The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

De novo missense variants in ZBTB47 are associated with developmental delays, hypotonia, seizures, gait abnormalities, and variable movement abnormalities.

The collection of known genetic etiologies of neurodevelopmental disorders continues to increase, including several syndromes associated with defects in zinc finger protein transcription factors (ZNFs) that vary in clinical severity from mild learning disabilities and developmental delay to refractory seizures and severe autism spectrum disorder. Here we describe a new neurodevelopmental disorder associated with variants in ZBTB47 (also known as ZNF651), which encodes zinc finger and BTB domain-containing protein 47. Exome sequencing (ES) was performed for five unrelated patients with neurodevelopmental disorders. All five patients are heterozygous for a de novo missense variant in ZBTB47, with p.(Glu680Gly) (c.2039A>G) detected in one patient and p.(Glu477Lys) (c.1429G>A) identified in the other four patients. Both variants impact conserved amino acid residues. Bioinformatic analysis of each variant is consistent with pathogenicity. We present five unrelated patients with de novo missense variants in ZBTB47 and a phenotype characterized by developmental delay with intellectual disability, seizures, hypotonia, gait abnormalities, and variable movement abnormalities. We propose that these variants in ZBTB47 are the basis of a new neurodevelopmental disorder.

Barriers to a successful healthcare transition for individuals with urea cycle disorders.

The pediatric to adult healthcare transition (HCT) is a process for individuals with chronic health conditions to gradually shift from a pediatric to an adult-oriented care system. Autonomy and self-management skills required for an individual's HCT readiness can be evaluated through the transition readiness assessment questionnaire (TRAQ). Despite general HCT preparation guidelines, little is known about the HCT experience of individuals with a urea cycle disorder (UCD). This is the first study to report the parent or guardian perception of the HCT process in children with a UCD by investigating the stages of transition readiness and transition outcome. We identify barriers to HCT readiness and planning, along with deficiencies in transition outcome for individuals with a UCD. For children that received special education services compared to those that did not, significantly lower transition readiness scores were identified in the total TRAQ score (p = 0.03) and in the domains of tracking health issues (p = 0.02), talking with providers (p = 0.03), and managing daily activities (p = 0.01). There was a lack of HCT preparation as most subjects did not have a HCT discussion with their healthcare provider before age 26. Deficiencies in HCT outcome are demonstrated by individuals with a UCD reporting delays in needed medical care and dissatisfaction with their healthcare services. Considerations for facilitating a successful HCT for individuals with a UCD include providing individualized education, appointing a transition coordinator, allowing flexibility in HCT timing, and ensuring that the individual recognizes concerning UCD symptoms and knows when to seek medical care.

PAK1 c.1409 T > a (p. Leu470Gln) de novo variant affects the protein kinase domain, leading to epilepsy, macrocephaly, spastic quadriplegia, and hydrocephalus: Case report and review of the literature.

The p-21-activated kinase 1 (PAK1) protein, encoded by the PAK1 gene, is an evolutionarily conserved serine/threonine-protein kinase that regulates key cellular developmental processes. To date, seven de novo PAK1 variants have been reported to cause the Intellectual Developmental Disorder with Macrocephaly, Seizures, and Speech Delay (IDDMSSD). In addition to the namesake features, other common characteristics include structural brain anomalies, delayed development, hypotonia, and dysmorphic features. Here, we report a de novo PAK1 NM_002576.5: c.1409 T > A variant (p.Leu470Gln) identified by trio genome sequencing (GS) in a 13-year-old boy with postnatal macrocephaly, obstructive hydrocephalus, medically refractory epilepsy, spastic quadriplegia, white matter hyperintensities, profound developmental disabilities, and a horseshoe kidney. This is the first recurrently affected residue identified in the protein kinase domain. Combined assessment of the eight pathogenic PAK1 missense variants reveal that the variants cluster in either the protein kinase or autoregulatory domains. Although interpretation of the phenotypic spectrum is limited by the sample size, neuroanatomical alterations were found more often in individuals with PAK1 variants in the autoregulatory domain. In contrast, non-neurological comorbidities were found more often in individuals with PAK1 variants in the protein kinase domain. Together, these findings expand the clinical spectrum of PAK1-associated IDDMSSD and reveal potential correlations with the affected protein domains.

DNA methylation episignature in Gabriele-de Vries syndrome.

PURPOSE: Gabriele-de Vries syndrome (GADEVS) is a rare genetic disorder characterized by developmental delay and/or intellectual disability, hypotonia, feeding difficulties, and distinct facial features. To refine the phenotype and to better understand the molecular basis of the syndrome, we analyzed clinical data and performed genome-wide DNA methylation analysis of a series of individuals carrying a YY1 variant. METHODS: Clinical data were collected for 13 individuals not yet reported through an international call for collaboration. DNA was collected for 11 of these individuals and 2 previously reported individuals in an attempt to delineate a specific DNA methylation signature in GADEVS. RESULTS: Phenotype in most individuals overlapped with the previously described features. We described 1 individual with atypical phenotype, heterozygous for a missense variant in a domain usually not involved in individuals with YY1 pathogenic missense variations. We also described a specific peripheral blood DNA methylation profile associated with YY1 variants. CONCLUSION: We reported a distinct DNA methylation episignature in GADEVS. We expanded the clinical profile of GADEVS to include thin/sparse hair and cryptorchidism. We also highlighted the utility of DNA methylation episignature analysis for classification of variants of unknown clinical significance.

Mutations of the histone linker H1-4 in neurodevelopmental disorders and functional characterization of neurons expressing C-terminus frameshift mutant H1.4.

Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, bone abnormalities and dysmorphic facies. RMNS is caused by de novo heterozygous mutations in the histone linker gene H1-4; however, mechanisms underlying impaired neurodevelopment in RMNS are not understood. All reported mutations associated with RMNS in H1-4 are small insertions or deletions that create a shared frameshift, resulting in a H1.4 protein that is both truncated and possessing an abnormal C-terminus frameshifted tail (H1.4 CFT). To expand understanding of mutations and phenotypes associated with mutant H1-4, we identified new variants at both the C- and N-terminus of H1.4. The clinical features of mutations identified at the C-terminus are consistent with other reports and strengthen the support of pathogenicity of H1.4 CFT. To understand how H1.4 CFT may disrupt brain function, we exogenously expressed wild-type or H1.4 CFT protein in rat hippocampal neurons and assessed neuronal structure and function. Genome-wide transcriptome analysis revealed ~ 400 genes altered in the presence of H1.4 CFT. Neuronal genes downregulated by H1.4 CFT were enriched for functional categories involved in synaptic communication and neuropeptide signaling. Neurons expressing H1.4 CFT also showed reduced neuronal activity on multielectrode arrays. These data are the first to characterize the transcriptional and functional consequence of H1.4 CFT in neurons. Our data provide insight into causes of neurodevelopmental impairments associated with frameshift mutations in the C-terminus of H1.4 and highlight the need for future studies on the function of histone H1.4 in neurons.

Arrhythmogenic Ventricular Cardiomyopathy: Challenges With Complex Genetics and Variable Phenotypes.

After a 20-year-old woman suddenly died, autopsy showed characteristic findings of biventricular arrhythmogenic cardiomyopathy. Screening of her family members revealed the same desmoplakin gene mutation and imaging abnormalities predominantly involving the left ventricle. We describe the variable phenotypic expression in a family that shares a common gene variant. (Level of Difficulty: Advanced.).

A novel de novo intronic variant in ITPR1 causes Gillespie syndrome.

Gillespie syndrome (GLSP) is characterized by bilateral symmetric partial aplasia of the iris presenting as a fixed and large pupil, cerebellar hypoplasia with ataxia, congenital hypotonia, and varying levels of intellectual disability. GLSP is caused by either biallelic or heterozygous, dominant-negative, pathogenic variants in ITPR1. Here, we present a 5-year-old male with GLSP who was found to have a heterozygous, de novo intronic variant in ITPR1 (NM_001168272.1:c.5935-17G > A) through genome sequencing (GS). Sanger sequencing of cDNA from this individual's fibroblasts showed the retention of 15 nucleotides from intron 45, which is predicted to cause an in-frame insertion of five amino acids near the C-terminal transmembrane domain of ITPR1. In addition, qPCR and cDNA sequencing demonstrated reduced expression of both ITPR1 alleles in fibroblasts when compared to parental samples. Given the close proximity of the predicted in-frame amino acid insertion to the site of previously described heterozygous, de novo, dominant-negative, pathogenic variants in GLSP, we predict that this variant also has a dominant-negative effect on ITPR1 channel function. Overall, this is the first report of a de novo intronic variant causing GLSP, which emphasizes the utility of GS and cDNA studies for diagnosing patients with a clinical presentation of GLSP and negative clinical exome sequencing.

Transforming the clinical outcome in CRIM-negative infantile Pompe disease identified via newborn screening: the benefits of early treatment with enzyme replacement therapy and immune tolerance induction.

PURPOSE: To assess the magnitude of benefit to early treatment initiation, enabled by newborn screening or prenatal diagnosis, in patients with cross-reactive immunological material (CRIM)-negative infantile Pompe disease (IPD), treated with enzyme replacement therapy (ERT) and prophylactic immune tolerance induction (ITI) with rituximab, methotrexate, and intravenous immunoglobulin (IVIG). METHODS: A total of 41 CRIM-negative IPD patients were evaluated. Among patients who were treated with ERT + ITI (n = 30), those who were invasive ventilator-free at baseline and had ≥6 months of follow-up were stratified based on age at treatment initiation: (1) early (≤4 weeks), (2) intermediate (>4 and ≤15 weeks), and (3) late (>15 weeks). A historical cohort of 11 CRIM-negative patients with IPD treated with ERT monotherapy served as an additional comparator group. RESULTS: Twenty patients were included; five, seven, and eight in early, intermediate, and late treatment groups, respectively. Genotypes were similar across the three groups. Early-treated patients showed significant improvements in left ventricular mass index, motor and pulmonary outcomes, as well as biomarkers creatine kinase and urinary glucose tetrasaccharide, compared with those treated later. CONCLUSION: Our preliminary data suggest that early treatment with ERT + ITI can transform the long-term CRIM-negative IPD phenotype, which represents the most severe end of the Pompe disease spectrum.

Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing.

BACKGROUNDTranscriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq-guided method to diagnose individuals across a wide range of ages and clinical phenotypes.METHODSOne hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and skin fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network clinical site from 2014 to 2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis.RESULTSThe transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Skin fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts.CONCLUSIONSFor our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.TRIAL REGISTRATIONNot applicable.FUNDINGNIH Common Fund, BCM Intellectual and Developmental Disabilities Research Center, Eunice Kennedy Shriver National Institute of Child Health & Human Development.

Evidences of a Direct Relationship between Cellular Fuel Supply and Ciliogenesis Regulated by Hypoxic VDAC1-ΔC.

Metabolic flexibility is the ability of a cell to adapt its metabolism to changes in its surrounding environment. Such adaptability, combined with apoptosis resistance provides cancer cells with a survival advantage. Mitochondrial voltage-dependent anion channel 1 (VDAC1) has been defined as a metabolic checkpoint at the crossroad of these two processes. Here, we show that the hypoxia-induced cleaved form of VDAC1 (VDAC1-ΔC) is implicated in both the up-regulation of glycolysis and the mitochondrial respiration. We demonstrate that VDAC1-ΔC, due to the loss of the putative phosphorylation site at serine 215, concomitantly with the loss of interaction with tubulin and microtubules, reprograms the cell to utilize more metabolites, favoring cell growth in hypoxic microenvironment. We further found that VDAC1-ΔC represses ciliogenesis and thus participates in ciliopathy, a group of genetic disorders involving dysfunctional primary cilium. Cancer, although not representing a ciliopathy, is tightly linked to cilia. Moreover, we highlight, for the first time, a direct relationship between the cilium and cancer cell metabolism. Our study provides the first new comprehensive molecular-level model centered on VDAC1-ΔC integrating metabolic flexibility, ciliogenesis, and enhanced survival in a hypoxic microenvironment.

De Novo Variants in the ATPase Module of MORC2 Cause a Neurodevelopmental Disorder with Growth Retardation and Variable Craniofacial Dysmorphism.

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.

Clinical and laboratory interpretation of mitochondrial mRNA variants.

Interpretation of mitochondrial protein-encoding (mt-mRNA) variants has been challenging due to mitochondrial characteristics that have not been addressed by American College of Medical Genetics and Genomics guidelines. We developed criteria for the interpretation of mt-mRNA variants via literature review of reported variants, tested and refined these criteria by using our new cases, followed by interpreting 421 novel variants in our clinical database using these verified criteria. A total of 32 of 56 previously reported pathogenic (P) variants had convincing evidence for pathogenicity. These variants are either null variants, well-known disease-causing variants, or have robust functional data or strong phenotypic correlation with heteroplasmy levels. Based on our criteria, 65.7% (730/1,111) of variants of unknown significance (VUS) were reclassified as benign (B) or likely benign (LB), and one variant was scored as likely pathogenic (LP). Furthermore, using our criteria we classified 2, 12, and 23 as P, LP, and LB, respectively, among 421 novel variants. The remaining stayed as VUS (91.2%). Appropriate interpretation of mt-mRNA variants is the basis for clinical diagnosis and genetic counseling. Mutation type, heteroplasmy levels in different tissues of the probands and matrilineal relatives, in silico predictions, population data, as well as functional studies are key points for pathogenicity assessments.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deletion of VDAC1 Hinders Recovery of Mitochondrial and Renal Functions After Acute Kidney Injury.

Voltage-dependent anion channels (VDACs) constitute major transporters mediating bidirectional movement of solutes between cytoplasm and mitochondria. We aimed to determine if VDAC1 plays a role in recovery of mitochondrial and kidney functions after ischemia-induced acute kidney injury (AKI). Kidney function decreased after ischemia and recovered in wild-type (WT), but not in VDAC1-deficient mice. Mitochondrial maximum respiration, activities of respiratory complexes and FoF1-ATPase, and ATP content in renal cortex decreased after ischemia and recovered in WT mice. VDAC1 deletion reduced respiration and ATP content in non-injured kidneys. Further, VDAC1 deletion blocked return of activities of respiratory complexes and FoF1-ATPase, and recovery of respiration and ATP content after ischemia. Deletion of VDAC1 exacerbated ischemia-induced mitochondrial fission, but did not aggravate morphological damage to proximal tubules after ischemia. However, VDAC1 deficiency impaired recovery of kidney morphology and increased renal interstitial collagen accumulation. Thus, our data show a novel role for VDAC1 in regulating renal mitochondrial dynamics and recovery of mitochondrial function and ATP levels after AKI. We conclude that the presence of VDAC1 (1) stimulates capacity of renal mitochondria for respiration and ATP production, (2) reduces mitochondrial fission, (3) promotes recovery of mitochondrial function and dynamics, renal morphology, and kidney functions, and (4) increases survival after AKI.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of a new aggressive axis driven by ciliogenesis and absence of VDAC1-ΔC in clear cell Renal Cell Carcinoma patients.

Rationale: Renal cell carcinoma (RCC) accounts for about 2% of all adult cancers, and clear cell RCC (ccRCC) is the most common RCC histologic subtype. A hallmark of ccRCC is the loss of the primary cilium, a cellular antenna that senses a wide variety of signals. Loss of this key organelle in ccRCC is associated with the loss of the von Hippel-Lindau protein (VHL). However, not all mechanisms of ciliopathy have been clearly elucidated. Methods: By using RCC4 renal cancer cells and patient samples, we examined the regulation of ciliogenesis via the presence or absence of the hypoxic form of the voltage-dependent anion channel (VDAC1-ΔC) and its impact on tumor aggressiveness. Three independent cohorts were analyzed. Cohort A was from PREDIR and included 12 patients with hereditary pVHL mutations and 22 sporadic patients presenting tumors with wild-type pVHL or mutated pVHL; Cohort B included tissue samples from 43 patients with non-metastatic ccRCC who had undergone surgery; and Cohort C was composed of 375 non-metastatic ccRCC tumor samples from The Cancer Genome Atlas (TCGA) and was used for validation. The presence of VDAC1-ΔC and legumain was determined by immunoblot. Transcriptional regulation of IFT20/GLI1 expression was evaluated by qPCR. Ciliogenesis was detected using both mouse anti-acetylated α-tubulin and rabbit polyclonal ARL13B antibodies for immunofluorescence. Results: Our study defines, for the first time, a group of ccRCC patients in which the hypoxia-cleaved form of VDAC1 (VDAC1-ΔC) induces resorption of the primary cilium in a Hypoxia-Inducible Factor-1 (HIF-1)-dependent manner. An additional novel group, in which the primary cilium is re-expressed or maintained, lacked VDAC1-ΔC yet maintained glycolysis, a signature of epithelial-mesenchymal transition (EMT) and more aggressive tumor progression, but was independent to VHL. Moreover, these patients were less sensitive to sunitinib, the first-line treatment for ccRCC, but were potentially suitable for immunotherapy, as indicated by the immunophenoscore and the presence of PDL1 expression. Conclusion: This study provides a new way to classify ccRCC patients and proposes potential therapeutic targets linked to metabolism and immunotherapy.

Interpretation of mitochondrial tRNA variants.

PURPOSE: To develop criteria to interpret mitochondrial transfer RNA (mt-tRNA) variants based on unique characteristics of mitochondrial genetics and conserved structural/functional properties of tRNA. METHODS: We developed rules on a set of established pathogenic/benign variants by examining heteroplasmy correlations with phenotype, tissue distribution, family members, and among unrelated families from published literature. We validated these deduced rules using our new cases and applied them to classify novel variants. RESULTS: Evaluation of previously reported pathogenic variants found that 80.6% had sufficient evidence to support phenotypic correlation with heteroplasmy levels among and within families. The remaining variants were downgraded due to the lack of similar evidence. Application of the verified criteria resulted in rescoring 80.8% of reported variants of uncertain significance (VUS) to benign and likely benign. Among 97 novel variants, none met pathogenic criteria. A large proportion of novel variants (84.5%) remained as VUS, while only 10.3% were likely pathogenic. Detection of these novel variants in additional individuals would facilitate their classification. CONCLUSION: Proper interpretation of mt-tRNA variants is crucial for accurate clinical diagnosis and genetic counseling. Correlations with tissue distribution, heteroplasmy levels, predicted perturbations to tRNA structure, and phenotypes provide important evidence for determining the clinical significance of mt-tRNA variants.

Liver transplantation in propionic and methylmalonic acidemia: A single center study with literature review.

BACKGROUND: Organic acidemias, especially propionic acidemia (PA) and methylmalonic acidemia (MMA), may manifest clinically within the first few hours to days of life. The classic presentation in the newborn period includes metabolic acidosis, hyperlactatemia, and hyperammonemia that is precipitated by unrestricted protein intake. Implementation of newborn screening to diagnose and initiate early treatment has facilitated a reduction in neonatal mortality and improved survival. Despite early diagnosis and appropriate management, these individuals are prone to have recurrent episodes of metabolic acidosis and hyperammonemia resulting in frequent hospitalizations. Liver transplantation (LT) has been proposed as a treatment modality to reduce metabolic decompensations which are not controlled by medical management. Published reports on the outcome of LT show heterogeneous results regarding clinical and biochemical features in the post transplantation period. As a result, we evaluated the outcomes of LT in our institution and compared it to the previously published data. STUDY DESIGN/METHODS: We performed a retrospective chart review of nine individuals with PA or MMA who underwent LT and two individuals with MMA who underwent LT and kidney transplantation (KT). Data including number of hospitalizations, laboratory measures, cardiac and neurological outcomes, dietary protein intake, and growth parameters were collected. RESULTS: The median age of transplantation for subjects with MMA was 7.2 years with a median follow up of 4.3 years. The median age of transplantation for subjects with PA was 1.9 years with a median follow up of 5.4 years. The survival rate at 1 year and 5 years post-LT was 100%. Most of our subjects did not have any episodes of hyperammonemia or pancreatitis post-LT. There was significant reduction in plasma glycine post-LT. One subject developed mild elevation in ammonia post-LT on an unrestricted protein diet, suggesting that protein restriction may be indicated even after LT. CONCLUSION: In a large single center study of LT in MMA and PA, we show that LT may reduce the incidence of metabolic decompensation. Moreover, our data suggest that LT may be associated with reduced number of hospitalizations and improved linear growth in individuals with PA and MMA.