Transcriptome of iPSC-derived neuronal cells reveals a module of co-expressed genes consistently associated with autism spectrum disorder.

Evaluation of expression profile in autism spectrum disorder (ASD) patients is an important approach to understand possible similar functional consequences that may underlie disease pathophysiology regardless of its genetic heterogeneity. Induced pluripotent stem cell (iPSC)-derived neuronal models have been useful to explore this question, but larger cohorts and different ASD endophenotypes still need to be investigated. Moreover, whether changes seen in this in vitro model reflect previous findings in ASD postmortem brains and how consistent they are across the studies remain underexplored questions. We examined the transcriptome of iPSC-derived neuronal cells from a normocephalic ASD cohort composed mostly of high-functioning individuals and from non-ASD individuals. ASD patients presented expression dysregulation of a module of co-expressed genes involved in protein synthesis in neuronal progenitor cells (NPC), and a module of genes related to synapse/neurotransmission and a module related to translation in neurons. Proteomic analysis in NPC revealed potential molecular links between the modules dysregulated in NPC and in neurons. Remarkably, the comparison of our results to a series of transcriptome studies revealed that the module related to synapse has been consistently found as upregulated in iPSC-derived neurons-which has an expression profile more closely related to fetal brain-while downregulated in postmortem brain tissue, indicating a reliable association of this network to the disease and suggesting that its dysregulation might occur in different directions across development in ASD individuals. Therefore, the expression pattern of this network might be used as biomarker for ASD and should be experimentally explored as a therapeutic target.

Two novel pathogenic variants in MED13L: one familial and one isolated case.

BACKGROUND: Genetic variants involving the MED13L gene can lead to an autosomal dominant syndrome characterised by intellectual disability/developmental delay and facial dysmorphism. METHODS: We investigated two cases (one familial and one isolated) of intellectual disability with speech delay and dysmorphic facial features by whole-exome sequencing analyses. Further, we performed a literature review about clinical and molecular aspects of MED13L gene and syndrome. RESULTS: Two MED13L variants have been identified [MED13L(NM_015335.5):c.4417C>T and MED13L(NM_015335.5):c.2318delC] and were classified as pathogenic according to the ACMG (American College of Medical Genetics and Genomics) guidelines. One of the variants was present in sibs. CONCLUSIONS: The two pathogenic variants identified have not been previously reported. Importantly, this is the first report of a familial case of MED13L nonsense mutation. Although the parents of the affected children were no longer available for analysis, their apparently normal phenotypes were surmised from familial verbal descriptions corresponding to normal mental behaviour and phenotype. In this situation, the familial component of mutation transmission might be caused by gonadal mosaicism of a MED13L mutation in a gonad from either the father or the mother. The case reports and the literature review presented in this manuscript can be useful for genetic counselling.

Esophageal IgG4 levels correlate with histopathologic and transcriptomic features in eosinophilic esophagitis.

BACKGROUND: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses. METHODS: This case-control study included pediatric subjects with EoE (≥15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts. RESULTS: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs 0.2 mg/g protein [0.1-0.9]; P < .0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = .0006); histologic grade (P = .0011) and stage (P = .0112) scores; and IL4, IL10, IL13, but not TGFB1, expression and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression. CONCLUSION: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease; is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines; and is likely produced locally.

Genomic copy number alterations in non-syndromic hearing loss.

Genetic heterogeneity has made the identification of genes related to hearing impairment a challenge. In the absence of a clear phenotypic aetiology, recurrence risk estimates are often based on family segregation and may be imprecise. We profiled by oligonucleotide array-CGH patients presenting non-syndromic hearing loss with presumptive autosomal recessive (n = 50) or autosomal dominant (n = 50) patterns of inheritance. Rare copy number variants (CNVs) were detected in 12 probands; four of the detected CNVs comprised genes previously associated with hearing loss (POU4F3, EYA4, USH2A, and BCAP31) and were considered causative, stressing the contribution of genomic imbalance to non-syndromic deafness. In six cases, segregation of the CNVs in pedigrees excluded them as causative. In one case, segregation could not be investigated, while in another case, a point mutation likely explains the phenotype. These findings show that the presumptive patterns of inheritance were incorrect in at least two cases, thereby impacting genetic counselling. In addition, we report the first duplication reciprocal to the rare ABCD1, BCAP31, and SLC6A8 contiguous deletion syndrome; as with most microduplication syndromes, the associated phenotype is much milder than the respective microdeletion and, in this case, was restricted to hearing impairment.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.

BACKGROUND: Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype-phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. METHODS: Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. RESULTS: The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype-phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. CONCLUSION: A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

High resolution DNA fiber-fish on yeast artificial chromosomes: direct visualization of DNA replication.

Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as 'Fiber-FISH', increase FISH-resolution to the DNA fiber, using decondensed nuclear DNA as hybridization target. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes, FISH has not been used successfully on yeast/YAC DNA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DNA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DNA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DNA cloned in yeast is capable of initiating its own replication.

[Imaging strategies for knee injuries]. / Bildgebungsstrategie bei Kniegelenkverletzungen.

Injuries of the knees are common. The Ottawa knee rule provides decisional support to determine whether radiographs are indicated or not. With the use of ultrasound it is possible to detect defects of the extensor ligaments and the anterior cruciate ligament. Furthermore, it is possible to detect indirect signs of an intra-articular fracture, e.g. lipohemarthrosis. In complex fractures, e.g. tibial plateau fractures, further diagnostic procedures with multislice computed tomography (CT) are needed for accurate classification and preoperative planning. Multislice CT with CT angiography enables three-dimensional reconstruction of the knee and non-invasive vascular imaging for detection of vascular injury. Magnetic resonance imaging (MRI) is the gold standard for detection of occult fractures and injuries of the ligaments and menisci. Higher field strengths can be used to improve the diagnostics of cartilage lesions. Virtual MR arthrography is superior to conventional MRI for detection of cartilage lesions especially after meniscus surgery.

[Laser ablation. Do we still need it?]. / Laserablation. Brauchen wir sie noch?

Laser ablation (LA) is momentarily the only invasive ablation procedure besides radiofrequency ablation (RFA) which can be performed entirely under magnetic resonance imaging (MRI) guidance. The long-term outcome and morbidity profiles are broadly identical for both modalities, excluding the RFA-specific prevalence for skin burns. The technical and logistic disadvantages of LA have been overcome since the introduction of miniaturized two-component applicators. The main advantage of LA is its superior MRI compatibility. Interference-free imaging during LA allows MR thermometric real-time therapy control without the need for RF filters. High-resolution thermometry in the target zone only makes sense without the extinction artifact of a metal probe and this condition is met only by the glass fibers of LA. An independent therapy monitoring is crucial in modern scenarios of oncologic quality management.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile.

INTRODUCTION: We hypothesised that gene expression in histologically normal (HN) epithelium (NlEpi) would differ between breast cancer patients and usual-risk controls undergoing reduction mammoplasty (RM), and that gene expression in NlEpi from cancer-free prophylactic mastectomy (PM) samples from high-risk women would resemble HN gene expression. METHODS: We analysed gene expression in 73 NlEpi samples microdissected from frozen tissue. In 42 samples, we used microarrays to compare gene expression between 18 RM patients and 18 age-matched HN (9 oestrogen receptor (ER)+, 9 ER-) and 6 PM patients. Data were analysed using a Bayesian approach (BADGE), and validated with quantitative real-time PCR (qPCR) in 31 independent NlEpi samples from 8 RM, 17 HN, and 6 PM patients. RESULTS: A total of 98 probe sets (86 genes) were differentially expressed between RM and HN samples. Performing hierarchical analysis with these 98 probe sets, PM and HN samples clustered together, away from RM samples. qPCR validation of independent samples was high (84%) and uniform in RM compared with HN patients, and lower (58%), but more heterogeneous, in RM compared with PM patients. The 86 genes were implicated in many processes including transcription and the MAPK pathway. CONCLUSION: Gene expression differs between the NlEpi of breast cancer cases and controls. The profile of cancer cases can be discerned in high-risk NlEpi from cancer-free breasts. This suggests that the profile is not an effect of the tumour, but may mark increased risk and reveal the earliest genomic changes of breast cancer.

Constitutional haploinsufficiency of tumor suppressor genes in mentally retarded patients with microdeletions in 17p13.1.

Chromosome microdeletions or duplications are detected in 10-20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of approximately 180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (approximately 1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer.

Chromosome imbalances in syndromic hearing loss.

The cause of hearing impairment has not been elucidated in a large proportion of patients. We screened by 1-Mb array-based comparative genomic hybridization (aCGH) 29 individuals with syndromic hearing impairment whose clinical features were not typical of known disorders. Rare chromosomal copy number changes were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage-sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2-q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but its role as a predisposition gene remains a possibility. Our results show that syndromic deafness is frequently associated with chromosome microimbalances (14-27%), and the use of aCGH for defining disease etiology is recommended.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Genomic imbalances associated with mullerian aplasia.

BACKGROUND: Aplasia of the müllerian ducts leads to absence of the uterine corpus, uterine cervix, and upper (superior) vagina. Patients with müllerian aplasia (MA) often exhibit additional clinical features such as renal, vertebral and cardiac defects. A number of different syndromes have been associated with MA, and in most cases its aetiology remains poorly understood. OBJECTIVE AND METHODS: 14 syndromic patients with MA and 46,XX G-banded karyotype were screened for DNA copy number changes by approximately 1 Mb whole genome bacterial artificial chromosome (BAC) array based comparative genomic hybridisation (CGH). The detected alterations were validated by an independent method and further mapped by high resolution oligo-arrays. RESULTS: Submicroscopic genomic imbalances affecting the 1q21.1, 17q12, 22q11.21, and Xq21.31 chromosome regions were detected in four probands. Presence of the alterations in the normal mother of one patient suggests incomplete penetrance and/or variable expressivity. CONCLUSION: 4 of the 14 patients (29%) were found to have cryptic genomic alterations. The imbalances on 22q11.21 support recent findings by us and others that alterations in this chromosome region may result in impairment of müllerian duct development. The remaining imbalances indicate involvement of previously unknown chromosome regions in MA, and point specifically to LHX1 and KLHL4 as candidate genes.

High frequency of submicroscopic chromosomal imbalances in patients with syndromic craniosynostosis detected by a combined approach of microsatellite segregation analysis, multiplex ligation-dependent probe amplification and array-based comparative genome hybridisation.

We present the first comprehensive study, to our knowledge, on genomic chromosomal analysis in syndromic craniosynostosis. In total, 45 patients with craniosynostotic disorders were screened with a variety of methods including conventional karyotype, microsatellite segregation analysis, subtelomeric multiplex ligation-dependent probe amplification) and whole-genome array-based comparative genome hybridisation. Causative abnormalities were present in 42.2% (19/45) of the samples, and 27.8% (10/36) of the patients with normal conventional karyotype carried submicroscopic imbalances. Our results include a wide variety of imbalances and point to novel chromosomal regions associated with craniosynostosis. The high incidence of pure duplications or trisomies suggests that these are important mechanisms in craniosynostosis, particularly in cases involving the metopic suture.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Tamoxifen's protection against breast cancer recurrence is not reduced by concurrent use of the SSRI citalopram.

Tamoxifen remains an important adjuvant therapy to reduce the rate of breast cancer recurrence among patients with oestrogen-receptor-positive tumours. Cytochrome P-450 2D6 metabolizes tamoxifen to metabolites that more readily bind the oestrogen receptor. This enzyme also metabolizes selective serotonin reuptake inhibitors (SSRI), so these widely used drugs - when taken concurrently - may reduce tamoxifen's prevention of breast cancer recurrence. We studied citalopram use in 184 cases of breast cancer recurrence and 184 matched controls without recurrence after equivalent follow-up. Cases and controls were nested in a population of female residents of Northern Denmark with stages I-III oestrogen-receptor-positive breast cancer 1985-2001 and who took tamoxifen for 1, 2, or most often for 5 years. We ascertained prescription histories by linking participants' central personal registry numbers to prescription databases from the National Health Service. Seventeen cases (9%) and 21 controls (11%) received at least one prescription for the SSRI citalopram while taking tamoxifen (adjusted conditional odds ratio=0.85, 95% confidence interval=0.42, 1.7). We also observed no reduction of tamoxifen effectiveness among regular citalopram users (>or=30% overlap with tamoxifen use). These results suggest that concurrent use of citalopram does not reduce tamoxifen's prevention of breast cancer recurrence.

Brief Report: Self-Injurious Behaviors in Preschool Children with Autism Spectrum Disorder Compared to Other Developmental Delays and Disorders.

We compared the prevalence of self-injurious behaviors (SIB) in preschoolers aged 30-68 months with autism spectrum disorder (ASD) (n = 691) versus other developmental delays and disorders (DD) (n = 977) accounting for sociodemographic, cognitive, and medical factors. SIB prevalence was higher in ASD versus all DD [adjusted odds-ratio (aOR) 2.13 (95% confidence interval (95% CI) 1.53, 2.97)]. In subgroup analyses, SIB prevalence was higher in ASD versus DD without ASD symptoms [aOR 4.42 (95% CI 2.66, 7.33)], but was similar between ASD and DD with ASD symptoms [aOR 1.09 (95% CI 0.68, 1.77)]. We confirmed higher prevalence of SIB in ASD versus DD, independent of confounders. In children with DD, SIB prevalence increased with more ASD symptoms. These findings are informative to clinicians, researchers, and policymakers.

Detection of monoclonal microsatellite alterations in atypical breast hyperplasia.

Atypical hyperplastic (AH) breast lesions are currently classified and treated as benign proliferative disorders, but their presence is associated with a four- to fivefold increased risk of developing breast cancer. Currently, it is not known if an AH lesion is a marker of increased risk, or is itself a premalignant lesion. To investigate this question, we used a series of 15 microsatellite loci to analyze 15 separate AH lesions microdissected from the archived pathology specimens of subjects with no coincident or previous breast malignancy. We found that a significant subset (6/15, or 40%) of these AH lesions demonstrated evidence of monoclonal microsatellite alterations, both length variation and allele loss. These monoclonal alterations suggest that the AH lesion has already undergone genetic changes conferring a growth advantage. Thus, these AH lesions may actually be early neoplasms. We also noted that monoclonality characterized AH lesions in younger as compared with older women (44 vs. 59 yrs, P < 0.05) and that a subset of monoclonal lesions (4/6) demonstrated microsatellite alterations at more than one locus, suggesting that an undetermined type of genetic instability may play a role early in the development of abnormal breast proliferations. These findings contribute to our understanding of the pathogenesis of AH lesions and may have implications regarding their relationship to breast tumors.

A 6Mb deletion in band 2q22 due to a complex chromosome rearrangement associated with severe psychomotor retardation, microcephaly and distinctive dysmorphic facial features.

High-resolution analyses of complex chromosome rearrangements (CCR) have demonstrated in individuals with abnormal phenotypes that not all seemingly balanced CCRs based on G-banding are completely balanced at breakpoint level. Here we report on an apparently balanced de novo CCR involving chromosomes 2, 3 and 5 present in a 6-month-old girl. She was referred for genetic evaluation because of severe psychomotor retardation, distinctive dysmorphic features and microcephaly. A 1Mb resolution array-CGH analysis of DNA from the patient revealed a deletion of about 6Mb for chromosome 2. FISH analysis showed that the deletion interval found in band 2q22 mapped at the translocation breakpoint, and that the ZFHX1B gene, which is known to be involved in the Mowat-Wilson syndrome, is located within the deletion interval. To our knowledge this is the first case of a complex chromosomal rearrangement associated with Mowat-Wilson syndrome. Our data illustrate the important role for high-resolution investigation of apparently balanced chromosome rearrangements in patients with unexplained psychomotor retardation and/or other clinical features, and should contribute to our understanding of the mechanisms involved in chromosome rearrangement.