Maternal germinal mosaicism of X‐linked agammaglobulinemia

X‐linked agammaglobulinemia (XLA) is an immunodeficiency caused by abnormalities in tyrosine kinase (BTK), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of BTK protein and analyzed the BTK gene (BTK) in peripheral blood mononuclear cells from two siblings with XLA and additional family members. Cytoplasmic expression of BTK protein in monocytes was not detected in either patient with XLA. A single base deletion (C563) in BTK‐exon 6, which encodes the TH domain, was identified in both XLA patients. However, normal cytoplasmic expression of BTK protein in monocytes was detected in their mother without any BTK mutation. These results strongly suggest germinal mosaicism in the mother. © 2001 Wiley‐Liss. Inc.     

X-linked agammaglobulinemia

Conclusions The identification of the gene responsible for XLA has made it possible to clarify the clinical and laboratory findings in this disorder. It has markedly improved our ability to provide informative genetic counseling for affected families and it has helped unmask disorders that are clinically similar to XLA but genotypically different. However, many significant questions remain unanswered. What are the factors that influence the severity of disease and can we manipulate these factors to the benefit of the patient? As an increasing proportion of patients with XLA reach middle age and old age, are there previously unidentified complications that we should be aware of? What are the biological mechanisms by which mutations in Btk result in a failure of B-cell development and are these mechanisms different at different stages of B-cell differentiation? The long term goal of patients with XLA, their families, and the physicians who provide care for them is improved treatment for this disorder. Perhaps it is not unreasonable to hope that we are on the brink of entering the molecular therapeutic are of immunology.     

X-linked agammaglobulinemia in northern Thailand

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by a failure to generate immunoglobulins of all isotypes due to the absence of mature B cells and plasma cells, secondary to mutations in the Bruton's tyrosine kinase (Btk) gene. We report six patients with XLA, confirmed by mutation analysis, from northern Thailand. The mean age of onset was 2.5 years and the mean age at diagnosis was 7.3 years. All patients had a history of otitis media, pneumonia and arthritis at the time of diagnosis, five patients had developed bronchiectasis and 3 patients septicemia. Other infections reported included sinusitis (5/6), pericarditis (1/6), meningitis (1/6) and pyoderma (1/6). Haemophilus influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were isolated on multiple occasions. One patient died of sepsis at the age of 16 years. These observations demonstrate that early diagnosis and treatment can improve prognosis and quality of life.     

Genotype/phenotype correlations in X-linked agammaglobulinemia

No clear genotype/phenotype correlations have been established in patients with X-linked agammaglobulinemia (XLA). To determine if the specific mutation in Btk might be one of the factors that influences the severity of disease or if polymorphic variants in Tec, a cytoplasmic tyrosine kinase that might substitute for Btk, could contribute to the clinical phenotype, we examined the age at diagnosis, the percentage of peripheral blood B cells and the plasma IgM in a large group of patients with XLA. The results demonstrated that polymorphic variants in Tec were not correlated with phenotypic markers; however, the specific mutation in Btk did influence disease severity. Mutations that conceivably allow the production of some Btk, amino acid substitutions or splice defects that occur at conserved but not invariant sites in the splice consensus sequence were associated with older age at diagnosis, a higher percentage of B cells in the peripheral circulation and higher concentrations of plasma IgM.     

Carrier detection and prenatal diagnosis of X-linked agammaglobulinemia

We investigated the pregnant mother of a boy with X-linked agammaglobulinemia (XLA) but with no family history of immune disease. The X-inactivation pattern was found, using a methylation-sensitive probe, to be skewed in the maternal B cells but random in the polymorphonuclear cells, indicating carrier status and a 50% risk of inheritance for her male fetus. Using probes assigned to regions on either side of the XLA locus and defining RFL polymorphism, we excluded for the first time a diagnosis of XLA on a chorionic villus sample, with a risk of error <0.003. Immunological studies performed at the 19th week of gestation and 3 days after birth confirmed normality. Carrier detection based on the X-chromosome inactivation pattern, together with prenatal studies using probes close to the disease locus, thus permits prenatal diagnosis in families with isolated cases of XLA. © 1992 Wiley-Liss, Inc.     

Potential application of gene therapy to X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA), or Bruton's disease, is the most common human primary humoral immunodeficiency. XLA is caused by mutations of the Bruton's tyrosine kinase (BTK), a key regulator of B-cell physiology. Since the mid 80's, substitutive therapy by intravenous gammaglobulin infusions has significantly improved XLA patient survival and quality of life. Nevertheless, some frequent affections persist despite treatment, and lead to handicapping and further to morbid clinical complications for XLA individuals. Development of gene therapy by transfer of the BTK gene into hematopoietic progenitors could represent an alternative strategy for the treatment of Bruton's disease, with the advantage of a definitive cure for XLA patients. Gene therapy of XLA could be considered as a paradigm for future expansion of gene therapy approaches for many other diseases, since future utilization may be strictly dependent on a marked improvement of risk-benefit ratio compared to pre-existing treatments.     

Stem cell transplants for patients with X-linked agammaglobulinemia

Six young patients with X-linked agammaglobulinemia and proven mutations in Btk were treated with cord blood or bone marrow transplants from HLA-matched siblings. Complete blood counts, serum chemistries, serum immunoglobulin concentrations, lymphocyte cell surface markers, and physical findings were evaluated at 3- to 5-day intervals for the first 2 weeks after transplant and then every 3 to 6 months. The first three patients were not given any preparative regimen or antirejection drugs and at 24 to 42 months posttransplant these patients have shown no benefit or harm related to the transplants. The second three patients were not given a preparative regimen but were treated with cyclosporine A (70 days) and mycophenolate mophetil (28 days) after transplant. Two of these patients have developed normal sized, nontender cervical lymph nodes 3 to 12 months after transplant but none of the three patients have shown an increase in serum IgM or an increase in the number of peripheral blood B cells. It is likely that successful engraftment will require more aggressive immunosupressive medications.     

Immunoglobulin heavy chain gene rearrangements in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) appears to involve a defect in human B lymphocyte differentiation which is manifested at the pre-B cell stage. The defect segregates as an X-linked recessive trait but is not a single genetic entity. IgM-producing B cell clones were established by Epstein-Barr virus transformation of peripheral blood mononuclear cells of patients with the XLA defect linked to the DXS3 and DXS17 chromosomal loci. Individual XLA B cell clones were demonstrated to have rearrangements of the JH regions of both immunoglobulin VH region loci. The rearranged JH regions of the B cell clone ALA 19 were molecularly cloned and their nucleotide sequence was determined. Both JH-associated rearrangements (designated 191 and 192) resulted from the juxtaposition of variable (VH), diversity (D) and joining (JH) segments (VHDJH rearrangements). The 191 rearrangement employed a VH segment belonging to VH subgroup III and a JH4 segment. The 192 rearrangement employed a VHII and a JH6 segment. The D191 and D192 segments encompassed 21 and 28 nucleotides, respectively, and showed little homology to each other or to previously reported human D sequences. Surprisingly, both VHDJH complexes had open reading frames. However, in accord with principles of allelic exclusion, only the 191 allele was detectably expressed in the total RNA of the cell. A possible mechanism for the lack of expression of the 192 allele is discussed. We conclude that the DXS3-DXS17-linked XLA defect does not preclude VH to DJH rearrangements or the expression of VH containing heavy chain molecules.     

Germline mosaicism in X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM; OMIM310400) is a congenital muscle disorder characterized by severe hypotonia and respiratory insufficiency. The disorder was mapped to Xq28 by linkage studies and the MTM1 gene was isolated by positional cloning. The gene product is a 603 amino acid protein named myotubularin. A small domain in its sequence shows high homology to a consensus active site of tyrosine phosphatases, a diverse class of proteins involved in signal transduction, control of cell growth, and differentiation. In this report, two brothers affected with XLMTM are shown to have a point mutation (G1187A) in exon 11 of the MTM1 gene. Surprisingly, their mother does not have this mutation in her lymphocytes. Therefore, she likely has a germline mosaicism. As this is the third report of germline mosaicism in XLMTM, the finding has important implications for genetic counseling.     

Intravenous immunoglobulin replacement therapy in X-linked agammaglobulinemia]

Intravenous immunoglobulin (IVIG) replacement therapy is the mainstay of therapy for X linked agammaglobulinemia (XLA). This study was attempted to investigate how patients with XLA were treated with IVIG in Japan. Data were complied from questionnaires filled in by the physicians. One hundred eighteen medical records had been given from 134 patients. One hundred eleven patients had been treated with IVIG. Most patients had been administered IVIG every 2 to 4 weeks, and maintained serum IgG trough levels of 400 mg/dl. Some of patients had adverse effects of IVIG. Antibiotics, especially macrolides, had been administered in some of patients. A few patients, despite the maintenance of higher trough levels, were associated with infections. The present study suggests that IVIG should be administered dependent on personal infection histories.     

Carrier detection and prenatal diagnosis of X-linked agammaglobulinemia.

We investigated the pregnant mother of a boy with X-linked agammaglobulinemia (XLA) but with no family history of immune disease. The X-inactivation pattern was found, using a methylation-sensitive probe, to be skewed in the maternal B cells but random in the polymorphonuclear cells, indicating carrier status and a 50% risk of inheritance for her male fetus. Using probes assigned to regions on either side of the XLA locus and defining RFL polymorphism, we excluded for the first time a diagnosis of XLA on a chorionic villus sample, with a risk of error less than 0.003. Immunological studies performed at the 19th week of gestation and 3 days after birth confirmed normality. Carrier detection based on the X-chromosome inactivation pattern, together with prenatal studies using probes close to the disease locus, thus permits prenatal diagnosis in families with isolated cases of XLA.     

X-linked agammaglobulinemia: a survey of 33 Iranian patients

In order to determine the clinical and laboratory features of X-linked agammaglobulinemia, the records of 33 male patients with XLA were reviewed during 22 years (1980-2002) in the Iranian referral center of primary immunodeficiency disorders. The patients' ages ranged from 20 to 360 months (median 113 months). The median age at the onset of the disease was 8 months and the median age of diagnosis was 48 months, with a median diagnosis delay of 33 months. Almost all of the patients presented common infectious diseases, which were: pneumonia, otitis, diarrhea, sinusitis, and arthritis. During the course of illness, infections in the respiratory tract, gastrointestinal tract, central nervous system, and musculoskeletal system were seen in 93.9%, 75.8%, 33.3%, and 21.2% of XLA patients, respectively. The most common complications of these patients were chronic infections in 75.8% of them, including: chronic otitis media, chronic sinusitis, chronic diarrhea, and bronchiectasis.     

Females with a disorder phenotypically identical to X-linked agammaglobulinemia

Clinical and laboratory findings in two girls with a disorder phenotypically indistinguishable from typical X-linked agammaglobulinemia (XLA) are described. To examine the possibility that subtle defects in the X chromosome might explain the findings, detailed genetic studies were performed on one of these patients. Cytogenetic studies showed a normal 46XX karyotype. Southern blot analysis of her DNA showed that she had inherited a maternal and a paternal allele at sites flanking the locus for typical XLA at Xq22, making a microdeletion or uniparental disomy unlikely. To determine whether both of her X chromosomes could function as the active X, somatic-cell hybrids that selectively retained the active X were produced from her activated T cells. A normal random pattern of X inactivation was seen. Of 21 T-cell hybrids, 3 retained both X chromosomes, 7 had one X as the active X, and 11 had the other X as the active X. We have interpreted these studies as indicating that there is an autosomal recessive disorder that is phenotypically identical to XLA.     

Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation

We used a recently developed strategy to analyze patterns of X-chromosome inactivation in human cell populations in order to study female members of families with X-linked agammaglobulinemia--i.e., to detect the carrier state and to test the hypothesis that the disorder results from a defect intrinsic in the development of B cells. According to this strategy, recombinant-DNA probes simultaneously detect restriction-fragment-length polymorphisms and patterns of methylation of X-chromosome genes. Random X-inactivation patterns were observed in isolated peripheral-blood granulocytes, T lymphocytes, and B lymphocytes of women who were not carriers. In contrast, one of the two X chromosomes was preferentially active in the peripheral B cells, but not the T cells or granulocytes, of three carriers of the disorder. This observation strongly supports the hypothesis that X-linked agammaglobulinemia results from an intrinsic defect in B-cell development. Moreover, the analysis described here can be used for direct identification of carriers in families with this disease.