Molecular cloning of a constitutional t(7;22) translocation associated with risk of hematological malignancy

We report the molecular characterization of a reciprocal constitutional translocation t(7;22)(p13;q11.2) carried by three family members who have each developed a hematological malignancy. The chromosome 7 breakpoint was localized to a single BAC clone, RP11-571N3, by sequential fluorescence in situ hybridization analysis of clones selected from the NCBI chromosome 7 map. This was further refined to a 739-bp region by Southern blot analysis of DNA from the two cell lines 1193 and 1194 digested with EcoRI, HindIII, PstI, and PvuII. A 2.8-kb fragment spanning the der(22) breakpoint was amplified by long-range inverse PCR. The sequence of this fragment was used to predict the composition of the der(7) breakpoint, and a 1.3-kb fragment was amplified by use of primers from both chromosomes 7 and 22 based on this prediction. The breakpoint on chromosome 22 is located between the 3rd and 4th V regions of the immunoglobulin lambda (IGL) locus, and the breakpoint on chromosome 7 is located 122 kb proximal to the insulin-like growth factor binding protein (IGFBP) 3 gene. Examination of both reciprocal junctions showed that four bases were lost from chromosome 22, whereas 75 bases were lost from chromosome 7. Small insertions of 46 bases and 13 bases were found at the der(22) and the der(7) junctions, respectively. As a consequence of this event, the entire IGL locus, less the first three Vlambda elements, is translocated to chromosome 7, whereas the three remaining Vlambda elements on the der(22) are juxtaposed with IGFBP3 and IGFBP1.     

Molecular analysis of human immunoglobulin Vλ germline genes: Subgroups VλIII and VλIV

Three human immunoglobulin Vλ germline genes have been isolated: two from the VλIV subgroup and one from the VλIII subgroup. The VλIII gene and one of the VλIV genes appear to be functional (each being utilized in at least two expressed Vλ genes), despite deviations from the reported consensus sequences in their promoter TATA-box and recombination signal sequence elements. The other VλIV gene is a pseudogene. Of the 20 human Vλ germline genes characterized to date, 45% are pseudogenes or vestigial genes.     

Polymorphism of immunoglobulin lambda constant region genes in populations from France, Lebanon and Tunisia

Polymorphism of immunoglobulin lambda constant region (IGLC) genes has been studied in French, Lebanese and Tunisian people. The human IGLC polymorphisms appear as EcoRI restriction fragment length variations-8, 13, 18 or 23 kb-, these polymorphic fragments being related to a number of IGLC genes varying from six to nine per haploid genome. DNAs digested with the endonucleases EcoRI and HindIII were hybridized to a human IGLC probe and an immunoglobulin lambda intervening sequence region probe containing the J lambda 2 gene segment. Restriction fragments detected in Southern hybridizations were assigned to the IGLC locus map. Family studies allowed us to confirm the allelic nature of four of the different EcoRI restriction fragments observed. Frequencies of the corresponding alleles in French, Lebanese and Tunisian populations were determined and compared. The decrease of the 8-kb fragment (allele A1) frequency and, conversely, the increase of that of the 13-kb and 18-kb fragments (alleles A2 and A3) seemed to be correlated to a Negroid African contribution in the gene pool more important in Tunisia than in Lebanon.     

Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assay

Multiple studies have underscored the importance of loss of tumor suppressor genes in the development of human cancer. To identify these genes, we used somatic cell hybrids in a functional assay for tumor suppression in vivo. A tumor suppressor gene in 11p15.5 was detected by transferring single human chromosomes into the G401 Wilms' tumor cell line. In order to better map this gene, we created a series of radiation-reduced t(X;11) chromosomes and characterized them at 24 loci between H-RAS and beta-globin. Interestingly, three of the chromosomes were indistinguishable as determined by genomic and cytogenetic analyses. Each contains an interstitial deletion with one breakpoint in 11p14.1 and the other breakpoint between the D11S601 and D11S648 loci in 11p15.5. PFGE analysis localized the 11p15.5 breakpoints to a 175 kb MluI fragment that hybridized to D11S601 and D11S648 probes. Genomic fragments from this 175 kb region were hybridized to DNA from mouse hybrid lines containing the delta t(X;11) chromosomes. This analysis detected the identical 11p15.5 breakpoint which disrupts a 7.8 kb EcoRI fragment in all three of the delta t(X;11) chromosomes, suggesting they are subclones of the same parent colony. Upon transfer into G401 cells, one of the chromosomes suppressed tumor formation in nude mice, while the other two chromosomes lacked this ability. Thus, our mapping data indicate that the gene in 11p15.5 which suppresses tumor formation in G401 cells must lie telomeric to the D11S601 locus. Koi et al. (Science 260: 361-364, 1993) have used a similar functional assay to localize a growth suppressor gene for the RD cell line centromeric to the D11S724 locus. The combination of functional studies by our lab and theirs significantly narrows the location of the tumor suppressor gene in 11p15.5 to the approximately 500 kb region between D11S601 and D11S724.      

Identification of genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders.     

Definition of the human immunoglobulin variable lambda (IGLV) gene subgroups

Comparison of 60 human immunoglobulin variable lambda (IGLV) sequences allowed us to define seven subgroups designated V lambda I to V lambda VII. We demonstrate that all lambda proteins sequenced so far fall into the subgroups I, II, III and VI, and that the lambda regions previously assigned to subgroups IV and V belong, in fact, to subgroups III and II, respectively. Four sequences not belonging to any of the subgroups I, II, III and VI define the new subgroups IV, V and VII. Interestingly, these subgroups show a higher homology to rabbit or mouse V lambda genes than to the other human V lambda subgroups. By comparison of the proteins either with the sequences deduced from the germ-line genes or with the consensus sequences, the rate of amino acid changes due to somatic mutations or allelic variations was evaluated in several lambda proteins. Framework and complementarity-determining regions of the human IGLV genes and proteins were delineated. Alignment of the lambda sequences shows that functional V-J rearrangement occurs, with or without deletion of nucleotides encoding one or two amino acids at the 3' end of the V gene. Diversity of the third complementarity-determining region is due to somatic mutations and to flexible V-J junction, but there is no evidence of N-diversity in the human lambda locus.     

Immunoglobulin lambda light chain orphons on human chromosome 8q11.2

We have identified two Vλ genes outside the major lambda locus on chromosome 22q11.2, and shown that they reside on chromosome 8q11.2. One gene (Orphéel), hybridizing strongly to the Vλ probes, was sequenced and found to belong to the Vλ8 family; the other gene (Orphée2) only hybridized weakly. Orphée1 was present in all individuals tested (140) from three different populations, and was also found in gorillas. We envisage that these genes were generated by duplication and translocation of the Vλ8a gene (and a Vλ pseudogene) from the major locus, and that this event occurred before the evolutionary divergence of humans and gorillas. As there is no other evidence for Vλ genes outside the major locus, it appears that the human lambda locus has undergone considerably less evolutionary shuffling than either the human light chain kappa locus or the heavy chain locus.     

Two pseudogenes among three rat immunoglobulin lambda chain genes

In order to examine the number and organization of the immunoglobulin lambda light chain genes of the rat, we have used mouse lambda chain cDNA probes to isolate hybridizing fragments from a partial EcoRI rat liver DNA library. We have determined the partial nucleotide sequence of two such clones. One clone, containing a 5.8 kb EcoRI insert which hybridizes to both mouse C lambda 1 and C lambda 2 probes, includes an apparently expressible C lambda 2-like gene as well as a C lambda 1-like pseudogene (psi C lambda 1.1), arranged similarly to the mouse C lambda gene complexes. Sequence analysis of a second cloned EcoRI fragment (1.15 kb in length) revealed part of a second C lambda 1-like pseudogene (psi C lambda 1.2), the coding regions of both pseudogenes being interrupted by multiple frame-shifting size differences. In the case of psi C lambda 1.2, the degree of sequence identity with mouse C lambda 1 drops abruptly immediately following the termination codon, suggesting that translocation events have played a role in its generation. These two rat pseudogenes, and the mouse C lambda 4 pseudogene, clearly have been rendered unexpressible by separate evolutionary events. Comparisons between C lambda coding and non-coding regions of rats and mice indicate that some of the unusual patterns of divergence we have observed in recently diverged Ck genes may exist in C lambda genes as well.     

Frequent deletions at 12q14.3 chromosomal locus in adult acute lymphoblastic leukemia

Cytogenetic abnormalities at the 12q12-q14 chromosomal locus are rarely detected in acute lymphoblastic leukemia (ALL). To examine submicroscopic deletions at this locus, we analyzed 78 adult precursor B- and T-cell ALL cases [27 with Philadelphia chromosome (Ph)-negative B-cell ALL, 20 with Ph-negative B-cell ALL with expression of one or two myeloid markers, 18 with Ph-positive B-cell ALL, and 13 with T-cell ALL] using a panel of 13 microsatellite (MST) markers that span the 12q12-q14.3 region. The status of MST markers was evaluated by use of polymerase chain reaction performed with fluorescence-labeled primers and automated fragment analysis. The MST marker analyses showed submicroscopic deletions at the 12q14.3 locus in 20 of the 78 ALL cases (26%). The frequency of deletions was highest in Ph-negative B-cell ALL (13 of 27, 48%) compared with that in Ph-negative B-cell ALL with expression of myeloid markers (4 of 20, 20%), Ph-positive B-cell ALL (2 of 18, 11%), and T-cell ALL (1 of 13, 8%). Deletion frequencies of MST markers along the 12q12-q14.3 locus suggest that the targeted gene of deletion is located within a 170-kb region bordered by the markers D12S1504 (approximately 65 kb upstream of HMGA2) and D12S1509 (in intron 3 of HMGA2) at the 12q14.3 locus. These submicroscopic deletions at the 12q14.3 locus may play a role in the pathogenesis of ALL, particularly in Ph-negative precursor B-cell ALL.     

Deletion mapping of chromosomes 8, 10, and 16 in human prostatic carcinoma

In an allelotyping study prostatic carcinoma, we found the highest frequency of allelic deletions on chromosomes 8, 10, 16, and 18. In all cases with allelic deletions, at least one of the chromosomes 8, 10, and 16 were involved. A detailed deletion mapping of these chromosomes in 18 cases was carried out with probes that detect restriction fragment length polymorphisms (RFLP) on chromosomes 8 (6 probes), 10 (11 probes), and 16 (9 probes). The highest frequency of allelic deletions were found on 8p (65%), where the minimally deleted region was between the PLAT locus and pter. The long arm of chromosome 16 had allelic deletions in 56% of informative cases, with three different break points, the most distal being located between D16S4 and D16S7. Chromosome 10 exhibited a complex deletion pattern with terminal deletions of the p or the q arm (2 cases each), a deletion pattern that could be interpreted as nonreciprocal translocations of the q arm (2 cases), or allelic losses on all informative loci, suggesting monosomy (2 cases). Our data suggest that tumor suppressor genes involved in the oncogenesis of prostatic carcinoma may be localized between 8 pter and the PLAT locus and that additional/alternative tumor suppressor genes may be localized on chromosome 10 and on the long arm of chromosome 16 distal to the D16S4 locus.     

Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom cytogenetic abnormality associated with acute myelogenous leukemia of the M2 subtype (FAB classification). The 8q- and 21q+ derivative chromosomes have previously been isolated in somatic cell hybrids and used to map the anonymous sequences D21S65 and D21S17, which were proximal and distal, respectively, to the breakpoint on chromosome 21. DNA from a series of 12 t(8;21) patients and 7 controls was analyzed by pulsed field gel electrophoresis. Physical linkage of probes D21S65 and D21S17 on a 2100 kb NruI fragment was established by partial digestion experiments. In all the patients, the translocation generated a rearranged D21S65 NruI fragment of 650 to 750 kb, suggesting heterogeneity in the breakpoints. This heterogeneity was confirmed by using BssHII, SacII, and EagI enzymes. Our results are consistent with the presence of a 100 Kb breakpoint cluster region on chromosome 21 encompassing the AML1 gene. Interestingly, in half of the patients, demethylation of an NruI site located 7 kb proximal to the last exon of the AML1 gene occurred on the nontranslocated chromosome 21.     

Evidence for subtelomeric exchange of 3.3 kb tandemly repeated units between chromosomes 4q35 and 10q26: implications for genetic counselling and etiology of FSHD1

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy, clinically characterized by asymmetric weakness of muscles in the face, shoulder girdle and upper arm. Deletion of an integral number of 3.3 kb repeated units within a highly polymorphic EcoRI fragment at chromosome 4q35, generating a relatively short EcoRI fragment (< 35 kb), has been shown to cause FSHD1. Probe p13E-11 detects these short fragments in FSHD1 patients, and has therefore been used for diagnostic DNA analysis. However, the reliability of this analysis has been hampered by cross-hybridization of p13E-11 to chromosome 10q26-linked EcoRI fragments of comparable size, which also contain a variable number of 3.3 kb repeated units. Recently, a BinI restriction site was identified within each of the repeated units derived from chromosome 10q26, which enables differentiation of the two polymorphic p13E-11 loci in most cases without haplotype analysis. Remarkably, applying the differential analysis to screen DNA of 160 Dutch cases referred to us for FSHD1 diagnosis, we obtained evidence for subtelomeric exchange of 3.3 kb repeated units between chromosomes 4q35 and 10q26 in affected and unaffected individuals. Subsequently, analysis of 50 unrelated control samples indicated such exchange between chromosomes 4q35 and 10q26 in at least 20% of the population. These subtelomeric rearrangements have generated a novel interchromosomal polymorphism, which has implications for the specificity and sensitivity of the differential restriction analysis for diagnostic purposes. Moreover, the high frequency of the interchromosomal exchanges of 3.3 kb repeated units suggests that they probably do not contain (part of) the FSHD1 gene, and supports position effect variegation as the most likely mechanism for FSHD1.      

Cloning of a functional human adenine phosphoribosyltransferase (APRT) gene: Identification of a restriction fragment length polymorphism and preliminary analysis of DNAs from APRT-deficient families and cell mutants

A complete human APRT gene has been isolated from a lambda phage genomic library using cloned mouse APRT DNA as a probe. The human gene, contained in a recombinant lambda phage designated Huap15, is functional by virtue of its capacity to transfer human APRT activity to Aprt^– mouse recipient cells after phage-mediated transfection. Digestion of Huap15 DNA with BamH1 generated a 2.2-kb fragment that is the only fragment of eight produced to hybridize with the mouse APRT gene. This 2.2-kb BamH1 fragment is a unique, single copy sequence, and has been used to identify a restriction fragment length polymorphism (RFLP) associated with the APRT locus. Taq1 digestion and Southern blot analysis of DNAs from 49 unrelated individuals produced three different patterns. DNAs of 30 individuals produced a restriction pattern of three labeled fragments about 500 bp, 600 bp, and 2.1 kb in size, which is characteristic for individuals homozygous for the more common allele. Two individuals homozygous for the less frequent allele displayed labeled fragments of 500 bp and 2.7 kb. The remaining 17 DNA samples produced all four labeled bands as expected for heterozygous individuals. The frequency of heterozygotes in the population is about 35%, while the frequency of the less common allele is about 0.21. Restriction enzyme analysis of DNAs from two APRT-deficient brothers and from an unrelated heterozygote revealed no gross deletions or rearrangements, nor the Taq1 polymorphism.     

Nomenclature of the human immunoglobulin lambda (IGL) genes

'Nomenclature of the Human Immunoglobulin Lambda (IGL) Genes', the 18th report of the 'IMGT Locus in Focus' section, provides the first complete list of all the human IGL genes. The total number of human IGL genes per haploid genome is 87--96 (93--102 if the orphons are included), of which 37--43 genes are functional. IMGT/Human Genome Organization (HUGO) gene names and definitions of the human IGL genes on chromosome 22q11.2 and IGL orphons on chromosomes 8 and 22 are provided with the gene functionality and the number of alleles, according to the rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences and with the accession ID of the Genome Database GDB and NCBI LocusLink databases, in which all the IMGT human IGL genes have been entered. The tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.     

Molecular mapping of the human T cell receptor gamma (TRG) genes and linkage of the variable and constant regions

In the human T cell receptor gamma (TRG) locus, fourteen variable (TRGV) genes belonging to four subgroups have been identified upstream of two constant region (TRGC) genes. Three joining segments, JP1, JP and J1, have been localized upstream of TRGC1, and two others, JP2 and J2, upstream of TRGC2. In this report, we demonstrate that a unique Xho I fragment of 120 kilobases (kb) contains the fourteen TRGV genes and that the hybridization of that fragment in pulsed-field gel electrophoresis (PFGE) allows linkage of the variable region to the constant region locus. We also show that the variable and the constant regions are remarkably close to each other since the distance between V11, the most 3' V gamma gene, and JP1, the most 5' J gamma segment, is only 16 kb. With its 14 V gamma genes, spanning 100 kb, the two C gamma genes and 5 joining segments covering less than 40 kb and only 16 kb separating the most 3' V gene from the most 5' J segment, the human TRG locus spans 160 kb of genomic DNA and represents a particularly condensed locus compared to the other rearranging gene loci.     

Mapping and functional analysis of regulatory sequences in the mouse lambda5-VpreB1 domain

The lambda5 and VpreB genes encode the components of the surrogate light-chain which forms part of the pre-B cell receptor and plays a key role in B cell development. In the mouse, the lambda5 and VpreB1 genes are closely linked and are co-regulated by a multi-component locus control region. To identify the sequences that regulate lambda5 and VpreB1 expression during B cell development, we have comprehensively mapped the DNaseI hypersensitive sites (HS) in the lambda5-VpreB1 functional domain. The active domain contains 12 HS that are distributed at high density across the 18.3 kb region that forms the lambda5 and VpreB1 functional unit. Analysis of a reporter gene driven by the VpreB1 promoter in transgenic mice identified a novel enhancer associated with two HS located upstream of lambda5. The lambda5-VpreB1 locus was also found to be closely linked to the ubiquitously expressed Topoisomerase-3beta (Topo3beta) gene. The VpreB1 and Topo3beta genes have entirely different expression patterns despite the fact that the two promoters are separated by a distance of only 1.5 kb.