Joris A. Veltman

A de novo paradigm for mental retardation.

The per-generation mutation rate in humans is high. De novo mutations may compensate for allele loss due to severely reduced fecundity in common neurodevelopmental and psychiatric diseases, explaining a major paradox in evolutionary genetic theory. Here we used a family based exome sequencing approach to test this de novo mutation hypothesis in ten individuals with unexplained mental retardation. We identified and validated unique non-synonymous de novo mutations in nine genes. Six of these, identified in six different individuals, are likely to be pathogenic based on gene function, evolutionary conservation and mutation impact. Our findings provide strong experimental support for a de novo paradigm for mental retardation. Together with de novo copy number variation, de novo point mutations of large effect could explain the majority of all mental retardation cases in the population.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes

BACKGROUND Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Diagnostic Genome Profiling in Mental Retardation

Mental retardation (MR) occurs in 2%-3% of the general population. Conventional karyotyping has a resolution of 5-10 million bases and detects chromosomal alterations in approximately 5% of individuals with unexplained MR. The frequency of smaller submicroscopic chromosomal alterations in these patients is unknown. Novel molecular karyotyping methods, such as array-based comparative genomic hybridization (array CGH), can detect submicroscopic chromosome alterations at a resolution of 100 kb. In this study, 100 patients with unexplained MR were analyzed using array CGH for DNA copy-number changes by use of a novel tiling-resolution genomewide microarray containing 32,447 bacterial artificial clones. Alterations were validated by fluorescence in situ hybridization and/or multiplex ligation-dependent probe amplification, and parents were tested to determine de novo occurrence. Reproducible DNA copy-number changes were present in 97% of patients. The majority of these alterations were inherited from phenotypically normal parents, which reflects normal large-scale copy-number variation. In 10% of the patients, de novo alterations considered to be clinically relevant were found: seven deletions and three duplications. These alterations varied in size from 540 kb to 12 Mb and were scattered throughout the genome. Our results indicate that the diagnostic yield of this approach in the general population of patients with MR is at least twice as high as that of standard GTG-banded karyotyping.

Genome sequencing identifies major causes of severe intellectual disability

Severe intellectual disability (ID) occurs in 0.5% of newborns and is thought to be largely genetic in origin. The extensive genetic heterogeneity of this disorder requires a genome-wide detection of all types of genetic variation. Microarray studies and, more recently, exome sequencing have demonstrated the importance of de novo copy number variations (CNVs) and single-nucleotide variations (SNVs) in ID, but the majority of cases remain undiagnosed. Here we applied whole-genome sequencing to 50 patients with severe ID and their unaffected parents. All patients included had not received a molecular diagnosis after extensive genetic prescreening, including microarray-based CNV studies and exome sequencing. Notwithstanding this prescreening, 84 de novo SNVs affecting the coding region were identified, which showed a statistically significant enrichment of loss-of-function mutations as well as an enrichment for genes previously implicated in ID-related disorders. In addition, we identified eight de novo CNVs, including single-exon and intra-exonic deletions, as well as interchromosomal duplications. These CNVs affected known ID genes more frequently than expected. On the basis of diagnostic interpretation of all de novo variants, a conclusive genetic diagnosis was reached in 20 patients. Together with one compound heterozygous CNV causing disease in a recessive mode, this results in a diagnostic yield of 42% in this extensively studied cohort, and 62% as a cumulative estimate in an unselected cohort. These results suggest that de novo SNVs and CNVs affecting the coding region are a major cause of severe ID. Genome sequencing can be applied as a single genetic test to reliably identify and characterize the comprehensive spectrum of genetic variation, providing a genetic diagnosis in the majority of patients with severe ID.

Array-Based Comparative Genomic Hybridization for the Genomewide Detection of Submicroscopic Chromosomal Abnormalities

Microdeletions and microduplications, not visible by routine chromosome analysis, are a major cause of human malformation and mental retardation. Novel high-resolution, whole-genome technologies can improve the diagnostic detection rate of these small chromosomal abnormalities. Array-based comparative genomic hybridization allows such a high-resolution screening by hybridizing differentially labeled test and reference DNAs to arrays consisting of thousands of genomic clones. In this study, we tested the diagnostic capacity of this technology using approximately 3,500 flourescent in situ hybridization-verified clones selected to cover the genome with an average of 1 clone per megabase (Mb). The sensitivity and specificity of the technology were tested in normal-versus-normal control experiments and through the screening of patients with known microdeletion syndromes. Subsequently, a series of 20 cytogenetically normal patients with mental retardation and dysmorphisms suggestive of a chromosomal abnormality were analyzed. In this series, three microdeletions and two microduplications were identified and validated. Two of these genomic changes were identified also in one of the parents, indicating that these are large-scale genomic polymorphisms. Deletions and duplications as small as 1 Mb could be reliably detected by our approach. The percentage of false-positive results was reduced to a minimum by use of a dye-swap-replicate analysis, all but eliminating the need for laborious validation experiments and facilitating implementation in a routine diagnostic setting. This high-resolution assay will facilitate the identification of novel genes involved in human mental retardation and/or malformation syndromes and will provide insight into the flexibility and plasticity of the human genome.

De novo mutations in human genetic disease

New mutations have long been known to cause genetic disease, but their true contribution to the disease burden can only now be determined using family-based whole-genome or whole-exome sequencing approaches. In this Review we discuss recent findings suggesting that de novo mutations play a prominent part in rare and common forms of neurodevelopmental diseases, including intellectual disability, autism and schizophrenia. De novo mutations provide a mechanism by which early-onset reproductively lethal diseases remain frequent in the population. These mutations, although individually rare, may capture a significant part of the heritability for complex genetic diseases that is not detectable by genome-wide association studies.

STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous Candidiasis

BACKGROUND Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to candida infection of skin, nails, and mucous membranes. Patients with recessive CMC and autoimmunity have mutations in the autoimmune regulator AIRE. The cause of autosomal dominant CMC is unknown. METHODS We evaluated 14 patients from five families with autosomal dominant CMC. We incubated their peripheral-blood mononuclear cells with different combinations of stimuli to test the integrity of pathways that mediate immunity, which led to the selection of 100 genes that were most likely to contain the genetic defect. We used an array-based sequence-capture assay, followed by next-generation sequencing, to identify mutations. RESULTS The mononuclear cells from the affected patients were characterized by poor production of interferon-γ, interleukin-17, and interleukin-22, suggesting that the defect lay within the interleukin-12 receptor and interleukin-23 receptor signaling pathways. We identified heterozygous missense mutations in the DNA sequence encoding the coiled-coil (CC) domain of signal transducer and activator of transcription 1 (STAT1) in the patients. These mutations lead to defective responses in type 1 and type 17 helper T cells (Th1 and Th17). The interferon-γ receptor pathway was intact in these patients. CONCLUSIONS Mutations in the CC domain of STAT1 underlie autosomal dominant CMC and lead to defective Th1 and Th17 responses, which may explain the increased susceptibility to fungal infection. (Funded by the Netherlands Organization for Scientific Research and others.).

Disruption of the neurexin 1 gene is associated with schizophrenia

Deletions within the neurexin 1 gene (NRXN1; 2p16.3) are associated with autism and have also been reported in two families with schizophrenia. We examined NRXN1, and the closely related NRXN2 and NRXN3 genes, for copy number variants (CNVs) in 2977 schizophrenia patients and 33 746 controls from seven European populations (Iceland, Finland, Norway, Germany, The Netherlands, Italy and UK) using microarray data. We found 66 deletions and 5 duplications in NRXN1, including a de novo deletion: 12 deletions and 2 duplications occurred in schizophrenia cases (0.47%) compared to 49 and 3 (0.15%) in controls. There was no common breakpoint and the CNVs varied from 18 to 420 kb. No CNVs were found in NRXN2 or NRXN3. We performed a Cochran-Mantel-Haenszel exact test to estimate association between all CNVs and schizophrenia (P = 0.13; OR = 1.73; 95% CI 0.81-3.50). Because the penetrance of NRXN1 CNVs may vary according to the level of functional impact on the gene, we next restricted the association analysis to CNVs that disrupt exons (0.24% of cases and 0.015% of controls). These were significantly associated with a high odds ratio (P = 0.0027; OR 8.97, 95% CI 1.8-51.9). We conclude that NRXN1 deletions affecting exons confer risk of schizophrenia.

A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism

Submicroscopic genomic copy number changes have been identified only recently as an important cause of mental retardation. We describe the detection of three interstitial, overlapping 17q21.31 microdeletions in a cohort of 1,200 mentally retarded individuals associated with a clearly recognizable clinical phenotype of mental retardation, hypotonia and a characteristic face. The deletions encompass the MAPT and CRHR1 genes and are associated with a common inversion polymorphism.

Disease gene identification strategies for exome sequencing

Next generation sequencing can be used to search for Mendelian disease genes in an unbiased manner by sequencing the entire protein-coding sequence, known as the exome, or even the entire human genome. Identifying the pathogenic mutation amongst thousands to millions of genomic variants is a major challenge, and novel variant prioritization strategies are required. The choice of these strategies depends on the availability of well-phenotyped patients and family members, the mode of inheritance, the severity of the disease and its population frequency. In this review, we discuss the current strategies for Mendelian disease gene identification by exome resequencing. We conclude that exome strategies are successful and identify new Mendelian disease genes in approximately 60% of the projects. Improvements in bioinformatics as well as in sequencing technology will likely increase the success rate even further. Exome sequencing is likely to become the most commonly used tool for Mendelian disease gene identification for the coming years.