Gene‐specific features enhance interpretation of mutational impact on acid α‐glucosidase enzyme activity

We present a computational model for predicting mutational impact on enzymatic activity of human acid α‐glucosidase (GAA), an enzyme associated with Pompe disease. Using a model that combines features specific to GAA with other general evolutionary and physiochemical features, we made blind predictions of enzymatic activity relative to wildtype human GAA for >300 GAA mutants, as part of the Critical Assessment of Genome Interpretation 5 GAA challenge. We found that gene‐specific features can improve the performance of existing impact prediction tools that mostly rely on general features for pathogenicity prediction. Majority of the poorly predicted mutants that lower wildtype GAA enzyme activity occurred on the surface of the GAA protein. We also found that gene‐specific features were uncorrelated with existing methods and provided orthogonal information for interpreting the origin of pathogenicity, particular in variants that are poorly predicted by existing general methods. Specific variants in GAA, when investigated in the context of its protein structure, suggested gene‐specific information like the disruption of local backbone torsional geometry and disruption of particular sidechain‐sidechain hydrogen bonds as some potential sources for pathogenicity.     

Functional interrogation of Lynch syndrome‐associated MSH2 missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in‐cellulo functional assay in which we engineered site‐specific MSH2 VUS using clustered regularly interspaced short palindromic repeats‐Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild‐type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell‐based assay system for functional testing of MMR gene VUS will facilitate the identification of high‐risk LS patients.     

Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc‐1‐Phosphotransferase

The lysosomal storage disorder ML III γ is caused by defects in the γ subunit of UDP‐GlcNAc:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase, the enzyme that tags lysosomal enzymes with the mannose 6‐phosphate lysosomal targeting signal. In patients with this disorder, most of the newly synthesized lysosomal enzymes are secreted rather than being sorted to lysosomes, resulting in increased levels of these enzymes in the plasma. Several missense mutations in GNPTG, the gene encoding the γ subunit, have been reported in mucolipidosis III γ patients. However, in most cases, the impact of these mutations on γ subunit function has remained unclear. Here, we report that the variants c.316G>A (p.G106S), c.376G>A (p.G126S), and c.425G>A (p.C142Y) cause misfolding of the γ subunit, whereas another variant, c.857C>T (p.T286M), does not appear to alter γ subunit function. The misfolded γ subunits were retained in the ER and failed to rescue the lysosomal targeting of lysosomal acid glycosidases.     

Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic,clinical, and biological implications

Mucopolysaccharidosis (MPS) types IIIA, B, C, and D are a group of autosomal recessive lysosomal storage diseases caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. The progressive lysosomal storage of heparan sulfate eventually results in the clinical onset of disease, which is predominantly characterized by severe central nervous system degeneration. MPS‐IIIA and MPS‐IIIB involve deficiencies of heparan sulfate sulfamidase (SGSH) and α‐N‐acetylglucosaminidase (NAGLU), respectively. Both the SGSH and NAGLU genes have been cloned and characterized, thereby permitting mutation analysis of MPS‐IIIA and MPS‐IIIB patients. A total of 62 mutations have now been defined for MPS‐IIIA consisting of 46 missense/nonsense mutations, 15 small insertions/deletions, and one splice site mutation. A total of 86 mutations have been identified in the NAGLU gene of MPS‐IIIB patients; 58 missense/nonsense mutations, 27 insertions/deletions, and one splice site mutation. Most of the identified mutations in the SGSH and NAGLU genes are associated with severe clinical phenotypes. Many of the missense, nonsense, and insertion/deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on SGSH and NAGLU activity and intracellular processing and trafficking. For MPS‐IIIA and MPS‐IIIB many of the reported mutations are unique making screening the general population difficult. However, molecular characterization of MPS‐IIIA patients has revealed a high incidence of particular mutations of different geographical origins, which will be beneficial for the molecular diagnosis of MPS‐IIIA. Hum Mutat 18:264–281, 2001. © 2001 Wiley‐Liss, Inc.     

Characterization of new arylsulfatase A gene mutations reinforces genotype‐phenotype correlation in metachromatic leukodystrophy

Metachromatic Leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The disease manifests itself with a broad spectrum of clinical variants, all characterized by progressive neurodegeneration in the central and peripheral nervous systems. The correlation between mutations in the ARSA gene, residual enzymatic activity associated with the mutated alleles and patients' phenotype, which has been extensively drawn for common ARSA mutations, has recently been expanded to rare ones. In this context, functional studies on the rare allelic variances acquire particular relevance for patients' prognostic evaluation. Here we have characterized eight newly identified ARSA mutations, through lentiviral vector‐based expression studies on cell lines and ARSA defective murine fibroblasts. In each case, the residual activity associated with the new mutant allele correlates well with the patient's phenotype. Therefore, our results confirm the importance of functional characterization of mutant alleles for a precise genotype‐based classification and definition of prognosis in MLD patients, which is particularly relevant for pre‐symptomatic diagnosis. © 2009 Wiley‐Liss, Inc.     

Protective effect of γ‐tocopherol‐enriched diet on N‐methyl‐N‐nitrosourea‐induced epithelial dysplasia in rat ventral prostate

Despite recent advances in understanding the biological basis of prostate cancer (PCa), the management of this disease remains a challenge. Chemoprotective agents have been used to protect against or eradicate prostate malignancies. Here, we investigated the protective effect of γ‐tocopherol on N‐methyl‐N‐nitrosourea (MNU)‐induced epithelial dysplasia in the rat ventral prostate (VP). Thirty‐two male Wistar rats were divided into four groups (n = 8): control (CT): healthy control animals fed a standard diet; control+γ‐tocopherol (CT+γT): healthy control animals without intervention fed a γ‐tocopherol‐enriched diet (20 mg/kg); N‐methyl‐N‐nitrosourea (MNU): rats that received a single dose of MNU (30 mg/kg) plus testosterone propionate (100 mg/kg) and were fed a standard diet; and MNU+γ‐tocopherol (MNU+γT): rats that received the same treatment of MNU plus testosterone and were fed with a γ‐tocopherol‐enriched diet (20 mg/kg). After 4 months, the VPs were excised to evaluate morphology, cell proliferation and apoptosis, as well as cyclooxygenase‐2 (Cox‐2), glutathione‐S‐transferase‐pi (GST‐pi) and androgen receptor (AR) protein expression, and matrix metalloproteinase‐9 (MMP‐9) activity. An increase in the incidence of epithelial dysplasias, such as stratified epithelial hyperplasia and squamous metaplasia, in the MNU group was accompanied by augmented cell proliferation, GST‐pi and Cox‐2 immunoexpression and pro‐MMP‐9 activity. Stromal thickening and inflammatory foci were also observed. The administration of a γ‐tocopherol‐enriched diet significantly attenuated the adverse effects of MNU in the VP. The incidence of epithelial dysplasia decreased, along with the cell proliferation index, GST‐pi and Cox‐2 immunoexpression. The gelatinolytic activity of pro‐MMP‐9 returned to the levels observed for the CT group. These results suggest that γ‐tocopherol acts as a protective agent against MNU‐induced prostatic disorders in the rat ventral prostate.     

Functional and Clinical Consequences of Novel α‐Galactosidase A Mutations in Fabry Disease

Fabry disease (FD) is a rare metabolic disorder of glycosphingolipid storage caused by mutations in the GLA gene encoding lysosomal hydrolase α‐galactosidase A (α‐gal A). Recently, the diagnostic procedure for FD has advanced in several ways, through the development of a specific biomarker (lyso‐Gb3) and the implementation of newborn screenings, which acted as a catalyst to augment general awareness of the disease. Heterologous over‐expression of α‐gal A variants and subsequent in vitro measurement of enzyme activity provided molecular data to elucidate the relationship between mutation, enzyme damage, lyso‐Gb3 biomarker levels, and clinical phenotype. This knowledge is the foundation for improved counseling with regard to prognosis and therapeutic decisions. Herein, we resume the approach of in vitro characterization, with a further 73 mainly novel GLA gene mutations. Patient lyso‐Gb3 data were available for most of the mutations. All mutations were tested for responsiveness to pharmacological chaperone treatment and phenotypic data for 61 hemizygous male and 116 heterozygous female patients carrying a mutation associated with ≥20% residual activity, formerly classified as “mild” variant, were collected in order to evaluate the pathogenicity. We conclude that a mild GLA variant is typically characterized by high residual enzyme activity and normal biomarker levels. We found evidence that these variants can still be classified as a distinctive, but milder, sub‐type of FD.     

D‐2‐hydroxyglutaric aciduria Type I: Functional analysis of D2HGDH missense variants

D‐2‐hydroxyglutaric aciduria Type I (D‐2‐HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D‐2‐HGDH catalytic activity. Site‐directed mutagenesis was used to introduce 31 missense variants in the pCMV5‐D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D‐2‐HGDH enzyme activity was evaluated based on the conversion of [²H₄]D‐2‐HG to [²H₄]2‐ketoglutarate, which was subsequently converted into [²H₄]L‐glutamate and the latter quantified by LC‐MS/MS. Eighteen variants resulted in almost complete ablation of D‐2‐HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.     

BRCA1‐ and BRCA2‐specific in silico tools for variant interpretation in the CAGI 5 ENIGMA challenge

BRCA1 and BRCA2 (BRCA1/2) germline variants disrupting the DNA protective role of these genes increase the risk of hereditary breast and ovarian cancers. Correct identification of these variants then becomes clinically relevant, because it may increase the survival rates of the carriers. Unfortunately, we are still unable to systematically predict the impact of BRCA1/2 variants. In this article, we present a family of in silico predictors that address this problem, using a gene‐specific approach. For each protein, we have developed two tools, aimed at predicting the impact of a variant at two different levels: Functional and clinical. Testing their performance in different datasets shows that specific information compensates the small number of predictive features and the reduced training sets employed to develop our models. When applied to the variants of the BRCA1/2 (ENIGMA) challenge in the fifth Critical Assessment of Genome Interpretation (CAGI 5) we find that these methods, particularly those predicting the functional impact of variants, have a good performance, identifying the large compositional bias towards neutral variants in the CAGI sample. This performance is further improved when incorporating to our prediction protocol estimates of the impact on splicing of the target variant.     

Large‐scale in vitro functional testing and novel variant scoring via protein modeling provide insights into alkaline phosphatase activity in hypophosphatasia

Hypophosphatasia (HPP) is a rare metabolic disorder characterized by low tissue‐nonspecific alkaline phosphatase (TNSALP) typically caused by ALPL gene mutations. HPP is heterogeneous, with clinical presentation correlating with residual TNSALP activity and/or dominant‐negative effects (DNE). We measured residual activity and DNE for 155 ALPL variants by transient transfection and TNSALP enzymatic activity measurement. Ninety variants showed low residual activity and 24 showed DNE. These results encompass all missense variants with carrier frequencies above 1/25,000 from the Genome Aggregation Database. We used resulting data as a reference to develop a new computational algorithm that scores ALPL missense variants and predicts high/low TNSALP enzymatic activity. Our approach measures the effects of amino acid changes on TNSALP dimer stability with a physics‐based implicit solvent energy model. We predict mutation deleteriousness with high specificity, achieving a true‐positive rate of 0.63 with false‐positive rate of 0, with an area under receiver operating curve (AUC) of 0.9, better than all in silico predictors tested. Combining this algorithm with other in silico approaches can further increase performance, reaching an AUC of 0.94. This study expands our understanding of HPP heterogeneity and genotype/phenotype relationships with the aim of improving clinical ALPL variant interpretation.     

Mucolipidosis II‐Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc‐1‐Phosphotransferase Precursor Protein (GNPTAB)

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β‐subunit precursor protein of GlcNAc‐1‐phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α‐subunit‐specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild‐type and mutant α/β‐subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β‐subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc‐1‐phosphotransferase that may help to explain the clinical phenotype of MLII patients.     

β‐Catenin (CTNNB1) mutations and clinicopathological features of mesenteric desmoid‐type fibromatosis

Aims and methods: Desmoid‐type fibromatosis (desmoid) is a fibroblastic tumour that shows locally aggressive growth. Mesenteric desmoid is a rare lesion that shares morphological and biological features with fibromatoses occurring in the abdominal wall or in extraabdominal sites, but differs in terms of gross appearance and clinical presentation. We report on a series of 56 cases of mesenteric desmoids from our consultation files and compare them with cases of non‐mesenteric desmoids and retroperitoneal fibrosis. Results: Primary diagnosis of desmoid‐type fibromatosis was correct in 42%, and gastrointestinal stromal tumour was a common misdiagnosis. Nuclear expression of β‐catenin was detected in 91.6% of all desmoids. Mutational analysis of exon 3 of the β‐catenin gene (CTNNB1) revealed that mesenteric desmoids carried mutations significantly more often (51/56, 91.1%) than non‐mesenteric tumours (20/28; 71.4%; P = 0.027). p.T41A occurred significantly more frequently in mesenteric fibromatoses (80.4%) than in abdominal wall and extra‐abdominal fibromatoses (46.4%; P = 0.002). Two novel mutations (p.S45C and p.D32G) were found. In retroperitoneal fibrosis, mutations and nuclear β‐catenin expression were absent. β‐Catenin‐negative desmoids either carried a CTNNB1 mutation or were associated with Gardner syndrome. Conclusions:  Our study provides evidence that some clinical and genetic features of mesenteric desmoids differ from those of non‐mesenteric fibromatosis, and corroborates the usefulness of mutational analysis, especially in diagnosing β‐catenin‐negative mesenteric desmoids.     

Assessing computational predictions of the phenotypic effect of cystathionine‐beta‐synthase variants

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine‐beta‐synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.     

IFN‐α‐mediated increase in cytolytic activity of maturing NK cell upon exposure to HSV‐infected myelomonocytes

Impaired control of chronic pathogen replication may be associated to alterations of NK‐cell function. Whether mechanisms underlying this dysfunction involve perturbations of differentiating NK cells is still unknown. We studied an “in vitro” model of differentiation from CD34⁺Lin^? precursors growing only myelomonocytes and maturing NK cells and where myelomonocytes could be suitably infected with HSV, HIV, or vaccinia. Cultures were evaluated by cytofluorometry and cytotoxicity assays for perturbations in differentiating NK cells. Increased expression of natural cytotoxicity receptors on maturing NK cells with increased cytolytic activity was observed with HSV‐1 infection, and with vaccinia while no modulation of NK‐cell phenotype nor cytotoxic activity were evident with an ssRNA lentivirus (HIV‐1). In the presence of constant IL‐12 and IL‐15 concentrations, the observed effect did not require cell contact, involved IFN‐αand was not reproduced by the addition of TLR9 agonist, nor blocked by TLR9 antagonists. Virus replication at sites of NK‐cell precursor development may have different outcomes depending on the interaction between invading viruses and maturing NK cells. Thus, NK‐cell precursors may be involved in the immune response to dsDNA viruses and possibly contribute to efficient control of virus infection.     

Functional and structural analysis of rare SLC2A2 variants associated with Fanconi‐Bickel syndrome and metabolic traits

Deleterious variants in SLC2A2 cause Fanconi‐Bickel Syndrome (FBS), a glycogen storage disorder, whereas less common variants in SLC2A2 associate with numerous metabolic diseases. Phenotypic heterogeneity in FBS has been observed, but its causes remain unknown. Our goal was to functionally characterize rare SLC2A2 variants found in FBS and metabolic disease‐associated variants to understand the impact of these variants on GLUT2 activity and expression and establish genotype‐phenotype correlations. Complementary RNA‐injected Xenopus laevis oocytes were used to study mutant transporter activity and membrane expression. GLUT2 homology models were constructed for mutation analysis using GLUT1, GLUT3, and XylE as templates. Seventeen FBS variants were characterized. Only c.457_462delCTTATA (p.Leu153_Ile154del) exhibited residual glucose uptake. Functional characterization revealed that only half of the variants were expressed on the plasma membrane. Most less common variants (except c.593 C>A (p.Thr198Lys) and c.1087 G>T (p.Ala363Ser)) exhibited similar GLUT2 transport activity as the wild type. Structural analysis of GLUT2 revealed that variants affect substrate‐binding, steric hindrance, or overall transporter structure. The mutant transporter that is associated with a milder FBS phenotype, p.Leu153_Ile154del, retained transport activity. These results improve our overall understanding of the underlying causes of FBS and impact of GLUT2 function on various clinical phenotypes ranging from rare to common disease.     

Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study

BRCA1 and BRCA2 screening in women at high‐risk of breast cancer results in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). We examined a population‐based sample of young women with contralateral breast cancer (CBC, n=705) or unilateral breast cancer (UBC, n=1398). We identified 470 unique sequence variants, of which 113 were deleterious mutations. The remaining 357 VUS comprised 185 unique missense changes, 60% were observed only once, while 3% occurred with a frequency of >10%. Deleterious mutations occurred three times more often in women with CBC (15.3%) than in women with UBC (5.2%), whereas combined, VUS were observed in similar frequencies in women with CBC and UBC. A protein alignment algorithm defined 16 rare VUS, occurring at highly conserved residues and/or conferring a considerable biochemical difference, the majority located in the BRCA2 DNA‐binding domain. We confirm a multiplicity of BRCA1 and BRCA2 VUS that occur at a wide range of allele frequencies. Although some VUS inflict chemical differences at conserved residues, suggesting a deleterious effect, the majority are not associated with an increased risk of CBC. © 2010 Wiley‐Liss, Inc.     

Evidence for genetic heterogeneity in D‐2‐hydroxyglutaric aciduria

We performed molecular, enzyme, and metabolic studies in 50 patients with D ‐2‐hydroxyglutaric aciduria (D ‐2‐HGA) who accumulated D ‐2‐hydroxyglutarate (D ‐2‐HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D ‐2‐hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D ‐2‐hydroxyglutarate dehydrogenase (D ‐2‐HGDH). Enzyme assay of D ‐2‐HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D ‐2‐HGA whose enzyme activity was normal did not have mutations. Significantly lower D ‐2‐HG concentrations in body fluids were observed in mutation‐positive D ‐2‐HGA patients than in mutation‐negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D ‐2‐HG. Accordingly, we suggest a new classification: D ‐2‐HGA Type I associates with D ‐2‐HGDH deficiency, whereas idiopathic D ‐2‐HGA manifests with normal D ‐2‐HGDH activity and higher D ‐2‐HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D ‐2‐HGA patients with diverse genetic loci will be revealed in future studies. Hum Mutat 31:1–5, 2010. © 2009 Wiley‐Liss, Inc.     

Reduced immunogenicity of Plasmodium falciparum gamete surface antigen (Pfs48/45) in mice after disruption of disulphide bonds – evaluating effect of interferon‐γ‐inducible lysosomal thiol reductase

Sexual stages of Plasmodium are critical for malaria transmission and stage‐specific antigens are important targets for development of malaria transmission‐blocking vaccines. Plasmodium falciparum gamete surface antigen (Pfs48/45) is important for male gamete fertility and is being pursued as a candidate vaccine antigen. Vaccine‐induced transmission‐blocking antibodies recognize reduction‐sensitive conformational epitopes in Pfs48/45. Processing and presentation of such disulphide‐bond‐constrained epitopes is critical for eliciting the desired immune responses. Mice lacking interferon‐γ‐inducible lysosomal thiol reductase (GILT), an enzyme that mediates reduction of S‐S bonds during antigen processing, were employed to investigate immunogenicity of Pfs48/45. It has been well established that the ability to reduce S‐S bonds in antigens guides effective T‐cell immune responses; however, involvement of GILT in the induction of subsequent B‐cell responses has not been explored. We hypothesized that the ability to reduce S‐S bonds in Pfs48/45 will impact the generation of T‐cell epitopes, and so influence helper T‐cell responses required for specific B‐cell responses. Non‐reduced and reduced and alkylated forms of Pfs48/45 were employed to evaluate immune responses in wild‐type and GILT knockout mice and studies revealed important differences in several immune response parameters, including differences in putative T‐cell epitope recognition, faster kinetics of waning of Pfs48/45‐specific IgG1 antibodies in knockout mice, differential patterns of interferon‐γ and interleukin‐4 secretions by splenocytes, and possible effects of GILT on induction of long‐lived plasma cells and memory B cells responsible for antigen‐recall responses. These studies emphasize the importance of antigen structural features that significantly influence the development of effective immune responses.