The impact of clinical genome sequencing in a global population with suspected rare genetic disease.

There is mounting evidence of the value of clinical genome sequencing (cGS) in individuals with suspected rare genetic disease (RGD), but cGS performance and impact on clinical care in a diverse population drawn from both high-income countries (HICs) and low- and middle-income countries (LMICs) has not been investigated. The iHope program, a philanthropic cGS initiative, established a network of 24 clinical sites in eight countries through which it provided cGS to individuals with signs or symptoms of an RGD and constrained access to molecular testing. A total of 1,004 individuals (median age, 6.5 years; 53.5% male) with diverse ancestral backgrounds (51.8% non-majority European) were assessed from June 2016 to September 2021. The diagnostic yield of cGS was 41.4% (416/1,004), with individuals from LMIC sites 1.7 times more likely to receive a positive test result compared to HIC sites (LMIC 56.5% [195/345] vs. HIC 33.5% [221/659], OR 2.6, 95% CI 1.9-3.4, p < 0.0001). A change in diagnostic evaluation occurred in 76.9% (514/668) of individuals. Change of management, inclusive of specialty referrals, imaging and testing, therapeutic interventions, and palliative care, was reported in 41.4% (285/694) of individuals, which increased to 69.2% (480/694) when genetic counseling and avoidance of additional testing were also included. Individuals from LMIC sites were as likely as their HIC counterparts to experience a change in diagnostic evaluation (OR 6.1, 95% CI 1.1-∞, p = 0.05) and change of management (OR 0.9, 95% CI 0.5-1.3, p = 0.49). Increased access to genomic testing may support diagnostic equity and the reduction of global health care disparities.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

Developmental delay can precede neurologic regression in early onset metachromatic leukodystrophy.

OBJECTIVE: Metachromatic leukodystrophy (MLD) is a rare neurodegenerative disorder. Emerging therapies are most effective in the presymptomatic phase, and thus defining this window is critical. We hypothesize that early development delay may precede developmental plateau. With the advent of presymptomatic screening platforms and transformative therapies, it is essential to define the onset of neurologic disease. METHODS: The specific ages of gain and loss of developmental milestones were captured from the medical records of individuals affected by MLD. Milestone acquisition was characterized as: on target (obtained before the age limit of 90th percentile plus 2 standard deviations compared to a normative dataset), delayed (obtained after 90th percentile plus 2 standard deviations), or plateau (skills never gained). Regression was defined as the age at which skills were lost. LI-MLD was defined by age at onset before 2.5 years. RESULTS: Across an international cohort, 351 subjects were included (n = 194 LI-MLD subcohort). The median age at presentation of the LI-MLD cohort was 1.4 years (25th-75th %ile: 1.0-1.5). Within the LI-MLD cohort, 75/194 (39%) had developmental delay (or plateau) prior to MLD clinical presentation. Among the LI-MLD cohort with a minimum of 1.5 years of follow-up (n = 187), 73 (39.0%) subjects never attained independent ambulation. Within LI-MLD + delay subcohort, the median time between first missed milestone target to MLD decline was 0.60 years (maximum distance from delay to onset: 1.9 years). INTERPRETATION: Early developmental delay precedes regression in a subset of children affected by LI-MLD, defining the onset of neurologic dysfunction earlier than previously appreciated. The use of realworld data prior to diagnosis revealed an early deviation from typical development. Close monitoring for early developmental delay in presymptomatic individuals may help in earlier diagnosis with important consequences for treatment decisions.

Utility of genome sequencing in exome-negative pediatric patients with neurodevelopmental phenotypes.

Exome sequencing (ES) has emerged as an essential tool in the evaluation of neurodevelopmental disorders (NDD) of unknown etiology. Genome sequencing (GS) offers advantages over ES due to improved detection of structural, copy number, repeat number and non-coding variants. However, GS is less commonly utilized due to higher cost and more intense analysis. Here, we present nine cases of pediatric NDD that were molecularly diagnosed with GS between 2017 and 2022, following non-diagnostic ES. All individuals presented with global developmental delay or regression. Other features present in our cohort included epilepsy, white matter abnormalities, brain malformation and dysmorphic features. Two cases were diagnosed on GS due to newly described gene-disease relationship or variant reclassification (MAPK8IP3, CHD3). Additional features missed on ES that were later detected on GS were: intermediate-size deletions in three cases who underwent ES that were not validated for CNV detection, pathogenic variants within the non-protein coding genes SNORD118 and RNU7-1, pathogenic variant within the promoter region of GJB1, and a coding pathogenic variant within BCAP31 which was not sufficiently covered on ES. GS following non-diagnostic ES led to the identification of pathogenic variants in this cohort of nine cases, four of which would not have been identified by reanalysis alone.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Psychometric outcome measures in beta-propeller protein-associated neurodegeneration (BPAN).

BACKGROUND: Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurodegenerative disorder characterized by iron accumulation in the brain with spectrum of neurodevelopmental and movement phenotypes. In anticipation of future clinical trials and to inform clinical care, there is an unmet need to capture the phenotypic diversity of this rare disorder and better define disease subtypes. METHODS: A total of 27 individuals with BPAN were included in our natural history study, from which traditional outcome measures were obtained in 18 subjects. Demographic and diagnostic information, along with acquisition of basic developmental skills and overall neurologic severity were extracted from the medical records. Functional outcome measures were administered at the time of the evaluation or applied retrospectively at the last clinical encounter for patients who were not able to travel for in person. Based on age and functional level, the following assessments were administered: Leiter-3, Gross Motor Function Measure (GMFM)-66 Item Sets, Vineland-3, and Peabody-2. RESULTS: Overall, cognitive function was more impaired compared to gross motor function. Onset of symptoms of BPAN within the first 6 months of life was associated with decreased gain of ambulation and gain of spoken language (ambulation: log-rank test p = 0.0015; gain of first word: p = 0.0015). There was no difference in age at seizure onset by age at initial symptom onset (p = 0.8823). Collection of prospective outcome measures was limited by attention and behavior in our patient population, reinforcing the complexity of phenotype assessment and inadequacy of available standardized tests. Overall, gross motor and adaptive behavior assessments were better able to capture the dynamic range of function across the BPAN population than the fine motor and non-verbal cognitive tests. Floor effects were noted across outcome measures in a subset of individuals for cognitive and adaptive behavior tests. CONCLUSION: Our data suggest the distinct phenotypes of BPAN: a severe, early onset form and an attenuated form with higher cognitive capabilities. Early age at onset was a key factor in predicting future neurologic impairment.

Genome sequencing identifies three molecular diagnoses including a mosaic variant in the COL2A1 gene in an individual with Pol III-related leukodystrophy and Feingold syndrome.

Undiagnosed genetic disease imposes a significant burden on families and health-care resources, especially in cases with a complex phenotype. Here we present a child with suspected leukodystrophy in the context of additional features, including hearing loss, clinodactyly, rotated thumbs, tapered fingers, and simplified palmar crease. Trio genome sequencing (GS) identified three molecular diagnoses in this individual: compound heterozygous missense variants associated with polymerase III (Pol III)-related leukodystrophy, a 4-Mb de novo copy-number loss including the MYCN gene associated with Feingold syndrome, and a mosaic single-nucleotide variant associated with COL2A1-related disorders. These variants fully account for the individual's features, but also illustrate the potential for superimposed and unclear contributions of multiple diagnoses to an individual's overall presentation. This report demonstrates the advantage of GS in detection of multiple variant types, including low-level mosaic variants, and emphasizes the need for comprehensive genetic analysis and detailed clinical phenotyping to provide individuals and their families with the maximum benefit for clinical care and genetic counseling.

A molecular switch orchestrates enzyme specificity and secretory granule morphology.

Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. Here, we identify a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. Our study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, our in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.