Unclassified white matter disorders: A diagnostic journey requiring close collaboration between clinical and laboratory services.

BACKGROUND: Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended. AIM: This study aimed to identify the genetic causes for a group of individuals with unclassified white matter disorders with suspected genetic aetiology and highlight the investigations required when the initial testing is non-diagnostic. METHODS: Twenty-six individuals from 22 families with unclassified white matter disorders underwent deep phenotyping and genome sequencing performed on trio, or larger, family groups. Functional studies and transcriptomics were used to resolve variants of uncertain significance with potential clinical relevance. RESULTS: Causative or candidate variants were identified in 15/22 (68.2%) families. Six of the 15 implicated genes had been previously associated with white matter disease (COL4A1, NDUFV1, SLC17A5, TUBB4A, BOLA3, DARS2). Patients with variants in the latter two presented with an atypical phenotype. The other nine genes had not been specifically associated with white matter disease at the time of diagnosis and included genes associated with monogenic syndromes, developmental disorders, and developmental and epileptic encephalopathies (STAG2, LSS, FIG4, GLS, PMPCA, SPTBN1, AGO2, SCN2A, SCN8A). Consequently, only 46% of the diagnoses would have been made via a current leukodystrophy gene panel test. DISCUSSION: These results confirm the importance of broad genomic testing for patients with white matter disorders. The high diagnostic yield reflects the integration of deep phenotyping, whole genome sequencing, trio analysis, functional studies, and transcriptomic analyses. CONCLUSIONS: Genetic white matter disorders are genetically and phenotypically heterogeneous. Deep phenotyping together with a range of genomic technologies underpin the identification of causes of unclassified white matter disease. A molecular diagnosis is essential for prognostication, appropriate management, and accurate reproductive counseling.

Quantitative prediction of repeat dose toxicity values using GenRA.

Computational approaches have recently gained popularity in the field of read-across to automatically fill data-gaps for untested chemicals. Previously, we developed the generalized read-across (GenRA) tool, which utilizes in vitro bioactivity data in conjunction with chemical descriptor information to derive local validity domains to predict hazards observed in in vivo toxicity studies. Here, we modified GenRA to quantitatively predict point of departure (POD) values obtained from US EPA's Toxicity Reference Database (ToxRefDB) version 2.0. To evaluate GenRA predictions, we first aggregated oral Lowest Observed Adverse Effect Levels (LOAEL) for 1,014 chemicals by systemic, developmental, reproductive, and cholinesterase effects. The mean LOAEL values for each chemical were converted to log molar equivalents. Applying GenRA to all chemicals with a minimum Jaccard similarity threshold of 0.05 for Morgan fingerprints and a maximum of 10 nearest neighbors predicted systemic, developmental, reproductive, and cholinesterase inhibition min aggregated LOAEL values with R2 values of 0.23, 0.22, 0.14, and 0.43, respectively. However, when evaluating GenRA locally to clusters of structurally-similar chemicals (containing 2 to 362 chemicals), average R2 values for systemic, developmental, reproductive, and cholinesterase LOAEL predictions improved to 0.73, 0.66, 0.60 and 0.79, respectively. Our findings highlight the complexity of the chemical-toxicity landscape and the importance of identifying local domains where GenRA can be used most effectively for predicting PODs.

Intestinal crypt lesions associated with protein-losing enteropathy in the dog.

Six dogs were diagnosed with protein losing enteropathy (PLE). There was no evidence of inappropriate inflammatory infiltrates or lymphangiectasia in multiple mucosal biopsies of the small intestine of 4 of the dogs. The 5th and 6th dogs had obvious lymphangiectasia and a moderate infiltrate of inflammatory cells in the intestinal mucosa. All 6 dogs had a large number of dilated intestinal crypts that were filled with mucus, sloughed epithelial cells, and/or inflammatory cells. Whether PLE occurs in these dogs because of protein lost from the dilated crypts into the intestinal lumen or whether the dilated crypts are a mucosal reaction due to another undetermined lesion that is responsible for alimentary tract protein loss is unknown. However, when large numbers of dilated intestinal crypts are present, they appear to be associated with PLE even if there are no other remarkable lesions in the intestinal mucosa.

In vitro percutaneous absorption in mouse skin: influence of skin appendages.

Skin appendages are often envisaged as channels that bypass the stratum corneum barrier and are generally thought to facilitate the dermal absorption of topical agents. However, the significance of this transappendageal pathway in percutaneous absorption remains to be assessed experimentally. With the use of a skin organ culture penetration chamber system, the influence of skin appendages on the in vitro permeation of topically applied benzo[a]pyrene and testosterone (5 micrograms/2 cm2) was examined in skin preparations from both haired and hairless mice. Haired mice examined included the C57BL6, C3H, DBA2, Balbc, and Sencar strains and the hairless mice were the HRS and SKH. In all mouse strains examined, the overall permeation of testosterone (greater than 65% of applied dose) 16 hr following in vitro topical application was greater than that of benzo[a]pyrene (less than 10%). No strain differences were observed with respect to the percutaneous permeation of testosterone; however, percutaneous permeation of benzo[a]pyrene in the haired mice (7-10% of applied dose) was higher than that in the hairless mice (2%). In an in-house derived mouse strain which showed three phenotypic variants due to hair densities, the permeability to both compounds was highest in the skin of the haired phenotype (testosterone 67%, benzo[a]pyrene 7%), lowest in the hairless phenotype (35 and 1%, respectively) and intermediate in the fuzzy-haired animal (57 and 3%, respectively). Examination by fluorescence microscopy of cryosections of skin, prepared 1 hr after topical benzo[a]pyrene, showed areas of intense fluorescence deep within the nonfluorescing dermis of skin from the haired phenotype. These fluorescent areas were correlated with follicular ducts and sebaceous glands. In contrast, skin from the hairless phenotype showed no evidence of fluorescence in the dermis and intermediate was the fluorescence observed in the skin from the fuzzy-haired animal. These observations showed that transappendageal penetration could contribute significantly to the overall skin absorption of topical agents. They also suggest that regional distribution of skin appendages could influence the percutaneous fate of topically applied chemicals.