Peter Gustavsson

Copy Number Variation Characteristics in Subpopulations of Patients With Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a heterogeneous group of disorders with a complex genetic etiology. We used high‐resolution whole genome array‐based comparative genomic hybridization (array‐CGH) to screen 223 ASD patients for gene dose alterations associated with susceptibility for autism. Clinically significant copy number variations (CNVs) were identified in 18 individuals (8%), of which 9 cases (4%) had de novo aberrations. In addition, 20 individuals (9%) were shown to have CNVs of unclear clinical relevance. Among these, 13 cases carried rare but inherited CNVs that may increase the risk for developing ASDs, while parental samples were unavailable in the remaining seven cases. Classification of all patients into different phenotypic and inheritance pattern groups indicated the presence of different CNV patterns in different patient groups. Clinically relevant CNVs were more common in syndromic cases compared to non‐syndromic cases. Rare inherited CNVs were present in a higher proportion of ASD cases having first‐ or second‐degree relatives with an ASD‐related neuropsychiatric phenotype in comparison with cases without reported heredity (P = 0.0096). We conclude that rare CNVs, encompassing potential candidate regions for ASDs, increase the susceptibility for the development of ASDs and related neuropsychiatric disorders giving us further insight into the complex genetics underlying ASDs.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt−/− mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Rapp-Hodgkin and AEC syndromes due to a new frameshift mutation in the TP63 gene

Increases in the number of allelic malformation syndromes (due to mutations in a single gene) have led to their classification according to their pathogenesis rather than their clinical specific phenotype. TP63 mutations have been identified in several such syndromes characterised by autosomal dominant transmission and various combinations of ectodermal dysplasia, limb malformations, and orofacial clefting. The TP63 gene is a TP53 homologue,1–8 part of a family composed of only three members. The third member (TP73) is more similar to TP63 than to TP53 in both structure and function.9–13 Like p53, p63 has a transactivating (TA), a DNA binding (DB), and a polymerisation domain; it exerts p53-like activities in various contexts, such as binding canonical p53 sites, transactivating p53 target genes, and inducing apoptosis.1,2 Unlike TP53, which expresses one major transcript, TP63 contains four separate transcription initiation sites that direct expression of two fundamentally different isotypes that retain (TA products) or lack (ΔN products) the TA domain.14 Alternative splicing generates additional complexity at the C terminus. ΔN isoforms lack TA activity and may also suppress the TA isoforms, either by simple competition for the DNA target sites or by acting as dominant negatives through oligomerisation. By contrast to p53, the C terminus in p63 is longer and contains a SAM domain and a TID (transactivation inhibitory domain). SAM domains are involved in protein-protein interactions and probably have regulatory functions in p63,15–17 since its TA-α isoform shows a lower TA activity than the γ form, which lacks the SAM but retains the TA domain. The TID has been mapped within the α tail downstream to the SAM domain.14 The differences at the C terminus identify three transcripts which have different properties and functions: α, β, and γ isoforms. The α isoforms have …

Grainyhead genes and mammalian neural tube closure

BACKGROUND Grainyhead genes encode a family of transcription factors that are well conserved from fly to human. The three mammalian homologues, Grainyhead-like-1, -2, and -3 are expressed in various ectodermal and endodermal cell types during embryonic development and in adult skin. Gene targeting in mice has demonstrated functional roles for Grhl1 and Grhl3 in epidermal integrity and wound healing ability of the epidermis, which appear functionally related to the role of Drosophila grainyhead in production and healing of the epidermal cuticle. Importantly, targeted null mice for Grhl3 also display NTDs, comprising severe spina bifida as well as occasional exencephaly. The chromosomal location of Grhl3 and the finding of NTDs in null embryos suggested that Grhl3 could be allelic with the mouse mutant curly tail, a well known model for NTDs. Expression analysis and transgenic rescue suggest that curly tail is a hypomorphic allele of Grhl3. The functional role and downstream mediators of Grhl3 in neural tube closure are largely unknown. However, the developmental and cellular basis of NTDs in curly tail mutants is well established, involving a proliferation defect in the hindgut endoderm. CONCLUSIONS On this basis, it is possible that Grhl3 has a direct regulatory function in cell proliferation in the hindgut endoderm. Identification of the transcriptional targets of Grhl3 will serve not only to further our understanding of the mechanisms of mammalian neural tube closure, but also to identify potential molecular factors involved in the pathogenesis of NTDs in human.

Different mutations in PDE4D associated with developmental disorders with mirror phenotypes

Background Point mutations in PDE4D have been recently linked to acrodysostosis, an autosomal dominant disorder with skeletal dysplasia, severe brachydactyly, midfacial hypoplasia and intellectual disability. The purpose of the present study was to investigate clinical and cellular implications of different types of mutations in the PDE4D gene. Methods We studied five acrodysostosis patients and three patients with gene dose imbalances involving PDE4D clinically and by whole exome sequencing, Sanger sequencing and array comparative hybridisation. To evaluate the functional consequences of the PDE4D changes, we used overexpression of mutated human PDE4D message and morpholino-based suppression of pde4d in zebrafish. Results We identified three novel and two previously described PDE4D point mutations in the acrodysostosis patients and two deletions and one duplication involving PDE4D in three patients suffering from an intellectual disability syndrome with low body mass index, long fingers, toes and arms, prominent nose and small chin. When comparing symptoms in patients with missense mutations and gene dose imbalances involving PDE4D, a mirror phenotype was observed. By comparing overexpression of human mutated transcripts with pde4d knockdown in zebrafish embryos, we could successfully assay the pathogenicity of the mutations. Conclusions Our findings indicate that haploinsufficiency of PDE4D results in a novel intellectual disability syndrome, the 5q12.1-haploinsufficiency syndrome, with several opposing features compared with acrodysostosis that is caused by dominant negative mutations. In addition, our results expand the spectrum of PDE4D mutations underlying acrodysostosis and indicate that, in contrast to previous reports, patients with PDE4D mutations may have significant hormone resistance with consequent endocrine abnormalities.

Lamin B1 Polymorphism Influences Morphology of the Nuclear Envelope, Cell Cycle Progression, and Risk of Neural Tube Defects in Mice

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression.

Chimerism Resulting From Parthenogenetic Activation and Dispermic Fertilization

Whole‐body human chimerism is the result of two zygotes giving rise to one individual, and is a rarely detected condition. We have studied the molecular background and discuss the likely mechanism for the chimerism in a patient with a 46,XX/47,XY,+14 karyotype and ambiguous genitalia, cryptorchidism, pigment anomalies, and normal psychomotor development. We have used karyotyping, interphase‐FISH and array‐CGH analysis as well as molecular analysis of polymorphic markers from 48 loci in order to define the origin and percentage of 47,XY,+14 cells in different tissues. Based on the findings of two paternal alleles and the detection of homozygous maternal alleles without evidence of crossing‐over, and the fact that four alleles were never detected, our results indicate that the chimerism in our patient is the result of dispermic fertilization of a parthenogenetically activated oocyte. Our report underlines that cytogenetic findings suggesting mosaicism might actually indicate chimerism as an underlying mechanism in patients. It also highlights the difficulties in predicting the clinical outcome in patients with genetic aberrations in mosaic or chimeric form.

Folate metabolite profiling of different cell types and embryos suggests variation in folate one-carbon metabolism, including developmental changes in human embryonic brain.

Folates act as co-factors for transfer of one-carbon units for nucleotide production, methylation and other biosynthetic reactions. Comprehensive profiling of multiple folates can be achieved using liquid chromatography tandem mass spectrometry, enabling determination of their relative abundance that may provide an indication of metabolic differences between cell types. For example, cell lines exposed to methotrexate showed a dose-dependent elevation of dihydrofolate, consistent with inhibition of dihydrofolate reductase. We analysed the folate profile of E. coli sub-types as well as cell lines and embryonic tissue from both human and mouse. The folate profile of bacteria differed markedly from those of all the mammalian samples, most notably in the greater abundance of formyl tetrahydrofolate. The overall profiles of mouse and human fibroblasts and mid-gestation mouse embryos were broadly similar, with specific differences. The major folate species in these cell types was 5-methyl tetrahydrofolate, in contrast to lymphoblastoid cell lines in which the predominant form was tetrahydrofolate. Analysis of embryonic human brain revealed a shift in folate profile with increasing developmental stage, with a decline in relative abundance of dihydrofolate and increase in 5-methyl tetrahydrofolate. These cell type-specific and developmental changes in folate profile may indicate differential requirements for the various outputs of folate metabolism.

Mutation screening and array comparative genomic hybridization using a 180K oligonucleotide array in VACTERL association.

In order to identify genetic causes of VACTERL association (V vertebral defects, A anorectal malformations, C cardiac defects, T tracheoesofageal fistula, E esophageal atresia, R renal anomalies, L limb deformities), we have collected DNA samples from 20 patients diagnosed with VACTERL or with a VACTERL-like phenotype as well as samples from 19 aborted fetal cases with VACTERL. To investigate the importance of gene dose alterations in the genetic etiology of VACTERL association we have performed a systematic analysis of this cohort using a 180K array comparative genomic hybridization (array-CGH) platform. In addition, to further clarify the significance of PCSK5, HOXD13 and CHD7 genes in the VACTERL phenotype, mutation screening has been performed. We identified pathogenic gene dose imbalances in two fetal cases; a hemizygous deletion of the FANCB gene and a (9;18)(p24;q12) unbalanced translocation. In addition, one pathogenic mutation in CHD7 was detected, while no apparent disease-causing mutations were found in HOXD13 or PCSK5. Our study shows that although large gene dose alterations do not seem to be a common cause in VACTERL association, array-CGH is still important in clinical diagnostics to identify disease cause in individual cases.

Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation

Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low‐coverage mate pair whole‐genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX‐male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error‐prone replication‐based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.