Mono and biallelic variants in HCN2 cause severe neurodevelopmental disorders.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

Megalencephalic leukoencephalopathy with subcortical cysts: a variant update and review of the literature.

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.

Erythromelalgia caused by the missense mutation p.Arg220Pro in an alternatively spliced exon of SCN9A (NaV1.7).

Erythromelalgia (EM), is a familial pain syndrome characterized by episodic 'burning' pain, warmth, and erythema. EM is caused by monoallelic variants in SCN9A, which encodes the voltage-gated sodium channel (NaV) NaV1.7. Over 25 different SCN9A mutations attributed to EM have been described to date, all identified in the SCN9A transcript utilizing exon 6N. Here we report a novel SCN9A missense variant identified in seven related individuals with stereotypic episodes of bilateral lower limb pain presenting in childhood. The variant, XM_011511617.3:c.659G>C;p.(Arg220Pro), resides in the exon 6A of SCN9A, an exon previously shown to be selectively incorporated by developmentally regulated alternative splicing. The mutation is located in the voltage-sensing S4 segment of domain I, which is important for regulating channel activation. Functional analysis showed the p.Arg220Pro mutation altered voltage-dependent activation and delayed channel inactivation, consistent with a NaV1.7 gain-of-function molecular phenotype. These results demonstrate that alternatively spliced isoforms of SCN9A should be included in all genomic testing of EM.

Atypical splicing variants in PKD1 explain most undiagnosed typical familial ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of kidney failure and is primarily associated with PKD1 or PKD2. Approximately 10% of patients remain undiagnosed after standard genetic testing. We aimed to utilise short and long-read genome sequencing and RNA studies to investigate undiagnosed families. Patients with typical ADPKD phenotype and undiagnosed after genetic diagnostics were recruited. Probands underwent short-read genome sequencing, PKD1 and PKD2 coding and non-coding analyses and then genome-wide analysis. Targeted RNA studies investigated variants suspected to impact splicing. Those undiagnosed then underwent Oxford Nanopore Technologies long-read genome sequencing. From over 172 probands, 9 met inclusion criteria and consented. A genetic diagnosis was made in 8 of 9 (89%) families undiagnosed on prior genetic testing. Six had variants impacting splicing, five in non-coding regions of PKD1. Short-read genome sequencing identified novel branchpoint, AG-exclusion zone and missense variants generating cryptic splice sites and a deletion causing critical intron shortening. Long-read sequencing confirmed the diagnosis in one family. Most undiagnosed families with typical ADPKD have splice-impacting variants in PKD1. We describe a pragmatic method for diagnostic laboratories to assess PKD1 and PKD2 non-coding regions and validate suspected splicing variants through targeted RNA studies.

Integrated multi-omics for rapid rare disease diagnosis on a national scale.

Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner.

Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

Australian Genomics: Outcomes of a 5-year national program to accelerate the integration of genomics in healthcare.

Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

ahctf1 and kras mutations combine to amplify oncogenic stress and restrict liver overgrowth in a zebrafish model of hepatocellular carcinoma.

The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks krasG12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107-160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Participant Choice towards Receiving Potential Additional Findings in an Australian Nephrology Research Genomics Study.

The choices of participants in nephrology research genomics studies about receiving additional findings (AFs) are unclear as are participant factors that might influence those choices. Methods: Participant choices and factors potentially impacting decisions about AFs were examined in an Australian study applying research genomic testing following uninformative diagnostic genetic testing for suspected monogenic kidney disease. Results: 93% of participants (195/210) chose to receive potential AFs. There were no statistically significant differences between those consenting to receive AFs or not in terms of gender (p = 0.97), median age (p = 0.56), being personally affected by the inherited kidney disease of interest (p = 0.38), or by the inheritance pattern (p = 0.12-0.19). Participants were more likely to choose not to receive AFs if the family proband presented in adulthood (p = 0.01), if there was family history of another genetic disorder (p = 0.01), and where the consent process was undertaken by an adult nephrologist (p = 0.01). Conclusion: The majority of participants in this nephrology research genomics study chose to receive potential AFs. Younger age of the family proband, family history of an alternate genetic disorder, and consenting by some multidisciplinary team members might impact upon participant choices.

Generation of human induced pluripotential stem cells from individuals with complex heterozygous, isogenic corrected, and homozygous Bloc1s1 mutations.

Genetic studies show that BLOC1S1 modulates mitochondrial and endosome-lysosome function (Wu et al., 2021a). Furthermore, Bloc1s1 mutations are linked to leukodystrophy (Bertoli-Avella et al., 2021). The Vanderver laboratory identified additional individuals with leukodystrophy that harbored either complex heterozygous (Bloc1s1 c.206A > C and c.359G > A), or homozygous (Bloc1s1 c.185 T > C) point mutations. We generated induced pluripotential stem cell (iPSC) lines from these subjects, from parents of the complex heterozygous mutations patient, and from CRISPR isogenic (c.206A > C and c.359G > A) corrected iPSC-line. These complex heterozygous, homozygous, and isogenic-corrected Bloc1s1 lines were phenotypically normal and were capable of differentiation towards the three germ layers.

Heterozygous NOTCH1 Variants Cause CNS Immune Activation and Microangiopathy.

NOTCH1 belongs to the NOTCH family of proteins that regulate cell fate and inflammatory responses. Somatic and germline NOTCH1 variants have been implicated in cancer, Adams-Oliver syndrome, and cardiovascular defects. We describe 7 unrelated patients grouped by the presence of leukoencephalopathy with calcifications and heterozygous de novo gain-of-function variants in NOTCH1. Immunologic profiling showed upregulated CSF IP-10, a cytokine secreted downstream of NOTCH1 signaling. Autopsy revealed extensive leukoencephalopathy and microangiopathy with vascular calcifications. This evidence implicates that heterozygous gain-of-function variants in NOTCH1 lead to a chronic central nervous system (CNS) inflammatory response resulting in a calcifying microangiopathy with leukoencephalopathy. ANN NEUROL 2022;92:895-901.

The HIDDEN Protocol: An Australian Prospective Cohort Study to Determine the Utility of Whole Genome Sequencing in Kidney Failure of Unknown Aetiology.

Early identification of genetic kidney disease allows personalised management, clarification of risk for relatives, and guidance for family planning. Genetic disease is underdiagnosed, and recognition of genetic disease is particularly challenging in patients with kidney failure without distinguishing diagnostic features. To address this challenge, the primary aim of this study is to determine the proportion of genetic diagnoses amongst patients with kidney failure of unknown aetiology, using whole genome sequencing (WGS). A cohort of up to 100 Australian patients with kidney failure of unknown aetiology, with onset <50 years old and approved by a panel of study investigators will be recruited via 18 centres nationally. Clinically accredited WGS will be undertaken with analysis targeted to a priority list of ∼388 genes associated with genetic kidney disease. The primary outcome will be the proportion of patients who receive a molecular diagnosis (diagnostic rate) via WGS compared with usual -care (no further diagnostic investigation). Participant surveys will be undertaken at consent, after test result return and 1 year subsequently. Where there is no or an uncertain diagnosis, future research genomics will be considered to identify candidate genes and new pathogenic variants in known genes. All results will be relayed to participants via the recruiting clinician and/or kidney genetics clinic. The study is ethically approved (HREC/16/MH/251) with local site governance approvals in place. The future results of this study will be disseminated and inform practical understanding of the potential monogenic contribution to kidney failure of unknown aetiology. These findings are anticipated to impact clinical practice and healthcare policy. Study Registration: [https://dora.health.qld.gov.au], identifier [HREC/16/MH/251].

Feasibility of Screening for Chromosome 15 Imprinting Disorders in 16 579 Newborns by Using a Novel Genomic Workflow.

Importance: Newborn screening for Angelman syndrome (AS), Prader-Willi syndrome (PWS), and chromosome 15 duplication syndrome (Dup15q) may lead to benefit from early diagnosis and treatment. Objective: To examine the feasibility of newborn screening for these chromosome 15 imprinting disorders at population scale. Design, Setting, and Participants: In this diagnostic study, the validation data set for the first-tier SNRPN test, called methylation-specific quantitative melt analysis (MS-QMA), included 109 PWS, 48 AS, 9 Dup15q, and 1190 population control newborn blood spots (NBS) and peripheral tissue samples from participants recruited from January 2000 to December 2016. The test data set included NBS samples from 16 579 infants born in 2011. Infants with an NBS identified as positive for PWS, AS, or Dup15q by the first-tier test were referred for droplet digital polymerase chain reaction, real-time polymerase chain reaction, and low-coverage whole-genome sequencing for confirmatory testing. Data analyses were conducted between February 12, 2015, and August 15, 2020. Results: In the validation data set, the median age for the 77 patients with PWS was 3.00 years (IQR, 0.01-44.50 years); for the 46 patients with AS, 2.76 years (IQR, 0.028 to 49.00 years); and for the 9 patients with Dup15q, 4.00 years (IQR, 1.00 to 28.00 years). Thirty-eight patients (51.4%) in the PWS group, 20 patients (45.5%) in the AS group, and 6 patients (66.7%) in the Dup15q group who had sex reported were male. The validation data set showed MS-QMA sensitivity of 99.0% for PWS, 93.8% for AS, and 77.8% for Dup15q; specificity of 100% for PWS, AS, and Dup15q; positive predictive and negative predictive values of 100% for PWS and AS; and a positive predictive value of 87.5% and negative predictive value of 100% for Dup15q. In the test data set of NBS samples from 16 579 infants, 92 had a positive test result using a methylation ratio cut-off of 3 standard deviations from the mean. Of these patients, 2 were confirmed to have PWS; 2, AS; and 1, maternal Dup15q. With the use of more conservative PWS- and AS-specific thresholds for positive calls from the validation data set, 9 positive NBS results were identified by MS-QMA in this cohort. The 2 PWS and 2 AS calls were confirmed by second-tier testing, but the 1 Dup15q case was not confirmed. Together, these results provided prevalence estimates of 1 in 8290 for both AS and PWS and 1 in 16 579 for maternal Dup15q, with positive predictive values for first-tier testing at 67.0% for AS, 33.0% for PWS, and 44.0% for combined detection of chromosome 15 imprinting disorders for the validation data set. Conclusions and Relevance: The findings of this diagnostic study suggest that it is feasible to screen for all chromosome 15 imprinting disorders using SNRPN methylation analysis, with 5 individuals identified with these disorders out of 16 579 infants screened.

Early-Onset Vascular Leukoencephalopathy Caused by Bi-Allelic NOTCH3 Variants.

OBJECTIVE: Heterozygous NOTCH3 variants are known to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), with patients typically presenting in adulthood. We describe three patients presenting at an early age with a vascular leukoencephalopathy. Genome sequencing revealed bi-allelic variants in the NOTCH3 gene. METHODS: Clinical records and available MRI and CT scans of three patients from two unrelated families were retrospectively reviewed. RESULTS: The patients presented at 9 to 14 months of age with developmental delay, seizures, or both. The disease course was characterized by cognitive impairment and variably recurrent strokes, migraine attacks, and seizures. MRI findings pointed at a small vessel disease, with extensive cerebral white matter abnormalities, atrophy, lacunes in the basal ganglia, microbleeds, and microcalcifications. The anterior temporal lobes were spared. Bi-allelic cysteine-sparing NOTCH3 variants in exons 1, 32, and 33 were found. INTERPRETATION: This study indicates that bi-allelic loss-of-function NOTCH3 variants may cause a vascular leukoencephalopathy, distinct from CADASIL.

Lessons learnt from multifaceted diagnostic approaches to the first 150 families in Victoria's Undiagnosed Diseases Program.

BACKGROUND: Clinical exome sequencing typically achieves diagnostic yields of 30%-57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals. AIM: We share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases. METHODS: We enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis. RESULTS: In 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis. CONCLUSION: We share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease-gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

Pathogenic variants in nucleoporin TPR (translocated promoter region, nuclear basket protein) cause severe intellectual disability in humans.

The nuclear pore complex (NPC) is a multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Genetic variants in components of the NPC have been shown to cause a range of neurological disorders, including intellectual disability and microcephaly. Translocated promoter region, nuclear basket protein (TPR) is a critical scaffolding element of the nuclear facing interior of the NPC. Here, we present two siblings with biallelic variants in TPR who present with a phenotype of microcephaly, ataxia and severe intellectual disability. The variants result in a premature truncation variant, and a splice variant leading to a 12-amino acid deletion respectively. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR levels, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, and decreased global RNA intensity in the nucleus. The discovery of variants that partly disable TPR function provide valuable insight into this essential protein in human disease, and our findings suggest that TPR variants are the cause of the siblings' neurological disorder.