Genotype-phenotype Relationships Research Articles

A different approach to the evaluation of the genotype-phenotype relationship in biotinidase deficiency: repeated measurement of biotinidase enzyme activity.

In the present study, we aimed to evaluate the genotype-phenotype relation in patients with biotinidase enzyme deficiency based on repeated biotinidase enzyme measurements. The hospital file information of patients with biotinidase, enzyme deficiency was assessed retrospectively, and the relationship between the BTD gene mutations analysis results and biotinidase enzyme activity following the first and repeated enzyme activity assessments was analyzed. One-hundred-ten patients were included. In the first enzyme evaluation, profound biotinidase enzyme deficiency was identified in 15 (13.6 %), partial biotinidase enzyme deficiency in 63 (57.3 %), and heterozygous biotinidase enzyme deficiency in 32 (29.1 %) of the patients. The BTD genetic analysis revealed 42 (38.2 %) homozygous, 42 (38.2 %) heterozygous, and 26 (23.6 %) compound heterozygous variants. The most common homozygous variant, p.Asp444His, was evaluated with 130 repeated enzyme measurements and was consistent with a partial biotinidase enzyme deficiency in 55.4 % of cases, heterozygous biotinidase enzyme deficiency in 43.8 % of cases, and profound biotinidase enzyme deficiency in one (0.8 %) case. Clinical symptoms developed in 17 patients during follow-up, of which 70.6 % were related to neurodevelopment. The most common variant was homozygous p.Asp444His (29.4 %) among the patients who developed symptoms. This is the first study to date to evaluate thegenotype-phenotype relationship in patients with biotinidase deficiency through repeated measurements of biotinidase enzyme activity. The study reveals that biotinidase enzyme activity alone is inadequate for diagnosing biotinidase enzyme deficiency or evaluating disease severity, asgenetic investigations are also required for a definitive diagnosis of biotinidase enzyme deficiency.

Transcriptional linkage analysis with in vivo AAV-Perturb-seq.

The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.

CCNK Gene Deficiency Influences Neural Progenitor Cells Via Wnt5a Signaling in CCNK-Related Syndrome.

Rare variants of CCNK (cyclin K) give rise to a syndrome with intellectual disability. The purpose of this study was to describe the genotype-phenotype spectrum of CCNK-related syndrome and the underlying molecular mechanisms of pathogenesis. We identified a number of de novo CCNK variants in unrelated patients. We generated patient-induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs) as disease models. In addition, we constructed NPC-specific Ccnk knockout (KO) mice and performed molecular and morphological analyses. We identified 2 new patients harboring CCNK missense variants and followed-up 3 previous reported patients, which constitute the largest patient population analysis of the disease. We demonstrate that both the patient-derived NPC models and the Ccnk KO mouse displayed deficient NPC proliferation and enhanced apoptotic cell death. RNA sequencing analyses of these NPC models uncovered transcriptomic signatures unique to CCNK-related syndrome, revealing significant changes in genes, including WNT5A, critical for progenitor proliferation and cell death. Further, to confirm WNT5A's role, we conducted rescue experiments using NPC and mouse models. We found that a Wnt5a inhibitor significantly increased proliferation and reduced apoptosis in NPCs derived from patients with CCNK-related syndrome and NPCs in the developing cortex of Ccnk KO mice. We discussed the genotype-phenotype relationship of CCNK-related syndrome. Importantly, we demonstrated that CCNK plays critical roles in NPC proliferation and NPC apoptosis in vivo and in vitro. Together, our study highlights that Wnt5a may serve as a promising therapeutic target for the disease intervention. ANN NEUROL 2023;94:1136-1154.

Identification of genetic variants associated with a wide spectrum of phenotypes clinically diagnosed as Sanfilippo and Morquio syndromes using whole genome sequencing.

Mucopolysaccharidoses (MPSs) are inherited lysosomal storage disorders (LSDs). MPSs are caused by excessive accumulation of mucopolysaccharides due to missing or deficiency of enzymes required for the degradation of specific macromolecules. MPS I-IV, MPS VI, MPS VII, and MPS IX are sub-types of mucopolysaccharidoses. Among these, MPS III (also known as Sanfilippo) and MPS IV (Morquio) syndromes are lethal and prevalent sub-types. This study aimed to identify causal genetic variants in cases of MPS III and MPS IV and characterize genotype-phenotype relations in Pakistan. We performed clinical, biochemical and genetic analysis using Whole Genome Sequencing (WGS) in 14 Pakistani families affected with MPS III or MPS IV. Patients were classified into MPS III by history of aggressive behaviors, dementia, clear cornea and into MPS IV by short trunk, short stature, reversed ratio of upper segment to lower segment with a short upper segment. Data analysis and variant selections were made based on segregation analysis, examination of known MPS III and MPS IV genes, gene function, gene expression, the pathogenicity of variants based on ACMG guidelines and in silico analysis. In total, 58 individuals from 14 families were included in the present study. Six families were clinically diagnosed with MPS III and eight families with MPS IV. WGS revealed variants in MPS-associated genes including NAGLU, SGSH, GALNS, GNPTG as well as the genes VWA3B, BTD, and GNPTG which have not previously associated with MPS. One family had causal variants in both GALNS and BTD. Accurate and early diagnosis of MPS in children represents a helpful step for designing therapeutic strategies to protect different organs from permanent damage. In addition, pre-natal screening and identification of genetic etiology will facilitate genetic counselling of the affected families. Identification of novel causal MPS genes might help identifying new targeted therapies to treat LSDs.

The natural history, clinical outcomes, and genotype-phenotype relationship of otoferlin-related hearing loss: a systematic, quantitative literature review.

Congenital hearing loss affects one in 500 newborns. Sequence variations in OTOF, which encodes the calcium-binding protein otoferlin, are responsible for 1-8% of congenital, nonsyndromic hearing loss and are the leading cause of auditory neuropathy spectrum disorders. The natural history of otoferlin-related hearing loss, the relationship between OTOF genotype and hearing loss phenotype, and the outcomes of clinical practices in patients with this genetic disorder are incompletely understood because most analyses have reported on small numbers of cases with homogeneous OTOF genotypes. Here, we present the first systematic, quantitative literature review of otoferlin-related hearing loss, which analyzes patient-specific data from 422 individuals across 61 publications. While most patients display a typical phenotype of severe-to-profound hearing loss with prelingual onset, 10-15% of patients display atypical phenotypes, including mild-to-moderate, progressive, and temperature-sensitive hearing loss. Patients' phenotypic presentations appear to depend on their specific genotypes. For example, non-truncating variants located in and immediately downstream of the C2E calcium-binding domain are more likely to produce atypical phenotypes. Additionally, the prevalence of certain sequence variants and their associated phenotypes varies between populations due to evolutionary founder effects. Our analyses also suggest otoacoustic emissions are less common in older patients and those with two truncating OTOF variants. Critically, our review has implications for the application and limitations of clinical practices, including newborn hearing screenings, hearing aid trials, cochlear implants, and upcoming gene therapy clinical trials. We conclude by discussing the limitations of available research and recommendations for future studies on this genetic cause of hearing loss.

Molecular Genetic Analysis of Russian Patients with Coagulation Factor FVII Deficiency.

Coagulation factor VII (proconvertin) is one of the proteins starting the blood coagulation cascade. Plasma FVII concentration is regulated by different factors. A low level of FVII could also be a result of FVII deficiency (MIM# 227500), the rare autosomal recessive inherited disease caused by pathogenic variants in the F7 gene. The aim of this study was to describe a mutation spectrum of the F7 gene and genotype-phenotype relationship in patients with FVII deficiency in Russia for the first time. We studied the primary structure of the F7 gene of 54 unrelated patients with FVII deficiency by direct Sanger sequencing. Pathogenic variants in the F7 gene were detected in 37 (68.5%) of them. We identified 24 different mutations located mostly in the serine protease domain. Five pathogenic variants had never been reported before. A major mutation in the Russian population was c.1391delC (p. Pro464Hisfs*32), linked with rs36209567 and rs6046 functional polymorphisms, that is widely distributed in East Europe. As in other countries, the F7 genotypes poorly correlated with the severity of clinical manifestations but were quite well associated with FVII levels. Minor alleles of functional polymorphisms rs510335, rs5742910, rs561241, rs36209567, and rs6046 could also participate in the F7 genotype and influence FVII levels.

Moderating effects of impulsivity and morning cortisol on the genotype-phenotype relationship of attention deficit hyperactivity disorder in young adults.

Dysregulation of the morning cortisol response in young adults with attention deficit hyperactivity disorder (ADHD) has been shown to underlie several of the alterations present in their lives. Thus, the interaction of this mechanism with genetic and behavioural characteristics could explain a large proportion of the aetiology of ADHD in this population. For these reasons, the present study explores the associations of 30 single nucleotide polymorphisms (SNPs) previously identified as significant (after correction for multiple comparisons) in the aetiology of ADHD with an assessment of morning cortisol and impulsivity traits in a group of 120 adults aged 18-24years. Participants were recruited through private centres of neuropsychology and psychiatry, as well as through events in local universities. Morning cortisol within 30min of awakening and motor impulsivity traits were shown to moderate the effect of SNP rs10129500 on the severity of the symptoms of ADHD measured by the Adult Self-Report Scale. This variant associated with cortisol-binding globulin would explain the low concentrations of this hormone found in young adults with high symptoms of ADHD, which is accentuated when there are high levels of impulsivity. The proposed model allows for transferring the theoretical relationships between the dimensions that explain the aetiology of ADHD to an applied exploratory model with good performance.

Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

Pediatric Cardiomyopathies: Establishing Genotype-Phenotype Relationship as a Determinant in Prognosis and Therapy: A Review of the Literature

Pediatric Cardiomyopathies: Establishing Genotype-Phenotype Relationship as a Determinant in Prognosis and Therapy: A Review of the Literature

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

Cognitive-Behavioral Profile in Pediatric Patients with Syndrome 5p-; Genotype-Phenotype Correlationships.

(1) Background: 5p minus Syndrome (S5p-) is a neurodevelopmental disorder caused by a deletion in the short arm of chromosome 5. Among the phenotypic characteristics of S5p-, the most characteristic and representative element is a monochromatic cry with a high-pitched tone reminiscent of a cat's meow. Individuals may also show great phenotypic heterogeneity and great genetic variability. Regarding cognitive-behavioral aspects of the syndrome, the studies are scarce and do not establish a general profile of the main cognitive-behavioral particularities that this syndrome presents. The main objective of this work was to describe the development profile of a cohort of 45 children with 5p minus Syndrome, concerning the biomedical, genetic, cognitive, and behavioral aspects. Establishing putative genotype-phenotype (cognitive-behavioral profiles) relationships in our cohort, from an interdisciplinary approach. (2) Methods: A selection of instruments of measures was selected for neuropsychological assessment (3) Results: In general, children with S5p- have a higher cognitive level than a communicative and motor level. Language difficulties, especially expressive ones, influence the frequency and severity of the most frequent behavioral problems in S5p. The most significant problem behavior of children with S5p-, especially girls, is self-harm. Compulsive behavior, limited preferences, and interest in monotony are significantly more frequent in subjects with better cognitive levels. We also find a significant correlation between the size of the loss of genetic material on 5p and the cognitive level of the subjects. (4) Conclusions: We described for the first time, the cognitive-behavioral profile of a cohort of minors with S5p-. Remarkably, it was found that language, especially of an expressive nature, modulates the most frequent behavioral aspects in subjects with lower cognitive levels, so it is essential to develop verbal or alternative communication strategies adjusted to these individuals.

The genotype–phenotype relationship and evolutionary genetics in the light of the Metabolic Control Analysis

Metabolic control analysis has long been used as a systemic model of the genotype–phenotype (GP) relationship. By considering kinetic parameters and enzyme concentrations as reflecting the genotype level and metabolic fluxes or pools as phenotypes related to fitness, MCA has given a biological basis to the relationship between these two levels. The non-linear and concave relationship between enzymes and fluxes can account for common genetic effects that reductionist approaches have been powerless to explain, such as the dominance of active alleles over less active alleles, the various types of epistasis and heterosis, and reveals the structural links between these genetic effects. The summation property of the flux control coefficients accounts for the L-shaped distribution of Quantitative Trait Locus (QTL) effects, irrespective of other possible causes. Metabolic models of response to selection results in evolutionary scenarios that are markedly different from those derived from the classical infinitesimal model of quantitative genetics. In particular, evolution towards selective neutrality appears to be a consequence of the diminishing return of the flux-enzyme relationship. In this paper, we survey the historical and recent achievements of MCA in genetics, quantitative genetics and evolution, focusing on epistasis and the evolution of flux in relation to enzyme concentrations.

Frameshift coding sequence variants in the LPL gene: identification of two novel events and exploration of the genotype–phenotype relationship for variants reported to date

BackgroundLipoprotein lipase (LPL) is the rate-limiting enzyme for triglyceride hydrolysis. Homozygous or compound heterozygous LPL variants cause autosomal recessive familial chylomicronemia syndrome (FCS), whereas simple heterozygous LPL variants are associated with hypertriglyceridemia (HTG) and HTG-related disorders. LPL frameshift coding sequence variants usually cause complete functional loss of the affected allele, thereby allowing exploration of the impact of different levels of LPL function in human disease.MethodsAll exons and flanking intronic regions of LPL were Sanger sequenced in patients with HTG-related acute pancreatitis (HTG-AP) or HTG-AP in pregnancy. Previously reported LPL frameshift coding sequence variants were collated from the Human Gene Mutation Database and through PubMed keyword searching. Original reports were manually evaluated for the following information: zygosity status of the variant, plasma LPL activity of the variant carrier, disease referred for genetic analysis, patient’s age at genetic analysis, and patient’s disease history. SpliceAI was employed to predict the potential impact of collated variants on splicing.ResultsTwo novel rare variants were identified, and 53 known LPL frameshift coding sequence variants were collated. Of the 51 variants informative for zygosity, 30 were simple heterozygotes, 12 were homozygotes, and 9 were compound heterozygotes. Careful evaluation of the 55 variants with respect to their clinical and genetic data generated several interesting findings. First, we conclude that 6–7% residual LPL function could significantly delay the age of onset of FCS and reduce the prevalence of FCS-associated syndromes. Second, whereas a large majority of LPL frameshift coding sequence variants completely disrupt gene function through their "frameshift" nature, a small fraction of these variants may act wholly or partly as "in-frame" variants, leading to the generation of protein products with some residual LPL function. Third, we identified two candidate LPL frameshift coding sequence variants that may retain residual function based on genotype–phenotype correlation or SpliceAI-predicted data.ConclusionsThis study reported two novel LPL variants and yielded new insights into the genotype–phenotype relationship as it pertains to LPL frameshift coding sequence variants.

HCN1 epilepsy: From genetics and mechanisms to precision therapies.

Pathogenic variation in HCN1 is now an established cause of epilepsy and intellectual disability. Variation in HCN1 causes a spectrum of disease with a genotype-phenotype relationship emerging. De novo pathogenic variants that occur in the transmembrane domains of the channel typically cause a cation 'leak' that associates with severe developmental and epileptic encephalopathy (DEE). Genotype-phenotype associations for variants that fall outside of the transmembrane domains are less well established but do include milder forms of epilepsy that can be either de novo or inherited. HCN1 DEE mouse models have been generated which recapitulate the seizures and learning difficulties seen in human patients. These mice have also acted as powerful preclinical models which share pharmacoresponsiveness with human HCN1 DEE patients. Data from these mouse models support the conclusion that anti-seizure medications with sodium channel block as their primary mechanism of action should be used with caution in HCN1 DEE. Other comorbidities of HCN1 DEE including retinal dysfunction have also been modelled in HCN1 DEE mice, suggesting HCN1 variants can cause a dramatically reduced sensitivity to light with limited ability to process temporal information. Our understanding of the genetics and pathophysiological mechanisms underlying HCN1 epilepsy has progressed significantly and is already influencing therapy. However, more research effort is needed to fully understand the natural histories of HCN1 epilepsies and to develop precision therapeutic approaches.

Abstract P1096: Computational Motion Artifact Removal Acts As Virtual Blebbistatin In Ipsc-derived Cardiomyocytes And Micro-tissues

Introduction: Drugs like blebbistatin that paralyze tissues are critical for accurate optocardiography but directly affect sarcomeres, making it challenging to study structure-function relationships. To overcome this, we applied Direct Deformation Estimation (DDE), a robust strain estimation tool, to enable computational motion removal from videos of beating monolayers and engineered heart tissues formed from human Induced Pluripotent Stem Cell derived cardiomyocytes (hiPSC-CM). Methods: hiPSC-CM and miniaturized engineered heart muscles were stained with the far-red voltage sensitive dye, BeRST-1. Videos of overall motion (bright-field), Ca 2+ (endogenous GCamP6f reporter) and voltage were captured at 100 Hz. Temporal median filtering and Contrast-Limited Adaptive Histogram Equalization were applied as pre-processing steps to improve convergence. The inverse of conformal mappings estimated from DDE were used to “unwarp” videos to artificially reduce motion. Removal of motion was assessed in bright-field videos by performing optical flow analysis. Improvements in optical mapping accuracy were estimated by calculating the spatial distribution of APD 80 (voltage) and time to 75% decay from peak (τ 75 ; Ca 2+ ). Results: Virtual blebbistatin removed >90% of motion from monolayers and engineered tissues without affecting sarcomere architecture (sarcomeric α-Actinin staining), in contrast to chemical blebbistatin. Virtual blebbistatin further reduced spatial dispersion in both APD 80 and τ 75, leading to overall distributions that were less skewed by artificially low repolarization times. Interestingly, when directly assessing Ca 2+ -contraction coupling in plots of motion-corrected Ca 2+ vs. contraction magnitude, we observed that engineered tissues had more uniform and consistent coupling compared to monolayers. Conclusions: Superior uniformity in calcium-contraction coupling of engineered tissues as opposed to monolayers is consistent with observations that engineered tissues are more likely to recapitulate genotype-phenotype relationships and drug responsiveness. Virtual blebbistatin is a promising tool for non-invasive analysis of Ca 2+ -contraction coupling and robust optocardiography.

Abstract P1118: Engineering A Patient-specific Heart On A Chip Model Of Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is the most expensive congenital cardiac disease and is characterized by underdevelopment of the left side of the heart. The surgical intervention leads to the developmentally disadvantaged right ventricle performing systemic circulation. As such, a subset of HLHS patients develops progressive heart failure that results in limited transplant-free survival. There are no animal models to study the molecular and functional properties of HLHS cardiomyocytes, limiting our understanding of disease mechanisms and human genotype-phenotype relationships. To address this, we are creating a physiologically and functionally relevant ”heart on a chip” model of HLHS hiPSC-derived cardiomyocytes (CMs). Induced Pluripotent Stem Cells (iPSCs) were derived from cord blood mononuclear cells isolated from the umbilical cord blood from 8 antenatally diagnosed HLHS children at birth. Control (iPSCs) from 3 healthy subjects were derived from peripheral blood mononuclear cells. We then differentiated ventricular CMs from these iPSCs using StemDiff ventricular CM kit. We observed HLHS iPSCs formed ~50% fewer CMs compared to healthy controls. We studied calcium handling by iPSC-CMs with Fluo-4 fluorescent calcium dye in HLHS and Healthy iPSC-CMs. We observed that calcium transients in HLHS-CMs have half the beat rate, 1.4 times longer rise time to rise, 1.5 times higher time constant (Tau), compared to Healthy CMs, suggesting that HLHS CMs have dysfunctional calcium handling compared to healthy CMs. Our results demonstrate that in an in vitro model of iPSC-derived CM, HLHS patient CMs demonstrate reduced formation and defective calcium handling, which may potentially explain the pathogenesis of heart failure in HLHS patients. In ongoing experiments, we are evaluating diastolic and systolic stress generation in these cells. We will also establish the underlying molecular mechanisms of functional defects in HLHS CMs and screen potential drug therapies for single-ventricle patients.

Graph embedding-based link prediction for literature-based discovery in Alzheimer’s Disease

Objective:We explore the framing of literature-based discovery (LBD) as link prediction and graph embedding learning, with Alzheimer’s Disease (AD) as our focus disease context. The key link prediction setting of prediction window length is specifically examined in the context of a time-sliced evaluation methodology. Methods:We propose a four-stage approach to explore literature-based discovery for Alzheimer’s Disease, creating and analyzing a knowledge graph tailored to the AD context, and predicting and evaluating new knowledge based on time-sliced link prediction. The first stage is to collect an AD-specific corpus. The second stage involves constructing an AD knowledge graph with identified AD-specific concepts and relations from the corpus. In the third stage, 20 pairs of training and testing datasets are constructed with the time-slicing methodology. Finally, we infer new knowledge with graph embedding-based link prediction methods. We compare different link prediction methods in this context. The impact of limiting prediction evaluation of LBD models in the context of short-term and longer-term knowledge evolution for Alzheimer’s Disease is assessed. Results:We constructed an AD corpus of over 16 k papers published in 1977–2021, and automatically annotated it with concepts and relations covering 11 AD-specific semantic entity types. The knowledge graph of Alzheimer’s Disease derived from this resource consisted of ∼11 k nodes and ∼394 k edges, among which 34% were genotype-phenotype relationships, 57% were genotype-genotype relationships, and 9% were phenotype-phenotype relationships. A Structural Deep Network Embedding (SDNE) model consistently showed the best performance in terms of returning the most confident set of link predictions as time progresses over 20 years. A huge improvement in model performance was observed when changing the link prediction evaluation setting to consider a more distant future, reflecting the time required for knowledge accumulation. Conclusion:Neural network graph-embedding link prediction methods show promise for the literature-based discovery context, although the prediction setting is extremely challenging, with graph densities of less than 1%. Varying prediction window length on the time-sliced evaluation methodology leads to hugely different results and interpretations of LBD studies. Our approach can be generalized to enable knowledge discovery for other diseases. Availability:Code, AD ontology, and data are available at https://github.com/READ-BioMed/readbiomed-lbd.

First Three Years’ Experience of Mucopolysaccharidosis Type-I Newborn Screening in California

To report on the first 3years of mucopolysaccharidosis type I (MPS I) newborn screening (NBS) in the large and diverse state of California. The California Genetic Disease Screening Program began universal NBS for MPS I on August 29, 2018. The screening uses a 2-tiered approach: an α-L-iduronidase (IDUA) enzyme activity assay followed by DNA sequencing for variants in the IDUA gene. As of August 29, 2021, 1 295 515 California newborns were screened for MPS I. In tier 1 of screening, 329 (0.025%) had an IDUA enzyme measurement below the cutoff and underwent tier-2 IDUA DNA sequencing. After tier 2, 146 (0.011%) newborns were screen positive, all of whom were referred to a metabolic Special Care Center for follow-up. After long-term follow-up, 7 cases were resolved as severe MPS I (Hurler syndrome) and 2 cases as attenuated MPS I for an MPS I birth prevalence of 1/143 946. DNA sequencing identified 107 unique IDUA variants among a total of 524 variants; 65% were known pseudodeficiency alleles, 25% were variants of uncertain significance, and 10% were pathogenic variants. As a result of a 2-tiered NBS approach, 7 newborns diagnosed with Hurler syndrome had received early treatment for MPS I. Continuation of California's long-term follow-up program will be crucial for further understanding the complex genotype-phenotype relationships of MPS I.

Clinic Heterogeneity and Management of Pediatric Patients With Germline RET Proto-oncogene Mutation: Single-center Experience.

Inherited forms of medullary thyroid carcinoma (MTC) can cause serious problems in diagnosis and follow-up. Family screening is performed, and prophylactic thyroidectomy at an appropriate age can be life-saving. This study aimed to investigate the diagnostic, clinical, laboratory characteristics, and treatment methods of cases with rearranged during transfection ( RET) mutation in the childhood age group. Patients diagnosed with hereditary MTC and patients who were evaluated by detecting MTC and/or RET mutations in their families were included in this study. Nine cases from 6 families were included in the study. Seven patients were evaluated as a result of screening, whereas 2 patients, one of whom was MEN2B, were symptomatic. Prophylactic thyroidectomy was performed in 7 cases. Medullary microcarcinoma was found in all, and additional papillary thyroid carcinoma in one. An inoperable tumor was detected in one patient, and sorafenib treatment was applied. A very heterogeneous clinical presentation can be seen in a group of pediatric patients with RET mutation. In rare RET mutations, the genotype-phenotype relationship is still unclear, and different clinical pictures can be seen. Although prophylactic thyroidectomy is life-saving, it can cause iatrogenic hypothyroidism and hypoparathyroidism. Concomitant papillary microcarcinomas may occur in very young children with germline RET mutation.

Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria

Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.