The patient with 41 reports: Analysis of laboratory exome sequencing reporting of a "virtual patient".

PURPOSE: Few studies have systematically analyzed the structure and content of laboratory exome sequencing reports from the same patient. METHODS: We merged 8 variants from patients into "normal" exomes to create virtual patient-parent trios. We provided laboratories worldwide with the data and patient phenotype information (developmental delay, dysmorphic features, and cardiac hypertrophy). Laboratories analyzed the data and issued a diagnostic exome report. Reports were scored using a coding matrix developed from existing guidelines. RESULTS: In total, 41 laboratories representing 17 countries issued reports. Reporting of quality control statistics and technical information was poor (46.3%). Although 75.6% of the reports clearly stated the classification of all reported variants, few reports listed extensive evidence supporting variant classification. Only 53.1% of laboratories that reported unsolicited or secondary findings gave advice regarding health-related follow-up and 20.5% gave advice regarding cascade testing for relatives. Of the 147 variants reported, 105 (71.4%) were classified in agreement with classifications based on American College of Medical Genetics and Genomics/Association for Molecular Pathology and Association for Clinical Genomic Science guidelines. Concordance was higher for known pathogenic variants (86.3%) than for novel unpublished variants (56.8%). CONCLUSION: The considerable variability identified in the components that laboratories included in their reports and their classification of variants suggests that existing guidelines are not being used consistently with significant implications for patient care.

Genetics-first approach improves diagnostics of ESKD patients <50 years old.

BACKGROUND: Often only chronic kidney disease (CKD) patients with high likelihood of genetic disease are offered genetic testing. Early genetic testing could obviate the need for kidney biopsies, allowing for adequate prognostication and treatment. To test the viability of a 'genetics-first' approach for CKD, we performed genetic testing in a group of kidney transplant recipients aged <50 years, irrespective of cause of transplant. METHODS: From a cohort of 273 transplant patients, we selected 110 that were in care in the University Medical Center Utrecht, had DNA available and were without clear-cut non-genetic disease. Forty patients had been diagnosed with a genetic disease prior to enrollment; in 70 patients, we performed a whole-exome sequencing-based 379 gene panel analysis. RESULTS: Genetic analysis yielded a diagnosis in 51%. Extrapolated to the 273 patient cohort, who did not all fit the inclusion criteria, the diagnostic yield was still 21%. Retrospectively, in 43% of biopsied patients, the kidney biopsy would not have had added diagnostic value if genetic testing had been performed as a first-tier diagnostic. CONCLUSIONS: The burden of monogenic disease in transplant patients with end-stage kidney disease (ESKD) of any cause prior to the age of 50 years is between 21% and 51%. Early genetic testing can provide a non-invasive diagnostic, impacting prognostication and treatment, and obviating the need for an invasive biopsy. We conclude that in patients who expect to develop ESKD prior to the age of 50 years, genetic testing should be considered as first mode of diagnostics.

Analysis of laboratory reporting practices using a quality assessment of a virtual patient.

PURPOSE: Existing research suggests that while some laboratories report variants of uncertain significance, unsolicited findings (UF), and/or secondary findings (SF) when performing exome sequencing, others do not. METHODS: To investigate reporting differences, we created virtual patient-parent trio data by merging variants from patients into "normal" exomes. We invited laboratories worldwide to analyze the data along with patient phenotype information (developmental delay, dysmorphic features, and cardiac hypertrophy). Laboratories issued a diagnostic exome report and completed questionnaires to explain their rationale for reporting (or not reporting) each of the eight variants integrated. RESULTS: Of the 39 laboratories that completed the questionnaire, 30 reported the HDAC8 variant, which was a partial cause of the patient's primary phenotype, and 26 reported the BICD2 variant, which explained another phenotypic component. Lack of reporting was often due to using a filter or a targeted gene panel that excluded the variant, or because they did not consider the variant to be responsible for the phenotype. There was considerable variation in reporting variants associated with the cardiac phenotype (MYBPC3 and PLN) and reporting UF/SF also varied widely. CONCLUSION: This high degree of variability has significant impact on whether causative variants are identified, with important implications for patient care.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability.

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

Origin and clinical relevance of chromosomal aberrations other than the common trisomies detected by genome-wide NIPS: results of the TRIDENT study.

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA in maternal blood is highly sensitive for detecting fetal trisomies 21, 18, and 13. Using a genome-wide approach, other chromosome anomalies can also be detected. We report on the origin, frequency, and clinical significance of these other chromosome aberrations found in pregnancies at risk for trisomy 21, 18, or 13.MethodsWhole-genome shallow massively parallel sequencing was used and all autosomes were analyzed.ResultsIn 78 of 2,527 cases (3.1%) NIPS was indicative of trisomy 21, 18, or 13, and in 41 (1.6%) of other chromosome aberrations. The latter were of fetal (n = 10), placental (n = 22), maternal (n = 1) or unknown (n = 7). One case lacked cytogenetic follow-up. Nine of the 10 fetal cases were associated with an abnormal phenotype. Thirteen of the 22 (59%) placental aberrations were associated with fetal congenital anomalies and/or poor fetal growth (

Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies.

BACKGROUND: Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious. OBJECTIVE: We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS). METHODS: We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs. RESULTS: This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs. CONCLUSION: This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders.

Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo).

BACKGROUND: The turkey (Meleagris gallopavo) is an important agricultural species and the second largest contributor to the world's poultry meat production. Genetic improvement is attributed largely to selective breeding programs that rely on highly heritable phenotypic traits, such as body size and breast muscle development. Commercial breeding with small effective population sizes and epistasis can result in loss of genetic diversity, which in turn can lead to reduced individual fitness and reduced response to selection. The presence of genomic diversity in domestic livestock species therefore, is of great importance and a prerequisite for rapid and accurate genetic improvement of selected breeds in various environments, as well as to facilitate rapid adaptation to potential changes in breeding goals. Genomic selection requires a large number of genetic markers such as e.g. single nucleotide polymorphisms (SNPs) the most abundant source of genetic variation within the genome. RESULTS: Alignment of next generation sequencing data of 32 individual turkeys from different populations was used for the discovery of 5.49 million SNPs, which subsequently were used for the analysis of genetic diversity among the different populations. All of the commercial lines branched from a single node relative to the heritage varieties and the South Mexican turkey population. Heterozygosity of all individuals from the different turkey populations ranged from 0.17-2.73 SNPs/Kb, while heterozygosity of populations ranged from 0.73-1.64 SNPs/Kb. The average frequency of heterozygous SNPs in individual turkeys was 1.07 SNPs/Kb. Five genomic regions with very low nucleotide variation were identified in domestic turkeys that showed state of fixation towards alleles different than wild alleles. CONCLUSION: The turkey genome is much less diverse with a relatively low frequency of heterozygous SNPs as compared to other livestock species like chicken and pig. The whole genome SNP discovery study in turkey resulted in the detection of 5.49 million putative SNPs compared to the reference genome. All commercial lines appear to share a common origin. Presence of different alleles/haplotypes in the SM population highlights that specific haplotypes have been selected in the modern domesticated turkey.

Impaired activity of the fusogenic micropeptide Myomixer causes myopathy resembling Carey-Fineman-Ziter syndrome.

Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9-mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.

Accurate detection of circulating tumor DNA using nanopore consensus sequencing.

Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ~60×, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in clinical workflows.

Regional differences in recombination hotspots between two chicken populations.

BACKGROUND: Although several genetic linkage maps of the chicken genome have been published, the resolution of these maps is limited and does not allow the precise identification of recombination hotspots. The availability of more than 3.2 million SNPs in the chicken genome and the recent advances in high throughput genotyping techniques enabled us to increase marker density for the construction of a high-resolution linkage map of the chicken genome. This high-resolution linkage map allowed us to study recombination hotspots across the genome between two chicken populations: a purebred broiler line and a broiler x broiler cross. In total, 1,619 animals from the two different broiler populations were genotyped with 17,790 SNPs. RESULTS: The resulting linkage map comprises 13,340 SNPs. Although 360 polymorphic SNPs that had not been assigned to a known chromosome on chicken genome build WASHUC2 were included in this study, no new linkage groups were found. The resulting linkage map is composed of 31 linkage groups, with a total length of 3,054 cM for the sex-average map of the combined population. The sex-average linkage map of the purebred broiler line is 686 cM smaller than the linkage map of the broiler x broiler cross. CONCLUSIONS: In this study, we present a linkage map of the chicken genome at a substantially higher resolution than previously published linkage maps. Regional differences in recombination hotspots between the two mapping populations were observed in several chromosomes near the telomere of the p arm; the sex-specific analysis revealed that these regional differences were mainly caused by female-specific recombination hotspots in the broiler x broiler cross.

Partial duplication of the PRLR and SPEF2 genes at the late feathering locus in chicken.

BACKGROUND: One of the loci responsible for feather development in chickens is K. The K allele is partially dominant to the k+ allele and causes a retard in the emergence of flight feathers at hatch. The K locus is sex linked and located on the Z chromosome. Therefore, the locus can be utilized to produce phenotypes that identify the sexes of chicks at hatch. Previous studies on the organization of the K allele concluded the integration of endogenous retrovirus 21 (ev21) into one of two large homologous segments located on the Z chromosome of late feathering chickens. In this study, a detailed molecular analysis of the K locus and a DNA test to distinguish between homozygous and heterozygous late feathering males are presented. RESULTS: The K locus was investigated with quantitative PCR by examining copy number variations in a total of fourteen markers surrounding the ev21 integration site. The results showed a duplication at the K allele and sequence analysis of the breakpoint junction indicated a tandem duplication of 176,324 basepairs. The tandem duplication of this region results in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Sequence analysis revealed that the duplication is similar in Broiler and White Leghorn. In addition, twelve late feathering animals, including Broiler, White Leghorn, and Brown Layer lines, contained a 78 bp breakpoint junction fragment, indicating that the duplication is similar in all breeds. The breakpoint junction was used to develop a TaqMan-based quantitative PCR test to allow distinction between homozygous and heterozygous late feathering males. In total, 85.3% of the animals tested were correctly assigned, 14.7% were unassigned and no animals were incorrectly assigned. CONCLUSION: The detailed molecular analysis presented in this study revealed the presence of a tandem duplication in the K allele. The duplication resulted in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Furthermore, a DNA test was developed to distinguish between homozygous and heterozygous late feathering males.

Discordant NIPT result in a viable trisomy-21 pregnancy due to prolonged contribution to cfDNA by a demised trisomy-14 cotwin.

One of the confounders in noninvasive prenatal testing (NIPT) is the vanishing twin phenomenon. Prolonged contribution to the maternal Cell-free DNA (cfDNA) pool by cytotrophoblasts representing a demised, aneuploid cotwin may lead to a false-positive outcome for a normal, viable twin. We show that a vanishing trisomy-14 twin contributes to cfDNA for more than 2 weeks after demise.

Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case.

Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-free fetal DNA and yields a biological explanation for falsely reassuring NIPT results.

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects.

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.

Signatures of selection in the genomes of commercial and non-commercial chicken breeds.

Identifying genomics regions that are affected by selection is important to understand the domestication and selection history of the domesticated chicken, as well as understanding molecular pathways underlying phenotypic traits and breeding goals. While whole-genome approaches, either high-density SNP chips or massively parallel sequencing, have been successfully applied to identify evidence for selective sweeps in chicken, it has been difficult to distinguish patterns of selection and stochastic and breed specific effects. Here we present a study to identify selective sweeps in a large number of chicken breeds (67 in total) using a high-density (58 K) SNP chip. We analyzed commercial chickens representing all major breeding goals. In addition, we analyzed non-commercial chicken diversity for almost all recognized traditional Dutch breeds and a selection of representative breeds from China. Based on their shared history or breeding goal we in silico grouped the breeds into 14 breed groups. We identified 396 chromosomal regions that show suggestive evidence of selection in at least one breed group with 26 of these regions showing strong evidence of selection. Of these 26 regions, 13 were previously described and 13 yield new candidate genes for performance traits in chicken. Our approach demonstrates the strength of including many different populations with similar, and breed groups with different selection histories to reduce stochastic effects based on single populations.