The modification of the renal carcinogenicity of dimethylnitrosamine by actinomycin D and a protein deficient diet.

The effect of a single treatment with 30 mg dimethylnitrosamine (DMN) and 6 mug actinomycin D (ACT), given at different time intervals (ACT application to DMN, 2 h before, simultaneously, 5, 9 or 48 h later), was tested in female Sprague-Dawley rats in relation to renal carcinogenesis; additionally, the animals were fed either a normal or a protein deficient diet. The ACT treatment did not significantly modify either the kidney tumour incidence or the survival time in the different groups fed a normal diet. Nevertheless, there are indications that additional ACT application may shorten the latency period for DMN induced renal neoplasms or, when administered 5 h later than DMN, a slightly decreased and delayed tumour induction can be assumed. In groups fed a protein deficient diet, a significantly higher percentage of kidney tumour bearing animals as well as a shortened latency period were found when compared with the DMN group on normal diet, but these differences were independent of the additional ACT treatment 9 h later than DMN and were due to the protein deprivation. Morphologically, the tumours were of epithelial and mesenchymal type with a clear preponderance of the former type. Biochemical and morphological aspects are discussed

Non-Penetrance for Ocular Phenotype in Two Individuals Carrying Heterozygous Loss-of-Function ZEB1 Alleles

ZEB1 loss-of-function (LoF) alleles are known to cause a rare autosomal dominant disorder—posterior polymorphous corneal dystrophy type 3 (PPCD3). To date, 50 pathogenic LoF variants have been identified as disease-causing and familial studies have indicated that the PPCD3 phenotype is penetrant in approximately 95% of carriers. In this study, we interrogated in-house exomes (n = 3616) and genomes (n = 88) for the presence of putative heterozygous LoF variants in ZEB1. Next, we performed detailed phenotyping in a father and his son who carried a novel LoF c.1279C>T; p.(Glu427*) variant in ZEB1 (NM_030751.6) absent from the gnomAD v.2.1.1 dataset. Ocular examination of the two subjects did not show any abnormalities characteristic of PPCD3. GnomAD (n = 141,456 subjects) was also interrogated for LoF ZEB1 variants, notably 8 distinct heterozygous changes presumed to lead to ZEB1 haploinsufficiency, not reported to be associated with PPCD3, have been identified. The NM_030751.6 transcript has a pLI score ≥ 0.99, indicating extreme intolerance to haploinsufficiency. In conclusion, ZEB1 LoF variants are present in a general population at an extremely low frequency. As PPCD3 can be asymptomatic, the true penetrance of ZEB1 LoF variants remains currently unknown but is likely to be lower than estimated by the familial led approaches adopted to date

Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD is biochemically characterized by increased C4-carnitine in plasma and ethylmalonic acid in urine. The diagnosis of SCADD is confirmed by DNA analysis showing SCAD gene mutations and/or variants. SCAD gene variants are present in homozygous form in approximately 6% of the general population and considered to confer susceptibility to development of clinical disease. Clinically, SCADD generally appears to present early in life and to be most frequently associated with developmental delay, hypotonia, epilepsy, behavioral disorders, and hypoglycemia. However, these symptoms often ameliorate and even disappear spontaneously during follow-up and were found to be unrelated to the SCAD genotype. In addition, in some cases, symptoms initially attributed to SCADD could later be explained by other causes. Finally, SCADD relatives of SCADD patients as well as almost all SCADD individuals diagnosed by neonatal screening remained asymptomatic during follow-up. This potential lack of clinical consequences of SCADD has several implications. First, the diagnosis of SCADD should never preclude extension of the diagnostic workup for other potential causes of the observed symptoms. Second, patients and parents should be clearly informed about the potential lack of relevance of the disorder to avoid unfounded anxiety. Furthermore, to date, SCADD is not an optimal candidate for inclusion in newborn screening programs. More studies are needed to fully establish the relevance of SCADD and solve the question as to whether SCADD is involved in a multifactorial disease or represents a nondisease

Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease

Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing

While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS

Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing

While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS

The Congenital Cataract-Linked G61C Mutation Destabilizes γD-Crystallin and Promotes Non-Native Aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation