Understanding pre-mRNA splicing is crucial to accurately diagnosing and treating genetic diseases. However, mutations that alter splicing can exert highly diverse effects. Of all the known types of splicing mutations, perhaps the rarest and most difficult to predict are those that activate pseudoexons, sometimes also called cryptic exons. Unlike other splicing mutations that either destroy or redirect existing splice events, pseudoexon mutations appear to create entirely new exons within introns. Since exon definition in vertebrates requires coordinated arrangements of numerous RNA motifs, one might expect that pseudoexons would only arise when rearrangements of intronic DNA create novel exons by chance. Surprisingly, although such mutations do occur, a far more common cause of pseudoexons is deep-intronic single nucleotide variants, raising the question of why these latent exon-like tracts near the mutation sites have not already been purged from the genome by the evolutionary advantage of more efficient splicing. Possible answers may lie in deep intronic splicing processes such as recursive splicing or poison exon splicing. Because these processes utilize intronic motifs that benignly engage with the spliceosome, the regions involved may be more susceptible to exonization than other intronic regions would be. We speculated that a comprehensive study of reported pseudoexons might detect alignments with known deep intronic splice sites and could also permit the characterisation of novel pseudoexon categories. In this report, we present and analyse a catalogue of over 400 published pseudoexon splice events. In addition to confirming prior observations of the most common pseudoexon mutation types, the size of this catalogue also enabled us to suggest new categories for some of the rarer types of pseudoexon mutation. By comparing our catalogue against published datasets of non-canonical splice events, we also found that 15.7% of pseudoexons exhibit some splicing activity at one or both of their splice sites in non-mutant cells. Importantly, this included seven examples of experimentally confirmed recursive splice sites, confirming for the first time a long-suspected link between these two splicing phenomena. These findings have the potential to improve the fidelity of genetic diagnostics and reveal new targets for splice-modulating therapies.

/pdf/analysis-of-pathogenic-pseudoexons-reveals-novel-mechanisms-vkpzr4le.pdf

Analysis of Pathogenic Pseudoexons Reveals Novel Mechanisms Driving Cryptic Splicing

ABSTRACT We describe a child from a consanguineous family born with a rare autosomal recessive disorder affecting junctional adhesion molecule 3 (JAM3) causing profound neurological and ophthalmological injury known as haemorrhagic brain destruction, subependymal calcifications, and congenital cataracts (HDBSCC; MIM# 613730). She was the product of an unremarkable pregnancy and was born near to term but was noted shortly after birth to have congenital cataracts, poor vision, increased muscle tone, seizures, and developmental delay. Her older sister had an identical syndrome and had previously been documented to have homozygous mutations in JAM3. Examination in our patient, although difficult because of bilateral central cataracts, revealed very poor vision, attenuated retinal vessels, optic atrophy, and a retinal haemorrhage in the right eye, implying that abnormal development of the retinas and/or optic nerves may at times play a significant role in the poor vision noted in children with HDBSCC.

Retinal Findings in Haemorrhagic Destruction of the Brain, Subependymal Calcification, and Congenital Cataracts (HDBSCC): Case Report and Review

Genome sequencing (GS) is a powerful test for the diagnosis of rare genetic disorders. Although GS can enumerate most non-coding variation, determining which non-coding variants are disease-causing is challenging. RNA sequencing (RNA-seq) has emerged as an important tool to help address this issue, but its diagnostic utility remains understudied, and the added value of a trio design is unknown. We performed GS plus RNA-seq from blood using an automated clinical-grade high-throughput platform on 97 individuals from 39 families where the proband was a child with unexplained medical complexity. RNA-seq was an effective adjunct test when paired with GS. It enabled clarification of putative splice variants in three families, but it did not reveal variants not already identified by GS analysis. Trio RNA-seq decreased the number of candidates requiring manual review when filtering for de novo dominant disease-causing variants, allowing for the exclusion of 16% of gene-expression outliers and 27% of allele-specific-expression outliers. However, clear diagnostic benefit from the trio design was not observed. Blood-based RNA-seq can facilitate genome analysis in children with suspected undiagnosed genetic disease. In contrast to DNA sequencing, the clinical advantages of a trio RNA-seq design may be more limited. 

https://doi.org/10.1016/j.ajhg.2023.03.006

Trio RNA sequencing in a cohort of medically complex children

Complex genetic disease mechanisms, such as structural or non-coding variants, currently pose a substantial difficulty in frontline diagnostic tests. They thus may account for most unsolved rare disease patients regardless of the clinical phenotype. However, the clinical diagnosis can narrow the genetic focus to just a couple of genes for patients with well-established syndromes defined by prominent physical and/or unique biochemical phenotypes, allowing deeper analyses to consider complex genetic origin. Then, clinical-diagnosis-driven genome sequencing strategies may expedite the development of testing and analytical methods to account for complex disease mechanisms as well as to advance functional assays for the confirmation of complex variants, clinical management, and the development of new therapies.

/pdf/the-power-of-clinical-diagnosis-for-deciphering-complex-nhbcxszk.pdf

The Power of Clinical Diagnosis for Deciphering Complex Genetic Mechanisms in Rare Diseases

Newborn screening (NBS) for severe combined immunodeficiency (SCID) utilizing T‐cell receptor excision circles (TRECs) has been implemented in all 50 states as of December 2018 and has been transformative for the clinical care of SCID patients. Though having high sensitivity for SCID, NBS‐SCID has low specificity, therefore is able to detect other causes of lymphopenia in newborns including many inborn errors of immunity (IEIs). In a recent study, three of six newborns later diagnosed with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome were found to have a low TRECs and lymphopenia at birth. This presents an opportunity to increase the detection and diagnosis of WHIM syndrome by NBS‐SCID with immunological follow‐up along with a combination of flow cytometry for immune cell subsets, absolute neutrophil count, and genetic testing, extending beyond the conventional bone marrow studies. Coupled with emerging technologies such as next‐generation sequencing, transcriptomics and proteomics, dried blood spots used in NBS‐SCID will promote earlier detection, diagnosis, and therefore treatment of IEIs such as WHIM syndrome.

Can we identify WHIM in infancy? Opportunities with the public newborn screening process

Variants in JAM3 have been reported in four families manifesting a severe autosomal recessive disorder characterized by hemorrhagic destruction of the brain, subependymal calcification, and cataracts. We describe a 7-year-old male with a similar presentation found by research-based quad genome sequencing to have two novel splicing variants in trans in JAM3, including one deep intronic variant (NM_032801.4: c.256+1260G>C) not detectable by standard exome sequencing. Targeted sequencing of RNA isolated from transformed lymphoblastoid cell lines confirmed that each of the two variants has a deleterious effect on JAM3 mRNA splicing. The role for genome sequencing as a clinical diagnostic test extends to those patients with phenotypes strongly suggestive of a specific Mendelian disorder, especially when the causal genetic variant(s) are not found by a more targeted approach. Barriers to diagnosis via identification of pathogenic deep intronic variation include lack of laboratory consensus regarding in silico splicing prediction tools and limited access to clinically validated confirmatory RNA experiments.

Melissa Bradley

Papers

Genome sequencing for detection of pathogenic deep intronic variation: A clinical case report illustrating opportunities and challenges.