Prader-Willi syndrome

Twenty-seven subjects with the Prader-Willi syndrome (PWS) were studied. Sixteen (59%) had a cytogenetic deletion involving chromosome lSqll-13. Nine were non-deletional and two patients had structural rearrangements of chromosome 15: 47,XY, + del(15)(pter-.q12), var(15)(pll) and 45,XX,t(14ql5q). At the DNA level, a greater proportion of patients (74%) showed loss ofone chromosome 15ql1-13 allele using a combination of densitometry and RFLP analysis. Deletion sizes were variable with 13 of 20 detectable both cytogenetically and with probe pML34 (D15S9). The remaining seven had microdeletions at the pML34 locus. Heterogeneity was further seen in three subjects who had cytogenetic deletions but normal DNA studies. In one patient there was evidence of a duplication at the pML34 locus. A new molecular rearrangement was identified with probe p3-21 (DISS1O) in two patients Department of Molecular Genetics, Royal Prince Alfred Hospital, Camperdown, NSW 2050, and Department of Medicine, University of Sydney, NSW 2006, Australia. R J Trent, F Volpato, R Lindeman, M-K Wong Cytogenetics Unit, Oliver Latham Laboratory, Department of Health, North Ryde, NSW, Australia. A Smith Endocrinology and Diabetes Department, Royal Children's Hospital, Melbourne, Victoria, Australia. G Warne Department of Medical Genetics, Adelaide Children's Hospital, Adelaide, SA, Australia. E Haan Correspondence to Professor Trent. and their mothers. Fifteen family studies were performed. In all 10 families where there was a molecular deletion, this was shown to have arisen de novo. DNA mapping confirmed that the paternal 15q allele was lost in three patients with PWS. The Prader-Willi syndrome (PWS) is a rare multisystem disorder. Clinical features include hypotonia, hypogonadism, hypopigmentation, mental retardation, and obesity. The latter two are particularly significant in the natural history of this disorder. Mental retardation is usually not severe (mean IQ 60 with a range of 20 to 80), but obesity is a major clinical and psychosocial problem. ' The newborn with PWS is floppy, sucks poorly, and fails to thrive. However, appetite increases rapidly at approximately 3 to 4 years of age. By the teens, excessive eating produces gross obesity. The diagnosis of PWS may be suggested in the neonatal period but cannot be made conclusively until early childhood. Thus, there is delay in initiating appropriate management.2 The aetiology of the PWS is unknown. Almost invariably only one member of the family is affected. However, some cases of familial recurrence have been described.3 Cytogenetic deletions involving the long arm of chromosome 15 were first described in 1981 and approximately half the patients with PWS have an interstitial deletion of 15ql 1-13.4 Five percent of patients have duplications or translocations while the remainder have normal karyotypes.56 DNA probes specific for the 15qll-13 region are now available.78 These have been used to characterise further the putative PWS locus called PWCR (Prader-Willi chromosome region). Recent results using these probes have provided further evidence that genomic imprinting secondary to loss of the paternal 15q locus is implicated in the pathogenesis of PWS.9 The present study describes DNA and Received for publication 17 September 1990. Revised version accepted for publication 19 February 1991. 649 Trent, Volpato, Smith, Lindeman, Wong, Warne, Haan cytogenetic findings in 27 patients with PWS, which is the largest cohort investigated in this way. Patients and methods PATIENTS This group comprised 27 patients in whom clinical histories and phenotypes were entirely consistent with PWS. These included early stage I phase of neonatal hypotonia and failure to thrive, hypogenitalism in the male, followed by hyperphagia in early childhood, mental retardation, short stature, typical facies, small hands and feet, and a behavioural disorder. Twenty-four patients had been followed over a period of time. '0 They were recently reviewed and three additional patients were added to this study. DNA STUDIES Detection of a new DNA restriction map DNA was isolated from buffy coats obtained from 10 ml of blood. Aliquots of DNA (10 jig) were digested with restriction endonucleases BamHI, EcoRI, HindIII, and BglII. Additional restriction enzymes were used where indicated below. Digested DNA was electrophoresed on 0-8% agarose gels and then transferred to nylon membranes by Southern blotting. Membranes were hybridised to 32P labelled DNA probes and washed using stringent conditions (0 1 x SSC at 65°C for 60 minutes). "I PWCR specific DNA probes included pML34 (D1SS9), p3-21 (D15S10) (donated by the late Professor S Latt, Boston), and 85(2 4) (DISS53) (donated by Professor F-T Kao, Denver). DNA polymorphisms identifiable with probes pML34 (ScaI, allele frequencies: 6-5 kb 0-72; 6-3 kb 0-28), p3-21 (TaqI, allele frequencies: 9-0kb 0-83; 8-2kb 0-17), and a multiallelic (CMW1) probe (EcoRI) were sought. "'14 The CMW1 probe was provided by Dr D Ledbetter (Houston). The 3'oa HVR probe (a gift from Dr D R Higgs, Oxford) was used for paternity testing. '5 For the latter, filters were hybridised at 55°C and washed in 5 x SSC. Detection of loss of one PWCR specific allele Membranes containing DNA digested with the restriction endonuclease HindIII were hybridised simultaneously to three DNA probes, pML34, OP1, and a j3 globin gene probe (Pst 0) located on chromosome 11p15 (Pst i was donated by Professor T Maniatis, Boston). Probe OPI is a 2i4kb HincII insert in plasmid pBluescript. It was isolated from an EMBL3 phage library prepared from normal DNA partially digested with MboI. The library was screened with probe p3-21 and so probe OP1 is located within 20 to 25 kb of p3-21 (Lindeman et al, in preparation). Band sizes obtained for the above HindIII digests were 6-0 kb (pML34), 3 0 kb (OPI), and 7-8 kb (3 globin). Autoradiographs were scanned in an LKB 2202 Ultroscan Laser Densitometer. Normal DNA samples were included in all membranes to control for hybridisation and washing conditions. Autoradiograph band intensities and densitometric traces of bands identified by probes pML34 and OP1 were scored by three observers (RJT, RL, M-KW) as representing normal, reduced, or increased intensities compared to the control , globin. Family studies Both parents of 15 patients were studied. KARYOTYPE ANALYSIS Peripheral blood cultures were set up for high resolution banding after the method of Yunis'6; 0-3 ml of blood were added to 4-5 ml of medium (either Ham's F10 or Iscoves, Flow Laboratory). After 48 hours, the cultures were blocked with 0 05 ml of a methotrexate solution (10-5 mol/l) and reincubated for 17 hours ovemight at 37°C. The following morning the methotrexate was washed out and the block released with fresh medium to which 0-05 ml (10-3 mol/l) thymidine were added. The cultures were then incubated at 37°C for a further six hours. Harvesting was performed with colcemid (final concentration 1 jg/ml) for 15 minutes, hypotonic 0 075 mol/l KC1 for 10 minutes, and fixed for one hour with 3:1 methanol:acetic acid. Slides were placed in a 37°C incubator for two to three days before GTG banding. '7 A total of 15 to 20 cells was examined in each case. On scanning under the microscope, metaphase spreads were chosen for the criteria of a band stage of resolution greater than 750 bands plus readily distinguishable chromosomes 15 which were reasonably straight and not involved in a crossover. Chromosome 15 was scored as deleted when the q12 band was not seen and was not based on the absolute length of the ql 1-13 region (fig 1). Other banding techniques were used as indicated.