Detecting cryptic clinically relevant structural variation in exome-sequencing data increases diagnostic yield for developmental disorders.

Structural variation (SV) describes a broad class of genetic variation greater than 50 bp in size. SVs can cause a wide range of genetic diseases and are prevalent in rare developmental disorders (DDs). Individuals presenting with DDs are often referred for diagnostic testing with chromosomal microarrays (CMAs) to identify large copy-number variants (CNVs) and/or with single-gene, gene-panel, or exome sequencing (ES) to identify single-nucleotide variants, small insertions/deletions, and CNVs. However, individuals with pathogenic SVs undetectable by conventional analysis often remain undiagnosed. Consequently, we have developed the tool InDelible, which interrogates short-read sequencing data for split-read clusters characteristic of SV breakpoints. We applied InDelible to 13,438 probands with severe DDs recruited as part of the Deciphering Developmental Disorders (DDD) study and discovered 63 rare, damaging variants in genes previously associated with DDs missed by standard SNV, indel, or CNV discovery approaches. Clinical review of these 63 variants determined that about half (30/63) were plausibly pathogenic. InDelible was particularly effective at ascertaining variants between 21 and 500 bp in size and increased the total number of potentially pathogenic variants identified by DDD in this size range by 42.9%. Of particular interest were seven confirmed de novo variants in MECP2, which represent 35.0% of all de novo protein-truncating variants in MECP2 among DDD study participants. InDelible provides a framework for the discovery of pathogenic SVs that are most likely missed by standard analytical workflows and has the potential to improve the diagnostic yield of ES across a broad range of genetic diseases.

Making new genetic diagnoses with old data: iterative reanalysis and reporting from genome-wide data in 1,133 families with developmental disorders.

PURPOSE: Given the rapid pace of discovery in rare disease genomics, it is likely that improvements in diagnostic yield can be made by systematically reanalyzing previously generated genomic sequence data in light of new knowledge. METHODS: We tested this hypothesis in the United Kingdom-wide Deciphering Developmental Disorders study, where in 2014 we reported a diagnostic yield of 27% through whole-exome sequencing of 1,133 children with severe developmental disorders and their parents. We reanalyzed existing data using improved variant calling methodologies, novel variant detection algorithms, updated variant annotation, evidence-based filtering strategies, and newly discovered disease-associated genes. RESULTS: We are now able to diagnose an additional 182 individuals, taking our overall diagnostic yield to 454/1,133 (40%), and another 43 (4%) have a finding of uncertain clinical significance. The majority of these new diagnoses are due to novel developmental disorder-associated genes discovered since our original publication. CONCLUSION: This study highlights the importance of coupling large-scale research with clinical practice, and of discussing the possibility of iterative reanalysis and recontact with patients and health professionals at an early stage. We estimate that implementing parent-offspring whole-exome sequencing as a first-line diagnostic test for developmental disorders would diagnose >50% of patients.

Genetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data.

BACKGROUND: Human genome sequencing has transformed our understanding of genomic variation and its relevance to health and disease, and is now starting to enter clinical practice for the diagnosis of rare diseases. The question of whether and how some categories of genomic findings should be shared with individual research participants is currently a topic of international debate, and development of robust analytical workflows to identify and communicate clinically relevant variants is paramount. METHODS: The Deciphering Developmental Disorders (DDD) study has developed a UK-wide patient recruitment network involving over 180 clinicians across all 24 regional genetics services, and has performed genome-wide microarray and whole exome sequencing on children with undiagnosed developmental disorders and their parents. After data analysis, pertinent genomic variants were returned to individual research participants via their local clinical genetics team. FINDINGS: Around 80,000 genomic variants were identified from exome sequencing and microarray analysis in each individual, of which on average 400 were rare and predicted to be protein altering. By focusing only on de novo and segregating variants in known developmental disorder genes, we achieved a diagnostic yield of 27% among 1133 previously investigated yet undiagnosed children with developmental disorders, whilst minimising incidental findings. In families with developmentally normal parents, whole exome sequencing of the child and both parents resulted in a 10-fold reduction in the number of potential causal variants that needed clinical evaluation compared to sequencing only the child. Most diagnostic variants identified in known genes were novel and not present in current databases of known disease variation. INTERPRETATION: Implementation of a robust translational genomics workflow is achievable within a large-scale rare disease research study to allow feedback of potentially diagnostic findings to clinicians and research participants. Systematic recording of relevant clinical data, curation of a gene-phenotype knowledge base, and development of clinical decision support software are needed in addition to automated exclusion of almost all variants, which is crucial for scalable prioritisation and review of possible diagnostic variants. However, the resource requirements of development and maintenance of a clinical reporting system within a research setting are substantial. FUNDING: Health Innovation Challenge Fund, a parallel funding partnership between the Wellcome Trust and the UK Department of Health.

Recurrent duplications of 17q12 associated with variable phenotypes.

The ability to identify the clinical nature of the recurrent duplication of chromosome 17q12 has been limited by its rarity and the diverse range of phenotypes associated with this genomic change. In order to further define the clinical features of affected patients, detailed clinical information was collected in the largest series to date (30 patients and 2 of their siblings) through a multi-institutional collaborative effort. The majority of patients presented with developmental delays varying from mild to severe. Though dysmorphic features were commonly reported, patients do not have consistent and recognizable features. Cardiac, ophthalmologic, growth, behavioral, and other abnormalities were each present in a subset of patients. The newly associated features potentially resulting from 17q12 duplication include height and weight above the 95th percentile, cataracts, microphthalmia, coloboma, astigmatism, tracheomalacia, cutaneous mosaicism, pectus excavatum, scoliosis, hypermobility, hypospadias, diverticulum of Kommerell, pyloric stenosis, and pseudohypoparathryoidism. The majority of duplications were inherited with some carrier parents reporting learning disabilities or microcephaly. We identified additional, potentially contributory copy number changes in a subset of patients, including one patient each with 16p11.2 deletion and 15q13.3 deletion. Our data further define and expand the clinical spectrum associated with duplications of 17q12 and provide support for the role of genomic modifiers contributing to phenotypic variability.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

Distinct effects of allelic NFIX mutations on nonsense-mediated mRNA decay engender either a Sotos-like or a Marshall-Smith syndrome.

By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.

Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.

Many patients suffering from developmental disorders harbor submicroscopic deletions or duplications that, by affecting the copy number of dosage-sensitive genes or disrupting normal gene expression, lead to disease. However, many aberrations are novel or extremely rare, making clinical interpretation problematic and genotype-phenotype correlations uncertain. Identification of patients sharing a genomic rearrangement and having phenotypic features in common leads to greater certainty in the pathogenic nature of the rearrangement and enables new syndromes to be defined. To facilitate the analysis of these rare events, we have developed an interactive web-based database called DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) which incorporates a suite of tools designed to aid the interpretation of submicroscopic chromosomal imbalance, inversions, and translocations. DECIPHER catalogs common copy-number changes in normal populations and thus, by exclusion, enables changes that are novel and potentially pathogenic to be identified. DECIPHER enhances genetic counseling by retrieving relevant information from a variety of bioinformatics resources. Known and predicted genes within an aberration are listed in the DECIPHER patient report, and genes of recognized clinical importance are highlighted and prioritized. DECIPHER enables clinical scientists worldwide to maintain records of phenotype and chromosome rearrangement for their patients and, with informed consent, share this information with the wider clinical research community through display in the genome browser Ensembl. By sharing cases worldwide, clusters of rare cases having phenotype and structural rearrangement in common can be identified, leading to the delineation of new syndromes and furthering understanding of gene function.

Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

aCGH.Spline--an R package for aCGH dye bias normalization.

MOTIVATION: The careful normalization of array-based comparative genomic hybridization (aCGH) data is of critical importance for the accurate detection of copy number changes. The difference in labelling affinity between the two fluorophores used in aCGH-usually Cy5 and Cy3-can be observed as a bias within the intensity distributions. If left unchecked, this bias is likely to skew data interpretation during downstream analysis and lead to an increased number of false discoveries. RESULTS: In this study, we have developed aCGH.Spline, a natural cubic spline interpolation method followed by linear interpolation of outlier values, which is able to remove a large portion of the dye bias from large aCGH datasets in a quick and efficient manner. CONCLUSIONS: We have shown that removing this bias and reducing the experimental noise has a strong positive impact on the ability to detect accurately both copy number variation (CNV) and copy number alterations (CNA).

Distinct genetic architectures for syndromic and nonsyndromic congenital heart defects identified by exome sequencing.

Congenital heart defects (CHDs) have a neonatal incidence of 0.8-1% (refs. 1,2). Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (∼2.7%), suggesting a considerable role for de novo mutations (DNMs) and/or incomplete penetrance. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of 'syndromic' patients with extra-cardiac manifestations. We exome sequenced 1,891 probands, including both syndromic CHD (S-CHD, n = 610) and nonsyndromic CHD (NS-CHD, n = 1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs but not inherited PTVs in known CHD-associated genes, consistent with recent findings. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study finds evidence for distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

Discovery of four recessive developmental disorders using probabilistic genotype and phenotype matching among 4,125 families.

Discovery of most autosomal recessive disease-associated genes has involved analysis of large, often consanguineous multiplex families or small cohorts of unrelated individuals with a well-defined clinical condition. Discovery of new dominant causes of rare, genetically heterogeneous developmental disorders has been revolutionized by exome analysis of large cohorts of phenotypically diverse parent-offspring trios. Here we analyzed 4,125 families with diverse, rare and genetically heterogeneous developmental disorders and identified four new autosomal recessive disorders. These four disorders were identified by integrating Mendelian filtering (selecting probands with rare, biallelic and putatively damaging variants in the same gene) with statistical assessments of (i) the likelihood of sampling the observed genotypes from the general population and (ii) the phenotypic similarity of patients with recessive variants in the same candidate gene. This new paradigm promises to catalyze the discovery of novel recessive disorders, especially those with less consistent or nonspecific clinical presentations and those caused predominantly by compound heterozygous genotypes.

Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome.

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

An atypical facial appearance and growth pattern in a child with Cornelia de Lange Syndrome: an intragenic deletion predicting loss of the N-terminal region of NIPBL.

Cornelia de Lange Syndrome (CdLS) is a multisystem disorder with a live birth prevalence of approximately one per 15 000. Clinical diagnosis is based on a characteristic facies ­ low frontal hair line, short nose, triangular nasal tip, crescent shaped mouth, upturned nose, and arched eyebrows ­ characteristic limb defects and a distinctive pattern of growth and development. Approximately half of all classical cases of CdLS have heterozygous loss of-function mutations in the gene encoding NIPBL, a component of the cohesion-loading apparatus (Dorsett and Krantz, 2009). Herein we describe a patient with a rare intragenic deletion of NIPBL who has typical microcephaly and developmental problems but atypical growth pattern and facial features.

Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants.

We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.

Germline rates of de novo meiotic deletions and duplications causing several genomic disorders.

Meiotic recombination between highly similar duplicated sequences (nonallelic homologous recombination, NAHR) generates deletions, duplications, inversions and translocations, and it is responsible for genetic diseases known as 'genomic disorders', most of which are caused by altered copy number of dosage-sensitive genes. NAHR hot spots have been identified within some duplicated sequences. We have developed sperm-based assays to measure the de novo rate of reciprocal deletions and duplications at four NAHR hot spots. We used these assays to dissect the relative rates of NAHR between different pairs of duplicated sequences. We show that (i) these NAHR hot spots are specific to meiosis, (ii) deletions are generated at a higher rate than their reciprocal duplications in the male germline and (iii) some of these genomic disorders are likely to have been underascertained clinically, most notably that resulting from the duplication of 7q11, the reciprocal of the deletion causing Williams-Beuren syndrome.