Clinical and Molecular Analysis of 49 Patients With X-linked Agammaglobulinemia From A Single Center in Argentina

Introduction  Argentina has a large number of patients with definite diagnosis of X-linked agammaglobulinemia reported in the Latin-American registry. Forty-nine of them were seen in our referral pediatric hospital, between 1987 and 2005. Results and Discussion  A retrospective study of clinical, laboratory, and molecular data showed that respiratory tract infections were the most frequent initial clinical presentation and the most common among all manifestations prior to diagnosis (69%). Up to diagnosis, we found a high frequency of severe infections (sepsis, 14% and meningitis, 16%) and a high proportion of patients with chronic lung disease. During follow-up, the development of chronic lung disease was significantly related with age at diagnosis and inappropriate treatment. Conclusion  Although molecular diagnosis has been available in our center for the past 10 years, there is no doubt that awareness for early recognition of immunodeficiency should be improved through broader and more comprehensive education programs emphasizing characteristics of patients with immunodeficiencies. Natalia Basile and Silvia Danielian contributed equally to this work     

Mutation of the <Emphasis Type="Italic">BTK</Emphasis> Gene and Clinical Feature of X-Linked Agammaglobulinemia in Mainland China

Introduction  X-Linked agammaglobulinemia is a prototypical humoral immunodeficiency with the mutation of the Bruton’s tyrosine kinase gene. Methods  We investigated the gene mutation and clinical features of 30 Chinese X-linked agammaglobulinemia (XLA) patients from 27 families. There were 26 mutations, including 11 novel and 15 recurrent mutations, distributing over the entire gene. The nucleotide and amino acid aberration, 1129C>T(H333Y) and 1196T>A(I355N), in SH2 have not been reported before. Five (I355N, W124R, R520X, I590F, G594E) of the 24 mutations not detected in the mothers receiving gene analysis were determined to be de novo. Two mutations occurred within intronic splice-site sequences (intron5(−2)A>G, intron17(−2)A>T). Results and Discussion  There are eight mutations in the PH domain, two mutations in the SH3 domain, three mutations in the SH2 domain, one mutation in the TH domain, and other 16 mutations in the TK domain. The mutations of protein domain is most common in TK (53%) domain and then in PH(8%) domain. Missense and nonsense mutations were found equal in 46% of the detected mutations. All of the patients are alive, but one died of liver cancer. Clinical features and serum Igs levels range variedly and were not correlated with genotypes. Our results demonstrated molecular genetic characteristics of XLA in mainland China.     

Safety and Efficacy of Privigen®, a Novel 10% Liquid Immunoglobulin Preparation for Intravenous Use,in Patients with Primary Immunodeficiencies

Purpose

The present study was designed to evaluate the efficacy and safety of a novel, 10% liquid formulation of intravenous immunoglobulin, stabilized with 250 mmol/L l-proline (Privigen®), in patients with primary immunodeficiency disease.

Materials and Methods

Eighty adults and children diagnosed with common variable immunodeficiency or X-linked agammaglobulinemia received intravenous Privigen® infusions (200–888 mg/kg) at 3- or 4-week intervals over a 12-month period, according to their previously established maintenance dose. The primary endpoint was the annual rate of acute serious bacterial infections.

Results

There were six episodes of acute serious bacterial infections, corresponding to an annual rate of 0.08; the annual rate for all infections was 3.55. Mean serum IgG trough levels were between 8.84 and 10.27 g/L. A total of 1,038 infusions were administered, most of them at the maximum rate permitted (8.0 mg kg^?1 min^?1). Temporally associated adverse events, possibly or probably related to study drug, occurred in 9% of infusions, either during or within 72 h after infusion end.

Conclusion

Privigen® is well tolerated and effective for the treatment of primary immunodeficiency.

     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X‐linked agammaglobulinemia

X‐linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B‐cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800‐1821del, 1843‐1847del, 1288‐1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA. Hum Mutat 18:356, 2001. © 2001 Wiley‐Liss, Inc.     

Cohort of Iranian Patients with Congenital Agammaglobulinemia: Mutation Analysis and Novel Gene Defects

Objectives: Impairment in early B-cell development can cause a predominantly antibody deficiency with severe depletion of peripheral B-cells. Mutations in the gene encoding for Bruton’s-tyrosine-kinase (BTK) and the components of the pre-B-cell receptor complex or downstream signaling molecules have been related to this defect in patients with agammaglobulinemia.

Methods: Iranian patients with congenital agammaglobulinemia were included and the correlation between disease-causing mutations and parameters such as clinical and immunologic phenotypes were evaluated in available patients.

Results: Out of 87 patients, a molecular investigation was performed on 51 patients leading to identification of 39 cases with BTK (1 novel mutation), 5 cases of µ-heavy chain (3 novel mutations) and 1 case of Igα-deficiencies.

Conclusion: Although there is no comprehensive correlation between type of responsible BTK mutation and severity of clinical phenotype, our data suggest that BTK-deficient and autosomal recessive agammaglobulinemia patients differ significantly regarding clinical/immunologic characteristics.     

Molecular analysis of Bruton’s tyrosine kinase gene in Spain

Mutations in Bruton’s tyrosine kinase (BTK) gene result in X linked agammaglobulinemia (XLA). Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing 21 mutations were found in 27 patients with an XLA phenotype from 21 unrelated families. We identified 13 novel and 8 known mutations: seven missense (R288W, R544G, P566S, K430E; K374N, L512P, R544S), 5 nonsense (Q196X, Y361X, L249X, Q612X, Q466X), 2 deletions of one nucleotide (A207fsX216, Q612fsX648), 2 deletion‐insertions (V219fsX227, K218fsX228), one insertion of two nucleotides (S572fsX587) and 4 point mutations in donor/acceptor splice sites (g.IVS1+1G>C, g.IVS6+5G>A, g.IVS10+1G>T, g.IVS13‐1GG>CT). Carrier detection was performed in 18 mothers. Only in one case the mutation was found to be de novo. Additionally, BTK mutations were not found in four patients without family history, but with XLA‐compatible phenotype. Hum Mutat 18:84, 2001. © 2001 Wiley‐Liss, Inc.     

Detection of a novel mutation in the SRC homology domain 2 (SH2) of Bruton's tyrosine kinase and direct female carrier evaluation in a family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease with a block in differentiation from pre-B to B cells resulting in a selective defect in the humoral immune response. Affected males have very low concentrations of serum immunoglobulins leading predominantly to recurrent bacterial infections beginning at age 6 to 18 months. The gene responsible for XLA was identified recently to encode a cytoplasmatic tyrosine kinase (Bruton's tyrosine kinase, BTK). We have analyzed the BTK gene in a large family in which two brothers presented with the severe phenotype of XLA. Genomic DNA of affected boys and from healthy relatives was amplified by PCR with primers specific for the putative promoter region and for all 19 exons, including flanking intron boundaries, and subsequently screened for mutations using single strand conformation polymorphism (SSCP) analysis. Altered single strand band patterns were found using primers specific for exon 10, 15, and 18. Direct cycle-sequencing of these BTK segments detected two known polymorphisms in intron 14 and in exon 18. Sequencing of exon 10 from two boys with XLA demonstrated a novel point mutation in the SH2 domain of BTK. Direct identification of healthy female carriers in three generations was performed by amplification mutagenesis using PCR with a modified first primer. This method can easily be applied also to prenatal diagnosis. © 1996 Wiley-Liss, Inc.     

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains

Background: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton’s Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations.

Methods: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient.

Results: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes.

Conclusions: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.     

Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.     

Progressive neurodegeneration in patients with primary immunodeficiency disease on IVIG treatment.

We have identified 14 patients with diverse primary immunodeficiencies who have developed progressive neurodegeneration of unknown etiology. All patients had received immunoglobulin replacement therapy for a mean duration of 6.5 years (range of 0.5-13.5 years) at the time of first neurological symptoms. Diagnostic tests of blood and cerebrospinal fluid analyses included chemistry, cultures, PCR for viral genomes, and cytology. In addition, neuroimaging and electrophysiologic studies were performed. Brain tissue histology (n = 5) revealed nonspecific encephalitis with microglial infiltration and neuronal loss. Twelve patients died 6 months to 15 years (median 4.3 years) after onset of neurologic findings. No evidence of any infectious disease that could have explained our patients' progressive encephalopathy was found either during their lifetimes or postmortem. These patients may have had an unusual manifestation of primary immunodeficiency diseases, an autoimmune reaction against neuronal tissue, a yet undefined infectious agent, or a complication of IVIG therapy. To help determine the etiology of this rare complication, an international surveillance system for primary immunodeficiency patients who develop progressive neurodegeneration of unknown cause is recommended.     

Polyclonal B-Cell Expansion in the Cerebrospinal Fluid of Patients with Psedotumor Cerebri

To investigate the hypothesis that pseudotumor cerebri (PTC) is associated with humoral immunity, we analyzed immunoglobulin heavy chain variable region (Ig-V_H) genes of B cells in the cerebrospinal fluid (CSF) of 10 patients with PTC. Using RT-PCR and sequencing techniques, intrathecal B-cell Ig-V_H genes were amplified in 6 of 10 PTC samples. Sequence analysis of complementarity-determining region 3 (CDR 3) and V_H genes revealed a polyclonal intrathecal B-cell expansion in these patients. The nucleotide sequences showed that one-third of analyzed sequences had a high replacement to silent nucleotide substitution ratio, indicating an antigen-driven T-cell-dependent intrathecal B-cell proliferation. Moreover, other one-third had germline V_H genes without or with a few nucleotide mutations, suggesting a T-cell-independent natural B-cell-mediated humoral immunity in the CNS of these patients. Our results suggest that both T-cell-dependent and T-cell-independent humoral immunity are present in the CSF of PTC.     

Evaluation of Serum Galactomannan Enzyme Immunoassay at Two Different Cut-offs for the Diagnosis of Invasive Aspergillosis in Patients with Febrile Neutropenia

Background: Invasive aspergillosis (IA) is an increasingly common and fatal opportunistic fungal infection in patients with haematological diseases. Early diagnosis is difficult as mycological culture techniques have low sensitivity and the radiological tools have low specificity. Galactomannan enzyme immunoassay (GEI) detects galactomannan in the human serum with a reported sensitivity and specificity between 30% and 100%. Aims: The aim of this study was to analyse the role of GEI in diagnosis of IA in patients with febrile neutropenia and to evaluate the role of GEI in the diagnosis of IA as per the revised (2008) European Organization for Research and Treatment of Cancer–Mycoses Study Group (EORTC–MSG) criteria at two different optical density (OD) cut-offs of 0.5 and 1.0. Setting: This prospective study was conducted in Safdarjung Hospital, New Delhi, India. Methods: GEI testing was performed in adult patients of febrile neutropenia with evidence of IA. Results at two different OD indices (ODIs) of 0.5 and 1.0 were analysed. The evaluation of the diagnostic parameter, that is, GEI was measured in terms of sensitivity, specificity and positive and negative predictive value and was validated with the revised (2008) EORTC–MSG diagnostic criteria of IA. Results: One hundred and eleven patients had evidence of IA, of which 79 patients were GEI positive when cut-off ODI was 0.5, whereas with cut-off ODI 1.0, 55 patients were GEI positive. Conclusion: ODI of 1.0 should be considered as positive while in patients with OD between 0.5 and 1.0, repeat sampling from the patient is recommended.     

A recurrent 1992delCT mutation of the type X collagen gene in a Japanese patient with Schmid metaphyseal chondrodysplasia

Summary We report here a recurrent frameshift mutation within the carboxyl-terminal noncollagenous domain coding region of the type X collagen gene (COL10A1) in a Japanese patient with Schmid metaphyseal chondrodysplasia. The mutation involves deletion of a CT dinucleotide from position 1992 (1992delCT), and produces a frameshift which creates a premature termination codon close to the site of the deletion. The predicted length of the mutant polypeptide is 664 amino acids, which is shorter than the wild type polypeptide (680 amino acids). A 1992delCT mutation ofCOL10A1 has been previously reported in one family. The independent occurrence ofde novo mutation of this specific dinucleotide repeat suggests that this region is a possible mutational hot spot onCOL10A1.     

Relationship between breath-hold time and physical performance in patients with cystic fibrosis

Rehabilitation including physiotherapy is an important part of the treatment used to help improve the quality of life of patients with cystic fibrosis (CF). The aim of this study was to determine the value of the breath-hold time as an index of exercise tolerance in patients with CF. Eighteen patients in different states of CF were included. The breath-hold time was measured in all patients. The fitness level was assessed by means of a progressive exercise test on a cycle-ergometer. During the test, oxygen uptake (VO₂) and carbon dioxide elimination (VCO₂) were measured breath by breath. The VO₂ and working capacity (WC) were computed at the anaerobic threshold (AT) and at peak. Duration of breath-hold was 24.7±2.87 (mean ± SEM) seconds, varying between 10 and 58. The breath-hold time (BHT) displayed a significant correlation with VO₂ (r=0.898), WC (r=0.899) at the AT, and the peak VO₂ (r=0.895). Regression equations were: VO₂ at the AT (ml/kg)=5.53+0.42×BHT and WC at the AT (watt/kg)=0.56+0.38×BHT Our results suggest that the voluntary breath-hold time might be a useful index for prediction of the exercise tolerance of CF patients.The institutional review board was the Human Ethics Committee of The Hospital of Chest Diseases of the Protestant Church of Hungary, Mosdós. The rights of human subjects were fully protected.     

An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver

The main pathological feature of liver fibrosis is the accumulation of extracellular matrix associated with hyperplasia and activation of perisinusoidal (Ito) cells (PSC) to myofibroblast-like cells. Interleukin-1 enhances collagen synthesis by increasing the proliferative activity of cultured PSC and has been implicated in the pathogenesis of hepatic fibrosis.Interleukin-1 receptor antagonist (IL-1ra) can block the binding of IL-1 to its receptors and act as a natural inhibitor of IL-1. We have examined whether the administration of IL-1ra can interfere with the development of experimental cirrhosis induced by dimethylnitrosamine (DMN). Rats were divided in three groups and received respectively DMN, DMN+IL-1ra and IL-1ra. For each group the collagen content of the hepatic tissue and the volume density of the inflammatory infiltrate were measured. Immunostaining for laminin and alpha-smooth muscle actin were also performed.In animals given DMN+IL-1ra we observed a decreased deposition of laminin and collagen, and a decreased number of laminin-positive PSC and of alpha-smooth muscle actin reactive cells, compared with animals receiving DMN alone. The present findings suggest that the early activation of PSC in vivo is at least in part mediated by IL-1 and confirm that the administration of IL-1ra may be of interest in modifying the biological effects of IL-1.     

Influence of blood corpuscles on the ultrafiltration of various proteins

The ultrafiltration characteristics of a commercial dialyzer were examined by measuring the sieving coefficient (s.c.) for three proteins, i.e., cytochrome c, chymotrypsinogen, and albumin, in a bovine blood system. Test blood was passed through a filter at a blood flow rate of 200 ml/min and filtrate was collected at an ultrafiltration flow rate of 10 ml/min from a port located near the blood inlet. The s.c. for cytochrome c and chymotrypsinogen in bovine blood sharply increased at the beginning of the experiment and reached a steady value that was about 60% of that obtained in an aqueous solution system. This may have been caused by the masking effect of the erythrocytes. The s.c. for albumin at steady state in bovine blood that contained added albumin only was almost the same as that obtained in the aqueous solution system. When all three proteins were added to bovine blood, however, the s.c. for albumin showed a steady value (after a slight decrease at the beginning of the experiment) the absolute value of which was higher than that found in the aqueous solution system. This phenomenon may have been caused by the stirring effect of erythrocytes that would disturb the concentration polarization of albumin formed near the membrane.     

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V_HDJ_H, VκJκ and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V_HDJ_H, VκJκ, and c-myc gene sequences. Tumor-site specific V_HDJ_H, VκJκ, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V_HDJ_H, VκJκ, and c-myc mutations displayed predominance of transitions over transversions. The “neoplastic” V_HDJ_H sequence was expressed by about 10⁻⁵ cells in the bone marrow, and contained two of the three orbital, but none of the testicular V_HDJ_H mutations. The nature and distribution of the Ig V(D)J mutations found in the κ chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V_HDJ_H, VκJκ, and c-myc genes with comparable efficiency and modalities.     

Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13 gene of Japanese patients with citrin deficiency

Deficiency of citrin, a liver-type mitochondrial aspartate-glutamate carrier (AGC), encoded by the SLC25A13 gene on chromosome 7q21.3, causes autosomal recessive disorders: adult-onset type II citrullinemia (CTLN2) and neonatal hepatitis associated with intrahepatic cholestasis (NICCD). So far, we have described 12 SLC25A13 mutations: 11 were from Japan and one from Israel. Three mutations found in Chinese and Vietnamese patients were the same as those in Japanese patients. In the present study, we identified a novel mutation IVS6+1G>C in a Japanese CTLN2 patient and widely screened 12 SLC25A13 mutations found in Japanese patients in control individuals from East Asia to confirm our preliminary results that the carrier frequency was high in Asian populations. Mutations 851–854del and 1638–1660dup were found in all Asian countries tested, and 851–854del associated with 290-haplotype in microsatellite marker D7S1812 was especially frequent. Other mutations frequently detected were IVS11+1G>A in Japanese and Korean, S225X in Japanese, and IVS6+5G>A in Chinese populations. We found a remarkable difference in carrier rates in China (including Taiwan) between north (1/940) and south (1/48) of the Yangtze River. We detected many carriers in Chinese (64/4169 = 1/65), Japanese (20/1372 = 1/69) and Korean (22/2455 = 1/112) populations, suggesting that over 80,000 East Asians are homozygotes with two mutated SLC25A13 alleles.