Previous mapping studies have by means of fluorescent in situ hybridization (FISH) demonstrated that terminal 15q-deletion frequently results in loss of one IGF1R allele. It has been postulated that a dosage effect of the IGF1R gene might be direct responsible for the intrauterine growth retardation seen in patients monosomic for 15q26 and for the postnatal overgrowth seen in patients trisomic for 15q25-qter [15]. However in vitro studies on fibroblast cells have been ambiguous. For instance Siebler et al. (1999) failed to provide conclusive evidence for a biological response to lowered IGFR1 expression due to distal deletion of 15q whereas Okubo et al. demonstrated accelerated and decreased growth in cells with three or one copy of the gene, respectively [5,8].

An initial analysis of the derivative chromosome detected in the patient was conducted with a whole chromosome paint (WCP) probe and a centromere probe specific for chromosome 15 (Vysis, Downers Grove, US) as well as with a probe specific for the nucleolus organizing regions (NOR) of the acrocentric chromosomes (Chrombios, Raubling, Germany). A probe specific for the P-W/A region (LSI D15S10/CEP15/LSI PML; Vysis) was also used. For a refined analysis of the derivative chromosome, a total of 12 bacterial artificial chromosome (BAC) probes targeting the 15q11.2-q14 region and the 15q25.1-q26.3 region, were selected from the UCSC Genome Browser [16] (Table 1). BAC DNA was extracted by alkaline lysis with SDS using standard protocols and labeled with Cy3-dUTP (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ) or Fluorescein-12-dUTP (FITC) (Roche, Mannheim, Germany) using the Megaprime DNA Labelling System (GE Healthcare Bio-Sciences Corporation). Fixed metaphase spreads were aged over night at 60°C then pretreated with 20 mg/ml pepsin (Serva, Heidelberg, Germany), postfixed in 1% formaldehyde and finally dehydrated in an ethanol series (70%, 85%, 100%). Commercial probes were hybridized according to the manufacturers' recommendations. For each BAC probe, a total of 90 ng labeled DNA was hybridized. After application of the probe solution and sealing of the metaphase spread, the preparations were denaturated on a hot plate for 4 min at 74°C and then incubated over-night in a humid chamber at 37°C. Post-hybridization washes were done in 0.4× SSC/0.05% Tween-20 (Sigma-Aldrich, St Louis, US) followed by dehydration in an ethanol series. For detection of the nuclei and evaluation of the fluorescent signals, the spreads were counterstained with 0.5 mg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and mounted in a 2% 1,4-diazabicyclo-[2,2,2]-octane (DABCO) solution (Sigma-Aldrich). Conventional epifluorescence microscopy was carried out with a Zeiss Axioplan2 imaging microscope (Zeiss, Jena, Germany) linked to a CytoVision Ultra system (Applied Imaging, San José, US). A minimum of 11 metaphases (median 17) were analyzed for each probe mixture.

DNA was extracted from the peripheral blood cells obtained at the time of diagnosis. Male genomic DNA (Promega, Madison, WI) was used as reference in the hybridization. The 32 k slides used, containing 32 433 tiling BAC clones covering at least 98% of the human genome, were produced at the SWEGENE DNA microarray resource center at Lund University, Sweden. Labeling of DNA, slide preparation, and hybridization were performed as described [17] with minor modifications. Analysis of the microarray images were performed with the GenePix Pro 4.0 software (Axon Instruments, Foster City, CA). For each spot, the median pixel intensity minus the median local background for both dyes was used to obtain the ratio of test gene copy number to reference gene copy number. Data normalization was performed for each array subgrid using lowess curve fitting with a smoothing factor of 0.33 [18]. The sex chromosomes were excluded when calculating the correction factor in the normalization of the data set. All normalizations and analyses were performed in the Bioarray Software Environment database (BASE) [19]. To identify imbalances, the MATLAB toolbox CGH plotter and the TM4 software suite [20] was applied, using a moving mean average over three clones and log2 limits of ≥0.2. Classification as gain or loss was based on identification as such by the CGH plotter and also by visual inspection of the log2 ratios. Ratios +/- ≥0.5 in three adjacent clones were classified as abnormal, with ratios between 0.5 and 1.0 interpreted as duplications/hemizygous deletions and ≥1.0 classified as amplifications/homozygous deletions.

In this study we used array CGH to delineate the breakpoint to position 93.86 Mb on chromosome 15. This was consistent with the preceding FISH analysis that localized the most proximally deleted BAC as clone RP11-79C10, positioned within subband 15q26.2, and encompassing the area detected by array CGH [21]. This approach of using tiling BACs to cover the whole genome in a single hybridization gives a resolution standard of 150–350 kb, applying the exclusion criteria that three contiguous clones must display abnormal log2 ratios to be considered to signal an aberration. This study, characterizing in detail a constitutional 15q26-deletion, is one of the first ones using molecular cytogenetic characterization with submegabase resolution on a syndrome related to this chromosomal region. Klaassens et al. (2005) used a similar approach relying on array CGH and FISH, but focused on cases presenting with distal 15q breakpoints resulting from ring chromosome formation or unbalanced translocations to determine a critical region associated with congenital diaphragmatic hernia (CDH). The platform used was a 1-Mb Human BAC array, giving resolution standards tenfold smaller than 32 k arrays [11]. However, a later study used array techniques with submegabase resolution on a similar patient [22]. In addition Slavotinek et al. (2005) have previously identified microdeletions at 15q26.2 to be associated with Fryns syndrome, a CDH-related autosomal recessive syndrome with relatively homogenous phenotype, using 2.4 k array [14].

The origin of the dicentric chromosome in the present case remains unknown since both parents displayed normal karyotypes and directed microsatellite analysis was not performed. However, it has been demonstrated that a de novo 15q-deletion can be of both paternal and maternal origin [4,6]. Studies on terminal chromosomal deletions have suggested that de novo telomere addition, by telomerase activity or recombinant mechanisms, work to stabilize the affected chromosome [23]. It could be speculated that the latter event might explain the formation of the dic(15) in the present case, but involving centromeres rather than telomeres. It is noteworthy that no duplication of near-centromeric q-arm material could be detected on the log2 ratio plot of chromosome 15 (Figure (Figure4).4). The preceding FISH analysis had confirmed that the duplication included the centromeric region, as well as p-arm material, both being located in regions not covered by the array.

The vast majority of patients with deletions of distal 15q have displayed pre- and postnatal growth retardation, cardiac defects, developmental delay, ear abnormalities, and clinodactyly. A variety of other different dysmorphic features have however been reported in association with 15q26 deletions such as café-au-lait spots, cholestatic liver disease, diaphragmatic hernia, eye and face abnormalities, equinovarus, high arched palate, hypertelorism, hypoplastic kidneys, hypotonia, lung hypoplasia, microcephaly, micrognathia, and spinal cord abnormality. Although high resolution cytogenetic mapping for all reported cases are lacking this wide range of phenotypic manifestations probably reflect size heterogeneity of distal 15q-deletions. More precise data on 15q-breakpoints might enable an elucidation of different candidate regions for different phenotypic manifestations. For instance, patients suffering from CDH in association with 15q26-deletion have been extensively studied and a critical region associated with CDH was assessed to a region ranging from distal 15q26.1 to proximal 15q26.2 [11]. The 15q-breakpoint in the present case mapped outside of the suggested critical region and in accordance did not suffer from CDH. Deletions of 15q26 have also been reported in association with Russel-Silver syndrome, which share many of the clinical features displayed in patients with deletions of distal 15q and it is possible that these cases should be categorized as an subgroup of the latter syndrome [24,25].

Considering all the reported cases with deletion of 15q26, the salient finding must be that the majority report loss of IGFR1, a gene that contributes to a wide range of developmental processes [26]. The relation between IGFR1 and pre- and postnatal growth in association with distal 15q-deletions has been studied without conclusive results [5,8]. In relation to the developmental delay commonly seen in patients with distal 15q-deletions, the IGF1 receptor gene plays an important function in the development of the central nervous system, which could partly explain this feature [27]. Studies on the effect of IGFR1 on the cardiovascular system indicate that it is an important player in heart development [24]. Monozygosity of this gene could therefore partly contribute to the complex cardiac defects so often seen in patients with this deletion. Obviously, all suggested clinical features postulated to relate to loss of one copy of the IGFR gene remains to be elucidated, preferentially by further gene expression studies on diagnostic material.

MEF2A have been suggested as a candidate gene for the cardiac defects [11] included in the phenotype of 15q26-deletions since the gene encode for DNA-binding regulatory protein that enhances the differentiation of mesodermal precursor cells to myoblast and therefore is key player in cardiac myocyte development [28]. The MEF2-family is an ancient mediator of signal-dependant transcription and diverse developmental programs and MEF2A was quite recently demonstrated to be involved in morphogenesis of postsynaptic neurons [29] which implicate that it could also contribute to the developmental delay seen in the patient.

AC performed the FISH analyses and drafted the manuscript.

GB treated the patient and wrote the clinical case report

MS participated in genetic counseling, designed the study and drafted the clinical case report.