Abstract Background Beckwith–Wiedemann syndrome (BWS) is a rare overgrowth syndrome characterized by congenital malformations and predisposition to embryonic tumors. Loss of methylation of imprinting center 2 (IC2) is the most frequent alteration and rarely associated with tumors compared to paternal uniparental disomy of chromosome 11 (UPD(11)pat) and gain of methylation of imprinting center 1. Methods Our study aimed to describe the clinical, histopathological and genetic characteristics of two patients and establish genotype‐phenotype correlations. The clinical diagnosis was based on the criteria defined by the international expert consensus of BWS. Molecular study of 11p15.5 methylation status was assessed using methylation‐specific‐multiplex ligation probe amplification (MS‐MLPA). Results Patients were aged 12 months and 3 months and fulfilled the clinical score of BWS. MS‐MLPA showed molecular alterations consisting of loss of methylation in IC2 (IC2‐LOM) at the maternal allele for one patient and a mosaic UPD(11)pat for the second patient in whom follow‐up at 6months revealed adrenocortical carcinoma (ACC) with low grade of malignancy. Molecular subtypes guide the follow‐up and tumor surveillance, our major concern. Conclusion We have to take into account the psychological impact of a possible tumor whatever the underlying mechanism is. Nevertheless, the tumor risk remains high for UPD(11)pat. Our study extended the phenotype of BWS with absence of macrosomia in Tunisian patients, contrasting with literature, and added a supplementary case of ACC in the tumor spectrum of BWS patients with UPD(11)pat.

Angelman syndrome (AS) is a rare neurogenetic imprinting disorder caused by the loss of function of UBE3A . In ~3–5% of AS patients, the disease is due to an imprinting defect (ID). These patients lack DNA methylation of the maternal SNRPN promotor so that a large SNRPN sense/ UBE3A antisense transcript ( SNHG14 ) is expressed, which silences UBE3A . In very rare cases, the ID is caused by a deletion of the AS imprinting centre (AS-IC). To search for sequence alterations, we sequenced this region in 168 patients without an AS-IC deletion, but did not detect any sequence alteration. However, the AS-IC harbours six common variants (five single nucleotide variants and one TATG insertion/deletion variant), which constitute five common haplotypes. To determine if any of these haplotypes is associated with an increased risk for an ID, we investigated 119 informative AS-ID trios with the transmission disequilibrium test, which is a family-based association test that measures the over-transmission of an allele or haplotype from heterozygous parents to affected offspring. By this we observed maternal over-transmission of haplotype H-AS3 ( p = 0.0073). Interestingly, H-AS3 is the only haplotype that includes the TATG deletion allele. We conclude that this haplotype and possibly the TATG deletion, which removes a SOX2 binding site, increases the risk for a maternal ID and AS. Our data strengthen the notion that the AS-IC is important for establishing and/or maintaining DNA methylation at the SNRPN promotor and show that common genetic variation can affect genomic imprinting.

Pseudohypoparathyroidism 1A (PHP1A) consists of signs of Albright hereditary osteodystrophy (AHO) and multiple, variable hormonal resistances. Elevated PTH levels are the biochemical hallmark of the disease. Short stature in PHP1A may be caused by a form of accelerated chondrocyte differentiation leading to premature growth plate closure, possibly in combination with GH deficiency in some patients. Treatment of short stature with recombinant growth hormone (rhGH) in pediatric patients may improve final height if started during childhood. The 10 11/12-year-old boy with clinical signs of AHO presented for evaluation of short stature [height standard deviation score (SDS) -2.72]. Clinically his mother was affected by AHO as well. A heterozygous mutation c.505G>A (p.E169K) in exon 6 of the GNAS gene confirmed a diagnosis of PHP1A in the boy. However, hormonal assessment was unremarkable except for low serum IGF-1 (SDS -2.67). On follow-up, GH deficiency due to GHRH resistance was suspected and confirmed by clonidine and arginine stimulation tests. Treatment with rhGH (0.035 mg/kg) for 2 years resulted in catch-up growth (height SDS -1.52). At age 15 years the PTH levels and bone age of the patient remain within the normal range. In patients with PHP1A, short stature is caused by the effects of Gs-α deficiency on the growth plate. However, resistance to GHRH and the resulting GH deficiency might also contribute. Recombinant GH treatment increases growth in these patients. Diagnostic workup for GH deficiency as a factor contributing to short stature is recommended even in the absence of other hormonal resistances.

This article is an update of the best practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes published in 2010 in BMC Medical Genetics [1]. The update takes into account developments in terms of techniques, differential diagnoses and (especially) reporting standards. It highlights the advantages and disadvantages of each method and moreover, is meant to facilitate the interpretation of the obtained results - leading to improved standardised reports.

Beckwith–Wiedemann syndrome (BWS; OMIM #130650) is an imprinting disorder caused by genetic or epigenetic alterations of one or both imprinting control regions on chromosome 11p15.5. Hypomethylation of the centromeric imprinting control region (KCNQ1OT1:TSS-DMR, ICR2) is the most common molecular cause of BWS and is present in about half of the cases. Based on a BWS family with a maternal deletion of the 5’ part of KCNQ1 we have recently hypothesised that transcription of KCNQ1 is a prerequisite for the establishment of methylation at the KCNQ1OT1:TSS-DMR in the oocyte. Further evidence for this hypothesis came from a mouse model where methylation failed to be established when a poly(A) truncation cassette was inserted into this locus to prevent transcription through the DMR. Here we report on a family where a balanced translocation disrupts the KCNQ1 gene in intron 9. Maternal inheritance of this translocation is associated with hypomethylation of the KCNQ1OT1:TSS-DMR and BWS. This finding strongly supports our previous hypothesis that transcription of KCNQ1 is required for establishing the maternal methylation imprint at the KCNQ1OT1:TSS-DMR.

Abstract: Introduction: According to the international literature, DNA methylation analysis of the promoter region of SNRPN locus is the most efficient way to start genetic investigation in patients with suspected Prader–Willi syndrome. Aim: Our aim was to develop a simple, reliable first-tier diagnosis to confirm Prader–Willi syndrome, therefore to compare our self-designed simple, cost-efficient high-resolution melting analysis and the most commonly used methylation-specific multiplex ligation-dependent probe amplification to confirm Prader–Willi syndrome. Method: We studied 17 clinically suspected Prader–Willi syndrome children and their DNA samples. With self-designed primers, bisulfite-sensitive polymerase chain reaction, high-resolution melting analysis and, as a control, methylation-specific multiplex ligation-dependent probe amplification were performed. Results: Prader–Willi syndrome was genetically confirmed in 6 out of 17 clinically suspected Prader–Willi syndrome patients. The results of high-resolution melting analysis and methylation-specific multiplex ligation-dependent probe amplification were equivalent in each case. Conclusion: Using our self-designed primers and altered bisulfite-specific PCR conditions, high-resolution melting analysis appears to be a simple, fast, reliable and effective method for primarily proving or excluding clinically suspected Prade-Willi syndrome cases. Orv Hetil. 2018; 159(2): 64–69.

The analysis of DNA methylation has become routine in the pipeline for diagnosis of imprinting disorders, with many publications reporting aberrant methylation associated with imprinted differentially methylated regions (DMRs). However, comparisons between these studies are routinely hampered by the lack of consistency in reporting sites of methylation evaluated. To avoid confusion surrounding nomenclature, special care is needed to communicate results accurately, especially between scientists and other health care professionals. Within the European Network for Human Congenital Imprinting Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission. The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deletion of the maternally inherited 15q11q13 region, paternal uniparental disomy 15 [upd(15)pat], an imprinting defect of the maternal chromosome region 15q11q13, or a pathogenic mutation of the maternal UBE3A allele. Predisposing factors for upd(15)pat, such as nonhomologous robertsonian translocation involving chromosome 15, have been discussed, but no evidence for this predisposition has been published. In the present study, chromosomal analysis was performed in a child with AS, both parents, and the maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was employed on DNA of the index individual, and microsatellite analysis was carried out on DNA of the index individual and his parents. The cytogenetic analysis showed that the mother and maternal grandfather are carriers of a rob(14;15). The index individual has a numerically normal karyotype, but MS-MLPA and microsatellite analyses confirmed the clinical diagnosis of AS and revealed a pattern highly suggestive of isodisomic upd(15)pat. This is the first report of an AS-affected individual with isodisomic upd(15)pat and a numerically normal karyotype that most likely results from a rob(14;15)-associated meiotic error in the maternal germline followed by monosomy 15 rescue in the early embryo.

Bevezetés: A nemzetközi szakirodalmi adatok alapján az SNRPN génlocus promoter régiójának DNS-metilációs vizsgálata jelenleg a legérzékenyebb és leghatékonyabb kezdeti lépés a Prader–Willi-szindróma-gyanús betegek genetikai vizsgálatakor. Célkitűzés: Célunk egy egyszerű, megbízható, könnyen hozzáférhető, elsődlegesen diagnosztikus, DNS-metiláción alapuló eljárás kidolgozása volt Prader–Willi-szindróma igazolására. Módszer: Vizsgálatunk során az általunk módosított, költséghatékony, metilációszenzitív, nagy felbontású olvadáspont-elemzéses technikát hasonlítottuk össze a leginkább elterjedt, költséges metilációspecifikus multiplex ligatiofüggő próbaamplifikációs technikával. Klinikailag a Prader–Willi-szindróma több tünetét mutató 17 gyermek DNSmetilációs vizsgálatát végeztük el saját tervezésű primerekkel: biszulfitszekvenáló polimeráz-láncreakció, metilációszenzitív nagy felbontású olvadáspont-elemzés és kontrollként metilációspecifikus multiplex ligatiofüggő próbaamplifikáció történt. Eredmények: A metilációszenzitív nagy felbontású olvadáspont-elemzés és a metilációspecifikus multiplex ligatiofüggő próbaamplifikáció eredményei minden esetben megegyeztek. A 17 esetből 6 esetben igazolódott a Prader–Williszindróma. Következtetés: Az általunk használt DNS-metiláción alapuló módszer, amelyben együttesen alkalmaztunk egyedi tervezésű primereket és módosított biszulfitszekvenáló polimeráz-láncreakciót, egyszerű, gyors, megbízható és hatékony vizsgálatnak bizonyult a Prader–Willi-szindróma elsődleges igazolására. Orv Hetil. 2018; 159(2): 64–69.

Introduction: According to the international literature, DNA methylation analysis of the promoter region of SNRPN locus is the most efficient way to start genetic investigation in patients with suspected Prader-Willi syndrome. Aim: Our aim was to develop a simple, reliable first-tier diagnosis to confirm Prader-Willi syndrome, therefore to compare our self-designed simple, cost-efficient high-resolution melting analysis and the most commonly used methylation-specific multiplex ligation-dependent probe amplification to confirm Prader-Willi syndrome. Method: We studied 17 clinically suspected Prader-Willi syndrome children and their DNA samples. With self-designed primers, bisulfite-sensitive polymerase chain reaction, high-resolution melting analysis and, as a control, methylation- specific multiplex ligation-dependent probe amplification were performed. Results: Prader-Willi syndrome was genetically confirmed in 6 out of 17 clinically suspected Prader-Willi syndrome patients. The results of high-resolution melting analysis and methylation-specific multiplex ligation-dependent probe amplification were equivalent in each case. Conclusion: Using our self-designed primers and altered bisulfite-specific PCR conditions, high-resolution melting analysis appears to be a simple, fast, reliable and effective method for primarily proving or excluding clinically suspected Prade-Willi syndrome cases.

Absztrakt: Bevezetes: A nemzetkozi szakirodalmi adatok alapjan az SNRPN genlocus promoter regiojanak DNS-metilacios vizsgalata jelenleg a legerzekenyebb es leghatekonyabb kezdeti lepes a Prader–Willi-szindroma-gyanus betegek genetikai vizsgalatakor. Celkitűzes: Celunk egy egyszerű, megbizhato, konnyen hozzaferhető, elsődlegesen diagnosztikus, DNS-metilacion alapulo eljaras kidolgozasa volt Prader–Willi-szindroma igazolasara. Modszer: Vizsgalatunk soran az altalunk modositott, koltseghatekony, metilacioszenzitiv, nagy felbontasu olvadaspont-elemzeses technikat hasonlitottuk ossze a leginkabb elterjedt, koltseges metilaciospecifikus multiplex ligatiofuggő probaamplifikacios technikaval. Klinikailag a Prader–Willi-szindroma tobb tunetet mutato 17 gyermek DNS-metilacios vizsgalatat vegeztuk el sajat tervezesű primerekkel: biszulfitszekvenalo polimeraz-lancreakcio, metilacioszenzitiv nagy felbontasu olvadaspont-elemzes es kontrollkent metilaciospecifikus multiplex ligatiofuggő probaamplifikacio tortent. Eredmenyek: A metilacioszenzitiv nagy felbontasu olvadaspont-elemzes es a metilaciospecifikus multiplex ligatiofuggő probaamplifikacio eredmenyei minden esetben megegyeztek. A 17 esetből 6 esetben igazolodott a Prader–Willi-szindroma. Kovetkeztetes: Az altalunk hasznalt DNS-metilacion alapulo modszer, amelyben egyuttesen alkalmaztunk egyedi tervezesű primereket es modositott biszulfitszekvenalo polimeraz-lancreakciot, egyszerű, gyors, megbizhato es hatekony vizsgalatnak bizonyult a Prader–Willi-szindroma elsődleges igazolasara. Orv Hetil. 2018; 159(2): 64–69. | Abstract: Introduction: According to the international literature, DNA methylation analysis of the promoter region of SNRPN locus is the most efficient way to start genetic investigation in patients with suspected Prader–Willi syndrome. Aim: Our aim was to develop a simple, reliable first-tier diagnosis to confirm Prader–Willi syndrome, therefore to compare our self-designed simple, cost-efficient high-resolution melting analysis and the most commonly used methylation-specific multiplex ligation-dependent probe amplification to confirm Prader–Willi syndrome. Method: We studied 17 clinically suspected Prader–Willi syndrome children and their DNA samples. With self-designed primers, bisulfite-sensitive polymerase chain reaction, high-resolution melting analysis and, as a control, methylation-specific multiplex ligation-dependent probe amplification were performed. Results: Prader–Willi syndrome was genetically confirmed in 6 out of 17 clinically suspected Prader–Willi syndrome patients. The results of high-resolution melting analysis and methylation-specific multiplex ligation-dependent probe amplification were equivalent in each case. Conclusion: Using our self-designed primers and altered bisulfite-specific PCR conditions, high-resolution melting analysis appears to be a simple, fast, reliable and effective method for primarily proving or excluding clinically suspected Prade-Willi syndrome cases. Orv Hetil. 2018; 159(2): 64–69.

Background Genomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance. Methods Whole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found. Results We now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss. Conclusion The identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.

Temple syndrome (TS14) is a rare imprinting disorder caused by genetic and epigenetic alterations on chromosome 14q32. A subset of these patients shows an imprinting defect (ID) where the paternal allele harbors a maternal epigenotype thus silencing the paternally expressed genes and leading to an increased expression of the maternally expressed genes. We investigated the grandparental origin of the incorrectly imprinted chromosome 14 in a cohort of 13 TS14 ID patients and their families. In seven families grandmaternal and, in six families, grandpaternal inheritance was observed. These results indicate that the ID occurred after imprint erasure in the paternal germ line. While the complete lack of methylation as observed in the majority of TS14 ID patients may be due to an imprint establishment error in the paternal germ line, cases with methylation mosaicism suggest that in general many IDs (TS14, AS, BWS, and SRS) are in fact of somatic origin in the early or late embryo.

Angelman syndrome (AS) is characterized by severe intellectual disability, limited, or absent speech and a generally happy demeanor. The four known etiological mechanisms; deletions, uniparental disomy, imprinting defects, and UBE3A mutation all affect expression of the UBE3A gene at 15q11-q13. An atypical phenotype is seen in individuals who are mosaic for a chromosome 15q11-q13 imprinting defect on the maternal allele. These patients present with a milder phenotype, often with hyperphagia and obesity or non-specific intellectual disability. Unlike typical AS syndrome, they can have a vocabulary up to 100 words and speak in sentences. Ataxia and seizures may not be present, and the majority of individuals do not have microcephaly. Here we review the current literature and present three individuals with atypical AS caused by a mosaic imprinting defect to demonstrate why DNA methylation analysis at the SNRPN locus needs to be considered in a broader clinical context. © 2017 Wiley Periodicals, Inc.

BACKGROUND:Maternal uniparental disomy of chromosome 6 (upd(6)mat) is a rare finding and its clinical relevance is currently unclear. Based on clinical data from two new cases and patients from the literature, the pathogenetic significance of upd(6)mat is delineated.METHODS:Own cases were molecularly characterized for isodisomic uniparental regions on chromosome 6. For further cases with upd(6)mat, a literature search was conducted and genetic and clinical data were ascertained.RESULTS:Comparison of isodisomic regions between the new upd(6)mat cases and those from four reports did not reveal any common isodisomic region. Among the patients with available cytogenetic data, five had a normal karyotype in lymphocytes, whereas a trisomy 6 (mosaicism) was detected prenatally in four cases. A common clinical picture was not obvious in upd(6)mat, but intrauterine growth restriction (IUGR) and preterm delivery were frequent.CONCLUSION:A common upd(6)mat phenotype is not obvious, but placental dysfunction due to trisomy 6 mosaicism probably contributes to IUGR and preterm delivery. In fact, other clinical features observed in upd(6)mat patients might be caused by homozygosity of recessive mutations or by an undetected trisomy 6 cell line. Upd(6)mat itself is not associated with clinical features, and can rather be regarded as a biomarker. In case upd(6)mat is detected, the cause for the phenotype is identified indirectly, but the UPD is not the basic cause.

The chromosomal region 14q32 contains several imprinted genes, which are expressed either from the paternal (DLK1 and RTL1) or the maternal (MEG3, RTL1as and MEG8) allele only. Imprinted expression of these genes is regulated by two differentially methylated regions (DMRs), the germline DLK1/MEG3 intergenic (IG)-DMR (MEG3/DLK1: IG-DMR) and the somatic MEG3-DMR (MEG3: TSS-DMR), which are methylated on the paternal and unmethylated on the maternal allele. Disruption of imprinting in the 14q32 region results in two clinically distinct imprinting disorders, Temple syndrome (TS14) and Kagami-Ogata syndrome (KOS14). Another DMR with a yet unknown function is located in intron 2 of MEG8 (MEG8-DMR, MEG8: Int2-DMR). In contrast to the IG-DMR and the MEG3-DMR, this somatic DMR is methylated on the maternal chromosome and unmethylated on the paternal chromosome. We have performed extensive methylation analyses by deep bisulfite sequencing of the IG-DMR, MEG3-DMR and MEG8-DMR in different prenatal tissues including amniotic fluid cells and chorionic villi. In addition, we have studied the methylation pattern of the MEG8-DMR in different postnatal tissues. We show that the MEG8-DMR is hypermethylated in each of 13 non-deletion TS14 patients (seven newly identified and six previously published patients), irrespective of the underlying molecular cause, and is always hypomethylated in the four patients with KOS14, who have different deletions not encompassing the MEG8-DMR itself. The size and the extent of the deletions and the resulting methylation pattern suggest that transcription starting from the MEG3 promoter may be necessary to establish the methylation imprint at the MEG8-DMR.

Angelman syndrome is a rare neurogenetic disorder that is characterized by microcephaly, severe intellectual deficit, speech impairment, epilepsy, EEG abnormalities, ataxic movements, tongue protrusion, paroxysms of laughter, abnormal sleep patterns, and hyperactivity. Angelman syndrome results from loss of function of the imprinted UBE3A (ubiquitin-protein ligase E3A) gene on chromosome 15q11.2-q13. This loss of function can be caused by a mutation on the maternal allele, a 5-7 Mb deletion of the maternally inherited chromosomal region, paternal uniparental disomy of chromosome 15, or an imprinting defect. The chromosomal deletion tends to cause the most severe symptoms, possibly owing to co-deletion of GABA receptor genes. UBE3A mutations and imprinting defects can be associated with a high risk of recurrence within families. Disruption of UBE3A function in neurons seems to inhibit synapse formation and experience-dependent synapse remodelling. Clinical diagnosis of Angelman syndrome in infants and young children is sometimes difficult, but can be verified by genetic tests. At present, treatment of symptoms such as seizures is the only medical strategy, but genetic therapies aimed at activating the silent copy of UBE3A on the paternal allele are conceivable.

Beckwith–Wiedemann syndrome (BWS; OMIM #130650) is an overgrowth syndrome caused by different genetic or epigenetic alterations affecting imprinted regions on chromosome 11p15.5. Here we report a family with multiple offspring affected with BWS including giant omphalocoeles in which maternal transmission of a chromosomal rearrangement including an inversion and two deletions leads to hypomethylation of the imprint control region 2 (ICR2). As the deletion includes the promoter and 5′ part of the KCNQ1 gene, we suggest that transcription of this gene may be involved in establishing the maternal methylation imprint of the ICR2, which is located in intron 10 of KCNQ1.

In approximately 20% of individuals with Kagami-Ogata syndrome (KOS14, MIM 608149), characterized by a bell-shaped thorax with coat-hanger configuration of the ribs, joint contractures, abdominal wall defects and polyhydramnios during the pregnancy, the syndrome is caused by a maternal deletion of the imprinted gene cluster in chromosome 14q32.2. Most deletions reported so far included one or both of the differentially methylated regions (DMRs) - DLK1/MEG3 IG-DMR and MEG3-DMR. We present two unrelated families with two affected siblings each, presenting with classical KOS14 due to maternally inherited microdeletions. Interestingly, all four patients have lived through to adulthood, even though mortality rates for patients with KOS14 due to a microdeletion are relatively high. In the first family, none of the DMRs is included in the deletion and the methylation status is identical to that of controls. Deletions that do not encompass the DMRs in this region are thus sufficient to elicit the full KOS14 phenotype. In the second family, a partially overlapping deletion including both DMRs and MEG3 was detected. In summary, we show that patients with KOS14 can live into adulthood, that causal deletions do not have to include the DMRs and that consequently a normal methylation pattern does not exclude KOS14.