Bi-allelic variants in HMGCR cause an autosomal-recessive progressive limb-girdle muscular dystrophy.

Statins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000 U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy.

Semiautomated approach focused on new genomic information results in time and effort-efficient reannotation of negative exome data.

Most rare disease patients (75-50%) undergoing genomic sequencing remain unsolved, often due to lack of information about variants identified. Data review over time can leverage novel information regarding disease-causing variants and genes, increasing this diagnostic yield. However, time and resource constraints have limited reanalysis of genetic data in clinical laboratories setting. We developed RENEW, (REannotation of NEgative WES/WGS) an automated reannotation procedure that uses relevant new information in on-line genomic databases to enable rapid review of genomic findings. We tested RENEW in an unselected cohort of 1066 undiagnosed cases with a broad spectrum of phenotypes from the Mayo Clinic Center for Individualized Medicine using new information in ClinVar, HGMD and OMIM between the date of previous analysis/testing and April of 2022. 5741 variants prioritized by RENEW were rapidly reviewed by variant interpretation specialists. Mean analysis time was approximately 20 s per variant (32 h total time). Reviewed cases were classified as: 879 (93.0%) undiagnosed, 63 (6.6%) putatively diagnosed, and 4 (0.4%) definitively diagnosed. New strategies are needed to enable efficient review of genomic findings in unsolved cases. We report on a fast and practical approach to address this need and improve overall diagnostic success in patient testing through a recurrent reannotation process.

Impact of integrated translational research on clinical exome sequencing.

PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Recurrent ganglioneuroma in PTPN11-associated Noonan syndrome: A case report and literature review.

Noonan syndrome (NS) is an autosomal dominant condition with variable expressivity most commonly due to a germline pathogenic variant in PTPN11, which encodes the protein tyrosine phosphatase SHP-2. Gain-of-function variants in PTPN11 are known to promote oncogenic behavior in affected tissues. We report the clinical description of a young adult male presenting with relapsing ganglioneuromas, dysmorphic features, cardiac abnormalities, and multiple lentigines, strongly suspicious for NS. Solid tumor testing identified the recurrent pathogenic c.922G>A (p.Asn308Asp) in PTPN11. Proband and parental blood sampling testing confirmed c.922G>A as a de novo germline alteration. Comprehensive literature review of solid tumors specifically associated to PTPN11, indicates that this is the first documentation of ganglioneuroma and its clinical recurrence after resection in conjunction with a genetically confirmed NS diagnosis. The findings in our patient further extend the list of neuroblastic and neural crest-derived neoplasms associated with this condition.

Next-Generation Sequencing of CYP2C19 in Stent Thrombosis: Implications for Clopidogrel Pharmacogenomics.

PURPOSE: Describe CYP2C19 sequencing results in the largest series of clopidogrel-treated cases with stent thrombosis (ST), the closest clinical phenotype to clopidogrel resistance. Evaluate the impact of CYP2C19 genetic variation detected by next-generation sequencing (NGS) with comprehensive annotation and functional studies. METHODS: Seventy ST cases on clopidogrel identified from the PLATO trial (n = 58) and Mayo Clinic biorepository (n = 12) were matched 1:1 with controls for age, race, sex, diabetes mellitus, presentation, and stent type. NGS was performed to cover the entire CYP2C19 gene. Assessment of exonic variants involved measuring in vitro protein expression levels. Intronic variants were evaluated for potential splicing motif variations. RESULTS: Poor metabolizers (n = 4) and rare CYP2C19*8, CYP2C19*15, and CYP2C19*11 alleles were identified only in ST cases. CYP2C19*17 heterozygote carriers were observed more frequently in cases (n = 29) than controls (n = 18). Functional studies of CYP2C19 exonic variants (n = 11) revealed 3 cases and only 1 control carrying a deleterious variant as determined by in vitro protein expression studies. Greater intronic variation unique to ST cases (n = 169) compared with controls (n = 84) was observed with predictions revealing 13 allele candidates that may lead to a potential disruption of splicing and a loss-of-function effect of CYP2C19 in ST cases. CONCLUSION: NGS detected CYP2C19 poor metabolizers and paradoxically greater number of so-called rapid metabolizers in ST cases. Rare deleterious exonic variation occurs in 4%, and potentially disruptive intronic alleles occur in 16% of ST cases. Additional studies are required to evaluate the role of these variants in platelet aggregation and clopidogrel metabolism.

Deciphering the DNA code for the function of the Drosophila polydactyl zinc finger protein Suppressor of Hairy-wing.

Polydactyl zinc finger (ZF) proteins have prominent roles in gene regulation and often execute multiple regulatory functions. To understand how these proteins perform varied regulation, we studiedDrosophila Suppressor of Hairy-wing [Su(Hw)], an exemplar multifunctional polydactyl ZF protein. We identified separation-of-function (SOF) alleles that encode proteins disrupted in a single ZF that retain one of the Su(Hw) regulatory activities. Through extended in vitro analyses of the Su(Hw) ZF domain, we show that clusters of ZFs bind individual modules within a compound DNA consensus sequence. Through in vivo analysis of SOF mutants, we find that Su(Hw) genomic sites separate into sequence subclasses comprised of combinations of modules, with subclasses enriched for different chromatin features. These data suggest a Su(Hw) code, wherein DNA binding dictates its cofactor recruitment and regulatory output. We propose that similar DNA codes might be used to confer multiple regulatory functions of other polydactyl ZF proteins.

Micropapillary adenocarcinoma of lung: Morphological criteria and diagnostic reproducibility among pulmonary pathologists.

CONTEXT: Invasive micropapillary adenocarcinoma (MPC) is an aggressive variant of lung adenocarcinoma, frequently manifesting with advanced stage lymph node metastasis and decreased survival. OBJECTIVE: Identification of this morphology is important, as it is strongly correlated with poor prognosis regardless of the amount of MPC component. To date, no study has investigated the morphological criteria used to objectively diagnose it. DESIGN: Herein, we selected 30 cases of potential MPC of lung, and distributed 2 digital images per case among 15 pulmonary pathology experts. Reviewers were requested to diagnostically interpret, assign the percentage of MPC component, and record the morphological features they identified. The noted features included: columnar cells, elongated slender cell nests, extensive stromal retraction, lumen formation with internal epithelial tufting, epithelial signet ring-like forms, intracytoplasmic vacuolization, multiple nests in the same alveolar space, back-to-back lacunar spaces, epithelial nest anastomosis, marked pleomorphism, peripherally oriented nuclei, randomly distributed nuclei, small/medium/large tumor nest size, fibrovascular cores, and spread through air-spaces (STAS). RESULTS: Cluster analysis revealed three subgroups with the following diagnoses: "MPC", "combined papillary and MPC", and "others". The subgroups correlated with the reported median percentage of MPC. Intracytoplasmic vacuolization, epithelial nest anastomosis/confluence, multiple nests in the same alveolar space, and small/medium tumor nest size were the most common criteria identified in the cases diagnosed as MPC. Peripherally oriented nuclei and epithelial signet ring-like forms were frequently identified in both the "MPC" and "combined papillary and MPC" groups. CONCLUSIONS: Our study provides objective diagnostic criteria to diagnose MPC of lung.

A case of YY1-associated syndromic learning disability or Gabriele-de Vries syndrome with myasthenia gravis.

Exome sequencing is being used increasingly to evaluate patients with intellectual disability. YY1 is a ubiquitously distributed transcription factor belonging to the GLIKruppel class of zinc finger proteins recently recognized as the causative gene in 23 patients for the Gabriele-de Vries syndrome. We report a new case with similar features and a novel variant in YY1, in a region of the gene, which has not previously been reported. A 25 year old female was referred to clinical genetics with a diagnosis of autoimmune myasthenia gravis, facial dysmorphism and learning disability. Chromosomal microarray and gene panel test for congenital myasthenic syndrome was negative. Whole exome sequencing (WES) revealed a presumed pathogenic de novo novel, heterozygous, truncating variant in the YY1 gene, c.860_864delTTAAAA, p.Ile287Argfs*3. The Ile287 residue is conserved across species and is situated in the transcription repressor domain of the protein. This variant is novel and lies in a domain of the protein where no previously reported variants occur. The phenotypic features of our case closely match those of the reported patients. Autoimmune myasthenia gravis has not been reported in these patients and may constitute an expansion of this phenotypic spectrum or perhaps more likely a second unrelated diagnosis.

Functional validation of a novel AAAS variant in an atypical presentation of Allgrove syndrome.

BACKGROUND: Achalasia-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. METHODS: Whole-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. RESULTS: Functional 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. CONCLUSIONS: Review of the patient's clinical, pathological, and genetic findings resulted in a diagnosis of Triple A syndrome.

A novel missense variant and multiexon deletion causing a delayed presentation of xeroderma pigmentosum, group C.

Pathogenic variants in the XPC complex subunit, DNA damage recognition, and repair factor (XPC) are the cause of xeroderma pigmentosum, group C (MIM: 278720). Xeroderma pigmentosum is an inherited condition characterized by hypersensitivity to ultraviolet (UV) irradiation and increased risk of skin cancer due to a defect in nucleotide excision repair (NER). Here we describe an individual with a novel missense variant and deletion of exons 14-15 in XPC presenting with a history of recurrent melanomas. The proband is a 39-yr-old female evaluated through the Mayo Clinic Department of Clinical Genomics. Prior to age 36, she had more than 60 skin biopsies that showed dysplastic nevi, many of which had atypia. At age 36 she presented with her first melanoma in situ, and since then has had more than 10 melanomas. The proband underwent research whole-exome sequencing (WES) through the Mayo Clinic's Center for Individualized Medicine and a novel heterozygous variant of uncertain significance (VUS) in XPC (c.1709T > G, p.Val570Gly) was identified. Clinical confirmation pursued via XPC gene sequencing and deletion/duplication analysis of XPC revealed a pathogenic heterozygous deletion of ∼1 kb within XPC, including exons 14 and 15. Research studies determined the alterations to be in trans Although variants in XPC generally result in early-onset skin cancer in childhood, the proband is atypical in that she did not present with her first melanoma until age 36. Review of the patient's clinical, pathological, and genetic findings points to a diagnosis of delayed presentation of xeroderma pigmentosum.

Interpretation challenges of novel dual-class missense and splice-impacting variant in POLR3A-related late-onset hereditary spastic ataxia.

BACKGROUND: RNA polymerase III (Pol III)-related disorders are autosomal recessive neurodegenerative disorders caused by variants in POLR3A or POLR3B. Recently, a novel phenotype of adult-onset spastic ataxia was identified in individuals with the c.1909+22G>A POLR3A variant in compound heterozygosity. METHODS: Whole-exome sequencing was performed in the proband and parents. Variants were confirmed by Sanger sequencing. RNA sequencing was performed to evaluate splicing implications. RESULTS: A 42-year-old female was evaluated for unexplained neurological findings with a slow progressive decline in gait and walking speed since adolescence. WES revealed a novel missense variant (c.3593A>C, p.Lys1198Arg) in exon 27 of POLR3A in compound heterozygosity with the c.1909+22G>A variant. Summary of previously reported clinical features from individuals with pathogenic biallelic alterations in POLR3A and adult-onset phenotype is consistent with our findings. RNA analysis revealed c.3593A>G drives the production of four RNA transcript products each with different functional impacts. CONCLUSION: The novel dual-class c.3593A>C variant in POLR3A causes an amino acid substitution and complex disruption of splicing. Our report supports the need to investigate variants near splice junctions for proper interpretation. Current interpretation guidelines need to address best practices for inclusion of predicted or measured transcriptional disruption pending functional activity or reliable transcript abundance estimates.

Developmental brain abnormalities and acute encephalopathy in a patient with myopathy with extrapyramidal signs secondary to pathogenic variants in MICU1.

Mitochondria play a variety of roles in the cell, far beyond their widely recognized role in ATP generation. One such role is the regulation and sequestration of calcium, which is done with the help of the mitochondrial calcium uniporter (MCU) and its regulators, MICU1 and MICU2. Genetic variations in MICU1 and MICU2 have been reported to cause myopathy, developmental disability and neurological symptoms typical of mitochondrial disorders. The symptoms of MICU1/2 deficiency have generally been attributed to calcium regulation in the metabolic and biochemical roles of mitochondria. Here, we report a female child with heterozygous MICU1 variants and multiple congenital brain malformations on MRI. Specifically, she shows anterior perisylvian polymicrogyria, dysmorphic basal ganglia, and cerebellar dysplasia in addition to white matter abnormalities. These novel findings suggest that MICU1 is necessary for proper neurodevelopment through a variety of potential mechanisms, including calcium-mediated regulation of the neuronal cytoskeleton, Miro1-MCU complex-mediated mitochondrial movement, or enhancing ATP production. This case provides new insight into the molecular pathogenesis of MCU dysfunction and may represent a novel diagnostic feature of calcium-based mitochondrial disease.

Correlation Between Programmed Death Receptor-1 Expression in Tumor-Infiltrating Lymphocytes and Programmed Death Ligand-1 Expression in Non-Small Cell Lung Carcinoma.

CONTEXT.­: The interaction between programmed death ligand-1 (PD-L1) and programmed death receptor-1 (PD-1) on activated T cells sends an inhibitory signal that dampens the immune response. Tumors can express PD-L1 and evade the immune system. In advanced non-small cell lung carcinoma, expression of PD-1 in tumor-infiltrating lymphocytes (TILs) correlates with PD-L1 expression in tumor cells (TCs). However, this relationship has not been thoroughly explored in early disease. OBJECTIVE.­: To investigate the correlation of PD-1 and PD-L1 in non-small cell lung carcinoma tumor samples, with emphasis on stage I disease. DESIGN.­: Whole tissue sections from non-small cell lung carcinoma tumors were retrospectively evaluated by immunohistochemistry for PD-1 and PD-L1 expression. The scoring was based on the percentage of cells positive for PD-1 in TILs and PD-L1 in TCs and tumor-infiltrating immune cells (ICs). RESULTS.­: Expression of PD-1 in TILs was observed in 147 of 161 non-small cell lung carcinoma cases (91%). The majority of cases negative for PD-1 also lacked PD-L1 in TCs. The 68 cases with highest PD-1 expression in TILs included 33 (49%) with expression of PD-L1 in TCs and ICs. Strong correlations were observed in patients with elevated PD-1 expression in TILs and PD-L1 in TCs ( P = .01) and ICs ( P = .003). Expression of PD-1 also correlated with increased PD-L1 in TCs and ICs when the 2 were grouped together ( P < .001). Finally, stage I patients with negative PD-1 and PD-L1 expression showed trends toward increased disease-specific survival. CONCLUSIONS.­: Expression of PD-1 in TILs correlates with PD-L1 expression in both TCs and ICs. Furthermore, negative expression of PD-1 and PD-L1 suggest trends toward disease-specific survival, even in early disease stages.

A Critical Review of Repurposing Apomorphine for Smoking Cessation.

Tobacco use disorder is the leading cause of preventable death and disability in the United States, with one in five Americans currently smoking cigarettes. Only two non-nicotine medications are FDA approved for treating tobacco use disorder, and advances in drug discovery are profoundly outpaced by the morbidity and mortality caused by tobacco dependence. Drug repurposing may provide an approach for addressing this health hazard, offering hope to tobacco users attempting to quit who have failed existing therapies. The focus of this review is to evaluate the potential role of apomorphine (APO) in treating tobacco dependence. Previously described in the literature as a non-specific dopamine agonist effective in treating Parkinson's disease and erectile dysfunction, APO's dopaminergic targeting activity may be effective in counteracting the modified response arising from tobacco use. Here, the literature describing APO's activity is reviewed and presented in the context of known nicotine-induced response in neurotransmitter systems. Based on these data, whether APO may be an effective smoking cessation agent by ameliorating a tobacco user's anhedonic state is critically appraised, along with withdrawal symptoms and the chemical reinforcement associated with drug-seeking behaviors.