Reinhilde Thoelen

Mosaicism del(8p)/inv dup(8p) in a dysmorphic female infant: a mosaic formed by a meiotic error at the 8p OR gene and an independent terminal deletion event

Various chromosomal rearrangements are associated with the distal 8p region. Among them are the inv dup(8p),1 del(8p22),2–5 and del(8)(pter).6 The cardinal phenotypic features of the inv dup(8p) are brain malformations, severe mental retardation with specific involvement of speech, and minor facial dysmorphisms.7 Deletions (8p22) are associated with congenital heart malformations, thought to be caused by haploinsufficiency of GATA4.2 Other features are microcephaly, intrauterine growth retardation, mental retardation, and a characteristic hyperactive impulsive behaviour. The patients with del(8p) present with variable severe malformations dependent on the size of the deletion. The largest terminal 8p deletion which has been detected in a liveborn is del(8)(pter→21.1).8 Molecular analysis of both 8p duplications and 8p interstitial deletions showed that all cases shared similar chromosomal break points. This finding led to the hypothesis that these rearrangements were caused by ectopic recombination at misaligned duplicons. Recently, Giglio et al 4 showed the presence of olfactory gene clusters ( OR clusters) at the sites where the interstitial 8p deletions occur. Whereas these interstitial deletions could thus be explained by misalignments of the OR clusters during meiosis, they also presented an elegant explanation for the origin of the 8p duplications. An inversion polymorphism between these OR clusters, present in 20% of the population, abrogates correct pairing between the OR clusters which causes susceptibility for an intrachromosomal crossover between the OR repeats. Ectopic recombination at these sites can lead to a dicentric intermediate which on breakage can lead to a duplicated chromosome 8p, the inv dup(8p). The complement of this breakage event was speculated to be a terminal deleted chromosome del(8p). In this paper we describe a girl with a mosaic del(8p)/inv dup(8p) in blood lymphocytes. Mosaics with two cell lines carrying two different rearranged chromosomes are extremely rare. Two reports describe the …

No evidence for a parental inversion polymorphism predisposing to rearrangements at 22q11.2 in the DiGeorge/Velocardiofacial syndrome

No evidence for a parental inversion polymorphism predisposing to rearrangements at 22q11.2 in the DiGeorge/Velocardiofacial syndrome

Direct fluorescent labelling of clones by DOP PCR

BackgroundArray Comparative Genomic Hybridisation (array CGH) is a powerful technique for the analysis of constitutional chromosomal anomalies. Chromosomal duplications or deletions detected by array CGH need subsequently to be validated by other methods. One method of validation is Fluorescence in situ Hybridisation (FISH). Traditionally, fluorophores or hapten labelling is performed by nick translation or random prime labelling of purified Bacterial Artificial Chromosome (BAC) products. However, since the array targets have been generated from Degenerate Oligonucleotide Primed (DOP) amplified BAC clones, we aimed to use these DOP amplified BAC clones as the basis of an automated FISH labelling protocol. Unfortunately, labelling of DOP amplified BAC clones by traditional labelling methods resulted in high levels of background.ResultsWe designed an improved labelling method, by means of degenerate oligonucleotides that resulted in optimal FISH probes with low background.ConclusionWe generated an improved labelling method for FISH which enables the rapid generation of FISH probes without the need for isolating BAC DNA. We labelled about 900 clones with this method with a success rate of 97%.

Biallelic inactivation of NF1 in a sporadic plexiform neurofibroma

Plexiform neurofibromas are a major cause of morbidity in individuals with neurofibromatosis type 1 (NF1). Sporadically, these tumors appear as an isolated feature without other signs of NF1. A role for the NF1 gene in solitary plexiform neurofibromas has never been described. In this study, we report a 13‐year‐old boy who was diagnosed with a plexiform neurofibroma, without other NF1 diagnostic criteria. The tumor was partially resected and analyzed using different techniques: karyotyping, fluorescence in situ hybridization (FISH), and microarray comparative genomic hybridization (aCGH). Tumor Schwann cell culture and subsequent karyotyping showed a rearrangement involving chromosomes 1 and 17, namely an insertion of chromosomal bands 1p36‐35 at 17q11.2. FISH demonstrated that the insertion interrupted the NF1 gene. In addition, a deletion was detected affecting the other NF1 allele. Whole‐genome aCGH analysis of the resected tumor confirmed the presence of an 8.28 Mb deletion including the NF1 gene locus in ∼15–20% of tumor cells. We conclude that biallelic NF1 inactivation was at the origin of the isolated plexiform neurofibroma in this patient. The insertion is most likely the “first hit” and the large deletion the “second hit.”

Detection of an unusual 17p13.3 microdeletion by array comparative genomic hybridisation in a patient with lissencephaly

To the Editor: The term lissencephaly (LIS) [MIM #607432], literally meaning smooth’ brain, refers to rare malformations with reduced gyration of the cerebral cortex (1). It is the result of defective neuronal migration occurring between weeks 12 and 16 of fetal development. Children with LIS have swallowing and feeding difficulties, severe seizures and psychomotor delay and sometimes dystonia. Brain magnetic resonance imaging (MRI) shows a thickened cortex (10–20 mm), and on microscopy, four rather than six layers are seen (2). LIS variants are characterized by extra-cortical anomalies including total or partial agenesis of the corpus callosum and severe cerebellar hypoplasia, mainly of the vermis (3). Five genes have been identified that cause or contribute to LIS in humans: LIS1 (17p13.3) (4), 14-3-3e (17p13.3) (5), DCX (Xq22.3-23) (6, 7), RELN (7q22) (8), and ARX (Xp22.13) (9). We report a female patient with isolated LIS and an unusual deletion of the LIS1 gene. She was born at term to healthy non-related parents after a normal pregnancy. There was no family history of developmental or neurological disorders. From early childhood, she developed severe epilepsy and psychomotor delay. She never learned to talk or walk and is a wheelchair user. General physical examination showed a narrow forehead, a high nasal bridge, a short upper lip and a small mouth with downturned corners (Fig. 1a). Her head circumference was below the 3rd centile. Brain MRI showed LIS grade 3 with agyria of the occipital lobes transitioning to pachygyria anteriorly, with a cell-sparse layer most prominent posteriorly. In addition, there was a mild dilation of the posterior horns of the lateral ventricles (10). High-resolution G-banding karyotype and fluorescence in situ hybridisation (FISH) investigations carried out using the Smith–Magenis/ Miller–Dieker diagnostic dual commercial probe (Cytocell LPU077, Cytocell Technologies Ltd, Cambridge, UK) were normal. Subsequent sequencing of the LIS1 and DCX genes did not show any mutation. Using array comparative genomic hybridisation (CGH) at a 1 Mb resolution as previously described (11, 12), we detected a deletion of BAC clone RP11-135N5 in the Miller–Dieker syndrome (MDS) critical region on chromosome 17p13.3 (Fig. 1b). This deletion encompasses the 5#UTR and exons 1 and 2 of the LIS1 gene. Subsequent FISH analysis using the RP11-135N5 clone confirmed the deletion of maximal size 1.4 Mb on one chromosome 17 (Fig. 1c). Neither parent had the deletion. Subsequent screening with the RP11-135N5 probe of 15 patients with the same clinical condition (LIS without a microdeletion, using the commercial FISH probe, and with no mutation in LIS1 or DCX) did not detect this deletion. In comparison with the case reported by Cardoso et al. (13), the deletion in our patient is much smaller and does not extend further towards the telomere of chromosome 17p. Patients with MDS have more severe LIS, suggesting that other genes distal to LIS1 might be involved in brain development and in producing the characteristic MDS dysmorphism (1, 4, 14). Toyo-oka et al. (5) found the 14-3-3e gene to be the best candidate for the more pronounced phenotype in MDS by demonstrating that mice compound heterozygous for 14-3-3e and LIS1 show more severe defects of neuronal migration than either heterozygous mutant mouse alone. The small deleted region in this patient does not encompass the 14-3-3e gene and could explain the milder phenotype of isolated LIS. The atypical deletion found probably represents a sporadic finding because it was not found in 15 other patients with the same clinical