. 2024 Apr 11;5(2):100273.

doi: 10.1016/j.xhgg.2024.100273. Epub 2024 Jan 30.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features

Morad Ansari¹, Kamli N W Faour², Akiko Shimamura³, Graeme Grimes⁴, Emeline M Kao⁵, Erica R Denhoff⁵, Ana Blatnik⁶, Daniel Ben-Isvy⁷, Lily Wang⁷, Benjamin M Helm⁸, Helen Firth⁹, Amy M Breman⁸, Emilia K Bijlsma¹⁰, Aiko Iwata-Otsubo⁸, Thomy J L de Ravel¹¹, Vincent Fusaro¹², Alan Fryer¹³, Keith Nykamp¹², Lara G Stühn¹⁴, Tobias B Haack¹⁴, G Christoph Korenke¹⁵, Panayiotis Constantinou¹⁶, Kinga M Bujakowska¹⁷, Karen J Low¹⁸, Emily Place¹⁷, Jennifer Humberson¹⁹, Melanie P Napier²⁰, Jessica Hoffman²⁰, Jane Juusola²⁰, Matthew A Deardorff²¹, Wanqing Shao²², Shira Rockowitz²³, Ian Krantz²⁴, Maninder Kaur²⁴, Sarah Raible²⁴, Victoria Dortenzio²⁴, Sabine Kliesch²⁵, Moriel Singer-Berk²⁶, Emily Groopman²⁷, Stephanie DiTroia²⁶, Sonia Ballal²⁸, Siddharth Srivastava²⁹, Kathrin Rothfelder³⁰, Saskia Biskup³¹, Jessica Rzasa³², Jennifer Kerkhof³², Haley McConkey³², Bekim Sadikovic³², Sarah Hilton³³, Siddharth Banka³⁴, Frank Tüttelmann³⁵, Donald F Conrad³⁶, Anne O'Donnell-Luria³⁷, Michael E Talkowski³⁸, David R FitzPatrick⁴, Philip M Boone³⁹

Affiliations

¹ South East Scotland Genetic Service, Western General Hospital, Edinburgh, UK; MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
² Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA.
³ Division of Hematology and Oncology, Boston Children's Hospital, Boston, MA, USA.
⁴ MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
⁵ Institutional Centers for Clinical and Translational Research, Boston Children's Hospital, Boston, MA, USA.
⁶ MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK; Department of Clinical Cancer Genetics, Institute of Oncology Ljubljana, Ljubljana, Slovenia.
⁷ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Division of Medical Sciences, Harvard Medical School, Boston, MA, USA.
⁸ Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA.
⁹ Clinical Genetics, Addenbrooke's Hospital, Cambridge University Hospitals, Cambridge, UK.
¹⁰ Department of Clinical Genetics, Leiden University Medical Centre, Leiden, the Netherlands.
¹¹ Centre for Human Genetics, UZ Leuven/Leuven University Hospitals, Leuven, Belgium.
¹² Invitae, San Francisco, CA, USA.
¹³ Department of Clinical Genetics, Alder Hey Children's Hospital Liverpool, Liverpool, UK.
¹⁴ Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.
¹⁵ Department of Neuropaediatric and Metabolic Diseases, University Children's Hospital Oldenburg, Oldenburg, Germany.
¹⁶ West of Scotland Centre for Genomic Medicine, Queen Elizabeth University Hospital, Glasgow, UK.
¹⁷ Massachusetts Eye and Ear Infirmary, Boston, MA, USA.
¹⁸ University Hospitals Bristol and Weston NHS Foundation Trust, Bristol, UK; University of Bristol, Bristol, UK.
¹⁹ University of Virginia Health System, Charlottesville, VA, USA.
²⁰ GeneDx, Gaithersburg, MD, USA.
²¹ Departments of Pathology and Pediatrics, Children's Hospital Los Angeles and University of Southern California, Los Angeles, CA, USA.
²² Research Computing, Information Technology, Boston Children's Hospital, Boston, MA, USA.
²³ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Research Computing, Information Technology, Boston Children's Hospital, Boston, MA, USA; The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA, USA.
²⁴ Children's Hospital of Philadelphia, Philadelphia, PA, USA.
²⁵ Department of Clinical and Surgical Andrology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany.
²⁶ Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
²⁷ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
²⁸ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Division of Gastroenterology, Boston Children's Hospital, Boston, MA, USA.
²⁹ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Divison of Neurology, Boston Children's Hospital, Boston, MA, USA.
³⁰ Zentrum für Humangenetik, Tübingen, Germany.
³¹ Zentrum für Humangenetik, Tübingen, Germany; Center for Genomics and Transcriptomics (CeGaT), Tübingen, Germany.
³² Molecular Diagnostics Program and Verspeeten Clinical Genome Centre, London Health Sciences Centre, London, ON, Canada.
³³ Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester, UK.
³⁴ Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester, UK; Division of Evolution, Infection, and Genomics, School of Biological Sciences, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UK.
³⁵ Institute of Reproductive Genetics, University of Münster, Münster, Germany.
³⁶ Division of Genetics, Oregon National Primate Research Center, Oregon Health and Science University, Portland, OR, USA; Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland, OR, USA.
³⁷ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³⁸ Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
³⁹ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address: philip.boone@childrens.harvard.edu.

PMID: 38297832
PMCID: PMC10876629
DOI: 10.1016/j.xhgg.2024.100273

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features

Morad Ansari et al. HGG Adv. 2024.

. 2024 Apr 11;5(2):100273.

doi: 10.1016/j.xhgg.2024.100273. Epub 2024 Jan 30.

Authors

Affiliations

¹ South East Scotland Genetic Service, Western General Hospital, Edinburgh, UK; MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
² Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA.
³ Division of Hematology and Oncology, Boston Children's Hospital, Boston, MA, USA.
⁴ MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
⁵ Institutional Centers for Clinical and Translational Research, Boston Children's Hospital, Boston, MA, USA.
⁶ MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK; Department of Clinical Cancer Genetics, Institute of Oncology Ljubljana, Ljubljana, Slovenia.
⁷ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Division of Medical Sciences, Harvard Medical School, Boston, MA, USA.
⁸ Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA.
⁹ Clinical Genetics, Addenbrooke's Hospital, Cambridge University Hospitals, Cambridge, UK.
¹⁰ Department of Clinical Genetics, Leiden University Medical Centre, Leiden, the Netherlands.
¹¹ Centre for Human Genetics, UZ Leuven/Leuven University Hospitals, Leuven, Belgium.
¹² Invitae, San Francisco, CA, USA.
¹³ Department of Clinical Genetics, Alder Hey Children's Hospital Liverpool, Liverpool, UK.
¹⁴ Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.
¹⁵ Department of Neuropaediatric and Metabolic Diseases, University Children's Hospital Oldenburg, Oldenburg, Germany.
¹⁶ West of Scotland Centre for Genomic Medicine, Queen Elizabeth University Hospital, Glasgow, UK.
¹⁷ Massachusetts Eye and Ear Infirmary, Boston, MA, USA.
¹⁸ University Hospitals Bristol and Weston NHS Foundation Trust, Bristol, UK; University of Bristol, Bristol, UK.
¹⁹ University of Virginia Health System, Charlottesville, VA, USA.
²⁰ GeneDx, Gaithersburg, MD, USA.
²¹ Departments of Pathology and Pediatrics, Children's Hospital Los Angeles and University of Southern California, Los Angeles, CA, USA.
²² Research Computing, Information Technology, Boston Children's Hospital, Boston, MA, USA.
²³ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Research Computing, Information Technology, Boston Children's Hospital, Boston, MA, USA; The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA, USA.
²⁴ Children's Hospital of Philadelphia, Philadelphia, PA, USA.
²⁵ Department of Clinical and Surgical Andrology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany.
²⁶ Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
²⁷ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
²⁸ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Division of Gastroenterology, Boston Children's Hospital, Boston, MA, USA.
²⁹ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Divison of Neurology, Boston Children's Hospital, Boston, MA, USA.
³⁰ Zentrum für Humangenetik, Tübingen, Germany.
³¹ Zentrum für Humangenetik, Tübingen, Germany; Center for Genomics and Transcriptomics (CeGaT), Tübingen, Germany.
³² Molecular Diagnostics Program and Verspeeten Clinical Genome Centre, London Health Sciences Centre, London, ON, Canada.
³³ Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester, UK.
³⁴ Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester, UK; Division of Evolution, Infection, and Genomics, School of Biological Sciences, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester, UK.
³⁵ Institute of Reproductive Genetics, University of Münster, Münster, Germany.
³⁶ Division of Genetics, Oregon National Primate Research Center, Oregon Health and Science University, Portland, OR, USA; Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland, OR, USA.
³⁷ Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³⁸ Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
³⁹ Cornelia de Lange Syndrome and Related Disorders Clinic, Boston Children's Hospital, Boston, MA, USA; Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address: philip.boone@childrens.harvard.edu.

PMID: 38297832
PMCID: PMC10876629
DOI: 10.1016/j.xhgg.2024.100273

Abstract

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

Keywords: CdLS3; Cornelia de Lange syndrome; LoF; SMC3; cohesin; loss-of-function.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests M.E.T. is supported by research funding and/or reagents from Illumina, Microsoft, Ionis Therapeutics, and Levo Therapeutics. M.P.N., J.H., and J.J. are employees of GeneDx.

Figures

**Figure 1**
*SMC3* pLoF variants (A) pLoF SNVs mapped to the SMC3 protein sequence. Subject cases are in black (top). The c.430-1G>T splice variant (individual 1) immediately precedes exon 8, which, if skipped, would result in a shift of reading frame; however, splicing may be rescued (see text). The p.(Gly1217MetfsTer39) variant (individual 10) is in the final exon and is predicted to escape nonsense-mediated decay. gnomAD pLoF variants passing quality filters are in gray (bottom). UKBB pLoF variants are in brown (bottom). Domains via UniProt. Arrowhead denotes the C-terminal end (equivalent to codon 211) of the minor transcript described in Figure 3. The diamond denotes the point after which nonsense-mediated decay may be escaped (codon 1,176). The 3′ region homologous to the *SMC3P1* pseudogene (codons 974–1,217) is in orange. (B) Somatic *SMC3* pLoF SNVs among 13,714 tumors from the NCI GDC collection (https://portal.gdc.cancer.gov/genes/ENSG00000108055). (C) Regional missense constraint over *SMC3*, via an analysis of gnomAD. The transition point is in codon 390 (exon 13 of 29). (D) Case deletions (based on genome build GRCh37/hg19). Of the OMIM phenotype-associated genes in this region (green), none are validated dominant haploinsufficient disease genes for disorders that include developmental delay or dysmorphic features.

**Figure 2**
Growth and developmental milestones associated with *SMC3* pLoF compared with pathogenic missense/in-frame indels (A) Growth measurements adjusted for age and sex (Z score) are shown for *SMC3* pLoF and missense/in-frame indel cases. Postnatal measurements are at the time of study enrollment/sampling. (B) Age at which developmental milestones were reached for the 2 categories of *SMC3* variants (LoF vs. pathogenic missense/in-frame indel). The 25th/50th/75th centiles of population data (Denver II, via DECIPHER) are shown at the right of each figure. Missense/in-frame indel data were obtained from Reference . Plots are standard boxplots. Statistical comparisons are the Mann-Whitney U test (Wilcoxon).

**Figure 3**
Gene expression in WT and *SMC3*^+/− tissues (A) GTEx data demonstrate that only a single, full-length *SMC3* isoform (ENST00000361804.4) is expressed at an appreciable level in any adult human tissue represented (https://gtexportal.org/home/gene/SMC3). (B) pLoF *SMC3* variants (LoF; nonsense, frameshift indel, splice site) in hematopoietic and lymphoid tumors correlate with decreased *SMC3* gene expression, to a mean of 71.6% of WT (p = 0.018, Mann-Whitney U test). Data derived from the CCLE (http://xenabrowser.net). Plot is standard boxplot. (C and D) Comparison of genes with altered expression in early postnatal *Smc3*^+/− mouse cortex and those altered in a late prenatal *Nipbl*^+/− mouse whole brain. (C) Fifty of 406 (12.3%) DEGs at any age (postnatal day [P]1, P3, P7, P14, P21) in *Smc3*^+/− mice overlap with 3,507 DEGs in E17.5 *Nipbl*^+/− mice (top); 9 of 58 (15.5%) DEGs in 2+ ages in *Smc3*^+/− mice overlap with those same 3,507 genes (bottom). (D) Neither of these is significantly different from chance (3,507/30,686 total genes; 11.4%) (any age p = 0.632, chi-square test of proportions; 2+ ages p = 0.441). See subjects and methods for derivation of denominator of 30,686 genes and discussion for a commentary on methodological differences between the 2 models. (E) Minimal displacement embedding of pseudobulk-averaged, z normalized expression profiles from a genome-wide Perturb-Seq experiment in K562 CML cells (plot generated via https://gwps.wi.mit.edu/). Each dot is 1 of 1,973 genes with strong transcriptomic signatures. The *SMC3* knockdown transcriptional disturbances group tightly with those of other cohesin genes (pink) and even an epigenetic regulator occasionally mutated in CdLS-like subjects (*EP300*).

**Figure 4**
Global DNA methylation pattern of *SMC3* pLoF compared with that of CdLS Multidimensional scaling plot of global methylation signature (episignature) of blood-derived DNA. *SMC3* pLoF subject 5 (p.Arg360Ter, purple) and an additional individual (c.2635C>T, p.(Arg879Ter), orange) do not plot with individuals clinically diagnosed with CdLS (red), instead plotting among the control population (green).

**Figure 5**
Hypothesized phenotypic spectra of *SMC3* variant types Missense (ms)/in-frame (i.f.) indel variants, via an apparent dominant-negative mechanism as supported by prior literature, carry the most severe *SMC3*-associated phenotype, with many subjects appearing to have atypical CdLS. LoF variants generally carry a less severe phenotype, with only some individuals being recognized to have CdLS and some individuals lacking key phenotypic features (growth retardation, developmental delay, characteristic facial dysmorphism). We hypothesize that nullimorphic missense and hypomorphic missense variants also exist.

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Heterozygous loss-of-function SMC3 variants are associated with variable and incompletely penetrant growth and developmental features.
Ansari M, Faour KNW, Shimamura A, Grimes G, Kao EM, Denhoff ER, Blatnik A, Ben-Isvy D, Wang L, Helm BM, Firth H, Breman AM, Bijlsma EK, Iwata-Otsubo A, de Ravel TJL, Fusaro V, Fryer A, Nykamp K, Stühn LG, Haack TB, Korenke GC, Constantinou P, Bujakowska KM, Low KJ, Place E, Humberson J, Napier MP, Hoffman J, Juusola J, Deardorff MA, Shao W, Rockowitz S, Krantz I, Kaur M, Raible S, Kliesch S, Singer-Berk M, Groopman E, DiTroia S, Ballal S, Srivastava S, Rothfelder K, Biskup S, Rzasa J, Kerkhof J, McConkey H, O'Donnell-Luria A, Sadikovic B, Hilton S, Banka S, Tüttelmann F, Conrad D, Talkowski ME, FitzPatrick DR, Boone PM. Ansari M, et al. medRxiv [Preprint]. 2023 Sep 28:2023.09.27.23294269. doi: 10.1101/2023.09.27.23294269. medRxiv. 2023. Update in: HGG Adv. 2024 Apr 11;5(2):100273. doi: 10.1016/j.xhgg.2024.100273. PMID: 37808847 Free PMC article. Updated. Preprint.

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Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features

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Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features

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