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Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Syrhptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

Proceedings of the Institute for Genome Research Computational Genomics Conference (TIGR), November 2000., Baltimore, Maryland, USA

The most common form of primary immunodeficiency is IgA deficiency (IgAD). However, the molecular basis of this disease remains elusive. Therefore, to address this issue we made a systematic analysis of the molecular events leading to IgA production. B lymphocytes that produce IgA have undergone somatic rearrangement that joins the switch (S) mu to S alpha region with deletion of the intervening sequences. Examination of the resulting S mu/S alpha junctions in unstimulated PBMC from IgAD patients by nested primer PCR revealed a significant decrease in the number of the S mu/S alpha fragments. To obtain the antisense primers to generate the S mu/S alpha fragments, we sequenced the human S alpha 1 and the downstream region extending to the C alpha 1 locus. Similar to previously reported switch sequences, we also found the S alpha 1 to be predominantly composed of pentameric repeats GAGCT and GGGCT. The decrease in the number of S mu/S alpha fragments is consistent with a profound decrease in the C alpha membrane mRNA expression in unstimulated PBMC, as well as in the C alpha mRNA levels and IgA production in PWM-stimulated PBMC. Sequence analysis of the switch junctions from IgA-producing cell lines, control donors, and an IgAD patient showed direct joining in 8 of 9 cases examined. TGF-beta 1, previously shown to be the switch factor for human and mouse IgA, was also examined. No difference in the TGF-beta 1 mRNA levels in unstimulated PBMC between the control subjects and the IgAD patients were detected. Our findings indicate that the failure to switch to IgA-producing B lymphocytes, or an impaired survival of such cells, may be an important molecular mechanism in IgAD.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.

Previous in vitro studies suggest that transcription of the unrearranged immunoglobulin switch region and its 5' flanking region precedes isotype switching. These transcripts, which are devoid of a variable region, contain unique exons and are called germ-line (GL) mRNA. A crucial point in this regard is whether such transcripts could be detected in vivo, and if their expression correlates with immunoglobulin class switching in health and disease. To understand the in vivo role of this transcriptional activity we have adapted the reverse transcription-polymerase chain reaction (PCR) to analyse the GL transcripts from unstimulated peripheral blood mononuclear cells (PBMC) in healthy individuals and in different immunological diseases. Furthermore, mononuclear cells from different human organs were also analysed. We report here that GL (I alpha, I gamma and I epsilon used to designate the GL mRNA for IgA, IgG and IgE, respectively) mRNA are expressed differentially during ontogeny of B cells. Unexpectedly, no difference of I alpha mRNA expression between the PBMC and the secondary lymphoid organs was detected. Rather, the levels of GL transcripts were correlated to the number of sIgM+ cells. GL mRNA of all three isotypes could be detected in PBMC from healthy donors, whereas there was a decrease of specific GL transcript synthesis in individuals with immunoglobulin deficiency. Furthermore, during the in vivo immune response in a parasitic infection, we could demonstrate an induction of GL I epsilon mRNA during in vivo immune response. Concomitantly, there was also increased synthesis of productive epsilon transcripts. These findings implicate a potential role of GL transcription during in vivo immunoglobulin class switching.

Immunoglobulin isotype switching is preceded by transcription of exons located 5' of the immunoglobulin switch regions. These exons are referred to as 'I-exons' and transcription of these regions is believed to be an essential step in switch recombination. Such mRNA species lack a variable portion and are denoted 'germline' transcripts. We have previously identified I-regions in man and we have now investigated the in vivo expression of human germline transcripts. Germline alpha transcripts (containing an I alpha exon) were expressed in various lymphoid organs, including peripheral blood mononuclear cells. Decreased levels of germline transcripts were found in immunoglobulin deficiency diseases. These findings are compatible with an in vivo role of germline transcription in isotype switching and may contribute to the understanding of mechanisms underlying deficiency diseases.