Search

Many human genetic diseases are associated with gross mutations such as aneuploidies, deletions, duplications, or inversions. For these “structural” disorders, conventional gene therapy, based on viral vectors and/or on programmable nuclease-mediated homologous recombination, is still unsatisfactory. To correct such disorders, chromosome transplantation (CT), defined as the perfect substitution of an endogenous defective chromosome with an exogenous normal one, could be applied. CT re-establishes a normal diploid cell, leaving no marker of the procedure, as we have recently shown in mouse pluripotent stem cells. To prove the feasibility of the CT approach in human cells, we used human induced pluripotent stem cells (hiPSCs) reprogrammed from Lesch-Nyhan (LN) disease patients, taking advantage of their mutation in the X-linked HPRT gene, making the LN cells selectable and distinguishable from the resistant corrected normal cells. In this study, we demonstrate, for the first time, that CT is feasible in hiPSCs: the normal exogenous X chromosome was first transferred using an improved chromosome transfer system, and the extra sex chromosome was spontaneously lost. These CT cells were functionally corrected and maintained their pluripotency and differentiation capability. By inactivation of the autologous HPRT gene, CT paves the way to the correction of hiPSCs from several X-linked disorders., Graphical Abstract

Acrofrontofacionasal dysostosis type 1 (AFFND1) is an extremely rare disorder characterized by several dysmorphic features, skeletal abnormalities and intellectual disability, and described only in seven patients in the literature. A biallelic variant in the Neuroblastoma Amplified Sequence (NBAS) gene was recently identified in two Indian patients with AFFND1. Here we report genetic investigation of AFFND1 in the originally described Brazilian families and the identification of an extremely rare, recessively-inherited, intronic variant in the Phosphatidylinositol Glycan class B (PIGB) gene NC_000015.10 (NM_004855.4): c.795-19T > G) in the affected individuals. The PIGB gene encodes an enzyme involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is required for the post-translational modification of a large variety of proteins, enabling their correct cellular localization and function. Recessive variants in PIGB have previously been reported in individuals with a neurodevelopmental syndrome having partial overlap with AFFND1. In vitro assays demonstrated that the intronic variant leads to exon skipping, suggesting the Brazilian AFFND1 patients may be null for PIGB, in agreement with their severe clinical phenotype. These data increase the number of pathogenic variants in the PIGB gene, place AFFND1 among GPI deficiencies and extend the spectrum of phenotypes associated with GPI biosynthesis defects.

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).

Biallelic variants in neuroblastoma-amplified sequence (NBAS) cause an extremely broad spectrum of phenotypes. Clinical features range from isolated recurrent episodes of liver failure to multisystemic syndrome including short stature, skeletal osteopenia and dysplasia, optic atrophy, and a variable immunological, cutaneous, muscular, and neurological abnormalities. Hemizygous variants in CUL4B cause syndromic X-linked intellectual disability characterized by limitations in intellectual functions, developmental delays in gait, cognitive, and speech functioning, and other features including short stature, dysmorphism, and cerebral malformations. In this study, we report on a 4.5-month-old preterm infant with a complex phenotype mainly characterized by placental-related severe intrauterine growth restriction, post-natal growth failure with spontaneous bone fractures, which led to a suspicion of osteogenesis imperfecta, and lethal bronchopulmonary dysplasia with pulmonary hypertension. Whole exome sequencing identified compound heterozygosity for a known frameshift and a novel missense variant in NBAS and hemizygosity for a known CUL4B nonsense mutation. In vitro functional studies on the novel NBAS missense substitution demonstrated altered Golgi-to-endoplasmic reticulum retrograde vesicular trafficking and reduced collagen secretion, likely explaining part of the patient's phenotype. We also provided a comprehensive overview of the phenotypic features of NBAS and CUL4B deficiency, thus updating the recently emerging NBAS genotype-phenotype correlations. Our findings highlight the power of a genome-first approach for an early diagnosis of complex phenotypes.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.

Background Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. Methods We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. Results We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. Conclusion Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies., Highlights • Ex vivo corrected autologous HSCs might cure Autosomal Recessive Osteopetrosis (ARO). • There is no animal model to prove in vivo functional rescue of corrected human cells. • NSG oc/oc mice display osteoclast-rich cell-autonomous ARO and immunodeficiency. • Human CD34+ cell-transplanted NSG oc/oc mice show human cell chimerism in the BM. • Further improvements will allow in vivo evaluating corrected patient-derived cells.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes. © 2015 American Society for Bone and Mineral Research.

Mesenchymal stem cells (MSCs) are recognized as an attractive tool owing to their self-renewal and differentiation capacity, and their ability to secrete bioactive molecules and to regulate the behavior of neighboring cells within different tissues. Accumulating evidence demonstrates that cells prefer three-dimensional (3D) to 2D culture conditions, at least because the former are closer to their natural environment. Thus, for in vitro studies and in vivo utilization, great effort is being dedicated to the optimization of MSC 3D culture systems in view of achieving the intended performance. This implies understanding cell–biomaterial interactions and manipulating the physicochemical characteristics of biomimetic scaffolds to elicit a specific cell behavior. In the bone field, biomimetic scaffolds can be used as 3D structures, where MSCs can be seeded, expanded, and then implanted in vivo for bone repair or bioactive molecules release. Actually, the union of MSCs and biomaterial has been greatly improving the field of tissue regeneration. Here, we will provide some examples of recent advances in basic as well as translational research about MSC-seeded scaffold systems. Overall, the proliferation of tools for a range of applications witnesses a fruitful collaboration among different branches of the scientific community.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Mutations in recombination activating genes (RAG) 1 and 2 have been found to cause Omenn syndrome (OS), a severe combined immunodeficiency (SCID) with a peculiar phenotype. Here we report the prenatal diagnosis performed in three OS patients. Mutations were detected in the probands as well as in their parents by genomic sequencing of the complete coding regions of both RAG 1 and RAG 2, which are contained in a single exon. All the three probands had RAG 1 mutations in both alleles, at least one of which was a missense substitution. Of the three fetuses tested, one had a wild type sequence on both alleles, while the other two had one mutated allele. None of the three patients were predicted to be affected and this was confirmed at birth. Detection of RAG genes mutations on fetal samples by direct sequencing is an easy and effective way to investigate fetuses from families affected with RAG-dependent SCID and OS families affected by RAG-dependent SCID and OS.

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2–mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4+/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.

We have determined the genomic structure of the humanLHX1 gene, a member of the LIM/homeobox (Lhx) gene family. The transcript is assembled from five exons, which are separated by introns ranging in size from 93 nt to 2.3 kb. The two LIM domains are entirely contained in the first and second exons, respectively, while the homeodomain is split into exons three and four. This structure closely parallels the organization of other mouse and human Lhx genes whose genomic structure is known. An exception is the mouse and humanisl1 genes, whose homeodomain does not contain introns. An intron at the same position also occurs in theXlim1 gene as well as in other homeobox genes, such asevx1 andevx2, suggesting that this intron insertion represents an ancestral event, from which homeobox genes of different families originated. In this context, evolution of theLhxgene family probably involved the shuffling of this intron-containing homeobox in the proximity of a LIM-only gene, whileIsletgenes were formed either by the shuffling of an intronless homeobox to the same LIM domain or, alternatively, by intron loss during their evolution.

We have determined the exon-intron structure of the human TFE3 gene located on Xp11.22-23. By designing PCR primers, we were able to amplify various segments of the TFE3 genomic region, thus establishing that this gene is composed of seven exons, the first six of which are small (from 56 to 159 nt). The 5' UT region is contained entirely in the first exon, while the 3' UT region is contained in the seventh exon. The comparison of the genomic and the published cDNA versions revealed that the deduced amino acid sequence of TFE3 in the C-terminus region is 125 amino acids shorter than previously reported. This eliminates most of the putative proline- and arginine-rich domain and makes the human sequence more similar to its mouse homolog. The activation domain at the N-terminus is contained in exon 2, as has been described for the mouse. The basic helix-loop-helix (BHLH) motif is spread over exons 4 to 6, while the leucine zipper (LZ) is almost all contained in the last portion of exon 6. This split BHLH is different from other BHLH-LZ genes whose genomic structures have been determined up to now.

The region between DXS52 and Factor VIII gene in the human Xq28 chromosomal band contains a G + C-rich isochore to which many genes have been mapped. We report here the isolation and characterization of a transcript mapping about 50 kb telomeric from the vasopressin type 2 receptor gene in a 180-kb YACs/cosmid contig containing the L1CAM gene at its centromeric end. The determined transcribed sequence from a human fetal brain library is identical to that of the recently identified accessory protein HCFC1 (host cell factor, also called C1) that activates herpes simplex virus VP16 (alpha TIF) transactivator protein for association with the octamer motif-binding protein Oct-1 (Cell 74: 115, 1993). The gene is expressed in a ubiquitous pattern and a larger transcript of approximately 10 kb is present in all the tissues tested, while an alternatively spliced RNA of approximately 8.0 kb is present in muscle and heart tissues. Genomic sequencing allowed us to determine that the sequenced transcript is assembled from 26 exons spread over a relatively small genomic region of approximately 24 kb. This allowed us to determine that a previously reported cDNA clone arises from the splicing out of an internal portion of exon 8 which does not change the reading frame. Ah together these results raise the possibility that alternative mRNA processing could partly contribute to the diversity of the polypeptide HCFC1 family in a subset of tissues. (C) 1994 Academic Press, Inc.

Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, we have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, we have defined at the genomic level the CD40L gene abnormalities for three previously described HIGM1 patients who demonstrated clustered deletions in the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, we have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Our knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease.

Although long contigs have been assembled in Xq28, the interval between the anonymous probe St14 and the color vision locus is still incompletely defined. We report here that the recently cloned gene for type 2 vasopressin receptor (V2R) is physically linked to L1CAM using YACs and cosmids across about 180 kb of the region. Since it is known that L1CAM maps near the color pigment genes, this finding locates V2R in Xq28 in the area where nephrogenic diabetes insipidus (NDI) has been mapped by linkage analysis. The PFGE analysis of the clones positions V2R about 40 kb from the L1CAM gene in a region that appears to contain other unknown genes, since at least four putative CpG islands were identified by restriction analysis with rare cutter enzymes.

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2R229Q knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2R229Q knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.

We have previously reported (Villa et al. (1993), Genomics 18: 223) the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain an ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and orangutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existence of at least two genes in cows and horses, suggests a relatively high conservation of this gene family.

X-linked thrombocytopenia (XLT) is a rare recessive hereditary disorder characterized by isolated thrombocytopenia with small-sized platelets. The XLT locus has been located to chromosome Xp11 by linkage analysis, which is also where the recently cloned Wiskott-Aldrich syndrome (WAS) gene, maps. The relationship between XLT and WAS has long been debated; they might be due to different mutations of the same gene or to mutations in different genes. We now show that mutations in the WAS gene, different from those found in WAS patients, are present in three unrelated male patients with isolated thrombocytopenia and small-sized platelets. Our results demonstrate that XLT and WAS are allelic forms of the same disease, but the causes of the differences need to be further investigated.

We have previously mapped a zinc finger genomic motif (ZNF75) to the Xq26 cytogenetic band by using a hybrid panel. Here, we report the isolation of the transcribed counterpart in a cDNA clone and its further localization. The cDNA clone, from a lung fibroblast library, is assembled from three exons, including a 289 amino acid (AA) long open reading frame containing a recently described motif, the Kruppel-associated box, 42 AA long, in exon 2. By comparison with other reported members of the subfamily, the exon-intron boundaries also appear to be very well conserved. Further analysis allowed us to map this gene 1 Mb downstream from the HPRT gene in the published YAC contig that extends across Xq26. Two other motifs, 87 and 78% homologous to ZNF75 at the amino acid level, were identified by PCR on total human DNA, but map outside Xq24-qter.

Istituto di Tecnologie Biomediche Avanzate, Consiglio Nazionale delle Ricerche, Milan, Italy In the last few years our laboratory has been involved in the identification and sequencing of genes in Xq24-qter. (1-4) During this work, a large number of data was obtained that could be analyzed with ad hoc software tools. From this analysis it was possible to identify specific fragments that had a high probability of containing coding sequences; however, direct proof of the existence of transcript still needed the labor-intensive approach of screening the cDNA libraries. In addition, the pattern of tissue expression of these putative transcripts was not known beforehand, and several tissues had to be tested, via Northern analysis, to detect the appropriate cell type, developmental stage, or tissue of expression. Here, we report a rapid PCR-based method of identifying cDNAs from genomic sequences with a high probability of being transcribed. Prescreening cDNA libraries by PCR amplification with primers designed in putative exons is an efficient way of determining which library has to be screened for the isolation of the desired mRNAs. Additionally, performing PCR assays on aliquots of a discrete number of cDNA clones from nonamplified libraries, instead of performing traditional hybridization screening directly on millions of phages, is both fast and economical and allows the systematic screening of cDNA libraries too, as it has been reported for YAC and k genomic DNA libraries. (s'6) These aliquots can be stored easily, used for an indefinite number of screenings, and maintained for an indeterminate period of time. The setting up of this procedure and its application using three cDNA libraries will be discussed.

Mutations in the CD40 ligand (CD40L) are responsible for human hyper immunoglobulin M (IgM) syndrome. The absence of the interaction between CD40L, expressed by T lymphocytes, and the CD40 receptor present on the surface of B cells is responsible for the inability of B cells to carry out the isotype switch from IgM to the other Ig classes. This leads to a fatal immunodeficiency for which no cure exists. For these reasons, the CD40L gene is a good candidate for gene therapy studies. To investigate the possible effects of the expression of this tightly regulated gene in vivo, we produced transgenic mice in which CD40L expression was deregulated. Widespread ectopic expression appears to be lethal. Overexpression in mature T cells is compatible with life, but in one-third of the cases, mice developed atypical lymphoid proliferations which, occasionally, progressed into frank lymphomas. Even though gene therapy is one of the most promising approaches to cure human hyper IgM syndrome, these results suggest that when we modify very tightly regulated genes such as cytokines or other growth factors, particular care has to be taken to avoid excessive stimulation of the target cells.