Variable Phenotype Research Articles

Analysis of perrault syndrome caused by pathogenic variants in LARS2 and HARS2 genes

Objective: To investigate the molecular etiology of Perrault syndrome by analyzing the clinical phenotype and pathogenic gene variants of 2 male patients with bilateral severe sensorineural deafness. Methods: Two male patients with Perrault syndrome characterized by severe sensonrineual deafness adimitted to the First Affiliated Hospital of Zhengzhou University between February 2021 and March 2022 were selected, and the clinical phenotype and pathogenic gene variants of them and their family members were summarized. The whole exome sequencing technology was used to screen the pathogenic variants of the probands, and the candidate variants were determined by combining with clinical phenotype. The probands and their family members were verified by the Sanger sequencing method. Results: The whole exome sequencing results showed that the proband of family 1 had a compound heterozygous variants of the LARS2 (NM_015340.4) gene c.1565C>A (p.Thr522Asn) and c.1079T>C (p.Ile360Thr). The reported pathogenic variant c.1565C>A came from the mother, and the novel variant c.1079T>C came from the father. The second proband harbored compound heterozygous variants of HARS2 gene (NM_012208.4) c.1273C>T (p.Arg425Trp) and c.1403G>C (p.Gly468Ala), with the former from the proband's mother, the latter from the father. The c.1273C>T was novel and c.1403G>C was the reported pathogenic variant. All above variants were respectively classified as pathogenic, uncertain significance, uncertain significance and likely pathogenic based on the ACMG guidelines. Conclusion: This study expands the mutational spectrum of LARS2 and HARS2 genes, which highlights that genetic testing plays an important role in the early diagnosis of syndromic deafness.

Mercurial risk from dental amalgam use in a population of Moroccan dentists: A latent class regression approach.

Dentists using dental amalgam are chronically exposed to low doses of elemental mercury. The complex toxico-kinetics of this systemic toxicant results in polymorphic and variable clinical phenotypes. In this context, adapted statistical methods are required to highlight potential adverse effects of occupational mercury exposure on dentists' health. The present study aims to analyze the distribution of self-reported subjective symptoms, commonly associated with chronic mercury poisoning, according to occupational mercury exposure in a population of Moroccan liberal dentists. In order to achieve the defined objectives, a three-step latent class regression was fitted. First a latent class analysis was performed to cluster the studied population according to their declared symptoms. Dentists were then classified in the defined latent classes based on their posterior probabilities. Finally, a logistic regression is fitted to identify predictors associated with latent classes' membership. The final obtained model showed acceptable calibration and discrimination. Its interpretation revealed that the increase of the frequency of amalgam use was associated with significant higher odds of belonging to the high risk latent class. The present study represents an initial step towards the development of diagnosis model that predict clinical profiles according to occupational mercury exposure.

High phenotypic variation found within the offspring of each mother tree in Fagus sylvatica regardless of the environment or source population

AbstractAimClimate change challenges temperate forest trees by increasingly irregular precipitation and rising temperatures. Due to long generation cycles, trees cannot quickly adapt genetically. Hence, the persistence of tree populations in the face of ongoing climate change depends largely on phenotypic variation, that is the capability of a genotype to express variable phenotypes under different environmental conditions, known as plasticity. We aimed to quantify phenotypic variation of central Europe's naturally dominant forest tree across various intraspecific scales (individuals, mother trees (families), populations) to evaluate its potential to respond to changing climatic conditions.LocationEurope.Time Period2016–2019.Major Taxa StudiedEuropean beech (Fagus sylvatica L.).MethodsWe conducted a fully reciprocal transplantation experiment with more than 9000 beech seeds from seven populations across a Europe‐wide gradient. We compared morphological (Specific Leaf Area), phenological (leaf unfolding) and fitness‐related (growth, survival) traits across various biological scales: within single mother trees, within populations and across different populations under the contrasting climates of the translocation sites.ResultsThe experiment revealed significant phenotypic variation within the offspring of each mother tree, regardless of geographic origin. Initially, seedling height growth varied among mother trees and populations, likely due to maternal effects. However, the growth performance successively aligned after the first year. In summary, we observed a consistent growth response in different beech populations to diverse environments after initial maternal effects.Main ConclusionsThe study strikingly demonstrates the importance of considering intraspecific variation. Given the surprisingly broad spectrum of phenotypes each mother tree holds within its juvenile offspring, we conclude that Fagus sylvatica might have the potential for medium‐term population persistence in face of climate change, provided that this pattern persists into later life stages. Hence, we also suggest further investigating the inclusion of passive adaptation and natural dynamics in the adaptive management of forests.

Prediction of molecular phenotypes for novel SCN1A variants from a Turkish genetic epilepsy syndromes cohort and report of two new patients with recessive Dravet syndrome.

Dravet syndrome and genetic epilepsy with febrile seizures plus (GEFS+) are both epilepsy syndromes that can be attributed to deleterious mutations occurring in SCN1A, the gene encoding the pore-forming α-subunit of the NaV 1.1 voltage-gated sodium channel predominantly expressed in the central nervous system. In this research endeavor, our goal is to expand our prior cohort of Turkish patients affected by SCN1A-positive genetic epilepsy disorders. This will be accomplished by incorporating two recently discovered and infrequent index cases who possess a novel biallelic (homozygous) SCN1A missense variant, namely E158G, associated with Dravet syndrome. Furthermore, our intention is to use computational techniques to predict the molecular phenotypes of each distinct SCN1A variant that has been detected to date within our center. The correlation between genotype and phenotype in Dravet syndrome/GEFS+ is intricate and necessitates meticulous clinical investigation as well as advanced scientific exploration. Broadened mechanistic and structural insights into NaV 1.1 dysfunction offer significant promise in facilitating the development of targeted and effective therapies, which will ultimately enhance clinical outcomes in the treatment of epilepsy.

Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells

Epidermolysis Bullosa Simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most of EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient’s keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon1. Through the CRISPR/Cas9 system we performed a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Prognostic Value of Genotype-Phenotype Correlations in X-Linked Myotubular Myopathy and the Use of the Face2Gene Application as an Effective Non-Invasive Diagnostic Tool.

X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy resulting from dysfunction of the protein myotubularin encoded by the MTM1 gene. XLMTM has a high neonatal and infantile mortality rate due to a severe myopathic phenotype and respiratory failure. However, in a minority of XLMTM cases, patients present with milder phenotypes and achieve ambulation and adulthood. Notable facial dysmorphia is also present. We investigated the genotype-phenotype correlations in newly diagnosed XLMTM patients in a patients' cohort (previously published data plus three novel variants, n = 414). Based on the facial gestalt difference between XLMTM patients and unaffected controls, we investigated the use of the Face2Gene application. Significant associations between severe phenotype and truncating variants (p < 0.001), frameshift variants (p < 0.001), nonsense variants (p = 0.006), and in/del variants (p = 0.036) were present. Missense variants were significantly associated with the mild and moderate phenotype (p < 0.001). The Face2Gene application showed a significant difference between XLMTM patients and unaffected controls (p = 0.001). Using genotype-phenotype correlations could predict the disease course in most XLMTM patients, but still with limitations. The Face2Gene application seems to be a practical, non-invasive diagnostic approach in XLMTM using the correct algorithm.

Patients with Gaucher Disease Type 1 and Their Outcome: A Case Series

Background: Gaucher disease is the most common lysosomal storage disease. Defective activity of the acid β-glucosidose which is encoded by the GBA1 gene, leads to the accumulation of excess amounts of the glucosyl ceramide lipid. Gaucher disease is clinically classified into three variants based on the relative degree and progression of neurological involvement. Gaucher disease type 1 manifests markedly variable phenotypes ranging from asymptomatic individuals to children who have massive hepatosplenomegaly, pancytopenia, and severe skeletal abnormalities.&#x0D; Case Report: We report a case series of four patients with Gaucher disease type 1 with variable clinical manifestations treated with enzyme replacement therapy (ERT). In follow-up, three patients showed visceral and hematological improvements, while one case did not respond to ERT and died due to liver failure.&#x0D; Conclusion: Gaucher disease should be considered with differential diagnosis of patients with unexplained organomegaly, who bruise easily and have bone pain which can be treated with ERT.

Genotype Phenotype characterisation, Imaging profile and novel genetic mutations from a large cohort of Frontotemporal Dementia from India

AbstractBackgroundDegenerative dementia like Frontotemporal dementia (FTD) are complex heterogeneous disorders with variable phenotypes, patterns of atrophy and pathogenicity. Advanced genetic studies from India are sparse and earlier techniques mostly reported negative results. Current study aims to evaluate the clinical, radiological and genetic profile of FTD/FTD overlap syndromes, to perform whole exome sequencing and to explore genotype phenotype correlations.MethodSubjects with dementia fulfilling standard criteria’s for FTD/FTD Overlap and AD were recruited from Dept. of Neurology, NIMHANS, India Demography, pedigree analysis, clinical, cognitive and neuropsychological evaluation and blood sampling for genetic analysis by next generation sequencing and MRI as per ADNI protocol were performed. RT‐PCR was done for analysing c9orf.Results133 FTD/FTD overlap were recruited. Mean age at onset was 56 ± 9.8 years in FTD There were 87 men and 75 women. Family history was documented in 43%. 42% had family history in first degree relatives, 11% had history in second degree and 4% in third degree relatives. Mean ACE scores were 43.8± 24.7. Frontotemporal atrophy was noted in 90% in MRI (n = 115). 41% had Grade 1 (mild) global cortical atrophy score .MRI also revealed characteristic features like diffusion restriction, white matter lesions (CSF1R) basal ganglia calcification with bone cysts(TYROBP) and Corpus callosal atrophy(TREM2)11 novel gene mutations including one each of CSF1R, TREM2, TYROBP, SQSTM1, GRN, MAPT, PSEN2 and four cases of C9orf were detected (Pathogenic in 11 and Variant of unknown significance in 26), out of the 100 samples sequenced and analysed (ACMG guidelines). VUS like GBA, PANK2, PLAG26, PRNP, LRRK2, DCNT etc. were also identified. Sanger validation confirmed the pathogenic mutations. Novel variants of CSF1R, TREM2 and TYROBP, primary microgliopathy as causative genes are being first time described from India. SQSTM1 presented as bvFTD without ALS overlap, GRN as non‐fluent aphasia, MAPT as bvFTD and PSEN2 as non‐fluent PPA and two patients with C9orf presented as bvFTD and two others as FTD‐ALS.ConclusionGenotype‐phenotype correlations along with neuroimaging and pedigree analysis has aided in early diagnosis, prognostication and novel and unique genetics causes shedding light on underlying FTD mechanism from our cohort.

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function or gain-of-function properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12) and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for loss-of-function and gain-of-function variants. Gain-of-function variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age: 1 month), hypertonic and more often had movement disorders, including hyperekplexia. Patients with loss-of-function variants were older at the time of seizure onset (median age: 16 months), hypotonic and had sleeping disturbances. Loss-of-function and gain-of-function variants were disease-causing in both sexes but affected males often carried de novo or hemizygous loss-of-function variants inherited from healthy mothers, whereas affected females had mostly de novo heterozygous gain-of-function variants.

Biomarkers of Alzheimer’s disease pathology in atypical non‐amnestic clinical phenotypes

AbstractBackgroundAlzheimer’s disease (AD) is most commonly presented with memory loss features. However, there are atypical clinical phenotypes including predominant visual, language, executive, behavioural, or motor dysfunction. The goal of our study was to estimate the prevalence of Alzheimer’s pathology in non‐amnestic clinical variants.MethodWe examined 40 patients (17 males, 23 females, 66.1±8.3 yrs) with atypical phenotypes of AD (Table 1). Seven patients had the phenotype of logopenic variant of primary progressive aphasia (lvPPA), 3 patients had the diagnosis of posterior cortical atrophy (PCA), 19 had corticobasal syndrome (CBS), and 11 cases met research criteria of clinical behavioral variant of AD (bvAD). All patients underwent lumbar puncture. CSF biomarkers Abeta‐42 (cutoff 150 pg/mL) and p‐tau181 (cutoff 600 pg/mL) were analyzed. According to the recommendations of International Working Group (2021) we defined probable and possible AD based on clinical phenotype and studied CSF biomarkers.ResultThe overall prevalence of AD pathology in the studied groups was 42.5% (20% for probable diagnosis and 22.5% for possible). All results are presented in Table 1. The highest prevalence of AD pathology in atypical clinical phenotypes was found in the group of PCA (66.7%), the lowest ‐ in the group of bvAD (36.4%). Almost a half of patients in both lvPPA and CBS groups had AD pathology based on biomarkers – 42.9% and 42.2%, respectively.ConclusionThe results showed the importance of verifying AD pathology biomarkers in common (PCA, lvPPA) and uncommon (CBS) phenotypes. Behavioral variant, considered as a rare AD phenotype, in our studied cohort was characterized by relatively high occurrence of positive AD biomarkers. Increasing the use of AD biomarkers in clinical practice and greater recognition of atypical phenotypes can ensure early diagnosis, treatment and care for these patients.

Evolutionary cell biology: New roles for Arp2/3 complex evolution in eukaryotic diversification

The actin cytoskeleton is a protein polymer system that underlies a wide variety of eukaryotic phenotypes. A new study reports that diversity in a key actin regulator, the Arp2/3 complex, drives species-specific sperm development within the Drosophila lineage.

Role of miR‐802 in brain insulin signaling and its impact on Down syndrome

AbstractBackgroundDown syndrome (DS) individuals are characterized by a variety of pathological phenotypes that manifest with wide variability in the different tissues. At the level of the central nervous system, the accelerated aging phenotype is associated with the risk to develop Alzheimer‐like dementia. A central aspect of neurodegeneration is the close association between metabolic disorders and cognitive decline. Multiple studies have suggested a link between metabolic disorder and microRNAs (miR), small non‐coding RNAs acting as post‐transcriptional regulators of a plethora of genes. Among triplicated miRNAs on chromosome 21, we focus on miR‐802 because recent studies demonstrated its association with development on insulin resistance in obesity and diabetes. Considering these findings and based on the “gene dosage hypothesis” of DS, the goal of the study is to decipher how miR‐802 may contribute to aberrant insulin signaling (IS) and, in parallel to the risk to develop dementia early in life in DS. MethodThe miR‐802 expression, protein levels and activation state of main components of the IS were evaluated (i) in the brain of autoptic cases from DS, DSAD and age‐matched controls and (ii) in the brain of euploid and Ts65Dn mice (a model of DS). Further, using bioinformatic tools we identified miR‐802 predicted target genes that are involved in the IS (PTEN and GSK‐3β).ResultThe IS alterations worsen in the transition from DS to DS/AD and similar findings were collected inTs65Dn mice where IS dysregulation persists with aging where neurodegeneration becomes significant. Intriguingly, these latter changes were driven by the over‐expression of miR‐802, which negatively regulates PTEN and GSK‐3β mRNA in the brain.ConclusionIn this picture, the identification of specific targets modulated by miR‐802 and involved in IS pathway, will provide molecular basis to develop novel therapeutic strategies to prevent/delay the onset of brain insulin resistance in DS.

Cardio-Facio-Cutaneous Syndrome with an Unusual Presentation of Respiratory Failure Requiring Tracheostomy and Long-term Ventilation

Cardio-facio-cutaenous (CFC) syndrome is a phenotypically diverse disorder that is caused by mutations in the RAS/MEK/ERK signaling pathway, sharing many clinical similarities with Noonan and Costello syndromes. Classic findings of CFC include congenital cardiac anomalies, dysmorphic facial features, dermatological manifestations, and other multisystemic abnormalities. Laryngomalacia, generalized muscle weakness, and hypotonia have been reported phenotypical variants of the disorder. We describe a case of a male newborn with a genetic diagnosis of CFC syndrome who presented with respiratory failure requiring tracheostomy placement and chronic ventilation

Echocardiographic Phenotypes of Subclinical Organ Damage: Clinical and Prognostic Value in the General Population. Findings from the Pamela Study.

Subclinical alterations in cardiac structure and function include a variety of abnormal phenotypes of established adverse prognostic significance such as left ventricular hypertrophy (LVH), alterations of LV geometry, left atrial (LA) enlargement, and aortic root (AR) dilatation. The excess cardiovascular (CV) risk associated with these phenotypes has been consistently demonstrated in different clinical settings such in patients with systemic hypertension, coronary heart disease, diabetes mellitus, chronic kidney disease, heart failure and in geneal population samples. The Pressioni Monitorate e Loro Associazioni (PAMELA), a longitudinal population-based study originally designed to assess the normality values, prognostic significance of office, home and 24-hour blood pressure, including among the many clinical and laboratory variables the collection of echocardiographic data, allowed to gather important information on the clinical prognostic significance of subclinical cardiac damage during a long follow-up period. This article summarizes the original findings provided by the PAMELA study on the clinical correlates and prognostic significance of echocardiographic markers of subclinical organa damage namely LVH, left atrial enlargement (LA) and AR dilatation at the community level.

Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity

BackgroundBiallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship.MethodsUsing an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity.ResultsA cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy.ConclusionsBased on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as “OGDHL-related disorders”.

Investigating Health Literacy in Thalassemia: Founding a Patient-Led Research Approach

Background: Thalassemia is a hereditary hemolytic anemia (HHA) with a variable phenotype and clinical severity, and constantly changing demographic profile. Despite advances in management, there are many unmet needs in this diverse population, with needs varying among different groups. The Thalassemia Advocacy Advisory Council (Thal AAC) was founded to define and better understand the unmet needs of the thalassemia patient community. This ten-person group consists of patients, caregivers, advocates, and healthcare professionals (HCPs) from six countries (USA N=5, Cyprus N=1, Italy N=1, Greece N=1, Taiwan N=1, Brazil N=1). We describe how the Thal AAC sought to better define the global community's unmet needs following a multi-step process of collecting, reviewing, and prioritizing the unmet needs experienced by patients with thalassemia (Figure). The process was coordinated, moderated, and supported by Agios Pharmaceuticals. Method: Thal AAC members shared their personal perspective on community unmet needs through a qualitative survey, which included open-ended questions aiming to collect each member's perspective on the top three unmet needs for patients and HCPs in their region and globally. Results were consolidated and reviewed by the AAC in a series of workshops. Two key unmet needs were highlighted: Among patients, there is a lack of understanding of the disease and both long- and short-term complications, which can lead to poor adherence to standard of careAmongst HCPs, improved education and advocacy are needed to support patient access to care, including innovative therapy options and research Based on these findings, the group aligned on the need for better education and understanding of the disease from a patient and HCP perspective, with health literacy (HL) being the central theme that tied these points together. With the goal of broadening understanding of patient HL, the Thal AAC defined four key sub-topics to investigate further: Patients' knowledge of the diseaseChallenges faced by patients in improving their HLPossible motivators for improving HLChannels and formats through which patients seek disease information. A non-exhaustive evidence audit of English-language literature published in the last 10 years on HL in thalassemia was conducted within PubMed and Cochrane databases using key search terms targeting thalassemia, similar hereditary hemolytic diseases, and HL. The published literature, was supplemented with patient advocacy group knowledge and data from other community-based research. Results: The audit showed that there is limited published literature or evidence for HL in the context of thalassemia and related hemolytic anemias (N=58). The format and delivery of information emerged as an important research topic in the context of rare diseases generally, but not thalassemia specifically. Although barriers and challenges to improving HL were studied in the literature (N=9), thalassemia-related items make up only ~10% (N=1), and there is relatively no research into the motivators for improving HL related to disease and outcomes. Thus, patients and their HCPs share unmet informational and educational needs, particularly regarding the long-term implications of inadequate treatment and care. Conclusion: The findings of the audit indicate a clear gap in thalassemia HL research. This gap may create scenarios in which not every patient with thalassemia accesses optimal care, which may have consequences for their long-term health. This provided the impetus for the AAC to launch a community-based, multi-national patient advocacy research initiative, comprising of a global survey and follow-up qualitative interviews, to better understand the community's perspective on HL in thalassemia. Insights from this survey will be used to share learnings and calls to action with community stakeholders, with an aim of contributing to the improvement of thalassemia patient outcomes and care. Thus, the AAC's findings have led to new insights which, in turn, have formed the foundation of a community-based research project.

M41 and Non-M41 UBA1 Variants Differ in Their Alteration of Hematopoiesis

Background: UBA1 variants have recently been described as causative to the somatic hematoinflammatory disease VEXAS Syndrome. Previously, the features of M41 and non-M41 variants regarding their diagnoses and inflammatory transcriptome in comparison to other patients (pts) diagnosed with hematological malignancies (HM) have been described ( Leukemia 37, 1080-1091, 2023). Given that UBA1 impairment also affects hematopoiesis with no evidence of malignancy, we characterized the effect of UBA1 variants on hematopoiesis including symptomatic pts without malignant diagnoses. Aim: (1) Evaluate the hematological phenotype of UBA1 M41 and non-M41 variants detected in an unbiased cohort of symptomatic pts; (2) compare the transcriptomic profiles of M41 and non-M41 variants with healthy and pathogenic hematopoiesis. Patients and method: Screening of 12,643 pts suspected of HM for UBA1 variants (complete coding region) by targeted sequencing. Patients were diagnosed with HM based on 2022 WHO Classification. Whole transcriptome sequencing was performed for 1297 samples (Control n = 56, CCUS n = 218, MDS n = 771, CMML n = 228, UBA1 M41 n = 16, non-M41 n = 8). UBA1 samples were selected based on availability of sample material with preference given to pts without co-mutations. K-means clustering followed by filtering with consensus score (&amp;gt;0.6) by ConsensusClusterPlus was used to define transcriptomic clusters. DEG was defined as |logFC| &amp;gt; 1.5 and adjusted p &amp;lt; 0.05. ToppFun was used for GO analysis with default settings. Results: We identified 116 pts harboring UBA1 putative somatic variants (0.9%), consisting of 64 pts (0.5%) harboring M41 variants (M41 pts) and 52 pts (0.4%) harboring non-M41 UBA1 variants (non-M41 pts). As known, the majority (60/64 [94%]) of M41 pts were only suspected of HM (n = 51) or were diagnosed with MDS (n = 9) and other WHO entities such as MM were the minority. The tendency was weaker in non-M41 pts (36/52 [69 %], consisting of suspected HM n = 25, MDS n = 11; 16/52 [31%], other WHO entities). Of interest, ring sideroblasts (RS) were observed in 9/36 (25%) M41 pts and 11/28 (39%) non-M41 pts with staining available. SF3B1 was negative in 7/9 M41 pts and 8/11 non-M41 pts. Furthermore, RS ≥ 15% were observed in 4 non-M41 pts, two of which had no other co-mutations, suggesting the involvement of non-M41 variants in RS generation. To understand the effect of UBA1 in hematopoiesis, we compared the expression of marker genes of hematopoiesis of M41 and non-M41 pts with healthy individuals, pts diagnosed with CCUS, MDS, or CMML. The reference groups were constructed by k-means clustering based on 212 genes standardly recognized as relevant in hematopoiesis, and pts with the strongest differentiation bias are shown (Figure). The known myeloid bias and B-cell decrease of M41 pts were observable, and half of the M41 pts had at least a weak membership to clusters with some myeloid bias (Cluster 2 and 4). The observed myeloid bias could either be granulocytic or monocytic and the tendency was not homogenous among M41 pts. In contrast, half (n = 4) of the non-M41 pts belonged to the erythroid bias Cluster 5, which is enriched in pts carrying SF3B1 variants, and 3 belonged to Cluster 5 without SF3B1 variants. Neither M41 nor non-M41 pts clustered with transcriptomic groups of uni-lineage differentiation bias (Cluster 1, 3 and 6), and only one non-M41 pt clustered with the healthy controls, suggesting low risk but pathogenic hematopoiesis. To further understand the erythroid bias in non-M41 variants, we performed DEG and GO analysis. As erythroid bias comparators, we chose male pts in Cluster 5 diagnosed with MDS- SF3B1 (n = 14) and Cluster 6 diagnosed with MDS-bi TP53 (n = 8), and male controls (n = 30). The pt with a SF3B1 variant was excluded from the non-M41 group. Compared to controls, both the non-M41 group and Cluster 5- SF3B1 had upregulated heme synthesis, sharing upregulation of SF3B1-targets ABCB6 and PPOX. Cluster6-bi TP53 was more characterized by the downregulation of myeloid genes, which was not shared with non-M41 pts. Prognostic marker WT1 was upregulated in non-M41 group compared to Cluster 5- SF3B1 but not to controls or Cluster6- biTP53. Conclusions: UBA1 M41 variants confer myeloid bias but do not lead to the uni-lineage differentiation bias seen in pts with high risk mutations. Isolated UBA1 non-M41 pts can have RS ≥ 15%, and manifest erythroid bias similar to MDS- SF3B1 but higher expression of prognostic marker WT1.

Deep computational phenotyping of genomic variants impacting the SET domain of KMT2C reveal molecular mechanisms for their dysfunction.

Introduction: Kleefstra Syndrome type 2 (KLEFS-2) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier type 2 lysine methyltransferase KMT2C have been identified to be causative in KLEFS-2 individuals. Methods: This work reports a translational genomic study that applies a multidimensional computational approach for deep variant phenotyping, combining conventional genomic analyses, advanced protein bioinformatics, computational biophysics, biochemistry, and biostatistics-based modeling. We use standard variant annotation, paralog annotation analyses, molecular mechanics, and molecular dynamics simulations to evaluate damaging scores and provide potential mechanisms underlying KMT2C variant dysfunction. Results: We integrated data derived from the structure and dynamics of KMT2C to classify variants into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). When compared with controls, these variants show values reflecting alterations in molecular fitness in both structure and dynamics. Discussion: We demonstrate that our 3D models for KMT2C variants suggest distinct mechanisms that lead to their imbalance and are not predictable from sequence alone. Thus, the missense variants studied here cause destabilizing effects on KMT2C function by different biophysical and biochemical mechanisms which we adeptly describe. This new knowledge extends our understanding of how variations in the KMT2C gene cause the dysfunction of its methyltransferase enzyme product, thereby bearing significant biomedical relevance for carriers of KLEFS2-associated genomic mutations.

Novel COX11 Mutations Associated with Mitochondrial Disorder: Functional Characterization in Patient Fibroblasts and Saccharomyces cerevisiae

Genetic defects in the nuclear encoded subunits and assembly factors of cytochrome c oxidase (mitochondrial complex IV) are very rare and are associated with a wide variety of phenotypes. Biallelic pathogenic variants in the COX11 protein were previously identified in two unrelated children with infantile-onset mitochondrial encephalopathies. Through comprehensive clinical, genetic and functional analyses, here we report on a new patient harboring novel heterozygous variants in COX11, presenting with Leigh-like features, and provide additional experimental evidence for a direct correlation between COX11 protein expression and sensitivity to oxidative stress. To sort out the contribution of the single mutations to the phenotype, we employed a multi-faceted approach using Saccharomyces cerevisiae as a genetically manipulable system, and in silico structure-based analysis of human COX11. Our results reveal differential effects of the two novel COX11 mutations on yeast growth, respiration, and cellular redox status, as well as their potential impact on human protein stability and function. Strikingly, the functional deficits observed in patient fibroblasts are recapitulated in yeast models, validating the conservation of COX11’s role in mitochondrial integrity across evolutionarily distant organisms. This study not only expands the mutational landscape of COX11-associated mitochondrial disorders but also underscores the continued translational relevance of yeast models in dissecting complex molecular pathways.

A nonsense mutation in VHL causing Von Hippel-Lindau syndrome in a large Chinese family—a genetic study of familial neoplastic disease

Von Hippel-Lindau (VHL) syndrome is a multi-organ neoplastic disease characterized by highly vascular and cystic tumors in the central nervous system (CNS), retina, and visceral lesions, which are mainly caused by germline mutations in VHL. We aimed to detect novel mutations in VHL gene in families with VHL. Here, a large consanguineous four-generation family with variant phenotypes of VHL syndrome was recruited, and its molecular genetics were tested via Sanger sequencing. And various tools and databases were used to predict the variant pathogenicity, frequency, and protein function. Genetic investigation detected a c.351G > A nonsense mutation in VHL that altered the downstream reading frame and created a premature TGA stop signal, resulting in severely truncated pVHL (p.Trp117Ter). This mutation is absent from most public databases, and functional prediction bioinformatic tools demonstrated that this residue is conserved and that this variant is highly likely to be deleterious. The c.315G > A nonsense mutation in VHL is the causal mutation of this kindred that may lead to clear familial aggregation of VHL syndrome because of the dysfunction of the truncated pVHL.