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Biomedical subjects

K Sperling

Publications and source records attributed to K Sperling.

At least 37 records · Page 2Linked to original sources
PubMed1999

Nijmegen breakage syndrome: consequences of defective DNA double strand break repair.

The autosomal recessive genetic disorder, Nijmegen Breakage Syndrome, is characterised by an excessively high risk for the development of lymphatic tumours and an extreme sensitivity towards ionising radiation. The most likely explanation for these characteristics, a deficiency in the repair of DNA lesions, has been greatly substantiated by the recent cloning of the gene mutated in Nijmegen Breakage Syndrome patients and the analysis of its protein product, nibrin. The direct involvement of this protein in the processing of DNA double strand breaks caused by ionising radiation and those also necessary for normal DNA metabolism can be correlated with many of the cellular and clinical aspects of the disease, including the cancer predisposition of patients and their heterozygous relatives.

Amino Acid Sequence↗
PubMed1999

Proving the authenticity of ancient DNA by comparative genomic hybridization.

In PCR-supported amplification of ancient, degraded DNA, contamination with contemporary DNA can lead to false-positive results, which frequently give rise to discussions in which the mere existence of ancient DNA is doubted. Our confirmation of ancient DNA using comparative genome hybridization (CGH) eliminates these doubts. Unlike PCR methods, CGH requires no amplification of the DNA to be analyzed if adequate amounts of specimen DNA is used. Thus, false results traceable to contaminations are practically ruled out. The examples provided here prove the authenticity of ancient DNA for a 250-year-old and a 3,000-year-old sample. At the same time, the CGH of ancient DNA offers the chance to gain insight into the pattern of DNA degradation and to monitor the preservation of certain chromosomal segments.

Chromosomes, Human↗
PubMed1999

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

Ataxia Telangiectasia↗
PubMed1998

Identification of mouse crystallins in 2D protein patterns by sequencing and mass spectrometry. Application to cataract mutants.

The eye lens proteins of the mouse were separated into 1940 polypeptide spots by two-dimensional electrophoresis in large gels. All 16 crystallins ubiquitous in mammals were identified by protein sequencing and mass spectrometry except for (gamma)-F, which shows an almost identical sequence with (gamma)-E. Two crystallins, (beta)-A2 and (gamma)-S, were shown for the first time to occur in the mouse lens. An investigation of the murine cataract mutant Cat2(nop)((gamma)-B gene) demonstrated that a monogenic mutation might affect a broad spectrum of proteins.

Amino Acid Sequence↗
PubMed1998

Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. We describe the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks.

Amino Acid Sequence↗
PubMed1998

Creation of monosomic derivatives of human cultured cell lines.

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

Anaphase↗
PubMed1998

[Small abnormality of the middle ear--a genetically-induced defect?].

Conductive and sensorineural hearing losses are of genetic origin in 20% to 60% of cases. In general, genetic abnormalities are more often expressed as a sensorineural hearing loss than as a conductive hearing loss. At present several genes for sensorineural hearing loss have been isolated. The most common genetically transmitted forms for isolated conductive hearing losses are otosclerosis and small malformations of the ossicles. To date no genes responsible for these deformations have been isolated. We present a family with four siblings having conductive hearing losses caused by ossification of the stapedial tendon. This finding is suggestive of an autosomal recessive inheritance. The early diagnosis of an hereditary conductive hearing loss contains the possibility for permitting normal development of speech.

Adult↗
PubMed1998

Beta-heterochromatin in mammals: evidence from studies in Microtus agrestis based on the extensive accumulation of L1 and non-L1 retroposons in the heterochromatin.

The heterochromatin of Microtus agrestis contains two retroposons, one L1 and one non-L1, which were cloned and analyzed with respect to their structure and genomic organization. These sequences have accumulated in the heterochromatin and exhibit a complex interspersed organization of relatively recent origin. In contrast, the pericentromeric heterochromatin is composed of simple, repetitive, tandemly organized elements. From the underlying sequences, the dinucleotide frequencies, the sequence organization, and its transcriptional activity, the heterochromatin of M. agrestis strongly resembles the beta-heterochromatin first described by Heitz and well characterized in Drosophila. This is evidence that this class of heterochromatin, presumably with its own distinct physiological and functional role, is indeed also present in the mammalian genome.

Animals↗
PubMed1997

Possible association of the allele status of the CS.7/HhaI polymorphism 5' of the CFTR gene with postnatal female survival.

Cystic fibrosis (CF) patients show a high degree of linkage disequilibrium between the CF transmembrane conductance regulator (CFTR) gene and polymorphisms 5' of that gene. To determine whether the region 5' of CFTR contains biologically important sequences, the allele frequencies of six CFTR-linked polymorphisms (metH/MspI, XV-2c/TaqI, CS.7/HhaI, KM19/PstI, MP6d9/MspI, J44/XbaI) were determined in 417 randomly selected elderly individuals (over 75 years of age) from the Czech population. The elderly individuals were considered "escapees" of strong selective pressures that had operated during their lifetime, prior to the introduction of modern health care since 1950. The pooled allele frequencies of the analyzed marker polymorphisms in the elderly did not significantly differ from published data. However, when analyzed by ex, the allele frequencies of markers CS.7/HhaI and KM19/PstI differed significantly (P < 0.05) between elderly females and males. The allele frequencies of the six polymorphisms were then determined in 646 newborns and 345 young adults of reproductive age; these individuals were selected in a similar manner and drawn from the same population. In these control groups, the studied marker polymorphisms exhibited no statistically significant differences between sexes and/or between individuals of the same sex, only between different age groups. A gradual relative increase in the frequency of allele "2" of marker CS.7/HhaI was observed from newborn females to elderly women, the overall difference in allele frequencies of this marker polymorphism between newborn females and elderly women reaching statistical significance (P < 0.05). Interestingly, allele "2" is the major constituent of the extended "B-haplotype", which is in strong linkage disequilibrium with common CF alleles. Taken together, our data suggest that the region spanning markers CS.7 and KM19 is associated with a genetic factor that influences postnatal female survival, providing a possible mechanism for increasing the frequency of particular mutations in the adjacent CFTR gene.

Adult↗
PubMed1997

Different mechanisms and recurrence risks of imprinting defects in Angelman syndrome.

Angelman syndrome (AS) is a neurogenetic disorder that appears to be caused by the loss of function of an imprinted gene expressed from maternal chromosome 15 only. Approximately 6% of patients have a paternal imprint on the maternal chromosome. In the few cases, this is due to an inherited microdeletion, in the 15q11-q13 imprinting center (IC), that blocks the paternal-->maternal imprint switch in the maternal germ line. We have determined the segregation of 15q11-q13 haplotypes in nine families with AS and with an imprinting defect. One family, with two affected siblings, has a microdeletion affecting the IC transcript. In the other eight patients, no mutation was found at this locus. In two families, the patient and a healthy sibling share the same maternal alleles. In one of these families and in two others, grandparental DNA samples were available, and the chromosomes with the imprinting defect were found to be of grandmaternal origin. These findings suggest that germ-line mosaicism or de novo mutations account for a significant fraction of imprinting defects, among patients who have an as-yet-undetected mutation in a cis-acting element. Alternatively, these data may indicate that some imprinting defects are caused by a failure to maintain or to reestablish the maternal imprint in the maternal germ line or by a failure to replicate the imprint postzygotically. Depending on the underlying cause of the imprinting defect, different recurrence risks need to be considered.

Angelman Syndrome↗
PubMed1997

Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation.

The functionality of the p53-mediated pathway, activated in response to DNA damage, has been assessed in primary fibroblast cell cultures and Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Nijmegen breakage syndrome (NBS) patients. This autosomal recessive disease is characterized by microcephaly, growth and mental retardation, chromosomal instability, radiosensitivity, and high cancer incidence. The recent mapping of the NBS gene to chromosome 8q21 demonstrates that NBS is genetically distinct from ataxia telangiectasia (AT). Changes in p53 protein levels were significantly reduced and delayed in all the NBS fibroblast cell cultures and lymphoblastoid cell lines examined compared to normal cultures over a 4-h period postirradiation (5 Gy). The transcriptional activation of p21(WAF1/CIP1) mRNA was also lower in 12 NBS fibroblast cultures examined. In agreement with an abrogated p53 function, NBS cells exposed to ionizing radiation show an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase. In contrast, exposure to the alkylating agent methyl methanesulfonate results in similar increases of p53 and p21(WAF1/CIP1) mRNA in both cell types. The ATM gene transcript was found to be expressed at similar levels in NBS and normal cells, whereas it was strongly reduced in the AT homozygote cells examined. These results suggest that the ATM gene product cannot substitute for that of the NBS gene in the signaling of cellular damage produced by ionizing radiation and that both are involved in the activation of p53. The suboptimal p53-mediated response could contribute to the high cancer risk and radiosensitivity seen in NBS patients.

Ataxia Telangiectasia↗
PubMed1997

Assignment of the chicken MAX gene to chromosome 5p by fluorescence in situ hybridization.

It has been shown that the protein encoded by the MAX gene plays an important role in the physiological activity of Myc oncoproteins. In this study, we determined the chromosome location of the chicken MAX gene via fluorescence in situ hybridization. Hybridization of two biotinylated cloned fragments of 5.7 kb and 12.0 kb derived from the chicken MAX locus localized the gene to chromosome 5p. It is the third gene marker to be assigned to this telocentric macrochromosome. Since the MAX sequence is highly conserved both at the nucleotide and at the amino acid level in a wide range of vertebrate species, our data may provide evidence for the existence of a segmental homology between human and chicken chromosomes.

Animals↗
PubMed1997

The gene for the ataxia-telangiectasia variant, Nijmegen breakage syndrome, maps to a 1-cM interval on chromosome 8q21.

Nijmegen breakage syndrome (NBS; Seemanová II syndrome) and Berlin breakage syndrome (BBS), also known as ataxia-telangiectasia variants, are two clinically indistinguishable autosomal recessive familial cancer syndromes that share with ataxia-telangiectasia similar cellular, immunological, and chromosomal but not clinical findings. Classification in NBS and BBS was based on complementation of their hypersensitivity to ionizing radiation in cell-fusion experiments. Recent investigations have questioned the former classification into two different disease entities, suggesting that NBS/BBS is caused by mutations in a single radiosensitivity gene. We now have performed a whole-genome screen in 14 NBS/BBS families and have localized the gene for NBS/BBS to a 1-cM interval on chromosome 8q21, between markers D8S271 and D8S270, with a peak LOD score of 6.86 at D8S1811. This marker also shows strong allelic association to both Slavic NBS and German BBS patients, suggesting the existence of one major mutation of Slavic origin. Since the same allele is seen in both former complementation groups, genetic homogeneity of NBS/BBS can be considered as proved.

Alleles↗
PubMed1996

Phenotypic differences in Angelman syndrome patients: imprinting mutations show less frequently microcephaly and hypopigmentation than deletions.

Angelman syndrome (AS) is a relatively frequent disorder of psychomotor development caused by loss of function of a gene from chromosome 15q11-q13, a region subject to genomic imprinting. The AS gene(s) is exclusively expressed from the maternal chromosome. Several kinds of mutations have been found to cause AS. More than half of the cases exhibit a deletion of the maternal 15q11-q13 region. Recently, we and others described a new mutation type, the imprinting mutation, characterised by normal, biparental inheritance but aberrant methylation patterns of the entire chromosomal region. In AS, a paternal imprint is found on the maternal chromosome probably leading to functional inactivation of the AS gene(s). We have now compared the phenotype of 9 AS patients with imprinting mutation to that of nine age-matched ones with a maternally derived deletion. Both groups were evaluated for 19 common AS symptoms. All patients, independently of their molecular findings, showed classical AS symptoms such s mental retardation, delayed motor development, and absent speech. In contrast, for two signs, hypopigmentation and microcephaly, a different distribution among both groups was observed. Only one of nine AS patients with an imprinting mutation, but seven of nine in the deletion control group showed either symptom. Our results suggest that imprinting mutations, in contrast to deletions, cause only incomplete loss of gene function or that maternally derived deletions affect also genes not subject to genomic imprinting. We conclude that AS is caused by loss of function of a major gene that is imprinted but that there are also other genes that contribute to the phenotype when in hemizygous condition.

Albinism, Oculocutaneous↗
PubMed1996

Identification of a HeLa mRNA fraction which corrects the mitomycin C sensitivity of irs1 cells.

The hamster cell mutant irs1 is defective in its response to DNA lesions caused by a variety of mutagens, particularly cross-linking agents. These cells have been assigned to complementation group 2 of X-ray-sensitive mutants and the mutated gene is called XRCC2(X-ray repair cross complementing). We have identified, by microinjection, a human mRNA fraction which can transiently correct the sensitivity of these cells to cross-linking agents. This fraction contains mRNAs of 3.5 kb (+/- 0.25) including, therefore, the transcript of the XRCC2 gene.

Animals↗
PubMed1996

Partial trisomy/monosomy 6q in fetal cells and CVS long-term culture not present in CVS short-term culture.

A cytogenetic discrepancy in chorionic villi with implications for prenatal diagnosis is described. Chromosome analysis revealed a normal karyotype in banded metaphases from short-term culture and a chromosome count of 46 in cells of the long-term culture. After the birth of a malformed infant, a structurally aberrant chromosome 6 was found in lymphocytes and skin fibroblasts. Re-analysis of chorionic villi confirmed the result from short-term culture but disclosed the presence of the structural aberration in cells of the long-term culture. This type of inconsistency is reported for the first time and stresses the importance of a numerical and structural analysis of both short-term and long-term culture. The application of three techniques, chromosome banding, comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) analysis, was essential to prove that the derivative chromosome carried a combined partial trisomy/monosomy for 6q. The findings are discussed with respect to the origin of the structural aberration and to the consequences for prenatal diagnosis on chorionic villi and genetic counselling.

Adult↗
PubMed1996

Molecular analysis of Fanconi anaemia.

The autosomal recessive genetic disease, Fanconi anaemia, is perceived as another manifestation of defective cellular DNA repair, just as in the autosomal recessive disease Xeroderma pigmentosum. The biochemistry and cellular biology of Xeroderma pigmentosum have been convincingly elucidated, but the same has not been true for Fanconi anaemia. In this review we consider the pleiotropic nature of Fanconi anaemia, its clinical and cellular variability and its genetic heterogeneity. We take into account the wealth of experimental findings available and offer a novel hypothesis involving feedback control of DNA replication during S phase of the cell cycle to explain the basic defect in the disease.

Cell Cycle↗