Of over 7000 patients referred to a diagnostic laboratory, 28% had diagnoses based on DNA sequencing, 5% of whom had two or more diagnoses. Their phenotypes could be better understood by considering ...whether the implicated genes affect independent biologic processes or organ systems.
Medical genetics focuses on the relationship between observed phenotypes and their underlying genotypes, modes of transmission, and risks of recurrence. Expected patterns of mendelian inheritance are often used to confirm the identification of disease genes, and deviations from mendelian expectations have led to the discovery of more complicated genetic underpinnings of disease (Fig. S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).
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Multiple (or dual) molecular diagnoses involve more than one clinical diagnosis and more than one genetic locus (Figure 1), each segregating independently.
Diagnostic whole-exome sequencing affords opportunities for providing insights into relationships . . .
Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the ...paucity of information about the clinical utility of exome sequencing in the prenatal setting.
We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive "fetal exomes" performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher's exact test.
Prenatal exome yielded an overall molecular diagnostic rate of 32% (n = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders (n = 23/46), 41% were autosomal recessive disorders (n = 19/46), and 9% were X-linked disorders (n = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families.
Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Improved resolution of molecular diagnostic technologies enabled detection of smaller sized exonic level copy-number variants (CNVs). The contribution of CNVs to autosomal recessive (AR) conditions ...may be better recognized using a large clinical cohort.
We retrospectively investigated the CNVs’ contribution to AR conditions in cases subjected to chromosomal microarray analysis (CMA, N=~70,000) and/or clinical exome sequencing (ES, N=~12,000) at Baylor Genetics; most had pediatric onset neurodevelopmental disorders.
CNVs contributed to biallelic variations in 87 cases, including 81 singletons and three affected sibling pairs. Seventy cases had CNVs affecting both alleles, and 17 had a CNV and a single-nucleotide variant (SNV)/indel in trans. In total, 94.3% of AR-CNVs affected one gene; among these 41.4% were single-exon and 35.0% were multiexon partial-gene events. Sixty-nine percent of homozygous AR-CNVs were embedded in homozygous genomic intervals. Five cases had large deletions unmasking an SNV/indel on the intact allele for a recessive condition, resulting in multiple molecular diagnoses.
AR-CNVs are often smaller in size, transmitted through generations, and underrecognized due to limitations in clinical CNV detection methods. Our findings from a large clinical cohort emphasized integrated CNV and SNV/indel analyses for precise clinical and molecular diagnosis especially in the context of genomic disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating ...protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels by counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insights into endosomal trafficking, illuminate the cooperativity between an ubiquitin ligase and a deubiquitinating enzyme, and establish a role for USP7 in human neurodevelopmental disease.
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•USP7 is part of the MAGE-L2-TRIM27 ubiquitin ligase and enables endosomal recycling•USP7 protects TRIM27 from auto-ubiquitination and proteasomal degradation•USP7 buffers WASH ubiquitination levels to maintain proper endosomal actin levels•Mutation of USP7 causes a human neurodevelopmental syndrome, including autism
Hao et al. describe a function of the USP7 deubiquitinating enzyme in regulation of WASH/retromer-mediated endosomal protein recycling. USP7 functions as a molecular rheostat to prevent auto-ubiquitination and proteasomal degradation of TRIM27 E3 ubiquitin ligase, but also deubiquitinates WASH. Genetic studies identify cases of USP7 mutation/deletion resulting in a human neurodevelopmental disorder that overlaps with MAGE-L2 mutation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions ...and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.
Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic ...fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed.
To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics.
We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes.
In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal ...microduplication syndrome described—the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (∼3.7 Mb), 13 subjects (∼37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive
RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mendelian and early-onset severe psychiatric phenotypes often involve genetic variants having a large effect, offering opportunities for genetic discoveries and early therapeutic interventions. Here, ...the index case is an 18-year-old boy, who at 14 years of age had a decline in cognitive functioning over the course of a year and subsequently presented with catatonia, auditory and visual hallucinations, paranoia, aggression, mood dysregulation, and disorganized thoughts. Exome sequencing revealed a stop-gain mutation in RCL1 (NM_005772.4:c.370 C > T, p.Gln124Ter), encoding an RNA 3'-terminal phosphate cyclase-like protein that is highly conserved across eukaryotic species. Subsequent investigations across two academic medical centers identified eleven additional cases of RCL1 copy number variations (CNVs) with varying neurodevelopmental or psychiatric phenotypes. These findings suggest that dosage variation of RCL1 contributes to a range of neurological and clinical phenotypes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The bromodomain adjacent to zinc finger 2B gene (BAZ2B) encodes a protein involved in chromatin remodeling. Loss of BAZ2B function has been postulated to cause neurodevelopmental disorders. To ...determine whether BAZ2B deficiency is likely to contribute to the pathogenesis of these disorders, we performed bioinformatics analyses that demonstrated a high level of functional convergence during fetal cortical development between BAZ2B and genes known to cause autism spectrum disorder (ASD) and neurodevelopmental disorder. We also found an excess of de novo BAZ2B loss‐of‐function variants in exome sequencing data from previously published cohorts of individuals with neurodevelopmental disorders. We subsequently identified seven additional individuals with heterozygous deletions, stop‐gain, or de novo missense variants affecting BAZ2B. All of these individuals have developmental delay (DD), intellectual disability (ID), and/or ASD. Taken together, our findings suggest that haploinsufficiency of BAZ2B causes a neurodevelopmental disorder, whose cardinal features include DD, ID, and ASD.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, ...constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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