Homozygosity for a Novel DOCK7 Variant Due to Segmental Uniparental Isodisomy of Chromosome 1 Associated with Early Infantile Epileptic Encephalopathy (EIEE) and Cortical Visual Impairment.

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.

Probing Vasoreactivity and Hypoxic Phenotype in Different Tumor Grafts Grown on the Chorioallantoic Membrane of the Chicken Embryo In Ovo Using MRI.

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.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases.

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.

Genotype-Phenotype Analysis of a Novel Recessive and a Recurrent Dominant SNRNP200 Variant Causing Retinitis Pigmentosa.

Purpose: To compare phenotype variability in retinitis pigmentosa patients with recessive and dominant mutations in the SNRNP200 gene. Methods: In a retrospective study, patients of two unrelated families were identified: family A, five patients aged 36 to 77 years; family B, one patient aged 9 years and his asymptomatic parents and sister. All patients received a comprehensive eye examination with a detailed retinal functional and morphologic assessment. Genetic testing was performed by whole exome sequencing (WES) in the index patient from each family. Genes described to be involved in eye diseases (n > 450) were screened for rare variants and segregation analysis was performed. Results: A known heterozygous missense variant (c.3260C>T, p.(Ser1087Leu)) in the SNRNP200 gene was identified in the index patient of family A while a novel homozygous missense mutation (c.1634G>A, p.(Arg545His)) was found in the index patient of family B. Nyctalopia and photophobia were reported by 6/6 and 2/6 patients, respectively. The phenotype associated with the dominant mutation was characterized by variable disease onset (early childhood to the sixth decade of life), disease severity (visual acuity of 20/20-20/200 in the seventh to eighth decade), and advanced rod-cone dysfunction. Characteristics of recessive disease included distinct fundus changes of dot-like hypopigmentation together with retinal atrophy and severe rod-cone dysfunction. Conclusions: The phenotype characteristics in autosomal dominant and recessive SNRNP200 mutations show distinct features, with earlier severe disease in the recessive case and a variable disease expression in the dominant inheritance pattern.

A new in vivo magnetic resonance imaging method to noninvasively monitor and quantify the perfusion capacity of three-dimensional biomaterials grown on the chorioallantoic membrane of chick embryos.

Adequate vascularization in biomaterials is essential for tissue regeneration and repair. Current models do not allow easy analysis of vascularization of implants in vivo, leaving it a highly desirable goal. A tool that allows monitoring of perfusion capacity of such biomaterials noninvasively in a cheap, efficient, and reliable in vivo model would hence add great benefit to research in this field. We established, for the first time, an in vivo magnetic resonance imaging (MRI) method to quantify the perfusion capacity of a model biomaterial, DegraPol(®) foam scaffold, placed on the embryonic avian chorioallantoic membrane (CAM) in ovo. Perfusion capacity was assessed through changes in the longitudinal relaxation rate before and after injection of a paramagnetic MRI contrast agent, Gd-DOTA (Dotarem(®); Guerbet S.A.). Relaxation rate changes were compared in three different regions of the scaffold, that is, at the interface to the CAM, in the middle and on the surface of the scaffold (p<0.05). The highest relaxation rate changes, and hence perfusion capacities, were measured in the interface region where the scaffold was attached to the CAM, whereas the surface of the scaffold showed the lowest relaxation rate changes. A strong positive correlation was obtained between relaxation rate changes and histologically determined vessel density (R(2) = 0.983), which corroborates our MRI findings. As a proof-of-principle, we measured the perfusion capacity in different scaffold materials, silk fibroin either with or without human dental pulp stem cells. For these, three to four times larger perfusion capacities were obtained compared to DegraPol; demonstrating that our method is sensitive to reveal such differences. In summary, we present a novel in vivo method for analyzing the perfusion capacity in three-dimensional-biomaterials grown on the CAM, enabling the determination of the perfusion capacity of a large variety of bioengineered materials.

The SPTLC3 subunit of serine palmitoyltransferase generates short chain sphingoid bases.

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C(16)-sphinganine and C(16)-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C(16)-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C(16) backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C(16)-sphingoid bases and that sphingolipids with a C(16) backbone constitute a significant proportion of human plasma sphingolipids.