Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy

Abstract

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning – incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.

Introduction

Despite the fundamental nature of DNA replication and cell division, inherited variants in genes involved in these processes are an underlying cause of human disease. Whilst these syndromes usually display unique clinical features that define them diagnostically, they typically exhibit common neurodevelopmental deficits, such as severe microcephaly and pre- and post-natal growth retardation^1,2,3. As such, many of these syndromes can be collectively referred to as microcephalic dwarfism (MD) disorders. This constellation of conditions includes Meier-Gorlin Syndrome, Seckel Syndrome Spectrum Disorders, Bloom Syndrome and Microcephalic Osteodysplastic Primordial Dwarfism type II and can be broadly classified as having deficiencies in one of three cellular processes: DNA replication, DNA repair, and mitotic cell division^1,2,3,4. Although mechanistically distinct, the common clinical phenotypes exhibited by these diseases are thought to result from a reduction in cellular proliferation and/or excessive cell death in the developing embryo, which reduces the number of cells available to maintain normal foetal growth⁵. Cells from these patients often exhibit signs of increased genome instability, such as micronuclei and/or elevated chromosome breakage. A distinct subgroup of these syndromes exhibit rare cytogenetic anomalies, for example, mosaic variegated aneuploidy syndrome (MVA)^6,7,8 caused by variants in the spindle assembly checkpoint genes BUB1B, CEP57 and TRIP13, or rail-road chromosomes and premature chromatid separation (PCS) associated with Warsaw Breakage Syndrome (WABS) and Cornelia de Lange syndrome, caused by variants in the helicase DDX11 and components of SMC1/3 cohesin complex respectively^9,10. Whilst, the presence of these chromosomal abnormalities is a useful diagnostic tool they can also help dissect the cellular mechanisms underlying the disease pathology.

Here, we report 11 patients with a neurodevelopmental disorder overlapping clinically with MVA and Fanconi Anemia (FA) with pathogenic variants in SLF2 and SMC5, two components of the recently discovered RAD18-SLF1/2-SMC5/6 genome stability pathway¹¹. The precise function of the SMC5/6 complex remains enigmatic, however, it has been linked to a number of fundamental processes, including DNA transcription, DNA replication, DNA repair and chromosome segregation^12,13. Evidence suggests that the primary function of this complex occurs during DNA replication to stabilize stalled forks, suppress the activity of pro-recombination factors and promote efficient replication through difficult-to-replicate and/or repetitive regions of the genome, such as rDNA and telomeres¹⁴. In contrast, the function of SLF1 and SLF2 remain unclear, other than a reported role in recruiting the SMC5/6 complex to sites of DNA damage¹¹.

Analysis of SLF2 and SMC5 patient-derived cell lines revealed spontaneous replication stress and multiple mitotic abnormalities that give rise to a unique, diagnostically relevant, genome instability phenotype consisting of segmented, dicentric and rail-road chromosomes, and mosaic variegated hyperploidy (MVH). The underlying basis for this chromosomal instability is not fully understood, but our data suggest that it may arise, in part, from the failed resolution of aberrant DNA structures during S-phase, such as G-quadruplexes (G4), potentially leading to a combination of under-replicated DNA and unresolved recombination intermediates persisting through to mitosis. Together, these data demonstrate that despite a hitherto unknown role as a core component of the SMC5/6 complex, SLF2 is essential for the SMC5/6 cohesin-like complex to maintain genome stability by regulating both DNA replication and cell division.

Results

Patients with microcephaly and short stature have biallelic SLF2 (FAM178A) and SMC5 variants

Whole exome sequencing (WES) was carried out on seven  patients (P1, P2, P3, P4-1, P4-2, P5 and P6) from five families, presenting with microcephaly, short stature, mild to severe developmental delay and spontaneous chromosome breakage. After aligning WES reads to the reference genome, variant calling, and filtering for rare variants (MAF <0.005), analysis under a recessive model of inheritance identified biallelic variants in SLF2 (FAM178A) in all seven patients. All identified SLF2 variants segregated amongst family members (with the exception of patients P1 and P5 where parental material was unavailable) and were present at a frequency of <0.5% in the gnomAD database (Fig. 1a, c; Supplementary Data 1–7; Supplementary Fig. 1a). Comparative genomic hybridization (CGH) array analysis carried out on gDNA from patient P5 confirmed the homozygosity of the identified SLF2 variant…