Wierenga, Klaas J.

ABSTRACT Polyglucosan body myopathy type 2 (PGBM2; OMIM #616199) is an autosomal recessive myopathy caused by biallelic variants in GYG1 , which encodes glycogenin‐1. It is characterized by progressive muscle weakness and PAS‐positive, diastase‐resistant polyglucosan inclusions on muscle biopsy. We report a 64‐year‐old woman who developed progressive proximal weakness beginning in her early 50s, with polyglucosan bodies identified on muscle histopathology. Genome sequencing (GS) detected a single heterozygous pathogenic splice‐site variant, NM_004130.4( GYG1 ):c.143+3G>C. Given strong histopathologic evidence of PGBM2, GS data were reanalyzed, revealing an additional rare deep intronic variant, NM_004130.4( GYG1 ):c.7+992T>G, predicted to activate a cryptic exon. RNA sequencing (RNA‐seq) confirmed aberrant splicing from both variants, demonstrating exon 2 skipping associated with c.143+3G>C and cryptic exon inclusion caused by c.7+992T>G. Variant phasing posed a diagnostic challenge due to unavailable parental DNA, and long‐range PCR results were inconclusive. Long‐read GS definitively demonstrated that the two variants were in trans , establishing compound heterozygosity and confirming the molecular diagnosis. These findings enabled reclassification of the deep intronic variant as likely pathogenic. This case highlights the diagnostic limitations of conventional phasing approaches in adult‐onset recessive disorders and demonstrates the clinical utility of integrating genome reanalysis, RNA‐seq, and long‐read GS for resolving complex phasing challenges.

Abstract Alpha-tubulin 4A encoding gene (TUBA4A) has been associated with familial amyotrophic lateral sclerosis and frontotemporal dementia, based on identification of likely pathogenic variants in patients from distinct amyotrophic lateral sclerosis and frontotemporal dementia cohorts. By screening a multicentric French cohort of 448 unrelated probands presenting with cerebellar ataxia, we identified ultra-rare TUBA4A missense variants, all being absent from public databases and predicted pathogenic by multiple in silico tools. In addition, gene burden analyses in the 100 000 Genomes project (100KGP) showed enrichment of TUBA4A rare variants in the inherited ataxia group compared to controls [odds ratio: 57.0847 (10.2−576.7); P = 4.02 ×10−7]. Taken together, we report 12 patients presenting with spasticity and/or cerebellar ataxia and harbouring a predicted pathogenic TUBA4A missense mutation, including five confirmed de novo cases and a mutation previously reported in a large family presenting with spastic ataxia. Cultured fibroblasts from three patients harbouring distinct TUBA4A missense showed significant alterations in microtubule organization and dynamics, providing insight of TUBA4A variants pathogenicity. Our data confirm the identification of a hereditary spastic ataxia disease gene with variable age of onset, expanding the clinical spectrum of TUBA4A associated phenotypes.

(1) Background: Mutations in NFκB1, a transcriptional regulator of immunomodulating proteins, are a known cause of inborn errors of immunity. Our proband is a 22-year-old male with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Genetic studies identified a novel, single-point mutation variant in NFκB1, c. T638A p. V213E. (2) Methods: Next-generation panel sequencing of the patient uncovered a novel single-point mutation in the NFκB1 gene that was modeled using the I-TASSER homology-modeling software, and molecular dynamics were assessed using the YASARA2 software (version 20.14.24). (3) Results: This variant replaces valine with glutamic acid at position 213 in the NFκB1 sequence. Molecular modeling and molecular dynamic studies showed altered dynamics in and around the rel homology domain, ankyrin regions, and death domain of the protein. We postulate that these changes alter overall protein function. (4) Conclusions: This case suggests the pathogenicity of a novel variant using protein-modeling techniques and molecular dynamic simulations.

Abstract BACKGROUND Mutations of NFkB1 are a known cause of inborn errors of immunity resulting in immunodeficiency due to the role of NFkB1 as a transcriptional regulator of immunomodulating proteins. We present a patient with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Our proband, a 22-year-old male, presents with a clinical picture of immunodeficiency, and has a maternal cousin with a similar gene mutation but differing phenotype. Next generation panel sequencing (NGPS) was utilized to discover a novel, single point mutation variant in NFkB1, V213E, that is present in our proband. METHODS The novel single-point mutation V213E in the NFkB gene was modeled in-silico using the I-TASSER homology modeling software. Molecular dynamics simulations of both the wild type and variant mutant form fitted to accommodate the novel mutation were conducted and analyzed to assess for possible pathogenicity of the novel mutation. RESULTS This variant replaces Valine with Glutamic Acid at position 213 in the transcription regulator’s sequence. We perform molecular modeling to analyze the proteomic impact and difference in molecular dynamics (MDS) which depicted suppressed dynamics in the ankyrin region and death domain of the protein complex. Analysis of the proband’s family showed no significant family history except for the maternal cousin with a mutation in NFkB1. CONCLUSION This case both highlights the heterogeneity in phenotype of NFkB1 pathogenic variants as shown in this patient, full penetrance of the variant mutation, and suggests the pathogenicity of a new variant through protein modeling techniques and molecular dynamic simulations.

Abstract Background In the United States, rare disease (RD) is defined as a condition that affects fewer than 200,000 individuals. Collectively, RD affects an estimated 30 million Americans. A significant portion of RD has an underlying genetic cause; however, this may go undiagnosed. To better serve these patients, the Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD) was created under the auspices of the Center for Individualized Medicine (CIM) aiming to integrate genomics into subspecialty practice including targeted genetic testing, research, and education. Methods Patients were identified by subspecialty healthcare providers from 11 clinical divisions/departments. Targeted multi-gene panels or custom exome/genome-based panels were utilized. To support the goals of PRaUD, a new clinical service model, the Genetic Testing and Counseling (GTAC) unit, was established to improve access and increase efficiency for genetic test facilitation. The GTAC unit includes genetic counselors, genetic counseling assistants, genetic nurses, and a medical geneticist. Patients receive abbreviated point-of-care genetic counseling and testing through a partnership with subspecialty providers. Results Implementation of PRaUD began in 2018 and GTAC unit launched in 2020 to support program expansion. Currently, 29 RD clinical indications are included in 11 specialty divisions/departments with over 142 referring providers. To date, 1,152 patients have been evaluated with an overall solve rate of 14.1% and as high as 66.7% depending on the phenotype. Noteworthy, 42.7% of the solved or likely solved patients underwent changes in medical management and outcome based on genetic test results. Conclusion Implementation of PRaUD and GTAC have enabled subspecialty practices advance expertise in RD where genetic counselors have not historically been embedded in practice. Democratizing access to genetic testing and counseling can broaden the reach of patients with RD and increase the diagnostic yield of such indications leading to better medical management as well as expanding research opportunities.

AbstractBackgroundAchalasia‐addisonianism‐alacrima syndrome, frequently referred to as Allgrove syndrome or Triple A syndrome, is a multisystem disorder resulting from homozygous or compound heterozygous pathogenic variants in the gene encoding aladin (AAAS). Aladin is a member of the WD‐repeat family of proteins and is a component of the nuclear pore complex. It is thought to be involved in nuclear import and export of molecules. Here, we describe an individual with a paternally inherited truncating variant and a maternally inherited, novel missense variant in AAAS presenting with alacrima, achalasia, anejaculation, optic atrophy, muscle weakness, dysarthria, and autonomic dysfunction.MethodsWhole‐exome sequencing was performed in the proband, sister, and parents. Variants were confirmed by Sanger sequencing. The localization of aladin to the nuclear pore was assessed in cells expressing the patient variant.ResultsFunctional testing of the maternally inherited variant, p.(Arg270Pro), demonstrated decreased localization of aladin to the nuclear pore in cells expressing the variant, indicating a deleterious effect. Follow‐up testing in the proband's affected sister revealed that she also inherited the biallelic AAAS variants.ConclusionsReview of the patient's clinical, pathological, and genetic findings resulted in a diagnosis of Triple A syndrome.

Background Musculocontractural Ehlers−Danlos syndrome is caused by biallelic loss-of-function variants in CHST14 (mcEDS-CHST14) or DSE (mcEDS-DSE). Although 48 patients in 33 families with mcEDS-CHST14 have been reported, the spectrum of pathogenic variants, accurate prevalence of various manifestations and detailed natural history have not been systematically investigated. Methods We collected detailed and comprehensive clinical and molecular information regarding previously reported and newly identified patients with mcEDS-CHST14 through international collaborations. Results Sixty-six patients in 48 families (33 males/females; 0–59 years), including 18 newly reported patients, were evaluated. Japanese was the predominant ethnicity (27 families), associated with three recurrent variants. No apparent genotype–phenotype correlation was noted. Specific craniofacial (large fontanelle with delayed closure, downslanting palpebral fissures and hypertelorism), skeletal (characteristic finger morphologies, joint hypermobility, multiple congenital contractures, progressive talipes deformities and recurrent joint dislocation), cutaneous (hyperextensibility, fine/acrogeria-like/wrinkling palmar creases and bruisability) and ocular (refractive errors) features were observed in most patients (>90%). Large subcutaneous haematomas, constipation, cryptorchidism, hypotonia and motor developmental delay were also common (>80%). Median ages at the initial episode of dislocation or large subcutaneous haematoma were both 6 years. Nine patients died; their median age was 12 years. Several features, including joint and skin characteristics (hypermobility/extensibility and fragility), were significantly more frequent in patients with mcEDS-CHST14 than in eight reported patients with mcEDS-DSE. Conclusion This first international collaborative study of mcEDS-CHST14 demonstrated that the subtype represents a multisystem disorder with unique set of clinical phenotypes consisting of multiple malformations and progressive fragility-related manifestations; these require lifelong, multidisciplinary healthcare approaches.