Genomic testing for differences of sex development: Practices and perceptions of clinicians.

OBJECTIVES: To investigate the approach taken by clinicians involved in the diagnosis and management of individuals with Differences of Sex Development (DSD), particularly with regard to genomic testing, and identify perceived gaps/strengths/barriers in current practice. DESIGN AND METHODS: An anonymous online survey was developed, with questions exploring demographics, perceptions of genomic testing, availability of genetics services and opinions on the role and utility of genomic testing in DSD. All responses were anonymous. Clinicians involved in the diagnosis and management of individuals with DSD were recruited from relevant societies and departments across Australia and New Zealand. RESULTS: 79 eligible clinicians commenced the survey, with 63 completing it and 16 providing a partial response. The perceived benefit of having a genetic diagnosis for DSD was almost unanimous (97%). Almost half (48%) of respondents reported barriers in genomic testing. 81% of respondents reported they order genomic tests currently. Approaches to genomic testing when faced with four different clinical scenarios varied across respondents. Clinicians perceived genomic testing to be underutilised (median 36 on sliding scale from 0 to 100). CONCLUSIONS: Despite 97% of respondents reporting benefit of a genetic diagnosis for individuals with DSD, this was not reflected throughout the survey with regard to clinical implementation. When faced with clinical scenarios, the recommendations for genomic testing from respondents was much lower, indicating the discrepancy between perception and clinical practice. Genomic testing in the context of DSD is seen as both beneficial and desired, yet there are multiple barriers impacting its integration into standard clinical care.

The presence of broadly neutralizing anti-SARS-CoV-2 RBD antibodies elicited by primary series and booster dose of COVID-19 vaccine.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design.

Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Intellectual Functioning of Children With Isolated PRS, PRS-Plus, and Syndromic PRS.

OBJECTIVE: Describe the intelligence quotient (IQ) of children with Pierre Robin sequence (PRS). DESIGN: Prospective cohort study. SETTING: Neurodevelopmental follow-up clinic within a hospital. PATIENTS: Children with PRS (n = 45) who had been in the Neonatal Intensive Care Unit (NICU) were classified by a geneticist into 3 subgroups of isolated PRS (n = 20), PRS-plus additional medical features (n = 8), and syndromic PRS (n = 17) based on medical record review and genetic testing. MAIN OUTCOME MEASURE: Children with PRS completed IQ testing at 5 or 8 years of age with the Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III) or Fourth Edition (WPPSI-IV) or the Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV) or Fifth Edition (WISC-V). RESULTS: IQ scores were more than 1 to 2 standard deviations below the mean for 36% of the overall sample, which was significantly greater compared to test norms (binomial test P = .001). There was a significant association between PRS subtype and IQ (Fisher's exact P = .026). While only 20% of children with isolated PRS were within 1 standard deviation below average and 35% of children with syndromic PRS were below 1 to 2 standard deviations, 75% of PRS-plus children scored lower than 1 to 2 standard deviations below the mean. CONCLUSION: PRS subgroups can help identify children at risk for cognitive delay. The majority of children with PRS-plus had low intellectual functioning, in contrast to the third of children with syndromic PRS who had low IQ and the majority of children with isolated PRS who had average or higher IQ.

Co-occurrence of dual lineages within Simulium (Gomphostilbia) atratum De Meijere in the Indonesian Archipelago along Wallace's Line.

Mitochondrial cytochrome c oxidase subunit I (COI) sequences were utilized to infer the population genetic structure of Simulium (Gomphostilbia) atratum De Meijere, an endemic simulid species to Indonesia. Both median-joining haplotype network and maximum-likelihood tree revealed two genetic lineages (A and B) within the species, with an overlap distribution in Lombok, which is situated along Wallace's line. Genetic differentiation and gene flow with varying frequencies (FST = 0.02-0.967; Nm = 0.01-10.58) were observed between populations of S. (G.) atratum, of which population pairs of different lineages showed high genetic differentiation. Notably, the high genetic distance of up to 5.92 % observed within S. (G.) atratum in Lombok was attributed to the existence of two genetically distinct lineages. The co-occurrence of distinct lineages in Lombok indicated that Wallace's line did not act as faunistic border for S. (G.) atratum in the present study. Moreover, both lineages also exhibited unimodal distributions and negative values of neutrality tests, suggesting a pattern of population expansion. The expansion and divergence time estimation suggested that the two lineages of S. (G.) atratum diverged and expanded during the Pleistocene era in Indonesia.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation.

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.