Distal Loci Research Articles

Physical and Genetic Characterization Reveals a Pseudogene, an Evolutionary Junction, and Unstable Loci in Distal Xq28

A large portion of human Xq28 has been completely characterized but the interval between G6PD and Xqter has remained poorly understood. Because of a lack of stable, high-density clone coverage in this region, we constructed a 1.6-Mb bacterial and P1 artificial chromosome (BAC and PAC, respectively) contig to expedite mapping, structural and evolutionary analysis, and sequencing. The contig helped to reposition previously mismapped genes and to characterize the XAP135 pseudogene near the int22h-2 repeat. BAC clones containing the distal int22h repeats also demonstrated spontaneous rearrangements and sparse coverage, which suggested that they were unstable. Because the int22h repeats are involved in genetic diseases, we examined them in great apes to see if they have always been unstable. Differences in copy number among the apes, due to duplications and deletions, indicated that they have been unstable throughout their evolution. Taking another approach toward understanding the genomic nature of distal Xq28, we examined the homologous mouse region and found an evolutionary junction near the distal int22h loci that separated the human distal Xq28 region into two segments on the mouse X chromosome. Finally, haplotype analysis showed that a segment within Xq28 has resisted excessive interchromosomal exchange through great ape evolution, potentially accounting for the linkage disequilibrium recently reported in this region. Collectively, these data highlight some interesting features of the genomic sequence in Xq28 and will be useful for positional cloning efforts, mouse mutagenesis studies, and further evolutionary analyses.

Functional and binding studies of HS3.2 of the beta-globin locus control region

The distal locus control region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the β-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1–5) mark the LCR. Sequence conservation and direct experimental evidence have implicated sequences within and between the HS cores in function of the LCR. In this report we confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences.

Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7.

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.

An integrated 5-Mb physical, genetic, and radiation hybrid map of a 1p36.1 region implicated in neuroblastoma pathogenesis

Common genetic aberrations of neuroblastoma are deletions of the short arm of chromosome 1 (1p36) and MYCN amplification. Our deletion analysis of 25 tumor cell lines and 171 tumors strongly suggests that 1p harbors several tumor suppressor loci. Distinct loci are involved in MYCN single-copy versus MYCN-amplified neuroblastoma. Deletions in MYCN single-copy tumors have a shortest region of overlap (SRO) of 20 cM at 1p36.3. MYCN-amplified tumors have large deletions with an SRO of about 60 cM, from 1p36.1 to the telomere. This SRO is defined by D1S7 (1p36.1), which was the most distal locus retained. Therefore, a suppressor gene associated with MYCN-amplified tumors probably maps within a few megabases distal of D1S7. In order to map this locus, we further refined this SRO. We mapped the breakpoint of the MYCN-amplified neuroblastoma with the smallest 1p deletion between 56.6 and 57.2 cM from 1pter. Pulsed-field gel electrophoresis and radiation hybrid mapping were used to construct a 5-Mb physical map of this region. The map includes the region from 82.73 till 92.89 cR from 1pter. About half of it was isolated in P1 and PAC clones. The region harbors the genes FGR, SLC9A1, HMG17, EXTL1, AML2, RH, OP18, four ESTs, and a newly identified gene with a transcript size of approximately 7 Kb. Several of the mapped genes have a putative role in cell growth, differentiation, and morphogenesis. Genes Chromosomes Cancer 27:143-152, 2000.

Sex-of-offspring-specific transmission ratio distortion on mouse chromosome X.

During our study of the DDK syndrome, we observed sex ratio distortion in favor of males among the offspring of F(1) backcrosses between the C57BL/6 and DDK strains. We also observed significant and reproducible transmission ratio distortion in favor of the inheritance of DDK alleles at loci on chromosome X among female offspring but not among male offspring in (C57BL/6 x DDK)F(1) x C57BL/6 and (C57BL/6-Pgk1(a) x DDK)F(1) x C57BL/6 backcrosses. The observed transmission ratio distortion is maximum at DXMit210 in the central region of chromosome X and decreases progressively at proximal and distal loci, in a manner consistent with the predictions of a single distorted locus model. DXMit210 is closely linked to two distortion-controlling loci (Dcsx1 and Dcsx2) described previously in interspecific backcrosses. Our analysis suggests that the female-offspring-specific transmission ratio distortion we observe is likely to be the result of the death of embryos of particular genotypic combinations. In addition, we confirm the previous suggestion that the transmission ratio distortion observed on chromosome X in interspecific backcrosses is also the result of loss of embryos.

Common regions of deletion on chromosome 22q12.3-q13.1 and 22q13.2 in human astrocytomas appear related to malignancy grade.

Approximately 30% of human astrocytomas have been reported to display allelic loss of the long arm of chromosome 22, suggesting the presence of a chromosome 22q astrocytoma suppressor gene. To define the most likely location for this putative tumor suppressor, we performed deletion mapping on 141 tumors using 16 chromosome 22q microsatellite markers. Allelic loss of 22q was observed in 2/12 (17%) of astrocytomas, 9/29 (31%) of anaplastic astrocytomas, and 38/100 (38%) of glioblastomas, consistent with a role for chromosome 22q loss in astrocytoma progression as well as formation. Twenty-two tumors exhibited allelic loss at every informative locus, consistent with loss of the entire arm of 22q. Twenty-seven tumors showed partial deletions, with one common region of deletion at 22q12.3-q13.1 between markers D22S280 and D22S282, and a second candidate region at 22q13.2 near the marker D22S1170. For the proximal candidate region, the incidence of allelic loss was similar between grades; for the distal locus, the incidence increased with grade, raising the possibility that the distal locus is involved in a later stage of astrocytoma tumorigenesis.

A Radiation Hybrid Map of Mouse Chromosome 13

A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci areD13Mit132andD13Mit35.The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes.

Temporal and Multiple Quantitative Trait Loci Analyses of Resistance to Bacterial Wilt in Tomato Permit the Resolution of Linked Loci

Ralstonia solanacearum is a soil-borne bacterium that causes the serious disease known as bacterial wilt in many plant species. In tomato, several QTL controlling resistance have been found, but in different studies, markers spanning a large region of chromosome 6 showed strong association with the resistance. By using two different approaches to analyze the data from a field test F3 population, we show that at least two separate loci approximately 30 cM apart on this chromosome are most likely involved in the resistance. First, a temporal analysis of the progression of symptoms reveals a distal locus early in the development of the disease. As the disease progresses, the maximum LOD peak observed shifts toward the proximal end of the chromosome, obscuring the distal locus. Second, although classical interval mapping could only detect the presence of one locus, a statistical "two-QTL model" test, specifically adapted for the resolution of linked QTL, strongly supported the hypothesis for the presence of two loci. These results are discussed in the context of current molecular knowledge about disease resistance genes on chromosome 6 and observations made by tomato breeders during the production of bacterial wilt-resistant varieties.

Fine mapping of distal 1p loci reveals TP73 at D1S468

In the present study we establish a FISH fine-map of 1p36.3 loci. This region is frequently altered in different types of human tumors suggesting the existence of cancer-related genes. Identification of cosmids carrying both D1S468 and TP73 sequences leads to the assignment of TP73 to the most frequently deleted locus in colon and breast cancer and integrates this gene in human genetic maps. Localization of other distal loci was determined as follows: distal–CDC2L1–D1Z2–D1S94–TP73/D1S468–D1S1615–proximal. D1S1615, earlier reported as a telomeric sequence, is considerably more proximal than previously thought.

Pseudo-exclusion from paternity due to maternal uniparental disomy 16.

The investigation of a case of disputed paternity revealed indirect exclusion of the alleged father in the haptoglobin system and in the DNA single-locus system D16S309/Hinf I (MS205). The paternity index for the non-exclusion systems was > 10(6). Since both exclusion systems (HP and MS205) are located on chromosome 16, we investigated 10 microsatellite loci covering this chromosome with 10-20 cM resolution. Analysis of the child's chromosome showed only alleles of maternal origin and lack of inheritance of paternal alleles for five informative loci. The markers close to the centromere of chromosome 16 were heterozygous, whereas distal loci were either heterozygous or homozygous for maternal alleles. This is consistent with a maternal meiosis I nondisjunction of chromosome 16 leading to maternal uniparental heterodisomy. This case emphasizes that the opinion of non-paternity should be based on the absence of paternal alleles at genetic systems located on at least two different chromosomes.

Substance P induces brief, localized increase in [Ca2+]i in dorsal horn neurons.

We determined the spatial and temporal dynamics of the increase in intracellular Ca2+ levels [Ca2+]i produced by substance P (SP) in dorsal horn neurons. A microinjection technique was used to apply minute amounts of SP to small areas of cultured neurons loaded with the Ca2+ indicator fura-2. Five successive applications of SP to the soma produced short-lasting (< 50 s) increases in [Ca2+]i that became gradually smaller, indicating receptor desensitization. Focal application of SP to a distal locus in a neurite produced a brief (12 s) increase in [Ca2+]i that travelled down the dendrite but did not spread into cell soma. Prolonged application of SP to these neurons caused the appearance of varicosities in their dendrites.

Genome plasticity in the distal tail fiber locus of the T-even bacteriophage: recombination between conserved motifs swaps adhesin specificity

The adsorption specificity of the T-even phages is determined by the protein sequence near the tip of the long tail fibers. These adhesin sequences are highly variable in both their sequence and specificity for bacterial receptors. The tail fiber adhesin domains are located in different genes in closely related phages of the T-even type. In phage T4, the adhesin sequence is encoded by the C-terminal domain of the large tail fiber gene (gene 37), but in T2, the adhesin is a separate gene product (gene 38) that binds to the tip of T2 tail fibers. Analysis of phage T6 and Ac3 sequences reveals additional variant forms of this locus. The tail fiber host specificity determinants can be exchanged, although the different loci have only limited homology. Chimeric fibers can be created by crossovers either between small homologies within the structural part of the fiber gene or in conserved motifs of the adhesin domain. For example, the T2 adhesin determinants are flanked by G-rich DNA motifs and exchanges involving these sequences can replace the specificity determinants. These features of the distal tail fiber loci genetically link their different forms and can mediate acquisition of diverse host range determinants, including those that allow it to cross species boundaries and infect taxonomically distant hosts.

Welander distal myopathy – an overview

Welander distal myopathy has an autosomal dominant inheritance and a late onset. The onset of symptoms is in the hands and gradually distal muscles of the lower extremities are involved. The most-affected muscles are the long extensors of the hands and feet. CK-values are normal or slightly elevated. There is never any cardiac involvement in Welander distal myopathy. Neurophysiological findings are of both myopathic and neuropathic character. Histopathological findings in muscle biopsies are mainly of myopathic type and include rimmed vacuoles which correspond to autophagic vacuoles on the ultrastructural level. Tubulo-filamentous inclusions with a diameter of 16–21 nm, i.e. of the same type as found in patients with Inclusion Body Myositis, are found in the sarcoplasm and in myofibre nuclei. A neurogenic component in Welander distal myopathy has been suggested, on the grounds of a sensory dysfunction, neuropathic findings on neurophysiology and muscle biopsy and a decrease of A- σ nerve fibres on sural nerve biopsy. Genetic analysis has excluded linkage to other defined distal myopathies and hereditary Inclusion Body Myopathy loci.

Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: evidence for multiple critical loci.

Complete or partial deletions of the long arm of chromosome 7 (7q- and -7) are nonrandom abnormalities seen in primary and therapy-induced myelodysplasia (MDS) and acute myelogenous leukemia (AML). Monosomy 7, occurring as the sole cytogenetic anomaly in a small but significant number of cases, may denote a dominant mechanism involving critical tumor suppressor gene(s). We have determined the extent of allele loss in cytogenetically prescreened MDS and AML patients for microsatellite markers from chromosome 7q22 and 7q31. Whereas >80% of these cases revealed allele loss for the entire region, a rare case of the 7q- chromosome showed allele loss for only the proximal 7q31.1 loci flanked by the markers D7S486 and D7S2456, and a case of monosomy 7 revealed allele loss for loci at both 7q31 and 7q22 with retention of sequences between these sets of loci. Furthermore, a case of AML with no cytogenetic anomaly of chromosome 7 revealed a submicroscopic allelic imbalance for a third distal locus, D7S677. These findings suggest the presence of three distinct critical loci that may contribute alone or in combination to the evolution of MDS and AML. The data also provide molecular evidence for unbalanced translocation with noncontiguous deletions, as an alternate mechanism underlying monosomy 7.

Short stature and azoospermia in a patient with Y chromosome long arm deletion

We report on a 42-year old male with short stature, azoospermia and a wide deletion of long arm of Y chromosome. On physical examination, the patient showed height of 149 cm (< 1 degree centile) and reduced volume (3 ml) and consistency of the testes. On hormonal evaluation, he showed increased serum gonadotropins and normal serum testosterone levels though its HCG stimulated levels were limited. Serum thyroid hormones were normal. Serum GH levels in baseline evaluation as well as after GHRH and GHRH + pyridostigmine administration were normal. Serum IGF I levels were lower than normal in baseline evaluation whereas its response to the GH administration was in the normal range. The bilateral testicular biopsy showed tubular atrophy, hyalinosis, interstitial sclerosis and a histological picture of a Sertoli cell only syndrome. Moreover the patient showed arthropathy, otopathy, small chin, small mouth and truncal obesity. On genetic evaluation, the patient showed a 46,X,delY (pter--q11.1:) karyotype and loss of several DNA loci on Yq. In fact he preserved short arm SRY, centromeric DYZ3 and more proximal euchromatic region Yq loci, including DYS270, DYS271, DYS272, DYS11, DYS273, DYS274, DYS148, DYS275, and missed more distal DNA loci from DYS246 to DYZ2. These results disclosed a wide Y long arm deletion, including all hypothized Yq azoospermia loci (except for AZFa and probably for one of the RBM genes, which lie proximally to the deletion) and possibly the Y-specific growth control region (GCY), mapped between DYS11 and DYS246 loci. This deletion is responsible for the complete azoospermia of the patient and probably also for his short stature, even if other factors could be implicated in the statural impairment. It further possibly allowed to relate the GCY gene(s) to the control of GH or IGF-I receptor or post-receptor pathway, being the alteration of this gene(s) consistent with the hormonal pattern of the patient.

Molecular analysis of the region of distal 1p commonly deleted in neuroblastoma.

Cellular, cytogenetic, and molecular evidence indicates that chromosome band 1p36 is often deleted in neuroblastoma cell lines and tumours, suggesting the presence of one or more tumour suppressor genes in this region. We used a multifaceted approach to analyse the commonly deleted region, 28 distal 1p-specific polymorphic loci were used to detect loss of heterozygosity (LOH) in a panel of primary neuroblastoma tumours. Thirty-two of 122 tumours (26%) demonstrated LOH at three or more loci. In addition, a patient with a constitutional deletion of 1p36.2-.3 and two neuroblastoma cell lines with 1p36 abnormalities were characterised by FISH. When combined with the LOH data, a single consensus region of deletion was defined proximally by PLOD and distally by D1S80, a region spanning approximately five megabases. Several proposed candidate tumour suppressor genes, including ID3, CDC2L1, DAN, PAX7, E2F2, TNFR2 and TCEB3, map outside of this region; however, the transcription factor HKR3 cannot be excluded. LOH for 1p is correlated with adverse clinical and biological features and a poor prognosis, but 1p LOH is not an independent predictor of overall survival. To identify additional candidate genes, an integrated physical map of 1p35-36 is being constructed. The current map includes 445 polymerase chain reaction (PCR)-formatted markers and 608 YACs. This map will help identify region-specific transcripts by direct selection and sequencing.

Allelic loss of 8p sequences in prostatic intraepithelial neoplasia and carcinoma

Objectives Previous work has suggested that prostatic intraepithelial neoplasia (PIN) may be a premalignant lesion important in tumorigenesis of the prostate. However, to adequately test this hypothesis at the genetic level, it is necessary to determine whether lesions in close proximity demonstrate similar genetic alterations and, hence, whether an “evolutionary” relationship might exist between PIN and tumor in the same prostate. Therefore, the purpose of this study was to examine at least two PIN lesions per prostate (one adjacent to and another distant from malignant lesions in the same prostate) for similarities or differences in the types and frequencies of genetic alterations. Methods To accomplish this goal, DNA was extracted from microdissected PIN, tumor and normal epithelial tissue samples from 48 radical prostatectomies and amplified using polymerase chain reaction techniques at highly polymorphic microsatellite repeat sequences at proximal ( D8S87, 8p12) and distal ( NEFL, 8p21) loci on the short arm of chromosome 8. PIN specimens were either adjacent to (within one high-power microscopic field [HPF]) or distant from (separated by two or more HPFs) tumor specimens from the same patients. Results Similar fractional allelic loss frequencies were observed for informative tumor (10 [35%] of 29) and PIN (6 [21%] of 29) samples at the NEFL locus, but allelic loss at the D8S87 locus was observed only in tumors (8 [22%] of 36 informative samples). Moreover, allelic loss at the NEFL locus involved the same allele in 4 cases and different alleles in 3 cases. Interestingly, all 4 cases with the same allele loss were from adjacent PIN and tumor tissues and all 3 with different allele loss were from distant PIN and tumor. Conclusions These results suggest that PIN and invasive cancer share common genetic events (eg, deletion at the NEFL locus) along the same pathway of development in the prostrate.

A High-Resolution STS, EST, and Gene-Based Physical Map of the Hereditary Paraganglioma Region on Chromosome 11q23

The genes responsible for hereditary paragangliomas (glomus tumors, MIM No. 168000) have been mapped to two distinct loci on the long arm of chromosome 11. Most of the informative families appear to be linked to the distal locus on chromosome 11q23 (PGL1), which has been previously confined to a 2-cM interval by haplotype analysis in an extended Dutch pedigree. To facilitate the identification of the PGL1 disease gene, we constructed an approximately 4-Mb ordered clone contig map of Sequence tagged sites, expressed sequence tags (ESTs), and known genes that spans the PGL1 critical region on chromosome 11q23. Among 29 new positional candidate ESTs, only two (EST100999 and EST241777) mapped within the PGL1 critical region. We further characterized the genomic organization of the promyelocytic leukemia zinc finger (PLZF) gene that maps within the PGL1 critical region and physically excluded the serotonin receptor type 3 (5HT3R) gene. Finally, we identified a common, silent, single-base substitution polymorphism in the 5HT3R gene and characterized the allele sets of two new highly polymorphic microsatellite repeats within the PGL1 critical region.

An interstitial telomere array proximal to the distal telomere of mouse chromosome 13.

Lambda clones of mouse DNA from BALB/c and C57BL/10, each containing an array of telomere hexamers, were localized by FISH to a region close to the telomere of Chr 13. Amplification of mouse genomic DNA with primers flanking SSRs within the cloned DNA showed several alleles, which were used to type eight sets of RI strains. The two lambda clones contained allelic versions of the interstitial telomere array, Tel-rs4, which is 495 bp in C57BL/10 and which includes a variety of sequence changes from the consensus telomere hexamer. Comparison of the segregation of the amplification products of the SSRs with the segregation of other loci in an interspecies backcross (C57BL/6JEi x SPRET/Ei) F1 x SPRET/Ei shows recombination suppression, possibly associated with ribosomal DNA sequences present on distal Chr 13 in Mus spretus, when compared with recombination in an interstrain backcross, (C57BL/6J x DBA/J) F1 x C57BL/6J, and with the MIT F2 intercross. Analysis of recombination in females using a second interstrain backcross, (ICR/Ha x C57BL/6Ha) F1 x C57BL/6Ha, also indicates recombination suppression when compared with recombination in males of the same strains, using backcross C57BL/6Ha x (ICR/Ha x C57BL/6Ha) F1. Thus, more than one cause may contribute to recombination suppression in this region. The combined order of the loci typed was D13Mit37-D13Mit30-D13Mit148-(D13Rp1, 2, 3, 4, Tel-rs4)-D13Mit53-D13Mit196-D13Mit77-(D13Mit7 8, 35). Data from crosses where apparently normal frequencies of recombination occur suggest that the telomere array is about 6 map units proximal to the most distal loci on Chr 13. This distance is consistent with evidence from markers identified in two YAC clones obtained from the region.

Welander distal myopathy is not linked to other defined distal myopathy gene loci

Welander distal myopathy is an autosomal dominant disorder with late onset that affects extensor muscles of the hands and the feet. The disorder is considered as the most prevalent of the distal myopathies and is almost only seen in Sweden and in some parts of Finland. On clinical, morphological and genetical grounds the disorder is clearly separated from other distal myopathies. We have performed linkage analysis with the MLINK program in a total of six families with microsatellite markers dispersed throughout the genome and report exclusion for the localisation of the gene of 64% of the human genome. These studies have clearly separated Welander distal myopathy from previously mapped forms of distal myopathy such as the Miyoshi myopathy by excluding linkage to chromosome 2. The region on 14q that has been suggested to house the gene of the distal myopathy described by Laing et al. (Am J Hum Genet 1995;56:422–7), has as well been excluded by several markers.