrating the usefulness of our X-arr ay. After validation, the array was used to screen a population of 108 patie nts with idiopathic MR and 15 genomic aberrations were found in 14 patie nts (13%); amongst these are 2 deletions and 13 duplications with variab le length (0.1-2.7 Mb) (part 2). To determine whether an aberration o n the X chromosome can be considered causal, we proposed the following c riteria: de novo aberration, segregation of the aberration in the family with the disease, absent in control individuals and nonrandom X-i nactivation in asymptomatic female carriers. Also, the presence of a kno wn (N)SXLMR gene can be a valuable indication. As such the aberration wa s suggested to be causal for the MR phenotype in 5 patients (4.6%). From this screening effort it was hypothesized that not only deletion, b ut also duplication of dosage-sensitive (N)SXLMR genes can result in XLM R. To test this hypothesis, we focused on the duplication of MECP2 , which was identified in four unrelated families. Via mRNA expression a nalysis we demonstrated a 2-fold higher expression of MECP2 in EBV -PBLs of patients with the duplication when compared to controls. Notabl y, it was reported that slight overexpression of MECP2 in Mecp2 -null mice resulted in neurological abnormalities, which underscores the biological relevance of our finding. Also, this dosage-sensitive rol e of MeCP2 in neuronal plasticity and function was observed in brain sli ces of patients with neurological abnormalities. As such, we defined gen e duplication of known (N)SXLMR genes as a new disease mechanism in XLMR (part 3). Several other causal duplications were later described by our group. The discovery of causal genomic duplications and deletions in disease in troduced the concept of genomic disorders, which are characterized by th e quantitative alteration of a dosage sensitive gene or genes via genomi c rearrangements. Since several unrelated patients often harbor a simila r genomic rearrangement, the question arises whether there is a common m echanism driving these rearrangements. We tackled this question via a st udy of the rather frequent nonrecurrent MECP2 duplication events. Bre akpoint mapping, in silico breakpoint analysis in 16 patients and seq uencing of the breakpoints in three patients revealed that the distinct complex genomic architecture at Xq28 can trigger the common NAHR and NHE J mechanisms but can also activate a combination of alternative repair p rocesses to restore genomic integrity, yielding either a simple or compl ex rearrangement (part 4). From this research project it is clear that a wide variety of molecular techniques is used to unravel the genetic causes underlying XLMR. Despit e extensive mutation screening via positional cloning, sequence analysis and array-CGH, the causal factor remains to be determined in >50% of th e XLMR patients. With the recent development of the high-throughput deep sequencing technologies, identification of additional genetic factors w ill speed up the mutation detection rate. Molecular passports will be ge nerated at base pair resolution; not only at the genomic but also at the epigenomic level because there is growing evidence for the involvement of the epigenome in neurological diseases and cognition. Large amounts o f raw data will be generated and it will be crucial to validate or compl ement those with accurate biological and functional data. In addition, o ther disease-associated mechanisms are hunted for and great efforts are currently being made to understand the involvement of regulatory element s and noncoding RNA s, or to investigate the importance of higher-order chromatin structure in normal development and disease.status: publishe