Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity.

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary ß subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

Experimental demonstration of the mechanism of steady-state microbunching.

The use of particle accelerators as photon sources has enabled advances in science and technology1. Currently the workhorses of such sources are storage-ring-based synchrotron radiation facilities2-4 and linear-accelerator-based free-electron lasers5-14. Synchrotron radiation facilities deliver photons with high repetition rates but relatively low power, owing to their temporally incoherent nature. Free-electron lasers produce radiation with high peak brightness, but their repetition rate is limited by the driving sources. The steady-state microbunching15-22 (SSMB) mechanism has been proposed to generate high-repetition, high-power radiation at wavelengths ranging from the terahertz scale to the extreme ultraviolet. This is accomplished by using microbunching-enabled multiparticle coherent enhancement of the radiation in an electron storage ring on a steady-state turn-by-turn basis. A crucial step in unveiling the potential of SSMB as a future photon source is the demonstration of its mechanism in a real machine. Here we report an experimental demonstration of the SSMB mechanism. We show that electron bunches stored in a quasi-isochronous ring can yield sub-micrometre microbunching and coherent radiation, one complete revolution after energy modulation induced by a 1,064-nanometre-wavelength laser. Our results verify that the optical phases of electrons can be correlated turn by turn at a precision of sub-laser wavelengths. On the basis of this phase correlation, we expect that SSMB will be realized by applying a phase-locked laser that interacts with the electrons turn by turn. This demonstration represents a milestone towards the implementation of an SSMB-based high-repetition, high-power photon source.

Missing trial results: analysis of the current publication rate of studies in pediatric dialysis from 2003 to 2020.

BACKGROUND: Decision-making in the field of pediatric dialysis requires evidence from clinical trials, but, similar to other fields of pediatric medicine, might be affected by a low trial publication rate. METHODS: We analyzed the current publication rate, the time to publication, and factors that might be associated with both rate of and time to publication in pediatric dialysis studies registered as completed on ClinicalTrials.gov from 2003 until November 2020. RESULTS: Fifty-three respective studies were identified. These enrolled 7287 patients in total. 28 of 53 studies (52.8%) had results available. We identified a median time to publication of 20.5 months (range, 3-67). Studies published after the FDA Amendments Act establishment in 2007 were published faster (P = 0.025). There was no trend toward a higher publication rate of studies completed more recently (P = 0.431). 26 of 53 studies (49.1%) focused on medication and control of secondary complications of kidney failure. 12 of 53 studies (22.6%) enrolled only children, were published faster (P = 0.029) and had a higher 5-year publication rate (P = 0.038) than studies enrolling both children and adults. 25 of 53 studies (47.1%) were co-funded by industry. These were published faster (P = 0.025). CONCLUSIONS: Currently, only 52.8% of all investigated studies in pediatric dialysis have available results, and the overall median time to publication did not meet FDA requirements. This might introduce a publication bias into the field, and it might negatively impact clinical decision-making in this critical subspecialty of pediatric medicine. A higher resolution version of the Graphical abstract is available as Supplementary information.

Quantitative longitudinal natural history of 8 gangliosidoses-conceptual framework and baseline data of the German 8-in-1 disease registry. A cross-sectional analysis.

PURPOSE: Gangliosidoses are a group of inherited neurogenetic autosomal recessive lysosomal storage disorders usually presenting with progressive macrocephaly, developmental delay, and regression, leading to significant morbidity and premature death. A quantitative definition of the natural history would support and enable clinical development of specific therapies. METHODS: Single disease registry of 8 gangliosidoses (NCT04624789). Cross-sectional analysis of baseline data in N = 26 patients. Primary end point: disease severity assessed by the 8-in-1 score. Secondary end points: first neurologic sign or symptom observed (1) by parents and (2) by physicians, diagnostic delay, as well as phenotypical characterization. Tertiary end points: neurologic outcomes (development, ataxia, dexterity) and disability. RESULTS: The 8-in-1 score quantitatively captured severity of disease. Parents recognized initial manifestations (startle reactions) earlier than physicians (motor developmental delay and hypotonia). Median diagnostic delay was 3.16 (interquartile range 0.69-6.25) years. In total, 8 patients presented with late-infantile phenotypes. CONCLUSION: Data in this registry raise awareness of these rare and fatal conditions to accelerate diagnosis, inform counseling of afflicted families, define quantitative end points for clinical trials, and can serve as historical controls for future therapeutic studies. We provide further insight into the rare late-infantile phenotype for GM2-gangliosidosis. Longitudinal follow up is planned.

Publication rate and research topics of studies in pediatric kidney transplantation.

BACKGROUND: The quality of medical care for pediatric kidney transplant recipients depends on sound evidence from published clinical trials. METHODS: We examined the publication rate, time to publication, and factors associated with publication of studies in pediatric kidney transplantation registered on ClinicalTrials.gov from 1999 to 2020. RESULTS: We identified 136 studies with an overall enrollment of 36255 study participants, of which only 58.8% have been published yet. Unpublished studies included data from 14 350 participants. The median time to publication was 25 months (range, 0-117) with a significantly shorter time to publication in more recent years. The most frequently investigated research topic was immunosuppressants (49.3%), followed by perioperative management (11.0%) and infectiology (10.3%). The percentage of published studies was highest for the topic steroid withdrawal (87.5%), followed by infectiology (78.6%), and nutrition, sports and quality of life (71.4%). Studies, which were co-funded by industry, showed a significantly higher 5-year publication rate (p = 0.019). CONCLUSIONS: In conclusion, nearly half of all studies in pediatric kidney transplantation remain unpublished. Non-publication of studies might lead to a publication bias with a negative impact on clinical decision-making.

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.

Cross-sectional quantitative analysis of the natural history of TUBA1A and TUBB2B tubulinopathies.

PURPOSE: TUBA1A and TUBB2B tubulinopathies are rare neurodevelopmental disorders characterized by cortical and extracortical malformations and heterogenic phenotypes. There is a need for quantitative clinical endpoints that will be beneficial for future diagnostic and therapeutic trials. METHODS: Quantitative natural history modeling of individuals with TUBA1A and TUBB2B tubulinopathies from clinical reports and database entries of DECIPHER and ClinVar. Main outcome measures were age at disease onset, survival, and diagnostic delay. Phenotypical, neuroradiological, and histopathological features were descriptively illustrated. RESULTS: Mean age at disease onset was 4 (TUBA1A) and 6 months (TUBB2B), respectively. Mortality was equally estimated with 7% at 3.2 (TUBA1A) and 8.0 years (TUBB2B). Diagnostic delay was significantly higher in TUBB2B (12.3 years) compared with TUBA1A tubulinopathy (4.2 years). We delineated the isotype-dependent clinical, neuroradiological, and histopathological phenotype of affected individuals and present brain malformations associated with epilepsy and an unfavorable course of disease. CONCLUSION: The natural history of tubulinopathies is defined by the genotype and associated brain malformations. Defined data on estimated survival, diagnostic delay, and disease characteristics of TUBA1A and TUBB2B tubulinopathy will help to raise disease awareness and encourage future clinical trials to optimize genetic testing, family counseling, and supportive care.

Quantitative retrospective natural history modeling for orphan drug development.

The natural history of most rare diseases is incompletely understood and usually relies on studies with low level of evidence. Consistent with the goals for future research of rare disease research set by the International Rare Diseases Research Consortium in 2017, the purpose of this paper is to review the recently developed method of quantitative retrospective natural history modeling (QUARNAM) and to illustrate its usefulness through didactically selected analyses examples in an overall population of 849 patients worldwide with seven (ultra-) rare neurogenetic disorders. A quantitative understanding of the natural history of the disease is fundamental for the development of specific interventions and counseling afflicted families. QUARNAM has a similar relationship to a published case study as a meta-analysis has to an individual published study. QUARNAM relies on sophisticated statistical analyses of published case reports focusing on four research questions: How long does it take to make the diagnosis? How long do patients live? Which factors predict disease severity (eg, genotypes, signs/symptoms, biomarkers)? Where can patients be recruited for studies? Useful statistical techniques include Kaplan-Meier estimates, cluster analysis, regression techniques, binary decisions trees, word clouds, and geographic mapping. In comparison to other natural history study methods (prospective studies or retrospective studies such as chart reviews), QUARNAM can provide fast information on hard clinical endpoints (ie, survival, diagnostic delay) with a lower effort. The choice of method for a particular drug development program may be driven by the research question and may encompass combinatory approaches.

Disease awareness or subtle product placement? Orphan diseases featured in the television series "House, M.D." - a cross-sectional analysis.

BACKGROUND: Approximately 7% of the general population is affected by an orphan disease, which, in the United States, is defined as affecting fewer than 1 in 1500 people. Disease awareness is often low and time-to-diagnosis delayed. Different legislations worldwide have created incentives for pharmaceutical companies to develop drugs for orphan diseases. A journalistic article in Bloomberg Businessweek has claimed that pharmaceutical companies have tried marketing orphan drugs by placing a specific disease into the popular television series "House, M.D." which features diagnostic journeys and was produced between 2004 and 2012. This study aimed to describe the presentation of orphan diseases in the television series "House, M.D.", to test in an exploratory fashion the hypothesis that treatable orphan conditions are overrepresented in "House, M.D." and to discuss whether such marketing practices may or may not be ethical. METHODS: A list of all medical cases depicted in the television series "House, M.D." was obtained and classified as orphan or non-orphan according to the Orphanet database. The ratios of orphan diseases among all diseases, such with an orphan drug designation and such with an orphan drug approval by the FDA were then compared with conservative approximations of real world conditions (chi-squared tests for equality of proportions). STROBE criteria were respected. RESULTS: Out of a total of n = 181 different medical diagnoses, n = 42 (23.2%) were orphan diseases. The difference in percentages in between "House, M.D." and reality was not statistically significant for orphan diseases overall (p = 0.96), yet was statistically significantly higher for both orphan diseases with one or more orphan drug designations (p = 0.0192) and such with one or more approved orphan drugs (p < 0.0001). CONCLUSIONS: Orphan diseases with a designated and/or approved orphan drug were overrepresented in the television series "House, M.D." with statistical significance while orphan diseases overall were not. This may be explained by (so far) undocumented efforts of pharmaceutical companies to place their orphan drugs in the television series, as described in the article in Bloomberg Businessweek. Further research is needed into marketing practices in popular and emerging media formats.

A cross-sectional quantitative analysis of the natural history of free sialic acid storage disease-an ultra-orphan multisystemic lysosomal storage disorder.

PURPOSE: Quantitative definition of the natural history of free sialic acid storage disease (SASD, OMIM 604369), an orphan disorder due to the deficiency of the proton-driven carrier SLC17A5. METHODS: Analysis of published cases with SASD (N = 116) respecting STROBE criteria. MAIN OUTCOME PARAMETERS: survival and diagnostic delay. Phenotype, phenotype-biomarker associations, and geographical patient distribution were explored. RESULTS: Median age at disease onset was 0.17 years. Median age at diagnosis was 3 years with a median diagnostic delay of 2.5 years. Median survival was 11 years. The biochemical phenotype clearly predicted the disease course: patients with a urinary free sialic acid excretion below 6.37-fold or an intracellular free sialic acid storage in fibroblasts below 7.37-fold of the mean of normal survived longer than patients with biochemical values above these thresholds. Cluster analysis of disease features suggested a continuous phenotypic spectrum. Patient distribution was panethnic. CONCLUSION: Combination of neurologic symptoms, visceromegaly, and dysmorphic features and/or nonimmune hydrops fetalis should prompt specific tests for SASD, reducing diagnostic delay. The present quantitative data inform clinical studies and may stimulate and accelerate development of specific therapies. Biomarker-phenotype association is particularly important for both counseling parents and study design.

Clinical characteristics of 248 patients with Krabbe disease: quantitative natural history modeling based on published cases.

PURPOSE: Krabbe disease (OMIM 245200) is an orphan neurometabolic disorder caused by a deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hard clinical endpoints and biomarker-phenotype correlations are useful for future clinical trials. METHODS: We performed a quantitative analysis of published cases (N = 248) with Krabbe disease, stratified by age at disease onset: early infantile (age 0-6 months), late infantile (age 7-36 months), juvenile/adolescent (age 37-180 months), and adult onset (>180 months). Main outcome measures were age of disease onset and survival. Cerebrospinal fluid (CSF) protein concentrations were explored as a potential predictor of survival. STROBE criteria were respected. RESULTS: Median age of onset was 4 months (early infantile), 14 months (late infantile), 48 months (juvenile), and 384 months (adult). Age of disease onset and therefore disease subtype determined survival rates. CSF protein concentrations predicted age at onset and survival rates in Krabbe disease. Patients with a CSF protein content ≤61.5 mg/dl survived significantly longer than patients with CSF protein values above this threshold. CONCLUSION: We define the estimated survival in published Krabbe disease cases and demonstrate an association of CSF protein concentration with disease severity. These data inform patient care and clinical trials.

Quantitative natural history characterization in a cohort of 142 published cases of patients with galactosialidosis-A cross-sectional study.

Galactosialidosis (GS; OMIM #256540) is a rare multisystemic inborn glycoprotein storage disease caused by biallelic mutations in the cathepsin A gene resulting in combined deficiency of the lysosomal enzymes ß-galactosidase and α-neuraminidase. The precise understanding of the natural course of the disease is limited. Development of enzyme replacement therapy is at the preclinical stage. The purpose of this research project was to quantitatively characterize the natural history of the condition. Quantitative analysis of all published cases in the literature with sufficient data (N = 142 patients) was carried out. Main outcome variables were survival, diagnostic delay, description of symptoms, biomarker-phenotype associations, and radiological findings. STROBE criteria were respected. Median survival age of the cohort was 48 years. Median age of onset was 4.25 years with interquartile range (IQR) 1 to 16 years. Median age at diagnosis was 19 (IQR: 8.92-29) years, with median diagnostic delay of 8 (IQR: 4-12) years. Patients with residual ß-galactosidase activity of more than 8.6% (leukocytes) survived significantly longer than patients with lower enzyme activities.

Ultra-orphan lysosomal storage diseases: A cross-sectional quantitative analysis of the natural history of alpha-mannosidosis.

Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disorder caused by a deficiency of the enzyme alpha-mannosidase. Recently, enzyme replacement therapy was approved in the European Union for the treatment of alpha-mannosidosis, but evaluation regarding long-term efficacy and safety is hard to assess due to missing quantitative natural history data, in particular survival. We performed a quantitative analysis of published cases (N = 111) with alpha-mannosidosis. Main outcome measures were age of disease onset, diagnostic delay and survival (overall and by subgroup exploration). Residual alpha-mannosidase activity and age of onset were explored as potential predictors of survival. STROBE criteria were respected. Median age of onset was 12 months. Median diagnostic delay was 6 years. At the age of 41 years 72.3% of patients were alive (N = 111). Residual alpha-mannosidase activity (N = 34) predicted survival: Patients with a residual alpha-mannosidase activity below or equal to 4.5% of normal in fibroblasts had a median survival of 3.5 years, whereas patients with alpha-mannosidase activity above this threshold all survived during the observation period reported. Patients with age of onset above 7 years survived significantly longer than patients with age of onset below or equal to 7 years. Patient distribution was panethnic with hotspots in the United States and Germany. We defined age of onset, diagnostic delay, and survival characteristics in a global cohort of 111 patients with alpha-mannosidosis by retrospective quantitative natural history modeling. These data expand the quantitative understanding of the clinical phenotype.

Corrigendum: Quantitative clinical characteristics of 53 patients with MPS VII: a cross-sectional analysis.

This corrects the article DOI: 10.1038/gim.2017.10.

A cross-sectional quantitative analysis of the natural history of Farber disease: an ultra-orphan condition with rheumatologic and neurological cardinal disease features.

PurposeFarber disease (OMIM 22800) is an ultrarare progressive multisystemic neurodevelopmental storage disorder caused by a deficiency of the lysosomal enzyme acid ceramidase (AC). Hard clinical end points for future clinical trials remain to be defined.MethodsWe quantitatively analyzed published cases with Farber disease (N = 96). The main outcome variables were survival and diagnostic delay. As a potential predictor of survival, the influence of residual AC enzyme activity was investigated. The analysis was performed in compliance with STROBE criteria.ResultsThe median survival period of the study population was 3 years. The median age at disease onset was 3 months, and the median age at diagnosis was 17 months. The median diagnostic delay was 13.75 months. Patients with residual AC activity in fibroblasts at more than 5.1% of the normal level survived significantly longer than patients with residual AC activity below this threshold. In addition, higher residual AC activity was associated with a later onset of symptoms.ConclusionFarber disease onset is in infancy. Diagnostic delay is typically substantial. Our data suggest a phenotype-biomarker association with implications for future clinical and therapeutic trials. In the absence of a prospective multicenter natural-history study protocol, we believe that our modeling approach, based on published case descriptions, is the best and most timely approximation for generalizability.

Cognitive and Behavioral Consequences of Pediatric Delirium: A Pilot Study.

OBJECTIVES: To investigate the long-term impact of postoperative delirium in children. DESIGN: Single-center point prevalence study. SETTING: Twenty-two bed PICU. PATIENTS: Forty-seven patients 1-16 years old. INTERVENTIONS: Standardized neuropsychologic follow-up investigation after a mean time of 17.7 ± 2.9 months after PICU discharge. MEASUREMENTS AND MAIN RESULTS: Pediatric delirium did not have significant long-term impact on global cognition, executive functions, or behavior. Severity of delirium did not influence the outcome. Different predictors were identified for later cognitive functioning, executive functions, and behavioral problems. Younger age was confirmed to be a relevant risk factor for delirium as well as for the cognitive and behavioral outcome. CONCLUSIONS: Contrary to the findings in adults, there was no clear association between pediatric delirium and long-term cognition or behavior in this cohort. However, this is a first pilot study with several limitations that should promote more comprehensive prospective trials.

Quantitative clinical characteristics of 53 patients with MPS VII: a cross-sectional analysis.

PURPOSE: The main purpose of the study was to provide quantitative data regarding survival and diagnostic delay. Mucopolysaccharidosis (MPS) type VII (OMIM 253220) is a progressive neurometabolic disorder caused by deficiency of the lysosomal enzyme ß-glucuronidase (GUS). Hard clinical end points have not been quantitatedMethods:We quantitatively analyzed published cases with MPS VII (N = 53/88 with sufficient data). Main outcome measures were onset of disease and survival. The role of biomarkers such as GUS residual enzyme activity and levels of storage material assessed as urinary excretion of glucosaminoglycans (GAG) as potential predictors of clinical outcomes were investigated. The analysis was conducted according to STROBE criteria. RESULTS: Median survival of the postnatally diagnosed population was up to 360 months . Median age of disease onset was the first day of life; median age at diagnosis was 11 months. Hydrops fetalis was frequent. Patients with residual GUS activity in fibroblasts more than 1.4% or urinary GAG excretion less than 602% of normal survived longer than patients with GUS enzyme activity below or GAG excretion above these thresholds. CONCLUSION: MPS VII has its disease onset prenatally. In the absence of a prenatal diagnosis, most cases are clinically apparent at birth. Our data corroborate a phenotype-biomarker association in MPS VII. The survival data characterize the natural history with important implications for therapeutic studies.Genet Med advance online publication 06 April 2017.

Enhancing the sensitivity of nano-FTIR spectroscopy.

Synchrotron radiation-based nano-FTIR spectroscopy utilizes the highly brilliant and ultra-broadband infrared (IR) radiation provided by electron storage rings for the infrared spectroscopic characterization of samples at the nanoscale. In order to exploit the full potential of this approach we investigated the influence of the properties of the radiation source, such as the electron bunch shape and spectral bandwidth of the emitted radiation, on near-field infrared spectra of silicon-carbide (SiC). The adapted configuration of the storage ring optics enables a modification of the transverse electron bunch profile allowing an increase of the measured near-field signal amplitude. Additionally, the decay of the signal amplitude due to the decreasing storage ring current is also eliminated. Further options for improving the sensitivity of nano-FTIR spectroscopy, which can also be applied to other broadband radiation sources, are the adaption of the spectral bandwidth to the wavelength range of interest or the use of polarization optics. The sensitivity enhancement emerging from these options is verified by comparing near-field spectra collected from crystalline SiC samples. The improvement in sensitivity by combining these approaches is demonstrated by acquiring nano-FTIR spectra from thin organic films, which show weak resonances in the IR-regime.

Enzyme replacement therapy and beyond-in memoriam Roscoe O. Brady, M.D. (1923-2016).

Lysosomal storage disorders are strong candidates for the development of specific innovative therapies. The discovery of enzyme deficiencies is an important milestone in understanding the underlying cause of disease. Being able to replace the first missing enzyme in a lysosomal storage required three decades of dedicated research. Successful drug development for lysosomal storage disorders was fostered by the U.S. Orphan Drug Act. Various optimization strategies have the potential to overcome the current limitations of enzyme replacement therapies. In addition, substrate reduction therapies are an alternative approach to treat lysosomal storage disorders, chemical chaperones enhance residual enzyme activity, and small molecules can facilitate substrate transport through subcellular compartments. Bone-marrow derived multipotent stem cells and gene therapies have received FDA orphan drug designation status. The science of small clinical trials played an essential role: non-neurological endpoints, biomarker, and regulatory alignment are key factors in successful drug development for lysosomal storage disorders. Being able to treat brain disease is the next frontier. This review is dedicated to the memory of Roscoe O. Brady, an early pioneer in the research of lysosomal storage diseases.

Critical appraisal of genotype assessment in molybdenum cofactor deficiency.

INTRODUCTION: Molybdenum cofactor deficiency (MoCD) is an ultra-orphan, life-threatening disease. Substrate substitution therapy has successfully been performed in single cases of MoCD type A and clinical trials are underway for drug registration. We present an innovative approach for classification of genotype severity to test the hypothesis that milder sequence variants in MoCD result in a less severe disease phenotype quantitated by patient survival. METHODS: All available worldwide published cases with clinical and genetic data were included (n = 40). We stratified the already published disease causing sequence variants as mild or severe with the use of in silico prediction programs, where possible and assessed the possible impact of the variants on the expression of the gene or function of the expressed protein. In a compound heterozygous situation the mildest sequence variant determined the genotype. Subsequently, clinical manifestations and outcomes of both groups were compared. RESULTS: Patients with a severe genotype showed a median survival of 15 months and had a lower probability of survival compared to patients with mild genotypes who were all alive at last reported follow-up (p = 0.0203, Log-rank test). DISCUSSION: The severity of the genotype assessed by in silico prediction and further classification explained survival in molybdenum cofactor deficiency and may therefore be considered a confounder for the outcome of therapeutic clinical trials requiring adjustment in the clinical trial design or analysis. These results should further be investigated by future in vitro or in vivo functional studies. Caution should be taken with this approach for the classification of variants in molecular genetic diagnostics or genetic counseling.