De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia

Abstract

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Keywords: POLR2A; RNA polymerase II complex; RPB1; de novo variants; desert Z score; desert regions; dominant-negative effect; haplo-insufficiency; infantile-onset hypotonia; neurodevelopmental syndrome.

Figure 1

Photos and Attainment of Developmental Milestones of Individuals with POLR2A Variants (A) Photos of 13 of the 16 individuals (ind.) harboring a de novo variant in POLR2A, in order of severity score. (B) Attained and unattained developmental milestones during the first 18 months of life for 14 of the 16 individuals harboring a de novo variant in POLR2A, in order of severity score. For each milestone, Z scores were calculated indicating the delay in attaining that milestone. The higher the Z scores, the greater the developmental delay. The figure demonstrates that in individuals with a mild phenotype, developmental Z scores are mostly <10, in individuals with a moderate phenotype, developmental Z scores are <20, and in individuals with a severe phenotype, developmental Z scores can even reach 30. In addition, among the individuals with a severe phenotype, relatively more (and more early) developmental milestones were not attained by the individual. Individual 3 (p.Asn531Ser) was not included in this figure because the pregnancy was terminated.

Figure 2

Structural Evaluation of POLR2A Variants (A) RPB1 is represented; the core and the C-terminal heptad repeats are shown as bars. Functional elements are highlighted in dark blue and are labeled underneath. The positions of gained stop codons, frameshifts, in-frame deletions (IF deletion), missense mutations, and synonymous mutations at the protein level, as reported in the gnomAD database, are indicated by vertical black strokes. The length of the stroke represents the number of different mutations observed at the DNA level in the corresponding codon. The individuals’ variants are indicated by red strokes and are labeled. Stretches devoid of missense mutations are highlighted by a yellow background and are labeled by Roman numerals. The degree of conservation in humans and S. cerevisiae is depicted underneath, whereby each identical amino acid is represented by a vertical black stroke. (B) Conservation across species of amino acid residues that are changed in the individuals affected by missense mutations. p.Arg1603His is not conserved and is localized in the region between the pol II core and the heptad repeats, and p.Pro1767fs is localized in the heptad repeat region. For these regions, alignments are not reliable. (C) A space-filling model of the elongating form of pol II from S. cerevisiae with TFIIS in ribbon representation in red. Subunits other than RPB1 are shown in dark gray. RPB1 is shown in light gray; the positions of individuals’ variants are highlighted in magenta, and stretches devoid of missense variants are shown in shades of yellow labeled by Roman numerals as in (A). The incoming nucleotide is shown as a space-filling model in orange. The template strand, the coding strand, and the nascent mRNA are shown as backbone trace in gray, black, and orange, respectively. (D) Same as in (C), but omitting subunits other than RPB1. Only the RPB1 stretches devoid of missense variants and the positions of individuals’ variants are shown, and all other parts of RPB1 have been omitted. The 3D-envelope of RPB1 is projected in light gray on the background plane. (E) Same as in (D), but additionally omitting the RPB1 stretches devoid of missenses variants. The Cα-atoms of amino acid residues affected in individuals’ variants are shown as magenta spheres. (F) Detailed view at the catalytic center of pol II. The coding strand in dark gray, the template strand in light gray, the nascent mRNA in orange, and the incoming nucleotide in red are shown as a space-filling model. The bridge helix is shown as a rod, and selected other parts of RPB1 as backbone trace. The trigger loop is shown in its open and closed conformation in light and dark blue, respectively. Side chains of amino acid residues affected in individuals with a missense variant and of some residues known to be of catalytic importance are shown in ball-and-stick representation. The Cα-atoms of amino acid residues affected by IF deletions or the gain of a stop codon are shown as spheres in magenta. (G) The environment of residue Pro357. Elements from RPB1, subunit 2, and the pol II subunit RPB11 are depicted in light blue, green, and orange, respectively. Hydrogen bonds are indicated by dotted lines (H) The environment of residue Asn517. Elements of RPB1 are shown in blue (I) The environment of residue Asn1232. Elements of RPB1 and TFIIS are shown in blue and red, respectively. Putative hydrogen bonding partners for Asn1232 in TFIIS are marked by an asterisk. (J) A table assigning amino acid residues that are affected in individuals to the corresponding residues of RPB1 in S. cerevisiae.

Figure 3

Functional Evaluation of POLR2A Variants (A) A growth assay in S. cerevisiae of six rpb1 mutants: p.Ile457Thr (p.Leu443Thr), p.Asn531Ser (p.Asn517Ser), p.Thr736Met (p.Ser713Met), p.Ser755del (p.Leu732del), p.Leu1124Pro (p.Leu1101Pro), and p.Asn1251Ser (p.Asn1232Ser) are compared to wild type (WT). p.Ala301Asp demonstrates a positive control for reduced transcriptional activity, and p.Glu1230Lys demonstrates a positive control for reduced genetic interaction with TFIIS. YPD 30°C depicts growth under normal circumstances, and YPD 37°C depicts growth under stress induced by increased temperature. YPRaf/Gal day 4 and day 6 depict growth under stress induced by increased inappropriate GAL 3′ termination on day 4 and day 6 of culture, respectively. SC-LEU + MPA day 4 and day 6 depict growth under stress induced by the lack of leucine and the addition of mycophenolic acid (MPA) on day 4 and day 6 of culture, respectively. (B) HeLa cell viability. The individuals’ variants were introduced in an α-amanitin-resistant version of POLR2A (ama^R) and expressed from a doxycycline-inducible promoter as a GFP fusion construct in HeLa cells. After the induction of expression, endogenous pol II was left untreated (dark gray bars) or inhibited by the application of α-amanitin (light gray bars), and cell viability was determined. Error bars represent the standard deviation. p.Lys812^∗ was used as a representative of p.Gln700^∗ and p.Gln735^∗ because it is similarly truncated (see Material and Methods). (C) Interactome analysis of wild-type (WT) and mutant versions of RPB1 using mass spectrometry. Nuclear extracts from HeLa cells expressing GFP-tagged WT or mutant versions of RPB1 were used for precipitations with control agarose beads (−) or GFP-affinity beads (+) in triplicate. In total, 1,133 proteins were identified by more than two peptides via intensity-based absolute quantification (IBAQ) mass spectroscopy. Known members of pol II (highlighted) and a random selection of 5% of the other proteins are shown. The summed peptide intensities of identified proteins were logarithmically transformed to the basis of ten and are represented by shades of gray. Note, intensities smaller than 10⁴ are not obtained due to technical constrains. p.Lys812^∗ was used as a representative of p.Gln700^∗ and p.Gln735^∗.