Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia

Abstract

Isolated congenital asplenia (ICA) is characterized by the absence of a spleen at birth in individuals with no other developmental defects. The patients are prone to life-threatening bacterial infections. The unbiased analysis of exomes revealed heterozygous mutations in RPSA in 18 patients from eight kindreds, corresponding to more than half the patients and over one-third of the kindreds studied. The clinical penetrance in these kindreds is complete. Expression studies indicated that the mutations carried by the patients-a nonsense mutation, a frameshift duplication, and five different missense mutations-cause autosomal dominant ICA by haploinsufficiency. RPSA encodes ribosomal protein SA, a component of the small subunit of the ribosome. This discovery establishes an essential role for RPSA in human spleen development.

Fig. 1

RPSA heterozygous coding mutations are the most frequent genetic etiology of ICA. (A) Manhattan plot showing the p-value for tests of the hypothesis that “mutations in a given gene were not specific to the ICA cohort”. Each dot represents one gene. X axis: physical position of each gene on the chromosome. Y axis: −log10(p). p was calculated for Fisher’s exact test comparing 23 exomes from 23 ICA kindreds and 508 exomes from patients with phenotypes other than invasive bacterial disease. The gray dashed line indicates threshold for statistical significance (0.05/4,222=1.2×10⁻⁵) (B) Familial segregation of all RPSA coding mutations. Mutations are described in red. Capital letters represent the kindred code. When available, the genotype of RPSA is indicated under each symbol. WT, wild-type; M, mutant. Black, ICA; gray, probable ICA.

Fig. 2

Haploinsufficiency at the RPSA locus. (A) RPSA cDNA was obtained from activated T cells of patients ICA-C-I.2, ICA-C-II.3 and ICA-C-II.4. Sequences of WT and mutant cDNA are shown. The deduced frequency of each mRNA is indicated in the diagram on the right. (B) Relative levels of RPSA mRNA in activated T cells from three patients, a healthy member of kindred C (ICA-C-I.1), and four unrelated healthy controls. PBMCs were activated with PHA for 5 days. A mean of four independent experiments is shown. Error bars indicate the SEM. ***, p<0.001. (C) Immunoblot showing the levels of the WT and mutant RPSA proteins following overproduction in HEK293T cells. GAPDH, loading control; GFP, transfection contol. The blot shown is representative of 4 independent experiments. Below: intensity of the bands corresponding to the FLAG antibody normalized with respect to the band from the GFP immunoblot. Error bars indicate the SEM. (D) Genome-wide distribution of the strength of purifying selection acting in 14,993 human genes. A low f estimate (13) indicates that the gene is particularly constrained.