It is proposed that transgenerational modulation of gene expression might be possible, if the metabolic response of the parent to some physiological or social stress modified imprint setting. Transcription regulators could theoretically mediate this process. The nature of imprinted genes poised, as it were, between a transcriptionally active and silent state, makes them good candidates for incorporation into the evolution of transgenerational adaption systems where coordinated changes in gene expression over the generations is a selective advantage. The coordination of human fetal (head) growth with the existing size of the mother's pelvis is suggested as just such a circumstance. The reduced birth weight of Dutch babies where their grandmothers suffered acute starvation in mid pregnancy, supports the notion of transgenerational adaption to nutrition, as does the secular change (increase) in child growth over the last century. The recent indication that there may be functional polymorphism in the imprinting of the human IGF2 and IGF2R genes suggests these ideas could be explored using association studies at the population and individual level.

The origins and development of human, medical and clinical genetics are interwoven and yet each of these disciplines follows its own path.

The beginnings of a systematic human genetics can be traced to the middle of the 19th century, but it took human genetics almost 100 years to mature fully and influence medicine. Its origins can be traced to the work of three scientists: Galton, Pearson and Bateson.

In 1865, 6 years after Darwin had published Origin of Species in London, and the year when Mendel's paper, “Experiments on Plant Hybrids”, was published in Brunn, Galton, also in London, set out his first ideas on human heredity. His thinking then developed in two directions. The first laid the foundations for the scientific study of human heredity through biometrics and quantitative genetics. This part of Galton's thinking is summarized in his epitaph: “the dominant idea of his life's work was to measure the influence of heredity on the mental and physical attributes of mankind”.

Galton's other line dealt with the application of heredity through eugenics, a word that Galton coined to signify “well bred”. He wanted, I quote his words: “to produce a highly gifted race of Man by judicious marriage through several generations”. Families of merit should be identified and positively encouraged to breed; conversely, the “ weak could find a welcome … in celebrate monasteries”.

In the beginning the dogma was that sex determination in man followed the Drosophila pattern in which XO is male, XXY female, and the Y chromosome has no direct influence on the determination of sex. On the grounds of specific anomalies with which they presented, females with Turner Syndrome were sex chromatin tested and found to be chromatin negative [1]. This result, confirmed in 1956 by the male frequency of red-green colour blindness in these subjects which indicated that they carried only one X chromosome in spite of their female phenotype, suggested that therefore they might be XO, and, so, hinted that sex determination in man might not follow the then accepted pattern [2]. In 1959 chromosome studies confirmed that XOs were female [3] and showed that subjects with the symmetrical XXY sex-chromosome anomaly were with Klinefelter syndrome [4]. In the same year, by showing that XOs were females also in mice [5] it became accepted that the Y chromosome was the determiner of the formation of the testis in the mammalian embryo, and so was the key element in primary sexual differentation. It would seem appropriate to call this formal model of chromosomal sex determination the Malandrium pattern [6].

In 1966 Jacobs and Ross [7], from work on males with Y chromosome deletions narrowed down the testis determining function of the Y chromosome to its short arm. Then, in 1975, Wachtel and collaborators [8] were the first to formulate a hypothesis on the sex determining gene, or, more precisely on the nature of its product. They suggested that this developmental role might be played by the H-Y antigen, a weak histocompatibility antigen which had been known to be involved in the rejection of male skin grafted onto otherwise histocompatible female mice. The idea had run into technical difficulties and a major problem was related to the significance that should be attached to the results of two different ways for demonstrating the antigen, namely the cell-mediated cytotoxicity test or the serological test. Efforts were made to keep the H-Y hypothesis alive, largely because there was a certain elegance about it [9, 10]. However eventually XX male mice, lacking H-Y by either test, spelt the end of the candidature of H-Y as the testis determining mechanism [11, 12].

The fact that all individuals are not identical in their risk for certain cancers has been known for many years. The factors responsible for the increased risk observed in certain individuals, families, or populations may be either environmental or genetic. The genetic risk factors identified to date generally result in a greatly increased relative risk – on the order of ten to one hundred thousand-fold in the case of familial retinoblastoma, for example. The major reason for the identification of genetic factors that result in such large increases in relative risk is that the transmission of such genes in families results in an easily identifiable pattern of inheritance and the concommitant ability to locate the gene resulting in increased risk by genetic linkage analysis. From our study of sporadic and familial retinoblastoma, we have identified a genetic factor whose action appears to result in a more modest increase in relative risk – on the order of one-hundred to one-thousand-fold. We believe that a locus that is responsible for this increase in relative risk lies on the human X-chromosome. Our data indicate that aberrant alleles at this locus not only appear to increase an individual's risk for a number of cancers, but also participate in the process of genome imprinting.

The epidemiological and genetic arguments in favor of the involvement of an X-linked “imprintor, mutator” gene in the generation of new, heritable, RB-1 mutations are given in detail in Naumova and Sapienza (1994). The pertinent data may be summarized as follows:

1. there is a significant excess-of-males among patients with bilateral sporadic but not unilateral sporadic retinoblastoma;

2. there is preferential retention of paternally-derived RB-1 alleles in bilateral, but not unilateral, sporadic retinoblastoma;

3. sex ratio bias in favor of males is observed among the offspring of male founders but not female founders of retinoblastoma pedigrees (i.e. the male founders fail to transmit their X-chromosomes with the expected frequency;

4. transmission ratio distortion in favor of affected individuals is observed among the offspring of male founders but not female founders, and

5. the affected male offspring of these male founders have families in which offspring are distributed equally among affected and unaffected boys and girls, as expected for an autosomal dominant trait, i.e. sex-ratio and transmission-ratio distortion are observed only among the offspring of male founders of retinoblastoma pedigrees.

Uniparental disomy (UPD), the inheritance of both homologues from one chromosome from the same parent, was first proposed in 1980 by Erik Engel [1] to be a potential cause of congenital developmental defects in hymans. First hints from the premolecular era towards its existence came from instances where a pericentric inversion was present on one homologue in a parent and on both in one offspring [2] and where there was transmission of an interhomologous Robertsonian translocation (of chromosome 22) from a healthy mother to healthy offspring [3-4]. In mice, UPD was experimentally produced by crossing two mice lines with different Robertsonian translocations both involving the same chromosome [for 2 review see ref. 5].

Through this approach, it was possible to define imprinted regions, chromosomes and chromosomal segments for which either maternal or paternal or both types of uniparental disomy led to phenotypic abnormalities. The latter are explained by genomic imprinting, the differential silencing of a gene or genes from one of the parents (the mother or the father) during any stage of embryogenesis or later in life. If, for example, the maternal homologue of a given gene is imprinted (and hence only the paternal allele is active), maternal UPD would lead to loss of the active allele and thus might cause consequences due to loss of function.

Uniparental disomy (UPD) can be caused by various genetic mechanisms such as gamete complementation, chromosome duplication in a monosomic zygote or postzygotic aneuploid correction. This latter mechanism has been recently well documented in human reproduction and seems to be strictly related to placental mosaicism. We have therefore studied some aspects of confined placental mosaicism (CPM) which are useful to clarify one of the most common sources of UPD in humans.

Abnormal distribution of chromosomes in postzygotic mitotic cell divisions may result in a mosaic condition with two or more cell lines showing different chromosome constitutions. The effects on fetal phenotype and pregnancy development depend on the chromosomes involved, the distribution of the abnormal cells among tissues and on the precise stage at which chromosome mutation occurs.

As shown in Fig. 1, when the mutational event occurs in the blastocyst, prior to the differentiation of embryonic and chorionic compartments, the mosaicism is found in both the placental and fetal tissues. In contrast, when the chromosome mutation occurs at a later stage, after embryonic and chorionic compartment separation, the abnormal cells may be confined to the placenta or to the embryo, and are not necessarily found in both.

The fragile X mutation is the result of an abnormal expansion of a CGG repeat sequence in the FMR-1 gene.

Molecular techniques enable the detection of the mutation and also of the exact length of this DNA sequence, allowing the classification of the tested subjects as normal, carrier or affected.

We propose a protocol of analysis that combines a method of non-radioactive PCR, Southern blotting and cytogenetic testing.

This protocol can be used for screening programme of selected groups of mentally retarded individuals and for prevention studies in families at risk.

A girl carrying a de novo balanced 13-14 robertsonian translocation showed a clinical phenotype with severe hypotonia, hyperextensible joints, frontal bossing, asymmetric face, no mental retardation, severe scoliosis and motor delay. In situ hybridization analysis on chromosome spreads revealed the presence of the two centromeres in the rearranged chromosomes. Molecular analysis on genomic DNA showed the presence in the proposita of two chromosomes 14 of maternal origin and no chromosome 14 from the father indicating a maternal monocentric uniparental disomy for chromosome 14 (mUPD14). Our patient shows several similarities with other reported cases of mUPD14, suggesting imprinting of a region(s) of chromosome 14 and defining a possible mUPD14 Syndrome.

Neuroblastoma is the most common solid tumor in children. It derives from the neural crest and originates from the sympathetic neuronal lineage [1-3]. At least two distinct biological-clinical entities can be distinguished [3-6]. One favorable subset occurs exclusively in infants and consists of early stages (I and II) as well as widespread disease (stage IV-S) at diagnosis. These tumors are commonly characterized by a hyperdiploid or pseudotriploid karyotype, but lack structural chromosome abnormalities. In particular, 1p abnormalities or N-myc gene amplification are not observed. Virtually all tumors identified with mass screening have belonged to these lower stages [4, 7, 8]. These patients show an excellent clinical outcome despite no or only minimal therapy. The other group of unfavorabled neuroblastomas is associated with older age and advanced stages (stages III and IV), and pseudodiploid karyotypes including lp deletions and N-myc oncogene amplification [2, 9]. Their outcome remains poor despite aggressive multimodality therapy and bone marrow transplantation. It is interesting to note that favorable neuroblastomas rarely, if ever, evolve into unfavorable disease [3].

Due to DNA technology, it is now apparent that the mechanisms of genetic disease are more complex than the model of a gene with biallelic expression in the diploid state. If a gene is imprinted, monoallelic expression is the norm when the chromosomes of a pair are inherited normally from each parent. Uniparental disomy (UPD) is the abnormal situation where both chromosomes of a pair come from the same parent. When the chromosome contains an imprinted gene, UPD may result in nullisomy or disomy for a functional copy of that gene. If there are two imprinted loci on the same chromosome, UPD for that chromosome results in nullisomy for one imprinted gene but functional disomy for the other a “diploid overdose” (DO). This situation has been well demonstrated in the Prader-Willi syndrome (PWS) which is the nullisomic phenotype for the PWS gene(s) on chromosome 15q 11-13. Chromosome 15q11-13 also contains the gene for Angelman syndrome (AS) which has a phenotype distinct from PWS. Both loci are subject to imprinting – in PWS, the imprint is on the maternal chromosome 15, in AS it is on the paternal chromosome 15. All individuals with PWS due to maternal UPD, while functionally nullisomic for the PWS locus, are functionally disomic for the AS locus – a DO situation. Assuming that biallelic expression of an imprinted gene is harmful, one would expect DO for an imprinted gene to produce a phenotypic effect. Cases of PWS due to UPD do not appear to differ from those due to deletion (hypopigmentation in deletional cases can be explained by loss of D15S12 downstream from the critical region). There is no good evidence of DO for the AS locus in PWS due to UPD. Why then was it ‘necessary’ in evolutionary terms to imprint the AS locus and maintain the imprint faithfully for life. A similar situation of two imprinted genes on the same chromosome occurs with IGF2 and H19 on chromosome 11p15. Maternal imprinting for IGF2 and paternal imprinting for H19 is the norm. Paternal UPD in this situation does lead to a DO effect, namely Beckwith-Wiedemann syndrome. The possibility of a DO effect needs to be considered when assessing the phenotypic spectrum of UPD for other chromosomes currently under investigation.

Since the original description of the Prader-Willi syndrome (PWS) in 1956 [1], and the recognition of the involvement of the proximal region of chromosome 15 in this disorder [2], understanding of the molecular basis of the genetic defect in PWS has progressed rapidly. A set of clinical criteria has been defined [3], although the diagnosis on clinical grounds alone remains difficult in the first year of life. Research has focussed both on improving the diagnostic molecular and cytogenetic tests for PWS and on identifying and defining the functions of genes whose expression is altered in this neurobehavioral disorder. Furthermore, this region is known to be subject to genomic imprinting effects, so that expression of genes involved in PWS is expected to be exclusively from the paternal allele.

A critical step in the definition of the region containing such genes was the identification of a subset of unusual patients affected with either PWS or the Angelman syndrome, which also involves a gene or genes in the proximal region of chromosome 15. These unique patients, who have chromosome 15 translocations or deletions, helped to narrow the critical region to an interval containing less than 500 kb of DNA [4-6] (Fig. 1). As will be discussed, below, regulatory elements exist in this 500 kb region which alter the expression of genes located outside this interval [7, 8].

In mammals, normal embryonic development requires differential genomic imprinting of male and female gametes [1, 2]. Many investigations have been directed towards the understanding of the molecular mechanisms of imprinting and the timing of establishment of the imprint during gametogenesis and its erasure during development.

Methylation is the focus of many of these studies as it has been known for some time that this epigenetic modification of the DNA correlates with the status of gene activity. So far, five imprinted genes, expressed from only one of the parental alleles, have been found to be differentially methylated in somatic tissue: mouse Igf2 [3] and Xist [4] and human SNRPN [5, 6] expressed from the paternal allele; mouse Igf2r [7] and H19 [8, 9] expressed from the maternal allele. However, so far, a gametic methylation imprint has been detected for only two of these genes: in an intron region of mouse Igf2r [7], and in the promoter region [10] and the first exon [11] of the Xist (X-inactivation-specific transcript [12, 13] gene.

The data accumulated for the Xist gene, during different phases of gametogenesis and development, provides the most comprehensive story about the role of methylation as a primary gametic imprint, and on the timing of its establishment during gametogenesis and erasure during development. Methylation studies have now been performed during oogenesis and spermatogenesis [Norris et al., 1994; 11] and in mature gametes and during early stages of development [10, 11]. In addition, expression of the gene has been described during gametogenesis [14-16] and throughout early development [4-17].

Several unique features distinguish imprinted from nonimprinted genes, including the unusual replication behavior the unequal methylation and the differential expression of imprinted alleles [1]. The replication timing in S phase of the two homologous alleles of a normal, nonimprinted gene is highly synchronous [2, 3]. Housekeeping genes replicate early, constitutive heterochromatic regions replicate late and tissue-specific genes replicate earlier when they are expressed than when they are not [2-4]. In contrast, imprinted genes which, by definition, display allele-specific expression replicate asynchronously [2-5].

The relative order of replication of homologous alleles as well as that of different loci can be elegantly compared with fluorescence in situ hybridization (FISH) on interphase nuclei [2-5]. Unreplicated DNA segments give singlet hybridization signals in normal diploid cells, while replicated loci are characterized by doublets. The distribution of these two patterns can be used to determine the S phase replication time of any DNA sequence. Moreover, determination of the singlet/doublet ratio allows a good estimation of the degree of replication asynchrony of two homologous alleles [2-5].

Using cell lines with deletions, disomies or associated FISH-detectable centromeric satellite polymorphisms, Kitsberg et al. [4] found that the paternal allele was the early replicating one in all the imprinted genes which they had analyzed. Subsequently, however, Knoll et al. [5] detected genes in the imprinted Prader-Willi region on chromosome 15, which also displayed other patterns. Therefore, it seems necessary to specify the relative timing of maternally and paternally derived alleles for each individual asynchronously replicating gene. Unfortunately, this is so far only feasible with a very restricted number of sequences.

We report on a Prader-Willi syndrome (PWS) patient carrier of a balanced 15q15q translocation and affected by a prolactin-secreting pituitary adenoma. Evidence provided by molecular studies indicates that the structural rearrangement is an isochromosome of maternal origin. According to the identification of isodisomy as the basis of the association of rare disorders and the recent report on chromosome 15 monosomy and nullisomy in pituitary adenoma, we suggest that in our case PWS and pituitary adenoma might be related.

Angelman syndrome (AS) is caused by the loss of function of yet unidentified gene(s) which map within 15q 11-13 and show monoallelic expression from the maternal allele. Lack of the maternal allele(s), due to either a deletion on the maternal chromosome 15 (about 70% of AS patients) or a paternal uniparental disomy (UPD)15 (<5%), are the most common molecular defects in AS. Prader-Willi syndrome (PWS) also maps to proximal 15q, but is caused by the loss of function of paternally expressed gen(s) [1]. Here we describe clinical, cytogenetic and molecular data for two non-related patients with AS who carry a nonmosaic extra cromosome inv dup(15).

Genetic complementation experiments have indicated that both a maternal and a paternal copy of the distal region of mouse chromosome 7 are essential for normal development [1]. This suggested the presence of genes whose expression is dependent on the gamete of origin in this chromosomal region. Two such imprinted genes, namely insulin-like growth factor II (Igf2) and H19, have been identified so far [2, 3]. The first encodes a peptide with mitotic activity towards several cell types, that contributes significantly to prenatal growth of mammals, whereas the second has, as yet, no defined role and seems not to encode any protein, but works as RNA. (Igf2) and H19 are located 90 kb apart, have similar expression patterns during development and are reciprocally imprinted, since the maternal Igf2 and the paternal H19 alleles are inactive in most fetal tissues [4, 5].

Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are well established as models of Genomic Imprinting in humans, since completely different phenotypes are generated by the absence of paternal (PWS) or maternal (AS) contribution to the q11-13 region of chromosome 15 as a result of deletion or uniparental disomy. We report a preliminary study based on our experience of more than 20 years of research into the genetics of PWS and AS syndromes.

Thirty nine subjects, referred from a number of Centers and Medical Doctors have been examined to either confirm or rule out a diagnosis of PWS or AS.

Patients were evaluated through the Clinical Genetics and Dysmorphology Program at the Human Genetics Center, Dept. of Paediatrics, University of Florence.

Clinical evaluation showed that 10 of these patients fulfilled diagnostic criteria for PWS and 8 for AS.

All patients were isolated cases and the 18 nuclear families were unrelated.

We adopted the staged diagnostic approach for all our families diagnosed PWS or AS families, moleucular using cytogenetic, genetic and molecular cytogenetic techniques.

An increasing number of clinical observations and genetic experiments have shown that some parts of the genome behave differently depending on whether they are of paternal or maternal origin. This phenomenon is known as “genomic imprinting” and has been defined as “a reversible process whereby a gamete-specific modification in the parental generation can sometimes lead to functional differences between maternal and paternal genomes in diploid cells of the offspring” [1]. The accumulating evidence for its important role in cancer predisposition syndromes as well as for the pathogenesis of certain types of sporadic tumors prompted us to investigate whether imprinting may also be instrumental in selecting particular parental chromosome regions involved in balanced rearrangements, such as the leukemia-specific translocation t(9;22) [2]. Several reports have analyzed the expression, the methylation and the replication patterns of the two genes, ABL and BCR, on chromosomes 9 and 22, respectively, which are affected by this translocation [3-6]. Although not directly comparable, the results of these studies seem to invalidate our cytogenetic observations. We therefore review this controversial issue and provide some possible explanations for the contradictory results.

Hyperdiploidy with a chromosome number between 51 and 65 and a mean peak at 55 occurs as a distinct karyotype pattern in approximately 25-30% of ALLs in childhood [1, 2]. It is considered a favorable prognostic factor. The most intriguing cytogenetic peculiarities of these leukemias are the nearly exclusive presence of nonrandom numerical abnormalities due to the gain of chromosomes 4, 6, 10, 14, 17, 18, 20, 21 and X [1, 2]. In contrast, chromosomes 1, 2, 3, 12 and 16 are rarely involved [1, 2]. Typically, the affected chromosomes are present in three copies, with chromosome 21 also often being tetrasomic.

Near-haploid cases, on the other hand, are extremely rare and have a bad prognosis [1, 2]. They contain at least one copy of each chromosome, a second copy of one of the sex chromosomes and both chromosomes 21 in most instances. In addition, two copies of chromosomes 10, 14 and 18 are commonly found.

In the majority of cases of hyperdiploid ALL, the mechanism leading to the increased number of chromosomes is unknown. However, once formed, the abnormal karyotype is uniform and stable in the malignant cell population. Molecular genetic studies performed by Onodera et al. [3] revealed that the hyperdiploid karyotype usually arises by a simultaneous event during a single abnormal cell division from a diploid karyotype. Occasionally, this can also occur by doubling of the chromosomes from a near-haploid karyotype [4]. In virtually all cases, tetrasomy of chromosome 21 was generated by a duplication of both the maternally and paternally derived homolog. This finding was one of the main arguments for the notion that hyperdiploidy cannot be caused by stepwise or sequential gains from a diploid karyotype or by consecutive losses from a tetraploid karyotype.

In previous work from this laboratory, an inverse dependence was established for the adenovirus type 2 E2A late promoter between sequence-specific DNA methylation and promoter activity [1-5; for reviews see ref. 6, 7]. The effect of DNA methylation on promoter activity was also assessed in the transgenic mice, which were obtained from microinjections of unmethylated or in vitro HpaII-premethylated pAd2E2AL-CAT DNA [1] into F2 zygotes from B6D2F, (C57BL/6 × DBA/2) hybrid mice. In CAT assays carried out on organ extracts from the pAd2E2AL-CAT mice, the inverse relationship was confirmed [2].

We studied the stability of the pAd2E2AL-CAT DNA methylation patterns in up to eight mouse generations and assessed the influence of the strain-specific genetic background. Three pAd2E2AL-CAT mouse lines were crossed with inbred DBA/2, C57BL/6 or B6D2F, mice. Parent-of-origin effects were controlled by exclusive hemizygous transgene transmission either via females or males. The founder animal of line 7-1 carried two groups of transgenes (A and B) on separate chromosomes. The transgene methylation patterns of the 7-1B transgenes and those of the lines 5-8 and 8-1 were stably transmitted.

Southern blot hybridization experiments [8, 9] revealed that the 7-1A transgene methylation pattern was a cellular mosaic. In mixed-genetic-background offspring from 7-1A animals, 10% carried transgenes with HpaII-DNA methylation levels that were reduced from 40 to 10-15%. This finding suggested that in this background the factors that supported high methylation levels were dominant. When inbred DBA/2 mice were the mates, 40% of the siblings carried demethylated transgenes, whereas this ratio amounted to only 10% in C57BL/6 offspring (comparable to B6D2F1 crossings). Transgene methylation patterns were not detectably influenced by the parent-of-origin.