Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells.

INTRODUCTION: RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR. It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. METHODS: Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. RESULTS: We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15. We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. DISCUSSION: We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15, a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol.

Recommendations by the ClinGen Rett/Angelman-like expert panel for gene-specific variant interpretation methods.

The genes MECP2, CDKL5, FOXG1, UBE3A, SLC9A6, and TCF4 present unique challenges for current ACMG/AMP variant interpretation guidelines. To address those challenges, the Rett and Angelman-like Disorders Variant Curation Expert Panel (Rett/AS VCEP) drafted gene-specific modifications. A pilot study was conducted to test the clarity and accuracy of using the customized variant interpretation criteria. Multiple curators obtained the same interpretation for 78 out of the 87 variants (~90%), indicating appropriate usage of the modified guidelines the majority of times by all the curators. The classification of 13 variants changed using these criteria specifications compared to when the variants were originally curated and as present in ClinVar. Many of these changes were due to internal data shared from laboratory members however some changes were because of changes in strength of criteria. There were no two-step classification changes and only 1 clinically relevant change (Likely pathogenic to VUS). The Rett/AS VCEP hopes that these gene-specific variant curation rules and the assertions provided help clinicians, clinical laboratories, and others interpret variants in these genes but also other fully penetrant, early-onset genes associated with rare disorders.

Hereditary Alpha-Tryptasemia: UK Prevalence and Variability in Disease Expression.

BACKGROUND: Hereditary alpha-tryptasemia (HAT) is a genetic trait caused by an increased alpha-tryptase tryptase alpha/beta 1 gene copy number. Basal serum mast cell tryptase (MCT) level is typically greater than or equal to 8.0 ng/mL. OBJECTIVES: To study the clinical disease spectrum of HAT and determine its UK prevalence. METHODS: Droplet digital PCR was used to determine tryptase alpha/beta 1 copy number in 432 DNA samples from an unselected UK birth cohort and in 70 patients referred with a basal MCT level greater than 8 ng/mL. Baseline MCT concentrations and clinical presentation were also assessed in 4283 samples sent to a regional immunology laboratory. RESULTS: Duplication in alpha copy number was present in 5% of the unselected British birth cohort, with all affected individuals having a basal MCT level of greater than or equal to 8.0 ng/mL. Basal MCT levels of greater than or equal to 8.0 ng/mL were also found in 5% of the 4283 individuals referred for MCT testing because of clinical symptoms. In 70 patients confirmed to have HAT (79% with a duplication; 21% with a higher alpha gene copy number), urticaria/angioedema (51%), skin flushing (41%), food intolerances (39%), and altered bowel habits (36%) were common presenting complaints. However, clinical manifestations were not more common in patients with gene triplications or quintuplications than in those with duplications. Some immediate family members with the same genetic trait and high basal MCT levels were asymptomatic. CONCLUSIONS: Five percent of people in the United Kingdom may have HAT. The diagnosis should be considered when basal MCT level is greater than or equal to 8 ng/mL. HAT has variable clinical penetrance. It may modify the expression of multifactorial allergic diseases rather than directly cause specific phenotypes.

Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.