Comprehensive multi-omic profiling of somatic mutations in malformations of cortical development.

Malformations of cortical development (MCD) are neurological conditions involving focal disruptions of cortical architecture and cellular organization that arise during embryogenesis, largely from somatic mosaic mutations, and cause intractable epilepsy. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat condition-related epilepsy. Here we report a genetic landscape from 283 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation including in utero electroporation of mice and single-nucleus RNA sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associated with distinct pathophysiological and clinical phenotypes. The unique single-cell level spatiotemporal expression patterns of mutated genes in control and patient brains indicate critical roles in excitatory neurogenic pools during brain development and in promoting neuronal hyperexcitability after birth.

Obesity Is Associated with Altered Tumor Metabolism in Metastatic Melanoma.

PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). EXPERIMENTAL DESIGN: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies. See related commentary by Smalley, p. 5.

Genotype-to-Phenotype Associations in the Aggressive Variant Prostate Cancer Molecular Profile (AVPC-m) Components.

The aggressive variant prostate cancer molecular profile (AVPC-m), composed of combined defects in TP53, RB1 and PTEN, characterizes a subset of prostate cancers linked to androgen indifference and platinum sensitivity. To contribute to the optimization of the AVPC-m assessment for inclusion in prospective clinical trials, we investigated the status of the AVPC-m components in 28 patient tumor-derived xenografts (PDXs) developed at MDACC. We subjected single formalin-fixed, paraffin-embedded (FFPE) blocks from each PDX to immunohistochemistry (IHC), targeted next-generation genomic sequencing (NGS) and Clariom-S Affymetrix human microarray expression profiling. Standard validated IHC assays and a 10% labeling index cutoff resulted in high reproducibility across three separate laboratories and three independent readers for all tumor suppressors, as well as strong correlations with loss-of-function transcriptional scores (LOF-TS). Adding intensity assessment to labeling indices strengthened the association between IHC results and LOF-TS for TP53 and RB1, but not for PTEN. For TP53, genomic alterations determined by NGS had slightly higher agreement scores with LOF-TS than aberrant IHC, while for RB1 and PTEN, NGS and IHC determinations resulted in similar agreement scores with LOF-TS. Nonetheless, our results indicate that the AVPC-m components can be assessed reproducibly by IHC using various widely available standardized assays.

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Identification of autosomal recessive novel genes and retinal phenotypes in members of the solute carrier (SLC) superfamily.

PURPOSE: This study aimed to investigate the clinical and genetic aspects of solute carrier (SLC) genes in inherited retinal diseases (IRDs). METHODS: Exome sequencing data were filtered to identify pathogenic variants in SLC genes. Analysis of transcript and protein expression was performed on fibroblast cell lines and retinal sections. RESULTS: Comprehensive analysis of 433 SLC genes in 913 exome sequencing IRD samples revealed homozygous pathogenic variants in 6 SLC genes, including 2 candidate novel genes, which were 2 variants in SLC66A1, causing autosomal recessive retinitis pigmentosa (ARRP), and a variant in SLC39A12, causing autosomal recessive mild widespread retinal degeneration with marked macular involvement. In addition, we present 4 families with ARRP and homozygous null variants in SLC37A3 that were previously suggested to cause retinitis pigmentosa, 2 of which cause exon skipping. The recently reported SLC4A7- c.2007dup variant was found in 2 patients with ARRP resulting in the absence of protein. Finally, variants in SLC24A1 were found in 4 individuals with either ARRP or congenital stationary night blindness. CONCLUSION: We report on SLC66A1 and SLC39A12 as candidate novel IRD genes, establish SLC37A3 pathogenicity, and provide further evidence of SLC4A7 as IRD genes. We extend the phenotypic spectrum of SLC24A1 and suggest that its ARRP phenotype may be more common than previously reported.

Spatially resolved transcriptomics of high-grade serous ovarian carcinoma.

Bulk and single-cell RNA sequencing do not provide full characterization of tissue spatial diversity in cancer samples, and currently available in situ techniques (multiplex immunohistochemistry and imaging mass cytometry) allow for only limited analysis of a small number of targets. The current study represents the first comprehensive approach to spatial transcriptomics of high-grade serous ovarian carcinoma using intact tumor tissue. We selected a small cohort of patients with highly annotated high-grade serous ovarian carcinoma, categorized them by response to neoadjuvant chemotherapy (poor or excellent), and analyzed pre-treatment tumor tissue specimens. Our study uncovered extensive differences in tumor composition between the poor responders and excellent responders to chemotherapy, related to cell cluster organization and localization. This in-depth characterization of high-grade serous ovarian carcinoma tumor tissue from poor and excellent responders showed that spatial interactions between cell clusters may influence chemo-responsiveness more than cluster composition alone.

Mouse-INtraDuctal (MIND): an in vivo model for studying the underlying mechanisms of DCIS malignancy.

Due to widespread adoption of screening mammography, there has been a significant increase in new diagnoses of ductal carcinoma in situ (DCIS). However, DCIS prognosis remains unclear. To address this gap, we developed an in vivo model, Mouse-INtraDuctal (MIND), in which patient-derived DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Similar to human DCIS, the cancer cells formed in situ lesions inside the mouse mammary ducts and mimicked all histologic subtypes including micropapillary, papillary, cribriform, solid, and comedo. Among 37 patient samples injected into 202 xenografts, at median duration of 9 months, 20 samples (54%) injected into 95 xenografts showed in vivo invasive progression, while 17 (46%) samples injected into 107 xenografts remained non-invasive. Among the 20 samples that showed invasive progression, nine samples injected into 54 xenografts exhibited a mixed pattern in which some xenografts showed invasive progression while others remained non-invasive. Among the clinically relevant biomarkers, only elevated progesterone receptor expression in patient DCIS and the extent of in vivo growth in xenografts predicted an invasive outcome. The Tempus XT assay was used on 16 patient DCIS formalin-fixed, paraffin-embedded sections including eight DCISs that showed invasive progression, five DCISs that remained non-invasive, and three DCISs that showed a mixed pattern in the xenografts. Analysis of the frequency of cancer-related pathogenic mutations among the groups showed no significant differences (KW: p > 0.05). There were also no differences in the frequency of high, moderate, or low severity mutations (KW; p > 0.05). These results suggest that genetic changes in the DCIS are not the primary driver for the development of invasive disease. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Single-cell evaluation reveals shifts in the tumor-immune niches that shape and maintain aggressive lesions in the breast.

There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.

PHENOTYPIC VARIABILITY OF RECESSIVE RDH12-ASSOCIATED RETINAL DYSTROPHY.

PURPOSE: To characterize the phenotypic variability and report the genetic defects in a cohort of Chinese patients with biallelic variants of the retinol dehydrogenase 12 (RDH12) gene. METHODS: The study included 38 patients from 38 unrelated families with biallelic pathogenic RDH12 variants. Systematic next-generation sequencing data analysis, Sanger sequencing validation, and segregation analysis were used to identify the pathogenic mutations. Detailed ophthalmic examinations, including electroretinogram, fundus photography, fundus autofluorescence and optical coherence tomography, and statistical analysis were performed to evaluate phenotype variability. RESULTS: Twenty-five different mutations of RDH12 were identified in the 38 families. Six of these variants were novel. Val146Asp was observed at the highest frequency (23.7%), and it was followed by Arg62Ter (14.5%) and Thr49Met (9.2%). Twenty-three probands were diagnosed with early-onset severe retinal dystrophy, 6 with Leber congenital amaurosis, 7 with autosomal recessive retinitis pigmentosa, and 2 with cone-rod dystrophy. Self-reported nyctalopia occurred in about a half of patients (55.3%) and was significantly more common among older patients (P < 0.01). Nyctalopia was not significantly associated with best-corrected visual acuity (P = 0.72), but older patients had significantly greater best-corrected visual acuity loss (P < 0.01). Only 15.8% of the patients had nystagmus, which was significantly more likely to occur among 36.8% of the patients with hyperopia >3D (P < 0.01) and/or in cases of reduced best-corrected visual acuity (P = 0.01), but was not associated with age (P = 0.87). CONCLUSION: Several high-frequency RDH12 variants were identified in patients with inherited retinal dystrophies, most of which were missense mutations. Variable but characteristic phenotypes of a progressive nature was observed. Overall, the findings indicated that biallelic RDH12 mutations are a common cause of early-onset retinal dystrophy and a rare cause of cone-rod dystrophy.

GRIPT: a novel case-control analysis method for Mendelian disease gene discovery.

Despite rapid progress of next-generation sequencing (NGS) technologies, the disease-causing genes underpinning about half of all Mendelian diseases remain elusive. One main challenge is the high genetic heterogeneity of Mendelian diseases in which similar phenotypes are caused by different genes and each gene only accounts for a small proportion of the patients. To overcome this gap, we developed a novel method, the Gene Ranking, Identification and Prediction Tool (GRIPT), for performing case-control analysis of NGS data. Analyses of simulated and real datasets show that GRIPT is well-powered for disease gene discovery, especially for diseases with high locus heterogeneity.

Whole-exome sequencing revealed HKDC1 as a candidate gene associated with autosomal-recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inheritable retina degenerative disease leading to blindness. Despite the identification of 70 genes associated with RP, the genetic cause of ∼40% of RP patients remains to be elucidated. Whole-exome sequencing was applied on the probands of a RP cohort of 68 unsolved cases to identify candidate genetic mutations. A homozygous missense variant (c.173C > T, p.T58 M) was found in HKDC1 in two unrelated families presenting late-onset retinal degeneration. This variant affects highly conserved amino acid residue and is very rare in several databases and absent in 4000 ethnic-matched controls. Mutant HKDC1 protein partially lost hexokinase activity. Hkdc1 is expressed in the mouse retina and localized to photoreceptor inner segments. To elucidate the in vivo roles of Hkdc1 in the retina, we generated Hkdc1 knockout (KO) mouse models using CRISPR/Cas9 technique. Two independent alleles were identified and backcrossed to C57BL/6 J for 6 generations. Absence of HKDC1 expression in the Hkdc1 KO retina was confirmed by western blot and immunostaning using HKDC1 antibody. Hkdc1 KO mice exhibited reduced scotopic electroretinogram response and thinner outer nuclear layer, similar to some of the human patient phenotypes. Loss of Hkdc1 led to mislocalization of rhodopsin to the inner segments and cell bodies of rods in some regions in the retina. Taken together, our data demonstrated that HKDC1 is associated with autosomal recessively inherited RP.

Folic acid exerts antidepressant effects by upregulating brain-derived neurotrophic factor and glutamate receptor 1 expression in brain.

Folic acid is a vitamin with a variety of pharmacological effects. The present study aims to explore the beneficial effects of folic acid on chronic unpredictable mild stress (CUMS)-induced depression-like behaviors and its possible mechanisms. The behavioral tests including open-field test, tail suspension test, and forced swimming test were used to evaluate the antidepressant effects of folic acid. Then the changes of brain 5-hydroxytryptamine (5-HT) concentration, brain-derived neurotrophic factor (BDNF), glutamate receptor 1 (GluR1) expression levels, and synaptic organization were assessed to explore the antidepressant mechanisms of folic acid. Our results showed that CUMS caused significant depression-like behaviors, neuropathological changes, and decreased brain 5-HT concentration, BDNF, and GluR1 expression in the hippocampus and association cortex. In conclusion, the results showed that folic acid significantly improved depression-like behaviors in CUMS-induced rats, and its antidepressant effects might be related to the increase of brain 5-HT concentration, BDNF and GluR1 expression, and repair of synaptic organization in the brain.

The phenotypic variability of HK1-associated retinal dystrophy.

Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of Mendelian disorders primarily affecting photoreceptor cells. The same IRD-causing variant may lead to different retinal symptoms, demonstrating pleiotropic phenotype traits influenced by both underlying genetic and environmental factors. In the present study, we identified four unrelated IRD families with the HK1 p.E851K variant, which was previously reported to cause autosomal dominant retinitis pigmentosa (RP), and described their detailed clinical phenotypes. Interestingly, we found that in addition to RP, this particular variant can also cause dominant macular dystrophy and cone-rod dystrophy, which primarily affect cone photoreceptors instead of rods. Our results identified pleiotropic effects for an IRD-causing variant and provide more insights into the involvement of a hexokinase in retinal pathogenesis.

Leveraging splice-affecting variant predictors and a minigene validation system to identify Mendelian disease-causing variants among exon-captured variants of uncertain significance.

The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice-affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT-AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice-disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines.

A Novel Dominant Mutation in SAG, the Arrestin-1 Gene, Is a Common Cause of Retinitis Pigmentosa in Hispanic Families in the Southwestern United States.

Purpose: To identify the causes of autosomal dominant retinitis pigmentosa (adRP) in a cohort of families without mutations in known adRP genes and consequently to characterize a novel dominant-acting missense mutation in SAG. Methods: Patients underwent ophthalmologic testing and were screened for mutations using targeted-capture and whole-exome next-generation sequencing. Confirmation and additional screening were done by Sanger sequencing. Haplotypes segregating with the mutation were determined using short tandem repeat and single nucleotide variant polymorphisms. Genealogies were established by interviews of family members. Results: Eight families in a cohort of 300 adRP families, and four additional families, were found to have a novel heterozygous mutation in the SAG gene, c.440G>T; p.Cys147Phe. Patients exhibited symptoms of retinitis pigmentosa and none showed symptoms characteristic of Oguchi disease. All families are of Hispanic descent and most were ascertained in Texas or California. A single haplotype including the SAG mutation was identified in all families. The mutation dramatically alters a conserved amino acid, is extremely rare in global databases, and was not found in 4000+ exomes from Hispanic controls. Molecular modeling based on the crystal structure of bovine arrestin-1 predicts protein misfolding/instability. Conclusions: This is the first dominant-acting mutation identified in SAG, a founder mutation possibly originating in Mexico several centuries ago. The phenotype is clearly adRP and is distinct from the previously reported phenotypes of recessive null mutations, that is, Oguchi disease and recessive RP. The mutation accounts for 3% of the 300 families in the adRP Cohort and 36% of Hispanic families in this cohort.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Mutations in the Spliceosome Component CWC27 Cause Retinal Degeneration with or without Additional Developmental Anomalies.

Pre-mRNA splicing factors play a fundamental role in regulating transcript diversity both temporally and spatially. Genetic defects in several spliceosome components have been linked to a set of non-overlapping spliceosomopathy phenotypes in humans, among which skeletal developmental defects and non-syndromic retinitis pigmentosa (RP) are frequent findings. Here we report that defects in spliceosome-associated protein CWC27 are associated with a spectrum of disease phenotypes ranging from isolated RP to severe syndromic forms. By whole-exome sequencing, recessive protein-truncating mutations in CWC27 were found in seven unrelated families that show a range of clinical phenotypes, including retinal degeneration, brachydactyly, craniofacial abnormalities, short stature, and neurological defects. Remarkably, variable expressivity of the human phenotype can be recapitulated in Cwc27 mutant mouse models, with significant embryonic lethality and severe phenotypes in the complete knockout mice while mice with a partial loss-of-function allele mimic the isolated retinal degeneration phenotype. Our study describes a retinal dystrophy-related phenotype spectrum as well as its genetic etiology and highlights the complexity of the spliceosomal gene network.