The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Tumor grafts grown on the chorioallantoic membrane (CAM) of chicken embryos represent a transition between cell culture and mammalian in vivo models. Magnetic resonance imaging (MRI) started to harness this potential. Functional gas challenge is feasible on the CAM. Using quantitative T1 and T2* mapping, we characterized the response of MC-38 colon, A549, and H460 adeno-carcinoma cell grafts to hypercapnic (HC) and hypercapnic-hyperoxic (HCHO) gas challenges, pertaining to the grafts’ vascular and oxygenation phenotypes. MR imaging revealed that larger T1 and T2* were located in the center of H460 and MC-38 tumors. Quantitative analysis showed a significant reduction in T1 and a significant increase in T2* in response to HCHO for A549 grafts, while H460 and MC-38 tumors did not respond to either gas challenge. Different tumor grafts respond differentially to HC and HCHO conditions. A549 tumor grafts, with higher vessel density and smaller tumor diameter compared with H460 and MC-38 grafts, had a significant response in T1 for HCHO and T2* increased slightly during HC and significantly under HCHO, consistent with a normoxic phenotype and functional vasoreactivity. Therefore, gas challenges enable differential characterization of tumor grafts with respect to their vascular and oxygenation status.

Early infantile epileptic encephalopathy (EIEE) is a severe neurologic and neurodevelopmental disease that manifests in the first year of life. It shows a high degree of genetic heterogeneity, but the genetic origin is only identified in half of the cases. We report the case of a female child initially diagnosed with Leber congenital amaurosis (LCA), an early-onset retinal dystrophy due to photoreceptor cell degeneration in the retina. The first examination at 9 months of age revealed no reaction to light or objects and showed wandering eye movements. Ophthalmological examination did not show any ocular abnormalities. The patient displayed mildly dysmorphic features and a global developmental delay. Brain MRI demonstrated pontine hypo-/dysplasia. The patient developed myoclonic epileptic seizures and epileptic spasms with focal and generalized epileptiform discharges on electroencephalogram (EEG) at the age of 16 months. Genetic screening for a potentially pathogenic DNA sequence variant by whole-exome sequencing (WES) revealed a novel, conserved, homozygous frameshift variant (c.5391delA, p.(Ala1798LeufsTer59)) in exon 42 of the DOCK7 gene (NM_001271999.1). Further analysis by SNP array (Karyomapping) showed loss of heterozygosity (LOH) in four segments of chromosome 1. WES data of the parents and the index patient (trio analysis) demonstrated that chromosome 1 was exclusively inherited from the mother. Four LOH segments of chromosome 1 alternately showed isodisomy (UPiD) and heterodisomy (UPhD). In WES data, the father was a noncarrier, and the mother was heterozygous for this DOCK7 variant. The DOCK7 gene is located in 1p31.3, a region situated in one of the four isodisomic segments of chromosome 1, explaining the homozygosity seen in the affected child. Finally, Sanger sequencing confirmed maternal UPiD for the DOCK7 variant. Homozygous or compound heterozygous pathogenic variants in the DOCK7 (dedicator of cytokinesis 7) gene are associated with autosomal recessive, early infantile epileptic encephalopathy 23 (EIEE23; OMIM #615,859), a rare and heterogeneous group of neurodevelopmental disorders diagnosed during early childhood. To our knowledge, this is the first report of segmental uniparental iso- and heterodisomy of chromosome 1, leading to homozygosity of the DOCK7 frameshift variant in the affected patient.