We present a new case of 11p15 duplication (trisomy 11p15) in a boy [46,XY,-21,+der(21), t(11;21)(p15.2;q22.3)] suffering from Beckwith-Wiedemann syndrome (BWS), whose phenotypically normal father carries a balanced translocation between chromosomes 11 and 21[46,XY, t(11;21)(p15.2;q22.3)]. The paternal grandmother has the same balanced translocation and is also clinically normal. BWS was suspected when the boy was 6 months old because of gigantism, macroglossia, visceromegaly, ear lobe creases and abdominal distention. Apart from the characteristic BWS phenotype, the boy has other features which are almost exclusively observed in 11p trisomy (high forehead with frontal upsweep of hair, wide central nose bridge, slightly beaked nose, chubby cheeks and severe mental retardation). So far, at least eight cases of 11p15 duplication have been described as patients with BWS. In six of these, the duplication was due to inheritance of a translocated or rearranged paternal chromosome. This was also the case in our patient. In the two other previously published cases, the 11p15 duplications were de novo, but in one of these, DNA analysis has subsequently shown that the duplication was of paternal origin. We discuss our observations in relation to the above-mentioned previous cases of 11p15 duplication and the possible role of genomic imprinting in the etiology of BWS.

Skewed X inactivation has been suspected as the genetic cause for some female carriers of Duchenne/Becker muscular dystrophy (DMD/BMD) presenting symptoms [1], as well as in manifesting females in other X-linked recessive diseases. No clear “parental source effect” – a difference in phenotype depending upon transmission of the mutated allele by either the father or the mother – has been observed [Cremer]. To date, most studies in this field have analysed the methylation status of flanking polymorphic sites (a measure of the maintenance of X inactivation and imprinting), not the expression of the causal gene itself.

To test the parental source effect on the protein expression of the dystrophin gene, we have set up crosses of mdx/mdx and mdx/y mice – the well-known DMD animal model [2] with the respective wild-type mice of the same strain (Table 1). The obligate heterozygous F1 females show a mosaic expression of dystrophin in skeletal and cardiac muscle upon immunehistological staining. Because the source of the mutation in the litters is well defined, the mosaic pattern should reflect a parental source effect, if any. Such an effect should also change the level of muscle enzymes in serum and may thus be easily testable in humans, too. Since DMD is genetically lethal in humans, one can test the hypothesis of a parental source effect in BMD families only.

A male child has been identified with Angelman syndrome. He has been shown to carry a de novo Robertsonian 15/15 translocation where both chromosome 15s have been derived from the father. Consequently the disease in this instance is due to paternal uniparental disomy.

Eight patients with Prader-Willi syndrome (PWS), 3 males and 5 females, who were shown to have uniparental maternal disomy after investigation with polymorphic DNA marker spanning chromosome 15, were investigated clinically.

Four patients displayed in addition to the clinical signs, some unusual clinical feature: two patients showed a hairshaft anomaly, one of whom, a female, suggested a Menkes'-like condition-an investigation of copper uptake showed slightly increased values. Further investigations are in progress. One patient died unexpectedly at the age of 1 year 4 months and adenovirus was isolated postmortem in the airway secretion. Two patients presented a seizure disorder.

The mean birth weight was 2,122 (1,195-3,170) g and the gestational age was 37.1 (32-42) weeks for this group, lower than expected for PWS. Mean maternal age was elevated [31.9 (25-39) year].

Since the recent report [1] of a case of Bloom syndrome in a PWS patient led to the mapping of the Bloom syndrome gene to chromosome 15, it has been suggested that isodisomy could lead to homozygosity for recessive genes. This opens up the possibility of mapping genes in a new way. We suggest that uniparental disomy PWS patients should be investigated for unusual features to identify other monogenic disorders with a locus on chromosome 15. Isodisomy defined by multiple DNA markers indicates potential regions of candidate genes.

Despite numerous studies, the clinical heterogeneity of Down syndrome has no explanation. We have attempted to investigate the role of genomic imprinting in the phenotype of liveborn Down syndrome patients. Hundred fifty eight patients were investigated for parental origin of the extra chromosome 21 with standard cytogenetic analyses and with DNA plymorphic markers. The extra chromosome 21 was of paternal origin in 8 cases and of maternal origin in 150 cases.

The phenotype of Down svndrome patients in whom the nondisjunction was of maternal origin, was not different from the phenotype of Down syndrome patients in whom the nondisjunction was of paternal origin.

We conclude that imprinting may probably not play a role in the heterogeneity of Down syndrome phenotype.

A group of 30 patients clinically described as having the Prader-Willi Syndrome (PWS) were studied using microsatellites from 15q11-13 and methylation analysis with probe PW71B (D15S63). The patients were categorized according to clinical symptoms. 80% of all patients were informative using molecular and cytogenetic methods. Among 8 patients with an atypical PWS phenotype, 2 showed uniparental disomy, and 2 had a mosaic deletion for 15q. The last 4 atypical and 2 typical patients had neither molecular defects confirmed by microsatellite analysis nor a parent-of-origin-specific methylation pattern for PWS. Our results confirm that methylation pattern analysis provides an additional and alternative microsatellite analysis to diagnose PWS.

Fluorescence in situ hybridization (FISH) has been used to visualize specific genomic DNA sequences in interphase nuclei. Timing of replication can be measured by FISH to interphase nuclei: nuclei with a sequence that has not replicated reveal two single signals (G1), whereas those in which the sequence has replicated show two signal doublets (G2). Asynchronous nuclei show a single signal on one allele and a double hybridization dot on the other homologue. In general, most sequences replicate synchronously on the two homologues, with only 10% of nuclei showing an asynchronous hybridization pattern. However, for the sequences known about to be imprinted, approximately 30% of nuclei reveal asynchronous replication. Little is known whether or not the proximal region of chromosome 22, involved in the DiGeorge syndrome [1], is imprinted. We have, therefore, examined the replication timing pattern of the DiGeorge critical region (DGCR).

Distal chromosome 2 is one of ten regions of the mouse genome subject to parental imprinting. Maternal duplication/paternal deficiency of distal chromosome 2 leads to hypokinetic mice with long flat-sided bodies and arched backs, which fail to suckle and die within a few hours of birth. The reciprocal type, paternal duplication/maternal deficiency, shows an opposite phenotype: the mice have short square bodies and broad flat backs and are distinctly hyperkinetic [1], The region is defined by the breakpoints of two reciprocal translocations, T(2;8)2Wa and T(2;16)28H, is estimated to be about 7.5 Mb in size and shows linkage conservation with human chromosome 20q13. We have tested for monoallelic expression of a number of candidate genes that are either likely or known to lie within the distal imprinting region. These included three-Cebpb, E2f1 and Tc/f4-that express transcription factors, two-Cyp24 and Pck1-that are involved in growth, six-Acra4, Edn3, Kcnb1, Mc3r, Ntsr and Ppgb-where a defect could lead to neurological and probably behavioural problems, three:-Cd40, Plcg1 and Rcad-that are doubtful candidates but whose sequence was available for testing their expression and finally, Gnas, which is our best candidate because there is clinical and biochemical evidence suggesting that its human homologue, GNAS1, is imprinted. On/off expression of each gene was tested by RT-PCR analysis of RNA extracted from tissues of mice with maternal duplication/paternal deficiency and its reciprocal for the distal region of chromosome 2. None of these genes was monoallelically expressed in the appropriate tissues before and after birth, suggesting that they are not imprinted. Evidence for the imprinting of GNAS1 in humans is based on studies of patients with Albright's hereditary osteodystrophy (AHO) who possess a dysfunctional GNAS1 allele. AHO somatic features together-with resistance to parathyroid hormone and other hormones that act via cAMP occur when the defective allele is inherited from the mother whereas AHO somatic features alone occur in paternally transmitted cases [2]. If genomic imprinting regulates Gnas expression in the mouse, then its effect must be specifically confined to hormone-responsive cells.

Recent reports have shown that in normal alleles, the CGG repeat at the FRAXA locus is interrupted by one or two AGG and that abnormal alleles seem to be generated by expansion of pure CGG repeats at the 3′ end [1].

It is therefore important to establish rapid, simple and low-cost sequencing procedures for determining not only the number of CGG repeats but also the presence of AGG triplets.

A one-lane sequencing procedure with PCR-amplified DNA, labelled at the 3′ or 5′ end with a single fluorochrome, has been recently developed in our laboratory [2, 3]. This methodology is particularly suitable for detecting mutations in family studies [4] and has been applied to the analysis of the sequence of triplet repeats at the FMR1 gene after PCR amplification, using one of the two primers fluorescently labelled at the 5′ end.

The sequence of the primers utilised in the PCR reaction is reported by Erster et al. [5]. The direct primer was fluorescently labelled at the 5′ end with 5-(6)-carboxyfluorescein (Fluoreprime, Pharmacia) and synthesised using an ABI 391 PCR-Mate-EP DNA synthesiser. The PCR amplification was performed on 0,5 ug DNA in a 50-μ1 PCR buffer containing 10% DMSO, 10% glycerol, 25 pmol of the two primers, 0,3 mM dNTPs and 1 U Taq DNA polymerase.

Each sample was submitted to amplification on a Perkin Elmer 9600 PCR reactor, with the following cycling profile: 5 min at 95 °C, 30 cycles of 30 s at 97 °C, 1 min at 55 °C and 1 min at 72 °C, followed by a 10-min terminal extension at 72 °C. After precipitation with 5 M ammonium acetate, the pellet was dissolved in 80% (vol/vol) formamide and aliquots were heated for 2 min at 90 °C or, alternatively, for sequence analysis, for 10 min at 110 °C. The samples were loaded on 6 or 8% PAGE sequencing gels and analysed using an ABI 373A automatic sequencer as previously described [2].

The c-fos promoter is negatively regulated by the retinoblastoma (Rb)-suceptibility-gene- encoded protein as well as by other genes involved in the control of transcription, cell cycle regulation and neoplastic transformation. We have examined by immunohistochemistry the c-Fos and c-Jun proteins in five cases of retinoblastoma in order to evaluate eventual alterations in their expression in vivo, possibly related to a gene mutation or to loss of Rb negative control.

In 1975, Miller, McKusick, Malvaux et al. reported a new form of low-birth weight dwarfism with normal intelligence, later called 3-M syndrome (after the initials of the first three authors). As of 1994 about 30 cases have been reported from different ethnic groups. The recurrence of the syndrome in sibs, with normal parents, provides evidence for an autosomal recessive disorder (MIM *273750). Diagnostic criteria are: prenatal onset dwarfism, dolicocephaly, joint hypermobility, slenderness of the shafts of the long bones and ribs and foreshortening of lumbar vertebral bodies, abnormal small pelvis, short femoral necks and vertical talus.

We report the occorrence of 3-M syndrome in two new unrelated families; the long follow-up offers important subjects for discussion.

In the first family the proposita at birth showed a Larsen-like phenotype with flat face, very short stature and generalized joint hypermobility. The clinical aspects greatly changed in the years and we were able to give the diagnosis of 3-M syndrome when she was 15 years old.

In the second family the propositus was a boy, showing obvious features since his birth: mild joint hypermobility, omphalocele, bilateral inguinal hernia and macrocephaly. At the age of 7 years the phenotype is still highly significant.

Two subsequent fetuses in the same sibship have been demonstrated affected by 3-M syndrome. The diagnosis was made after the 20th gestation week by means of ecography, and was confirmed after the termination, by both clinical and X-ray examination. Fetal parameters were not significant in the earlier stages of gestation. Of special interest are some-radiological aspects in both parents, whose stature is in the low normal range.

A search in the church registry, going back to 1704, revealed no parental consanguinity, but the same family name is present in both paternal and maternal pedigrees.

In the era of prevention and early diagnosis, mental retardation (MR) represents one of the most important challenges to modern medicine. Much needs to be done to restrict the number of different forms of this vast category of chronic handicaps for which accurate diagnoses are not yet available. The goal is to reduce the social burden and provide better care for patients and families.

The identification and characterisation of the molecular mechanisms which silence the FMR1 gene and which are responsible, in the majority of cases, for the fragile X syndrome (FRAXA) [1-4], the leading known cause of inherited mental retardation, led to the discovery of an extremely important new class of mutations: “dynamic mutations”. These are highly unstable interspersed repeats, located close to or within genes, which show a strong tendency to expand. This discovery has raised the possibility for direct molecular diagnosis of FRAXA and several other diseases based on the same molecular mechanism, including a different form of MR associated with a fragile site in Xq28, named FRAXE [5].

With these tools, we have started to study the structural characteristics and pattern of transmission of these mutations in a population of mentally retarded individuals mainly coming from north-eastern Italy. The aims of our study were (a) to establish the true incidence of FRAXA and FRAXE full mutations as a cause of mental retardation in our population, and (b) to re-evaluate families in which at least one individual had a cytogenetic fra(X) diagnosis, in order to identify mosaicisms and premutations that could not be identified cytogenetically, and to establish the carrier status of relatives of affected individuals.

A 30-year-old man with azoospermia was routinely investigated in the Department of Clinical Genetics at our hospital. His karyotype was analysed after phytohaemagglutinin stimulation of lymphocytes following short-term cultivation of peripheral blood.

Taking his history, it was ascertained that in childhood, Hodgkin's disease had been confirmed histologically. He began treatment at 7 years, receiving several series of combined cytostatic treatment supplemented with X-ray therapy. The patient's history is summarized in table 1.

For the last 14 years, the patient has felt well. He is not taking medication and has no problems with primary disease. He is examined by an oncologist twice a year (including scintigraphy) with negative findings. He has no complications, except for azoospermia.

The karyotype was analysed using standard methods. The chromosomes were stained by Giemsa following G-banding by the trypsin method.

The use of PCR (polymerase chain reaction) is having a profound impact on forensic science and medical diagnostics.

Limited amounts of biological material and/or degraded low molecular weight DNA can be anticipated for forensic identification and often also for diagnostic investigations in fetuses, stillbirths and children with birth defects.

In vitro amplification of DNA, via PCR, represents an important tool for overcoming some of the limitations. Nevertheless, the problem of availability of biological samples as DNA source is often critic.

In order to obtain a useful and rapid procedure of DNA analysis in such difficult situations, we have recently developed a simple DNA extraction method using Chelex 100 and a PCR based amplification technique, and using fingernail as an alternative source of DNA.

Chelex 100 procedures result in denaturated DNA samples, not suitable for RFLPS analysis. Nevertheless we demonstrated that no differences are detectable between the genotypes obtained by PCR amplification using the conventional phenol-chlorophorm or saline extraction and the Chelex based procedure.

A DNA sample of 3-5 micrograms weight was easily obtained from fingernail clippings, and enabled us to perform several tests by using DNA typing methodologies, both for personal identification (in various forensic, medical and social situations) and in disputed parentage.

Our laboratory uses as polymorphic markers a set of several VNTRs (variable number of tandem repeats) and, more recently, a series of unlinked STRs (short tandem repeats). The use of DNA typing with STR loci provides an accurate, highly sensitive and rapid assay for parentage testing, forensic identification and medical applications.

Fragile-X syndrome, which derives its name from the expression of a fragile site (FRAXA) at Xq27.3 associated with the phenotype, has achieved distinction as the most common inherited cause of mental retardation. It is the first disorder shown to be due to dynamic mutation in heritable instable DNA.

In 1991 the mutation responsible for Fragile-X syndrome was delineated as an expansion of the trinucleotide (CGG) sequence within an evolutionarily conserved gene, at the position of the fragile-X site.

The DNA of the promoter in the 5' UTR region of FMR-1 gene becomes abnormally methylated when the CGG sequence exceeds approximately 230 repeats, resulting in the transcriptional suppression of FMR-1. Based on the length of CGG repeat in the FMR-1 gene, the alleles are usually classified as normal, premutation or full mutation. CGG instability correlates with the length of repeats and number of AGGs within the FMR-1 CGG tract. In a minority of cases the Fragile-X syndrome may be due to deletion, or to point mutation in the FMR-1 gene.