Progress in rare inflammatory: Primary immunodeficiency diseases

The field of IEI is rapidly evolving after the introduction of next-generation sequencing (NGS) platforms, such as whole exome sequencing (WES) and whole genome sequencing (WGS). In recent years, the application of these tools has significantly increased the identification of genetic defects causing IEI. This expansion has enhanced our understanding of genotype-phenotype correlation and provided valuable pathogenetic and pathophysiological insights into IEI. However, further studies are needed to complete our understanding, especially in specific geographical populations where studies are conducted rarely such as in South-East Asia region. Furthermore, most modern genetic approaches, such as WES and WGS, are not affordable or available to a large portion of the global population, particularly in low-income developing countries. Even simple initial immunological tests such as immunoglobulin measurement, may not be accessible in such countries. Therefore, there is a need to develop or optimize accessible and affordable tests. Part I of this thesis describes the development of affordable techniques for the diagnosis of IEI. Chapter 2 represents the first study in which WES was applied for genetic testing in the Thai pediatric population suspected of IEI. Importantly, the study revealed novel variants associated with IEIs, which guided to a definite diagnosis and changed the treatment approaches for the tested patients, including the decision to perform the allogeneic stem cell transplantation. The results from this study expand our knowledge of the genotype-phenotype spectrum of IEI. However, utilizing WES and WGS for genetic analysis in a clinical setting has limitations. These techniques are time-consuming, costly, and involve complex data analysis and interpretation. Due to high costs and limited resources, NGS is not widely available in many developing countries. Therefore, there is a need to explore rapid, robust, and inexpensive molecular tools to address this gap. Chapter 3 describes the development of a rapid, low-cost customized microarray for genetic diagnosis in IEI. This technique demonstrated its promising potential as a first-line screening tool in developing countries. Further, the methods for microarray bioinformatics are reviewed in Chapter 4. While modern genetic testing methods provides important insights into the pathophysiology of IEI and guides to the therapeutic approach, the analysis starts with simple tests (“basic to the basics”) in order to investigate immunological changes in patients according to their phenotype. International consensus guidelines recommend prompt measurement of serum immunoglobulin (Ig)G in patients suspected of having an antibody deficiency. However, in many developing countries, even such tests are only available in referral centers and not easily accessible to a substantial number of people. Measurement of total serum globulin levels, routinely assessed in liver biochemistry testing, can be an attractive candidate for screening hypogammaglobulinemia in developing countries. Chapter 5 describes the development of a model that predicts immunoglobulin G levels in children using serum globulin levels combined with the patient's disease status. This model facilitates timely decision-making for initiating immunoglobulin therapy in pediatric patients suspected of hypogammaglobulinemia in developing countries.The presentation of IEI patients can be very diverse with atypical immunological manifestations and non-immunological comorbidities. Part II of the thesis describes different intriguing cases, demonstrating the diverse clinical spectrum of IEI patients. Chapter 6 describes a patient with hypomorphic DOCK8 variants who exhibited a milder phenotype compared to the typical DOCK8-deficient patients. Making a definite diagnosis is a challenge for such a patient. In comparison, DOCK8 deficiency was diagnosed in another more typical patient (chapter 7) who was experiencing recurrent episodes of different types of hematological malignancies, highlighting the role of DOCK8 in immune surveillance of malignancies. Chapter 8 describes the lifelong clinical course of two sisters with identical damaging NBAS variants, who suffered from combined immunodeficiency, autoimmunity, allergy and malignancy. Chapter 9 describes a patient in whom the innate immune system is affected. Genetic analysis revealed damaging FERMT3 variants. The patient was then diagnosed with leukocyte adhesion deficiency (LAD) type III. However, unexpectedly an impairment of the antibody production was found as well in this patient. This was never reported and indicated a defect in the adaptive immune system. Immunoglobulin replacement therapy led to a dramatic clinical improvement in this patient. Chapter 10 provides insights into a patient with congenital glycosylation disorder and a severe immune deficiency. Novel compound heterozygous variants in PGM3 were found. The longstanding history of this patient with immunodeficiency as well as the recovery time of PGM3 activity after a matched related donor stem cell transplantation is discussed.Traditionally, allergy has been regarded as a polygenic disease, influenced by genetic susceptibility in the form of single nucleotide polymorphisms (SNPs) that interact with immunomodulating factors in the environment. However, allergy has been recognized as a frequent immunological comorbidity in IEIs. Recently, a new subcategory of IEIs known as "primary atopic disorders" has been proposed, mainly referring to allergic patients who possess genomic variants contributing to their allergic manifestations. Part III Chapter 11 presents, for the first time, a family with a gain-of-function (GOF) variant in STAT6, causing an early onset severe allergic phenotype. This study provided important pathophysiological insights in patients with monogenic severe allergies early in life and sheds light on the role of STAT6 in allergic diseases.Finally, in Part IV, the general discussion of the entire thesis covers the current perspectives in IEIs. This knowledge expands further the recognition of the clinical spectrum of IEIs and emphasizes the importance of diagnosing IEI in patients who present with unusual clinical symptoms, enabling the implementation of mechanism-based therapeutic approaches.<br/

Progress in rare inflammatory: Primary immunodeficiency diseases

The field of IEI is rapidly evolving after the introduction of next-generation sequencing (NGS) platforms, such as whole exome sequencing (WES) and whole genome sequencing (WGS). In recent years, the application of these tools has significantly increased the identification of genetic defects causing IEI. This expansion has enhanced our understanding of genotype-phenotype correlation and provided valuable pathogenetic and pathophysiological insights into IEI. However, further studies are needed to complete our understanding, especially in specific geographical populations where studies are conducted rarely such as in South-East Asia region. Furthermore, most modern genetic approaches, such as WES and WGS, are not affordable or available to a large portion of the global population, particularly in low-income developing countries. Even simple initial immunological tests such as immunoglobulin measurement, may not be accessible in such countries. Therefore, there is a need to develop or optimize accessible and affordable tests. Part I of this thesis describes the development of affordable techniques for the diagnosis of IEI. Chapter 2 represents the first study in which WES was applied for genetic testing in the Thai pediatric population suspected of IEI. Importantly, the study revealed novel variants associated with IEIs, which guided to a definite diagnosis and changed the treatment approaches for the tested patients, including the decision to perform the allogeneic stem cell transplantation. The results from this study expand our knowledge of the genotype-phenotype spectrum of IEI. However, utilizing WES and WGS for genetic analysis in a clinical setting has limitations. These techniques are time-consuming, costly, and involve complex data analysis and interpretation. Due to high costs and limited resources, NGS is not widely available in many developing countries. Therefore, there is a need to explore rapid, robust, and inexpensive molecular tools to address this gap. Chapter 3 describes the development of a rapid, low-cost customized microarray for genetic diagnosis in IEI. This technique demonstrated its promising potential as a first-line screening tool in developing countries. Further, the methods for microarray bioinformatics are reviewed in Chapter 4. While modern genetic testing methods provides important insights into the pathophysiology of IEI and guides to the therapeutic approach, the analysis starts with simple tests (“basic to the basics”) in order to investigate immunological changes in patients according to their phenotype. International consensus guidelines recommend prompt measurement of serum immunoglobulin (Ig)G in patients suspected of having an antibody deficiency. However, in many developing countries, even such tests are only available in referral centers and not easily accessible to a substantial number of people. Measurement of total serum globulin levels, routinely assessed in liver biochemistry testing, can be an attractive candidate for screening hypogammaglobulinemia in developing countries. Chapter 5 describes the development of a model that predicts immunoglobulin G levels in children using serum globulin levels combined with the patient's disease status. This model facilitates timely decision-making for initiating immunoglobulin therapy in pediatric patients suspected of hypogammaglobulinemia in developing countries.The presentation of IEI patients can be very diverse with atypical immunological manifestations and non-immunological comorbidities. Part II of the thesis describes different intriguing cases, demonstrating the diverse clinical spectrum of IEI patients. Chapter 6 describes a patient with hypomorphic DOCK8 variants who exhibited a milder phenotype compared to the typical DOCK8-deficient patients. Making a definite diagnosis is a challenge for such a patient. In comparison, DOCK8 deficiency was diagnosed in another more typical patient (chapter 7) who was experiencing recurrent episodes of different types of hematological malignancies, highlighting the role of DOCK8 in immune surveillance of malignancies. Chapter 8 describes the lifelong clinical course of two sisters with identical damaging NBAS variants, who suffered from combined immunodeficiency, autoimmunity, allergy and malignancy. Chapter 9 describes a patient in whom the innate immune system is affected. Genetic analysis revealed damaging FERMT3 variants. The patient was then diagnosed with leukocyte adhesion deficiency (LAD) type III. However, unexpectedly an impairment of the antibody production was found as well in this patient. This was never reported and indicated a defect in the adaptive immune system. Immunoglobulin replacement therapy led to a dramatic clinical improvement in this patient. Chapter 10 provides insights into a patient with congenital glycosylation disorder and a severe immune deficiency. Novel compound heterozygous variants in PGM3 were found. The longstanding history of this patient with immunodeficiency as well as the recovery time of PGM3 activity after a matched related donor stem cell transplantation is discussed.Traditionally, allergy has been regarded as a polygenic disease, influenced by genetic susceptibility in the form of single nucleotide polymorphisms (SNPs) that interact with immunomodulating factors in the environment. However, allergy has been recognized as a frequent immunological comorbidity in IEIs. Recently, a new subcategory of IEIs known as "primary atopic disorders" has been proposed, mainly referring to allergic patients who possess genomic variants contributing to their allergic manifestations. Part III Chapter 11 presents, for the first time, a family with a gain-of-function (GOF) variant in STAT6, causing an early onset severe allergic phenotype. This study provided important pathophysiological insights in patients with monogenic severe allergies early in life and sheds light on the role of STAT6 in allergic diseases.Finally, in Part IV, the general discussion of the entire thesis covers the current perspectives in IEIs. This knowledge expands further the recognition of the clinical spectrum of IEIs and emphasizes the importance of diagnosing IEI in patients who present with unusual clinical symptoms, enabling the implementation of mechanism-based therapeutic approaches.<br/

Cost-effectiveness of therapeutic infant formulas for cow's milk protein allergy management

Cow's milk protein allergy (CMPA) is children's most common food allergy. Therapeutic infant formulas for CMPA lead to symptom-free and potentially benefit early tolerance induction and reducing the allergic march in non-breastfed babies. This study assessed the cost-effectiveness of CMPA management with different therapeutic infant formulas in Thailand, which may reflect situations in developing countries throughout Asia. An analytic decision model was developed to simulate the occurrence of eczema, urticaria, asthma, rhinoconjunctivitis, or being symptom-free in infants with CMPA over 36 months. Extensively hydrolyzed casein formula with added probiotic Lacticaseibacillus rhamnosus (previously Lactobacillus rhamnosus) strain GG (EHCF+LGG), extensively hydrolyzed whey formula (EHWF), soy protein-based formula (SPF), and amino acid formula (AAF) were compared from the healthcare payer perspective. The results from a prospective cohort study were used for comparative effectiveness measures, while local experts were interviewed to estimate the healthcare resource used in the management of CMPA. The costs of healthcare resources were obtained from standard, publicly available sources. The direct medical cost of CMPA management was lowest for EHCF+LGG (USD 1,720), followed by SPF (USD 2,090), EHWF (USD 2,791), and AAF (USD 7,881). Compared with other formulas, EHCF+LGG was expected to save USD 370 (SPF), USD 1,071 (EHWF), and USD 6,161 (AAF) in the total cost of CMPA management over 36 months. In conclusion, EHCF+LGG was the most cost-effective strategy for managing non-breastfed infants with CMPA. This strategy was associated with more children developing immune tolerance to cow's milk and being symptom-free, contributing to overall cost-saving potential

Rapid Low-Cost Microarray-Based Genotyping for Genetic Screening in Primary Immunodeficiency

Background: Genetic tests for primary immunodeficiency disorders (PIDs) are expensive, time-consuming, and not easily accessible in developing countries. Therefore, we studied the feasibility of a customized single nucleotide variant (SNV) microarray that we developed to detect disease-causing variants and copy number variation (CNV) in patients with PIDs for only 40 Euros. Methods: Probes were custom-designed to genotype 9,415 variants of 277 PID-related genes, and were added to the genome-wide Illumina Global Screening Array (GSA). Data analysis of GSA was performed using Illumina GenomeStudio 2.0, Biodiscovery Nexus 10.0, and R-3.4.4 software. Validation of genotype calling was performed by comparing the GSA with whole-genome sequencing (WGS) data of 56 non-PID controls. DNA samples of 95 clinically diagnosed PID patients, of which 60 patients (63%) had a genetically established diagnosis (by Next-Generation Sequencing (NGS) PID panels or Sanger sequencing), w

Transition practice for primary immunodeficiency diseases in Southeast Asia: a regional survey

IntroductionWith increased diagnostic capabilities and treatment modalities in the field of primary immunodeficiencies (PID), many pediatric patients survive beyond childhood and experience a change of care to the adult-oriented healthcare system. Unfortunately, the transition pathways for PID are less clearly defined, resulting in deterioration of quality of care in adulthood. Hence, this is the first regional study to address PID clinicians’ opinions on practices and challenges of transition care in 7 Southeast Asia (SEA) countries.MethodsWe adopted a cross-sectional study design through an online survey platform to enquire opinions of transition practices from expert representatives in 7 SEA countries.ResultsRegionally, 3 out 7 countries reported having no practice of transition care. Among cited challenges were reluctant adaptation by patients and caregivers to unfamiliarized adult healthcare systems, inadequate ratio of adult immunologists to patients and lack of facilities for transfer.Discussion and conclusionOur study provides evidence to advocate policy makers on the importance of standardized integration of transition practice towards betterment of transiting PID patients into adulthood

Targeted gene sanger sequencing should remain the first-tier genetic test for children suspected to have the five common X-linked inborn errors of immunity

DATA AVAILABILITY STATEMENT : The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited regions, our centre developed and offered free genetic testing for the most common IEI by Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of IEI were further improved with collaboration among centres caring for IEI patients from East and Southeast Asia. We also started to use whole exome sequencing (WES) for undiagnosed cases and further extended our collaboration with centres from South Asia and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744 identified to have disease-causing mutations (54.1%). The high diagnostic rate after just one round of targeted gene SS for each of the 5 common IEI (X-linked agammaglobulinemia (XLA) 77.4%, Wiskott–Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%) demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common X-linked IEI.The Hong Kong Society for Relief of Disabled Children and Jeffrey Modell Foundation.http://www.frontiersin.org/Immunologyam2023Paediatrics and Child Healt

Cost-effectiveness of therapeutic infant formulas for cow's milk protein allergy management.

Cow's milk protein allergy (CMPA) is children's most common food allergy. Therapeutic infant formulas for CMPA lead to symptom-free and potentially benefit early tolerance induction and reducing the allergic march in non-breastfed babies. This study assessed the cost-effectiveness of CMPA management with different therapeutic infant formulas in Thailand, which may reflect situations in developing countries throughout Asia. An analytic decision model was developed to simulate the occurrence of eczema, urticaria, asthma, rhinoconjunctivitis, or being symptom-free in infants with CMPA over 36 months. Extensively hydrolyzed casein formula with added probiotic Lacticaseibacillus rhamnosus (previously Lactobacillus rhamnosus) strain GG (EHCF+LGG), extensively hydrolyzed whey formula (EHWF), soy protein-based formula (SPF), and amino acid formula (AAF) were compared from the healthcare payer perspective. The results from a prospective cohort study were used for comparative effectiveness measures, while local experts were interviewed to estimate the healthcare resource used in the management of CMPA. The costs of healthcare resources were obtained from standard, publicly available sources. The direct medical cost of CMPA management was lowest for EHCF+LGG (USD 1,720), followed by SPF (USD 2,090), EHWF (USD 2,791), and AAF (USD 7,881). Compared with other formulas, EHCF+LGG was expected to save USD 370 (SPF), USD 1,071 (EHWF), and USD 6,161 (AAF) in the total cost of CMPA management over 36 months. In conclusion, EHCF+LGG was the most cost-effective strategy for managing non-breastfed infants with CMPA. This strategy was associated with more children developing immune tolerance to cow's milk and being symptom-free, contributing to overall cost-saving potential

MetGEMs Toolbox: Metagenome-scale models as integrative toolbox for uncovering metabolic functions and routes of human gut microbiome.

Investigating metabolic functional capability of a human gut microbiome enables the quantification of microbiome changes, which can cause a phenotypic change of host physiology and disease. One possible way to estimate the functional capability of a microbial community is through inferring metagenomic content from 16S rRNA gene sequences. Genome-scale models (GEMs) can be used as scaffold for functional estimation analysis at a systematic level, however up to date, there is no integrative toolbox based on GEMs for uncovering metabolic functions. Here, we developed the MetGEMs (metagenome-scale models) toolbox, an open-source application for inferring metabolic functions from 16S rRNA gene sequences to facilitate the study of the human gut microbiome by the wider scientific community. The developed toolbox was validated using shotgun metagenomic data and shown to be superior in predicting functional composition in human clinical samples compared to existing state-of-the-art tools. Therefore, the MetGEMs toolbox was subsequently applied for annotating putative enzyme functions and metabolic routes related in human disease using atopic dermatitis as a case study