Hypomorphic GINS3 variants alter DNA replication and cause Meier-Gorlin syndrome

The eukaryotic CDC45/MCM2-7/GINS (CMG) helicase unwinds the DNA double helix during DNA replication. The GINS subcomplex is required for helicase activity and is, therefore, essential for DNA replication and cell viability. Here, we report the identification of 7 individuals from 5 unrelated families presenting with a Meier-Gorlin syndrome–like (MGS-like) phenotype associated with hypomorphic variants of GINS3, a gene not previously associated with this syndrome. We found that MGS-associated GINS3 variants affecting aspartic acid 24 (D24) compromised cell proliferation and caused accumulation of cells in S phase. These variants shortened the protein half-life, altered key protein interactions at the replisome, and negatively influenced DNA replication fork progression. Yeast expressing MGS-associated variants of PSF3 (the yeast GINS3 ortholog) also displayed impaired growth, S phase progression defects, and decreased Psf3 protein stability. We further showed that mouse embryos homozygous for a D24 variant presented intrauterine growth retardation and did not survive to birth, and that fibroblasts derived from these embryos displayed accelerated cellular senescence. Taken together, our findings implicate GINS3 in the pathogenesis of MGS and support the notion that hypomorphic variants identified in this gene impaired cell and organismal growth by compromising DNA replication.


Supplementary
P2-4: A family with three children affected by an MGS-like phenotype and homozygous c.70G>A (p.D24N) GINS3 variants was identified through GeneMatcher. DNA of the three affected and eight unaffected siblings, as well as the mother, were genotyped using Axiom SNP Chip platform to determine the candidate autozygome as has been described previously (6).
Exome sequencing was performed on the index using TruSeq Exome Enrichment kit (Illumina) following the manufacturer's protocol. The coding/splicing homozygous exome sequencing variants that are within the candidate autozygome were considered as likely candidates if present with frequency of <0.1% in publicly available variant databases (1000 Genomes, NHLBI Exome Sequencing Project, Exome Variant Server, and Genome Aggregation Database [gnomAD]) and a database of in-house ethnically matched exomes (Saudi Human Genome Program; totaling 2,379 exomes), and predicted to be pathogenic by the two in-silico prediction modules PolyPhen, SIFT and had a CADD score of >15. P5-7: DNA libraries were constructed using Agilent SureSelect version 5 kits. Quality control for insert size and library representation were performed using an Agilent Tapestation and qPCR respectively. Sequencing (BGI Europe) was carried out using an Illumina HiSeq 4000 to an average depth of coverage of 150x with automated adapter trimming of the fastq sequences.
DNA sequence quality metrics were carried out using FASTQC version: 0.11.7. Alignment, quality filtering and variant identification were undertaken using commercially available algorithms. Human reference assemblies were aligned against GRCh37.p13. QIAGEN Clinical Insight -Interpret software was used in sequence analysis and interpretation. The application was  Table 2. Sequences of primers used in this study. Table 2 continued.

Primer name
GINS3cr-FLAG ORFs into the genome at a consistent location; once integrated, the GINS3cr-FLAG ORFs were under the control of a tetracycline/doxycycline-inducible promoter.
Selection for plasmid was maintained for 3 days using 1 µg/mL puromycin and then removed; induction of GINS3cr-FLAG by addition of 1µg/mL doxycycline every 48 hours was maintained continuously after CRISPR transfection. After 5 weeks, individual cells were isolated by flow cytometry using a BD Aria III Sorter (BD) in order to obtain isogenic cell lines.

Allelic balance and gene isoforms
Droplet digital PCR (ddPCR) was performed using the QX200 Droplet Digital PCR system (Bio-Rad Laboratories). Custom TaqMan probes (part 4331349, Life Technologies) were designed to using Primer Express 3.0 software (Life Technologies) to target the variant alleles c71A>G (GINS3c71-F, GINS3c71-R, GINS3c71-VIC, GINS3c71-FAM) and c245G>A The reaction mix consisted of ddPCR SuperMix for Probes (Bio-Rad Laboratories) and the GINS3c71 or GINSc245 SNP genotyping oligos. Assays were validated by temperature gradient to ensure optimal separation of alternate and reference-allele-containing droplets.
Cycling conditions for the reaction were 95C for 10 lysis buffer was added. The samples were then centrifuged at 16 500 x g for 10 minutes at 4°C. Fifty microliters of pre-washed Streptavidin Sepharose High Performance beads (Millipore Sigma) was added to each sample, after which samples were left to rotate overnight at 4°C.
Beads were recovered by centrifugation at 1000 x g for 5 minutes at 4°C and washed by resuspending in 1 ml of wash buffer (8M Urea, 50 mM HEPES, pH 7.4) with 8 minutes of rotation at room temperature. Beads were again recovered through a 2 min centrifugation at 1000 x g, and the washing steps repeated 4 times, after which the beads were transferred to fresh lowbinding tubes.

Protein digestion & LC-MS/MS analysis
Buffers were prepared using MS-grade water (ThermoFisher Scientific). Beads were enzymes were Trypsin (K/R not before P); variable modifications included in the analysis were methionine oxidation, protein N-terminal acetylation and protein carbamylation (K, N-terminal).
A mass tolerance of 10 ppm was used for precursor ions and a tolerance of 20 ppm was used for fragment ions. Identification values "PSM FDR", "Protein FDR" and "Site decoy fraction" were set to 0.05. Minimum peptide count was set to 1. Label-Free-Quantification (LFQ) was also selected with a LFQ minimal ratio count of 2. Both the "Second peptides" and "Match between runs" options were also allowed.
Results were sorted using Prostar (Proteomics statistical analysis with R). Proteins positive for at least one of the "Reverse", "Only.identified.by.site" or "Potential.contaminant" categories were eliminated, as well as proteins identified from a single peptide. An Mathworks, MA) was used. Briefly, an Otsu threshold was applied to DAPI images to generate a binary mask that could then be applied to all channels; small objects were removed, holes were filled, and adjacent nuclei were separated using a watershed algorithm. All masks were examined manually and any incorrectly identified nuclei were discarded. Masked images were smoothed using a bandpass algorithm, and foci were identified as local maxima. To measure intensity within foci, local maxima were expanded to a radius of 3 pixels and used to generate a binary mask for each nucleus; average focal intensity per nucleus was obtained by taking the average of all non-zero pixels. A minimum of 50 cells were analyzed for each cell line.

Yeast strain generation
To generate point mutations in the PSF3 gene, the kanMX6 gene cassette flanked by loxP sites was amplified from plasmid pOM10 (11)  Positive transformants were selected on SC-ura medium, and isolates were cultured on SC-ura medium containing galactose as a carbon source to induce the expression of Cre and catalyze the removal of the kanMX6 cassette from the 5' end of the PSF3 gene. After confirmation of kanMX6 cassette removal by PCR, isolates were transferred to minimal medium for sporulation.

Yeast doubling time & S phase progression
Yeast cultures were inoculated at 0.001 OD in YPAD in a 96-well plate, and culture density was measured at 630 OD every 30 minutes for 24 hours. Doubling time was calculated based on the rate of growth during exponential growth phase for 8 separate cultures.
To synchronize yeast cells in G1 phase, actively growing cultures were diluted to 0.18 OD and incubated 3 hours in YPAD with 5 µg/mL alpha factor at 30˚C with shaking; an additional 5 µg/mL alpha factor was added after 1.5 hours. Synchronization was confirmed by observing schmoo morphology in upwards of 50% of cells. To release from G1 arrest, cells were washed once with YPAD, then cultured in fresh YPAD medium with 50 µg/mL pronase at 0.75 OD and 30˚C with shaking; culture samples were collected at indicated time points.
To fix samples, ~0.2 OD of culture was mixed with ethanol (final concentration = 70%) and incubated at room temperature for a minimum of 20 minutes before being stored at 4˚C. To prepare samples for analysis by flow cytometry, samples were incubated 3 hours at 42˚C with 400 µg/mL RNAse, followed by 30 minutes at 50˚C with 1 mg/mL Proteinase K. Samples were sonicated 10 seconds at 30% cycle duty, then incubated with Sytox Green (Invitrogen) for a minimum of 10 minutes prior to flow cytometry using a FACSCalibur (BD Biosciences).
Analysis was performed using FlowJo software (BD Biosciences) with the Watson (Pragmatic) model for cell cycle analysis.

DNA combing analysis
One million cells were seeded in 100 mm dishes 24 hours prior to nucleotide analog