Chromosome polymorphism involving heterochromatic blocks in Chinese hamster chromosome 9

A chromosome polymorphism was detected between two early passage euploid Chinese hamster cell strains when a fluorescence shift of the small metacentric No. 9 chromosome was resolved by flow cytometry. The characteristics of the polymorphism were studied using cultures established from ear clippings taken from 16 additional hamsters from our breeding colony. Additional variants of chromosome 9 were detected using flow cytometry, and a subset of these variants were analyzed by G- and C-banding. An increase of fluorescence recorded by flow cytometry correlated with an increase of centromeric heterochromatin. Autosomal normalization of the flow karyotype from 18 different animals indicated three distinct peak positions for chromosome 9. The results indicate that a discrete block of constitutive heterochromatin may be present in one or two extra copies within the small inbred colony of hamsters studied. To determine the inheritance patterns, hamsters with known polymorphic No. 9 chromosomes were bred. The flow karyotypes derived from the offspring of these matings provide strong evidence that chromosomal polymorphisms are inherited in Mendelian fashion.     

Human chromosomal polymorphism. V. Chromosomal Q polymorphism in African populations

Summary The distribution pattern of Q-heterochromatin variants in seven autosomes (3, 4, 13–15, 21, and 22) was studied in three aboriginal Negroid populations of Africa (Mozambique, Angola, and Ethiopia). It was shown that among African Negroids there are no individuals completely lacking Q-heterochromatin bands with fluorescence levels 4 and 5. The mean number of Q variants per individual was 3.47, 4.80, and 4.85 in the Ethiopian, Mozambique, and Angola populations, respectively. The observed homo- and heteromorphic frequencies always agreed with those predicted by the law of Hardy-Weinberg. The populations of tropical lowland Negroids (Mozambique and Angola) proved to be significantly homogeneous both in the frequency of Q variants and the mean number of these variants per individual, so they were examined as a single group. However, comparative analysis of highland (Ethiopians) and lowland Negroids revealed statistically significant differences. The following questions are discussed: (1) the possible selective value of chromosomal Q heterochromatin material in the adaptation of human populations to high-altitude climate; (2) the possible existence of intraracial heterogeneity in Negroids living in different ecological zones of Africa; (3) the possible taxonomic value of an inverted Q-heterochromatin band in chromosome 3 in ethnic anthropology.     

The cytogenetics of 90 patients with idiopathic mental retardation/malformation syndromes and of 90 normal subjects

Summary A cytogenetic study, done on randomized coded slides, of 90 patients with idiopathic mental retardation and at least 3 other developmentally independent congenital anomalies and of 90 normal subjects is reported. Audiatorography, Q-banding and C-staining were used in the analysis of chromosomally abnormal cases. Eight patients were found to have chromosome abnormalities. Four had substantial chromosome aberrations that would be expected to cause abnormal phenotype. These were CD165 (46,18q-); CD25 (46,18q+) (partial trisomy of 10q); CD175 (46,4q+) and CD95 (46,mar22). In addition, 4 patients were found to have chromosomal anomalies that could not account for their conditions. Three of these were considered to have heterochromatic variants. Patient CD167 had an 9qh+ chromosome which had been inherited from her mother. Case CD137 had a No. 19 chromosome with additional centric heterochromatin. A similar chromosome was found in her mother, maternal grandmother and 2 of 3 half sibs. In patient CD125 a telocentric No. 13 was found. In addition, CD80 was shown to have an XYY constitution.In the normal subjects, no unbalanced chromosome rearrangements were found. Four persons, however, had minor chromosome anomalies. Three were considered to have heterochromatic variants. These were CD54 (46,22p+); CD149 (46,21p+) and CD19 (46,tel22). One normal subject (CD51) was found to be a balanced t(13q14q) carrier. The translocation chromosome had been inherited from his father.     

The segregation of C-band polymorphisms on chromosomes 1, 9, and 16.

Eleven normal families with at least four children were studied cytogenetically using the C-band technique to identify polymorphisms in the constitutive heterochromatin of chromosomes 1, 9 and 16. Thirteen individuals showed one or more variants in such chromosomes. The analysis of the segregation ratios in the 35 offspring of these 13 individuals showed that these marker chromosomes generally segregated according to the expected 50:50. However, one of these variants, chromosome no. 9 with an increased heterochromatin block in the secondary constriction, has an apparently preferential segregation, when the findings from this study are combined with those of other authors.     

Whole-genome sequencing of monozygotic twins discordant for schizophrenia indicates multiple genetic risk factors for schizophrenia

Schizophrenia is a common disorder with a high heritability, but its genetic architecture is still elusive.We implemented whole-genome sequencing(WGS) analysis of 8 families with monozygotic(MZ) twin pairs discordant for schizophrenia to assess potential association of de novo mutations(DNMs) or inherited variants with susceptibility to schizophrenia. Eight non-synonymous DNMs(including one splicing site) were identified and shared by twins, which were either located in previously reported schizophrenia risk genes(p.V24689 I mutation in TTN, p.S2506 T mutation in GCN1L1, IVS3+1G T in DOCK1) or had a benign to damaging effect according to in silico prediction analysis. By searching the inherited rare damaging or loss-of-function(LOF) variants and common susceptible alleles from three classes of schizophrenia candidate genes, we were able to distill genetic alterations in several schizophrenia risk genes, including GAD1, PLXNA2, RELN and FEZ1. Four inherited copy number variations(CNVs; including a large deletion at 16p13.11) implicated for schizophrenia were identified in four families, respectively. Most of families carried both missense DNMs and inherited risk variants, which might suggest that DNMs, inherited rare damaging variants and common risk alleles together conferred to schizophrenia susceptibility. Our results support that schizophrenia is caused by a combination of multiple genetic factors, with each DNM/variant showing a relatively small effect size.     

Population cytogenetics of Atractomorpha similis

Atractomorpha similis (2 n=19 ♂, 20 ♀) is a hygrophilous, tropical to temperate, species of pyrgomorphine grasshopper. We have sampled 70 populations covering the known distributional range of this species within Australia. All of them proved to be polymorphic for heterochromatin content as revealed by C-band analysis of embryonic neuroblasts. This polymorphism affects all ten members of the basic haploid set and includes variants involving differences in either the presence or the amount of procentric, interstitial and terminal C-blocks, as well as variation in the occurrence and nature of short arms on otherwise telocentric chromosomes. A majority of these variants appear to result from heterochromatin addition since the presumptive sibling, Atractomorpha australis, like other species of the genus that have been C-banded, is generally depauperate in heterochromatin. The net result of this extraordinary polymorphism is that each chromosome of A. similis exists in 10–50 distinct morphs. Consequently, there is a high level of chromosomal heterozygosity in all populations in terms of the number of heterozygous pairs present within a complement and an even higher level in terms of the total range of karyomorph patterns. There is also a wide range of total heterochromatin content, as measured by the percent of the total chromosome area occupied by C-band material, with values ranging from 13% to 44%. Specific marker chromosomes which predominate in particular geographical areas serve to distinguish six major cytotypes within A. similis. The two most southerly of these cytotypes show a narrower range of heterochromatin content but with higher values which reflect the more general occurrence of substantial terminal C-blocks within them. Finally, the populations from Fraser Island constitute a particularly distinctive cytotype characterised by the least number of morphs, the lowest level of chromosomal heterozygosity and a restricted range of heterochromatin content confined to the lower end of the known distributional spectrum.     

Cholinesterase unit establishment and issuing of >Warning Cards< for carriers of suxamethonium sensitive serum butyrylcholinesterase variants

Recognition of butyrylcholinesterase (EC 3.1.1.8) variants in human serum is essential to identify patients who may be susceptible to a prolonged reaction of suxamethonium and mivacurium, short-acting muscle relaxants. Thus they can be given appropriate advice along with their relatives who may be similarly affected. Therefore, Cholinesterase Unit for detection of individuals, carriers of inherited suxamethonium sensitive butyrylcholinesterase variants was established at the Institute for Clinical Chemistry of the Clinical Hospital >Merkur<, Zagreb, Croatia. A study was conducted on sera from patients referred to the Unit. Butyrylcholinesterase variants were determined by measuring the enzyme activity and inhibition by specific inhibitors in the sera of 384 patients and of the members of seven families. Cholinesterase Unit issued >Warning Cards< to the carriers of inherited serum butyrylcholinesterase variants in order to avoid prolonged apnea that suxamethonium might cause.     

Human chromosomal polymorphism. IV. Chromosomal Q polymorphism in Russians living in Kirghizia

Summary Chromosomal Q polymorphism was studied in 200 Russian individuals (94 females and 106 males) living in Kirghizia. Of the 200 individuals, 191 had chromosomal Q polymorphic variants, while nine (4.5%) had no Q bands with fluorescence levels 4 and 5. The mean number of Q variants per individual ranged from 0 to 7, with a mean of 2.9. There were no differences in the frequency of Q variants between sexes. The observed homo- and heteromorphic frequencies completely agreed with those predicted by the law of Hardy-Weinberg. Of the 200 individuals, 12 (6.0%) had pericentric inversion of the Q band in chromosome 3, one individual (0.5%) having a homomorphic form of this inversion. The possible selective value of chromosomal Q heterochromatin material in the adaptation of human populations to extreme environmental factors, in particular to cold, and the possible taxonomic value of inverted Q heterochromatin bands in chromosome 3 in ethnic anthropology, are discussed.     

Human chromosomal polymorphism

Chromosomal Q-polymorphism was studied in 198 Kirghiz subjects (98 males and 100 females) from one high-altitude isolate located in the south-eastern part of Kirghizia. Small samples of mountaineers (N = 37) and volunteer subjects (N = 34) were also studied. The samples studied did not differ significantly from each other in the relative frequencies of chromosomal variants in 12 loci of seven Q-polymorphic autosomes. The mean number of Q variants per individual in the populations ranged from 1.3 to 2.0. No sex differences were found in the frequencies of Q variants. The observed homo- and heteromorphic frequencies agreed with those predicted by the law of Hardy-Weinberg. The possible selective value of chromosomal Q heterochromatin material in the adaptation of human populations to high-altitude climate is discussed.     

Interindividual hyperpolymorphism of autosomal satellites III of human DNA

Hypervariability of DNA restriction fragments from pericentromeric heterochromatin detected by autosomal "classic" DNA of satellite III has been demonstrated in this work. Using hybridization probe of satellite III DNA localized predominantly on the chromosome 9 strong interindividual differences in the sets of polymorphic DNA restriction fragments inherited from both paternal and maternal sides as well as intraspecies amplifications of some variants of the satellite III were observed. The number and intensity of restriction bands are identical in both sexes. It is suggested that strong interindividual DNA variability may be largely specified by high level of DNA spot mutability in satellites III. Pericentromeric "classic" satellites III may serve as efficient markers for identification of individuals and for molecular-genetic mapping of human genome pericentromeric regions.     

Chromosome patterns of nontransformed variants from chemically transformed Balb-3T3 cells

Nine variant cell lines isolated from cloned 7,12-dimethylbenz(a) -ahthracene transformed Balb/3T3 mouse cells by treatment with FUdR had growth parameters closely resembling nontransformed cells. Chromosome analysis of the variant lines demonstrated that six variants had a diminished number and three variants had an increased number of chromosomes compared to the parental transformed cell line. All variants had unique marker chromosomes not present in the parental transformed Balb/3T3 cells. The distribution of marker chromosomes and heterochromatin suggested that the initial event in variant formation was a reduction in chromosome number with a subsequent polyploidization of the reduced chromosome complement.     

Genetic analysis of the heterochromatin of chromosome 3 in Drosophila melanogaster. I. Products of compound-autosome detachment

The heterochromatin of the third chromosome is the largest uncharacterized region of the Drosophila melanogaster genome, and the last major block of D. melanogaster heterochromatin to be thoroughly analyzed. In the present study, this region was genetically dissected by generating and analyzing a series of attached, detached and reattached third chromosomes. Separate detachment experiments were conducted for all 12 possible combinations of four newly synthesized sister-strand compound-3L and three newly synthesized sister-strand compound-3R chromosomes. A total of 443 recessive lethal detachment products carrying putative heterochromatic deficiencies were tested for complementation in a several-stage complementation analysis. The results revealed the presence of seven separable vital regions in the heterochromatin of chromosome 3. Attempts to reattach deficiency-carrying detachment products established that six of these vital regions are on the left arm, but only one is on the right arm. An analysis of the types and frequencies of detachment-product deficiencies generated in each detachment experiment permitted the genetic characterization of the progenitor compounds. It was also possible to determine the proximal-distal orientation of the genes on each arm, and to identify possible breakpoints for each lethal detachment product produced. The results of this study suggest that vital genes in the heterochromatin of the third chromosome are not randomly distributed between, nor within, the heterochromatic blocks of the left and right arms.     

Berenil-induced undercondensation in human heterochromatin

The aromatic diamidine berenil specifically inhibits the condensation of a subset of constitutive heterochromatin in human lymphocyte cultures. In the normal male chromosome complement, only the quinacrine-brilliant Y heterochromatin exhibits distinct undercondensation. The optimal culture conditions for inhibiting heterochromatin condensation are achieved when berenil is added at a final concentration of 150 micrograms/ml 24 h before cell harvest. Various examples of the use of berenil in the analysis of chromosome rearrangements involving quinacrine-brilliant heterochromatin are presented. A variant, giant-satellited chromosome 22 was found to respond to berenil treatment, although its enlarged and quinacrine-bright short-arm region did not contain Y heterochromatin. Southern blot analysis and chromosome in situ hybridization suggested that most chromosome 22 variants do not stem from Y; acrocentric translocations. The experimentally undercondensed Y heterochromatin is characterized by moderate C-band labeling, bright quinacrine fluorescence, and specific silver staining. At the ultrastructural level, undercondensation is associated with loosely packed, mutliply folded chromatin fibers with a diameter of approximately 250 A and organized probably as loops.     

Clonal variants of constitutive heterochromatin of human fibroblasts after recovery from mitomycin treatment

Presumptive clones of human skin fibroblast-like cells surviving mitomycin C treatments show a variety of intra- and interchromosomal rearrangements limited to the constitutive heterochromatic regions. Starting with a wild-type line polymorphic for chromosome no. 1 heterochromatin, we have observed clones with complete symmetry and varying degrees of asymmetry of the no. 1 heterochromatin, translocations of excess chromosome 1 heterochromatin to one no. 9 member, interstitial translocations to sites normally devoid of heterochromatin, and duplication of the Y chromosome long arm heterochromatin. In the case of the chromosome no. 1 pair, the extent of heterochromatin variation was quantitated to test the hypothesis that discrete classes of variants occur. There appear to be two types of variants: Those showing reciprocal changes between homologues (2 examples) and those showing a change in the amount of heterochromatin of a single homologue (5 examples). The latter group showed an approximately linear series of variants. Unequal cross-over following repair after damage from the alkylating agent is considered the most likely explanation for the observed changes, given the repetitive nature of DNA at these heterochromatic sites.     

Co-occurrences of polymorphic heterochromatin regions of chromosomes and effect on reproductive failure

Although the polymorphic heterochromatin regions of chromosomes (heteromorphisms) have been extensively studied for their phenotypic effects on humans, co-occurrences of chromosome 1, 9, 16 and Y heteromorphisms and of acrocentric variants have never been studied on humans with an objective scoring system. Here we compared the frequencies of individual heteromorphisms on a total of 602, 768 and 224 patients with the indications of infertility, recurrent miscarriage and in vitro fertilization (IVF) failure, respectively and on 272 controls. Then we examined whether there were significant co-occurrences between heteromorphisms within and between the groups. There were no statistically significant differences in the frequencies of heteromorphisms between the groups. Both statistically significant and non-significant correlations were observed within the non-acrocentric and certain acrocentric heteromorphisms in each group. When these co-occurrences were examined between the groups, a 2.2 fold increased risk of IVF failure in males in the presence of either chromosome 13 or chromosome 21 variants was observed (95 %CI:1.1–4.2). We conclude that the simultaneous manifestations of heteromorphisms have no effect on reproductive failure. There seems to be a correlation between the non-acrocentric heteromorphisms (1qh+, 9qh+, 16qh + and Yqh+/-), which might be the result of complex interactions of formation of these heterochromatin regions. The correlations observed between certain acrocentric chromosomes might be related to satellite association and nucleolus formation. The increased risk observed in males with IVF failure in the presence of either chromosome 13 or 21 variants should be interpreted cautiously due to the heterogeneity of the group.     

An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants

Somatic cell variants expressing an altered antigenic form of the H-2Kb molecule were isolated for the purpose of performing structure-function analysis of a class I MHC molecule. Over 25 independently isolated variants were derived from an Abelson virus transformed pre- B cell line (R8) by mutagenesis with ethyl methane sulfonate or ethyl nitrosourea. Negative selection was performed by complement-dependent cytotoxicity with anti-H-2Kb monoclonal antibodies subsequently followed by positive selection to separate the H-2Kb surface negative variants from structural variants. Biochemical characterization of a random selection of three independent variants indicated that the variant H-2Kb molecule was present in normal amounts in lysates, and was unchanged in size. Cytofluorometric analysis with the use of a panel of seven monoclonal antibodies against H-2Kb indicated that all of the variants had lost one or more alloantigenic determinants (monoclonal antibody binding sites). For these variants, the pattern of monoclonal antibody loss of recognition suggested that antibody defined alloantigenic determinants appear to be discretely localized to a single domain, either the alpha 1 or the alpha 2 domain, of the H-2Kb molecule. In contrast, CTL recognition of the Kb molecule of these variants depends on involvement of both alpha 1 and alpha 2 domains as shown in the companion paper.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.     

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.     

The role of cytologic NOR variants in the etiology of trisomy 21.

Silver-stained chromosomes from 29 couples with a trisomy 21 offspring and from 25 control couples were studied to determine whether there was an association of nucleolar-organizing-region variants in parents of children with trisomy 21. A reproducible scoring system for the analysis of silver-stained chromosomes was developed, and this was applied to the analysis of study participants in a blinded fashion. Seven of the 58 parents of children with trisomy 21 and seven of the 50 control parents were found to have variant NORs on silver staining. Therefore, we do not find a demonstrable risk for nondisjunction of chromosome 21 in individuals with silver-staining variants.