Canadian College of Medical Geneticists: clinical practice advisory document - responsibility to recontact for reinterpretation of clinical genetic testing.

BACKGROUND: Advances in technology and knowledge have facilitated both an increase in the number of patient variants reported and variants reclassified. While there is currently no duty to recontact for reclassified genetic variants, there may be a responsibility. The purpose of this clinical practice advisory document is to provide healthcare practitioners guidance for recontact of previously identified and classified variants, suggest methods for recontact, and principles to consider, taking account patient safety, feasibility, ethical considerations, health service capacity and resource constraints. The target audience are practitioners who order genetic testing, follow patients who have undergone genetic testing and those analysing and reporting genetic testing. METHODS: A multidisciplinary group of laboratory and ordering clinicians, patient representatives, ethics and legal researchers and a genetic counsellor from the Canadian Association of Genetic Counsellors reviewed the existing literature and guidelines on responsibility to recontact in a clinical context to make recommendations. Comments were collected from the Canadian College of Medical Geneticists (CCMG) Education, Ethics, and Public Policy, Clinical Practice and Laboratory Practice committees, and the membership at large. RESULTS: Following incorporation of feedback, and external review by the Canadian Association of Genetic Counsellors and patient groups, the document was approved by the CCMG Board of Directors. The CCMG is the Canadian organisation responsible for certifying laboratory and medical geneticists who provide medical genetics services, and for establishing professional and ethical standards for clinical genetics services in Canada. CONCLUSION: The document describes the ethical and practical factors and suggests a shared responsibility between patients, ordering clinician and laboratory practitioners.

Expansion of the clinical phenotype of the distal 10q26.3 deletion syndrome to include ataxia and hyperemia of the hands and feet.

Distal deletion of the long arm of chromosome 10 is associated with a dysmorphic craniofacial appearance, microcephaly, behavioral issues, developmental delay, intellectual disability, and ocular, urogenital, and limb abnormalities. Herein, we present clinical, molecular, and cytogenetic investigations of four patients, including two siblings, with nearly identical terminal deletions of 10q26.3, all of whom have an atypical presentation of this syndrome. Their prominent features include ataxia, mild-to-moderate intellectual disability, and hyperemia of the hands and feet, and they do not display many of the other features commonly associated with deletions of this region. These results point to a novel gene locus associated with ataxia and highlight the variability of the clinical presentation of patients with deletions of this region.

A duplication CNV that conveys traits reciprocal to metabolic syndrome and protects against diet-induced obesity in mice and men.

The functional contribution of CNV to human biology and disease pathophysiology has undergone limited exploration. Recent observations in humans indicate a tentative link between CNV and weight regulation. Smith-Magenis syndrome (SMS), manifesting obesity and hypercholesterolemia, results from a deletion CNV at 17p11.2, but is sometimes due to haploinsufficiency of a single gene, RAI1. The reciprocal duplication in 17p11.2 causes Potocki-Lupski syndrome (PTLS). We previously constructed mouse strains with a deletion, Df(11)17, or duplication, Dp(11)17, of the mouse genomic interval syntenic to the SMS/PTLS region. We demonstrate that Dp(11)17 is obesity-opposing; it conveys a highly penetrant, strain-independent phenotype of reduced weight, leaner body composition, lower TC/LDL, and increased insulin sensitivity that is not due to alteration in food intake or activity level. When fed with a high-fat diet, Dp(11)17/+ mice display much less weight gain and metabolic change than WT mice, demonstrating that the Dp(11)17 CNV protects against metabolic syndrome. Reciprocally, Df(11)17/+ mice with the deletion CNV have increased weight, higher fat content, decreased HDL, and reduced insulin sensitivity, manifesting a bona fide metabolic syndrome. These observations in the deficiency animal model are supported by human data from 76 SMS subjects. Further, studies on knockout/transgenic mice showed that the metabolic consequences of Dp(11)17 and Df(11)17 CNVs are not only due to dosage alterations of Rai1, the predominant dosage-sensitive gene for SMS and likely also PTLS. Our experiments in chromosome-engineered mouse CNV models for human genomic disorders demonstrate that a CNV can be causative for weight/metabolic phenotypes. Furthermore, we explored the biology underlying the contribution of CNV to the physiology of weight control and energy metabolism. The high penetrance, strain independence, and resistance to dietary influences associated with the CNVs in this study are features distinct from most SNP-associated metabolic traits and further highlight the potential importance of CNV in the etiology of both obesity and MetS as well as in the protection from these traits.

Enriched rearing improves behavioral responses of an animal model for CNV-based autistic-like traits.

Potocki-Lupski syndrome (PTLS; MIM #610883), characterized by neurobehavioral abnormalities, intellectual disability and congenital anomalies, is caused by a 3.7-Mb duplication in 17p11.2. Neurobehavioral studies determined that ∼70-90% of PTLS subjects tested positive for autism or autism spectrum disorder (ASD). We previously chromosomally engineered a mouse model for PTLS (Dp(11)17/+) with a duplication of a 2-Mb genomic interval syntenic to the PTLS region and identified consistent behavioral abnormalities in this mouse model. We now report extensive phenotyping with behavioral assays established to evaluate core and associated autistic-like traits, including tests for social abnormalities, ultrasonic vocalizations, perseverative and stereotypic behaviors, anxiety, learning and memory deficits and motor defects. Alterations were identified in both core and associated ASD-like traits. Rearing this animal model in an enriched environment mitigated some, and even rescued selected, neurobehavioral abnormalities, suggesting a role for gene-environment interactions in the determination of copy number variation-mediated autism severity.

Frequency of nonallelic homologous recombination is correlated with length of homology: evidence that ectopic synapsis precedes ectopic crossing-over.

Genomic disorders constitute a class of diseases that are associated with DNA rearrangements resulting from region-specific genome instability, that is, genome architecture incites genome instability. Nonallelic homologous recombination (NAHR) or crossing-over in meiosis between sequences that are not in allelic positions (i.e., paralogous sequences) can result in recurrent deletions or duplications causing genomic disorders. Previous studies of NAHR have focused on description of the phenomenon, but it remains unclear how NAHR occurs during meiosis and what factors determine its frequency. Here we assembled two patient cohorts with reciprocal genomic disorders; deletion associated Smith-Magenis syndrome and duplication associated Potocki-Lupski syndrome. By assessing the full spectrum of rearrangement types from the two cohorts, we find that complex rearrangements (those with more than one breakpoint) are more prevalent in copy-number gains (17.7%) than in copy-number losses (2.3%); an observation that supports a role for replicative mechanisms in complex rearrangement formation. Interestingly, for NAHR-mediated recurrent rearrangements, we show that crossover frequency is positively associated with the flanking low-copy repeat (LCR) length and inversely influenced by the inter-LCR distance. To explain this, we propose that the probability of ectopic chromosome synapsis increases with increased LCR length, and that ectopic synapsis is a necessary precursor to ectopic crossing-over.

Circadian abnormalities in mouse models of Smith-Magenis syndrome: evidence for involvement of RAI1.

Smith-Magenis syndrome (SMS; OMIM 182290) is a genomic disorder characterized by multiple congenital anomalies, intellectual disability, behavioral abnormalities, and disordered sleep resulting from an ~3.7 Mb deletion copy number variant (CNV) on chromosome 17p11.2 or from point mutations in the gene RAI1. The reciprocal duplication of this region results in another genomic disorder, Potocki-Lupski syndrome (PTLS; OMIM 610883), characterized by autism, intellectual disability, and congenital anomalies. We previously used chromosome-engineering and gene targeting to generate mouse models for PTLS (Dp(11)17/+), and SMS due to either deletion CNV or gene knock-out (Df(11)17-2/+ and Rai1(+/-) , respectively) and we observed phenotypes in these mouse models consistent with their associated human syndromes. To investigate the contribution of individual genes to the circadian phenotypes observed in SMS, we now report the analysis of free-running period lengths in Rai1(+/-) and Df(11)17-2/+ mice, as well as in mice deficient for another known circadian gene mapping within the commonly deleted/duplicated region, Dexras1, and we compare these results to those previously observed in Dp(11)17/+ mice. Reduced free-running period lengths were seen in Df(11)17-2/+, Rai1(+/-) , and Dexras1(-/-) , but not Dexras1(+/-) mice, suggesting that Rai1 may be the primary gene underlying the circadian defects in SMS. However, we cannot rule out the possibility that cis effects between multiple haploinsufficient genes in the SMS critical interval (e.g., RAI1 and DEXRAS1) either exacerbate the circadian phenotypes observed in SMS patients with deletions or increase their penetrance in certain environments. This study also confirms a previous report of abnormal circadian function in Dexras1(-/-) mice.

Opposing phenotypes in mice with Smith-Magenis deletion and Potocki-Lupski duplication syndromes suggest gene dosage effects on fluid consumption behavior.

A quantitative long-term fluid consumption and fluid-licking assay was performed in two mouse models with either an ∼2 Mb genomic deletion, Df(11)17, or the reciprocal duplication copy number variation (CNV), Dp(11)17, analogous to the human genomic rearrangements causing either Smith-Magenis syndrome [SMS; OMIM #182290] or Potocki-Lupski syndrome [PTLS; OMIM #610883], respectively. Both mouse strains display distinct quantitative alterations in fluid consumption compared to their wild-type littermates; several of these changes are diametrically opposing between the two chromosome engineered mouse models. Mice with duplication versus deletion showed longer versus shorter intervals between visits to the waterspout, generated more versus less licks per visit and had higher versus lower variability in the number of licks per lick-burst as compared to their respective wild-type littermates. These findings suggest that copy number variation can affect long-term fluid consumption behavior in mice. Other behavioral differences were unique for either the duplication or deletion mutants; the deletion CNV resulted in increased variability of the licking rhythm, and the duplication CNV resulted in a significant slowing of the licking rhythm. Our findings document a readily quantitated complex behavioral response that can be directly and reciprocally influenced by a gene dosage effect.

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment.

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through ß1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/ß1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.

A rare case of pediatric lipoma with t(9;12)(p22;q14) and evidence of HMGA2-NFIB gene fusion.

Lipoma is a benign tumor, typically of adulthood, with characteristic cytogenetic findings, including rearrangement of 12q13-15; these rearrangements often lead to the fusion of the HMGA2 gene at this locus to the transcriptional regulatory domain of its fusion partner, resulting in neomorphic activity that presumably facilitates the neoplastic process. Herein, we report a rare case of pediatric lipoma with t(9;12)(p22;q14) and evidence of HMGA2-NFIB gene fusion in a 9 year-old boy. This case provides further evidence of the link between NFIB rearrangement and early-onset, deep-seated lipomatous tumors.

Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation.

Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7(-/-) satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

A functional role for structural variation in metabolism.

A contribution of structural genomic variation to the heritability of complex metabolic phenotypes was illuminated by the recent characterization of chromosome-engineered mouse models for genomic disorders associated with metabolic dysfunction. Herein we discuss our study, "A duplication CNV that conveys traits reciprocal to metabolic syndrome and protects against diet-induced obesity in mice and men," which describes the opposing metabolic phenotypes of mouse models for two prototypical genomic disorders,1,2 Smith-Magenis syndrome (SMS) and Potocki-Lupski syndrome (PTLS). SMS and PTLS are caused by reciprocal deletion or duplication copy number variations (CNVs), respectively, on chromosome 17p11.2. The implications of the results of this study and the potential relevance of these findings for future studies in the field of metabolism are discussed.