PRIM1 deficiency causes a distinctive primordial dwarfism syndrome

In this study, Parry et al. report PRIM1, encoding the catalytic subunit of DNA primase, as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals and phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component.


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Given functional conservation between yeast and human PRIM1, and to investigate the cellular consequences of the C301R missense mutation we introduced orthologous variants at this codon into S. cerevisiae. Initially, we attempted to introduce both the L309C and L309R mutations into budding yeast Pri1, changing the equivalent residue to that of wild-type human PRIM1 or patient PRIM1 respectively. Despite several attempts, no colonies were found to carry the L309R mutation (0 out of 32 colonies), although 94% (30) of these did have the silent PAM site mutation. In contrast, 50% of colonies (8 out of 16) carried the L309C mutation, all of which showed normal growth (A). This suggested that the L309R mutation but not the L309C mutation may impact on viability. Lower colony numbers for each L309R CRISPR editing attempt (48±8% colonies compared to L309C, n=4 independent attempts) were consistent with this. Therefore, we instead employed a plasmid shuffle method (B, C), which confirmed lethality of this mutation in budding yeast.
(A) Growth is not affected by C-terminal Myc-tag, PAM-site mutant or introduction of the L309C substitution. Normal growth of independent yeast strains with C-terminally Myc-tagged Pri1p-L309C (MRY203/204) or silent PAM site mutations (PAM; MRY201/202), compared to non-tagged wild-type (BY4741) and Myc-tagged parental strains (WT, MRY175). Relative OD 600 values plotted for yeast grown in YPD medium at 30°C and kept in log phase (OD600 = 0.2-0.8), by diluting in fresh medium when necessary.
(B) Diagram outlining methodology for introducing the L309R mutation by CRISPR at the genomic PRI1 locus. Viability of this strain is maintained by expression of an episomal copy of the wild-type PRI1 gene, while the mutation is introduced at the endogenous locus by CRISPR. The episomal gene remains unedited due to a silent mutation at the PAM site preventing editing. Once the L309R mutation has been confirmed to be present, viability of the L309R mutation is tested by plating on 5-FOA medium, which leads to loss of the WT-PRI1 ("recue") plasmid due to negative selection for the URA3 selection cassette.
(C) Yeast strains expressing Pri1-L309R from the endogenous genetic locus and WT-PRI1 from a centromeric URA3 vector, grow normally on SD-Ura-Met plates, but cannot survive on 5-FOA containing medium, regardless of the presence or absence of a C-terminal Myc-tag. On 5-FOA medium only the genomic copy of PRI1 is expressed, due to negative selection against the URA3 vector. Colonies with the L309R mutation (MRY200, MRY206) or with a silent PAM site mutation only (WT; MRY199, MRY205) were streaked out after CRISPR-mediated genome editing of non-tagged (BY4741) and 13myc-tagged Pri1p yeast (MRY175), and grown at 30°C. The same results were obtained for all independent L309R and control strains (Supplemental Table S11). Figure S6: Minigene splicing assay of the c.103+1G>T variant.

Supplemental
(A) Schematic of minigene assay. Genomic DNA spanning exon1 (Ex1) and exon 2 (Ex2) of PRIM1 was cloned into minigene reporter construct and the c.103+1G>T variant introduced by site-directed mutagenesis. Arrows, PCR primers used to assay splicing from cDNA following transfection. Dotted line indicates alternate splicing from the cryptic splice donor (Fig. 3E).
(B) Agarose gel electrophoresis of PCR products generated from the minigene assay. UT, untransfected; WT, wild-type construct; donor, c.103+1G>T variant construct. Right, Schematic of the minigene construct mRNA after splicing. Nine nucleotides of intronic sequence are inserted (red) relative to the wild-type construct ( Figure 3G).  Supplemental Figure S12. The DNA replica�on fork and microcephalic primordial dwarfism (MPD) spectrum disorders. Schema�c of the DNA replica�on fork. Pol α-primase ini�ates DNA replica�on at replica�on origins by synthesis of an RNA primer by PRIM1, which is extended by the DNA synthesising cataly�c subunit of Pol α, POLA1. Pol δ con�nues DNA synthesis a�er the synthesis of the short RNA-DNA hybrid primer. On the leading strand Pol ε takes over from Pol δ to perform the bulk of leading strand DNA synthesis. Subunits of the polymerases with variants iden�fied as causing MPD disorders are underlined.

Relatedness and Inbreeding Coefficient Calculation
Cohort WGS variant data were filtered to include only SNVs. Variants that were rare (allele frequency < 1 % in all gnomAD population groupings), below the VQSR truth sensitivity threshold of 99.9% or overlapping low-complexity or segmentally duplicated regions were removed. Inbreeding coefficients were calculated using vcftools (Danecek et al. 2011) '--het' option. Relatedness was calculated using the KING algorithm (Manichaikul et al. 2010) also with vcftools, using the '--relatedness2' option. A minimum genotype quality score cutoff of 20 (--minGQ 20) was set for both vcftools commands.

Prediction of PRIM1 Substitutions on Protein Stability
Fourteen crystal structures of PRIM1 were obtained from PDBe (4BPU, 4BPW, 4BPX, 4LIK, 4LIL, 4MHQ, 4RR2, 5EXR, 6R4S, 6R4T, 6R4U, 6R5D, 6R5E and 6RB4). Each structure was converted using the FoldX (v5) RepairPDB command with default settings to find the energy minima for each structure. Each repaired PDB file was then analysed using the FoldX BuildModel command to assess the free energy change of the C301R, C301L and C301T substitutions with five replicates (--numberOfRuns=5) and the mean free energy change calculated for each substitution. Visualisation and solvent accessibility analysis was performed using pymol (Schrödinger 2015).

Multiple Sequence Alignments
Multiple sequence alignments of PRIM1 orthologs were generated using Clustal (Larkin et al. 2007) and formatted using Jalview (Waterhouse et al. 2009).

Yeast strains, media and growth conditions
Standard yeast culture media were used: Yeast extract Peptone Dextrose (YPD: 10 g/l yeast extract, 20 g/l bactopeptone, 20 g/l dextrose, 20 g/l agar), Synthetic Defined (SD: 6.7 g/l yeast nitrogen base without amino acids, complete supplement single/double dropout mixture (Formedium), 20 g/l dextrose, 20 g/l agar) and Synthetic Complete (SC: as SD, but with complete supplement mixture, Formedium). Where specified medium was supplemented with hygromycin B (Hyg, 300 mg/l; Calbiochem) or 5-fluoroorotic acid (5-FOA, 1 g/l, Formedium). S. cerevisiae cultures and plates were incubated at 30°C, unless otherwise specified. All strains used in this study were isogenic with BY4741 (Brachmann et al. 1998), and are listed in Supplemental Table S11. In MRY175, the endogenous PRI1 gene was C-terminally tagged with a 13myc tag, using hygromycin resistance for selection (Spiller et al. 2007), making use of a PCR product with 60 bp homology arms. To allow expression of Pri1-GFP from the MET25 promoter on pMAR782 by growing strains on SD medium without methionine, BY4741 and MRY175 were complemented with a 2.65 kbp PCR fragment (S288C genomic DNA used as template) covering MET15 (a.k.a. MET17/MET25), generating MRY215 and MRY216 respectively. Point mutations were introduced into PRI1 by CRISPR, using previously described methods (Laughery et al. 2015) along with pMAR780 or pMAR781 and annealed oligonucleotides (Supplemental Table S9
Immunodetection of proteins transferred to PVDF was performed using the following primary antibodies: anti-Myc (Cell Signaling, 9B11, 1:2,000) and anti-PGK1 antibody (Thermo Fisher Scientific, 22C5D8, 1:2,000; kindly provided by Aziz El Hage). Finally, detection was performed using Amersham ECL Prime Western Blotting Detection Reagent on the ImageQuantLAS4000 (GE Healthcare Life Sciences), or the Odyssey CLx Imaging System (LI-COR Biosciences). Quantifications were performed using ImageQuant TL 7.0 and Image Studio Lite 5.2, respectively.

Cycloheximide chase
Cycloheximide chase experiments were performed using a previously described method (Buchanan et al. 2016) with minor modifications. Briefly, yeast strains were grown in SD-Ura-Met medium at 30˚C until they reached mid log phase (OD600 0.6-0.8), cells pelleted and resuspended in fresh medium at 2.5 OD units per ml. Cycloheximide was added at 250 µg/ml (Alfa Aesar, J66901; stock at 20 mg/ml in ethanol), cells grown at 30˚C with continuous agitation (250 rpm) and 950 µl of cells collected at the appropriate time points. Cell suspensions were mixed with 50 µl ice-cold 20x stop mix (200 mM sodium azide, 5 mg/ml BSA), temporarily stored on ice and processed for immunoblotting. Cell pellets were resuspended in 100 μl distilled water, mixed with 100 μl 0.2 M NaOH and incubated at room temperature for 5 min. Cells were then pelleted, resuspended in 50 μl SDS-loading buffer (50 mM Tris.HCl pH6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.63 M β-mercaptoethanol, bromophenol blue), heated for 10 min at 95˚C and 4 μl of each sample used for SDS-PAGE and immunoblotting as described above.

P1 -female
6th child of 3rd cousin Hungarian parents. Brother also likely affected.
Born small for gestational age at 30 weeks gestation, weight 740 g, length 34 cm, OFC 25 cm. Ventilated for first 4 days of life, then nasal CPAP for a further 8 days.
At 4.5 months of age, severe growth retardation, consistent with severe primordial dwarfism, was evident: weight 1880g; length 40 cm; OFC 30 cm. While there was an absolute microcephaly, short stature was more extreme, leading to an appearance of relative macrocephaly. Examination findings were of a broad high forehead, a late-closing fontanelle, blepharophimosis, small eyes, low-set, underdeveloped ears and a flat nasal bridge. The mouth was straight and horizontal, with overreaching of the upper lip. She had a generalized hypotonia, along with small, lax hands and feet with puffy backs and tapering fingers. Absence of subcutaneous fat was also noted.
Developmentally, she was attentive, but did not attain any motor milestones.
From 5 months of age, she experienced persisting lower-airway obstructive symptoms and chronic cough unresponsive to therapy that progressed to end-stage acute respiratory distress syndrome (ARDS), with Chest X-ray demonstrating a homogenous, increased opacification of the lungs. Death occurred at 7 months of age, due to RSV and parainfluenza 2 bronchiolitis (despite initiation of passive RSV immunization one month prior). A severe sagittal craniosynostosis resulted in marked scaphocephaly and dilated lateral ventricles. This necessitated skull reconstruction surgery at 4 months, and ventriculo-peritoneal shunt at 7 months.
Morphologically, the patient had a high broad forehead with frontal bossing and flat supraorbital ridges. Skull veins were visible through the skin. Facially she had blepharophimosis, small eyes, epicanthus inversus, a broad flat nasal bridge, anteverted nares, a long philtrum and a large and horizontal mouth. The ears were low-set and malformed -aside from the tragi and helices, no other folding could be distinguished. The hands and feet were small with tapering fingers. Skin folds were visible on the limbs, joints were lax, and subcutaneous fat tissue underdeveloped.
Developmentally, she was significantly delayed, at 23 months of age being able to wave "bye", babble, clap hands and roll over. At approaching 5 years of age she was able to sit unsupported and speak 3-4 words. By 5 years growth remained markedly impaired with extreme microcephalic dwarfism evident: weight 6095 g, length 71 cm, OFC 41 cm.
Respiratory involvement appeared minimal, with only increased perihilar density on chest X-ray noted.
Infections accompanied by fever and raised C-reactive protein occurred on several occasions. These included an episode of ventriculitis treated with long-term antibiotics and neurosurgical revision; another due to Rotavirus and Clostridium difficile enteritis. During an episode of antibiotic-treated sepsis aspirin therapy was initiated for a transient thrombocytosis (thrombocytes 1154 x 10 9 /L (Ref.: 150-620 x 10 9 /L)).
Born at 35 weeks gestation, with severe growth retardation, birth weight 990 g, length 38 cm, OFC 28 cm. A diffusely hypertrophic myocardium with mildly dilated right ventricle was noted antenatally. Neonatal respiratory distress treated with surfactant, mechanical ventilation and intravenous antibiotics, (perinatal infection not confirmed), further complicated by a right upper lobe atelectasis on day 8. Neonatal hyperbilirubinemia resolved with phototherapy and anemia treated by red blood cell transfusion (Hgb 78 g/L, Ref: 110-170 g/L).
Developmentally, he had a persisting generalized hypotonia, such that by 1 year he was able to roll over but not able to sit or achieve head control, at which time he was able to smile, and recognize familiar faces. He was attentive and grasped objects. Growth continued to be severely restricted postnatally: at 15 months of age, weight 3000 g, length 55 cm, OFC 38 cm. Feeding was challenging and required occasional nasogastric tube-feeding.
Dysmorphic facial features evident at birth became more prominent with age. There was a relative macrocephaly, prominent occiput, triangular-shaped face with a broad high forehead and frontal bossing. Ears were low-set and very small, the nasal root depressed with a broad nasal base. He had a long philtrum, horizontal, relatively large mouth with prominent, pouting lips and micrognathia. Blepharophimosis was evident with short palpebral fissures. The hands and feet were small, the backs of the hands and feet were puffy, and the fingers were distally tapering. Patellae were palpable. There was cryptorchism.
Respiratory difficulties persisted postnatally, fluctuating in severity, with tachypnoea, wheeze and bilateral vesicular sounds evident on auscultation. Radiographically, increased right perihilar increased density was frequently evident.
Additionally, recurrent episodes of vomiting ± diarrhea ± fever started at 7 months of age and occurring on a total of nine occasions. At 2 years of age adenovirus infection led to acute respiratory distress syndrome (ARDS) and his death.
There was a persisting mild lymphopenia, lymphocyte absolute count 1.96 and at 8 months, abdominal ultrasonography revealed a slightly enlarged liver with inhomogeneous structure, with scattered 5-6 mm hypoechogenic islets. Abdominal MRI did not reveal pathological changes in the viscera. Brain MRI was in keeping with 'microcephaly with simplified gyri (MSG) (Adachi et al. 2011), with a medially thin corpus callosum, broad gyri with narrow sulci, and moderately thin cortex reported. Cardiac status improved postnatally, with normal cardiac function and wall-thickness at 20 months of age. Autopsy: Macronodular hepatic cirrhosis, atrophic large intestine mucosa. In the lung vascular congestion and granular changes in keeping with ARDS. Adrenal hemorrhage, and mild thickening of the left-ventricular wall (7 mm), reflecting a minimal grade myocardial hypertrophy.

P4 -male
Brother of P3, 4th child of Hungarian parents Born by elective Caesarian section at 38 weeks gestation weighing1640 g, length 40.5 cm, OFC 29 cm. Intrauterine growth restriction evident from 23rd week of the pregnancy. Primordial dwarfism and similarities to deceased brother, including cryptorchidism noted immediately after birth. Neonatally, supplemental nasogastric tube feeding failed to ameliorate failure to thrive. Hypothyroidism diagnosed at 5 weeks, sTSH 11.17 mu/L (Ref.: 6-10 mU/l), fT4, normal: 13.8 pmol/L. Cardiological assessment also identified a 3-4 mm secundum-type atrial defect and mildly dilated right ventricle.
Development was severely delayed, accompanied by generalized muscular hypotonia. Rolling onto to his side was achieved, but no other gross motor milestones were attained. Yet, the patient was attentive, his cognitive status appearing much better than his motor status. Severe growth restriction persisted with extreme microcephalic dwarfism evident at his last assessment at 16 months, weight 2740 g, length 54 cm, OFC 41 cm.
Morphologically, the patient was a phenocopy of his brother: exhibiting relative macrocephaly with frontal bossing, a broad and high forehead, prominent occiput, low-set very small ears, a depressed nasal root, broad nasal base, long philtrum, prominent 'pouting' lips and micrognathia, with limited opening of the mouth. Palpebral fissures were short. The hands and feet were small, the backs of the hands and feet were puffy, with distal tapering of fingers. The patellae were palpable.
From 3 months of age, home oxygen was prescribed because of progressive chronic respiratory difficulties. At 11 months, chest X-ray changes were evident with decreased perihilar and upper-lobe translucency suggestive of pulmonary interstitial disease, along with lower lobe consolidation. From 18 months of age pulmonary hypertension was present and to further clarify the nature of the underlying respiratory disorder, pulmonary biopsy was offered, but declined.
Recurrent episodes of vomiting, diarrhea and a mixed respiratory/metabolic acidosis started from 6 weeks postnatally, presumed infective, although stool cultures were negative. At week 7, rotavirus enteritis caused a life-threatening infection, at week 10, intensive care was needed again following vomiting and diarrhea. Hypogammaglobulinemia prompted initiation of regular IVIG therapy but did not prevent further infections. Death occurred at 19 months of age, thought to be due to rapid cardiorespiratory failure. Autopsy revealed foci of bronchopneumonia and thickened fibrotic alveolar walls. Accompanying this were broadened pulmonary vessel walls and concentric right ventricle hypertrophy, in keeping with pulmonary hypertension. Hepatic fibrosis and a still open anterior fontanelle were also reported.
Individual P5 -male 2 nd child born to unrelated British parents. No family history of note Intrauterine growth retardation was evident during pregnancy, and he was born by emergency Caesarian section at 38 weeks gestation. Birth weight 1.995 kg and OFC 32 cm. At birth, micrognathia, microphthalmia, and cryptorchidism were noted. Neonatal investigations documented bilateral intra-abdominal testes and small kidneys (3 cm length) by ultrasound, while cranial ultrasound,and cardiac echochardiography were normal. A supernumerary ring 20 chromosome (20q11.21-20q13.33) was detected in 3/30 cells from blood, however this was not subsequently detected on array CGH of a skin biopsy, nor by buccal FISH. This was judged insufficient to explain the child's phenotype and trio whole exome sequencing initiated through the DDD study.