A new paradigm emerges from the study of de novo mutations in the context of neurodevelopmental disease.

The study of de novo point mutations (new germline mutations arising from the gametes of the parents) remained largely static until the arrival of next-generation sequencing technologies, which made both whole-exome sequencing (WES) and whole-genome sequencing (WGS) feasible in practical terms. Single nucleotide polymorphism genotyping arrays have been used to identify de novo copy-number variants in a number of common neurodevelopmental conditions such as schizophrenia and autism. By contrast, as point mutations and microlesions occurring de novo are refractory to analysis by these microarray-based methods, little was known about either their frequency or impact upon neurodevelopmental disease, until the advent of WES. De novo point mutations have recently been implicated in schizophrenia, autism and mental retardation through the WES of case-parent trios. Taken together, these findings strengthen the hypothesis that the occurrence of de novo mutations could account for the high prevalence of such diseases that are associated with a marked reduction in fecundity. De novo point mutations are also known to be responsible for many sporadic cases of rare dominant mendelian disorders such as Kabuki syndrome, Schinzel-Giedion syndrome and Bohring-Opitz syndrome. These disorders share a common feature in that they are all characterized by intellectual disability. In summary, recent WES studies of neurodevelopmental and neuropsychiatric disease have provided new insights into the role of de novo mutations in these disorders. Our knowledge of de novo mutations is likely to be further accelerated by WGS. However, the collection of case-parent trios will be a prerequisite for such studies. This review aims to discuss recent developments in the study of de novo mutations made possible by technological advances in DNA sequencing.

Integrating next-generation sequencing into the diagnostic testing of inherited cancer predisposition.

The clinical application of next-generation sequencing (NGS) as a diagnostic tool has become increasingly evident. The coupling of NGS technologies with new genomic sequence enrichment methods has made the sequencing of panels of target genes technically feasible, at the same time as making such an approach cost-effective for diagnostic applications. In this article, we discuss recent studies that have applied NGS in the diagnostic setting in relation to hereditary cancer.

Clinical characterisation of 29 neurofibromatosis type-1 patients with molecularly ascertained 1.4 Mb type-1 NF1 deletions.

BACKGROUND: Large deletions of the NF1 gene region occur in approximately 5% of patients with neurofibromatosis type-1 (NF1) and are associated with particularly severe manifestations of the disease. However, until now, the genotype-phenotype relationship has not been comprehensively studied in patients harbouring large NF1 gene deletions of comparable extent (giving rise to haploinsufficiency of the same genes). METHOD: We have performed the most comprehensive clinical/neuropsychological characterisation so far undertaken in NF1 deletion patients, involving 29 patients with precisely determined type-1 NF1 (1.4 Mb) deletions. RESULTS: Novel clinical features found to be associated with type-1 NF1 deletions included pes cavus (17% of patients), bone cysts (50%), attention deficit (73%), muscular hypotonia (45%) and speech difficulties (48%). Type-1 NF1 deletions were found to be disproportionately associated with facial dysmorphic features (90% of patients), tall stature (46%), large hands and feet (46%), scoliosis (43%), joint hyperflexibility (72%), delayed cognitive development and/or learning disabilities (93%) and mental retardation (IQ<70; 38%), as compared with the general NF1 patient population. Significantly increased frequencies (relative to the general NF1 population) of plexiform neurofibromas (76%), subcutaneous neurofibromas (76%), spinal neurofibromas (64%) and MPNSTs (21%) were also noted in the type-1 deletion patients. Further, 50% of the adult patients exhibited a very high burden of cutaneous neurofibromas (N>or=1000). CONCLUSION: These findings emphasise the importance of deletion analysis in NF1 since frequent monitoring of tumour presence and growth could potentiate early surgical intervention thereby improving patient survival.

Revealing the human mutome.

The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the 'mutome'). With the advent of next-generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole-genome sequencing (WGS) has the potential to identify all disease-causing or disease-associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene-coding sequences for those disease-causing or disease-associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation-screening strategies but also for enhancing the interpretation of findings derived from genome-wide association studies, whole-exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology.

Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism.

A soluble lactose-binding lectin with subunit Mr of 14,500 is believed to function by interacting with extracellular glycoconjugates, because it has been detected extracellularly by immunohistochemistry. This localization has been questioned, however, since the lectin lacks a secretion signal sequence, which challenges the contention that it is secreted. We have demonstrated externalization of this lectin from C2 mouse muscle cells by both immunoprecipitation of metabolically labeled protein and immunohistochemical localization. We further show that externalization of the lectin is a developmentally regulated process that accompanies myoblast differentiation and that the lectin codistributes with laminin in myotube extracellular matrix. Immunohistochemical localization during intermediate stages of externalization suggests that the lectin becomes concentrated in evaginations of plasma membrane, which pinch off to form labile lectin-rich extracellular vesicles. This suggests a possible mechanism for lectin export from the cytosol to the extracellular matrix.

Endogenous muscle lectin inhibits myoblast adhesion to laminin.

L-14, a dimeric lactose-binding lectin with subunits of 14 kD, is expressed in a wide range of vertebrate tissues. Several functions have been postulated for this lectin, but definitive evidence for a specific biological role has been elusive. In muscle, L-14 is secreted during differentiation and accumulates with laminin in basement membrane surrounding each myofiber. Here we present evidence that laminin is a major glycoprotein ligand for L-14 in differentiating mouse C2C12 muscle cells and that binding of secreted L-14 to polylactosamine oligosaccharides of substrate laminin induces loss of cell-substratum adhesion. These results suggest that one function of L-14 is to regulate myoblast detachment from laminin during differentiation and fusion into tubular myofibers.

Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum.

The distribution of discoidin I and discoidin II, developmentally regulated lectins in Dictyostelium discoideum, was determined immunohistochemically at various stages of development. Discoidin I was first prominent as focal clumps in aggregating cells, then accumulated on the surface of aggregates and around them. Discoidin II became prominent later and ultimately localized in what appear to be prespore vesicles. The results indicate that discoidin I and discoidin II have different and possibly multiple functions.

Discoidin I, an endogenous lectin, is externalized from Dictyostelium discoideum in multilamellar bodies.

Discoidin I, a soluble lectin synthesized by aggregating Dictyostelium discoideum and implicated in their adhesion to the substratum, is localized in multilamellar bodies both intracellularly and upon externalization. These structures also contain a glycoconjugate that binds discoidin I. The multilamellar bodies apparently serve to package the lectin for externalization, and may then gradually release it to function extracellularly.

A new pathway for protein export in Saccharomyces cerevisiae.

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.

Mosaicism in sporadic neurofibromatosis type 1: variations on a theme common to other hereditary cancer syndromes?

Mosaicism constitutes a frequent complication of the genotype-phenotype relationship in genetic disease and is an important consideration for the estimation of transmission risk. Mosaicism has been identified in several hereditary cancer syndromes including retinoblastoma, familial adenomatous polyposis coli, von Hippel-Lindau disease and neurofibromatosis type 2. Recent data support the postulate that the frequency of mosaicism is increased in cancer predisposition syndromes characterised by high new mutation rates. Since the new mutation rate is very high in neurofibromatosis type 1 (NF1), mosaicism might reasonably be expected to be frequent among sporadic cases but this remains to be formally demonstrated. Here we summarise current knowledge of mosaicism in NF1, focusing on the types of mutations identified as well as their inferred developmental timing and representation in different cell types, and assess the potential impact of high frequency mosaicism on mutation screening in patients with apparent de novo NF1.

Comparative analysis of copy number variation in primate genomes.

Copy number differences between human and chimpanzee genomic sequences often overlap with regions of intraspecies copy number variation. Copy number variations (CNVs) that occur at orthologous sites in both humans and chimpanzees ('shared' CNVs) are likely to represent unstable genomic regions that have been prone to recurrent rearrangements during primate evolution. Lineage-specific CNVs are also important because they may have been subject to positive selection in specific primate lineages and hence could have contributed both to intra- and interspecies phenotypic diversity. In this study, we reinvestigated the data originally obtained by Perry et al. (2006) relating to chimpanzee CNVs and identified 24 genomic regions with the potential to be chimpanzee-specific CNVs. Since every putative chimpanzee-specific CNV was found in at least two of the 20 chimpanzees originally studied, it would appear as if these 24 CNVs are fairly frequent in chimpanzees. A combination of new mutation, genetic drift, and directional or balancing selection is likely to have influenced the maintenance of these CNVs in the chimpanzee population. Several genes map to the regions encompassed by the chimpanzee-specific CNVs. Some of their human orthologues are already known either to influence the phenotype (e.g., SLC24A4) or to be associated with inherited diseases (e.g., PAK3 and DTNBP1). Although the relatively small number of chimpanzee-specific CNVs (N = 24) among the 355 CNVs originally identified may be in part due to the use of a human BAC array to detect them, we nevertheless surmise that lineage-specific CNVs are not abundant in the chimpanzee. The thorough characterization of CNVs in the great ape genomes is a sine qua non for identifying the human-specific CNVs that may constitute genomic regions which have experienced either positive or negative selection during human evolution.

Chromosomal speciation of humans and chimpanzees revisited: studies of DNA divergence within inverted regions.

The human and chimpanzee karyotypes are distinguishable in terms of nine pericentric inversions. According to the recombination suppression model of speciation, these inversions could have promoted the process of parapatric speciation between hominoid populations ancestral to chimpanzees and humans. Were recombination suppression to have occurred in inversion heterozygotes, gene flow would have been reduced, resulting in the accumulation of genetic incompatibilities leading to reproductive isolation and eventual speciation. In an attempt to detect the molecular signature of such events, the sequence divergence of non-coding DNA was compared between humans and chimpanzees. Precise knowledge of the locations of the inversion breakpoints permitted accurate discrimination between inverted and non-inverted regions. Contrary to the predictions of the recombination suppression model, sequence divergence was found to be lower in inverted chromosomal regions as compared to non-inverted regions, albeit with borderline statistical significance. Thus, no signature of recombination suppression resulting from inversion heterozygosity appears to be detectable by analysis of extant human and chimpanzee non-coding DNA. The precise delineation of the inversion breakpoints may nevertheless still prove helpful in identifying potential speciation-relevant genes within the inverted regions.

Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients.

MECP2 mutations are identifiable in approximately 80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

The molecular genetics of familial venous thrombosis.

Inherited defects of antithrombin III, protein C, protein S, heparin cofactor II, plasminogen and the fibrinogens are thought to be responsible for between 10 and 15% of all patients presenting with recurrent venous thrombosis. The structure, function and expression of these genes and the nature of the gene lesions underlying the deficiency states are reviewed in detail.

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.

Promoter shuffling has occurred during the evolution of the vertebrate growth hormone gene.

Comparative studies of vertebrate gene promoter regions seldom detect gross rearrangements ('promoter shuffling') since such analyses usually employ relatively similar DNA sequences. Conversely, attempts to compare evolutionarily more divergent promoter sequences have been largely unsuccessful owing to the inability of conventional alignment procedures to deal with gross rearrangements. These limitations have been circumvented in the present study by using the novel technique of complexity analysis to identify modular components ('blocks') in the growth hormone (GH) gene promoter sequences of some 22 vertebrate species, from salmon to human. Significant rearrangement of blocks was found to have occurred, indicating that they have evolved as independent units. Some blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Considerable variation between orthologous GH gene promoters was apparent in terms of block length, copy number and relative location. It may be inferred that a wide variety of different mutational mechanisms have operated upon the GH gene promoter over evolutionary time. These include gross changes such as deletion, duplication, amplification, elongation, contraction, transposition, inversion and fusion, as well as the slow, steady accumulation of single base-pair substitutions. Thus the patchwork structure of the modular GH promoter region, and those of its paralogous GH2 and prolactin (PRL) counterparts, have continually been shuffled into new combinations through the rearrangement of pre-existing blocks. Although some of these changes may have had no influence on promoter function, others could have served to alter either the level of gene expression or the responsiveness of the promoter to external stimuli.

Multiple soluble vertebrate galactoside-binding lectins.

All vertebrates synthesize soluble galactoside-binding lectins. Many are expressed at high levels in the embryo and at lower levels in the adult, whereas others show an inverse pattern of expression. Most lectins tend to be concentrated in one or a number of specific cell types. In the past few years, the multiplicity of these lectins has become more apparent. For example, in Xenopus laevis 3 galactoside-binding lectins, 2 with a preference for alpha-galactosides, have been purified and partially characterized. They have subunit molecular weights ranging from 16,000 to 69,000. More detailed studies have been done in mammals. For example, rat lung contains 3 soluble beta-galactoside-binding lectins, RL-14.5, RL-18 and RL-29, with subunit molecular weights, respectively, of 14,500, 18,000 and 29,000. A notable feature of these lectins is that, although they all bind lactose about equally well, their carbohydrate-binding sites are actually quite different, as shown by competitive binding studies with a range of complex mammalian glycoconjugates. Human lung also contains several beta-galactoside-binding lectins, including HL-14, HL-22 and HL-29 with subunit molecular weights, respectively, of 14,000, 22,000 and 29,000. They too show significant differences in their carbohydrate-binding sites when analyzed with naturally occurring mammalian glycoconjugates. Sequencing of purified lectins and cDNA clones indicates that at least 4 distinct genes code for what appears to be a family of HL-14. Heterogeneity is also indicated from isoelectric focusing studies which resolve at least 6 acidic forms of HL-14 and 5 acidic forms of HL-29.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics.

The monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth factor-BB (PDGF-BB) and to lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.

Ectopic transcript analysis indicates that allelic exclusion is an important cause of type I protein C deficiency in patients with nonsense and frameshift mutations in the PROC gene.

Nonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through "allelic exclusion" at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3' to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons.