Cheryl Shuman

Beckwith|[ndash]|Wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS) is a model disorder for the study of imprinting, growth dysregulation, and tumorigenesis. Unique observations in this disorder point to an important embryonic developmental window relevant to the observations of increased monozygotic twinning and an increased rate of epigenetic errors after subfertility/assisted reproduction.

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.

GPC3 mutation analysis in a spectrum of patients with overgrowth expands the phenotype of Simpson-Golabi-Behmel syndrome.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome caused by deletions in glypican 3 (GPC3). SGBS is characterized by pre- and postnatal overgrowth, a characteristic facial appearance, and a spectrum of congenital malformations which overlaps that of other overgrowth syndromes. We performed GPC3 deletion screening on 80 male patients with somatic overgrowth in the following categories: SGBS (n = 19), possible SGBS (n = 26), including families in which individuals had previously been diagnosed with other overgrowth syndromes, and Wiedemann-Beckwith syndrome (WBS) (n = 35). Using exon-specific PCR and Southern blot analysis, we identified seven GPC3 deletions. In most cases a clear X-linked family history was not present. In two cases, GPC3 deletions were identified in patients belonging to pedigrees published previously as other overgrowth syndromes: one with a diagnosis of Sotos syndrome and the other Perlman syndrome with nephroblastomatosis. A third patient developed hepatoblastoma, a tumor type not previously described in SGBS. No GPC3 deletions were identified among the WBS patients. Direct sequencing of all GPC3 exons in the remaining 13 SGBS patients without GPC3 deletions did not identify any further mutations, raising the possibility of alternative silencing mechanisms and/or other genes in the pathogenesis of SGBS. Our results validate the clinical specificity of the facial appearance, skeletal/hand anomalies, and supernumerary nipples in patients with GPC3 deletions. Our data also suggest that nephroblastomatosis and hepatoblastoma are included in the phenotypic spectrum of GPC3 deletions and SGBS, underscoring the importance of tumor surveillance in these children.

Natural history of Alström syndrome in early childhood: onset with dilated cardiomyopathy.

Alström syndrome is an autosomal recessive disorder characterized by cone-rod dystrophy, obesity, hearing impairment, and diabetes caused by insulin resistance. By reviewing the charts of eight patients followed for periods of 2 to 22 years, we established the natural history of this syndrome during childhood. Five patients, in four families, were seen between the ages of 3 weeks and 4 months with a dilated cardiomyopathy, a previously unrecognized feature of the syndrome. Photophobia and nystagmus were first documented in the eight patients between the ages of 5 months and 15 months. In all patients, electroretinography initially showed a severe cone impairment with mild (2/8) or no (6/8) rod involvement. Electroretinograms, obtained again at ages 9 to 22 years for four patients, revealed extinguished rod-and-cone responses. Obesity developed during childhood in seven patients, in at least three of them before age 2 years. Hearing impairment (5/8) and diabetes/glucose intolerance (4/8) were diagnosed at the end of the first decade or during the second decade. This constellation of features should facilitate early diagnosis of the syndrome.

Molecular findings in Beckwith-Wiedemann syndrome.

Our understanding of Beckwith–Wiedemann syndrome (BWS) has recently been enhanced by advances in its molecular characterization. These advances have further delineated intricate (epi)genetic regulation of the imprinted gene cluster on chromosome 11p15.5 and the role of these genes in normal growth and development. Studies of the molecular changes associated with the BWS phenotype have been instrumental in elucidating critical molecular elements in this imprinted region. This review will provide updated information on the multiple new regulatory elements that have been recently found to contribute to in cis or in trans control of imprinted gene expression in the chromosome 11p15.5 region and the clinical expression of the BWS phenotype.

Whole-genome sequencing expands diagnostic utility and improves clinical management in paediatric medicine

The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold increase in CMA plus targeted gene sequencing (13%; P value=0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harbouring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counselling. Clinical implementation of WGS as a primary test will provide a higher diagnostic yield than conventional genetic testing and potentially reduce the time required to reach a genetic diagnosis.

Whole Genome Scanning: Resolving Clinical Diagnosis and Management Amidst Complex Data

Momentum around the era of genomic medicine is building, and with it, anticipation of the benefits that whole genome analysis (personalized or individualized genomics) will bring for the provision of health care. These technologies have the potential to revolutionize genetic diagnosis; however, the expansive data generated can lead to complex or unexpected findings, sometimes complicating clinical utility and patient benefit. Here, we use our experience with whole genome scanning microarrays, an early instance of whole genome analysis already in clinical use, to highlight fundamental challenges raised by these technologies and to discuss their medical, ethical, legal and social implications. We discuss issues that physicians and healthcare professionals will face, in particular, as the resolution of testing further increases toward full genome sequence determination. We emphasize that addressing these issues now, and starting to evolve our healthcare systems in response, will be pivotal in avoiding harms and realizing the promise of these new technologies.

Constitutional UPD for chromosome 11p15 in individuals with isolated hemihyperplasia is associated with high tumor risk and occurs following assisted reproductive technologies

Isolated hemihyperplasia (IH) refers to a distinct diagnosis involving asymmetric overgrowth of single or multiple organs or regions of the body and can result from various genomic changes including molecular alterations of 11p15; these are paternal uniparental disomy (UPD), and alterations of methylation at two imprinting centers at 11p15: IC1 (H19) and IC2 (KCNQ1OT1). As little information is available on the molecular basis of tumor development in IH, or on the frequency of tumors in children with different molecular subtypes of IH, molecular testing was undertaken on 51 patients with IH and revealed: 8 (16%) with UPD, 3 (6%) with hypomethylation at KCNQ1OT1, and 0 with hypermethylation at H19. Of the 8 patients with UPD, 4 had tumors (3 hepatoblastomas, 1 Wilms tumor); 0/3 patients with hypomethylation at KCNQ1OT1 had a tumor; of the remaining 40 with no molecular alterations, 6 had tumors (3 Wilms tumors, 2 neuroblastomas, 1 adrenocortical adenoma). The 50% tumor frequency in patients with IH and UPD was statistically significantly higher than the 15% tumor frequency in those with IH and no molecular alteration detected (Fishers exact test P = 0.047, OR 5.67). This is the first demonstration that UPD at 11p15 in patients with IH confers a higher tumor risk than in patients with IH without this molecular change. Of note, two of the eight patients with UPD and IH were conceived using assisted reproductive technologies (ART), thus raising the question whether ART might impact the rate of somatic recombination during embryonic development.

Renal Abnormalities in Beckwith-Wiedemann Syndrome Are Associated with 11p15.5 Uniparental Disomy

Beckwith-Wiedemann syndrome (BWS) is a somatic overgrowth syndrome characterized by a variable incidence of congenital anomalies, including hemihyperplasia and renal malformations. BWS is associated with disruption of genomic imprinting and/or mutations in one or more genes encoded on 11p15.5, including CDKN1C (p57(KIP2)). It was hypothesized that genotypic and epigenotypic abnormalities of the 11p15.5 region affecting CDKN1C were associated with renal abnormalities. Medical records for 159 individuals with BWS were reviewed. All underwent at least one abdominal ultrasonographic evaluation. Testing for paternal uniparental disomy (UPD) at 11p15.5, CDKN1C mutations, and imprinting defects at KvDMR1 was performed for 96, 32, and 47 patients, respectively. Of the 159 patients, 67 (42%) exhibited renal abnormalities, mainly nephromegaly (25%), collecting system abnormalities (11%), and renal cysts (10.5%). The frequency of renal lesions among patients who were tested for genetic abnormalities did not differ from that among patients who were not tested. Paternal UPD was demonstrated in 22 of 96 cases (23%), CDKN1C mutations in eight of 32 cases (25%), and KvDMR1 imprinting defects in 21 of 47 cases (45%). The 22 UPD-positive patients exhibited a significantly higher incidence of renal abnormalities (P = 0.0026). Surprisingly, the eight patients with CDKN1C mutations exhibited no significant increase in the incidence of renal lesions (P = 0.29). Imprinting defects at KvDMR1, which might downregulate CDKN1C, were also not associated with a significant difference in the incidence of renal disease. Whereas UPD at 11p15.5 in BWS was associated with a higher incidence of renal abnormalities, mutations at CDKN1C and KvDMR1 imprinting defects were not, suggesting that imprinted genes on 11p15.5 other than CDKN1C are critical for renal development.

Recommendations for the integration of genomics into clinical practice

The introduction of diagnostic clinical genome and exome sequencing (CGES) is changing the scope of practice for clinical geneticists. Many large institutions are making a significant investment in infrastructure and technology, allowing clinicians to access CGES, especially as health-care coverage begins to extend to clinically indicated genomic sequencing-based tests. Translating and realizing the comprehensive clinical benefits of genomic medicine remain a key challenge for the current and future care of patients. With the increasing application of CGES, it is necessary for geneticists and other health-care providers to understand its benefits and limitations in order to interpret the clinical relevance of genomic variants identified in the context of health and disease. New, collaborative working relationships with specialists across diverse disciplines (e.g., clinicians, laboratorians, bioinformaticians) will undoubtedly be key attributes of the future practice of clinical genetics and may serve as an example for other specialties in medicine. These new skills and relationships will also inform the development of the future model of clinical genetics training curricula. To address the evolving role of the clinical geneticist in the rapidly changing climate of genomic medicine, two Clinical Genetics Think Tank meetings were held that brought together physicians, laboratorians, scientists, genetic counselors, trainees, and patients with experience in clinical genetics, genetic diagnostics, and genetics education. This article provides recommendations that will guide the integration of genomics into clinical practice.Genet Med 18 11, 1075–1084.