Karin Geleijns

HLA class II alleles are not a general susceptibility factor in Guillain–Barré syndrome

Objective: To assess whether human leukocyte antigen (HLA)–DRB1 and HLA-DQB1 alleles confer susceptibility to Guillain-Barré syndrome (GBS) or are related to specific clinical or serologic subgroups of GBS. Methods: The HLA-DRB1 and HLA-DQB1 loci were genotyped by PCR amplification with sequence-specific primers in 164 well-documented Dutch patients with GBS and 207 healthy Dutch control subjects. Patients with GBS were divided into subgroups based on clinical features, severity of disease, antecedent infection, and anti-ganglioside antibodies. Data were compared with those of all case-control HLA studies in GBS performed previously. Results: In this case-control study, HLA-DRB1 and HLA-DQB1 alleles did not differ between GBS patients and control subjects. The frequency of HLA-DRB1*01 was increased in patients who needed mechanical ventilation (odds ratio 4.2; 95% CI 1.9 to 9.6; pc = 0.02). Multivariate logistic regression analysis showed that this association was independent of the severity of paresis and the presence of cranial nerve involvement (all p < 0.05). There was a tendency toward an association between certain HLA alleles and several anti-ganglioside antibodies. Conclusions: Human leukocyte antigen (HLA) class II antigens are not a general susceptibility factor in Guillain-Barré syndrome (GBS). However, HLA class II alleles may be a determinant in distinct subgroups of GBS, indicating the need for further exploration in large-scale studies.

Diagnostic value of anti-GM1 ganglioside serology and validation of the INCAT-ELISA

The Inflammatory Neuropathy and Treatment (INCAT) group developed a standardized ELISA method for the detection of serum anti-GM1 antibodies. The diagnostic value of anti-GM1 antibodies determined by this method has not yet been established in large groups of patients. We assessed the reproducibility, sources of variation, optimal cut-off values and evaluated the diagnostic relevance of the INCAT-ELISA in various groups of patients and controls (N=1232). The coefficient of variance was 11.2% for IgM and 3.8% for IgG. High IgG titers were only found in Guillain-Barré syndrome (GBS) and other inflammatory polyneuropathies. High IgM titers were associated with GBS and multifocal motor neuropathy. Low IgM titers had no additional diagnostic value. The INCAT-ELISA is a reliable test with additional diagnostic value in specific clinical situations.

Mannose-Binding Lectin Contributes to the Severity of Guillain-Barré Syndrome

In Guillain-Barré syndrome (GBS), complement activation plays a crucial role in the induction and extent of the postinfectious immune-mediated peripheral nerve damage. Mannose-binding lectin (MBL) activates the complement system via the lectin pathway after recognition of repetitive sugar groups on pathogens. We investigated whether the MBL2 genotype, serum MBL level, and MBL complex activity are associated with the development and severity of GBS. Single nucleotide polymorphisms in the promoter region (−550 H/L and −221 X/Y) and exon 1 (A/O) of the MBL2 gene were determined in 271 GBS patients and 212 healthy controls. The frequencies of the H allele, HY promoter haplotype, and HYA haplotype, which are related to high MBL activity, were all increased in GBS patients compared with healthy controls (p ≤ 0.03), particularly in severely affected GBS patients (MRC-sum score <40) (p ≤ 0.02). Severe weakness was also associated with high MBL concentrations and MBL complex activity in sera from GBS patients (p < 0.01). The MBL2 B allele was associated with functional deficiency and relatively mild weakness. These results support the hypothesis that complement activation mediated by MBL contributes to the extent of nerve damage in GBS, which is codetermined by the MBL2 haplotype.

Functional polymorphisms in LPS receptors CD14 and TLR4 are not associated with disease susceptibility or Campylobacter jejuni infection in Guillain–Barré patients

Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy preceded by infections. Campylobacter jejuni is the most frequent pathogen and its lipopolysaccharide (LPS) induces antibodies cross-reactive with gangliosides. In this study we assessed whether known functional polymorphisms in the LPS receptors CD14 and Toll-like receptor 4 (TLR4) are associated with an increased susceptibility for GBS or with C. jejuni serology or C. jejuni related clinical and serological features. Comparison of the genotypes of 242 GBS patients and 210 healthy subjects showed that polymorphisms in CD14 and TLR4 did not confer disease susceptibility and were not associated with C. jejuni infection.

Anti–GM1 IgG antibodies induce leukocyte effector functions via Fcγ receptors

Guillain–Barré syndrome (GBS) is an immune‐mediated neuropathy, in which leukocytes and humoral components of the immune system proposedly initiate localized inflammation. An important pathogenic role for anti–GM1 ganglioside antibodies has been suggested. Therefore, we evaluated anti‐GM1 IgG antibody‐induced leukocyte effector functions such as degranulation and phagocytosis using serum of 24 GBS patients. Serum without anti‐GM1 antibodies of 9 GBS patients as well as pooled serum from healthy individuals served as controls. Ten out of 15 (67%) of anti‐GM1 IgG positive sera were capable of inducing leukocyte degranulation, and 8 out of 15 (53%) of anti‐GM1 IgG positive sera were capable of inducing phagocytosis of GM1‐coated beads. In all of these sera anti‐GM1 antibody titers were ≥1:800. No leukocyte degranulation or phagocytosis was observed in control sera. Leukocyte activation was completely abrogated in the presence of IgG receptor (FcγR) blocking antibodies, suggesting a crucial role for leukocyte FcγR in GBS pathogenesis. No correlation of antibody titers with the extent of leukocyte activation, or severity of disease was observed. These data document the capacity of anti‐GM1 IgG antibodies to activate leukocyte inflammatory functions, and suggest an important role for anti‐ganglioside IgG antibodies in the pathogenesis of GBS.

The occurrence of Guillain–Barré syndrome within families

This report describes 12 Dutch families of which at least two members have had Guillain-Barré syndrome (GBS). The authors observed an earlier onset of GBS in successive generations. The occurrence of GBS within families suggests a role for genetic factors in the pathogenesis of GBS.

Subclass IgG to motor gangliosides related to infection and clinical course in Guillain–Barré syndrome

In 176 patients with Guillain-Barré syndrome the subclass and cross-reactivity of serum IgG antibodies to motor gangliosides was related to preceding infections and clinical phenotypes. Two subgroups of patients were identified. Presence of only IgG1 antibodies was related to diarrhea, positive Campylobacter serology, cross-reactive antibodies to C. jejuni lipo-oligosaccharides and poor outcome. In contrast, having both IgG1 and IgG3 antibodies was related to upper respiratory tract infections, cross-reactive antibodies to Haemophilus influenzae lipo-oligosaccharides and better outcome. These findings support a model in which C. jejuni and H. influenzae infections induce two distinct patterns of cross-reactive antibodies with different clinical outcome.

Screening for anti-ganglioside antibodies in hypocretin-deficient human narcolepsy.

Narcolepsy is a sleep disorder caused by defective hypocretin (orexin) neurotransmission. It is thought to result from an autoimmune destruction of hypocretin producing neurons. Recently, low hypocretin levels were found in patients with Guillain-Barré syndrome, a post-infectious immune-mediated disorder in which a variety of circulating antibodies against neuronal gangliosides are found. We therefore considered gangliosides to be candidate antigens in narcolepsy as well, and screened for the presence of a panel of serum anti-ganglioside antibodies in a group of 28 well-characterized narcoleptic patients. We did not find a correlation between increased titers of anti-ganglioside antibodies and hypocretin-deficient narcolepsy. This study does not support the hypothesis that an autoimmune response is involved in narcolepsy. However, as an autoimmune attack may be selective and/or transient, future studies are needed to ultimately refute or confirm the autoimmune hypothesis.

Susceptibility to Guillain–Barré syndrome is not associated with CD1A and CD1E gene polymorphisms

Immune responses to microbial glycolipids that cross-react to neural epitopes may trigger the Guillain-Barré syndrome (GBS). CD1 molecules are involved in antigen presentation of glycolipids and variation in CD1 genes was recently reported to confer susceptibility to develop GBS. This hypothesis was tested by comparing single nucleotide polymorphisms (SNPs) of CD1A and CD1E in 312 well defined GBS patients and 212 healthy controls. SNPs in CD1A and CD1E were not associated with GBS susceptibility, specific clinical subgroups, anti-ganglioside antibodies, antecedent infections and prognosis. Based on this study, CD1 polymorphisms are not a susceptibility or disease modifying factor in GBS.

Telomere length and telomerase complex mutations in pediatric acute myeloid leukemia

Pediatric acute myeloid leukemia (AML) is a heterogeneous malignant disease, characterized by abnormal proliferation and impaired differentiation of myeloid cells. Acquired cytogenetic aberrations such as t(8;21)(q22;q22)/AML-ETO, inv(16)(p13q22)/ CBFb-MYH11, t(15;17)(q22;q21)/PML-RARa, rearrangements involving MLL and molecular aberrations in genes such as FLT3, NPM1, CEBPA and WT1 are involved in AML pathogenesis and are prognostic factors for treatment outcome. Recently, constitutional loss-of-function mutations in telomerase complex genes have been implicated as risk factors for AML in adults. The telomerase complex is expressed in highly proliferative cells, and is responsible for maintaining telomeres, which cap the ends of chromosomes and protect genomic DNA from eroding during cell divisions. Impaired telomerase function can result in extremely short telomeres, limiting the proliferative capacity of progenitor cells, and may lead to chromosomal instability, thus predisposing to malignant transformation. This pathogenesis is manifested in dyskeratosis congenita, an inherited bone marrow (BM) failure syndrome in which patients are at increased risk for malignancies, including AML, and in which telomerase complex mutations are etiologic. To date, the significance of telomerase complex mutations as risk factor for AML in children is unknown. Furthermore, although extremely short telomeres in leukemic cells of adult AML patients have been reported to be associated with cytogenetic aberrations and complex karyotype, the clinical implications of these short telomeres are unknown. In the current study, we assessed the frequency of mutations in the telomerase complex genes TERT, telomerase reverse transcriptase, and TERC, the RNA template of the telomerase complex, in pediatric AML. Furthermore, we measured telomere length in pediatric AML patients at diagnosis and determined its relationship with clinical and molecular characteristics and outcome. The frequency of mutations in TERT and TERC was determined as previously described in diagnostic BM or peripheral blood (PB) samples obtained from a representative cohort (Supplementary Table S1) of 168 de novo pediatric AML cases, who were enrolled in consecutive DCOG, AML-BFM and LAME treatment protocols between 1987 and 2008. Samples contained, after enrichment, 80% or more leukemic cells. No mutations in TERC were detected. In TERT, three non-synonymous variants were present in a total of eight patients (carrier frequency 0.048). In one patient, a novel heterozygous variant in a well-conserved amino acid in exon 2 in the N-terminal extension of TERT, E327D (mRNA NM_198253.2: 1039G/T), was detected. Telomerase activity of this variant, as determined in reconstitution assays in telomerase-deficient VA13 cells, was similar to wild-type TERT (94.7±3.5% (mean±s.d.)). Furthermore, a heterozygous variant in exon 6 in the reverse transcriptase domain of TERT, T726M (mRNA NM_198253.2: 2235C/T), was found in another patient. T726M, located in the reverse transcriptase domain of telomerase, was not associated with reduced telomerase activity. Both mutations were absent among 406 Dutch adult blood donors. Remission material to confirm germline origin of these mutations was not available in the two patients. Six of 168 patients carried a heterozygous variant in the C-terminal extension of TERT, A1062T (mRNA NM_198253.2: 3242G/A; carrier frequency 0.036). A1062T was also the most common gene variant in adult AML (n1⁄4 594; minor allele frequency (MAF) 0.019), with a frequency three times higher than in ethnicity and/or geographically matched controls (n1⁄4 1110; MAF 0.006). In contrast, A1062T was present in a similar carrier frequency in pediatric AML patients (0.048) as in 406 Dutch controls (0.049), composed of two independent cohorts (6/192 (0.031) and 14/214 (0.065)). In three of the six patients carrying A1062T, remission material (o1% blasts by flow cytometry immunophenotyping) was available, confirming germline origin of the variant. Telomerase activity of A1062T was reported normal or decreased to 60% of wild type. Clinical characteristics of patients carrying TERT variants are described in Supplementary Table S2. Other variants, all reported in healthy controls and not associated with reduced telomerase function, were present in this cohort and are described in Supplementary Table S3. MAFs of two synonymous variants and variant A279T in patients were similar in controls, demonstrating comparability of both cohorts (Supplementary Table S3). Our data are in contrast to adult AML patients, in whom constitutional TERT mutations appear to be a risk factor for AML. Although variants E327D and T726M were absent in healthy controls, the relationship of these variants to the etiology of AML is dubious, as the variants showed normal telomerase function when assayed in vitro, karyotype was normal in both patients and telomere lengths of leukemic cells were within the third quartile of the total cohort. Furthermore, the most common variant in our cohort, A1062T, was present in a comparable frequency in geographically matched controls, and thus, at least in our pediatric AML cohort, it cannot be regarded as a pathologic variant. The carrier frequency of A1062T (0.049) in Dutch blood bank donors is remarkably higher than the MAF of 0.006 in controls in the adult AML study, but comparable to a carrier frequency of 0.036 in 600 German controls and 0.031 in 477 white controls from the NCI PLCO Cancer Screening Trial. This might suggest that in individuals of Caucasian descent, or within specific ethnic sub-populations, the A1062T variant may be less pathogenic than previously thought. Although mutations in the telomerase complex genes TERT and TERC are infrequent events in pediatric AML, we investigated whether short telomeres in leukemic cells are associated with AML characteristics or contribute to an adverse outcome. Telomere length was successfully determined by quantitative PCR, as previously described, with some modifications in leukemic cells derived from 167 pediatric AML patients at diagnosis. Median telomere length (T/S ratio) was 0.65 (range: 0.28–2.3). When compared with healthy control PB leukocytes, telomere length in leukemic cells was very short (Figure 1a), confirming previous reports. Because age at diagnosis was not associated with telomere length (Figure 1a), patients were categorized into quartiles based on telomere length without age-adjustment (Table 1). There was no difference in distribution of sex or white blood cell count among the quartiles. When evaluated as a continuous variable, there was no difference in telomere length among FAB types, nor among AML characteristic cytogenetic subgroups (Figures 1b and c). Telomere lengths between prognostic relevant molecular subgroups, determined as described,