CD36 expression and its relationship with obesity in blood cells from people with and without Prader-Willi syndrome

Although Prader-Willi syndrome (PWS) has been linked to the loss of function of imprinted genes in 15q11q13, very little is known about the pathogenesis. Using quantitative real-time PCR, we have confirmed the previous observation of an abnormality of CD36 expression in cells with maternal uniparental disomy 15, obtained from a proband with mosaicism for PWS, by demonstrating reduced expression levels in blood cells from a series of non-mosaic probands with PWS. Furthermore, we have extended these observations to show that CD36 expression in a non-PWS population is inversely correlated with body mass index but that this correlation does not hold in PWS. CD36 which maps to 7q11.2 is the first gene outside the 15q11q13 region whose level of expression appears to be reduced in people with PWS. Low CD36 expression levels in PWS point to an abnormal control of lipid and glucose homeostasis which may explain the insatiable hunger in these patients.     

Clinical features of maternal uniparental disomy 14 in patients with an epimutation and a deletion of the imprinted DLK1/GTL2 gene cluster

Maternal uniparental disomy 14 [upd(14)mat] is associated with a recognizable phenotype that includes pre- and postnatal growth retardation, neonatal hypotonia, feeding problems and precocious puberty. Chromosome 14 contains an imprinted gene cluster, which is regulated by a differentially methylated region (IG-DMR) between DLK1 and GTL2. Here we report on four patients with clinical features of upd(14)mat who show a maternal-only methylation pattern, but biparental inheritance for chromosome 14. In three of the patients loss of paternal methylation appears to be a primary epimutation, whereas the other patient has a paternally derived deletion of -1 Mb that includes the imprinted DLK1-GTL2 gene cluster. These findings demonstrate that the upd(14)mat phenotype is caused by altered expression of genes within this cluster.     

Evidence for imprinting on chromosome 16: the effect of uniparental disomy on the outcome of mosaic trisomy 16 pregnancies

Although a number of infants with maternal uniparental disomy of chromosome 16 (upd(16)mat) have been reported, the evidence for imprinting on chromosome 16 is not yet conclusive. To test the hypothesis that upd(16)mat has a distinct phenotype, which would support the existence of imprinted gene(s) on chromosome 16, statistical analysis was performed on a large series (n = 83) of mosaic trisomy 16 cases with molecular determination of uniparental disomy status. The incidence of upd(16)mat was 40%, which is consistent with the expected one third from random chromosome loss during trisomy rescue (P = 0.262). In pairwise comparisons, upd(16)mat was found to be associated with fetal growth restriction (P = 0.029) and with increased risk of major malformation (RR = 1.43; P = 0.053). Regression modeling showed that the effect of upd(16)mat on fetal/neonatal weight and malformation is independent of the degree of trisomy detected in the fetus. Regression modeling to control for the degree of trisomy detected in the placenta was not possible due to limited sample size. We conclude that upd(16)mat is associated with more severe growth restriction, and possibly, with higher risk of malformation. Our hypothesis is that imprinted gene(s) exist on chromosome 16 and that abnormal expression of these gene(s) in upd(16)mat cells during development results in decreased cell proliferation. Although we do not advocate prenatal testing for upd(16), studies on the long-term outcome of upd(16)mat neonates is necessary for counseling purposes.     

Maternal age effect on the development of Prader-Willi syndrome resulting from upd(15)mat through meiosis 1 errors

Prader-Willi syndrome (PWS) is primarily caused by deletions involving the paternally derived imprinted region at chromosome 15q11.2-q13 and maternal uniparental disomy 15 (upd(15)mat). The underlying mechanisms for upd(15)mat include trisomy rescue (TR), gamete complementation (GC), monosomy rescue and post-fertilization mitotic error, and TR/GC is mediated by non-disjunction at maternal meiosis 1 (M1) or meiosis 2 (M2). Of these factors involved in the development of upd(15)mat, M1 non-disjunction is a maternal age-dependent phenomenon. We studied 117 Japanese patients with PWS and identified deletions in 84 patients (Deletion group) and TR/GC type upd(15)mat through M1 non-disjunction in 15 patients (TR/GC (M1) group), together with other types of abnormalities. Maternal age was significantly higher in TR/GC (M1) group than in Deletion group (median (range), 37 (35-45) versus 30 (19-42); P=1.0 × 10(-7)). Furthermore, delayed childbearing age became obvious since the year 2003 in Japan, and relative frequency of TR/GC (M1) group was significantly larger in patients born since the year 2003 than in those born until the year 2002. The results imply that the advanced maternal age at childbirth is a predisposing factor for the development of upd(15)mat because of increased M1 errors.     

Is there a higher incidence of maternal uniparental disomy 14 [upd(14)mat]? Detection of 10 new patients by methylation-specific PCR

Maternal uniparental disomy for chromosome 14 [upd(14)mat] is associated with a characteristic phenotype including pre- and postnatal growth retardation, muscular hypotonia, feeding problems, motor delay, small hands and feet, precocious puberty and truncal obesity. Patients with upd(14)mat show features overlapping with Prader-Willi syndrome (PWS) and are probably underdiagnosed. Maternal upd(14) is frequently described in carriers of a Robertsonian translocation involving chromosome 14, but is also found in patients with a normal karyotype. Based on the above mentioned criteria we have identified six patients with upd(14)mat including two patients with a normal karyotype, one patient with a de novo Robertsonian translocation (14;21), one patient with a familial Robertsonian translocation (13;14) and two patients with a marker chromosome. In addition, we analyzed a cohort of 33 patients with low birth weight, feeding difficulties and consecutive obesity in whom PWS had been excluded by methylation analysis of SNRPN. In four of these patients (12%) we detected upd(14)mat. For rapid testing of upd(14)mat we analyzed the methylation status of the imprinted MEG3 locus. In conclusion, we recommend considering upd(14)mat in patients with low birth weight, growth retardation, neonatal feeding problems, muscular hypotonia, motor delay, precocious puberty and truncal obesity as well as in patients with a PWS like phenotype presenting with low birth weight, feeding difficulties and obesity.     

Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.      

Prader-Willi syndrome in a child with mosaic trisomy 15 and mosaic triplo-X: a molecular analysis.

A 3.3 year old girl with Prader-Willi syndrome (PWS) and mosaicism for two aneuploidies, 47,XXX and 47,XX,+15, is presented. The triplo-X cell line was found in white blood cells and fibroblasts, the trisomy 15 cell line in 50% of the fibroblasts. Using methylation studies of the PWS critical region and by polymorphic microsatellite analysis, the existence of uniparental maternal heterodisomy for chromosome 15 was shown in white blood cells. This provided a molecular explanation for the PWS in this child. In fibrolasts, an additional paternal allele was detected for markers on chromosome 15, which is in agreement with the presence of mosaicism for trisomy 15 in these cells. This example provides direct evidence for trisomic rescue by reduction to disomy as a possible basis for PWS. Whereas the trisomy 15 was caused by a maternal meiosis I error, the triplo-X resulted from a postzygotic gain of a maternal X chromosome, as shown by the finding of two identical maternal X chromosomes in the 47,XXX cell line. Because the triplo-X and the trisomy 15 were present in different cell lines, gain of an X chromosome occurred either in the same cell division as the trisomy 15 rescue or shortly before or after.     

Trisomy 7 mosaicism, maternal uniparental heterodisomy 7 and Hirschsprung's disease in a child with Silver-Russell syndrome

Prenatal trisomy 7 is usually a cell culture artifact in amniocytes with normal diploid karyotype at birth and normal fetal outcome. In the same way, true prenatal trisomy 7 mosaicism usually results in a normal child except when trisomic cells persist after birth or when trisomy rescue leads to maternal uniparental disomy, which is responsible for 5.5-7% of patients with Silver-Russell syndrome (SRS). We report here on the unusual association of SRS and Hirschsprung's disease (HSCR) in a patient with maternal uniparental heterodisomy 7 and trisomy 7 mosaicism in intestine and skin fibroblasts. HSCR may be fortuitous given its frequency, multifactorial inheritance and genetic heterogeneity. However, the presence of the trisomy 7 mosaicism in intestine as well as in skin fibroblasts suggests that SRS and HSCR might possibly be related. Such an association might result from either an increased dosage of a nonimprinted gene due to trisomy 7 mosaicism in skin fibroblasts (leading to SRS) and in intestine (leading to HSCR), or from an overexpression, through genomic imprinting, of maternally expressed imprinted allele(s) in skin fibroblasts and intestine or from a combination of trisomy 7 mosaicism and genomic imprinting. This report suggests that the SRS phenotype observed in maternal uniparental disomy 7 (mUPD(7)) patients might also result from an undetected low level of trisomy 7 mosaicism. In order to validate this hypothesis, we propose to perform a conventional and molecular cytogenetic analysis in different tissues every time mUPD7 is displayed.     

Maternal uniparental disomy of chromosome 16 [upd(16)mat]: clinical features are rather caused by (hidden) trisomy 16 mosaicism than by upd(16)mat itself

Maternal uniparental disomy of chromosome 16 [upd(16)mat] as the result of trisomy 16 is one of the most frequently reported uniparental disomies in humans, but a consistent phenotype is not obvious. Particularly, it is difficult to discriminate between features resulting from upd(16)mat and mosaic trisomy 16. By evaluating literature data (n = 74) and three own cases we aimed to determine whether the clinical features are due to upd(16)mat or to trisomy 16 mosaicism. While in single cases the clinical symptoms were caused by homozygosity of autosomal recessive mutations on chromosome 16, it turned out that clinical features in upd(16)mat are caused by (hidden) trisomy 16 mosaicism and a specific chromosome 16‐associated imprinting disorder does not exist. In trisomy 16/upd(16)mat pregnancies, the management should be based on the ultrasound results and on the clinical course of the pregnancy. In fact, mosaic trisomy 16 pregnancies require a close monitoring because of the higher risk for hypertensive disorders. Postnatal testing for upd(16)mat should be considered in case of homozygosity for an autosomal‐recessive mutation, in individuals carrying chromosome 16 aberrations and in phenotypes comprising features of the trisomy 16/upd(16)mat spectrum. Finally, upd(16)mat probably represents a bioindicator for a hidden trisomy 16 mosaicism.     

Maternal uniparental disomy of chromosome 21 in a normal child

Maternal uniparental disomy of chromosome 21 [upd(21)mat] was found previously in a normal female and in 2 cases of early embryonic failure. We present a phenotypically normal child with upd(21)mat due to a de novo der(21;21)(q10;10). This finding suggests that chromosome 21 is not imprinted in the maternal germline.     

The necdin gene is deleted in Prader-Willi syndrome and is imprinted in human and mouse

Human chromosome 15q11-q13 contains genes that are imprinted and expressed from only one parental allele. Prader-Willi syndrome (PWS) is due to the loss of expression of one or more paternally expressed genes on proximal human chromosome 15q, most often by deletion or maternal uniparental disomy. Several candidate genes and a putative imprinting centre have been identified in the deletion region. We report that the human necdin-encoding gene (NDN) is within the centromeric portion of the PWS deletion region, between the two imprinted genes ZNF127 and SNRPN. Murine necdin is a nuclear protein expressed exclusively in differentiated neurons in the brain. Necdin is postulated to govern the permanent arrest of cell growth of post-mitotic neurons during murine nervous system development. We have localized the mouse locus Ndn encoding necdin to chromosome 7 in a region of conserved synteny with human chromosome 15q11-q13, by genetic mapping in an interspecific backcross panel. Furthermore, we demonstrate that expression of Ndn is limited to the paternal allele in RNA from newborn mouse brain. Expression of NDN is detected in many human tissues, with highest levels of expression in brain and placenta. NDN is expressed exclusively from the paternally inherited allele in human fibroblasts. Loss of necdin gene expression may contribute to the disorder of brain development in individuals with PWS.      

Evidence from skewed X inactivation for trisomy mosaicism in Silver-Russell syndrome

The finding of maternal uniparental disomy for chromosome 7 (matUPD7) in approximately 7% of Silver-Russell syndrome (SRS) cases has lead to the assumption that imprinted gene(s) on chromosome 7 are responsible for at least some cases. However, the observation in a familial case that both maternal and paternal inheritance of proximal 7p results in an SRS-like phenotype suggests that the causative genes may not be imprinted, and that an extra copy of genes within this region cause SRS. As all cases of complete matUPD7 could have arisen by trisomy rescue, it is possible that undetected trisomy 7 mosaicism contributes towards the phenotype of SRS, and that the matUPD7 seen in some cases is a consequence of trisomy rescue. Previous studies in cases of trisomy rescue for a number of autosomes have shown a strong association with skewed X inactivation in diploid tissues. Thus, we hypothesised that if trisomy mosaicism was involved in SRS, the frequency of skewed X inactivation should be increased in a population of non-matUPD7 SRS patients. Consistent with this hypothesis, results showed a significant increase in the frequency of completely skewed X inactivation in SRS patients (three of 29) when compared to controls (three of 270), suggesting the possible presence of undetected trisomy 7 in SRS patients and/or their placentas.     

Novel deletions affecting the MEG3-DMR provide further evidence for a hierarchical regulation of imprinting in 14q32

The imprinted region on chromosome 14q32 harbors several maternally or paternally expressed genes as well as two DMRs (differentially methylated regions), the IG-DMR and the MEG3-DMR, which both act as imprinting control centers. Genetic aberrations affecting the imprinted gene cluster in 14q32 result in distinct phenotypes, known as maternal or paternal uniparental disomy 14 phenotypes (upd(14)mat, upd(14)pat). In both syndromes, three types of molecular alterations have been reported: uniparental disomy 14, deletions and epimutations. In contrast to uniparental disomy and epimutations, deletions affecting regulatory elements in 14q32 are associated with a high-recurrence risk. Based on two single deletion cases a functional hierarchy of the IG-DMR as a regulator for the methylation of the MEG3-DMR has been proposed. We have identified two novel deletions of maternal origin spanning the MEG3-DMR, but not the IG-DMR in patients with upd(14)pat syndrome, one de novo deletion of 165 kb and another deletion of 5.8 kb in two siblings. The 5.8 kb deletion was inherited from the phenotypically normal mother, who carries the deletion in a mosaic state on her paternal chromosome 14. The methylation at both DMRs was investigated by quantitative next generation bisulfite sequencing and revealed normal methylation patterns at the IG-DMR in all patients with the exception of certain CpG dinucleotides. Thus, we could confirm that deletions of the MEG3-DMR does not generally influence the methylation pattern of the IG-DMR, which strengthens the hypothesis of a hierarchical structure and distinct functional properties of the two DMRs.     

The phenomenology and diagnosis of psychiatric illness in people with Prader-Willi syndrome

BACKGROUND: Psychotic illness is strongly associated with the maternal uniparental disomy (mUPD) genetic subtype of Pradertwo-hit' hypothesis, involving imprinted genes on chromosome 15, for the development of affective psychosis in people with PWS, regardless of genetic subtype.     

Silver-Russell syndrome due to maternal uniparental disomy 7 and a familial reciprocal translocation t(7;13)

Behnecke A, Hinderhofer K, Jauch A, Janssen JWG, Moog U. Silver-Russell syndrome due to maternal uniparental disomy 7 and a familial reciprocal translocation t(7;13). Silver-Russell syndrome (SRS) is a genetically heterogeneous disorder characterized by intrauterine and postnatal growth retardation, typical facial features and a spectrum of additional features including body and limb asymmetry and clinodactyly. Maternal uniparental disomy for chromosome 7 (upd(7)mat) was shown to occur in 5-10% of patients with SRS. Maternal UPD7 is clinically often associated with mild SRS. Parents of an affected child are given a negligible recurrence risk as all reported cases with upd(7)mat have been sporadic so far. In general, chromosomal rearrangements-like translocations increase the likelihood of uniparental disomy (UPD) for the chromosomes involved. However, SRS as the result of a upd(7)mat in association with an inherited chromosomal translocation involving chromosome 7 has only been reported once before. Here, we describe the second case of SRS with upd(7)mat due to a familial reciprocal translocation t(7;13). This emphasizes the importance of chromosome analysis in SRS patients with upd(7)mat to rule out chromosomal rearrangements despite their rare occurrence as they are of great relevance for genetic counseling of SRS families.     

A rapid, PCR based test for differential molecular diagnosis of Prader-Willi and Angelman syndromes.

Approximately 98% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11-13, uniparental disomy for chromosomes 15 (UPD15), or mutations affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of mutation depends upon the parent of origin. Both disorders are characterised at the molecular level by abnormal methylation of imprinted genes at 15q11-q13 including the small nuclear ribonucleoprotein N gene (SNRPN). Current diagnostic strategies include high resolution cytogenetics, fluorescence in situ hybridisation (FISH), Southern blot hybridisation, or microsatellite typing. We have developed a novel and rapid diagnostic test for PWS and AS based on differential digestion of expressed (paternally imprinted) SNRPN sequences by the methylation sensitive endonuclease NotI or repressed (maternally imprinted) SNRPN sequences by the methylation requiring nuclease McrBC, followed by PCR amplification of the SNRPN promoter. We have evaluated this test by blinded analysis of 60 characterised DNA samples (20 PWS, 20 AS, and 20 unaffected controls). SNRPN sequences could not be amplified from PWS patient DNA which had been digested with McrBC, nor from AS patient DNA which had been digested with NotI. We were able to make a correct diagnosis of PWS, AS, or unaffected in all 60 samples tested. This novel test is rapid and has a high specificity and sensitivity for deletion and UPD15 cases. These features make this new test suitable as the initial step in a molecular diagnostic strategy for PWS/AS.     

A paternal deletion of MKRN3, MAGEL2 and NDN does not result in Prader�CWilli syndrome

The Prader–Willi syndrome (PWS) is caused by a 5–6 Mbp de novo deletion on the paternal chromosome 15, maternal uniparental disomy 15 or an imprinting defect. All three lesions lead to the lack of expression of imprinted genes that are active on the paternal chromosome only: MKRN3, MAGEL2, NDN, C15orf2, SNURF-SNRPN and more than 70 C/D box snoRNA genes (SNORDs). The contribution to PWS of any of these genes is unknown, because no single gene mutation has been described so far. We report on two patients with PWS who have an atypical deletion on the paternal chromosome that does not include MKRN3, MAGEL2 and NDN. In one of these patients, NDN has a normal DNA methylation pattern and is expressed. In another patient, the paternal alleles of these genes are deleted as the result of an unbalanced translocation 45,X,der(X)t(X;15)(q28;q11.2). This patient is obese and mentally retarded, but does not have PWS. We conclude that a deficiency of MKRN3, MAGEL2 and NDN is not sufficient to cause PWS.     

Segmental maternal uniparental disomy 7q associated with DLK1/GTL2 (14q32) hypomethylation

Aberrant methylation at different imprinted loci has been reported for several congenital imprinting disorders, that is, Silver-Russell syndrome (SRS), but the coincidental occurrence of aberrant methylation and uniparental disomy (UPD) has not yet been described. We report on a patient initially diagnosed with SRS carrying a segmental maternal UPD of chromosome 7 [upd(7q)mat]. By further screening the patient's DNA for methylation defects on other chromosomes we identified a hypomethylation of the paternally methylated DLK1/GTL2 locus in 14q32, an epigenotype typically associated with the upd(14)mat phenotype. Detailed clinical analysis confirmed the molecular finding in the patient indicating that the 14q32 epimutation was clinically preponderant. The parallel occurrence of upd(7q)mat and a DLK1/GTL2 hypomethylation in the same patient is a unique finding. Indeed, both disturbances might have occurred coincidentally, but it can also be hypothesized that the upd(7q)mat as the initial genomic mutation represents a trans-acting mutation causing an aberrant methylation in 14q32.     

Epimutation at human chromosome 14q32.2 in a boy with a upd(14)mat-like clinical phenotype

Recently, three reports described deletions and epimutations affecting the imprinted region at chromosome 14q32.2 in individuals with a phenotype typical for maternal uniparental disomy of chromosome 14 [upd(14)mat]. In this study, we describe another patient with upd(14)mat-like phenotype including low birth weight, neonatal feeding problems, muscular hypotonia, motor and developmental delay, small hands and feet, and truncal obesity. Conventional cytogenetic analyses, fluorescence in situ hybridization subtelomere screening, multiplex ligation-dependent probe amplification analysis of common microdeletion and microduplication syndromes, and methylation analysis of SNRPN all gave normal results. Methylation analysis at 14q32.2 revealed a gross hypomethylation of the differentially methylated regions (intergenic DMR and MEG3 -DMR). Further molecular studies excluded full or segmental upd(14)mat as well as a microdeletion within this region. Evidently, the upd(14)mat-like clinical phenotype is caused by an epimutation at 14q32.2. The clinical and molecular features of this novel case are discussed with respect to the recently published cases.     

Epimutation (hypomethylation) affecting the chromosome 14q32.2 imprinted region in a girl with upd(14)mat-like phenotype

Maternal uniparental disomy for chromosome 14 (upd(14)mat) causes clinically discernible features such as pre- and/or postnatal growth failure, hypotonia, obesity, small hands, and early onset of puberty. The monoallelic expression patterns at the 14q32.2 imprinted region are tightly related to methylation status of the DLK1-MEG3 intergenic differential methylation region (DMR) and the MEG3-DMR that are severely hypermethylated after paternal transmission and grossly hypomethylated after maternal transmission. We examined this imprinted region in a 2 2/12-year-old Japanese patient who was born with a normal birth size (length, +0.2 SD; weight, -0.5 SD) and showed postnatal growth failure (height, -3.1 SD; weight, -3.4 SD), hypotonia, frontal bossing, micrognathia, and small hands. Methylation analysis, genotyping analysis, and deletion analysis were performed with blood samples of the patient and the parents, showing that the DMRs of this patient were grossly hypomethylated in the absence of upd(14)mat and deletion of the DMRs. The results indicate the occurrence of an epimutation (hypomethylation) affecting the normally methylated DMRs of paternal origin, and imply that epimutations should be examined in patients with upd(14)mat-like phenotype.