A rapid and non-invasive proteomic analysis using DBS and buccal swab for multiplexed second-tier screening of Pompe disease and Mucopolysaccharidosis type I.

PURPOSE: Current newborn screening programs for Pompe disease (PD) and mucopolysaccharidosis type I (MPS I) suffer from a high false positive rate and long turnaround time for clinical follow up. This study aimed to develop a novel proteomics-based assay for rapid and accurate second-tier screening of PD and MPS I. A fast turnaround assay would enable the identification of severe cases who need immediate clinical follow up and treatment. METHODS: We developed an immunocapture coupled with mass spectrometry-based proteomics (Immuno-SRM) assay to quantify GAA and IDUA proteins in dried blood spots (DBS) and buccal swabs. Sensitivity, linearity, reproducibility, and protein concentration range in healthy control samples were determined. Clinical performance was evaluated in known PD and MPS I patients as well as pseudodeficiency and carrier cases. RESULTS: Using three 3.2 mm punches (~13.1 µL of blood) of DBS, the assay showed reproducible and sensitive quantification of GAA and IDUA. Both proteins can also be quantified in buccal swabs with high reproducibility and sensitivity. Infantile onset Pompe disease (IOPD) and severe MPS I cases are readily identifiable due to the absence of GAA and IDUA, respectively. In addition, late onset Pompe disease (LOPD) and attenuated MPS I patients showed much reduced levels of the target protein. By contrast, pseudodeficiency and carrier cases exhibited significant higher target protein levels compared to true patients. CONCLUSION: Direct quantification of endogenous GAA and IDUA peptides in DBS by Immuno-SRM can be used for second-tier screening to rapidly identify severe PD and MPS I patients with a turnaround time of <1 week. Such patients could benefit from immediate clinical follow up and possibly earlier treatment.

Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

"Doctors can read about it, they can know about it, but they've never lived with it": How parents use social media throughout the diagnostic odyssey.

Parents of children with undiagnosed conditions struggle to obtain information about how to treat and support their children. It can be particularly challenging to find communities and other parents who share their experiences and can provide emotional and informational support. This study sought to characterize how parents use social media, both throughout the diagnostic odyssey and post-diagnosis, to meet their informational, social, and emotional support needs. We conducted qualitative semi-structured interviews with 14 parents from the Stanford site of the Undiagnosed Diseases Network (UDN), including five whose children had received a diagnosis through study participation. Interview recordings were analyzed using inductive, team-based coding and thematic analysis based in grounded theory using Dedoose qualitative analysis software. Through this process, we identified four key themes related to social media use. First, parents struggled to find the "right" community, often seeking out groups of similar patients based on symptoms or similar conditions. Second, though they found much valuable information through social media about caring for their child, they also struggled to interpret the relevance of the information to their own child's condition. Third, the social support and access to other patients' and families' lived experiences were described as both highly valued and emotionally challenging, particularly in the case of poor outcomes for similar families. Finally, parents expressed the need to balance concerns about their child's privacy with the value of transparency and data sharing for diagnosis. Our results suggest that the needs and experiences of undiagnosed patients and families differ from those with diagnosed diseases and highlight the need for support in best utilizing social media resources at different stages of the diagnostic odyssey.

Attitudes Toward Hypothetical Uses of Gene-Editing Technologies in Parents of People with Autosomal Aneuploidies.

Researchers are exploring the use of gene-editing technologies to prevent and/or treat genetic conditions in humans. Stakeholder views, including those of patient and family populations, are important in the ongoing bioethical discussion. We conducted 27 semi-structured interviews with parents of people with trisomy 21 (T21; N = 10), trisomy 18 (T18; N = 8), and trisomy 13 (T13; N = 9)-conditions not previously studied in regard to attitudes toward hypothetical gene editing. While many discussions focus on the morality of gene editing, parents in our study focused on quality of life and concerns about changing their children's identity. All participants prioritized ameliorating life-threatening health issues when those were present; many also emphasized increasing their children's communication and cognitive ability. These results suggest that patient populations with the lived experience of genetic conditions have unique concerns that may differ from broader discourse.