Leveraging the T2T assembly to resolve rare and pathogenic inversions in reference genome gaps.

Chromosomal inversions (INVs) are particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage-sensitive genes in cis Short-read genome sequencing (srGS) can only resolve ∼70% of cytogenetically visible inversions referred to clinical diagnostic laboratories, likely due to breakpoints in repetitive regions. Here, we study 12 inversions by long-read genome sequencing (lrGS) (n = 9) or srGS (n = 3) and resolve nine of them. In four cases, the inversion breakpoint region was missing from at least one of the human reference genomes (GRCh37, GRCh38, T2T-CHM13) and a reference agnostic analysis was needed. One of these cases, an INV9 mappable only in de novo assembled lrGS data using T2T-CHM13 disrupts EHMT1 consistent with a Mendelian diagnosis (Kleefstra syndrome 1; MIM#610253). Next, by pairwise comparison between T2T-CHM13, GRCh37, and GRCh38, as well as the chimpanzee and bonobo, we show that hundreds of megabases of sequence are missing from at least one human reference, highlighting that primate genomes contribute to genomic diversity. Aligning population genomic data to these regions indicated that these regions are variable between individuals. Our analysis emphasizes that T2T-CHM13 is necessary to maximize the value of lrGS for optimal inversion detection in clinical diagnostics. These results highlight the importance of leveraging diverse and comprehensive reference genomes to resolve unsolved molecular cases in rare diseases.

Behavioral screening reveals a conserved residue in Y-Box RNA-binding protein required for associative learning and memory in C. elegans.

RNA-binding proteins (RBPs) regulate translation and plasticity which are required for memory. RBP dysfunction has been linked to a range of neurological disorders where cognitive impairments are a key symptom. However, of the 2,000 RBPs in the human genome, many are uncharacterized with regards to neurological phenotypes. To address this, we used the model organism C. elegans to assess the role of 20 conserved RBPs in memory. We identified eight previously uncharacterized memory regulators, three of which are in the C. elegans Y-Box (CEY) RBP family. Of these, we determined that cey-1 is the closest ortholog to the mammalian Y-Box (YBX) RBPs. We found that CEY-1 is both necessary in the nervous system for memory ability and sufficient to promote memory. Leveraging human datasets, we found both copy number variation losses and single nucleotide variants in YBX1 and YBX3 in individuals with neurological symptoms. We identified one predicted deleterious YBX3 variant of unknown significance, p.Asn127Tyr, in two individuals with neurological symptoms. Introducing this variant into endogenous cey-1 locus caused memory deficits in the worm. We further generated two humanized worm lines expressing human YBX3 or YBX1 at the cey-1 locus to test evolutionary conservation of YBXs in memory and the potential functional significance of the p.Asn127Tyr variant. Both YBX1/3 can functionally replace cey-1, and introduction of p.Asn127Tyr into the humanized YBX3 locus caused memory deficits. Our study highlights the worm as a model to reveal memory regulators and identifies YBX dysfunction as a potential new source of rare neurological disease.

Diagnostic utility of DNA methylation analysis in genetically unsolved pediatric epilepsies and CHD2 episignature refinement.

Sequence-based genetic testing identifies causative variants in ~ 50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases.

Exome sequencing implicates ancestry-related Mendelian variation at SYNE1 in childhood-onset essential hypertension.

Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Mind the gap: the relevance of the genome reference to resolve rare and pathogenic inversions.

Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180° and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis . Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.

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.

Behavioral screening of conserved RNA-binding proteins reveals CEY-1/YBX RNA-binding protein dysfunction leads to impairments in memory and cognition.

RNA-binding proteins (RBPs) regulate translation and plasticity which are required for memory. RBP dysfunction has been linked to a range of neurological disorders where cognitive impairments are a key symptom. However, of the 2,000 RBPs in the human genome, many are uncharacterized with regards to neurological phenotypes. To address this, we used the model organism C. elegans to assess the role of 20 conserved RBPs in memory. We identified eight previously uncharacterized memory regulators, three of which are in the C. elegans Y-Box (CEY) RBP family. Of these, we determined that cey-1 is the closest ortholog to the mammalian Y-Box (YBX) RBPs. We found that CEY-1 is both necessary in the nervous system for memory ability and sufficient to increase memory. Leveraging human datasets, we found both copy number variation losses and single nucleotide variants in YBX1 and YBX3 in individuals with neurological symptoms. We identified one predicted deleterious YBX3 variant of unknown significance, p.Asn127Tyr, in two individuals with neurological symptoms. Introducing this variant into endogenous cey-1 locus caused memory deficits in the worm. We further generated two humanized worm lines expressing human YBX3 or YBX1 at the cey-1 locus to test evolutionary conservation of YBXs in memory and the potential functional significance of the p.Asn127Tyr variant. Both YBX1/3 can functionally replace cey-1, and introduction of p.Asn127Tyr into the humanized YBX3 locus caused memory deficits. Our study highlights the worm as a model to reveal memory regulators and identifies YBX dysfunction as a potential new source of rare neurological disease.

Diagnostic Utility of Genome-wide DNA Methylation Analysis in Genetically Unsolved Developmental and Epileptic Encephalopathies and Refinement of a CHD2 Episignature.

Sequence-based genetic testing currently identifies causative genetic variants in ∼50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. Rare epigenetic variations ("epivariants") can drive disease by modulating gene expression at single loci, whereas genome-wide DNA methylation changes can result in distinct "episignature" biomarkers for monogenic disorders in a growing number of rare diseases. Here, we interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 516 individuals with genetically unsolved DEEs who had previously undergone extensive genetic testing. We identified rare differentially methylated regions (DMRs) and explanatory episignatures to discover causative and candidate genetic etiologies in 10 individuals. We then used long-read sequencing to identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and two copy number variants. We also identify pathogenic sequence variants associated with episignatures; some had been missed by previous exome sequencing. Although most DEE genes lack known episignatures, the increase in diagnostic yield for DNA methylation analysis in DEEs is comparable to the added yield of genome sequencing. Finally, we refine an episignature for CHD2 using an 850K methylation array which was further refined at higher CpG resolution using bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate genetic causes as ∼2% (10/516) for unsolved DEE cases.

Rare variants in PPFIA3 cause delayed development, intellectual disability, autism, and epilepsy.

PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

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.

Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Wide range of phenotypic severity in individuals with late truncations unique to the predominant CDKL5 transcript in the brain.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by heterozygous or hemizygous variants in CDKL5 and is characterized by refractory epilepsy, cognitive and motor impairments, and cerebral visual impairment. CDKL5 has multiple transcripts, of which the longest transcripts, NM_003159 and NM_001037343, have been used historically in clinical laboratory testing. However, the transcript NM_001323289 is the most highly expressed in brain and contains 170 nucleotides at the 3' end of its last exon that are noncoding in other transcripts. Two truncating variants in this region have been reported in association with a CDD phenotype. To clarify the significance and range of phenotypes associated with late truncating variants in this region of the predominant transcript in the brain, we report detailed information on two individuals, updated clinical information on a third individual, and a summary of published and unpublished individuals reported in ClinVar. The two new individuals (one male and one female) each had a relatively mild clinical presentation including periods of pharmaco-responsive epilepsy, independent walking and limited purposeful communication skills. A previously reported male continued to have a severe phenotype. Overall, variants in this region demonstrate a range of clinical severity consistent with reports in CDD but with the potential for milder presentation.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

An Integrated Phenotypic and Genotypic Approach Reveals a High-Risk Subtype Association for EBF3 Missense Variants Affecting the Zinc Finger Domain.

OBJECTIVE: Collier/Olf/EBF (COE) transcription factors have distinct expression patterns in the developing and mature nervous system. To date, a neurological disease association has been conclusively established for only the Early B-cell Factor-3 (EBF3) COE family member through the identification of heterozygous loss-of-function variants in individuals with autism spectrum/neurodevelopmental disorders (NDD). Here, we identify a symptom severity risk association with missense variants primarily disrupting the zinc finger domain (ZNF) in EBF3-related NDD. METHODS: A phenotypic assessment of 41 individuals was combined with a literature meta-analysis for a total of 83 individuals diagnosed with EBF3-related NDD. Quantitative diagnostic phenotypic and symptom severity scales were developed to compare EBF3 variant type and location to identify genotype-phenotype correlations. To stratify the effects of EBF3 variants disrupting either the DNA-binding domain (DBD) or the ZNF, we used in vivo fruit fly UAS-GAL4 expression and in vitro luciferase assays. RESULTS: We show that patient symptom severity correlates with EBF3 missense variants perturbing the ZNF, which is a key protein domain required for stabilizing the interaction between EBF3 and the target DNA sequence. We found that ZNF-associated variants failed to restore viability in the fruit fly and impaired transcriptional activation. However, the recurrent variant EBF3 p.Arg209Trp in the DBD is capable of partially rescuing viability in the fly and preserved transcriptional activation. INTERPRETATION: We describe a symptom severity risk association with ZNF perturbations and EBF3 loss-of-function in the largest reported cohort to date of EBF3-related NDD patients. This analysis should have potential predictive clinical value for newly identified patients with EBF3 gene variants. ANN NEUROL 2022;92:138-153.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

PRUNE1 c.933G>A synonymous variant induces exon 7 skipping, disrupts the DHHA2 domain, and leads to an atypical NMIHBA syndrome presentation: Case report and review of the literature.

Prune exopolyphosphatase-1 (PRUNE1) encodes a member of the aspartic acid-histidine-histidine (DHH) phosphodiesterase superfamily that regulates cell migration and proliferation during brain development. In 2015, biallelic PRUNE1 loss-of-function variants were identified to cause the neurodevelopmental disorder with microcephaly, hypotonia, and variable brain abnormalities (NMIHBA, OMIM#617481). NMIHBA is characterized by the namesake features and structural brain anomalies including thinning of the corpus callosum, cerebral and cerebellar atrophy, and delayed myelination. To date, 47 individuals have been reported in the literature, but the phenotypic spectrum of PRUNE1-related disorders and their causative variants remains to be characterized fully. Here, we report a novel homozygous PRUNE1 NM_021222.2:c.933G>A synonymous variant identified in a 6-year-old boy with intellectual and developmental disabilities, hypotonia, and spastic diplegia, but with the absence of microcephaly, brain anomalies, or seizures. Fibroblast RNA sequencing revealed that the PRUNE1 NM_021222.1:c.933G>A variant resulted in an in-frame skipping of the penultimate exon 7, removing 53 amino acids from an important protein domain. This case represents the first synonymous variant and the third pathogenic variant known to date affecting the DHH-associated domain (DHHA2 domain). These findings extend the genotypic and phenotypic spectrums in PRUNE1-related disorders and highlight the importance of considering synonymous splice site variants in atypical presentations.

EIF2AK2-related Neurodevelopmental Disorder With Leukoencephalopathy, Developmental Delay, and Episodic Neurologic Regression Mimics Pelizaeus-Merzbacher Disease.

OBJECTIVE: To demonstrate that de novo missense single nucleotide variants (SNVs) in EIF2AK2 cause a neurodevelopmental disorder with leukoencephalopathy resembling Pelizaeus-Merzbacher disease (PMD). METHODS: A retrospective chart review was performed of 2 unrelated males evaluated at a single institution with de novo EIF2AK2 SNVs identified by clinical exome sequencing (ES). Clinical and radiographic data were reviewed and summarized. RESULTS: Both individuals presented in the first year of life with concern for seizures and developmental delay. Common clinical findings included horizontal and/or pendular nystagmus during infancy, axial hypotonia, appendicular hypertonia, spasticity, and episodic neurologic regression with febrile viral illnesses. MRI of the brain demonstrated severely delayed myelination in infancy. A hypomyelinating pattern was confirmed on serial imaging at age 4 years for proband 1. In proband 2, repeat imaging at age 13 months confirmed persistent delayed myelination. These clinical and radiographic features led to a strong suspicion of PMD. However, neither PLP1 copy number variants nor pathogenic SNVs were detected by chromosomal microarray and trio ES, respectively. Reanalysis of trio ES identified heterozygous de novo EIF2AK2 missense variant c.290C>T (p.Ser97Phe) in proband 1 and c.326C>T (p.Ala109Val) in proband 2. CONCLUSIONS: The autosomal dominant EIF2AK2-related leukoencephalopathy, developmental delay, and episodic neurologic regression syndrome should be considered in the differential diagnosis for PMD and other hypomyelinating leukodystrophies (HLDs). A characteristic history of developmental regression with febrile illnesses may help distinguish it from other HLDs.

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.

De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy.

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.