A region-specific microdissection library for human chromosome 2p23–p25 and the analysis of an interstitial deletion of 2p23.3–p25.1

A region-specific library for human chromosome 2p23–p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50–600 base pairs (bp) with a mean of 200 bp. About 50%–60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3–p25.1 region — 46,XY, del(2) (pterp25.1::p23.3qter) — previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23–p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.     

Familial occurrence of 18q-

Summary An unusual segregation of the partial long arm deletion of a chromosome 18 is reported. This aberration was found in the feeble-minded mother and in her 4 daughters. The fifth child has XXY-Klinefelter's syndrome. The carriers of 18q — in this family reveal small stature, microcephaly, and mental deficiency in the range from feeble-minded to severe imbecility. Other characteristic features commonly found in patients with 18q — syndrome, as mid-face retraction, downward slanting mouth, heart defect, and atretic ear canals were not observed.     

Mapping of the α4 subunit gene (GABRA4) to human chromosome 4 defines an α2—α4—β1—γ1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster

We demonstrated previously that an α₁—β₂—γ₂ gene cluster of the γ-aminobutyric acid (GABA_A) receptor is located on human chromosome 5q34–q35 and that an ancestral α—β—γ gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the α₄ gene (GABRA4) maps to human chromosome 4p14–q12, defining a cluster comprising the α₂, α₄, β₁, and γ₁ genes. The existence of an α₂—α₄—β₁—γ₁ cluster on chromosome 4 and an α₁—α₆—β₂—γ₂ cluster on chromosome 5 provides further evidence that the number of ancestral GABA_A receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the α gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of α subunit should be located on human chromosome 15q11–q13 within an α₅—α_x—β₃—γ₃ gene cluster at the locus for Angelman and Prader—Willi syndromes.     

Cri-du-chat syndrome mimics Silver-Russell syndrome depending on the size of the deletion: a case report

Background

Silver-Russell Syndrome (SRS) is a rare growth-related genetic disorder mainly characterized by prenatal and postnatal growth failure. Although molecular causes are not clear in all cases, the most common mechanisms involved in SRS are loss of methylation on chromosome 11p15 (≈50%) and maternal uniparental disomy for chromosome 7 (upd(7)mat) (≈10%).

Case presentation

We present a girl with clinical suspicion of SRS (intrauterine and postnatal growth retardation, prominent forehead, triangular face, mild psychomotor delay, transient neonatal hypoglycemia, mild hypotonia and single umbilical artery). Methylation and copy number variations at chromosomes 11 and 7 were studied by methylation-specific multiplex ligation-dependent probe amplification and as no alterations were found, molecular karyotyping was performed. A deletion at 5p15.33p15.2 was identified (arr[GRCh37] 5p15.33p15.2(25942–11644643)×?1), similar to those found in patients with Cri-du-chat Syndrome (CdCS). CdCS is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-), whose main feature is a high-pitched mewing cry in infancy, accompanied by multiple congenital anomalies, intellectual disability, microcephaly and facial dysmorphism.

Conclusions

The absence of some CdCS features in the current patient could be due to the fact that in her case the critical regions responsible do not lie within the identified deletion. In fact, a literature review revealed a high degree of concordance between the clinical manifestations of the two syndromes.

     

G banding in cytogenetic study of hemoblastoses

Summary Two boys (W. M. and C. P.) are described, in each of whom a short-arm deleted C chromosome was apparently present. The clinical findings on W. M. are stenosis of the sagittal sutura associated with atrophy of the nervus opticus and mental retardation, and on C. P. osteogenesis imperfecta. An analysis of the G- and Q-banding patterns revealed in each patient a 12p—chromosome. The deletion involved most of band p12 as shown by length measurements on G-banded chromosomes. Both cases were compared to proven and presumed cases of 12p—from literature, but no common clinical phenotype could be demonstrated.     

Chromosome mapping in barley by means of telotrisomic analysis

Summary A total of 37 genetic markers located in chromosomes 2, 3, 4 and 5 were associated with specific arms by means of telotrisomic analysis in five telotrisomics (Triplo 2 L, 2 S, 3 S, 4 S, 5 L) of barley (Hordeum vulgare L.). The genes v, gp (= gp 2), li, gs 5, tr and msg2 showed a trisomic ratio with Triplo 2 L indicating that these genes were on the long arm of chromosome 2. A disomic ratio was obtained for genes wst 4, gs 5, and v with Triplo 2 S, confirming that these genes were on the long arm of chromosome 2(2 L). A disomic ratio was observed for genes e, f(= lg), sk, and gs6 with Triplo 2 L. Two genes, f(= lg) and gs6 showed a trisomic ratio with Triplo 2S. These results indicated that genes e, f(= lg), sk, and gs 6 are on the short arm of chromosome 2 (2S). Since only one telocentric chromosome was available for chromosome 3, 4 and 5, most of the well-mapped marker genes were tested with those telocentric chromosomes. The genes cu 2, uz, wst, als, gs 2, zb,f2, and cer-zn ³⁴⁸ showed trisomic ratio with the telocentric for chromosome 3. These genes were located on the short arm of chromosome 3 (Robertson 1971). This indicated that the telocentric chromosome is for the short arm of chromosome 3(3 S). A disomic ratio was obtained for genes yst, x _c, al, yst2, a _n, ari-a ⁶ and x _s, indicating that these genes are on the long arm of chromosome 3. Two genes, f9 and K, showed trisomic ratio with the telocentric chromosome for 4, while genes gl(= gl2), br2, yh, lg 3, lg 4 and lk 5 showed disomic ratios. This indicated that the telocentric chromosome is for the short arm of chromosome 4. Two genes, fs 2 and g, were studied with Triplo 5 L. Both showed trisomic ratio, indicating that fs 2 and g are located on Triplo 5 L. The centromere position (C) on chromosome 2, 3 and 4 was thus located as (the left side of C is the short arm and the right is the long arm): chromosome 2: f — sk — gs6 — e — C — gs5 — msg2 — wst4 — v — gp — li — tr; chromosome 3: f2 — cer-zn ³⁴⁸ — uz — gs2 — als — cu2 — wst — zb — C — yst — x _c — al — yst2 — a _n — ari-a ⁶ — x _s; chromosome 4: f9 — K — C — lg4 — lg ³ — gl2 — br2 — lk5 — yh. The centromere position on chromosome 5 was not precisely located.Contribution from the Department of Agronomy, Published with the approval of the director of the Colorado State University Experiment Station as Scientific Series Paper No. 2606. This research was supported in part by by NSF Grant GB 4482X and GB 30 493 to T. Tsuchiya and Colorado State University Experiment Station Hatch Project     

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development unitl their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (MCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in inter-specific backcross progeny revealed linkage of mCd19 with hemoglobin (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere — Hbb — mCd19 — H19 — Int-2 —telomere. The genetic distance between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C polypeptide.     

Molecular cytogenetic characterization of the first reported case of an inv dup (4p)(p15.1-pter) with a concomitant 4q35.1-qter deletion and normal parents

Inverted duplications associated with terminal deletions are complex anomalies described in an increasing of chromosome ends. We report on the cytogenetic characterization of the first de novo inv dup del(4) with partial 4p duplication and 4q deletion in a girl with clinical signs consistent with “recombinant 4 syndrome”. This abnormality was suspected by banding, but high-resolution molecular cytogenetic investigations allowed us to define the breakpoints of the rearrangement. The terminal duplicated region extending from 4p15.1 to the telomere was estimated to be 29.27 Mb, while the size of the terminal deletion was 3.114 Mb in the 4q35.1 region. Until now, 10 patients with duplicated 4p14-p15 and deleted 4q35 chromosome 4 have been described. In all cases the abnormal chromosome 4 was derived from a pericentric inversion inherited from one of the parents. In conclusion, we have identified the first case of inv dup del(4) with normal parents suggesting that, often, terminal duplications or terminal deletions mask complex rearrangements.     

Long range restriction map of the von Hippel-Lindau gene region on human chromosome 3p

Von Hippel-Lindau disease is a heritable tumour syndrome caused by the loss of the function of a tumour suppressor gene on the short arm of human chromosome 3. The interval RAF1-D3S18 (3p25–3p26) has been identfied by genetic linkage studies to harbour the von Hippel-Lindau gene. We have constructed a long range restriction map of this region and have succeeded in demonstrating the physical linkage of loci D3S726 (DNA probe LIB31-38), D3S18 (c-LIB-1, L162E5), D3S601 (LIB1963) and D3S587 (LIB 12–48). Since multipoint analysis has located D3S601 proximal to D3S726, the physical map should be oriented with D3S726 towards the telomere. The order and distances of probes within the von Hippel-Lindau gene region is as follows: telomere — LIB3138 — (<280 kb) — c-LIB-1 — (overlapping) — L162E5 — (900–1600 kb) — (LIB 19-63, LIB 12–48) — centromere. In tissues that included blood, semen and Epstein-Barrvirus-transformed lymphocytes, we detected a putative CpG island flanking D3S18.     

Rotifer genetics: integration of classic and modern techniques

Rotifer genetics has a long but sporadic history. There have been 4 major periods of research activity: (1) determining the environmental control of sexuality with inferences regarding genetics — early 1900's; (2) exploring the relationship between chromosome numbers and the rotifer life cycle — 1920's; (3) physiological and developmental genetics — 1960's; and (4) theoretical and experimental population genetics late 1970's. With newly developed molecular techniques, in conjunction with more traditional approaches, integration of these fields is beginning. Examples include investigation of gene expression involved in sexual reproduction by isolating glycoproteins responsible for mate recognition. Improvement of techniques for chromosome analysis has made it possible to verify haploidy in males and led to the discovery of polyploidy. The role of specialized proteins in the stress response is being elaborated with an accompanying search for the genetic elements which control them. Most recently the polymerase chain reaction (PCR) has been used to amplify ribosomal genes, and is a first step in using DNA sequences to define evolutionary relationships among the Rotifera.     

A specific chromosome element,the telomere of Drosophila polytene chromosomes

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 Å diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 Å, 200–250 Å or 400 Å. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 Å. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.     

Chromosome size and DNA content of species of anemone L. and related genera (Ranunculaceae)

Relative amounts of DNA were determined by Feulgen cytophotometry in 22 diploid species of Ranunculaceae (n=7, 8, 9) representing six genera, and exhibiting large differences in chromosome size, but no marked differences in karyotype pattern. Chemical determination of absolute amounts of DNA for six of these species, allowed conversion of all the photometric data into absolute units of DNA. The mean DNA content per nucleus varied from.13×10^–11gm in Aquilegia to 5.25×10^–11gm in species of Anemone in the section Homalocarpus. The DNA values obtained appeared to be quantized, and data for the majority of species fitted a non-geometrical series with the observed relative terms: 1—8—12—16—20—24—40. The magnitude of these variations in DNA content, the preservation of the karyotype and the tendency towards a simple numerical progression in DNA values, lead us to prefer an interpretation of the evolution of DNA content in terms of differential polynemy to one postulating changes in size of genetic units in an unchanging number of strands per chromosome.     

A comparative study on the effect of various detergents in human chromosome G-banding prior to tryptic digestion

The effect of treatment of chromosomes with various detergents prior to tryptic banding was investigated. The pre-treatment improved the resolution of banding for most of the chromosomes. The effectiveness of the detergents varied greatly. The number of chromosome pairs with optimal banding found after the use of different detergents was as follows: 19 of the possible 23 for emulphogene, 15 for Nonidet, 9 for Triton X-100, 10 for Tween 40, 4 for Tween 80, 4 for DOC and 18 for SDS compared to 3 for trypsin without using detergent. Optimal banding was as defined by the Paris Conference (1971) map. The improvement of banding was shown to be due to detergent-induced changes in the chromosomal proteins. When the chromosomes were treated first with trypsin followed by the detergent no improvement of chromosomal banding was observed. The detergents showed a degree of specificity towards individual chromosomes; certain chromosomes were found to be better banded with a particular detergent. Pretreatment of chromosomes with a combination of two detergents — simultaneously or consecutively — was found not to be additive. After such treatment the chromosome bands were disrupted. Pretreatment with different detergents sometimes changed the topography of chromosome banding, i.e., the relative location of the bands from the centromere. These findings suggest that the proteins attached to the DNA of the chromosomes were removed or loosened from different sites by the various detergents. — For chromosomes 8, 9, 20 and X, additional bands not reported previously were detected.On sabbatical leave from the Weizman Institute of Science, Rehovot, Israel     

Molecular definition of the smallest region of deletion overlap in the Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS), associated with a deletion of chromosome 4p, is characterized by mental and growth retardation and typical facial dysmorphism. A girl with clinical features of WHS was found to carry a subtle deletion of chromosome 4p. Initially suggested by high-resolution chromosome analysis, her deletion was confirmed by fluorescence in situ hybridization (FISH) with cosmid probes, E13 and Y2, of D4S113. To delineate this 4p deletion, we performed a series of FISH and pulsed-field gel electrophoresis analyses by using probes from 4p16.3. A deletion of approximately 2.5 Mb with the breakpoint at approximately 80 kb distal to D4S43 was defined in this patient and appears to be the smallest WHS deletion so far identified. To further refine the WHS critical region, we have studied three unrelated patients with presumptive 4p deletions, two resulting from unbalanced segregations of parental chromosomal translocations and one resulting from an apparently de novo unbalanced translocation. Larger deletions were identified in two patients with WHS. One patient who did not clinically present with WHS had a smaller deletion that thus eliminates the distal 100-300 kb from the telomere as being part of the WHS region. This study has localized the WHS region to approximately 2 Mb between D4S43 and D4S142.     

Chromosome 1 in human colorectal tumors

Summary The significance of short and long arm anomalies of chromosome 1 was investigated in 55 colorectal tumors comprising 41 carcinomas and 14 adenomas. The tumors were at various stages of transformation from adenoma to carcinoma. Our investigation was prompted by the observation of a p32-pter deletion on the short arm of chromosome 1 in a case of benign tubulovillous adenoma with mild dysplasia, as well as by frequent reports that chromosome 1 is involved in many neoplastic processes. Long arm anomalies were found in seven of the 41 carcinomas, six of which were in stage B2, and short arm anomalies in ten carcinomas at various stages. Three of the adenomas exhibited chromosome 1 anomalies, which in one case comprised a 1p32-pter deletion only. Overall, short arm anomalies especially concerned the p32–36 region. These results suggest that the cytogenetic anomalies respectively located on the short and long arms of chromosome 1 should be considered separately. Damage to the long arm might constitute a late non-specific event, whereas damage to the p32-pter region of the short arm might be involved in triggering colorectal tumor development.     

Fine cytogenetical analysis of the band 10A1-2 and the adjoining regions in the Drosophila melanogaster X chromosome

The X chromosome region 9F12-10A7 (7 bands removed by Df(1)v ^l3) was saturated with lethal, semi-lethal, visible and male sterile mutations. A total of 11 complementation groups were found. In the more narrow interval of Df(1)v ^l1 which removes 3 bands (10A1-2, 10A3, 10A4-5) 6 loci were localised. — The band 10A1-2 consists of a sereis of 5 different subunits: (i) silent DNA where no functions were found — at the distal edge of the band; (ii) and (iii) two genes: v and 1(1)BP4; (iv) silent DNA in middle of the band, (v) locus sev on the proximal edge of the band. About 70% of the band's DNA was found to be silent. — Using the set of chromosome rearrangements removing different parts of the band it was shown that these five sequences may function independently from each other.     

Molecular and cytogenetic analysis of an interstitial 20p deletion associated with syndromic intrahepatic ductular hypoplasia (Alagille syndrome)

Summary High-resolution chromosome analysis of a 19-year-old female proband with syndromic intrahepatic ductular hypoplasia (Alagille syndrome, AWS) revealed an interstitial deletion of chromosome 20p with breakpoints provisionally located in or close to p11.22 and p12.2. Southern blots from digests of DNA of the proband and her chromosomally normal parents were hybridized with the human DNA probes pR12.21, HuPrPcDNA2, and pDS6-SgI, which have been mapped to the region 20 (p12-pter), and rehybridized with the F IX probe for calibration. Comparing the hybridization signals of the normally sized DNA fragments of the familiy, we found no evidence for loss of any of the three tested distal chromosome 20p loci in our proband. Furthermore, in situ hybridization with HuPrPcDNA2 revealed a specific accumulation of grains at or around the faint distal G band suspected to represent all or most of band p12.3 of the proband's deleted 20p and at p12 of the normal chromosome 20. Thus the AWS of our proband is associated with an interstitial deletion that preserved the three tested distal loci on 20p. Since nine further reported cases of 20p deletion are clinically similar, we propose AWS as a further contiguous gene syndrome and assign it to an approximately 8-Mb-large chromosome 20p segment (provisionally, p11.23–p12.1).     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Fragile site long arm chromosome 16

Summary A fragile site at the long arms (q21) of chromosome 16 was found in two persons, each of whom became the parent of a child with a de novo structural chromosome abnormality—a balanced autosomal translocation and an autosomal deletion. The question of an increased risk of structural chromosome abnormalities in the offspring of persons with fragile site long arm 16 is discussed.