TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

A 9.8 Mb deletion at 7q31.2q31.31 downstream of FOXP2 in an individual with speech and language impairment suggests a possible positional effect.

Haploinsufficiency of FOXP2 causes FOXP2-related speech and language disorder. We report a 9.8 Mb deletion downstream of FOXP2 in a girl with speech and language impairment, developmental delay, and other features. We propose involvement of FOXP2 in pathogenesis of these phenotypes, likely due to positional effects on the gene.

KMT2D loss drives aggressive tumor phenotypes in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most lethal skin cancer. Due to ultraviolet light-induced damage, cSCCs have a high mutation rate, but some genes are more frequently mutated in aggressive cSCCs. Lysine-specific histone methyltransferase 2D (KMT2D) has a two-fold higher mutation frequency in metastatic cSCCs relative to primary non-metastatic associated cSCCs. The role of KMT2D in more aggressive phenotypes in cSCC is uncharacterized. Studies of other tumor types suggest that KMT2D acts to suppress tumor development. To determine whether KMT2D loss has an impact on tumor characteristics, we disrupted KMT2D in a cSCC cell line using CRISPR-cas9 and performed phenotypic analyses. KMT2D loss modestly increased cell proliferation and colony formation (1.4- and 1.6-fold respectively). Cells lacking KMT2D showed increased rates of migration and faster cell cycle progression. In xenograft models, tumors with KMT2D loss showed slight increases in mitotic indices. Collectively, these findings suggest that KMT2D loss-of-function mutations may promote more aggressive and invasive behaviors in cSCC, suggesting that KMT2D-related pathways could be targets for cancer therapies. Future studies to determine the downstream genes and mechanism of phenotypic effect are needed.

MicroRNA Expression Profiling of Cutaneous Squamous Cell Carcinomas Arising in Different Sites.

OBJECTIVE: To examine the microRNA (miRNA) expression profile of cutaneous squamous cell carcinoma (cSCC) tumors from aggressive head and neck locations compared with nonaggressive anatomic sites and normal controls. STUDY DESIGN: Retrospective analysis of miRNA expression. SETTING: Tertiary care center. SUBJECTS AND METHODS: Tissue samples were collected from 3 anatomic regions: aggressive head and neck sites (ie, ears/lip), nonaggressive locations (ie, extremities/trunk), and adjacent normal skin. RNA was isolated from tissue cores of 45 samples (18 aggressive sites, 15 nonaggressive sites, and 12 normal-adjacent skin). miRNA expression analysis was completed for approximately 800 miRNAs using the NanoString nCounter panel. Five candidate miRNAs were selected for validation. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on the original samples plus 30 additional tissue samples (7 aggressive sites, 14 nonaggressive sites, and 9 normal-adjacent skin). RESULTS: Five candidate miRNAs with significant differences in miRNA expression (P < 0 ≤ .001) from discovery samples were selected: miR-21, miR-31, let-7g, miR-93, and miR-22. Relative expression for these miRNAs using qRT-PCR in the new sample set did not reveal any significant differences using 1-way analysis of variance. When sets were combined, miR-21 showed increased expression in aggressive tumors relative to nonaggressive tumors (P = .009), but no others reached statistical significance. CONCLUSION: cSCC behaves more aggressively when originating from specific anatomical subsites of the head and neck. Of 5 miRNAs evaluated, only miR-21 showed significantly higher expression in tumors from aggressive sites relative to nonaggressive sites. Larger sample sizes are needed to evaluate other miRNAs.