Preferential retention of paternal alleles in human retinoblastoma: evidence for genomic imprinting

The origins of the initial mutations in sporadic retinoblastoma were explored using polymorphic markers from chromosome 13q. The paternal chromosome was maintained in 3 of 3 informative bilateral tumors which had undergone reduction to homozygosity for regions of this chromosome. The paternal chromosome was maintained in 7 of 8 informative unilateral tumors which likewise demonstrated a reduction of homozygosity. These data are in contrast to previously published studies of chromosome retention in unilateral retinoblastoma [Dryja, T. P., Mukai, S., Petersen, R., Rapaport, J. M., Walton, D., and Yandel, D. W. Nature (Lond.), 339: 556-558, 1989; Zhu, Z., Dunn, J. M., Phillips, R. A., Goddard, A. D., Paton, K. E., Becker, A., and Gallie, B. L. Nature (Lond.), 340: 312-313, 1989] and provide the first evidence that genomic imprinting may play a role in this disease.     

Maternal uniparental disomy 22 has no impact on the phenotype.

A 25-year-old normal healthy male was karyotyped because five of his wife's pregnancies terminated in spontaneous abortions at 6-14 wk of gestation. Cytogenetic investigation disclosed a de novo balanced Robertsonian t(22q;22q) translocation. Molecular studies revealed maternal only inheritance for chromosome 22 markers. Reduction to homozygosity for all informative markers indicates that the rearranged chromosome is an isochromosome derived from one of the maternal chromosomes 22. Except for the possibility of homozygosity for recessive mutations, maternal uniparental disomy 22 does not seem to have an adverse impact on the phenotype, apart from causing reproductive failure. It can be concluded that no maternally imprinted genes with major effect map to chromosome 22.     

Chromosome 15 uniparental disomy is not frequent in Angelman syndrome.

Genetic imprinting has been implicated in the etiology of two clinically distinct but cytogenetically indistinguishable disorders--Angelman syndrome (AS) and Prader-Willi syndrome (PWS). This hypothesis is derived from two lines of evidence. First, while the molecular extents of de novo cytogenetic deletions of chromosome 15q11q13 in AS and PWS patients are the same, the deletions originate from different parental chromosomes. In AS, the deletion occurs in the maternally inherited chromosome 15, while in PWS the deletion is found in the paternally inherited chromosome 15. The second line of evidence comes from the deletion of an abnormal parental contribution of 15q11q13 in PWS patients without a cytogenetic and molecular deletion. These patients have two maternal copies and no paternal copy of 15q11q13 (maternal uniparental disomy) instead of one copy from each parent. By qualitative hybridization with chromosome 15q11q13 specific DNA markers, we have now examined DNA samples from 10 AS patients (at least seven of which are familial cases) with no cytogenetic or molecular deletion of chromosome 15q11q13. Inheritance of one maternal copy and one paternal copy of 15q11q13 was observed in each family, suggesting that paternal uniparental disomy of 15q11q13 is not responsible for expression of the AS phenotype in these patients.     

Homozygosity mapping of the gene for Chediak-Higashi syndrome to chromosome 1q42-q44 in a segment of conserved synteny that includes the mouse beige locus (bg).

Chediak-Higashi syndrome (CHS) is an autosomal recessive disorder characterized by hypopigmentation or oculocutaneous albinism and severe immunologic deficiency with neutropenia and lack of natural killer (NK) cell function. Most patients die in childhood from pyogenic infections or an unusual lymphoma-like condition. A hallmark of the disorder is giant inclusion bodies seen in all granule-containing cells, including granulocytes, lymphocytes, melanocytes, mast cells, and neurons. Similar ultrastructural abnormalities occur in the beige mouse, which thus has been suggested to be homologous to human CHS. High-resolution genetic mapping has indicated that the bg gene region of mouse chromosome 13 is likely homologous to the distal portion of human chromosome 1q. Accordingly, we carried out homozygosity mapping using markers derived from distal human chromosome 1q in four inbred families or probands with CHS. Our results indicate that the human CHS gene maps to an 18.8-cM interval in chromosome segment 1q42-q44 and that human CHS therefore is very likely homologous to mouse bg.     

Genome-wide homozygosity mapping localizes a gene for autosomal recessive non-progressive infantile ataxia to 20q11-q13

Autosomal recessive ataxias represent genetic and clinical heterogeneity. Unsteady gait is often accompanied by poor coordination of limbs, speech, and eye movements. To date, seven genes have been identified. In addition, five chromosomal loci have been localized in non-related families. Here, we report homozygosity mapping of a novel locus to a 19.5-cM region on chromosome 20q11–q13 in a large inbred Norwegian family with infantile non-progressive ataxia.L. Tranebjaerg, T.M. Teslovich, and M. Jones contributed equally to this work     

A null allele of esterase D is a marker for genetic events in retinoblastoma formation

Summary The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14.11 is required for tumorigenesis. Observations reported by Benedict and colleagues in one case of bilateral retinoblastoma, LA-RB 69, provided early evidence in favor of this hypothesis. By examining levels of esterase D, an enzyme also mapping to 13q14.11, it was previously inferred that one chromosome 13 in this patient's somatic cells contained a submicroscopic deletion of the Rb and esterase D loci and that this chromosome was retained in her tumor while the normal chromosome 13 was lost. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, we have found that (1) low but detectable quantities of esterase D protein and enzymatic activity are present in tumor cells from LA-RB 69; (2) fibroblast from this patient contain two copies of the esterase D gene, indicated by heterozygosity at an ApaI polymorphic site within this gene; and (3) tumor cells from the same patient are homozygous at this site, indicating loss and reduplication of the esterase D locus. These results demonstrate that one of the two esterase D alleles in this patient acted as a null or silent allele — that is, was present in the genome with markedly decreased protein expression. This mutant allele acted as a marker for tumor-associated loss of chromosome 13 heterozygosity, in concordance with previous proposals.     

Refined genetic and comparative physical mapping of the canine copper toxicosis locus

Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR₃₀₀₀ region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR₃₀₀₀ is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb. Received: 16 December 1999 / Accepted: 23 February 2000     

A genetic model for the Prader-Willi syndrome and its implication for angelman syndrome

Summary Sporadic cases of Prader-Willi syndrome (PWS) are associated with the physical absence of the paternal Prader-Willi chromosome region (PWCR) by deletion 15q11–13, by segmental maternal heterodisomy or by chromosome rearrangements resulting in homozygosity for maternal PWCR. In isolated/familial cases, it is proposed that the expression of PWS depends on the functional absence caused by mutated gene(s) within the paternal PWCR. The same mutation on a maternally derived chromosome 15 is not able to express PWS. An epigenetic mechanism associated with the paternal meiosis is essential. In the Angelman syndrome (AS), inverse mechanisms are postulated. There is convincing evidence for specific PWS and AS genes or alleles within PWCR. This is compatible with the observations of interstitial chromosome deletions of the critical region in normal individuals or in probands with phenotypes other than PWS or AS. The new ideas of the model stated here are: (1) the proposed epigenetic mechanism in PWCR is obviously common in humans, but is usually of no phenotypic relevance; (2) interactions with specific chromosomal or gene mutations are required for the clinical expression of PWS or AS; (3) each factor alone is not able to produce an abnormal phenotype.     

A locus for Fanconi anemia on 16q determined by homozygosity mapping.

We report the results of a genomewide scan using homozygosity mapping to identify genes causing Fanconi anemia, a genetically heterogeneous recessive disorder. By studying 23 inbred families, we detected linkage to a locus causing Fanconi anemia near marker D16S520 (16q24.3). Although -65% of our families displayed clear linkage to D16S520, we found strong evidence (P = .0013) of genetic heterogeneity. This result independently confirms the recent mapping of the FAA gene to chromosome 16 by Pronk et al. Family ascertainment was biased against a previously identified FAC gene on chromosome 9, and no linkage was observed to this locus. Simultaneous search analysis suggested several additional chromosomal regions that could account for a small fraction of Fanconi anemia in our families, but the sample size is insufficient to provide statistical significance. We also demonstrate the strong effect of marker allele frequencies on LOD scores obtained in homozygosity mapping and discuss ways to avoid false positives arising from this effect.     

Loss of heterozygosity at chromosomal regions 3p and 13q in non-small-cell carcinoma of the lung represents low-frequency events

It was recently reported that loss of heterozygosity occurred at the chromosomal region 3p in small-cell as well as in non-small-cell carcinoma of the lung. A recent report also indicated genetic changes involving sequences on chromosomes 13q and 17p in small-cell and in non-small-cell carcinomas. In the present study normal and tumor DNAs representing mostly adeno-and squamous cell carcinomas of the lung were examined for loss of heterozygosity on chromosomes 3p, 13q, 11p, and 1p. With the exception of two non-small-cell carcinomas which demonstrated loss of alleles on chromosome 3p and one small-cell carcinoma which demonstrated loss of heterozygosity at chromosome 3p as well as at 13q, evidence for loss of alleles on chromosomes 3p, 13q, 11p, and 1p could not be obtained in greater than 75% of the non-small-cell carcinoma DNAs tested. Given this result it appears unlikely that a recessive gene is located on either chromosome 3p or 13q in the majority of non-small-cell carcinomas of the lung.     

Exclusion of human chromosome 13q34 as the site of the cystic fibrosis mutation.

We have studied a family in which both cystic fibrosis (CF) and an unbalanced translocation between chromosomes 6 and 13 are found. As CF occurs in the child who is effectively monosomic for the translocated part of the long arm of chromosome 13, it was suggested that the locus of the gene mutation causing CF is on chromosome 13q34. The gene for human coagulation factor X is located at 13q34, and we have found a restriction fragment length polymorphism (RFLP) that is revealed by a cloned cDNA coding for this protein. Linkage analysis in eight CF families shows no evidence of cosegregation between CF and the gene for factor X, strongly suggesting that the locus for the defect causing cystic fibrosis is not at 13q34.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

A Somatic Origin of Homologous Robertsonian Translocations and Isochromosomes

One t(14q14q), three t(15q15q), two t(21q21q), and two t(22q22q) nonmosaic, apparently balanced, de novo Robertsonian translocation cases were investigated with polymorphic markers to establish the origin of the translocated chromosomes. Four cases had results indicative of an isochromosome: one t(14q14q) case with mild mental retardation and maternal uniparental disomy (UPD) for chromosome 14, one t(15q15q) case with the Prader-Willi syndrome and UPD(15), a phenotypically normal carrier of t(22q22q) with maternal UPD(22), and a phenotypically normal t(21q21q) case of paternal UPD(21). All UPD cases showed complete homozygosity throughout the involved chromosome, which is supportive of a postmeiotic origin. In the remaining four cases, maternal and paternal inheritance of the involved chromosome was found, which unambiguously implies a somatic origin. One t(15q15q) female had a child with a ring chromosome 15, which was also of probable postmeiotic origin as recombination between grandparental haplotypes had occurred prior to ring formation. UPD might be expected to result from de novo Robertsonian translocations of meiotic origin; however, all de novo homologous translocation cases, so far reported, with UPD of chromosomes 14, 15, 21, or 22 have been isochromosomes. These data provide the first direct evidence that nonmosaic Robertsonian translocations, as well as isochromosomes, are commonly the result of a mitotic exchange.     

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

Normal phenotype with maternal isodisomy in a female with two isochromosomes: i(2p) and i(2q)

A 36-year-old normal healthy female was karyotyped because all of her five pregnancies had terminated in spontaneous abortions during the first 3 mo. Cytogenetic investigation disclosed a female karyotype with isochromosomes of 2p and 2q replacing the two normal chromosomes 2. Her husband and both of her parents had normal karyotypes. Molecular studies revealed maternal only inheritance for chromosome 2 markers. Reduction to homozygosity of all informative markers indicated that the isochromosomes derived from a single maternal chromosome 2. Except for the possibility of homozygosity for recessive mutations, maternal uniparental disomy 2 appears to have no adverse impact on the phenotype. Our data indicate that no maternally imprinted genes with major effect map to chromosome 2.     

Homozygosity mapping to chromosome 5p15 of a gene responsible for Hartnup disorder

Hartnup disorder is an autosomal recessive phenotype involving a transporter for monoamino-monocarboxylic acids. Genetic analysis of the mouse model mapped its locus to human chromosome 11q13 (8). We report here the results of linkage analysis in two Japanese first cousin-marriage families. In the first family, the proband had Hartnup disorder and his deceased older brother was reported to have had typical Hartnup symptoms. The younger brother of the proband was shown to have decreased tryptophan absorption by oral loading test. In the second family, a 6-year-old girl, the proband, had specific hyperaminoaciduria. DNA was isolated from either blood samples or umbilical cord stumps. Genome-wide screening by homozygosity mapping was conducted. Taking into account that the older brother was affected and the younger brother was a carrier in the first family, homozygosity mapping (LOD score = 3.55) and GENEHUNTER (LOD score = 3.28) locates the locus of the Hartnup disorder on 5p15.     

Loss of heterozygosity in hypotriploid cell cultures from testicular tumours

Summary We have established cell lines with a hypotriploid chromosome number from four testicular tumours. Each line had at least one Y chromosome and most of the informative centromere and enzyme markers were heterozygous implying that the tumours originated from germ cells before the first meiotic division. The small metacentric marker chromosome (i12p), specific for testicular tumours, was present in all tumour cell lines and up to three copies were found in some lines. Rearrangements of chromosome 1 and 11 were each found in three out of four tumours. The rearrangements of chromosome 1 all resulted in duplication of 1q and deletion of short-arm material from the same chromosome giving loss of heterozygosity for enzyme markers on 1p. Loss of satellite material from chromosome 13 and the centromere region of chromosome 9 were found in single cases. This study shows that even where the chromosome number of tumour cells is near triploid, regions of the genome can be deleted. The chromosomes most frequently involved in rearrangements, 1, 11, and 12 all contain sites of ras oncogenes and it is suggested that loss of normal alleles could result in homozygosity for mutant oncogenes which may play a part in tumour progression.     

Linkage of Bipolar Affective Disorder to Chromosome 18 Markers in a New Pedigree Series

Several groups have reported evidence suggesting linkage of bipolar affective disorder (BPAD) to chromosome 18. We have reported data from 28 pedigrees that showed linkage to marker loci on 18p and to loci 40 cM distant on 18q. Most of the linkage evidence derived from families with affected phenotypes in only the paternal lineage and from marker alleles transmitted on the paternal chromosome. We now report results from a series of 30 new pedigrees (259 individuals) genotyped for 13 polymorphic markers spanning chromosome 18. Subjects were interviewed by a psychiatrist and were diagnosed by highly reliable methods. Genotypes were generated with automated technology and were scored blind to phenotype. Affected sib pairs showed excess allele sharing at the 18q markers D18S541 and D18S38. A parent-of-origin effect was observed, but it was not consistently paternal. No robust evidence of linkage was detected for markers elsewhere on chromosome 18. Multipoint nonparametric linkage analysis in the new sample combined with the original sample of families supports linkage on chromosome 18q, but the susceptibility gene is not well localized.     

Complete paternal uniparental isodisomy for chromosome 1 revealed by mutation analyses of the<Emphasis Type="Italic"> TRKA (NTRK1)</Emphasis> gene encoding a receptor tyrosine kinase for nerve growth factor in a patient with congenital insensitivity to pain with anhidrosis

Uniparental disomy (UPD) is defined as the presence of a chromosome pair that derives from only one parent in a diploid individual. The human TRKA gene on chromosome 1q21-q22 encodes a receptor tyrosine kinase for nerve growth factor and is responsible for an autosomal recessive genetic disorder: congenital insensitivity to pain with anhidrosis (CIPA). We report here the second case of paternal UPD for chromosome 1 in a male patient with CIPA who developed normally at term and did not show overt dysmorphisms or malformations. He had only the usual features of CIPA with a homozygous mutation at the TRKA locus and a normal karyotype with no visible deletions or evidence of monosomy 1. Haplotype analysis of the TRKA locus and allelotype analyses of whole chromosome 1 revealed that the chromosome pair was exclusively derived from his father. Non-maternity was excluded by analyses of autosomes other than chromosome 1. Thus, we have identified a complete paternal isodisomy for chromosome 1 as the cause of reduction to homozygosity of the TRKA gene mutation, leading to CIPA. Our findings further support the idea that there are no paternally imprinted genes on chromosome 1 with a major effect on phenotype. UPD must be considered as a rare but possible cause of autosomal recessive disorders when conducting genetic testing.     

Fine-mapping chromosome 20 in 230 systemic lupus erythematosus sib pair and multiplex families: evidence for genetic epistasis with chromosome 16q12

The presence of systemic lupus erythematosus (SLE) susceptibility genes on chromosome 20 is suggested by the observation of genetic linkage in several independent SLE family collections. To further localize the genetic effects, we typed 59 microsatellites in the two best regions, as defined by genome screens. Genotypes were analyzed for statistical linkage and/or association with SLE, by use of a combination of nonparametric linkage methods, family-based tests of association (transmission/disequilibrium and pedigree disequilibrium tests), and haplotype-sharing statistics (haplotype runs test), in a set of 230 SLE pedigrees. Maximal evidence for linkage to SLE was to 20p12 (LOD = 2.84) and 20q13.1 (LOD = 1.64) in the white pedigrees. Subsetting families on the basis of evidence for linkage to 16q12 significantly improved the LOD scores at both chromosome 20 locations (20p12 LOD = 5.06 and 20q13 LOD = 3.65), consistent with epistasis. We then typed 162 single-nucleotide polymorphism markers across a 1.3-Mb candidate region on 20q13.1 and identified several SNPs that demonstrated significant evidence for association. These data provide additional support for linkage and association to 20p12 and 20q13.1 in SLE and further refine the intervals of interest. These data further suggest the possibility of epistatic relationships among loci within the 20q12, 20q13, and 16q12 regions in SLE families.