The Human-Specific BOLA2 Duplication Modifies Iron Homeostasis and Anemia Predisposition in Chromosome 16p11.2 Autism Individuals

Abstract

Human-specific duplications at chromosome 16p11.2 mediate recurrent pathogenic 600 kbp BP4-BP5 copy-number variations, which are among the most common genetic causes of autism. These copy-number polymorphic duplications are under positive selection and include three to eight copies of BOLA2, a gene involved in the maturation of cytosolic iron-sulfur proteins. To investigate the potential advantage provided by the rapid expansion of BOLA2, we assessed hematological traits and anemia prevalence in 379,385 controls and individuals who have lost or gained copies of BOLA2: 89 chromosome 16p11.2 BP4-BP5 deletion carriers and 56 reciprocal duplication carriers in the UK Biobank. We found that the 16p11.2 deletion is associated with anemia (18/89 carriers, 20%, p = 4e-7, OR = 5), particularly iron-deficiency anemia. We observed similar enrichments in two clinical 16p11.2 deletion cohorts, which included 6/63 (10%) and 7/20 (35%) unrelated individuals with anemia, microcytosis, low serum iron, or low blood hemoglobin. Upon stratification by BOLA2 copy number, our data showed an association between low BOLA2 dosage and the above phenotypes (8/15 individuals with three copies, 53%, p = 1e-4). In parallel, we analyzed hematological traits in mice carrying the 16p11.2 orthologous deletion or duplication, as well as Bola2^+/- and Bola2^-/- animals. The Bola2-deficient mice and the mice carrying the deletion showed early evidence of iron deficiency, including a mild decrease in hemoglobin, lower plasma iron, microcytosis, and an increased red blood cell zinc-protoporphyrin-to-heme ratio. Our results indicate that BOLA2 participates in iron homeostasis in vivo, and its expansion has a potential adaptive role in protecting against iron deficiency.

Keywords: 16p11.2 copy number variants; BOLA2; gene duplication; human evolution; human-specific segmental duplications; iron deficiency anemia.

Figure 1

The 16p11.2 and BOLA2 Locus Architecture (A) Gene content of the most common human haplotype of the 16p11.2 BP4-BP5 region (top) and comparison with the syntenic single-copy region rearranged in 16p11.2 CNV mouse models (bottom). Human single-copy genes are gray shaded; the BOLA2-SLX1-SULT1A human duplicon is green shaded; the NPIP human duplicon is orange shaded. At BP4, the BOLA2B, SLX1B, and SULT1A4 paralogs map; at BP5, two copies of the BOLA2A, SLX1A, and SULT1A3 map. Scale bar 100 kb. (B) Two examples of outcomes from non-allelic homologous recombination between copies of the BOLA2-SLX1-SULT1A-NPIP segment at BP4 and BP5 are presented. The BP4-BP5 structure corresponds to the most common H3 haplotype. For example, sequence misalignments could occur between one BP4 copy and either the distal (top panel) or proximal (bottom panel) BP5 copy. The resulting alleles with BP4-BP5 duplications and deletions have, respectively, more and fewer copies of BOLA2, SLX1, SULT1A, and NPIP (right panels). Numbers count the number of copies of the BOLA2-SLX1-SULT1A green segment in each haplotype. (C) BOLA2 copy number in 635 European individuals from the 1000 Genomes Project and Human Genome Diversity Project (Ctrl), 67 16p11.2 BP4-BP5 deletion carriers from the European cohort, and 63 deletion carriers from the SVIP cohort.

Figure 2

Hematological Parameters and Anemia Prevalence in 16p11.2 BP4-BP5 CNV Carriers (A) Red blood cell distribution width (RDW), mean reticulocyte volume (MRV), platelet count (PLTc), mean platelet volume (MPV), and platelet crit (PLTcrit) in 16p11.2 deletion and duplication carriers and control individuals in the UK Biobank (UKB). Beta and p values of linear models and p-values of paired t-tests are shown. (B) Prevalence of anemia in 379,385 control individuals and 89 16p11.2 deletion carriers in the UKB (top) and 83 16p11.2 deletion carriers in the Simons Variation in Individuals Project (SVIP) and European cohorts stratified by BOLA2 copy number (bottom).

Figure 3

Blood Traits of the Del/+ and Dup/+ Mice (A) Plasma iron level in Del/+ and Dup/+ mice and their wild-type littermates at different ages (f: female, m: male, w: weeks). (B) Red blood cell traits in Del/+ and Dup/+ mice and their wild-type littermates at the age of 29 weeks. These traits are significantly different in at least two longitudinal measurements (Tables S6 and S7). T-test p ≤ 0.1 are shown (MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, CH: red cell hemoglobin content, RDW: red cell distribution width, MCHCr: mean corpuscular hemoglobin concentration reticulocytes, CHr: red cell hemoglobin content reticulocytes).

Figure 4

Characterization of the Bola2^tm1 Knockout Mouse Line (A) Mouse mating strategy of the Swiss Bola2^tm1 line and gender and genotype ratios observed. (B) Longitudinal body weight profiles. Note: the x axis scale is not continuous. (C) Plasma iron level in Bola2^+/− and Bola2^−/− mice and wild-type littermates (f: female, m: male, w: weeks). Red dots represent mice housed in single cages. Beta and p values of linear models (female mice at 8 weeks of age and male mice at 22 weeks of age) are shown. (D) Red blood cell (HGB: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, CH: red cell hemoglobin content), reticulocyte (MCVr: mean corpuscular volume reticulocytes, MCHCr: mean corpuscular hemoglobin concentration reticulocytes, CHr: red cell hemoglobin content reticulocytes), and platelet (MPC: mean platelet component concentration, PCDW: platelet component distribution width, MPM: mean platelet mass) parameters of Bola2^+/− and Bola2^−/− mice and wild-type littermates at 17 weeks of age. Beta and p values of linear models with p ≤ 0.1 are shown. (E) Blood zinc protoporphyrin (ZPP) levels of Bola2^−/− mice and wild-type littermates at 8 weeks of age. P values of t-tests are shown.