Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.     

Confirmation of a susceptibility locus for diabetic nephropathy on chromosome 3q23-q24 by association study in Russian type 1 diabetic patients

Family-based studies and segregation analyses suggest that inherited factors play a significant role in susceptibility to diabetic nephropathy (DN). Moczulski et al. [Diabetes 47 (1998) 1164-1169] found a susceptibility locus for DN in type 1 diabetes covering a 20cM region on chromosome 3q, with a peak of linkage close to the angiotensin II type 1 receptor (AT1) gene. We examined eight polymorphic markers (D3S1512, D3S1550, D3S1557, D3S1744, D3S2326, D3S3599, D3S3694, and a (CA)(n) dinucleotide repeat polymorphism in the 3' flanking region of the AT1 gene) spanning about 6.2 megabases (Mb) in the region of maximal linkage with DN on chromosome 3q23-q24. The markers were used to genotype a total of 381 Russian type 1 diabetic subjects, 195 of whom had DN and 186 had no clinical nephropathy. Four of the markers tested, D3S1512, D3S1550, D3S2326, and D3S3599, showed an association with DN in type 1 diabetes mellitus. These markers are located within a 1.0Mb interval that starts about 4.4Mb centromeric to the AT1 gene. Thus, our results suggest the existence of the DN susceptibility locus previously described by Moczulski et al. on chromosome 3q.     

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.     

A gene-anchored map position of the rat warfarin-resistance locus, Rw, and its orthologs in mice and humans

The locus underlying hereditary resistance to the anticoagulant warfarin (symbol in the rat, Rw) was placed in relation to 8 positionally mapped gene-anchored microsatellite loci whose positions were known in the genome maps of the rat, mouse, and human. Rw segregated with the markers Myl2 (zero recombinants) and Itgam, Il4r, and Fgf2r (one recombinant each) during linkage analysis in a congenic warfarin- and bromadiolone-resistant laboratory strain of rats. Comparative ortholog mapping between rat, mouse, and human placed Rw onto mouse chromosome 7 at about 60 to 63 cM and onto one of the human chromosomes 10q25.3-26, 12q23-q24.3, and 16p13.1-p11. (Blood. 2000;96:1996-1998)     

Alpha-chain locus of the T-cell antigen receptor is involved in the t(10;14) chromosome translocation of T-cell acute lymphocytic leukemia.

Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C alpha [the constant region of TCRA (the alpha-chain locus of the T-cell antigen receptor gene)], but not for V alpha (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V alpha genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V alpha and the C alpha genes. These results suggest that the translocation of the C alpha locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which we propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.     

Evidence for linkage of familial Diamond-Blackfan anemia to chromosome 8p23.3-p22 and for non-19q non-8p disease

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia that usually presents early in infancy and is inherited in 10% to 20% of cases. Linkage analysis has shown that DBA in many of both dominant and recessive DBA families mapped to chromosome 19q13.2 leading to the cloning of a gene on chromosome 19q13.2 that encodes a ribosomal protein, RPS19. However, subsequently, mutations of the RPS19 gene have only been identified in 25% of all patients with DBA. This study analyzed 14 multiplex DBA families, 9 of which had 19q13.2 haplotypes inconsistent with 19q linkage. A genome-wide search for linked loci suggested the presence of a second DBA locus in a 26.4-centimorgan (cM) interval on human chromosome 8p. Subsequently, 24 additional DBA families were ascertained and all 38 families were analyzed with additional polymorphic markers on chromosome 8p. In total, 18 of 38 families were consistent with linkage to chromosome 8p with a maximal LOD score with heterogeneity of 3.55 at D8S277 assuming 90% penetrance. The results indicate the existence of a second DBA gene in the 26.4-cM telomeric region of human chromosome 8p23.3-p22, most likely within an 8.1-cM interval flanked by D8S518 and D8S1825. Seven families were inconsistent with linkage to 8p or 19q and did not reveal mutations in the RPS19 gene, suggesting further genetic heterogeneity. (Blood. 2001;97:2145-2150)     

A genome-wide search identifies potential new susceptibility loci for Crohn's disease

Chronic inflammatory bowel disease (IBD) presents as two major clinical forms, Crohn's disease (CD) and ulcerative colitis (UC). Genetic epidemiological studies and animal models suggest that inherited factors play significant roles in the susceptibility to both forms of IBD. From four genome-wide scans, putative susceptibility loci on chromosome 16 (IBD1 for CD), and on chromosomes 1, 3, 4, 6, 7, 10, and 12 for IBD, have been identified. Several other groups, including ours, have confirmed linkage to the loci on chromosomes 12 and 16. The aim of this study is to identify other potential susceptibility loci for CD with a genome-wide search approach. In our sample of 222 individuals from 46 families (20 Jewish and 26 non-Jewish), with a total of 65 sibpairs diagnosed with CD, we observed a novel locus with suggestive linkage [multipoint logarithm of the odds score (Mlod) > 2] at chromosome 14q11.2 (Mlod = 2.8, p = 0.0002). In addition, suggestive linkage was observed in our Jewish families at chromosome 17q21-q23 (Mlod = 2.1, p = 0.01) and chromosome 5q33-q35 (Mlod = 2.2, p = 0.0003). The syntenic regions of the latter locus are mapped within two putative loci on mouse chromosomes 11 and 18, which were identified in a mouse IBD model induced by dextran sulfate sodium (29). Our preliminary results provide potential evidence for several susceptibility loci contributing to the risk of CD. The observation of man-mouse synteny may accelerate the identification of CD susceptibility gene(s) on human chromosome 5.     

Identification of a major locus for age-related cortical cataract on chromosome 6p12-q12 in the Beaver Dam Eye Study

Age-related cataracts are one of the leading causes of visual impairment and blindness among the elderly worldwide. Among age-related cataracts, cortical opacities rank as the second most common type; however, little is known about their molecular pathogenesis or genetics. To identify susceptibility loci for cortical cataracts, we genotyped a subset of families (102 families; n = 224 sib pairs) from the Beaver Dam Eye Study and performed a model-free genome-wide linkage analysis for markers linked to a quantitative measure of cortical opacity. We obtained evidence for linkage at marker D1S1622 on chromosome 1p35 (P < 0.0002) and at marker D6S1053 on 6q12 (P < 0.00008) in the initial scan. Five additional regions on 1q31, 2p24, 2q11, 4q28, and 15q13 that are suggestive of linkage (P < or = 0.01 or logarithm of the likelihood ratio > or = 1.18) were observed. The region on chromosomes 6p12-q12 was selected for fine mapping, and the intermarker distance was reduced to 3 cM by adding 11 markers in the interval between D6S1017 and D6S1021. After fine mapping, significant evidence of linkage remained on chromosome 6p12-q12 at D6S1053 (P < 0.00005). The current genome scan for age-related cortical cataracts may lead to identification of novel genes, because few regions identified in the current scan have previously been implicated in congenital or age-related cataracts.     

Anisomastia associated with interstitial duplication of chromosome 16, mental retardation, obesity, dysmorphic facies, and digital anomalies: molecular mapping of a new syndrome by fluorescent in situ hybridization and microsatellites to 16q13 (D16S419-D16S503)

Anisomastia is a common problem among developing adolescent girls. We recently evaluated a 22-yr-old female patient who had severe anisomastia (which had been repaired by surgery), associated with moderate to severe mental retardation, a stocky body habitus with mild obesity, dysmorphic facies (prominent, upslanting palpebral fissures, beaked nose, and a prominent philtrum), webbed neck, low hairline, and severe bilateral clinodactyly of the third, fourth, and fifth fingers with acral (but not large joint) flexion contractures. A peripheral blood high resolution karyotype revealed additional chromosomal material within the long arm of chromosome 16. Densitometric analysis of amplified polymorphic sequence-tagged sites (STS) mapping to 16q suggested that the duplication is defined by the noninvolved markers D16S419 [16q12-cen, 66 centimorgan (cM) from 16p terminus] and D16S421 (16q13-q21, 84.4 cM), encompassing a maximum of 18.4 cM of genetic distance. The STS analysis showed that the duplication was on the maternally derived chromosome 16, resulting in two maternal (and one paternal) copies of that region of chromosome 16. The location was further confirmed by bacterial artificial chromosomes (BACs) that were obtained from a commercially available library, labeled, and used for fluorescence in situ hybridization. The BACs containing STSs D16S408, D16S3137, and D16S3032 (markers that correspond to 16q13) showed two regions of hybridization, indicating that these sites were duplicated, whereas a BAC containing the STS D16S512 (which corresponds to 16q21-q22) revealed one hybridization signal per 16q, indicating that the corresponding region was not involved in the duplication. The distance between the probe signals suggested a tandem duplication. We conclude that even though trisomy 16 is the most common autosomal trisomy in spontaneous abortions, few patients with unbalanced chromosome 16 abnormalities survive to adulthood; in this report we describe one such patient with an interstitial chromosome 16 duplication (at 16q13), who had a specific phenotype associated with abnormal breast size. There are clinical similarities between this patient and patients with other 16q abnormalities, although the breast findings were unique. Molecular cytogenetics, including fluorescence in situ hybridization and densitometric analysis of amplified STSs, provided useful tools for the precise mapping of the syndrome to 16q13, where the gene(s) responsible for this phenotype might be localized.     

Genetic analysis of phytosterolaemia

Two women with multiple xanthomas, intermittent arthritis and thrombocytopenia were diagnosed as phytosterolaemia, an autosomal-recessive lipid storage disease, based on their increased serum concentrations of -sitosterol, campesterol and sitostanol. The gene responsible for this disease is located within a distance of 18 cM between microsatellite markers of D2S1788 and D2S1352 at chromosome 2p21. We genotyped the patients and their family members with 16 microsatellite markers around this locus. The results from the homozygosity mapping of one family suggested that the gene was located within the distance of 12.6 cM between D2S2328 and D2S1352. We have shortened the genetic distance by 5.4 cM.     

Genome-wide search for genes related to the fat-free body mass in the Québec family study

Fat-free mass (FFM) consists mostly of skeletal muscle and bone tissues, and identification of the genes and molecular mechanisms involved in the control of FFM would have implications for the understanding of sarcopenia and potentially osteoporesis associated with aging, as well as the response to starvation, refeeding, anorexia, and any other conditions in which lean body mass is important. A genome-wide search for genes related to body leanness has been completed in the Quebec Family Study (QFS). Microsatellite markers (N = 292) from the 22 autosomal chromosomes were typed. The mean spacing of the markers was 11.9 centimorgans (cM) (range, <0.1 to 41). FFM was calculated from percent body fat, derived from underwater weighing, and body weight and was adjusted by regression for age and sex effects before analysis. A maximum of 336 sib pairs or 609 pairs of extended relatives were analyzed using single-point Haseman-Elston regression (SIBPAL and RELPAL) and multipoint variance component (SEGPATH) linkage analyses. Significant linkages were observed on chromosomes 15q25-q26 for a CA repeat within the insulin-like growth factor 1 receptor (IGF1R) gene (Lod score = 3.56) and at 18q12 with D18S877 (Lod score = 3.53) and D18S535 (Lod score = 3.58), 2 markers located 10 cM apart. A moderately significant linkage was also observed on chromosome 7p15.3 with the marker D7S1808 (Lod score = 2.72). The most obvious candidate genes within the regions identified by these linkages include the IGF1R on 15q and neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) receptor on 7p. On 18q, the melanocortin receptor 4 (MC4R) is not likely the candidate gene for the observed linkage. This study represents the first genome-wide search for genes that may be involved in the regulation of the lean component of body mass in humans.     

7q32-q36 translocations in childhood T cell leukemia: cytogenetic evidence for involvement of the T cell receptor beta-chain gene

Blast cell chromosomal rearrangements involving the long arm of chromosome 7 were identified in eight of 197 cases of childhood acute lymphoblastic leukemia (ALL). Breakpoints were variable but tended to cluster in either the proximal or the terminal 7q region, depending on the immunophenotype of the cells. The 7q32-q36 region, the locus of the T cell receptor beta-chain gene, was the site of breakpoints in four of 31 cases of T cell ALL but was not involved in any of the 166 cases originating from B cell precursors (P less than .0004). In three of the four T cell cases it was possible to identify the chromosomal segment that had been translocated to the 7q32-q36 region: 1p32, 2p21, and 6p21. The 1p32 and 6p21 bands are particularly interesting, as they contain the sites of two known protooncogenes, c-L-myc and hpim, respectively. Our findings suggest that the locus of the beta-chain gene of the T cell receptor is a preferential site for certain chromosomal rearrangements in leukemic T lymphoblasts, analogous to the T cell receptor alpha-chain gene on human chromosome 14. Translocation of proto-oncogenes to a site near the beta-chain regulatory sequences provides a potential mechanism for oncogene activation.     

Possible evidence for genomic imprinting in childhood acute myeloblastic leukaemia associated with monosomy for chromosome 7

Monosomy or deletion of chromosome 7 is a frequent finding in both de novo and secondary acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Based on analysis of deletions of chromosome 7 in such patients, it has been suggested that there is a critical region of the chromosome lying within bands q21-q31. We have examined bone marrow and peripheral blood samples from 10 patients with MDS, AML and biphenotypic acute leukaemia who had monosomy for or rearrangement of chromosome 7, seeking evidence of non-random allele loss that might suggest the presence of imprinted genes on the chromosome. Bone marrow cells from one patient with the infant monosomy 7 syndrome had loss of maternal alleles as did two patients with biphenotypic leukaemia. Five out of five patients with MDS and both patients with de novo AML had loss of paternal alleles. One of the latter patients had a del(7) (q31q36) rather than monosomy 7. These findings suggest that imprinting of a gene(s) on chromosome 7, within the bands q31-q36, may be of importance in MDS and AML. Despite the reported increased incidence of AML amongst relatives of patients with cystic fibrosis (CF) the gene for which lies in chromosome region 7q31, none of the patients nor parents studied here appeared to be carriers of the most common gene mutation seen in patients with CF, the delta F508.