RNA sequencing resolves novel DYNC2H1 variants causing short-rib thoracic dysplasia type 3: Case report.

BACKGROUND: Intronic variants outside the canonical splice site are challenging to interpret and therefore likely represent an underreported cause of human disease. Autosomal recessive variants in DYNC2H1 are associated with short-rib thoracic dysplasia 3 with or without polydactyly (SRTD3), a clinically heterogeneous disease generally presenting with short ribs, shortened tubular bones, narrow thorax and acetabular roof anomalies. We describe a case of SRTD3 with compound heterozygous frameshift and intronic variants and highlight the essential role of RNA sequencing (RNA-Seq) in variant interpretation. METHODS: Following inconclusive clinical genetic testing identifying a likely pathogenic frameshift variant and an intronic variant of uncertain significance (VUS) in DYNC2H1 in trans, the family enrolled in the Care4Rare Canada research program, where RNA-Seq studies were performed. RESULTS: The proband presented with post-axial polydactyly of all four limbs, a significantly small chest with a pectus excavatum and anterior flaring of the ribs. RNA-Seq investigations revealed a novel splice junction as a result of the intronic VUS and significantly decreased DYNC2H1 gene expression in the proband. CONCLUSION: This case demonstrates the diagnostic utility of RNA-Seq for variant interpretation following inconclusive clinical testing, which can ultimately lead to diagnosis for patients with rare disease.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

An RB-Condensin II Complex Mediates Long-Range Chromosome Interactions and Influences Expression at Divergently Paired Genes.

Interphase chromosomes are organized into topologically associated domains in order to establish and maintain integrity of transcriptional programs that remain poorly understood. Here, we show that condensin II and TFIIIC are recruited to bidirectionally transcribed promoters by a mechanism that is dependent on the retinoblastoma (RB) protein. Long-range chromosome contacts are disrupted by loss of condensin II loading, which leads to altered expression at bidirectional gene pairs. This study demonstrates that mammalian condensin II functions to organize long-range chromosome contacts and regulate transcription at specific genes. In addition, RB dependence of condensin II suggests that widespread misregulation of chromosome contacts and transcriptional alterations are a consequence of RB mutation.

RB1 Deletion in Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression.

Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.