J.P. Leek

A Locus for Isolated Cleft Palate, Located on Human Chromosome 2q32

We present evidence for the existence of a novel chromosome 2q32 locus involved in the pathogenesis of isolated cleft palate. We have studied two unrelated patients with strikingly similar clinical features, in whom there are apparently balanced, de novo cytogenetic rearrangements involving the same region of chromosome 2q. Both children have cleft palate, facial dysmorphism, and mild learning disability. Their karyotypes were originally reported as 46, XX, t(2;7)(q33;p21) and 46, XX, t(2;11)(q33;p14). However, our molecular cytogenetic analyses localize both translocation breakpoints to a small region between markers D2S311 and D2S116. This suggests that the true location of these breakpoints is 2q32 rather than 2q33. To obtain independent support for the existence of a cleft-palate locus in 2q32, we performed a detailed statistical analysis for all cases in the human cytogenetics database of nonmosaic, single, contiguous autosomal deletions associated with orofacial clefting. This revealed 2q32 to be one of only three chromosomal regions in which haploinsufficiency is significantly associated with isolated cleft palate. In combination, our data provide strong evidence for the location at 2q32 of a gene that is critical to the development of the secondary palate. The close proximity of these two translocation breakpoints should also allow rapid progress toward the positional cloning of this cleft-palate gene.

Novel translocation of the BCL10 gene in a case of mucosa associated lymphoid tissue lymphoma

Interest has focused on a recently identified gene, BCL10, thought to play an important role in the genesis of extranodal, marginal zone (MALT) lymphomas. This gene belongs to a family containing caspase recruitment domains (CARD), that are involved in the apoptotic pathway. Translocations of the BCL10 gene to the immunoglobulin heavy chain locus at 14q32 have been described. We report herein a case of MALT lymphoma showing t(1; 2)(p22; p12). The translocation was shown to involve the BCL10 gene and the immunoglobulin kappa light chain locus by fluorescence in situ hybridization.

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

BackgroundCerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.MethodsHere we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA).ResultsA novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals.ConclusionsThis result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts.Table 4GAD1 single nucleotide substitutions detected on mutation analysis and occurring in sequences submitted to NCBI SNP database and in the literature. This is not a definitive list, but includes those described at the time of the mutational analysis. *Nucleotide positions were not provided by Maestrini et al. [47].SourceSNP position in mRNA, from the translational start site (bp)Gene position of SNP(bp)Amino acid change(A)Lappalainen et al. (2002)A(-478)DelExon 0 (73)No substitution(B)Lappalainen et al. (2002)G(-147)AExon 0 (404)No substitution(C)Lappalainen et al. (2002)A(-39)CExon 1 (25)No substitution(D)Spastic CP patients family BG(36)CExon 1 (97)Ser(12)Cys(E)NCBI collated resourceG(48)CExon 1 (104)Pro(17)Ala(F)Control samples & family A NCBI collated resourceT(110)CExon 2 (29)No substitution(G)Kure et al. (1998)T(315)CExon 4 (14)No substitution(H)Bu and Tobin (1994) Kure et al. (1998)A(407)GExon 4 (105)No substitution(I)Maestrini et al. (2002)*G/CIntron 4No substitution(J)NCBI collated resourceC(696)TExon 6 (56)No substitution(K)Lappalainen et al. (2002)T/DelIntron 7 (35)No substitution(L)In control samples Lappalainen et al. (2002)T/CIntron 8 (185)No substitution(M)Maestrini et al. (2002)*C/TIntron 9No substitution

Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3 approximately p23.2, 2q21 approximately q34, 3p12.3 approximately p14.1, 3p22 approximately p26, 3q, 12q13.2 approximately q22, 13q12 approximately q14, 17q21.3 approximately q23, 20q11.2 approximately q13.1 and 21q11.2 approximately q21 as well as the deletion of 6p21.1, 6p23 approximately p25, 7q11.1 approximately q31, 7q35 approximately q36, 11p15, 11q24, 13q33, 17p, 18q12 approximately q21.1, 19p, 19q13.3, 22q13.1 approximately q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Co-localization of the ketohexokinase and glucokinase regulator genes to a 500-kb region of Chromosome 2p23

The glucokinase regulator (GCKR) is a 65-kDa protein that inhibits glucokinase (hexokinase IV) in liver and pancreatic islet. The role of glucokinase (GCK) as pancreatic β cell glucose sensor and the finding of GCK mutations in maturity onset diabetes of the young (MODY) suggest GCKR as a further candidate gene for type 2 diabetes. The inhibition of GCK by GCKR is relieved by the binding of fructose-1-phosphate (F-l-P) to GCKR. F-l-P is the end product of ketohexokinase (KHK, fructokinase), which, like GCK and GCKR, is present in both liver and pancreatic islet. KHK is the first enzyme of the specialized pathway that catabolizes dietary fructose. We have isolated genomic clones containing the human GCKR and KHK genes. By fluorescent in situ hybridization (FISH), KHK maps to Chromosome (Chr) 2p23.2-23.3, a new assignment corroborated by somatic cell hybrid analysis. The localization of GCKR, originally reported by others as 2p22.3, has been reassessed by high-resolution FISH, indicating that, like KHK, GCKR maps to 2p23.2-23.3. The proximity of GCKR and KHK was further demonstrated both by two-color interphase FISH, which suggests that the two genes lie within 500 kb of each other, and by analysis of overlapping YAC and PI clones spanning the interval between GCKR and KHK. A new microsatellite polymorphism was used to place the GCKR-KHK locus between D2S305 and D2S165 on the genetic map. The colocalization of these two metabolically connected genes has implications for the interpretation of linkage or allele association studies in type 2 diabetes. It also raises the possibility of coordinate regulation of GCKR and KHK by common as-acting regulatory elements.

Detection by fluorescence in situ hybridization of microdeletions at 1p36 in lymphomas, unidentified on cytogenetic analysis.

The chromosomal band 1p36 exhibits frequent loss of heterozygosity in a variety of human malignancies, suggesting the presence of an as yet unidentified tumor suppressor gene. The faint terminal subbands often make cytogenetic analysis of 1p36 particularly difficult. Small deletions at this locus may therefore escape detection on analysis by conventional cytogenetics, a hypothesis that we have explored using fluorescence in situ hybridization (FISH) in malignant lymphoma. The study cohort consisted of 20 cases of lymphoma of various subtypes without any 1p abnormality on G-banded karyotyping. FISH was performed using a human chromosome 1 paint and a bacterial artificial chromosome probe RP4-755G5 localizing to 1p36.33, the most telomeric subband of 1p36. Tumors demonstrating 1p36.33 deletions were additionally analyzed by FISH using a second probe from the proximal 1p36.1 subband, to further define the breakpoint. Eight cases of follicular lymphoma (FL), 5 diffuse large B-cell lymphomas (DLBCL), 2 Hodgkin disease, 2 B-cell small lymphocytic lymphomas, 2 T-cell lymphomas, and 1 marginal zone lymphoma were analyzed. FISH identified deletions at 1p36.33 in 5 of the 20 cases: 3 DLBCL and 2 FL. FISH is considerably more sensitive for identifying lymphoma genetic alterations than conventional cytogenetics. Deletion of the distal part of the 1p36 may be a much more common aberration than previously recognized in lymphoma.

Assignment of ANGPT4, ANGPT1, and ANGPT2 encoding angiopoietins 4, 1 and 2 to human chromosome bands 20p13, 8q22.3-->q23 and 8p23.1, respectively, by in situ hybridization and radiation hybrid mapping.

The recently discovered Angiopoietin family is comprised of four growth factors thought to be very important for vascular development (Davis et al., 1996; Suri et al., 1996; Maisonpierre et al., 1997; Suri et al., 1998; Valenzuela et al., 1999). All four angiopoietins bind to the endothelial cell-specific receptor Tie2 with similar affinity. The mechanism by which these proteins contribute to angiogenesis is thought to involve regulation of endothelial cell interactions with supporting perivascular cells (Jones, 1997). The recently described angiopoietin 3 and angiopoietin 4 appear to represent rapidly diverging orthologs in mouse and man, with disparate expression profiles and differing abilities to activate the Tie-2 receptor (Valenzuela et al., 1999). Materials and methods

Assignment1 of GALGT encoding β-1,4N-acetylgalactosaminyl-transferase (GalNAc-T) and KIF5A encoding neuronal kinesin (D12S1889) to human chromosome band 12q13 by assignment to ICI YAC 26EG10 and in situ hybridization

A locus including the genes GLI, DDIT3, LRP1, SAS and CDK4 on 12q13 is frequently amplified in gliomas and sarcomas (Reifenberger et al., 1994; Forus et al., 1993). We have now mapped two further genes, ß-1,4 N-acetylgalactosaminyltransferase (GALGT) and human neuronal kinesin (KIF5A, D12S1889), to the same region. More specifically, GALGT and KIF5A map to the same 200-kb yeast artificial chromosome as GLI and DDIT3. ß-1,4N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is an enzyme involved in the synthesis of complex gangliosides which have been suggested to have a role in neuronal functioning (Takamiya et al., 1996). Human neuronal kinesin is a member of the kinesin gene family. These proteins are involved in numerous cellular processes including organelle transport, maintenance of the endoplasmic reticulum, intermediate filament distribution, organisation of spindle microtubules, chromosome segregation, flagellar growth and positioning of developmental morphogens (Bloom and Endow, 1995). GLI is the human homologue of the Drosophila gene Cubitus interruptus (Ci), which is a transcription factor involved in the hedgehog signalling pathway, controlling cell fates and patterning in development (Altaba, 1997). It has recently been demonstrated in Drosophila that a cytoplasmic protein, Cos2 binds to microtubules and associates with Ci thereby directly controlling its activity (Sisson et al., 1997). It is of interest that the human homologue of Cos2 is actually human neuronal kinesin (or Kinesin HC) which we have now discovered maps to within, at most, 200 kb of GLI, its putative binding partner. ß-1,4N-Acetylgalactosaminyltransferase and neuronal kinesin are involved in neuronal cellular functioning and both map to a region known to be amplified in certain tumours. This data raises the possibility that these neuronally expressed genes are also amplified in malignancies, particularly gliomas.

A human ubiquitin conjugating enzyme, L-UBC, maps in the Alzheimer's disease locus on chromosome 14q24.3.

We have identified a novel ubiquitin conjugating enzyme gene, L-UBC, which maps to human Chromosome (Chr) 14q24.3. This is also the location of the major early onset familial Alzheimers disease gene (FAD3). L-UBC encodes a protein that demonstrates homology to the yeast ubiquitin conjugating enzyme, UBC-4, and human UbcH5. Their functions are to ubiquitinate specific proteins targeted for degradation. The protein also exhibits very strong homology to a rabbit protein, E2-F1, which mediates p53 degradation driven by papilloma virus E6 protein in vitro. The accumulation of specific proteins that have undergone aberrant processing in neurofibrillary tangles and amyloid plaques is the classic pathological feature in brains of Alzheimers disease patients. Abnormal ubiquitination has previously been suggested to play a role in the etiology of Alzheimers disease. This gene therefore represents a plausible candidate gene for FAD3.

Rapid isolation of genomic clones for individual members of human multigene families : identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family

We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.