Understanding the Variability of 22q11.2 Deletion Syndrome: The Role of Epigenetic Factors.

Initially described as a triad of immunodeficiency, congenital heart defects and hypoparathyroidism, 22q11.2 deletion syndrome (22q11.2DS) now encompasses a great amount of abnormalities involving different systems. Approximately 85% of patients share a 3 Mb 22q11.2 region of hemizygous deletion in which 46 protein-coding genes are included. However, the hemizygosity of the genes of this region cannot fully explain the clinical phenotype and the phenotypic variability observed among patients. Additional mutations in genes located outside the deleted region, leading to "dual diagnosis", have been described in 1% of patients. In some cases, the hemizygosity of the 22q11.2 region unmasks autosomal recessive conditions due to additional mutations on the non-deleted allele. Some of the deleted genes play a crucial role in gene expression regulation pathways, involving the whole genome. Typical miRNA expression patterns have been identified in 22q11.2DS, due to an alteration in miRNA biogenesis, affecting the expression of several target genes. Also, a methylation epi-signature in CpG islands differentiating patients from controls has been defined. Herein, we summarize the evidence on the genetic and epigenetic mechanisms implicated in the pathogenesis of the clinical manifestations of 22q11.2 DS. The review of the literature confirms the hypothesis that the 22q11.2DS phenotype results from a network of interactions between deleted protein-coding genes and altered epigenetic regulation.

Beckwith-Wiedemann syndrome and twinning: case report and brief review of literature.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS, OMIM #130,650) is a pediatric overgrowth disorder involving a predisposition to tumor development. Although the clinical management of affected patients is well established, it is less clear how to handle with the cases of siblings of affected patients, since the prevalence of the condition in twins (1:1000) is ten times higher than in singletones (1:10000). CASE PRESENTATION: We report the case of a premature twin patient who during her follow-up develops a clinical phenotype compatible with BWS, genetically confirmed in blood. However, the methylation alteration characteristic of the condition was also found in the almost phenotypically normal sibling, making it challening her management. CONCLUSION: Through our case report we highlight how the diagnosis of BWS can be made without any prenatal suspicion and we propose a review of the literature on how to manage siblings of affected patients in twinning situation.

Co-occurrence of Beckwith-Wiedemann syndrome and pseudohypoparathyroidism type 1B: coincidence or common molecular mechanism?

Imprinting disorders are congenital diseases caused by dysregulation of genomic imprinting, affecting growth, neurocognitive development, metabolism and cancer predisposition. Overlapping clinical features are often observed among this group of diseases. In rare cases, two fully expressed imprinting disorders may coexist in the same patient. A dozen cases of this type have been reported so far. Most of them are represented by individuals affected by Beckwith-Wiedemann spectrum (BWSp) and Transient Neonatal Diabetes Mellitus (TNDM) or BWSp and Pseudo-hypoparathyroidism type 1B (PHP1B). All these patients displayed Multilocus imprinting disturbances (MLID). Here, we report the first case of co-occurrence of BWS and PHP1B in the same individual in absence of MLID. Genome-wide methylation and SNP-array analyses demonstrated loss of methylation of the KCNQ1OT1:TSS-DMR on chromosome 11p15.5 as molecular cause of BWSp, and upd(20)pat as cause of PHP1B. The absence of MLID and the heterodisomy of chromosome 20 suggests that BWSp and PHP1B arose through distinct and independent mechanism in our patient. However, we cannot exclude that the rare combination of the epigenetic defect on chromosome 11 and the UPD on chromosome 20 may originate from a common so far undetermined predisposing molecular lesion. A better comprehension of the molecular mechanisms underlying the co-occurrence of two imprinting disorders will improve genetic counselling and estimate of familial recurrence risk of these rare cases. Furthermore, our study also supports the importance of multilocus molecular testing for revealing MLID as well as complex cases of imprinting disorders.

Co-Occurrence of Beckwith-Wiedemann Syndrome and Early-Onset Colorectal Cancer.

CRC is an adult-onset carcinoma representing the third most common cancer and the second leading cause of cancer-related deaths in the world. EO-CRC (<45 years of age) accounts for 5% of the CRC cases and is associated with cancer-predisposing genetic factors in half of them. Here, we describe the case of a woman affected by BWSp who developed EO-CRC at age 27. To look for a possible molecular link between BWSp and EO-CRC, we analysed her whole-genome genetic and epigenetic profiles in blood, and peri-neoplastic and neoplastic colon tissues. The results revealed a general instability of the tumor genome, including copy number and methylation changes affecting genes of the WNT signaling pathway, CRC biomarkers and imprinted loci. At the germline level, two missense mutations predicted to be likely pathogenic were found in compound heterozygosity affecting the Cystic Fibrosis (CF) gene CFTR that has been recently classified as a tumor suppressor gene, whose dysregulation represents a severe risk factor for developing CRC. We also detected constitutional loss of methylation of the KCNQ1OT1:TSS-DMR that leads to bi-allelic expression of the lncRNA KCNQ1OT1 and BWSp. Our results support the hypothesis that the inherited CFTR mutations, together with constitutional loss of methylation of the KCNQ1OT1:TSS-DMR, initiate the tumorigenesis process. Further somatic genetic and epigenetic changes enhancing the activation of the WNT/beta-catenin pathway likely contributed to increase the growth advantage of cancer cells. Although this study does not provide any conclusive cause-effect relationship between BWSp and CRC, it is tempting to speculate that the imprinting defect of BWSp might accelerate tumorigenesis in adult cancer in the presence of predisposing genetic variants.

Germline (epi)genetics reveals high predisposition in females: a 5-year, nationwide, prospective Wilms tumour cohort.

BACKGROUND: Studies suggest that Wilms tumours (WT) are caused by underlying genetic (5%-10%) and epigenetic (2%-29%) mechanisms, yet studies covering both aspects are sparse. METHODS: We performed prospective whole-genome sequencing of germline DNA in Danish children diagnosed with WT from 2016 to 2021, and linked genotypes to deep phenotypes. RESULTS: Of 24 patients (58% female), 3 (13%, all female) harboured pathogenic germline variants in WT risk genes (FBXW7, WT1 and REST). Only one patient had a family history of WT (3 cases), segregating with the REST variant. Epigenetic testing revealed one (4%) additional patient (female) with uniparental disomy of chromosome 11 and Beckwith-Wiedemann syndrome (BWS). We observed a tendency of higher methylation of the BWS-related imprinting centre 1 in patients with WT than in healthy controls. Three patients (13%, all female) with bilateral tumours and/or features of BWS had higher birth weights (4780 g vs 3575 g; p=0.002). We observed more patients with macrosomia (>4250 g, n=5, all female) than expected (OR 9.98 (95% CI 2.56 to 34.66)). Genes involved in early kidney development were enriched in our constrained gene analysis, including both known (WT1, FBXW7) and candidate (CTNND1, FRMD4A) WT predisposition genes. WT predisposing variants, BWS and/or macrosomia (n=8, all female) were more common in female patients than male patients (p=0.01). CONCLUSION: We find that most females (57%) and 33% of all patients with WT had either a genetic or another indicator of WT predisposition. This emphasises the need for scrutiny when diagnosing patients with WT, as early detection of underlying predisposition may impact treatment, follow-up and genetic counselling.

Performance Metrics of the Scoring System for the Diagnosis of the Beckwith-Wiedemann Spectrum (BWSp) and Its Correlation with Cancer Development.

Different scoring systems for the clinical diagnosis of the Beckwith-Wiedemann spectrum (BWSp) have been developed over time, the most recent being the international consensus score. Here we try to validate and provide data on the performance metrics of these scoring systems of the 2018 international consensus and the previous ones, relating them to BWSp features, molecular tests, and the probability of cancer development in a cohort of 831 patients. The consensus scoring system had the best performance (sensitivity 0.85 and specificity 0.43). In our cohort, the diagnostic yield of tests on blood-extracted DNA was low in patients with a low consensus score (~20% with a score = 2), and the score did not correlate with cancer development. We observed hepatoblastoma (HB) in 4.3% of patients with UPD(11)pat and Wilms tumor in 1.9% of patients with isolated lateralized overgrowth (ILO). We validated the efficacy of the currently used consensus score for BWSp clinical diagnosis. Based on our observation, a first-tier analysis of tissue-extracted DNA in patients with <4 points may be considered. We discourage the use of the consensus score value as an indicator of the probability of cancer development. Moreover, we suggest considering cancer screening for negative patients with ILO (risk ~2%) and HB screening for patients with UPD(11)pat (risk ~4%).

Different Mechanisms Cause Hypomethylation of Both H19 and KCNQ1OT1 Imprinted Differentially Methylated Regions in Two Cases of Silver-Russell Syndrome Spectrum.

Silver-Russell syndrome is an imprinting disorder characterised by pre- and post-natal growth retardation and several heterogeneous molecular defects affecting different human genomic loci. In the majority of cases, the molecular defect is the loss of methylation (LOM) of the H19/IGF2 differentially methylated region (DMR, also known as IC1) at the telomeric domain of the 11p15.5 imprinted genes cluster, which causes the altered expression of the growth controlling genes, IGF2 and H19. Very rarely, the LOM also affects the KCNQ1OT1 DMR (also known as IC2) at the centromeric domain, resulting in an SRS phenotype by an unknown mechanism. In this study, we report on two cases with SRS features and a LOM of either IC1 and IC2. In one case, this rare and complex epimutation was secondary to a de novo mosaic in cis maternal duplication, involving the entire telomeric 11p15.5 domain and part of the centromeric domain but lacking CDKN1C. In the second case, neither the no 11p15.5 copy number variant nor the maternal-effect subcortical maternal complex (SCMC) variant were found to be associated with the epimutation, suggesting that it arose as a primary event. Our findings further add to the complexity of the molecular genetics of SRS and indicate how the LOM in both 11p15.5 DMRs may result from different molecular mechanisms.

Epigenetics: An opportunity to shape innate and adaptive immune responses.

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post-translational modifications and the regulation of chromatin accessibility by non-coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental-driven nature, piloting myeloid and lymphoid cell fate decisions with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage-specific transcription factors and maintenance of cell type-specific epigenetic modifications, referred to as 'epigenetic memory', dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as 'trained immunity'. Here, we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases.

Clinical and molecular characterization of patients affected by Beckwith-Wiedemann spectrum conceived through assisted reproduction techniques.

The prevalence of Beckwith-Wiedemann spectrum (BWSp) is tenfold increased in children conceived through assisted reproductive techniques (ART). More than 90% of ART-BWSp patients reported so far display imprinting center 2 loss-of-methylations (IC2-LoM), versus 50% of naturally conceived BWSp patients. We describe a cohort of 74 ART-BWSp patients comparing their features with a cohort of naturally conceived BWSp patients, with the ART-BWSp patients previously described in literature, and with the general population of children born from ART. We found that the distribution of UPD(11)pat was not significantly different in ART and naturally conceived patients. We observed 68.9% of IC2-LoM and 16.2% of mosaic UPD(11)pat in our ART cohort, that strongly differ from the figure reported in other cohorts so far. Since UPD(11)pat likely results from post-fertilization recombination events, our findings allows to hypothesize that more complex molecular mechanisms, besides methylation disturbances, may underlie BWSp increased risk in ART pregnancies. Moreover, comparing the clinical features of ART and non-ART BWSp patients, we found that ART-BWSp patients might have a milder phenotype. Finally, our data show a progressive increase in the prevalence of BWSp over time, paralleling that of ART usage in the last decades.

Novel genetic variants of KHDC3L and other members of the subcortical maternal complex associated with Beckwith-Wiedemann syndrome or Pseudohypoparathyroidism 1B and multi-locus imprinting disturbances.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) and Pseudohypoparathyroidism type 1B (PHP1B) are imprinting disorders (ID) caused by deregulation of the imprinted gene clusters located at 11p15.5 and 20q13.32, respectively. In both of these diseases a subset of the patients is affected by multi-locus imprinting disturbances (MLID). In several families, MLID is associated with damaging variants of maternal-effect genes encoding protein components of the subcortical maternal complex (SCMC). However, frequency, penetrance and recurrence risks of these variants are still undefined. In this study, we screened two cohorts of BWS patients and one cohort of PHP1B patients for the presence of MLID, and analysed the positive cases for the presence of maternal variants in the SCMC genes by whole exome-sequencing and in silico functional studies. RESULTS: We identified 10 new cases of MLID associated with the clinical features of either BWS or PHP1B, in which segregate 13 maternal putatively damaging missense variants of the SCMC genes. The affected genes also included KHDC3L that has not been associated with MLID to date. Moreover, we highlight the possible relevance of relatively common variants in the aetiology of MLID. CONCLUSION: Our data further add to the list of the SCMC components and maternal variants that are involved in MLID, as well as of the associated clinical phenotypes. Also, we propose that in addition to rare variants, common variants may play a role in the aetiology of MLID and imprinting disorders by exerting an additive effect in combination with rarer putatively damaging variants. These findings provide useful information for the molecular diagnosis and recurrence risk evaluation of MLID-associated IDs in genetic counselling.

Epigenetic Alterations in Inborn Errors of Immunity.

The epigenome bridges environmental factors and the genome, fine-tuning the process of gene transcription. Physiological programs, including the development, maturation and maintenance of cellular identity and function, are modulated by intricate epigenetic changes that encompass DNA methylation, chromatin remodeling, histone modifications and RNA processing. The collection of genome-wide DNA methylation data has recently shed new light into the potential contribution of epigenetics in pathophysiology, particularly in the field of immune system and host defense. The study of patients carrying mutations in genes encoding for molecules involved in the epigenetic machinery has allowed the identification and better characterization of environment-genome interactions via epigenetics as well as paving the way for the development of new potential therapeutic options. In this review, we summarize current knowledge of the role of epigenetic modifications in the immune system and outline their potential involvement in the pathogenesis of inborn errors of immunity.

Variable Expressivity of the Beckwith-Wiedemann Syndrome in Four Pedigrees Segregating Loss-of-Function Variants of CDKN1C.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by prenatal and/or postnatal overgrowth, organomegaly, abdominal wall defects and tumor predisposition. CDKN1C is a maternally expressed gene of the 11p15.5 chromosomal region and is regulated by the imprinting control region IC2. It negatively controls cellular proliferation, and its expression or activity are frequently reduced in BWS. In particular, loss of IC2 methylation is associated with CDKN1C silencing in the majority of sporadic BWS cases, and maternally inherited loss-of-function variants of CDKN1C are the most frequent molecular defects of familial BWS. We have identified, using Sanger sequencing, novel CDKN1C variants in three families with recurrent cases of BWS, and a previously reported variant in a woman with recurrent miscarriages with exomphalos. Clinical evaluation of the patients showed variable manifestation of the disease. The frameshift and nonsense variants were consistently associated with exomphalos, while the missense variant caused a less severe phenotype. Pregnancy loss and perinatal lethality were found in the families segregating nonsense mutations. Intrafamilial variability of the clinical BWS features was observed, even between siblings. Our data are indicative of severe BWS phenotypes that, with variable expressivity, may be associated with both frameshift and nonsense variants of CDKN1C.

The number of the CTCF binding sites of the H19/IGF2:IG-DMR correlates with DNA methylation and expression imprinting in a humanized mouse model.

The reciprocal parent of origin-specific expression of H19 and IGF2 is controlled by the H19/IGF2:IG-DMR (IC1), whose maternal allele is unmethylated and acts as a CTCF-dependent insulator. In humans, internal IC1 deletions are associated with Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), depending on their parental origin. These genetic mutations result in aberrant DNA methylation, deregulation of IGF2/H19 and disease with incomplete penetrance. However, the mechanism linking the microdeletions to altered molecular and clinical phenotypes remains unclear. To address this issue, we have previously generated and characterized two knock-in mouse lines with the human wild-type (hIC1wt) or mutant (hIC1∆2.2) IC1 allele replacing the endogenous mouse IC1 (mIC1). Here, we report an additional knock-in line carrying a mutant hIC1 allele with an internal 1.8 kb deletion (hIC1∆1.8). The phenotype of these mice is different from that of the hIC1∆2.2-carrying mice, partially resembling hIC1wt animals. Indeed, proper H19 and Igf2 imprinting and normal growth phenotype were evident in the mice with maternal transmission of hIC1Δ1.8, while low DNA methylation and non-viable phenotype characterize its paternal transmission. In contrast to hIC1wt, E15.5 embryos that paternally inherit hIC1Δ1.8 displayed variegated hIC1 methylation. In addition, increased Igf2 expression, correlating with increased body weight, was found in one third of these mice. Chromatin immunoprecipitation experiments in mouse embryonic stem cells carrying the three different hIC1 alleles demonstrate that the number of CTCF target sites influences its binding to hIC1, indicating that in the mouse, CTCF binding is key to determining hIC1 methylation and Igf2 expression.

Mosaic Segmental and Whole-Chromosome Upd(11)mat in Silver-Russell Syndrome.

Molecular defects altering the expression of the imprinted genes of the 11p15.5 cluster are responsible for the etiology of two congenital disorders characterized by opposite growth disturbances, Silver-Russell syndrome (SRS), associated with growth restriction, and Beckwith-Wiedemann syndrome (BWS), associated with overgrowth. At the molecular level, SRS and BWS are characterized by defects of opposite sign, including loss (LoM) or gain (GoM) of methylation at the H19/IGF2:intergenic differentially methylated region (H19/IGF2:IG-DMR), maternal or paternal duplication (dup) of 11p15.5, maternal (mat) or paternal (pat) uniparental disomy (upd), and gain or loss of function mutations of CDKN1C. However, while upd(11)pat is found in 20% of BWS cases and in the majority of them it is segmental, upd(11)mat is extremely rare, being reported in only two SRS cases to date, and in both of them is extended to the whole chromosome. Here, we report on two novel cases of mosaic upd(11)mat with SRS phenotype. The upd is mosaic and isodisomic in both cases but covers the entire chromosome in one case and is restricted to 11p14.1-pter in the other case. The segmental upd(11)mat adds further to the list of molecular defects of opposite sign in SRS and BWS, making these two imprinting disorders even more specular than previously described.

Biallelic variant in cyclin B3 is associated with failure of maternal meiosis II and recurrent digynic triploidy.

BACKGROUND: Triploidy is one of the most common chromosome abnormalities affecting human gestation and accounts for an important fraction of first-trimester miscarriages. Triploidy has been demonstrated in a few cases of recurrent pregnancy loss (RPL) but its molecular mechanisms are unknown. This study aims to identify the genetic cause of RPL associated with fetus triploidy. METHODS: We investigated genomic imprinting, genotyped sequence-tagged site (STS) markers and performed exome sequencing in a family including two sisters with RPL. Moreover, we evaluated oocyte maturation in vivo and in vitro and effect of the candidate protein variant in silico. RESULTS: While features of hydatidiform mole were excluded, the presence of triploidy of maternal origin was demonstrated in the fetuses. Oocyte maturation was deficient and all the maternally inherited pericentromeric STS alleles were homozygous in the fetuses. A deleterious missense variant (p.V1251D) of the cyclin B3 gene (CCNB3) affecting a residue conserved in placental mammals and located in a region that can interact with the cyclin-dependent kinase 1 or cyclin-dependent kinase 2 cosegregated in homozygosity with RPL. CONCLUSION: Here, we report a family in which a damaging variant in cyclin B3 is associated with the failure of oocyte meiosis II and recurrent fetus triploidy, implicating a rationale for CCNB3 testing in RPL.

Both Epimutations and Chromosome Aberrations Affect Multiple Imprinted Loci in Aggressive Wilms Tumors.

The embryonal renal cancer Wilms tumor (WT) accounts for 7% of all children's malignancies. Its most frequent molecular defect is represented by DNA methylation abnormalities at the imprinted 11p15.5 region. Multiple imprinted methylation alterations dictated by chromosome copy-number variations have been recently demonstrated in adult cancers, raising the question of whether multiple imprinted loci were also affected in WT. To address this issue, we analyzed DNA methylation and chromosome profiles of 7 imprinted loci in 48 WT samples. The results demonstrated that methylation abnormalities of multiple imprinted loci occurred in 35% of the cases, but that they were associated with either chromosome aberrations or normal chromosome profiles. Multiple imprinted methylation changes were correlated with tumor stage and presence of metastasis, indicating that these epimutations were more frequent in highly aggressive tumors. When chromosome profiles were affected, these alterations were extended to flanking cancer driver genes. Overall, this study demonstrates the presence of multiple imprinted methylation defects in aggressive WTs and suggests that the mechanism by which they arise in embryonal and adult cancers is different.

Loss-of-function maternal-effect mutations of PADI6 are associated with familial and sporadic Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance.

BACKGROUND: PADI6 is a component of the subcortical maternal complex, a group of proteins that is abundantly expressed in the oocyte cytoplasm, but is required for the correct development of early embryo. Maternal-effect variants of the subcortical maternal complex proteins are associated with heterogeneous diseases, including female infertility, hydatidiform mole, and imprinting disorders with multi-locus imprinting disturbance. While the involvement of PADI6 in infertility is well demonstrated, its role in imprinting disorders is less well established. RESULTS: We have identified by whole-exome sequencing analysis four cases of Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance whose mothers are carriers of PADI6 variants. In silico analysis indicates that these variants result in loss of function, and segregation analysis suggests they act as either recessive or dominant-negative maternal-effect mutations. Genome-wide methylation analysis revealed heterogeneous and extensively altered methylation profiles of imprinted loci in the patients, including two affected sisters, but not in their healthy siblings. CONCLUSION: Our results firmly establish the role of PADI6 in imprinting disorders. We report loss-of-function maternal-effect variants of PADI6 that are associated with heterogeneous multi-locus imprinting disturbances in the progeny. The rare finding of two siblings affected by Beckwith-Wiedemann syndrome suggests that in some cases, familial recurrence risk of these variants may be high. However, the heterogeneous phenotypes of the other pedigrees suggest that altered oocyte PADI6 function results in stochastic maintenance of methylation imprinting with unpredictable consequences on early embryo health.

The phenotypic variations of multi-locus imprinting disturbances associated with maternal-effect variants of NLRP5 range from overt imprinting disorder to apparently healthy phenotype.

BACKGROUND: A subset of individuals affected by imprinting disorders displays multi-locus imprinting disturbances (MLID). MLID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (gDMRs) in early embryogenesis. Pedigrees of individuals with MLID also include siblings with healthy phenotype. However, it is unknown if these healthy individuals have MLID themselves or if their methylation patterns differ from those associated with imprinting disorders, and in general, if MLID affects the clinical phenotype. METHODS: We have investigated gDMR methylation by locus-specific and whole-genome analyses in a family with multiple pregnancy losses, a child with Beckwith-Wiedemann syndrome (BWS) and a further child with no clinical diagnosis of imprinting disorder or other pathologies. RESULTS: We detected MLID with different methylation profiles in the BWS-affected and healthy siblings. Whole-exome sequencing demonstrated the presence of novel loss-of-function variants of NLRP5 in compound heterozygosity in the mother. The methylation profiles of the two siblings were compared with those of other cases with MLID and control groups by principal component analysis and unsupervised hierarchical clustering, but while their patterns were clearly separated from those of controls, we were unable to cluster those associated with specific clinical phenotypes among the MLID cases. CONCLUSION: The identification of two novel maternal-effect variants of NLRP5 associated with poly-abortivity and MLID adds further evidence to the role of this gene in the maintenance of genomic imprinting in early embryos. Furthermore, our results demonstrate that within these pedigrees, MLID can also be present in the progeny with healthy phenotype, indicating that some sort of compensation occurs between altered imprinted loci in these individuals. The analysis of larger cohorts of patients with MLID is needed to formulate more accurate epigenotype-phenotype correlations.

Transcription alterations of KCNQ1 associated with imprinted methylation defects in the Beckwith-Wiedemann locus.

PURPOSE: Beckwith-Wiedemann syndrome (BWS) is a developmental disorder caused by dysregulation of the imprinted gene cluster of chromosome 11p15.5 and often associated with loss of methylation (LOM) of the imprinting center 2 (IC2) located in KCNQ1 intron 10. To unravel the etiological mechanisms underlying these epimutations, we searched for genetic variants associated with IC2 LOM. METHODS: We looked for cases showing the clinical features of both BWS and long QT syndrome (LQTS), which is often associated with KCNQ1 variants. Pathogenic variants were identified by genomic analysis and targeted sequencing. Functional experiments were performed to link these pathogenic variants to the imprinting defect. RESULTS: We found three rare cases in which complete IC2 LOM is associated with maternal transmission of KCNQ1 variants, two of which were demonstrated to affect KCNQ1 transcription upstream of IC2. As a consequence of KCNQ1 haploinsufficiency, these variants also cause LQTS on both maternal and paternal transmission. CONCLUSION: These results are consistent with the hypothesis that, similar to what has been demonstrated in mouse, lack of transcription across IC2 results in failure of methylation establishment in the female germline and BWS later in development, and also suggest a new link between LQTS and BWS that is important for genetic counseling.

Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.