Cardiomyopathy in mice with paternal uniparental disomy for chromosome 12

Mice inheriting both copies of MMU12 either maternally or paternally demonstrate imprinting effects. Whereas maternal uniparental disomy 12 (matUPD12) fetuses are growth retarded and die perinatally, paternal UPD12 (patUPD12) fetuses die during late gestation and exhibit placentomegaly and skeletal muscle maturation defects. To examine further the developmental consequences of UPD12, we intercrossed mouse stocks heterozygous for Robertsonian translocation chromosomes (8.12) and (10.12). We report that at 13.5-14.5 dg patUPD12 hearts exhibit increased ventricular diameter, thinner, less compact myocardium, and deep intertrabecular recesses when compared to controls. These data provide evidence for cardiac failure, a lethal condition, and suggest a role for an imprinted gene(s) in normal heart development.     

Skeletal defects in paternal uniparental disomy for chromosome 14 are re-capitulated in the mouse model (paternal uniparental disomy 12)

Human paternal uniparental disomy for chromosome 14 (upd(14)pat) presents with skeletal abnormalities, joint contractures, dysmorphic facial features and developmental delay/mental retardation. Distal human chromosome 14 (HSA14) is homologous to distal mouse chromosome 12 (MMU12) and both regions have been shown to contain imprinted genes. In humans, consistent radiographic findings include a narrow, bell-shaped thorax with caudal bowing of the anterior ribs, cranial bowing of the posterior ribs and flaring of the iliac wings without shortening or dysplasia of the long bones. Mice with upd(12)pat have thin ribs with delayed ossification of the sternum, skull and feet. In both mice and humans, the axial skeleton is predominantly affected. We hypothesize that there is an imprinted gene or genes on HSA14/MMU12 that specifically affects rib/thorax development and the maturation of ossification centers in the sternum, feet and skull with little effect on long bone development.     

Parental origin-specific developmental defects in mice with uniparental disomy for chromosome 12

Genetic analysis has shown that the distal portion of mouse chromosome 12 is imprinted; however, the developmental roles of imprinted genes in this region are not known. We have therefore generated conceptuses with uniparental disomy for chromosome 12, in which both copies of chromosome 12 are either paternally or maternally derived (pUPD12 and mUPD12, respectively). Both types of UPD12 result in embryos that are non-viable and that exhibit distinct developmental abnormalities. Embryos with pUPD12 die late in gestation, whereas embryos with mUPD12 can survive to term but die perinatally. The mUPD12 conceptuses are invariably growth-retarded while pUPD12 conceptuses exhibit placentomegaly. Skeletal muscle maturation defects are evident in both types of UPD12. In addition, embryos with paternal UPD12 have costal cartilage defects and hypo-ossification of mesoderm-derived bones. In embryos with mUPD12, the development of the neural crest-derived middle ear ossicles is defective. Some of these anomalies are consistent with those seen with uniparental disomies of the orthologous chromosome 14 region in humans. Thus, imprinted genes on chromosome 12 are essential for viability, the regulation of prenatal growth, and the development of mesodermal and neural crest-derived lineages.     

Maternal uniparental disomy 14 dissection of the phenotype with respect to rare autosomal recessively inherited traits, trisomy mosaicism, and genomic imprinting

The phenotype of maternal uniparental disomy of chromosome 14 (upd(14)mat) is characterized by pre and postnatal growth retardation, early onset of puberty, joint laxity, motor delay, and minor dysmorphic features of the face, hands, and feet. Based on a clinical analysis of 24 cases extracted from the literature the phenotype of upd(14)mat was dissected with respect to each symptom's most likely primary causative: trisomy mosaicism, rare autosomal recessively inherited traits, and the impact of known imprinted genes located on chromosome 14q32. As a result, primary factors are confined placental mosaicism for prenatal growth retardation and one or more imprinted genes, which contribute to the reduced final height by accelerated skeletal maturation. As a secondary effect the latter might also cause early onset of puberty. Other secondary effects might be postnatal adaptation problems associated with neurological deficits such as muscular hypotonia due to premature delivery and reduced birthweight and most dysmorphic features as a consequence of subtle skeletal abnormalities and muscular hypotonia. Considering the rarity of traits such as cleft palate, trisomy mosaicism in the fetus is more likely causative than homozygosity of autosomal recessively inherited mutations. Totally, the variable phenotype of upd(14)mat is mainly the consequence of trisomy mosaicism and genomic imprinting. Rare traits might be due to homozygosity of autosomal recessively inherited mutations.     

Paternal uniparental disomy for chromosome 1 revealed by molecular analysis of a patient with pycnodysostosis.

Molecular analysis of a patient affected by the autosomal recessive skeletal dysplasia, pycnodysostosis (cathepsin K deficiency; MIM 265800), revealed homozygosity for a novel missense mutation (A277V). Since the A277V mutation was carried by the patient's father but not by his mother, who had two normal cathepsin K alleles, paternal uniparental disomy was suspected. Karyotyping of the patient and of both parents was normal, and high-resolution cytogenetic analyses of chromosome 1, to which cathepsin K is mapped, revealed no abnormalities. Evaluation of polymorphic DNA markers spanning chromosome 1 demonstrated that the patient had inherited two paternal chromosome 1 homologues, whereas alleles for markers from other chromosomes were inherited in a Mendelian fashion. The patient was homoallelic for informative markers mapping near the chromosome 1 centromere, but he was heteroallelic for markers near both telomeres, establishing that the paternal uniparental disomy with partial isodisomy was caused by a meiosis II nondisjunction event. Phenotypically, the patient had normal birth height and weight, had normal psychomotor development at age 7 years, and had only the usual features of pycnodysostosis. This patient represents the first case of paternal uniparental disomy of chromosome 1 and provides conclusive evidence that paternally derived genes on human chromosome 1 are not imprinted.     

Evolution of genomic imprinting with biparental care: implications for Prader-Willi and Angelman syndromes

The term “imprinted gene” refers to genes whose expression is conditioned by their parental origin. Among theories to unravel the evolution of genomic imprinting, the kinship theory prevails as the most widely accepted, because it sheds light on many aspects of the biology of imprinted genes. While most assumptions underlying this theory have not escaped scrutiny, one remains overlooked: mothers are the only source of parental investment in mammals. But, is it reasonable to assume that fathers'' contribution of resources is negligible? It is not in some key mammalian orders including humans. In this research, I generalize the kinship theory of genomic imprinting beyond maternal contribution only. In addition to deriving new conditions for the evolution of imprinting, I have found that the same gene may show the opposite pattern of expression when the investment of one parent relative to the investment of the other changes; the reversion, interestingly, does not require that fathers contribute more resources than mothers. This exciting outcome underscores the intimate connection between the kinship theory and the social structure of the organism considered. Finally, the insight gained from my model enabled me to explain the clinical phenotype of Prader-Willi syndrome. This syndrome is caused by the paternal inheritance of a deletion of the PWS/AS cluster of imprinted genes in human Chromosome 15. As such, children suffering from this syndrome exhibit a striking biphasic phenotype characterized by poor sucking and reduced weight before weaning but by voracious appetite and obesity after weaning. Interest in providing an evolutionary explanation to such phenotype is 2-fold. On the one hand, the kinship theory has been doubted as being able to explain the symptoms of patients with Prader-Willi. On the other hand, the post-weaning symptoms remain as one of the primary concern of pediatricians treating children with Prader-Willi. In this research, I reconcile the clinical phenotype of Prader-Willi syndrome with the kinship theory, contending that paternal investment relative to maternal investment increases after weaning. I also propose a genetic composition of the PWS/AS cluster, discuss the effects of new types of mutations, and contemplate the potential side effects of reactivating silent genes for medical purposes.     

Analysis of imprinting in mice with uniparental duplication of proximal chromosomes 7 and 15 by use of a custom oligonucleotide microarray

We have developed an imprinting assay combining the use of mice carrying maternal or paternal duplication of chromosomal regions of interest with custom oligonucleotide microarrays. As a model system, we analyzed RNA from CNS tissue of neonatal mice carrying the reciprocal translocation T(7;15)9H and uniparental duplication of proximal Chr 7 and 15. The duplicated region includes the locus on proximal Chr 7 corresponding to the human Prader-Willi/Angelman Syndrome. The microarray contained 322 oligonucleotides, including probes to detect major genes involved in neural excitability and synaptic transmission, as well as known imprinted genes mapping to proximal Chr 7: Ndn, Snrpn, Mkrn3, Magel2, Peg3, and Ube3a. Imprinting of these genes in neonatal cortex and cerebellum was first confirmed by quantitative RT-PCR. Their inclusion on the microarray thus provided positive controls for evaluating the effect of background on the sensitivity of the assay, and for establishing the minimum level of expression required to detect imprinting. Our analysis extended previous work by revealing bi-allelic expression in CNS tissue of those queried genes mapping to proximal Chr 7 or 15, including the Gabrb3 gene, for which there have been conflicting reports. Microarray analysis also revealed no effect of the maternal or paternal disomy on expression levels of the unlinked genes detected, including those potentially implicated in the Prader-Willi or Angelman Syndrome. In addition, quantitative RT-PCR revealed a gene dosage effect in both cerebellum and cortex for all of the known imprinted genes assayed, except for Ube3a in cerebellum.     

Peg1/Mest locates distal to the currently defined imprinting region on mouse proximal chromosome 6 and identifies a new imprinting region affecting growth

Mice with maternal duplication for proximal chromosome 6 (Chr 6) die in utero before 11.5 dpc, an effect that can be attributed to genomic imprinting. Previous studies have defined the region of Chr 6 responsible as lying proximal to the T6Ad translocation breakpoint in G-band 6B3. Evidence presented here with a new Chr 6 translocation T77H has substantially reduced the size of the imprinting region, locating it between G-band 6A3.2 and the centromere. The paternally expressed imprinted gene Mest had been mapped within the original imprinting region and was therefore a candidate for the early embryonic lethality. FISH has shown that Mest locates distal to T77H and therefore outside the redefined imprinting region. This evidence confirms that Mest is not a candidate for the early embryonic lethality found with two maternal copies of proximal Chr 6. Furthermore mice with maternal duplication for Ch 6 distal to T77H (MatDp.dist6) were found to be growth retarded at birth, the weight reduction remaining similar until adulthood. It can be concluded that the growth retardation is established in utero and is maintained at a similar level from birth to adulthood. Therefore Mest locates in a new imprinting region, distal to G-band 6A3.2 which affects growth. A targeted mutation of Mest has been reported that exhibits growth retardation, reduced postnatal survival and abnormal maternal behaviour. Here the phenotype of MatDp.dist6 mice is compared to that of Mest-deficient mutant mice. Unlike the latter, MatDp.dist6 mice have good survival rates and females have normal maternal behaviour. Possible reasons for these differences are discussed.     

Localization of the Mas proto-oncogene to a densely marked region of mouse chromosome 17 associated with genomic imprinting.

The mouse homolog of the human proto-oncogene MAS was mapped by two interspecific backcrosses to the proximal portion of MMU17. Higher resolution mapping was accomplished through the analysis of genotypes duplicated or deleted for a megabase-size subregion within MMU17. The results demonstrate a map position for Mas in the close vicinity of Igf2r, which encodes another membrane receptor known to undergo genomic imprinting. The data provide further evidence for the clustering of genes in a 1-Mb region of chromosome 17, with the absence of any identified genes in a nearby region likely to be six times larger.     

Partial paternal uniparental disomy of chromosome 6 in an infant with neonatal diabetes, macroglossia, and craniofacial abnormalities

Neonatal diabetes, which can be transient or permanent, is defined as hyperglycemia that presents within the first month of life and requires insulin therapy. Transient neonatal diabetes mellitus has been associated with abnormalities of the paternally inherited copy of chromosome 6, including duplications of a portion of the long arm of chromosome 6 and uniparental disomy, implicating overexpression of an imprinted gene in this disorder. To date, all patients with transient neonatal diabetes mellitus and uniparental disomy have had complete paternal isodisomy. We describe a patient with neonatal diabetes, macroglossia, and craniofacial abnormalities, with partial paternal uniparental disomy of chromosome 6 involving the distal portion of 6q, from 6q24-qter. This observation demonstrates that mitotic recombination of chromosome 6 can also give rise to uniparental disomy and neonatal diabetes, a situation similar to that observed in Beckwith-Wiedemann syndrome, another imprinted disorder. This finding has clinical implications, since somatic mosaicism for uniparental disomy of chromosome 6 should also be considered in patients with transient neonatal diabetes mellitus.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Genomewide scan in families with schizophrenia from the founder population of Afrikaners reveals evidence for linkage and uniparental disomy on chromosome 1

We report on our initial genetic linkage studies of schizophrenia in the genetically isolated population of the Afrikaners from South Africa. A 10-cM genomewide scan was performed on 143 small families, 34 of which were informative for linkage. Using both nonparametric and parametric linkage analyses, we obtained evidence for a small number of disease loci on chromosomes 1, 9, and 13. These results suggest that few genes of substantial effect exist for schizophrenia in the Afrikaner population, consistent with our previous genealogical tracing studies. The locus on chromosome 1 reached genomewide significance levels (nonparametric LOD score of 3.30 at marker D1S1612, corresponding to an empirical P value of.012) and represents a novel susceptibility locus for schizophrenia. In addition to providing evidence for linkage for chromosome 1, we also identified a proband with a uniparental disomy (UPD) of the entire chromosome 1. This is the first time a UPD has been described in a patient with schizophrenia, lending further support to involvement of chromosome 1 in schizophrenia susceptibility in the Afrikaners.     

Maternal uniparental disomy for human chromosome 14, due to loss of a chromosome 14 from somatic cells with t(13;14) trisomy 14.

Uniparental disomy (UPD) for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans. We recently studied a 9-year-old female with a de novo Robertsonian translocation t(13;14), short stature, mild developmental delay, scoliosis, hyperextensible joints, hydrocephalus that resolved spontaneously during the first year of life, and hypercholesterolemia. To determine the parental origin of chromosomes 13 and 14 in the proband, we have studied the genotypes of DNA polymorphic markers due to (GT)n repeats in the patient and her parents' blood DNA. The genotypes of markers D14S43, D14S45, D14S49, and D14S54 indicated maternal UPD for chromosome 14. There was isodisomy for proximal markers and heterodisomy for distal markers, suggesting a recombination event on maternal chromosomes 14. In addition, DNA analysis first revealed--and subsequent cytogenetic analysis confirmed--that there was mosaic trisomy 14 in 5% of blood lymphocytes. There was normal (biparental) inheritance for chromosome 13, and there was no evidence of false paternity in genotypes of 11 highly polymorphic markers on human chromosome 21. Two cases of maternal UPD for chromosome 14 have previously been reported, one with a familial rob t(13;14) and the other with a t(14;14). There are several similarities among these patients, and a "maternal UPD chromosome 14 syndrome" is emerging; however, the contribution of the mosaic trisomy 14 to the phenotype cannot be evaluated. The study of de novo Robertsonian translocations of the type reported here should reveal both the extent of UPD in these events and the contribution of particular chromosomes involved in certain phenotypes.     

Supernumerary small marker chromosome (SMC) and uniparental disomy 22 in a child with confined placental mosaicism of trisomy 22: trisomy rescue due to marker chromosome formation

Trisomy rescue is one of various proposed mechanisms in formation of supernumerary small marker chromosomes (SMC) and uniparental disomy (UPD). In the present report a small de novo marker chromosome derived from chromosome 14 or 22 was diagnosed at prenatal diagnosis due to maternal age. Follow up investigations at birth revealed mosaicism 47,XX,+mar/46,XX. Using FISH, the marker was positive for the probe D14/22Z1, but negative for the probes midi 54 and D22Z4. Using three informative markers both chromosomes 22 were shown to be inherited from the mother (UPDmat). The results are consistent with nondisjunction at maternal meiosis I. The girl is 18 months old now and phenotypically normal. Cardiac and abdominal malformations were excluded by sonographic examinations. Motor and mental development is according to or ahead of developmental milestones (free walking with 10 months, first words at 12 months). The case confirms that maternal UPD 22 most likely is not associated with clinical abnormalities. According to FISH results, UPD 22, and 47,XX,+22 in the placenta, we conclude that the SMC was derived from alpha satellite sequences of chromosome 22. This case for the first time gives evidence that early postzygotic reduction of a chromosome to a small marker chromosome is a real existing mechanism to rescue a conceptus with trisomy.     

Equal parental origin of chromosome 22 losses in human sporadic meningioma: no evidence for genomic imprinting.

Inactivation of tumor suppressor genes can occur either by mutation at the gene locus or by loss of part or all of the chromosome region containing the gene. The latter is most frequently detected by DNA markers as loss of heterozygosity in the tumor tissue. In several reports, the paternal homologue was preferentially retained in embryonal tumors associated with loss of particular chromosomal regions, suggesting genomic imprinting of the corresponding tumor suppressor loci. To explore the generality of these findings and the possible role of genomic imprinting in adult tumors of the nervous system, we have determined the parental origin of chromosome 22 loss in sporadic meningioma. Of nine cases studied, five tumors retained the maternally derived chromosome 22 homologue while four retained the paternally derived chromosome 22. Thus, in contrast to the embryonal tumors, the meningioma locus on chromosome 22 is inactivated by random mutation in sporadic adult meningiomas.     

Establishment and maintenance of DNA methylation patterns in mouse Ndn: implications for maintenance of imprinting in target genes of the imprinting center

Ndn is located on chromosome 7C, an imprinted region of the mouse genome. Imprinting of Ndn and adjacent paternally expressed genes is regulated by a regional imprinting control element known as the imprinting center (IC). An IC also controls imprint resetting of target genes in the region of conserved synteny on human chromosome 15q11-q13, which is deleted or rearranged in the neurodevelopmental disorder Prader-Willi syndrome. Epigenetic modifications such as DNA methylation, which occur in gametes and can be stably propagated, are presumed to establish and maintain the imprint in target genes of the IC. While most DNA becomes substantially demethylated by the blastocyst stage, some imprinted genes have regions that escape global demethylation and may maintain the imprint. We have now analyzed the methylation of 39 CpG dinucleotide sequences in the 5' end of Ndn by sodium bisulfite sequencing in gametes and in preimplantation and adult tissues. While sperm DNA is completely unmethylated across this region, oocyte DNA is partially methylated. A distinctive but unstable maternal methylation pattern persists until the morula stage and is lost in the blastocyst stage, where low levels of methylation are present on most DNA strands of either parental origin. The methylation pattern is then substantially remodeled, and fewer than half of maternally derived DNA strands in adult brain resemble the oocyte pattern. We postulate that for Ndn, DNA methylation may initially preserve a gametic imprint during preimplantation development, but other epigenetic events may maintain the imprint later in embryonic development.     

SNP array karyotyping allows for the detection of uniparental disomy and cryptic chromosomal abnormalities in MDS/MPD-U and MPD

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder.     

Inverted distal duplication of the long arm of chromosome 8: borderline intelligence and discrete dysmorphic syndrome.

In this report, we describe an 11-years-old girl with inverted duplication in the distal part of the long arm of chromosome 8 (inv dup(8)(q24.11----q24.3). The most remarkable finding is her borderline intelligence and her nearly normal phenotype.