2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Population Pharmacokinetic Modeling and Determination of Individual Exposure to Avalglucosidase Alfa in Adolescent and Adult Patients With Late-Onset Pompe Disease: Analysis of Pooled Data From Phase I to III Clinical Trials.

BACKGROUND: Pompe disease is a rare genetic disorder caused by a deficiency of a lysosomal enzyme called acid alpha-glucosidase and is classified into infantile and late-onset forms. Since 2006, an enzyme replacement therapy involving alglucosidase alfa has been available. In 2021, a new enzyme replacement therapy involving avalglucosidase alfa demonstrated improved clinical benefits. In this article, the authors describe the pharmacokinetics of avalglucosidase alfa using a population pharmacokinetic approach. METHODS: The population pharmacokinetic model was developed using a data set that included 75 patients and 2042 plasma drug concentrations determined through enzymatic activity assay from 3 studies (phases I/II and III) and involved 3 dose levels (5, 10, and 20 mg/kg). The analysis was performed using NONMEM software. RESULTS: Two sequences were observed in the plasma drug concentration profile: the first kinetic driving exposure, and after 12 hours postdose, a slight rebound addressing very low concentrations that lasted up to 2 weeks. Following model screening, a model with a central compartment with parallel linear and nonlinear elimination and 2 concatenated peripheral compartments was proposed. A putative back-redistribution of a marginal fraction of the drug from the second peripheral compartment to the central compartment may explain the slight rebound in concentration. The final model's mean bias and precision for individual predictions were -2.66% and 30.7%, respectively, and -0.433% and 38.9%, respectively, for population predictions. CONCLUSIONS: A concatenated 3-compartment model was developed to describe the avalglucosidase alfa concentrations in patients with late-onset Pompe disease. None of the covariates tested could explain the interindividual variability.

Reverse triage in COVID surge planning: a case study of an allied health supported clinical care pathway in an acute hospital setting.

Objective This case study describes the development and outcomes of a new integrated and multidisciplinary care pathway. Spearheaded by allied health, the 'COVID community navigator team', applied established principles of reverse triage to create additional surge capacity. Methods A retrospective cohort study examined workflow patterns using electronic medical records of patients who received navigator input at the Royal Melbourne Hospital between 20 September 2021 and 20 December 2021. Results There were 437 eligible patient encounters identified. On average patients stayed 4.15 h in the emergency departments (ED) (s.d. = 4.31) and 9.5 h (s.d. = 10.9) in the short stay unit. Most patients were discharged into a 'low risk pathway' with community general practitioner follow up. Of discharged patients, only 38 re-presented to the ED with symptoms related to their initial COVID-19 diagnosis (34.9% of total re-admissions). Of these re-admissions, more than half did not require admission to a ward. Conclusion The findings presented here provide support for the clinical utility of a multidisciplinary reverse triage approach in surge planning for anticipated presentation peaks.

Selective Csp3-F Bond Functionalization with Lithium Iodide.

A highly efficient method for C-F bond functionalization of a broad variety of activated and unactivated aliphatic substrates with inexpensive lithium iodide is presented. Primary, secondary, tertiary, benzylic, propargylic and α-functionalized alkyl fluorides react in chlorinated or aromatic solvents at room temperature or upon heating to the corresponding iodides which are isolated in 91-99% yield. The reaction is selective for aliphatic monofluorides and can be coupled with in situ nucleophilic iodide replacements to install carbon-carbon, carbon-nitrogen and carbon-sulfur bonds with high yields. Alkyl difluorides, trifluorides, even in activated benzylic positions, are inert under the same conditions and aryl fluoride bonds are also tolerated.

Criteria of slope and endogenous level for selection of matrix in the quantitation of endogenous compounds by LC-MS/MS.

Selection of the appropriate matrix for standard curve is critical for an accurate and sensitive biomarker method. Slope of a standard curve is the key factor for parallelism assessment between tested matrix and authentic matrix for LC-MS/MS assays. Here the authors have established slope criteria using a generic equation and endogenous level criteria for achieving assay sensitivity. The slope difference criterion is from -13.0 to +17.6% for LC-MS assays with ± 15% bias criteria. When the ratio of endogenous concentration in the tested matrix to the lower limit of quantitation is <4.0, the lower limit of quantitation is achievable. If these criteria are met, the tested matrix can be used for assays. The utility of the two criteria has been illustrated with case studies.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Copper(ii) ketimides in sp3 C-H amination.

Commercially available benzophenone imine (HN[double bond, length as m-dash]CPh2) reacts with ß-diketiminato copper(ii) tert-butoxide complexes [CuII]-O t Bu to form isolable copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2. Structural characterization of the three coordinate copper(ii) ketimide [Me3NN]Cu-N[double bond, length as m-dash]CPh2 reveals a short Cu-Nketimide distance (1.700(2) Å) with a nearly linear Cu-N-C linkage (178.9(2)°). Copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2 readily capture alkyl radicals R˙ (PhCH(˙)Me and Cy˙) to form the corresponding R-N[double bond, length as m-dash]CPh2 products in a process that competes with N-N coupling of copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2 to form the azine Ph2C[double bond, length as m-dash]N-N[double bond, length as m-dash]CPh2. Copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CAr2 serve as intermediates in catalytic sp3 C-H amination of substrates R-H with ketimines HN[double bond, length as m-dash]CAr2 and t BuOO t Bu as oxidant to form N-alkyl ketimines R-N[double bond, length as m-dash]CAr2. This protocol enables the use of unactivated sp3 C-H bonds to give R-N[double bond, length as m-dash]CAr2 products easily converted to primary amines R-NH2 via simple acidic deprotection.

Development of Rabbit Monoclonal Antibodies for Quantitation of Therapeutic Human Antibodies in Preclinical Non-Human Primate Studies.

During preclinical studies, there is a great need to develop monoclonal antibodies (mAbs) that are specific to human immunoglobulin (IgG), without binding to monkey IgG, to detect therapeutic human mAb in non-human primates. We took advantage of the latest rabbit B cell cloning technology to develop six unique rabbit anti-human IgG mAb clones for this purpose. These clones are capable of binding to both human IgG and Fab with high affinity without nonspecific binding to cynomolgus monkey IgG. These clones have been evaluated as a generic capture reagent for the detection of human IgG and Fab, in the presence of cynomolgus monkey serum, by Gyrolab™ immunoassay. They may be used in singlet or as pairs for the detection of human IgG, in any host animal, to meet the need for therapeutic mAb development in preclinical studies.

Using Organizational Philosophy to Create a Self-Sustaining Compensation Plan Without Harming Academic Missions.

PROBLEM: Academic physician reimbursement has moved to productivity-based compensation plans. To be sustainable, such plans must be self-funding. Additionally, unless research and education are appropriately valued, faculty involved in these efforts will become disillusioned, yet revenue generation in these activities is less robust than for clinical care activities. APPROACH: Faculty at the Department of Medicine, University of Florida Health, elected a committee of junior and senior faculty and division chiefs to restructure the compensation plan in fiscal year (FY) 2011. This committee was charged with designing a new compensation plan based on seven principles of organizational philosophy: equity, compensation coupled to productivity, authority aligned with responsibility, respect for all academic missions, transparency, professionalism, and self-funding in each academic mission. OUTCOMES: The new compensation plan was implemented in FY2013. A survey administered at the end of FY2015 showed that 61% (76/125) of faculty were more satisfied with this plan than the previous plan. Since the year before implementation, clinical relative value units per faculty increased 7% (from 3,458 in FY2012 to 3,704 in FY2015, P < .002), incentives paid per faculty increased 250% (from $3,191 in FY2012 to $11,153 in FY2015, P ≤ .001), and publications per faculty increased 15% (from 2.6 in FY2012 to 3.0 in FY2015, P < .001). Grant submissions, external funding, and teaching hours also increased per faculty but did not reach statistical significance. NEXT STEPS: An important next step will be to incorporate quality metrics into the compensation plan, without affecting costs or throughput.

The influence of a polymorphism in the gene encoding angiotensin converting enzyme (ACE) on treatment outcomes in late-onset Pompe patients receiving alglucosidase alfa.

Correlations between angiotensin-converting enzyme (ACE) genotype (I/I, I/D, D/D), disease severity at baseline and response to enzyme replacement therapy (ERT) were assessed in the Pompe disease Late-Onset Treatment Study (LOTS). No correlations were observed between ACE genotype and disease severity at baseline. However, D/D patients appeared to have a reduced response to alglucosidase alfa treatment than I/I or I/D patients, suggesting that ACE polymorphisms may influence the response to alglucosidase alfa treatment and warrants further investigation.

Immunotoxicology: fifty years of global scientific progress.

The Immunotoxicology Specialty Section of the Society of Toxicology (SOT) celebrated the 50(th) Anniversary of the SOT by constructing a poster to highlight the milestones of Immunotoxicology during that half-century period. This poster was assembled by an ad hoc committee and intertwines in words, citations, graphics, and photographs our attempts to capture a timeline reference of the development and progressive movement of immunotoxicology across the globe. This poster was displayed during the 50(th) Annual SOT Meeting in Washington DC in March, 2011. The poster can be accessed by any Reader at the SOT Website via the link http://www.toxicology.org/AI/MEET/AM2011/posters_rcsigss.asp#imss. We dedicate this poster to all of the founders and the scientists that followed them who have made the discipline of Immunotoxicology what it is today.

One- and two-year outcomes and predictors of mortality following emergency laparotomy: a consecutive series from a United Kingdom teaching hospital.

BACKGROUND: Data on outcomes of patients who underwent emergency laparotomy (EML) are limited. This prospective observational study examined aspects of inpatient care and outcomes following EML with a view to identifying predictors of mortality. METHODS: Data collected from consecutive inpatients who underwent EML in a UK teaching hospital over a 3-month period included perioperative physiology, treatment, morbidity, and mortality (30-day, in-hospital, 12-month, and 24-month). Univariate and multiple logistic regression analyses were used to identify predictors of mortality. RESULTS: Eighty-five patients (44 male) with a mean ± SD age of 61 ± 18 years were studied. Postoperatively, 51 % of patients were admitted to the intensive care (ICU) or the high-dependency unit (HDU). 30-day, in-hospital, 12-month, and 24-month mortality was 14, 16.5, 22.4, and 25.9 %, respectively. After adjusting for confounding variables, age ≥70 years (odds ratio [OR] = 9.2, P = 0.004) and a need for postoperative ICU/HDU (OR = 15.0, P = 0.014) were independent predictors of 30-day mortality. Independent predictors of in-hospital mortality were age ≥70 years (OR = 18.2, P = 0.016), ASA ≥III (OR = 22.1, P = 0.034), preoperative sepsis (OR = 20.6, P = 0.045), and need for postoperative ICU/HDU (OR = 21.5, P = 0.038). Independent predictors of 12-month mortality were preoperative urea >7.5 mmol/L (OR = 3.5, P = 0.038) and need for postoperative ICU/HDU (OR = 3.7, P = 0.044). Age ≥70 years was the only independent predictor of 24-month mortality (OR = 4.5, P = 0.014). Almost all deaths recorded in the 24 months following surgery resulted from disseminated malignancy. CONCLUSION: Patients who underwent EML had favourable outcomes, with 2-year survival close to 75 %. Age ≥70 years and the need for postoperative ICU/HDU care were independent predictors of mortality.

Antibacterial agent triclosan suppresses RBL-2H3 mast cell function.

Triclosan is a broad-spectrum antibacterial agent, which has been shown previously to alleviate human allergic skin disease. The purpose of this study was to investigate the hypothesis that the mechanism of this action of triclosan is, in part, due to effects on mast cell function. Mast cells play important roles in allergy, asthma, parasite defense, and carcinogenesis. In response to various stimuli, mast cells degranulate, releasing allergic mediators such as histamine. In order to investigate the potential anti-inflammatory effect of triclosan on mast cells, we monitored the level of degranulation in a mast cell model, rat basophilic leukemia cells, clone 2H3. Having functional homology to human mast cells, as well as a very well defined signaling pathway leading to degranulation, this cell line has been widely used to gain insight into mast-cell driven allergic disorders in humans. Using a fluorescent microplate assay, we determined that triclosan strongly dampened the release of granules from activated rat mast cells starting at 2 µM treatment, with dose-responsive suppression through 30 µM. These concentrations were found to be non-cytotoxic. The inhibition was found to persist when early signaling events (such as IgE receptor aggregation and tyrosine phosphorylation) were bypassed by using calcium ionophore stimulation, indicating that the target for triclosan in this pathway is likely downstream of the calcium signaling event. Triclosan also strongly suppressed F-actin remodeling and cell membrane ruffling, a physiological process that accompanies degranulation. Our finding that triclosan inhibits mast cell function may explain the clinical data mentioned above and supports the use of triclosan or a mechanistically similar compound as a topical treatment for allergic skin disease, such as eczema.

Negotiated dying: a grounded theory of how nurses shape withdrawal of treatment in hospital critical care units.

BACKGROUND: The process of withdrawal of treatment in critical care environments has created ethical and moral dilemmas in relation to end of life care in the UK and elsewhere. Common within this discourse is the differing demands made on health professionals as they strive to provide care for the dying patient and family members. Despite reports that withdrawal of treatment is a source of tension between those nurses and doctors involved in the process, the role of the nurse in facilitating withdrawal of treatment has received relatively little attention. OBJECTIVES: To illustrate how differing dying trajectories impact on decision-making underpinning withdrawal of treatment processes, and what nurses do to shape withdrawal of treatment. DESIGN: Qualitative methods of enquiry using clinical vignettes and applying Charmaz's grounded theory method. METHODS AND SETTINGS: Single audio-recorded qualitative interviews with thirteen critical care nurses from four intensive care specialities: cardiac; general; neurological and renal were carried out. Interviews were facilitated by an end-of-life vignette developed with clinical collaborators. FINDINGS: Across critical care areas four key dying trajectories were identified. These trajectories were shaped by contested boundaries associated with delayed or stalled decision-making around how withdrawal of treatment should proceed. Nurses provided end of life care (including collaborative and action-oriented skills) to shape the dying trajectory of patients so as to satisfy the wishes of the patient and family, and their own professional aims. CONCLUSIONS: Differing views as to when withdrawal of treatment should commence and how it should be operationalised appeared to be underpinned by the requirements of the role that health professionals fulfil, with doctors focusing on making withdrawal of treatment decisions, and nurse's being tasked with operationalising the processes that constitute it. Multidisciplinary teams need a 'shared' understanding of each other's roles, responsibilities, aims, and motivations when planning and implementing the dying trajectory of withdrawal of treatment.

Inorganic arsenite inhibits IgE receptor-mediated degranulation of mast cells.

Millions of people worldwide are exposed to arsenic (As), a toxicant which increases the risk of various cancers, cardiovascular disease and several other health problems. Arsenic is a potent endocrine disruptor, including of the estrogen receptor. It was recently shown that environmental estrogen-receptor disruptors can affect the signaling of mast cells, which are important players in parasite defense, asthma and allergy. Antigen (Ag) or allergen crosslinking of IgE-bound receptors on mast cells leads to signaling, culminating in degranulation, the release of histamine and other mediators. Because As is an endocrine disruptor and because endocrine disruptors have been found to affect degranulation, here we have tested whether sodium arsenite affects degranulation. Using the rat basophilic leukemia (RBL) mast cell model, we have measured degranulation in a fluorescence assay. Arsenic alone had no effect on basal levels of degranulation. However, As strongly inhibited Ag-stimulated degranulation at environmentally relevant concentrations, in a manner that is very dependent on concentrations of both As and Ag. The concentrations of As effective at inhibiting degranulation were not cytotoxic. This inhibition may be a mechanism underlying the traditional Chinese medicinal use of As to treat asthma. These data indicate that As may inhibit the ability of humans to fight off parasitic disease.

Pompe disease in a Brazilian series: clinical and molecular analyses with identification of nine new mutations.

Pompe disease (glycogen storage disease type II or acid maltase deficiency) is an inherited autosomal recessive deficiency of acid alpha-glucosidase (GAA), with predominant manifestations of skeletal muscle weakness. A broad range of studies have been published focusing on Pompe patients from different countries, but none from Brazil. We investigated 41 patients with either infantile-onset (21 cases) or late-onset (20 cases) disease by muscle pathology, enzyme activity and GAA gene mutation screening. Molecular analyses identified 71 mutant alleles from the probands, nine of which are novel (five missense mutations c.136T > G, c.650C > T, c.1456G > C, c.1834C > T, and c.1905C > A, a splice-site mutation c.1195-2A > G, two deletions c.18_25del and c.2185delC, and one nonsense mutation c.643G > T). Interestingly, the c.1905C > A variant was detected in four unrelated patients and may represent a common Brazilian Pompe mutation. The c.2560C > T severe mutation was frequent in our population suggesting a high prevalence in Brazil. Also, eight out of the 21 infantile-onset patients have two truncating mutations predicted to abrogate protein expression. Of the ten late-onset patients who do not carry the common late-onset intronic mutation c.-32-13T > G, five (from three separate families) carry the recently described intronic mutation, c.-32-3C > A, and one sibpair carries the novel missense mutation c.1781G > C in combination with known severe mutation c.1941C > G. The association of these variants (c.1781G > C and c.-32-3C > A) with late-onset disease suggests that they allow for some residual activity in these patients. Our findings help to characterize Pompe disease in Brazil and support the need for additional studies to define the wide clinical and pathological spectrum observed in this disease.

Update of the Pompe disease mutation database with 107 sequence variants and a format for severity rating.

Pompe disease was named after the Dutch pathologist Dr JC Pompe who reported about a deceased infant with idiopathic hypertrophy of the heart. The clinical findings were failure to thrive, generalized muscle weakness and cardio-respiratory failure. The key pathologic finding was massive storage of glycogen in heart, skeletal muscle and many other tissues. The disease was classified as glycogen storage disease type II and decades later shown to be a lysosomal disorder caused by acid alpha-glucosidase deficiency. The clinical spectrum of Pompe disease appeared much broader than originally recognized. Adults with the same enzyme deficiency, alternatively named acid maltase deficiency, were reported to have slowly progressive skeletal muscle weakness and respiratory problems, but no cardiac involvement. The clinical heterogeneity is largely explained by the kind and severity of mutations in the acid alpha-glucosidase gene (GAA), but secondary factors, as yet unknown, have a substantial impact. The Pompe disease mutation database aims to list all GAA sequence variations and describe their effect. This update with 107 sequence variations (95 being novel) brings the number of published variations to 289, the number of non-pathogenic mutations to 67 and the number of proven pathogenic mutations to 197. Further, this article introduces a tool to rate the various mutations by severity, which will improve understanding of the genotype-phenotype correlation and facilitate the diagnosis and prognosis in Pompe disease.

Pompe disease (glycogen storage disease type II) in Argentineans: clinical manifestations and identification of 9 novel mutations.

Pompe disease is an autosomal recessive disorder caused by a deficiency in 1,4-alpha-glucosidase (EC.3.2.1.3), the enzyme required to hydrolyze lysosomal glycogen to glucose. While previous studies have focused on Pompe patients from Europe, the United States, and Taiwan, we have analyzed a group of South American Pompe patients to better understand the molecular basis of their disease. From 14 Argentinean patients diagnosed with either infantile or late-onset disease, we identified 14 distinct mutations in the acid alpha-glucosidase (GAA) gene including nine novel variants (c.236_246del, c.377G>A, c.1099T>C, c.1397T>G, c.1755-1G>A, c.1802C>G, c.1978C>T, c.2281delGinsAT, and c.2608C>T). Three different families displayed the c.377G>A allelic variant, suggesting a higher frequency among a subset of Argentineans. Comparison of patients with similar or identical variations in the GAA gene highlights the phenotypic diversity of late-onset disease and supports a role for other genetic and environmental factors in disease presentation.