The Association Between Stuttering Burden and Psychosocial Aspects of Life in Adults.

PURPOSE: Stuttering is a speech condition that can have a major impact on a person's quality of life. This descriptive study aimed to identify subgroups of people who stutter (PWS) based on stuttering burden and to investigate differences between these subgroups on psychosocial aspects of life. METHOD: The study included 618 adult participants who stutter. They completed a detailed survey examining stuttering symptomatology, impact of stuttering on anxiety, education and employment, experience of stuttering, and levels of depression, anxiety, and stress. A two-step cluster analytic procedure was performed to identify subgroups of PWS, based on self-report of stuttering frequency, severity, affect, and anxiety, four measures that together inform about stuttering burden. RESULTS: We identified a high- (n = 230) and a low-burden subgroup (n = 372). The high-burden subgroup reported a significantly higher impact of stuttering on education and employment, and higher levels of general depression, anxiety, stress, and overall impact of stuttering. These participants also reported that they trialed more different stuttering therapies than those with lower burden. CONCLUSIONS: Our results emphasize the need to be attentive to the diverse experiences and needs of PWS, rather than treating them as a homogeneous group. Our findings also stress the importance of personalized therapeutic strategies for individuals with stuttering, considering all aspects that could influence their stuttering burden. People with high-burden stuttering might, for example, have a higher need for psychological therapy to reduce stuttering-related anxiety. People with less emotional reactions but severe speech distortions may also have a moderate to high burden, but they may have a higher need for speech techniques to communicate with more ease. Future research should give more insights into the therapeutic needs of people highly burdened by their stuttering. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.25582980.

Haemoglobin thresholds to define anaemia from age 6 months to 65 years: estimates from international data sources.

BACKGROUND: Detection of anaemia is crucial for clinical medicine and public health. Current WHO anaemia definitions are based on statistical thresholds (fifth centiles) set more than 50 years ago. We sought to establish evidence for the statistical haemoglobin thresholds for anaemia that can be applied globally and inform WHO and clinical guidelines. METHODS: In this analysis we identified international data sources from populations in the USA, England, Australia, China, the Netherlands, Canada, Ecuador, and Bangladesh with sufficient clinical and laboratory information collected between 1998 and 2020 to obtain a healthy reference sample. Individuals with clinical or biochemical evidence of a condition that could reduce haemoglobin concentrations were excluded. We estimated haemoglobin thresholds (ie, 5th centiles) for children aged 6-23 months, 24-59 months, 5-11 years, and 12-17 years, and adults aged 18-65 years (including during pregnancy) for individual datasets and pooled across data sources. We also collated findings from three large-scale genetic studies to summarise genetic variants affecting haemoglobin concentrations in different ancestral populations. FINDINGS: We identified eight data sources comprising 18 individual datasets that were eligible for inclusion in the analysis. In pooled analyses, the haemoglobin fifth centile was 104·4 g/L (90% CI 103·5-105·3) in 924 children aged 6-23 months, 110·2 g/L (109·5-110·9) in 1874 children aged 24-59 months, and 114·4 g/L (113·6-115·2) in 1839 children aged 5-11 years. Values diverged by sex in adolescents and adults. In pooled analyses, the fifth centile was 122·2 g/L (90% CI 121·3-123·1) in 1741 female adolescents aged 12-17 years and 128·2 g/L (126·4-130·0) in 1103 male adolescents aged 12-17 years. In pooled analyses of adults aged 18-65 years, the fifth centile was 119·7 g/L (90% CI 119·1-120·3) in 3640 non-pregnant females and 134·9 g/L (134·2-135·6) in 2377 males. Fifth centiles in pregnancy were 110·3 g/L (90% CI 109·5-111·0) in the first trimester (n=772) and 105·9 g/L (104·0-107·7) in the second trimester (n=111), with insufficient data for analysis in the third trimester. There were insufficient data for adults older than 65 years. We did not identify ancestry-specific high prevalence of non-clinically relevant genetic variants that influence haemoglobin concentrations. INTERPRETATION: Our results enable global harmonisation of clinical and public health haemoglobin thresholds for diagnosis of anaemia. Haemoglobin thresholds are similar between sexes until adolescence, after which males have higher thresholds than females. We did not find any evidence that thresholds should differ between people of differering ancestries. FUNDING: World Health Organization and the Bill & Melinda Gates Foundation.

Stuttering associated with a pathogenic variant in the chaperone protein cyclophilin 40.

Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

Statistical haemoglobin thresholds to define anaemia across the lifecycle.

Detection of anaemia is critical for clinical medicine and public health. Current WHO values that define anaemia are statistical thresholds (5 th centile) set over 50 years ago, and are presently <110g/L in children 6-59 months, <115g/L in children 5-11 years, <110g/L in pregnant women, <120g/L in children 12-14 years of age, <120g/L in non-pregnant women, and <130g/L in men. Haemoglobin is sensitive to iron and other nutrient deficiencies, medical illness and inflammation, and is impacted by genetic conditions; thus, careful exclusion of these conditions is crucial to obtain a healthy reference population. We identified data sources from which sufficient clinical and laboratory information was available to determine an apparently healthy reference sample. Individuals were excluded if they had any clinical or biochemical evidence of a condition that may diminish haemoglobin concentration. Discrete 5 th centiles were estimated along with two-sided 90% confidence intervals and estimates combined using a fixed-effect approach. Estimates for the 5 th centile of the healthy reference population in children were similar between sexes. Thresholds in children 6-23 months were 104.4g/L [90% CI 103.5, 105.3]; in children 24-59 months were 110.2g/L [109.5, 110.9]; and in children 5-11 years were 114.1g/L [113.2, 115.0]. Thresholds diverged by sex in adolescents and adults. In females and males 12-17 years, thresholds were 122.2g/L [121.3, 123.1] and 128.2 [126.4, 130.0], respectively. In adults 18-65 years, thresholds were 119.7g/L [119.1, 120.3] in non-pregnant females and 134.9g/L [134.2, 135.6] in males. Limited analyses indicated 5 th centiles in first-trimester pregnancy of 110.3g/L [109.5, 111.0] and 105.9g/L [104.0, 107.7] in the second trimester. All thresholds were robust to variations in definitions and analysis models. Using multiple datasets comprising Asian, African, and European ancestries, we did not identify novel high prevalence genetic variants that influence haemoglobin concentration, other than variants in genes known to cause important clinical disease, suggesting non-clinical genetic factors do not influence the 5 th centile between ancestries. Our results directly inform WHO guideline development and provide a platform for global harmonisation of laboratory, clinical and public health haemoglobin thresholds.

Iron homeostasis governs erythroid phenotype in polycythemia vera.

Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Genetic aetiologies for childhood speech disorder: novel pathways co-expressed during brain development.

Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we aimed to identify molecular causation in 70 unrelated probands ascertained with CAS. We performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.

Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Self-reported impact of developmental stuttering across the lifespan.

AIM: To examine the phenomenology of stuttering across the lifespan in the largest prospective cohort to date. METHOD: Participants aged 7 years and older with a history of developmental stuttering were recruited. Self-reported phenotypic data were collected online including stuttering symptomatology, co-occurring phenotypes, genetic predisposition, factors associated with stuttering severity, and impact on anxiety, education, and employment. RESULTS: A total of 987 participants (852 adults: 590 males, 262 females, mean age 49 years [SD = 17 years 10 months; range = 18-93 years] and 135 children: 97 males, 38 females, mean age 11 years 4 months [SD = 3 years; range = 7-17 years]) were recruited. Stuttering onset occurred at age 3 to 6 years in 64.0%. Blocking (73.2%) was the most frequent phenotype; 75.9% had sought stuttering therapy and 15.5% identified as having recovered. Half (49.9%) reported a family history. There was a significant negative correlation with age for both stuttering frequency and severity in adults. Most were anxious due to stuttering (90.4%) and perceived stuttering as a barrier to education and employment outcomes (80.7%). INTERPRETATION: The frequent persistence of stuttering and the high proportion with a family history suggest that stuttering is a complex trait that does not often resolve, even with therapy. These data provide new insights into the phenotype and prognosis of stuttering, information that is critically needed to encourage the development of more effective speech therapies. WHAT THIS PAPER ADDS: Half of the study cohort had a family history of stuttering. While 75.9% of participants had sought stuttering therapy, only 15.5% identified as having recovered. There was a significant negative correlation between age and stuttering frequency and severity in adults.

Rare and low-frequency exonic variants and gene-by-smoking interactions in pulmonary function.

Genome-wide association studies have identified numerous common genetic variants associated with spirometric measures of pulmonary function, including forced expiratory volume in one second (FEV1), forced vital capacity, and their ratio. However, variants with lower minor allele frequencies are less explored. We conducted a large-scale gene-smoking interaction meta-analysis on exonic rare and low-frequency variants involving 44,429 individuals of European ancestry in the discovery stage and sought replication in the UK BiLEVE study with 45,133 European ancestry samples and UK Biobank study with 59,478 samples. We leveraged data on cigarette smoking, the major environmental risk factor for reduced lung function, by testing gene-by-smoking interaction effects only and simultaneously testing the genetic main effects and interaction effects. The most statistically significant signal that replicated was a previously reported low-frequency signal in GPR126, distinct from common variant associations in this gene. Although only nominal replication was obtained for a top rare variant signal rs142935352 in one of the two studies, interaction and joint tests for current smoking and PDE3B were significantly associated with FEV1. This study investigates the utility of assessing gene-by-smoking interactions and underscores their effects on potential pulmonary function.

A systematic analysis of protein-altering exonic variants in chronic obstructive pulmonary disease.

Genome-wide association studies (GWASs) have identified regions associated with chronic obstructive pulmonary disease (COPD). GWASs of other diseases have shown an approximately 10-fold overrepresentation of nonsynonymous variants, despite limited exonic coverage on genotyping arrays. We hypothesized that a large-scale analysis of coding variants could discover novel genetic associations with COPD, including rare variants with large effect sizes. We performed a meta-analysis of exome arrays from 218,399 controls and 33,851 moderate-to-severe COPD cases. All exome-wide significant associations were present in regions previously identified by GWAS. We did not identify any novel rare coding variants with large effect sizes. Within GWAS regions on chromosomes 5q, 6p, and 15q, four coding variants were conditionally significant (P < 0.00015) when adjusting for lead GWAS single-nucleotide polymorphisms A common gasdermin B (GSDMB) splice variant (rs11078928) previously associated with a decreased risk for asthma was nominally associated with a decreased risk for COPD [minor allele frequency (MAF) = 0.46, P = 1.8e-4]. Two stop variants in coiled-coil α-helical rod protein 1 (CCHCR1), a gene involved in regulating cell proliferation, were associated with COPD (both P < 0.0001). The SERPINA1 Z allele was associated with a random-effects odds ratio of 1.43 for COPD (95% confidence interval = 1.17-1.74), though with marked heterogeneity across studies. Overall, COPD-associated exonic variants were identified in genes involved in DNA methylation, cell-matrix interactions, cell proliferation, and cell death. In conclusion, we performed the largest exome array meta-analysis of COPD to date and identified potential functional coding variants. Future studies are needed to identify rarer variants and further define the role of coding variants in COPD pathogenesis.

Identification of genetic factors influencing metabolic dysregulation and retinal support for MacTel, a retinal disorder.

Macular Telangiectasia Type 2 (MacTel) is a rare degenerative retinal disease with complex genetic architecture. We performed a genome-wide association study on 1,067 MacTel patients and 3,799 controls, which identified eight novel genome-wide significant loci (p < 5 × 10-8), and confirmed all three previously reported loci. Using MAGMA, eQTL and transcriptome-wide association analysis, we prioritised 48 genes implicated in serine-glycine biosynthesis, metabolite transport, and retinal vasculature and thickness. Mendelian randomization indicated a likely causative role of serine (FDR = 3.9 × 10-47) and glycine depletion (FDR = 0.006) as well as alanine abundance (FDR = 0.009). Polygenic risk scoring achieved an accuracy of 0.74 and was associated in UKBiobank with retinal damage (p = 0.009). This represents the largest genetic study on MacTel to date and further highlights genetically-induced systemic and tissue-specific metabolic dysregulation in MacTel patients, which impinges on retinal health.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Severe childhood speech disorder: Gene discovery highlights transcriptional dysregulation.

OBJECTIVE: Determining the genetic basis of speech disorders provides insight into the neurobiology of human communication. Despite intensive investigation over the past 2 decades, the etiology of most speech disorders in children remains unexplained. To test the hypothesis that speech disorders have a genetic etiology, we performed genetic analysis of children with severe speech disorder, specifically childhood apraxia of speech (CAS). METHODS: Precise phenotyping together with research genome or exome analysis were performed on children referred with a primary diagnosis of CAS. Gene coexpression and gene set enrichment analyses were conducted on high-confidence gene candidates. RESULTS: Thirty-four probands ascertained for CAS were studied. In 11/34 (32%) probands, we identified highly plausible pathogenic single nucleotide (n = 10; CDK13, EBF3, GNAO1, GNB1, DDX3X, MEIS2, POGZ, SETBP1, UPF2, ZNF142) or copy number (n = 1; 5q14.3q21.1 locus) variants in novel genes or loci for CAS. Testing of parental DNA was available for 9 probands and confirmed that the variants had arisen de novo. Eight genes encode proteins critical for regulation of gene transcription, and analyses of transcriptomic data found CAS-implicated genes were highly coexpressed in the developing human brain. CONCLUSION: We identify the likely genetic etiology in 11 patients with CAS and implicate 9 genes for the first time. We find that CAS is often a sporadic monogenic disorder, and highly genetically heterogeneous. Highly penetrant variants implicate shared pathways in broad transcriptional regulation, highlighting the key role of transcriptional regulation in normal speech development. CAS is a distinctive, socially debilitating clinical disorder, and understanding its molecular basis is the first step towards identifying precision medicine approaches.

Meta-analysis of up to 622,409 individuals identifies 40 novel smoking behaviour associated genetic loci.

Smoking is a major heritable and modifiable risk factor for many diseases, including cancer, common respiratory disorders and cardiovascular diseases. Fourteen genetic loci have previously been associated with smoking behaviour-related traits. We tested up to 235,116 single nucleotide variants (SNVs) on the exome-array for association with smoking initiation, cigarettes per day, pack-years, and smoking cessation in a fixed effects meta-analysis of up to 61 studies (up to 346,813 participants). In a subset of 112,811 participants, a further one million SNVs were also genotyped and tested for association with the four smoking behaviour traits. SNV-trait associations with P < 5 × 10-8 in either analysis were taken forward for replication in up to 275,596 independent participants from UK Biobank. Lastly, a meta-analysis of the discovery and replication studies was performed. Sixteen SNVs were associated with at least one of the smoking behaviour traits (P < 5 × 10-8) in the discovery samples. Ten novel SNVs, including rs12616219 near TMEM182, were followed-up and five of them (rs462779 in REV3L, rs12780116 in CNNM2, rs1190736 in GPR101, rs11539157 in PJA1, and rs12616219 near TMEM182) replicated at a Bonferroni significance threshold (P < 4.5 × 10-3) with consistent direction of effect. A further 35 SNVs were associated with smoking behaviour traits in the discovery plus replication meta-analysis (up to 622,409 participants) including a rare SNV, rs150493199, in CCDC141 and two low-frequency SNVs in CEP350 and HDGFRP2. Functional follow-up implied that decreased expression of REV3L may lower the probability of smoking initiation. The novel loci will facilitate understanding the genetic aetiology of smoking behaviour and may lead to the identification of potential drug targets for smoking prevention and/or cessation.

Recessive variants in ZNF142 cause a complex neurodevelopmental disorder with intellectual disability, speech impairment, seizures, and dystonia.

PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.