Ines Müller

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

The genome of Erwinia tasmaniensis strain Et1/99, a non-pathogenic bacterium in the genus Erwinia

The complete genome of the bacterium Erwinia tasmaniensis strain Et1/99 consisting of a 3.9 Mb circular chromosome and five plasmids was sequenced. Strain Et1/99 represents an epiphytic plant bacterium related to Erwinia amylovora and E. pyrifoliae, which are responsible for the important plant diseases fire blight and Asian pear shoot blight, respectively. Strain Et1/99 is a non-pathogenic bacterium and is thought to compete with these and other bacteria when occupying the same habitat during initial colonization. Genome analysis revealed tools for colonization, cellular communication and defence modulation, as well as genes coding for the synthesis of levan and a not detected capsular exopolysaccharide. Strain Et1/99 may secrete indole-3-acetic acid to increase availability of nutrients provided on plant surfaces. These nutrients are subsequently accessed and metabolized. Secretion systems include the hypersensitive response type III pathway present in many pathogens. Differences or missing parts within the virulence-related factors distinguish strain Et1/99 from pathogens such as Pectobacterium atrosepticum and the related Erwinia spp. Strain Et1/99 completely lacks the sorbitol operon, which may also affect its inability to invade fire blight host plants. Erwinia amylovora in contrast depends for virulence on utilization of sorbitol, the dominant carbohydrate in rosaceous plants. The presence of other virulence-associated factors in strain Et1/99 indicates the ancestral genomic background of many plant-associated bacteria.

Comprehensive genotyping and clinical characterisation reveal 27 novel NKX2-1 mutations and expand the phenotypic spectrum

Background NKX2-1 encodes a transcription factor with large impact on the development of brain, lung and thyroid. Germline mutations of NKX2-1 can lead to dysfunction and malformations of these organs. Starting from the largest coherent collection of patients with a suspected phenotype to date, we systematically evaluated frequency, quality and spectrum of phenotypic consequences of NKX2-1 mutations. Methods After identifying mutations by Sanger sequencing and array CGH, we comprehensively reanalysed the phenotype of affected patients and their relatives. We employed electrophoretic mobility shift assay (EMSA) to detect alterations of NKX2-1 DNA binding. Gene expression was monitored by means of in situ hybridisation and compared with the expression level of MBIP, a candidate gene presumably involved in the disorders and closely located in close genomic proximity to NKX2-1. Results Within 101 index patients, we detected 17 point mutations and 10 deletions. Neurological symptoms were the most consistent finding (100%), followed by lung affection (78%) and thyroidal dysfunction (75%). Novel symptoms associated with NKX2-1 mutations comprise abnormal height, bouts of fever and cardiac septum defects. In contrast to previous reports, our data suggest that missense mutations in the homeodomain of NKX2-1 not necessarily modify its DNA binding capacity and that this specific type of mutations may be associated with mild pulmonary phenotypes such as asthma. Two deletions did not include NKX2-1, but MBIP, whose expression spatially and temporarily coincides with NKX2-1 in early murine development. Conclusions The high incidence of NKX2-1 mutations strongly recommends the routine screen for mutations in patients with corresponding symptoms. However, this analysis should not be confined to the exonic sequence alone, but should take advantage of affordable NGS technology to expand the target to adjacent regulatory sequences and the NKX2-1 interactome in order to maximise the yield of this diagnostic effort.

Expression of mouse Tbx22 supports its role in palatogenesis and glossogenesis

TBX22 belongs to the T‐box family of transcription factors and was originally found in an in silico approach designed to identify new genes on the human Xq12‐q21 region. Mutations in TBX22 have been reported in families with X‐linked cleft palate and ankyloglossia (CPX), but the underlying pathogenetic mechanism remained unknown. We have identified mouse Tbx22 and analyzed its expression during embryogenesis by reverse transcriptase‐polymerase chain reaction and in situ hybridization. In mouse embryos, it is expressed in distinct areas of the head, namely the mesenchyme of the inferior nasal septum, the posterior palatal shelf before fusion, the attachment of the tongue, and mesenchymal cells surrounding the eye anlage. The localization in the tongue frenulum perfectly correlates with the ankyloglossia phenotype in CPX. Furthermore, we identified positionally conserved binding sites for transcription factors, two of which have been implicated previously in palatogenesis (MSX1, PRX2). Developmental Dynamics 226:579–586, 2003.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

ABSTRACT Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the worlds largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.

Absence of TRIF Signaling in Lipopolysaccharide-Stimulated Murine Mast Cells

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

Chromosome aberrations involving 10q22: report of three overlapping interstitial deletions and a balanced translocation disrupting C10orf11.

Interstitial deletions of chromosome band 10q22 are rare. We report on the characterization of three overlapping de novo 10q22 deletions by high-resolution array comparative genomic hybridization in three unrelated patients. Patient 1 had a 7.9 Mb deletion in 10q21.3–q22.2 and suffered from severe feeding problems, facial dysmorphisms and profound mental retardation. Patients 2 and 3 had nearly identical deletions of 3.2 and 3.6 Mb, the proximal breakpoints of which were located at an identical low-copy repeat. Both patients were mentally retarded; patient 3 also suffered from growth retardation and hypotonia. We also report on the results of breakpoint analysis by array painting in a mentally retarded patient with a balanced chromosome translocation 46,XY,t(10;13)(q22;p13)dn. The breakpoint in 10q22 was found to disrupt C10orf11, a brain-expressed gene in the common deleted interval of patients 1–3. This finding suggests that haploinsufficiency of C10orf11 contributes to the cognitive defects in 10q22 deletion patients.

Identification of candidate genes for sporadic amyotrophic lateral sclerosis by array comparative genomic hybridization.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder of the central nervous system that leads to progressive loss of upper and lower motor neurons. Most cases are sporadic and of unknown aetiology. In this study, we screened 72 patients with sporadic ALS for the presence of DNA copy number variations, in order to identify novel candidate disease genes. We have used sub-megabase resolution BAC array comparative genomic hybridization to detect genomic imbalances in our ALS patient cohort. Aberrations with potential relevance for disease aetiology were verified by oligo array CGH. In 72 patients with sporadic ALS, we identified a total of six duplications and five deletions that scored above our threshold. Nine of these 11 variations were smaller than 1Mb, and five were observed exclusively in ALS patients. In conclusion, non-polymorphic sub-microscopic duplications and deletions observable by array CGH are frequent in patients with sporadic ALS. Analysis of such aberrations serves as a starting point in deciphering the aetiology of this complex disease, given that affected genes can be considered candidates for influencing disease susceptibility.

CDKL5 truncation due to a t(X;2)(p22.1;p25.3) in a girl with X-linked infantile spasm syndrome

To the Editor : X-linked infantile spasm syndrome (ISSX; OMIM 308350) occurs in 2–5 of 10,000 newborns and is characterized by early onset seizures, hypsarrhythmia, and severe mental retardation. Three girls with ISSX and an X;autosome translocation that disrupted the CDKL5 gene have been reported (1–3), whereas ∼50 different CDKL5 mutations have been found in >60 clinically similar patients with normal chromosomes (4–15). We report a girl with severe ISSX and a t(X;2) disrupting the CDKL5 gene in intron 3 and fusing CDKL5 and RPS7 genes. The proposita (Fig. 1a,b) was born to a 35year-old mother and a 45-year-old father. Since the age of 1 month, she presented myoclonic seizures with cyanosis that were partially controlled with several drugs. Her psychomotor development has been profoundly delayed. At the age of 2 years, she exhibited generalized hypotonia, lack of eye contact, poor pupilar reflex, weight of 7300 g [−5.5 standard deviation (SD)], length of 79 cm (−4.5 SD), occipital-frontal circumference (OFC) of 45 cm (−3 SD/−2.5 SD), narrow forehead, frontal hypertrichosis, synophrys, upslanted palpebral fissures, broad nasal bridge, small nose and diminished muscle bulk. Evoked visual and auditory potentials were consistent with central blindness and visual pathway immaturity, but showed normal hearing. Cranial computed tomography scan revealed fronto-temporal cortical atrophy and corpus callosum hypoplasia (Fig. 1c). Repeated electroencephalograms disclosed generalized paroxysmal activity and disorganization. The patient and her mother were karyotyped on GTG-banded chromosomes; the former had a 46,X,t(X;2)(p22.1;p25.3) karyotype assumed to be de novo (Fig. 1d) as the mother was 46,XX and the unlikelihood of the father (not sampled) being a carrier. A preferential inactivation of the normal X was found in 30 RBG-banded metaphases and confirmed by the human androgen receptor assay (HUMARA) (16) that also revealed a maternal origin for this chromosome (data not shown); i.e. the der(X) was the paternal homologue. Genescan analysis confirmed a ∼90% inactivation of the maternal allele (MA) (Fig. 1e). The X-chromosome inactivation in the mother was at random. Fluorescence in situ hybridization (FISH) with clones RP11-558P14 (AL109798) and RP1245G19 (Z92542) revealed that the X-chromosome breakpoint disrupted CDKL5 (Fig. 2a). Reverse transcriptase-polymerase chain reaction (RT-PCR) on patient RNA showed products with CDKL5 primer pairs spanning exons 9–12 (Fig. 2c) and exons 13–18 (data not shown). No product could be obtained using primers located in exons 2 and 8 (Fig. 2c). These results let us hypothesize that the chromosome 2 breakpoint mapped also within a gene and the rearrangement had produced fusion genes. To test this, we have fine mapped the breakpoints by array painting as described previously (17, 18). The results indicated that on der(X) the breakpoint lies within CDKL5 intron 3 (chrX:18,447,901-18,460,135) (UCSC, hg18) and on der(2) in a region that contained the 5′ segment, including exons 1 and 2, of the RPS7 gene (chr2:3,599,632-3,601,126). RT-PCR with CDKL5 exon 2 forward primer and RPS7 reverse primers for amplifying fusion transcripts from the der(2) resulted in products containing CDKL5 exons 2 and 3 spliced to chromosome 2 position 3,600,514 within a single exon EST (expressed sequence tags) (DA710611), followed by RPS7 exons 1–6 (shown in Fig. 2d). This result let us to conclude that the single exon EST, or at least a part of it, presents an extension of the RPS7 5′UTR. Therefore, the ORF of RPS7, which codes for a ribosomal protein of the small subunit, is most likely not affected by the translocation but the CDKL5–RPS7 fusion gene is very likely transcribed from the CDKL5 promoter. For der(X), a similar approach for amplifying

Diagnosis of a terminal deletion of 4p with duplication of Xp22.31 in a patient with findings of Opitz G/BBB syndrome and Wolf–Hirschhorn syndrome†

Opitz G/BBB syndrome (OS) is a congenital midline malformation syndrome characterized by hypertelorism, hypospadias, cleft lip/palate, laryngotracheoesophageal abnormalities, imperforate anus, developmental delay and cardiac defects. The X‐linked form is caused by mutations in the MID1 gene, while no gene has yet been identified for the autosomal dominant form. Here, we report on a 15‐year‐old boy who was referred for MID1 mutation analysis with findings typical of OS, including apparent hypertelorism, hypospadias, a history of feeding difficulties, dysphagia secondary to esophageal arteria lusoria, growth retardation and developmental delay. No MID1 mutation was found, but subsequent sub‐megabase resolution array CGH unexpectedly documented a 2.34 Mb terminal 4p deletion, suggesting a diagnosis of WHS, and a duplication in Xp22.31. Wolf–Hirschhorn syndrome (WHS) is a contiguous gene deletion syndrome involving terminal chromosome 4p deletions, in particular 4p16.3. WHS is characterized by typical facial appearance (“Greek helmet facies”), mental retardation, congenital hypotonia, and growth retardation. While the severity of developmental delay in this patient supports the diagnosis of WHS rather than OS, this case illustrates the striking similarities of clinical findings in seemingly unrelated syndromes, suggesting common or interacting pathways at the molecular and pathogenetic level. This is the first report of arteria lusoria (esophageal vascular ring) in a patient with WHS.