An 800 kb deletion at 17q23.2 including the MED13 (THRAP1) gene, revealed by aCGH in a patient with a SMC 17p^†

INTRODUCTION

The characterization of small supernumerary marker chromosomes (SMCs) discovered on karyotyping is a real challenge and is necessary for the establishment of genotype-phenotype correlations and for accurate genetic counseling. Indeed, only one third of SMCs are associated with a precise phenotype (e.g.: tetrasomy 12p and Pallister-Killian syndrome, chromosome 22 inversion-duplication, and cat-eye syndrome). This lack of phenotype–genotype correlation results from the extreme diversity of SMCs (degree of mosaicism, size, structure, origin, gene content) and the limitations of classical cytogenetic investigations [Crolla et al., 2005]. However, the development in recent years of cytogenetic tools, including array comparative genome hybridization (aCGH) in particular, has made it possible to characterize imbalanced chromosome abnormalities with a higher resolution (size, breakpoints), even in cases of mosaicism [Ballif et al., 2006; Menten et al., 2006]. Moreover, aCGH sometimes reveals other unexpected abnormalities associated with the markers [Baldwin et al., 2008; Tsuchiya et al., 2008] and the considerable phenotypic variability of patients with SMCs may be accounted for by these other “hidden” imbalanced rearrangements, which remained undetected before karyotyping. The case reported below illustrates this point.

We report here on clinical, cytogenetic and molecular studies in a girl with both a de novo SMC derived from the proximal short arm of chromosome 17 and a de novo 800 kb 17q23.2 deletion detected by aCGH. We also present additional evidence that the genes present on this SMC(17) probably have little, if any, phenotypic effect.

CLINICAL REPORT

This 7-year-old child was the second child of nonconsanguineous parents of French background with no informative family history. Her father was 34 and her mother was 31 years old at the time of conception. Intrauterine growth retardation was observed during pregnancy. The child was delivered normally at term. Her birth weight was 2,560 g (−2 SD), her birth length was 44.5 cm (−2 SD), and her head circumference was 32.2 cm (−2 SD). Blood lymphocyte karyotyping was performed at birth, due to possible sexual ambiguity (abnormality of the clitoris, subsequently considered to be an anatomic variant). A supernumerary marker chromosome was found in all the metaphase cells observed (40/40). Parental karyotypes were normal. Clinical follow-up showed significant feeding difficulties and recurrent bronchiolitis. Growth retardation (−2.5 SD), developmental delay (the patient sat unsupported at the age of 10 months walked at the age of 21 months), and delayed speech acquisition were noted at the age of three years. The patient also had a hearing loss, with prolonged latencies in auditory evoked potential tests. MRI scan showed bilateral semicircular canal dysplasia, with ossicular malformations. On clinical examination, we noted minor craniofacial abnormalities: temporal retraction, small ears with posterior rotation, unilateral preauricular fistula, small mouth, short nose, small hands and feet (no picture of the patient is provided because the required informed written consent could not be obtained).

On examination at the age of four years, the patient presented with pes equinus of the right foot, with loss of the Achilles and patellar tendon reflexes. Pes equinus was resolved by physiotherapy. A recent clinical examination of this child at the age of six years showed bilateral peripheral cataract and right conduction hearing loss (60–70 dB) requiring the use of a hearing aid. Speech and motor development improved with time. This patient had attention deficit and hyperactivity disorder (ADHD), which was improved by risperidone treatment. No sleep disorder was noted.

MATERIALS AND METHODS

RESULTS

The postnatal karyotype revealed the presence of two X chromosomes and a supernumerary small chromosome in all cells (40/40). This additional marker was too small for identification on the basis of its banding pattern (Fig. 1a). The karyotypes of the parents were normal. We therefore determined the origin of this de novo SMC by FISH with centromeric probes. The supernumerary chromosome hybridized with the chromosome 17 centromeric probe but not with the Smith–Magenis and Miller–Dieker microdeletion syndrome probes (Fig. 1c). We then carried out aCGH analysis, which revealed a gain of about 1.6 Mb of chromosomal material on 17p11.2, between the binding sites of oligonucleotides A_16_P40777061 and A_14_P113402 (respective positions 20,404,913 and 22,004,995 Mb; hg18, NCBI36; Fig. 1c), confirming partial trisomy 17p with the inclusion of euchromatic sequences. This result was confirmed by FISH (Fig. 1b). Interestingly, aCGH analysis also revealed the unexpected loss of 800 kb from the genomic sequence of 17q23.2, between the binding sites for oligonucleotides A_16_P03272064 and A_16_P20698245 (respective positions 57,372,913 and 58,206,595 Mb; hg18, NCBI36). This result was confirmed by qPCR with a primer binding to exon 8 of the MED13 gene and was shown to be a de novo modification (data not shown).

An analysis of parental samples with the BAC clone RP11-342K2 (17q23) revealed no major chromosome rearrangement, such as large inversions (data not shown). This clone was found to be absent from the patient's cells (data not shown).

The microsatellite analysis revealed that the deletion was of maternal origin. Unfortunately, the three microsatellites located in the same region of the genome as the SMC were not informative (Table I).

DISCUSSION

Eleven cases of chromosome 17-derived SMCs have been reported [Yatsenko et al., 2005; Kogan et al., 2009]. The 17p11.2 region is particularly rich in low-copy number repeats (LCRs). LCRs are DNA sequences involved in nonallelic homologous recombination (NAHR), a mechanism responsible for DNA rearrangements, such as deletions, duplications, inversions and SMCs. In the 17p11.2 region, the proximal and distal Smith–Magenis LCRs (SMS-REP) mediate the common deletion and reciprocal duplication resulting in the Smith–Magenis [Park et al., 2002] and Potocki–Lupski syndromes [Potocki et al., 2007], respectively. We describe here a case of SMC(17) with a distal breakpoint mapping to the centromere region and a proximal breakpoint mapping close to the proximal SMS-REP in the LCR17pD (Fig. 2). This SMC(17) therefore did not include the region generally implicated in Potocki–Lupski syndrome (PLS). To our knowledge, only two cases of SMC(17) that did not include the this region have been described to date [Shaw et al., 2004; Kogan et al., 2009].The case of Shaw et al. [2004] 2,170 had a distal breakpoint between the binding sites of clones CTD-2010G8 (19.9 Mb) and RP5-836L9 (20.1 Mb) and a centromeric proximal breakpoint. In our case, the distal breakpoint was located at 20.4 Mb, so the SMC had a lower euchromatin content (Fig. 2). In the other case, described by Kogan et al., there was a distal breakpoint in the proximal SMS-REP, as in our patient, but a proximal breakpoint in the 17q11.2 band. In addition to the mosaic SMC(17), the patient also displayed mosaicism for an SMC(13). The clinical features of these patients were not consistent with the phenotype of our patient. In particular, our patient presented with cataracts, severe hearing loss and semicircular canal dysplasia, which have never previously been reported in patients with SMC(17).

The duplicated region in our patient contained nine genes: MGC87631, USP22, DHRS7B, TMEM11, MGC33894, MAP2K3, KCNJ12, C17orf51, and FAM27L (RefSeqGenes, UCSC hg18). Six of these genes are encompassed by two copy number polymorphisms (CNPs) described by Redon et al. (CNPs 1218 and 1219). The other three genes (TMEM11, DHRS7B, and MGC33894) encode proteins with poorly characterized functions: TMEM11 encodes a protein thought to regulate the morphogenesis of mitochondria, particularly during cell stress (hypoxia, infection) [Rival et al., 2011]. DHRS7B (dehydrogenase/reductase member 7B) and MGC33894 encode proteins of unknown function (UCSC genome browser). In addition, a girl with a de novo min(17) encompassing the chromosomal region found in our patient was reported on Thomas Liehr's marker site (http://www.med.uni-jena.de/fish/sSMC/17.htm#Start17, case 17-O-p11.2/3-1). At the age of two years, the patient displayed no malformations, dysmorphism, or retardation of psychomotor development. We cannot entirely exclude the possibility that the SMC(17) plays a role in the intellectual disability of this patient, but we suggest that the phenotype observed in our patient, including the hearing loss and ocular abnormalities, was due to the de novo 800 kb 17q23.2 deletion rather than the SMC.

To our knowledge, no other small deletion has ever been described in this chromosomal region. This 17q23.2 deletion encompasses six genes (EFCAB3, MED13, METTL2A, TLK2, MRC2, and MARCH10, RefSeqGenes, UCSC hg18). A copy number polymorphism (CNP 1238) has been described in this region, but it is smaller than the observed deletion and includes only the EFCAB3 gene. The other five genes are absent from this CNP and are potential candidate genes underlying the clinical symptoms of this child. One of these genes, MED13 (OMIM 603808), is of particular interest with respect to the phenotype of the patient. Indeed, the product of this gene probably forms a subcomplex with the products of the MED12, CDK8, and Cyclin C genes—the human CDK8 subcomplex—which is thought to downregulate transcription [Knuesel et al., 2009]. These four genes appear to be essential for embryo development, but not for cell viability. In Drosophila, alteration to this complex cause malformations of the eyes and external sensory organs [Loncle et al., 2007]. Indeed, mutant Med13-Drosophilia display a lack of photoreceptor differentiation in the eye discs, leading to malformation of the ommatidia [Treisman, 2001]. These Med13-mutants also display downregulation of the bab gene in the leg discs. This gene is required for development of the proximo-distal leg axis and its downregulation results in distal leg shortening. A loss of bristle differentiation has also been observed in the adult notum: in Med13-mutants, expression of the sens gene, which is required for sensory organ precursor differentiation, is undetectable. Malformations of wings and antennas have also been noted. Interestingly, our patient presented with neurosensorial loss (hearing loss and cataracts). Moreover, one of the partners of MED13, MED12, interacts with Mediator, a protein involved in regulating neuronal gene expression [Ding et al., 2008]. MED12 mutations have been shown to be responsible for two X-linked intellectual disability disorders: the Opitz–Kaveggia and Lujan syndromes [Risheg et al., 2007; Schwartz et al., 2007].

Three other deletions of the MED13 gene were found in the DECIPHER database (Patients 249 237, 250 379, and 253 402). These three deletions were more proximal, but the only deleted region common to our patient contained only the MED13 locus. Two of the patients with these deletions presented mental retardation and deafness (Patients 249,237 and 250,379). No clinical data were available for case 253,402.

Our results provide no conclusive proof concerning the parental origin of the SMC. The chromosomal deletion was found to be of maternal origin, but our analyses of the parental samples found no chromosomal rearrangement involving the deleted region. We cannot exclude the possibility of a small parental inversion, but the presence of such an inversion could not account for the presence of both the SMC(17) and the 17q23 deletion. An in silico analysis of the deleted region with the TCAG website identified no highly similar duplicated sequences that might account for this deletion through an NAHR-based mechanism.

In summary, this patient's report highlights the complexity of phenotype-genotype correlations in microdeletion and microduplication syndromes. Our attention was initially drawn to the SMC(17), which was thought likely to be responsible for the patient's phenotype. However, our findings suggest that the basis of the abnormal phenotype in this patient, particularly as concerns the ocular and hearing abnormalities, is actually haploinsufficiency of the MED13 gene.

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