FRRS1L variants and ferriheme overload drive hyperpigmentation and systemic Iron overload in lanping black bone sheep

Abstract

Background: Hemoglobin metabolism disorder can result in systemic iron overload, leading to pigmentation in multiple organs. Although these disorders are often of genetic origin, the specific genes and mechanisms remain incompletely understood. Lanping black bone sheep (LP–BBS), a unique population from the high altitudes along the Hengduan Mountains in Yunnan province, exhibits hyperpigmentation in multiple tissues. Investigating the genetic and environmental factors underlying this phenotype provides a natural model to better understand hemoglobin metabolism disorder.

Results: LP-BBS were found to exhibit increased red blood cell counts, elevated hemoglobin levels, and systemic iron overload, evidenced by hyperpigmentation in various tissues. Histological and molecular analyses revealed that hyperpigmentation is driven by ferriheme overload, an inheritable quantitative trait influenced by both genetic variation and environmental factors. Genome-wide association studies identified FRRS1L as a candidate gene, with significant mutations in its 3′-untranslated region (3′-UTR) reducing FRRS1L expression. Functional assays demonstrated that insufficient FRRS1L expression promotes ferriheme accumulation in reticuloendothelial cells and macrophages, as confirmed in vitro using FRRS1L knockdown models. Ferriheme overload was associated with oxidative stress and systemic inflammation, causing pathological damage to critical organs such as the kidney, liver, and uterus.

Conclusion: This study identifies FRRS1L as a key contributor to ferriheme overload through aberrant hemoglobin metabolism in LP–BBS. These findings offer new insights into the genetic basis and pathological mechanisms of iron overload disorders, providing a potential target for therapeutic intervention. Moreover, LP–BBS serves as a valuable natural model for studying hematogenous pigment disorders and their interplay with environmental factors.

Supplementary Information: The online version contains supplementary material available at 10.1186/s13578-025-01426-6.

Keywords: Black bone sheep; Hyperpigmentation; Iron overload; Macrophage; Systemic inflammation.

Fig. 1

LP–BBS hyperpigmentation characterization. A Hyperpigmentation phenotype of inner tissues from LP–BBS. B Histological distribution of pigment in the inner tissues. Bar = 100 μm. C Ultrastructure of pigment (black arrow) capsuled with membrane (white arrow) in the renal tubular epithelial cells and collecting tubular epithelial cells. Different stages of melanosomes were observed in the melanocytes of Silky Fowl. N, nucleus. 1–4, one to four stages of melanosome. D More number of red blood cells and concentration of hemoglobin in blood. Data are mean ± SEM. Unpaired two-sided Student’s t-test (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). E Iron contents in the kidney and blood. F Level of transferrin in the blood. G Absorption peak value of pigment from LP–BBS by HPLC analysis. H Pigment showed similar peak value to the hemin

Fig. 2

Pathological analysis of LP–BBS. A Higher expressions of SEPW1 and GPX1 are significantly detected in the spleen and kidney of LP–BBS (n = 3–5) by qPCR. LP–NBS (n = 3–4) from same indigenous population; Data are mean ± SEM. Unpaired two-sided Student’s t-test (^*p < 0.05, ^**p < 0.01). B Higher levels of GSH-PX (p = 0.0106) and more contents of MDA (p = 0.0468) are detected in the blood of LP–BBS (n = 11). LP–NBS (n = 10) from same indigenous population; Data are mean ± SEM. Unpaired two-sided Student’s t-test. C Severe bursting of cell membranes and organelles shown as swollen, vacuolated and broken are in the renal tubular epithelial cells. D Obvious inflammatory cells infiltrated in the kidney, liver, lung and lymph node (arrow). E Follicular atresia and cell necrosis were in the ovary and uterus when ferriheme overload notably in the germinal epithelium and atretic follicle of the ovary, and in the lamina propria of uterus. Bar = 100 μm

Fig. 3

Ferriheme overload characterization in LP–BBS. A Numbers of DEGs identified in the spleen, kidney and liver. B Co-regulated genes in the spleen, kidney, and liver of LP–BBS. C Higher expressions of GPX1 were significantly detected in the liver, spleen and kidney of LP–BBS (n = 3–5). LP–NBS (n = 3–4) from same indigenous population; Data are mean ± SEM. Unpaired two-sided Student’s t-test. D Enriched GO terms from DEGs in the spleen. E Higher expressions of ALAS2 was detected in the spleen of LP–BBS by qRT-PCR; Data are mean ± SEM. Unpaired two-sided Student’s t-test (^*p < 0.05, ^**p < 0.01). F Heatmap of DEGs related to hemoglobin synthesis and metabolism. G Genes related to heme metabolism were enriched in the spleen, liver, and kidney of LP–BBS by GSEA

Fig. 4

The selection signals of LP–BBS genomes and FRRS1L locus. A Geographical distribution and sampling of LP–BBS in Lanping, Jianchuan, and Shangri-La, Yunnan province, along the HDMs with different high altitudes. The map is from Google Maps (map data © 2016 Google, SK Planet, ZENRIN). B Distribution of log₂ (hp ratios) and Z (F_ST) values calculated in 100-kb sliding windows with 50-kb increments between LP–BBS and LP–NBS, and 845 genes were identified. C Enriched terms of the screened genes by NGS. D Manhattan plots from determinate phenotypic individuals of hybrid population showed the significance and location distribution of SNPs with threshold as–log₁₀ (5e^− 8) and–log₁₀ (5e^− 9). E Manhattan plot of the results from the determinate phenotypic individuals by NGS. F The located genes FRRS1L with two SNPs, ASTN2, TMEM245, and CTNNAL1 in Chromosome 2. Population genetic analysis revealed gene variation frequencies of the two mutated sites in the FRRS1L 3′-UTR. One is a T/G mutation at the 4,852 bp from the end of the 3′-UTR (frequencies of gene variation is 0.92), and the other is an A/C mutation at the 1,016 bp from the end of the 3′-UTR (frequencies of gene variation is 0.79). HDMs, Hengduan Mountains; NGS, next generation sequencing

Fig. 5

The key variation of FRRS1L in LP–BBS genomes. A Six mutation sites in the FRRS1L 3′-UTR by mix pool sequencing in LP–BBS (n = 20) and LP–NBS (n = 5). B SNPs at the 1,016 bp and 4,852 bp from the end of 3′-UTR downregulated the reporter gene’s expression. n = 6 per group; Data are mean ± SEM. Paired two-sided Student’s t-test (^*p < 0.05, ^**p < 0.01). C Expressions of FRRS1L in the inner tissues by qRT-PCR. Data are mean ± SEM. Paired two–sided Student’s t-test (^*p < 0.05, ^**p < 0.01). D FRRS1L is markedly expressed in the lymph node (reticuloendothelial cell), kidney (renal tubular epithelial cell), and lung (macrophage and alveolar epithelial cell). E Significant lower expression of FRRS1L in LP–BBS (n = 6). Data are mean ± SEM. Paired two-sided Student’s t-test (^***p < 0.001, ^****p < 0.0001). IOD, integral optical density

Fig. 6

The association between FRRS1L and hyperpigmentation. A Ferriheme is present in macrophages from different inner tissues. Immunohistochemistry. Bar = 100 μm. B Ferriheme extracted from LP–BBS is engulfed by alveolar macrophages at 24 h in vitro. Control: no ferriheme treatment; Bar = 20 μm. C Macrophages express ALAS1, FECH, FRRS1, and FRRS1L, which are involved in heme synthesis and metabolism. Downregulated expression of FRRS1 and FRRS1L, ALAS1 and FECH significantly increases at 48 h. n = 3 per group; Data are mean ± SEM. Paired two-sided Student’s t-test (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). D Downregulating FRRS1 and FRRS1L expressions in macrophages via RNA interference induces pigmentation in the cytoplasm of macrophage (arrow). Bar = 20 μm. E Pigment accumulated in the macrophages was extracted via cell lysis (1, FRRS1–RNAi. 2, FRRS1L–RNAi. 3, Control–Negative RNAi). F Pigment in the macrophage was identified as being the same as ferriheme in LP–BBS using high–performance liquid chromatography