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Molecular genetic testing for the 11p15-associated imprinting disorders Silver-Russell and Beckwith-Wiedemann syndrome (SRS, BWS) is challenging because of the molecular heterogeneity and complexity of the affected imprinted regions. With the growing knowledge on the molecular basis of these disorders and the demand for molecular testing, it turned out that there is an urgent need for a standardized molecular diagnostic testing and reporting strategy. Based on the results from the first external pilot quality assessment schemes organized by the European Molecular Quality Network (EMQN) in 2014 and in context with activities of the European Network of Imprinting Disorders (EUCID.net) towards a consensus in diagnostics and management of SRS and BWS, best practice guidelines have now been developed. Members of institutions working in the field of SRS and BWS diagnostics were invited to comment, and in the light of their feedback amendments were made. The final document was ratified in the course of an EMQN best practice guideline meeting and is in accordance with the general SRS and BWS consensus guidelines, which are in preparation. These guidelines are based on the knowledge acquired from peer-reviewed and published data, as well as observations of the authors in their practice. However, these guidelines can only provide a snapshot of current knowledge at the time of manuscript submission and readers are advised to keep up with the literature.

Beckwith-Wiedemann and Silver-Russell syndromes (BWS/SRS) are two imprinting disorders (IDs) associated with disturbances of the 11p15.5 chromosomal region. In BWS, epimutations and genomic alterations within 11p15.5 are observed in >70% of patients, whereas in SRS they are observed in about 60% of the cases. In addition, 10% of the SRS patients carry a maternal uniparental disomy of chromosome 7 11p15.5. There is an increasing demand for prenatal testing of these disorders owing to family history, indicative prenatal ultrasound findings or aberrations involving chromosomes 7 and 11. The complex molecular findings underlying these disorders are a challenge not only for laboratories offering these tests but also for geneticists counseling affected families. The scope of counseling must consider the range of detectable disturbances and their origin, the lack of precise quantitative knowledge concerning the inheritance and recurrence risks for the epigenetic abnormalities, which are hallmarks of these developmental disorders. In this paper, experts in the field of BWS and SRS, including members of the European network of congenital IDs (EUCID.net; www.imprinting-disorders.eu), put together their experience and work in the field of 11p15.5-associated IDs with a focus on prenatal testing. Altogether, prenatal tests of 160 fetuses (122 referred for BWS, 38 for SRS testing) from 5 centers were analyzed and reviewed. We summarize the current knowledge on BWS and SRS with respect to diagnostic testing, the consequences for prenatal genetic testing and counseling and our cumulative experience in dealing with these disorders.

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are caused by imprinting defects on chromosome 11p15.5. Standard diagnostic tests for these syndromes include methylation analysis of the differential methylated regions of the H19 and KCNQ1OT1 genes. Traditionally this has been conducted by Southern blot analysis. PCR-based methods greatly improve the turn around time of the test and require less DNA. One of the newly emerging techniques for SNP genotyping and mutation scanning, high-resolution melting (HRM) analysis, has been shown to be also applicable for methylation analysis. We tested methylation-sensitive HRM analysis as a method for the detection of methylation defects in a group of 16 BWS and SRS patients with known methylation status (determined previously by Southern blotting), as well as 45 normal controls. HRM analysis was able to detect all methylation aberrations in the patients and appeared to be more sensitive than Southern blotting. Variation in normal controls is minimal and the presence of SNPs in the amplified fragment does not influence the outcome of the test. We conclude that methylation-sensitive HRM analysis is a robust, fast, sensitive and cost effective method for methylation analysis in BWS and SRS.

Properdin type I deficiency is characterised by complete absence of extracellular properdin, a positive regulator of the alternative pathway of complement activation. Properdin deficiency is associated with increased susceptibility to severe meningococcal disease. We have identified the genetic defect in 10 Dutch families. Six different mutations and one sequence polymorphism in the properdin gene were found. All amino acid substitutions were limited to conserved amino acids in exons 7 and 8 in contrast to the premature stops that were found in other exons. The missense mutations may alter the protein conformation in such a way that properdin will not be secreted and therefore catabolised intracellularly. The decreased properdin levels found in some healthy females carrying one mutated properdin gene were studied for X-inactivation. Most carriers with extreme low or high properdin levels showed preferential X-inactivation for the normal or mutated X chromosome, respectively. We observed some exceptions, suggesting additional regulation of properdin excretion apart from X-inactivation.

Cytogenetic and DNA analyses of patients with the Beckwith-Wiedemann syndrome (BWS) enabled us to refine the localization of the syndrome at 11p15.3-pter to two distinct regions. One chromosome region (BWSCR1) is near the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. The other region (BWSCR2) is more proximal near two sequences with zinc-binding finger motifs and a number of known and putative genes. This latter region, at least, seems to be associated with the development of childhood tumors. Our results strongly support the proposed involvement of parental imprinting in the etiology of BWS since all balanced chromosomal abnormalities in these patients were maternally transmitted while the mothers were phenotypically normal. We demonstrate that such an autosomal balanced rearrangement can lead to a specific maternal hypomethylation of the INS/IGF2 genes localized distal to the breakpoint. This underlines the role of these genes in the etiology of the syndrome.

Genomic imprinting has been implicated in the aetiology of an overgrowth cancer-prone syndrome, the Wiedemann-Beck-with syndrome (WBS). We have demonstrated uniparental disomy (UPD) for paternal chromosome 11p markers in 5 out of 25 sporadic cases (20%). Delineation of the extent of the disomy region may help in understanding the mechanism and the stage, meiotic or mitotic, of disomy formation in this disease and in associated tumours. Our current studies in WBS patients with seventeen 11p and one 11q markers reveal paternal isodisomy, not heterodisomy, in the five cases. For one case we demonstrate unambiguously that partial isodisomy for 11p and somatic mosaicism for UPD resulted from a post-fertilization event. The restriction of isodisomy to part of 11p in another case, and somatic mosaicism for UPD in three other cases, suggest a mitotic recombinational event that must have occurred after fertilization. Mosaic phenotypes reflect the timing of their origin and the fate of the cells involved, as well as the cell-specific pattern of imprinting. Somatic mosaicism for UPD in four cases may thus explain the incomplete forms of WBS. The association of hemihypertrophy in sporadic WBS and even some cases of isolated hemihypertrophy. This is in agreement with a recent report of paternal isodisomy for 11p markers in a patient with hemihypertrophy, Wilms' tumour and adrenocortical carcinoma. Moreover, the risk of developing a tumour seems higher (50%) for patients with paternal 11p UPD than for WBS patients in general (7.5%).