The IONA® Test: Development of an Automated Cell-Free DNA-Based Screening Test for Fetal Trisomies 13, 18, and 21 That Employs the Ion Proton Semiconductor Sequencing Platform.

OBJECTIVE: To develop a screening test for fetal trisomy 13, 18, and 21 using cell-free DNA from maternal blood with an automated workflow using the Ion Proton sequencing platform. METHODS: An automated next-generation sequencing workflow was developed using the Ion Proton sequencing platform and software developed for straightforward bioinformatic analysis. An algorithm was developed using 239 samples to determine the likelihood of trisomy, using DNA fragment counts and a fetal fraction validity check; the results were compared with those from invasive diagnostic procedures. A further 111 samples were used to assess the tests' sensitivity (detection rate) and specificity (1 minus false-positive rate). RESULTS: The 110 of a possible 111 valid samples used to verify the IONA® test gave 100% sensitivity and specificity, compared with invasive diagnostic procedures; one failed the fetal fraction validity check giving a sample failure rate of 0.29% across all 350 analysed samples. CONCLUSION: The data indicate that the IONA test provides a robust, accurate automated workflow suitable for use on maternal blood samples to screen for trisomies 13, 18, and 21. The test has the potential to reduce the number of unnecessary invasive procedures performed and facilitate testing by screening laboratories.

Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

The Mediator complex protein Med31 is required for embryonic growth and cell proliferation during mammalian development.

During development, the mammalian embryo must integrate signals to control growth and proliferation. A failure in the ability to respond to mitogenic stimuli can cause embryonic growth restriction. We have identified a mouse mutant, l11Jus15, from a mutagenesis screen that exhibits growth defects and late-gestation lethality. Here we demonstrate that this phenotype results from a mutation in the Mediator complex gene Med31, which causes degradation of Med31 protein. The Med31 mutant phenotype is not similar to other Mediator complex mouse mutants, and target genes of other Mediator proteins are expressed normally in Med31 mutants, suggesting that Med31 has distinct target genes required for mammalian development. Med31 mutant embryos have fewer proliferating cells than controls, especially in regions that expand rapidly during development such as the forelimb buds. Likewise, embryonic fibroblast cells cultured from mutant embryos have a severe proliferation defect, as well as reduced levels of the cell cycle protein Cdc2. Med31 mutants have normal limb bud patterning but defective or delayed chondrogenesis due to a lack of Sox9 and Col2a1 expression. As the Mediator complex is a transcriptional co-activator, our results suggest that Med31 functions to promote the transcription of genes required for embryonic growth and cell proliferation.