A second hotspot for pathogenic exon-skipping variants in CDC45.

Biallelic pathogenic variants in CDC45 are associated with Meier-Gorlin syndrome with craniosynostosis (MGORS type 7), which also includes short stature and absent/hypoplastic patellae. Identified variants act through a hypomorphic loss of function mechanism, to reduce CDC45 activity and impact DNA replication initiation. In addition to missense and premature termination variants, several pathogenic synonymous variants have been identified, most of which cause increased exon skipping of exon 4, which encodes an essential part of the RecJ-orthologue's DHH domain. Here we have identified a second cohort of families segregating CDC45 variants, where patients have craniosynostosis and a reduction in height, alongside common facial dysmorphisms, including thin eyebrows, consistent with MGORS7. Skipping of exon 15 is a consequence of two different variants, including a shared synonymous variant that is enriched in individuals of East Asian ancestry, while other variants in trans are predicted to alter key intramolecular interactions in α/ß domain II, or cause retention of an intron within the 3'UTR. Our cohort and functional data confirm exon skipping is a relatively common pathogenic mechanism in CDC45, and highlights the need for alternative splicing events, such as exon skipping, to be especially considered for variants initially predicted to be less likely to cause the phenotype, particularly synonymous variants.

A novel KNL1 intronic splicing variant likely destabilizes the KMN complex, causing primary microcephaly.

Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by head circumference of at least two standard deviations below the mean. Biallelic variants in the kinetochore gene KNL1 is a known cause of MCPH4. KNL1 is the central component of the KNL1-MIS12-NSL1 (KMN) network, which acts as the signaling hub of the kinetochore and is required for correct chromosomal segregation during mitosis. We identified biallelic KNL1 variants in two siblings from a non-consanguineous family with microcephaly and intellectual disability. The two siblings carry a frameshift variant predicted to prematurely truncate the transcript and undergo nonsense mediated decay, and an intronic single nucleotide variant (SNV) predicted to disrupt splicing. An in vitro splicing assay and qPCR from blood-derived RNA confirmed that the intronic variant skips exon 23, significantly reducing levels of the canonical transcript. Protein modeling confirmed that absence of exon 23, an inframe exon, would disrupt a key interaction within the KMN network and likely destabilize the kinetochore signaling hub, disrupting mitosis. Therefore, this splicing variant is pathogenic and, in trans with a frameshift variant, causes the MCPH phenotype associated with KLN1. This finding furthers the association of splicing variants as a common pathogenic variant class for KNL1.

Extending the PAX1 spectrum: a dominantly inherited variant causes oculo-auriculo-vertebral syndrome.

Oculo-auriculo-vertebral syndrome (OAVS) is a clinically heterogeneous disorder, with both genetic and environmental contributors. Multiple genes have been associated with OAVS and common molecular pathways, such as retinoic acid and the PAX-SIX-EYA-DACH (PSED) network, are being implicated in the disease pathophysiology. Biallelic homozygous nonsense or hypomorphic missense mutations in PAX1 cause otofaciocervical syndrome type 2 (OTFCS2), a similar but more severe multi-system disorder that can be accompanied by severe combined immunodeficiency due to thymic aplasia. Here we have identified a multi-generational family with mild features of OAVS segregating a heterozygous frameshift in PAX1. The four base duplication is expected to result in nonsense-mediated decay, and therefore cause a null allele. While there was full penetrance of the variant, expressivity of facial and ear features were variable. Our findings indicate there can be monoallelic and biallelic disorders associated with PAX1, and further implicate the PSED network in OAVS.

A synonymous variant in a non-canonical exon of CDC45 disrupts splicing in two affected sibs with Meier-Gorlin syndrome with craniosynostosis.

Disruption of the initiation of DNA replication is significantly associated with Meier-Gorlin syndrome (MGORS), an autosomal recessive condition of reduced growth, microtia and patellar a/hypoplasia. Biallelic mutations in CDC45, a member of the pre-initiation complex in DNA replication, cause a spectrum of phenotypes ranging from MGORS with craniosynostosis, through to isolated short stature and craniosynostosis. Here we report two affected sibs with MGORS and craniosynostosis, with biallelic variants in CDC45 identified by 10X Chromium whole genome sequencing. One variant is a frameshift mutation, predicted to be pathogenic, and is inherited in trans with a synonymous variant in a non-canonical exon (exon 7) of CDC45. An in vitro splicing assay showed that while the canonical CDC45 exon 6-exon 8 transcript (with skipping of exon 7; numbering as per NM001178010.2) remained as the predominant transcript, the variant allele induced the use of novel splice acceptor sites in intron 6, all of which produced transcripts harbouring premature stop codons. This perturbation of canonical splicing provides evidence that this synonymous variant is indeed a deleterious alteration in this family. This report adds to the initial patient cohort in which several synonymous variants were also described, further highlighting the contribution of this variant type in CDC45. It also reiterates the true potential pathogenicity of synonymous variants, which is a mutation type that is commonly ignored in variant prioritization strategies.

Complete Genome Sequences of the Escherichia coli Donor Strains ST18 and MFDpir.

Escherichia coli ST18 and MFDpir are donors commonly used to transfer oriT RP4-containing plasmids to diverse bacteria via conjugation. ST18 and MFDpir were constructed via multiple genetic manipulations involving several E. coli strains. Here, we used Illumina and Nanopore sequencing to determine the complete genomes of these widely used strains.