Transcriptome analysis of MBD5-associated neurodevelopmental disorder (MAND) neural progenitor cells reveals dysregulation of autism-associated genes.

MBD5-associated neurodevelopmental disorder (MAND) is an autism spectrum disorder (ASD) characterized by intellectual disability, motor delay, speech impairment and behavioral problems; however, the biological role of methyl-CpG-binding domain 5, MBD5, in neurodevelopment and ASD remains largely undefined. Hence, we created neural progenitor cells (NPC) derived from individuals with chromosome 2q23.1 deletion and conducted RNA-seq to identify differentially expressed genes (DEGs) and the biological processes and pathways altered in MAND. Primary skin fibroblasts from three unrelated individuals with MAND and four unrelated controls were converted into induced pluripotent stem cell (iPSC) lines, followed by directed differentiation of iPSC to NPC. Transcriptome analysis of MAND NPC revealed 468 DEGs (q < 0.05), including 20 ASD-associated genes. Comparison of DEGs in MAND with SFARI syndromic autism genes revealed a striking significant overlap in biological processes commonly altered in neurodevelopmental phenotypes, with TGFß, Hippo signaling, DNA replication, and cell cycle among the top enriched pathways. Overall, these transcriptome deviations provide potential connections to the overlapping neurocognitive and neuropsychiatric phenotypes associated with key high-risk ASD genes, including chromatin modifiers and epigenetic modulators, that play significant roles in these disease states.

Further delineation of the clinical spectrum of KAT6B disorders and allelic series of pathogenic variants.

PURPOSE: Genitopatellar syndrome and Say-Barber-Biesecker-Young-Simpson syndrome are caused by variants in the KAT6B gene and are part of a broad clinical spectrum called KAT6B disorders, whose variable expressivity is increasingly being recognized. METHODS: We herein present the phenotypes of 32 previously unreported individuals with a molecularly confirmed diagnosis of a KAT6B disorder, report 24 new pathogenic KAT6B variants, and review phenotypic information available on all published individuals with this condition. We also suggest a classification of clinical subtypes within the KAT6B disorder spectrum. RESULTS: We demonstrate that cerebral anomalies, optic nerve hypoplasia, neurobehavioral difficulties, and distal limb anomalies other than long thumbs and great toes, such as polydactyly, are more frequently observed than initially reported. Intestinal malrotation and its serious consequences can be present in affected individuals. Additionally, we identified four children with Pierre Robin sequence, four individuals who had increased nuchal translucency/cystic hygroma prenatally, and two fetuses with severe renal anomalies leading to renal failure. We also report an individual in which a pathogenic variant was inherited from a mildly affected parent. CONCLUSION: Our work provides a comprehensive review and expansion of the genotypic and phenotypic spectrum of KAT6B disorders that will assist clinicians in the assessment, counseling, and management of affected individuals.

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations.

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.

Triangulating the sexually dimorphic brain through high-resolution neuroimaging of murine sex chromosome aneuploidies.

Murine sex chromosome aneuploidies (SCAs) provide powerful models for charting sex chromosome influences on mammalian brain development. Here, building on prior work in X-monosomic (XO) mice, we use spatially non-biased high-resolution imaging to compare and contrast neuroanatomical alterations in XXY and XO mice relative to their wild-type XX and XY littermates. First, we show that carriage of a supernumerary X chromosome in XXY males (1) does not prevent normative volumetric masculinization of the bed nucleus of the stria terminalis (BNST) and medial amygdala, but (2) causes distributed anatomical alterations relative to XY males, which show a statistically unexpected tendency to be co-localized with and reciprocal to XO-XX differences in anatomy. These overlaps identify the lateral septum, BNST, ventral group thalamic nuclei and periaqueductal gray matter as regions with replicable sensitivity to X chromosome dose across two SCAs. We then harness anatomical variation across all four karyotype groups in our study--XO, XX, XY and XXY--to create an agnostic data-driven segmentation of the mouse brain into five distributed clusters which (1) recover fundamental properties of brain organization with high spatial precision, (2) define two previously uncharacterized systems of relative volume excess in females vs. males ("forebrain cholinergic" and "cerebelo-pontine-thalamo-cortical"), and (3) adopt stereotyped spatial motifs which delineate ordered gradients of sex chromosome and gonadal influences on volumetric brain development. Taken together, these data provide a new framework for the study of sexually dimorphic influences on brain development in health and disrupted brain development in SCA.

Molecular and clinical analyses of 16q24.1 duplications involving FOXF1 identify an evolutionarily unstable large minisatellite.

BACKGROUND: Point mutations or genomic deletions of FOXF1 result in a lethal developmental lung disease Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins. However, the clinical consequences of the constitutively increased dosage of FOXF1 are unknown. METHODS: Copy-number variations and their parental origin were identified using a combination of array CGH, long-range PCR, DNA sequencing, and microsatellite analyses. Minisatellite sequences across different species were compared using a gready clustering algorithm and genome-wide analysis of the distribution of minisatellite sequences was performed using R statistical software. RESULTS: We report four unrelated families with 16q24.1 duplications encompassing entire FOXF1. In a 4-year-old boy with speech delay and a café-au-lait macule, we identified an ~15 kb 16q24.1 duplication inherited from the reportedly healthy father, in addition to a de novo ~1.09 Mb mosaic 17q11.2 NF1 deletion. In a 13-year-old patient with autism and mood disorder, we found an ~0.3 Mb duplication harboring FOXF1 and an ~0.5 Mb 16q23.3 duplication, both inherited from the father with bipolar disorder. In a 47-year old patient with pyloric stenosis, mesenterium commune, and aplasia of the appendix, we identified an ~0.4 Mb duplication in 16q24.1 encompassing 16 genes including FOXF1. The patient transmitted the duplication to her daughter, who presented with similar symptoms. In a fourth patient with speech and motor delay, and borderline intellectual disability, we identified an ~1.7 Mb FOXF1 duplication adjacent to a large minisatellite. This duplication has a complex structure and arose de novo on the maternal chromosome, likely as a result of a DNA replication error initiated by the adjacent large tandem repeat. Using bioinformatic and array CGH analyses of the minisatellite, we found a large variation of its size in several different species and individuals, demonstrating both its evolutionarily instability and population polymorphism. CONCLUSIONS: Our data indicate that constitutional duplication of FOXF1 in humans is not associated with any pediatric lung abnormalities. We propose that patients with gut malrotation, pyloric or duodenal stenosis, and gall bladder agenesis should be tested for FOXF1 alterations. We suggest that instability of minisatellites greater than 1 kb can lead to structural variation due to DNA replication errors.

Syngnathia and obstructive apnea in a case of popliteal pterygium syndrome.

UNLABELLED: We describe an infant with popliteal pterygia, syngnathia, cleft lip and palate, and retrognathia diagnosed with popliteal pterygium syndrome (PPS). The neonatal course was complicated by severe obstructive apnea necessitating tracheostomy. CONCLUSION: This report illustrates the potential for airway compromise in PPS patients and the need for thorough neonatal airway assessment.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.

Heterozygous de novo and inherited mutations in the smooth muscle actin (ACTG2) gene underlie megacystis-microcolon-intestinal hypoperistalsis syndrome.

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a rare disorder of enteric smooth muscle function affecting the intestine and bladder. Patients with this severe phenotype are dependent on total parenteral nutrition and urinary catheterization. The cause of this syndrome has remained a mystery since Berdon's initial description in 1976. No genes have been clearly linked to MMIHS. We used whole-exome sequencing for gene discovery followed by targeted Sanger sequencing in a cohort of patients with MMIHS and intestinal pseudo-obstruction. We identified heterozygous ACTG2 missense variants in 15 unrelated subjects, ten being apparent de novo mutations. Ten unique variants were detected, of which six affected CpG dinucleotides and resulted in missense mutations at arginine residues, perhaps related to biased usage of CpG containing codons within actin genes. We also found some of the same heterozygous mutations that we observed as apparent de novo mutations in MMIHS segregating in families with intestinal pseudo-obstruction, suggesting that ACTG2 is responsible for a spectrum of smooth muscle disease. ACTG2 encodes γ2 enteric actin and is the first gene to be clearly associated with MMIHS, suggesting an important role for contractile proteins in enteric smooth muscle disease.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing.

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60,000 SNP probes, referred to as Chromosomal Microarray Analysis - Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.

A point mutation in the gene for asparagine-linked glycosylation 10B (Alg10b) causes nonsyndromic hearing impairment in mice (Mus musculus).

The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway.

Expanding the genotype-phenotype correlation in subtelomeric 19p13.3 microdeletions using high resolution clinical chromosomal microarray analysis.

Structural rearrangements of chromosome 19p are rare, and their resulting phenotypic consequences are not well defined. This is the first study to report a cohort of eight patients with subtelomeric 19p13.3 microdeletions, identified using clinical chromosomal microarray analysis (CMA). The deletion sizes ranged from 0.1 to 0.86 Mb. Detailed analysis of the patients' clinical features has enabled us to define a constellation of clinical abnormalities that include growth delay, multiple congenital anomalies, global developmental delay, learning difficulties, and dysmorphic facial features. There are eight genes in the 19p13.3 region that may potentially contribute to the clinical phenotype via haploinsufficiency. Moreover, in silico genomic analysis of 19p13.3 microdeletion breakpoints revealed numerous highly repetitive sequences, suggesting LINEs/SINEs-mediated events in generating these microdeletions. Thus, subtelomeric 19p13.3 appears important for normal embryonic and childhood development. The clinical description of patients with deletions in this genomic interval will assist clinicians to identify and treat individuals with similar deletions.

High resolution whole brain imaging of anatomical variation in XO, XX, and XY mice.

The capacity of sex to modify behavior in health and illness may stem from biological differences between males and females. One such difference--fundamental to the biological definition of sex--is inequality of X chromosome dosage. Studies of Turner Syndrome (TS) suggest that X-monosomy profoundly alters mammalian brain development. However, use of TS as a model for X chromosome haploinsufficiency is complicated by karyotypic mosaicism, background genetic heterogeneity and ovarian dysgenesis. Therefore, to better isolate X chromosome effects on brain development and identify how these overlap with normative sex differences, we used whole-brain structural imaging to study X-monosomic mice (free of mosaicism and ovarian dysgenesis) alongside their karyotypical normal male and female littermates. We demonstrate that murine X-monosomy (XO) causes (i) accentuation of XX vs XY differences in a set of sexually dimorphic structures including classical foci of sex-hormone action, such as the bed nucleus of the stria terminal and medial amygdala, (ii) parietal and striatal abnormalities that recapitulate those reported TS, and (iii) abnormal development of brain systems relevant for domains of altered cognition and emotion in both murine and human X-monosomy. Our findings suggest an unexpected role for X-linked genes in shaping sexually dimorphic brain development, and an evolutionarily conserved influence of X-linked genes on both cortical and subcortical development in mammals. Furthermore, our murine findings highlight the bed nucleus of the stria terminalis and periaqueductal gray matter as novel neuroanatomical candidates for closer study in TS. Integration of these data with existing genomic knowledge generates a set of novel, testable hypotheses regarding candidate mechanisms for each observed pattern of anatomical variation across XO, XX and XY groups.

Co-occurrence of recurrent duplications of the DiGeorge syndrome region on both chromosome 22 homologues due to inherited and de novo events.

BACKGROUND: Genomic rearrangements usually involve one of the two chromosome homologues. Homozygous microdeletion/duplication is very rare. The chromosome 22q11.2 region is prone to recurrent rearrangements due to the presence of low-copy repeats. A common 3 Mb microdeletion causes the well-characterised DiGeorge syndrome (DGS). The reciprocal duplication is associated with an extremely variable phenotype, ranging from apparently normal to learning disabilities and multiple congenital anomalies. METHODS AND RESULTS: We describe duplications of the DGS region on both homologues in five patients from three families, detected by array CGH and confirmed by both fluorescence in situ hybridisation and single nucleotide polymorphism arrays. The proband in the first family is homozygous for the common duplication; one maternally inherited and the other a de novo duplication that was generated by nonallelic homologous recombination during spermatogenesis. The 22q11.2 duplications in the four individuals from the other two families are recurrent duplications on both homologues, one inherited from the mother and the other from the father. The phenotype in the patients with a 22q11.2 tetrasomy is similar to the features seen in duplication patients, including cognitive deficits and variable congenital defects. CONCLUSIONS: Our studies that reveal phenotypic variability in patients with four copies of the 22q11.2 genomic segment, demonstrate that both inherited and de novo events can result in the generation of homozygous duplications, and further document how multiple seemingly rare events can occur in a single individual.

Small genomic rearrangements involving FMR1 support the importance of its gene dosage for normal neurocognitive function.

Fragile X syndrome, the most common form of X-linked intellectual disability, results from transcriptional silencing of the FMR1 gene. As of yet, the phenotypic consequences of the duplication of FMR1 have not been well characterized. In this report, we characterize the clinical features in two females with duplications involving only the FMR1 gene. In addition, we describe the phenotypes of two subjects with deletion of FMR1 and show that both loss and gain of FMR1 copy number can lead to overlapping neurodevelopmental phenotypes. Our report supports the notion that FMR1 gene dosage is important for normal neurocognitive function.

Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator.

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer's, Parkinson's, Huntington's, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer's disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background and recurred on reintroduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds, indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.

Mutation discovery in mice by whole exome sequencing.

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

Detection of clinically relevant exonic copy-number changes by array CGH.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

Mouse mutagenesis with the chemical supermutagen ENU.

The generation and analysis of germline mutations in the mouse is one of the cornerstones of modern biological research. The chemical supermutagen N-ethyl-N-nitrosourea (ENU) is the most potent known mouse mutagen and can be used to generate point mutations throughout the mouse genome. The progeny of ENU-mutagenized males can be screened for autosomal dominant phenotypes, or they can be used to generate multigeneration pedigrees to screen for autosomal recessive traits. The introduction of balancer chromosomes into the breeding scheme can allow for the selective capture of mutations in a specific chromosomal region. More recent work has demonstrated that the use of animals that already have a mutation of interest can lead to the successful isolation of additional mutations that modify the original mutant phenotype. Further, modern molecular techniques ensure that mutations can be readily identified. We describe here the procedures for mutagenizing male mice with ENU and explain the various types of screens that can be performed for different kinds of induced mutations. The currently published research on ENU mutagenesis in the mouse has only scratched the surface of what is possible with this powerful technique, and further work is certain to deepen our knowledge of the role of the individual components of the mouse genome and the myriad relationships between them.