Identification of SPOCK2 as a susceptibility gene for bronchopulmonary dysplasia

Abstract

Rationale: Bronchopulmonary dysplasia is the most common chronic respiratory disease in premature infants. Genetic factors might contribute to bronchopulmonary dysplasia susceptibility.

Objectives: To identify genetic variants involved in bronchopulmonary dysplasia through a genome-wide association study.

Methods: We prospectively evaluated 418 premature neonates (gestational age <28 wk), of whom 22% developed bronchopulmonary dysplasia. Two discovery series were created, using a DNA pooling strategy in neonates from white and African ancestry. Polymorphisms associated with the disease were confirmed in an independent replication population. Genes were then explored by fine mapping and associations were replicated in an external Finnish population of 213 neonates. Validated genes expression patterns were studied in rat lung, after air or hyperoxia exposure.

Measurements and main results: SPOCK2 gene was identified by both discovery series. The most significant polymorphism (rs1245560; P = 1.66 × 10(-7)) was confirmed by individual genotyping, and in the replication population (P = 0.002). Fine mapping confirmed the association of rs1245560 with bronchopulmonary dysplasia in both white and African populations with adjusted odds ratios of 2.96 (95% confidence interval [CI], 1.37-6.40) and 4.87 (95% CI, 1.88-12.63), respectively. In white neonates, rs1049269 was also associated with the disease (odds ratio, 3.21; 95% CI, 1.51-6.82). These associations were replicated in the Finnish population. In newborn rat lungs, SPOCK2 mRNA levels markedly increased during the alveolar stage of lung development. After rat exposure to hyperoxia, SPOCK2 expression increased relative to air-exposed controls.

Conclusions: We identified SPOCK2 as a new possible candidate susceptibility gene for bronchopulmonary dysplasia. Its lung expression pattern points toward a potential role in alveolarization.

Figure 1. Fine scale mapping of SPOCK2 locus in neonates of Caucasian ancestry

Individual genotyping of tag-SNPs in the Caucasian population (51 cases and 185 controls) confirmed the results obtained in DNA pooling studies. Statistical analyses identified 5 SNPs as being significantly associated with BPD (B). Only rs1245560 and rs1049269 remained significant after correction for multiple testing and adjustment for major clinical risk factors (A, white spots). LD plots were constructed using Haploview version 4.1. Red squares indicate regions of strong linkage disequilibrium (D′ given).

Figure 2. SPOCK2 mRNA expression patterns

Developmental SPOCK2 mRNA expression patterns in rat whole-lung tissue (A), lung fibroblasts (C) and AECs (D). Expression was quantified by real-time PCR from fetal life to adulthood, in three to six individual lung samples per stage. The birth level in whole lung tissue and the day-1 level in isolated cells were arbitrarily attributed a value of 100. Values are mean ± SEM. * p<0.05 versus reference level. B. Changes in lung SPOCK2 mRNA expression in newborn rats exposed to hyperoxia from day 5 to day 10. Values are mean ± SEM and results are expressed as a percentage of the control value. * p<0.05.

Figure 3. SPOCK2 immunofluorescence studies in rat lung tissue on postnatal day 14

Immunocolocalization of SPOCK2 (green labelling) and SP-B (red labelling, A) or collagen IV (red labelling, B) showed the presence of SPOCK2 throughout the ECM, including the basement membrane, as shown by superimposing the 2 fluorescent signals in some areas (B, white arrow). Negative control lung incubated with mouse and rabbit polyclonal IgG, showing fluorescence restricted to the nucleus (C.) (original magnification x126).