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. 1989 Nov 16;342(6247):281-5.
doi: 10.1038/342281a0.

Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome

R D Nicholls  1 , J H Knoll, M G Butler, S Karam, M Lalande
Affiliations

Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome

R D Nicholls et al. Nature. .

Abstract

Prader-Willi syndrome (PWS) is the most common form of dysmorphic genetic obesity associated with mental retardation. About 60% of cases have a cytological deletion of chromosome 15q11q13 (refs 2, 3). These deletions occur de novo exclusively on the paternal chromosome. By contrast, Angelman syndrome (AS) is a very different clinical disorder and is also associated with deletions of region 15q11q13 (refs 6-8), indistinguishable from those in PWS except that they occur de novo on the maternal chromosome. The parental origin of the affected chromosomes 15 in these disorders could, therefore, be a contributory factor in determining their clinical phenotypes. We have now used cloned DNA markers specific for the 15q11q13 subregion to determine the parental origin of chromosome 15 in PWS individuals not having cytogenetic deletions; these individuals account for almost all of the remaining 40% of PWS cases. Probands in two families displayed maternal uniparental disomy for chromosome 15q11q13. This is the first demonstration that maternal heterodisomy--the presence of two different chromosome 15s derived from the mother--can be associated with a human genetic disease. The absence of a paternal contribution of genes in region 15q11q13, as found in PWS deletion cases, rather than a mutation in a specific gene(s) in this region may result in expression of the clinical phenotype. Thus, we conclude that a gene or genes in region 15q11q13 must be inherited from each parent for normal human development.

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Figures

FIG. 1
FIG. 1
Maternal uniparental disomy in a PWS patient with a balanced Robertsonian translocation. a, Normal chromosome 15 and translocated homologue from patient PWS1 (left) and his mother M1 (right). Other unaffected maternal relatives, including the half-sister of PWS1 carry the same balanced chromosome 13q15q robertsonian translocation, b, Maternal origin of the two chromosomes 15 in patient PWS1. An 8.2-kb TaqI allele of probe 3–21 (D15S10) is shared by PWS1 (lane 3), his mother M1 (lane 2) and half-sister (lane 1), whereas an 8.9-kb band is shared by PWS1 and M1. PWS1 does not inherit a 9.0-kb paternal allele (F1, lane 4). Mixing experiments (lane 5, PWS1 and F1; lane 6, M1 and F1) show the identity of the three alleles in this new RFLP system, c, Presence of two maternal alleles only for CMW-1 (D15S24) in PWS1. PWS1 (lane 2) shares two TaqI restriction fragments, of 2.1 and 2.2 kb. with M1 (lane 3) but has neither of the 1.5- or 1.8-kb paternal alleles (F1, lane 1). The half-sister (lane 4) displays the maternal 2.1-kb and a 2.15-kb allele. The band at 1.7 kb is constant, C. d, Exclusion of non-paternity for PWS1. PvuII-digested DNA was hybridized to the α-globin 3’HVR marker (D16S85), one of the most highly polymorphic markers in the human genome. PWS1 (lane 2) shows mendelian inheritance of one paternal 3.9-kb allele (lane 1) and one maternal 4.3-kb allele (lane 3). The half-sister inherits her mother’s 5.3-kb allele (result not shown). METHODS. DNA was isolated from blood and/or cell lines from each individual as described previously. Hybridizations were carried out as previously described High-resolution chromosome analyses from peripheral blood lymphocytes were performed by Giemsa-trypsin banding.
FIG. 2
FIG. 2
Maternal uniparental disomy in a PWS patient with apparently normal chromosomes 15. a, Two pairs of intact chromosomes 15 of PWS2. Previous reports have indicated these chromosomes as being intact’3 or deleted 15q (ref. 14); in the deletion report a polymorphic shortening of 15q11 (ref. 7) was probably detected, because we show here at a higher level of resolution that the chromosomes are intact. b, Maternal origin of the two chromosomes 15 in PWS2. DNA was digested with SeaI. PWS2 (lane 1) inherits a maternal 6.3-kb allele (lane 3) but no paternal 6.5-kb allele (lane 2) for probe 34 (D15S9). Each individual is heterozygous for IR10-1 (D15S12; 16- and 12.5-kb alleles), which is from the same 15q11q13 region as probe 34 (ref. 10). The band at 10 kb is constant. C. for probe 34 in all individuals c. PWS2 inherits maternal alleles only for probe IR39d (D15S18). DNA was digested with SacI and hybridized with the 15q11q13-specific probe IR39d. PWS2 (lane 1) inherits his mother’s (lane 3) 14-kb alleles and not his father’s (lane 2) 8.5-kb alleles. d, Two copies of the maternal allele for probe 34 in PWS2. HindIII-digested DNAs from PWS2 (lane 2), mother M2 (lane 3), father F2 (lane 4) and a PWS patient displaying both a cytological and molecular deletion of 15q11q13 (ref. 5; lane 1. HS2) were hybridized simultaneously with probes 34 and H2-26 (D13S28) . Probe H2-26 from chromosome 13 serves as a reference for quantitation of copy number. Probe 34 shows a reduced hybridization intensity relative to H2-26 only in HS2, which is consistent with a deletion of 15q11q13 in this individual. No reduction in probe 34 hybridization intensity is observed in PWS2 or his parents. To quantitate the intensity of the hybridization bands, the autoradiogram was scanned with an LKB Ultrascan XL laser densitometer (Pharmacia LKB Biotechnology). The area ratio of the probe 34 to probe H2-26 peaks was calculated for each lane. The number of copies of probe 34 per genome was obtained by setting the ratio in lane 3 (M2, mother) to 2 and normalizing the values in the other lanes to this value. The number of copies of probe 34 per genome determined by this method were 1.0 in lane 1 (the HS2 deletion), 1.8 in lane 2 (PWS2) and 2.2 in lane 4 (F2, father). e, Exclusion of non-paternity for PWS2. One maternal 5.2-kb (lane 2) and one paternal 2.9-kb (lane 3) PvuII allele are inherited by PWS2 (lane 1) for the 3’HVR. METHODS. DNA and cytogenetic studies were as for Fig. 1. Probe IR39d is a subclone of IR39 (ref. 10), which lacks repetitive sequences. PWS2 has been described before as patient number 5 (ref. 14) and patient number 31 (ref. 13).
FIG. 3
FIG. 3
PFGE provides evidence for the lack of a submicroscopic deletion in PWS patients with uniparental disomy. NotI fragments are shown for probes IR4-3R (D15S11) (left) and 34 (right) in PWS2 (lane 3), M2 (lane 2) and F2 (lane 1). Multiple bands are indicative of partial cleavage at restriction sites when CpG is methylated and a similar pattern of multiple bands has been observed in other normal controls (results not shown). PWS2 shares a normal 2,500-kb band with his mother, although in the latter this results from partial digestion at a NotI site in this region of the genome. The same 2,500-kb band has been detected in other normal individuals. The larger three fragments are common to probes IR4-3R, 189-1 (results not shown) and 34, showing that these loci are closely linked in region 15q11q13. These probes and a 1,300-kb fragment detected with probes 3-21 and IR10-1 (unpublished data) are deleted in all PWS and AS patients with cytological deletions. Additional PFGE analysis (results not shown) also indicates that PWS2 displays PFGE restriction fragments of normal size for IR10-1 in a NotI digest (1,300 kb) and for IR39d in a BssHII digest (250 kb). PWS1 also seems to be intact for the probe 34 and 3-21 loci because the restriction fragments detected using NotI and BssHII are unaltered compared with those of normal individuals. METHODS. PFGE analyses were performed as described previously using a modification of techniques for DNA preparation and digestion in agarose blocks. Electrophoresis was performed in 0.8% agarose in TBE (89 mM Tris buffer, pH 8.0, 89 mM boric acid, 2 mM EDTA) at 22 °C and was divided into three 48-h intervals during which the forward polarity pulse durations were varied from 75-600 s, 600-2,500 s and 2,500-3,000 s and field intensities were set at +2, +1.7 and +1.3 V cm−1, respectively. The ratios of the duration and intensity of the inverse polarity pulse relative to the forward were 0.4 and 0.5, respectively.
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