Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping

Background The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. Methods Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. Results Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4 %). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for “cascade” screening and/or prenatal diagnosis. Conclusions Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals

Characteristics of soft-clipped breakpoint-containing reads.

<p>Characteristics of soft-clipped breakpoint-containing reads.</p

Chromosome 7 ideogram and breakpoint confirmation.

<p><b>(A)</b> Arrows showing the breakpoint locations. Greek letters facilitate interpretation of the resulting pericentric inversion. Sanger sequencing results for the normal and breakpoint spanning amplicons for <b>(B)</b> the 7p15 and <b>(C)</b> the 7q21 inversion boundaries. The vertical dashed read line highlights the breakpoint. For ease of comparison a dashed black line has been drawn onto the normal sequence. (+): sense strand sequence; (-): antisense strand sequence. The inversion has resulted in an AT dinucleotide duplication which is shown arbitrarily assigned to the 7p15 breakpoint.</p

Reads pairs with discordant reads mapping to 7p15 and 7q21 within 200 bp of each other.

<p>Reads pairs with discordant reads mapping to 7p15 and 7q21 within 200 bp of each other.</p

Additional file 2: Figure S1. of Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping

Schematic representation of the FishingCNV-defined deletions showing the minimum (red) and maximum (green) possible boundaries of the deletion breakpoints for (A) the intragenic AHI1 deletion and (B) the TMEM237 to MPP4 deletion. Exons are displayed in blue and numbering is in accordance with transcripts [GenBank:NM_001134830.1] (AHI1), [GenBank:NM_001044385.2] and [GenBank:NM_152388.3] (TMEM237) and [GenBank:NM_033066.2] (MPP4). (TIF 19237 kb

Additional file 1: Table S1. of Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping

Joubert and Meckel-Gruber syndrome genes included in the targeted exome analysis. Table S2. Summary performance metrics for each sample analysed. Table S3. The percentage of target nucleotides for each gene with a read depth ≥30. Table S4. Summary variant counts following data processing using the SNV/small indel detection pipeline. (DOCX 190 kb

Deficiency of the myogenic factor MyoD causes a perinatally lethal fetal akinesia

Background: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. Methods: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. Results: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. Conclusions: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme

Sacral Pressure injuries in bed confined ICU patients

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