The GENESIS database and tools: A decade of discovery in Mendelian genomics.

In the past decade, human genetics research saw an acceleration of disease gene discovery and further dissection of the genetic architectures of many disorders. Much of this progress was enabled via data aggregation projects, collaborative data sharing among researchers, and the adoption of sophisticated and standardized bioinformatics analyses pipelines. In 2012, we launched the GENESIS platform, formerly known as GEM.app, with the aims to 1) empower clinical and basic researchers without bioinformatics expertise to analyze and explore genome level data and 2) facilitate the detection of novel pathogenic variation and novel disease genes by leveraging data aggregation and genetic matchmaking. The GENESIS database has grown to over 20,000 datasets from rare disease patients, which were provided by multiple academic research consortia and many individual investigators. Some of the largest global collections of genome-level data are available for Charcot-Marie-Tooth disease, hereditary spastic paraplegia, and cerebellar ataxia. A number of rare disease consortia and networks are archiving their data in this database. Over the past decade, more than 1500 scientists have registered and used this resource and published over 200 papers on gene and variant identifications, which garnered >6000 citations. GENESIS has supported >100 gene discoveries and contributed to approximately half of all gene identifications in the fields of inherited peripheral neuropathies and spastic paraplegia in this time frame. Many diagnostic odysseys of rare disease patients have been resolved. The concept of genomes-to-therapy has borne out for a number of such discoveries that let to rapid clinical trials and expedited natural history studies. This marks GENESIS as one of the most impactful data aggregation initiatives in rare monogenic diseases.

A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.

Unravelling undiagnosed rare disease cases by HiFi long-read genome sequencing.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism.

ATP1A1 encodes a sodium-potassium ATPase that has been linked to several neurological diseases. Using exome and genome sequencing, we identified the heterozygous ATP1A1 variant NM_000701.8: c.2707G>A;p.(Gly903Arg) in two unrelated children presenting with delayed motor and speech development and autism. While absent in controls, the variant occurred de novo in one proband and co-segregated in two affected half-siblings, with mosaicism in the healthy mother. Using a specific ouabain resistance assay in mutant transfected HEK cells, we found significantly reduced cell viability. Demonstrating loss of ATPase function, we conclude that this novel variant is pathogenic, expanding the phenotype spectrum of ATP1A1.

Dominant NARS1 mutations causing axonal Charcot-Marie-Tooth disease expand NARS1-associated diseases.

Pathogenic variants in six aminoacyl-tRNA synthetase (ARS) genes are implicated in neurological disorders, most notably inherited peripheral neuropathies. ARSs are enzymes that charge tRNA molecules with cognate amino acids. Pathogenic variants in asparaginyl-tRNA synthetase (NARS1) cause a neurological phenotype combining developmental delay, ataxia and demyelinating peripheral neuropathy. NARS1 has not yet been linked to axonal Charcot-Marie-Tooth disease. Exome sequencing of patients with inherited peripheral neuropathies revealed three previously unreported heterozygous NARS1 variants in three families. Clinical and electrophysiological details were assessed. We further characterized all three variants in a yeast complementation model and used a knock-in mouse model to study variant p.Ser461Phe. All three variants (p.Met236del, p.Cys342Tyr and p.Ser461Phe) co-segregate with the sensorimotor axonal neuropathy phenotype. Yeast complementation assays show that none of the three NARS1 variants support wild-type yeast growth when tested in isolation (i.e. in the absence of a wild-type copy of NARS1), consistent with a loss-of-function effect. Similarly, the homozygous knock-in mouse model (p.Ser461Phe/Ser472Phe in mouse) also demonstrated loss-of-function characteristics. We present three previously unreported NARS1 variants segregating with a sensorimotor neuropathy phenotype in three families. Functional studies in yeast and mouse support variant pathogenicity. Thus, NARS1 is the seventh ARS implicated in dominant axonal Charcot-Marie-Tooth disease, further stressing that all dimeric ARSs should be evaluated for Charcot-Marie-Tooth disease.

Recurrent de-novo gain-of-function mutation in SPTLC2 confirms dysregulated sphingolipid production to cause juvenile amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) leads to paralysis and death by progressive degeneration of motor neurons. Recently, specific gain-of-function mutations in SPTLC1 were identified in patients with juvenile form of ALS. SPTLC2 encodes the second catalytic subunit of the serine-palmitoyltransferase (SPT) complex. METHODS: We used the GENESIS platform to screen 700 ALS whole-genome and whole-exome data sets for variants in SPTLC2. The de-novo status was confirmed by Sanger sequencing. Sphingolipidomics was performed using liquid chromatography and high-resolution mass spectrometry. RESULTS: Two unrelated patients presented with early-onset progressive proximal and distal muscle weakness, oral fasciculations, and pyramidal signs. Both patients carried the novel de-novo SPTLC2 mutation, c.203T>G, p.Met68Arg. This variant lies within a single short transmembrane domain of SPTLC2, suggesting that the mutation renders the SPT complex irresponsive to regulation through ORMDL3. Confirming this hypothesis, ceramide and complex sphingolipid levels were significantly increased in patient plasma. Accordingly, excessive sphingolipid production was shown in mutant-expressing human embryonic kindney (HEK) cells. CONCLUSIONS: Specific gain-of-function mutations in both core subunits affect the homoeostatic control of SPT. SPTLC2 represents a new Mendelian ALS gene, highlighting a key role of dysregulated sphingolipid synthesis in the pathogenesis of juvenile ALS. Given the direct interaction of SPTLC1 and SPTLC2, this knowledge might open new therapeutic avenues for motor neuron diseases.

Standards of NGS Data Sharing and Analysis in Ataxias: Recommendations by the NGS Working Group of the Ataxia Global Initiative.

The Ataxia Global Initiative (AGI) is a worldwide multi-stakeholder research platform to systematically enhance trial-readiness in degenerative ataxias. The next-generation sequencing (NGS) working group of the AGI aims to improve methods, platforms, and international standards for ataxia NGS analysis and data sharing, ultimately allowing to increase the number of genetically ataxia patients amenable for natural history and treatment trials. Despite extensive implementation of NGS for ataxia patients in clinical and research settings, the diagnostic gap remains sizeable, as approximately 50% of patients with hereditary ataxia remain genetically undiagnosed. One current shortcoming is the fragmentation of patients and NGS datasets on different analysis platforms and databases around the world. The AGI NGS working group in collaboration with the AGI associated research platforms-CAGC, GENESIS, and RD-Connect GPAP-provides clinicians and scientists access to user-friendly and adaptable interfaces to analyze genome-scale patient data. These platforms also foster collaboration within the ataxia community. These efforts and tools have led to the diagnosis of > 500 ataxia patients and the discovery of > 30 novel ataxia genes. Here, the AGI NGS working group presents their consensus recommendations for NGS data sharing initiatives in the ataxia field, focusing on harmonized NGS variant analysis and standardized clinical and metadata collection, combined with collaborative data and analysis tool sharing across platforms.

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies.

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies.

Deep structured learning for variant prioritization in Mendelian diseases.

Effective computer-aided or automated variant evaluations for monogenic diseases will expedite clinical diagnostic and research efforts of known and novel disease-causing genes. Here we introduce MAVERICK: a Mendelian Approach to Variant Effect pRedICtion built in Keras. MAVERICK is an ensemble of transformer-based neural networks that can classify a wide range of protein-altering single nucleotide variants (SNVs) and indels and assesses whether a variant would be pathogenic in the context of dominant or recessive inheritance. We demonstrate that MAVERICK outperforms all other major programs that assess pathogenicity in a Mendelian context. In a cohort of 644 previously solved patients with Mendelian diseases, MAVERICK ranks the causative pathogenic variant within the top five variants in over 95% of cases. Seventy-six percent of cases were solved by the top-ranked variant. MAVERICK ranks the causative pathogenic variant in hitherto novel disease genes within the first five candidate variants in 70% of cases. MAVERICK has already facilitated the identification of a novel disease gene causing a degenerative motor neuron disease. These results represent a significant step towards automated identification of causal variants in patients with Mendelian diseases.

Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

Mutations in MYO9B are associated with Charcot-Marie-Tooth disease type 2 neuropathies and isolated optic atrophy.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders caused by mutations in at least 100 genes. However, approximately 60% of cases with axonal neuropathies (CMT2) still remain without a genetic diagnosis. We aimed at identifying novel disease genes responsible for CMT2. METHODS: We performed whole exome sequencing and targeted next generation sequencing panel analyses on a cohort of CMT2 families with evidence for autosomal recessive inheritance. We also performed functional studies to explore the pathogenetic role of selected variants. RESULTS: We identified rare, recessive variants in the MYO9B (myosin IX) gene in two families with CMT2. MYO9B has not yet been associated with a human disease. MYO9B is an unconventional single-headed processive myosin motor protein with signaling properties, and, consistent with this, our results indicate that a variant occurring in the MYO9B motor domain impairs protein expression level and motor activity. Interestingly, a Myo9b-null mouse has degenerating axons in sciatic nerves and optic nerves, indicating that MYO9B plays an essential role in both peripheral nervous system and central nervous system axons, respectively. The degeneration observed in the optic nerve prompted us to screen for MYO9B mutations in a cohort of patients with optic atrophy (OA). Consistent with this, we found compound heterozygous variants in one case with isolated OA. CONCLUSIONS: Novel or very rare variants in MYO9B are associated with CMT2 and isolated OA.

BiP inactivation due to loss of the deAMPylation function of FICD causes a motor neuron disease.

PURPOSE: The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. METHODS: Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. RESULTS: We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. CONCLUSION: Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.

RFC1 repeat expansions: A recurrent cause of sensory and autonomic neuropathy with cough and ataxia.

BACKGROUND AND PURPOSE: Ataxia and cough are rare features in hereditary sensory and autonomic neuropathies (HSAN), a group of diseases of mostly unknown genetic cause. Biallelic repeat expansions in RFC1 are associated with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). This study aimed to investigate the prevalence of RFC1 repeat expansions in a cohort of HSAN patients. METHODS: After unremarkable whole-exome sequencing (WES) analysis, we performed repeat-primed PCR to detect intronic RFC1 expansions in 12 HSAN families, who all presented with chronic cough. RESULTS: In these patients, 75% carried biallelic expansions of the pathogenic AAGGG motif. Compared with RFC1-/- cases, RFC1+/+ cases presented more consistently with positive sensory and autonomic symptoms. Afferent ataxia was more severe in the RFC1+/+ cohort and cerebellar ataxia was a common feature (21%). CONCLUSIONS: We demonstrate that RFC1 is a frequent cause of (WES-negative) HSAN with chronic cough and ataxia. The diagnostic yield of RFC1 repeat-primed PCR was surprisingly high, given that HSAN is genetically poorly understood. This combination of HSAN, ataxia, and chronic cough symptoms represents a new nosological entity within the neuropathy-ataxia spectrum.

Heterozygous POLG variant Ser1181Asn co-segregating in a family with autosomal dominant axonal neuropathy, proximal muscle fatigability, ptosis, and ragged red fibers.

By whole-exome sequencing, we found the heterozygous POLG variant c.3542G>A; p.Ser1181Asn in a family of four affected individuals, presenting with a mixed neuro-myopathic phenotype. The variant is located within the active site of polymerase gamma, in a cluster region associated with an autosomal dominant inheritance. In adolescence, the index developed distal atrophies and weakness, sensory loss, afferent ataxia, double vision, and bilateral ptosis. One older sister presented with Charcot-Marie-Tooth-like symptoms, while the youngest sister and father reported exercise-induced muscle pain and proximal weakness. In none of the individuals, we observed any involvement of the central nervous system. Muscle biopsies obtained from the father and the older sister showed ragged-red fibers, and electron microscopy confirmed mitochondrial damage. We conclude that this novel POLG variant explains this family's phenotype.

De Novo and Dominantly Inherited SPTAN1 Mutations Cause Spastic Paraplegia and Cerebellar Ataxia.

BACKGROUND: Pathogenic variants in SPTAN1 have been linked to a remarkably broad phenotypical spectrum. Clinical presentations include epileptic syndromes, intellectual disability, and hereditary motor neuropathy. OBJECTIVES: We investigated the role of SPTAN1 variants in rare neurological disorders such as ataxia and spastic paraplegia. METHODS: We screened 10,000 NGS datasets across two international consortia and one local database, indicative of the level of international collaboration currently required to identify genes causative for rare disease. We performed in silico modeling of the identified SPTAN1 variants. RESULTS: We describe 22 patients from 14 families with five novel SPTAN1 variants. Of six patients with cerebellar ataxia, four carry a de novo SPTAN1 variant and two show a sporadic inheritance. In this group, one variant (p.Lys2083del) is recurrent in four patients. Two patients have novel de novo missense mutations (p.Arg1098Cys, p.Arg1624Cys) associated with cerebellar ataxia, in one patient accompanied by intellectual disability and epilepsy. We furthermore report a recurrent missense mutation (p.Arg19Trp) in 15 patients with spastic paraplegia from seven families with a dominant inheritance pattern in four and a de novo origin in one case. One further patient carrying a de novo missense mutation (p.Gln2205Pro) has a complex spastic ataxic phenotype. Through protein modeling we show that mutated amino acids are located at crucial interlinking positions, interconnecting the three-helix bundle of a spectrin repeat. CONCLUSIONS: We show that SPTAN1 is a relevant candidate gene for ataxia and spastic paraplegia. We suggest that for the mutations identified in this study, disruption of the interlinking of spectrin helices could be a key feature of the pathomechanism. © 2022 International Parkinson and Movement Disorder Society.

Homozygous N-terminal missense variant in PLEKHG5 associated with intermediate CMT: A case report.

Mutations in PLEKHG5, a pleckstrin homology domain containing member of the GEF family, are associated with distal spinal muscular atrophy and intermediate Charcot-Marie-Tooth disease. Here, we describe an isolated case with distal intermediate neuropathy with scapular winging. By whole exome sequencing, we identified the homozygous PLEKHG5 Arg97Gln missense mutation, located in the N-terminal region of the protein. This mutation resides between a zinc-finger motif and a RBD domain, involved in binding rnd3, a RhoA effector protein. We conclude that based on the characteristic phenotype presented by the patient and the supportive genetic findings, the PLEKHG5 mutation is the causative variant.

Biallelic ADPRHL2 mutations in complex neuropathy affect ADP ribosylation and DNA damage response.

ADP ribosylation is a reversible posttranslational modification mediated by poly(ADP-ribose)transferases (e.g., PARP1) and (ADP-ribosyl)hydrolases (e.g., ARH3 and PARG), ensuring synthesis and removal of mono-ADP-ribose or poly-ADP-ribose chains on protein substrates. Dysregulation of ADP ribosylation signaling has been associated with several neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Recessive ADPRHL2/ARH3 mutations are described to cause a stress-induced epileptic ataxia syndrome with developmental delay and axonal neuropathy (CONDSIAS). Here, we present two families with a neuropathy predominant disorder and homozygous mutations in ADPRHL2 We characterized a novel C26F mutation, demonstrating protein instability and reduced protein function. Characterization of the recurrent V335G mutant demonstrated mild loss of expression with retained enzymatic activity. Although the V335G mutation retains its mitochondrial localization, it has altered cytosolic/nuclear localization. This minimally affects basal ADP ribosylation but results in elevated nuclear ADP ribosylation during stress, demonstrating the vital role of ADP ribosylation reversal by ARH3 in DNA damage control.

Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation.

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.