Expanding the phenotypic spectrum of mutations in LRP2: a novel candidate gene of non-syndromic familial comitant strabismus

Abstract

Background: Comitant strabismus (CS) is a heterogeneous disorder that is a major contributing factor to unilateral childhood-onset visual impairment. Studies have confirmed that genetic factors play an important role in the development of CS. The aim of this study was to identify the genetic cause of non-syndromic familial CS.

Methods: Fourteen unrelated CS families were recruited for the study. Twelve affected and 2 unaffected individuals from a large four-generation family (CS08) were selected to perform whole genome-wide linkage analysis. Parallel whole-exome sequencing (WES) was conducted in the same family (9 patients and 1 unaffected member) and 31 additional CS cases from 13 other unrelated families. Sanger sequencing was used to determine whether any of the remaining variants co-segregated with the disease phenotype in the corresponding family.

Results: Based on linkage analysis, CS in family CS08 mapped to a novel region of 34.17 centimorgan (cM) on chromosome 2q22.3-2q32.1 between markers D2S151 and D2S364, with a maximum log odds (LOD) score of 3.54 (theta = 0) at D2S142. Parallel WES identified a heterozygous variant, LRP2 c.335 A > G (p.Q112R), located in such a linkage interval that completely co-segregated with the disease in the family. Furthermore, another novel heterozygous variant (c.7274A > G, p.D2425G) in LRP2 that co-segregated was detected in 2 additional affected individuals from another unrelated family by WES. Both variants are predicted to be damaging by PolyPhen-2, SIFT and MutationTaster, and were absent in 100 ethnically matched normal controls.

Conclusion: LRP2 is a novel candidate genetic cause of non-syndromic familial CS.

Keywords: Comitant strabismus; Linkage analysis; Mutation; Phenotype; Whole-exome sequencing.

Fig. 1

Pedigree of family CS08 and haplotype reconstruction for the mapped region on chromosome 2 and clinical evaluations of the proband in family CS08. a Affected and unaffected members are indicated by filled and open symbols respectively. The black arrow indicates the proband. Haplotypes for tested short tandem repeat (STR) markers and genotypes for LRP2 c.335, rs2683454 and rs2683454, are given for all participants. Black bars represent the ancestral haplotype associated with the disease. *Individuals on whom WES was performed, ^§Individuals on whom sanger sequencing was performed. Abbreviation: M1, mutation c.335A > G b Ocular positions and movements. White arrow, exotropia phenotype of right eye. c Fundus photograph of right eye. d Ocular MRI. e–f Brain MRI for patient III:19

Fig. 2

Multipoint linkage analysis results and analysis of mutation c.335 A > G (p.Q112R) in LPR2. a The locus for chromosomal region 2q22.3-2q32.1. Relative order of genotyped microsatellite markers is shown at the bottom next to an ideogram of chromosome 2. b Results from multipoint linkage analysis and genetic locations for the markers genotyped. The horizontal axis represents position of Chromosome 2. The vertical axis represents LOD score. bt., between. c Sanger sequencing showing heterozygous c.335 A > G mutation in III:19 (patient) and III:12 (unaffected) respectively. Reference sequences are given at the bottom. Abbreviation: Het., heterozygous; Ref., reference. d Conservation analysis of residue p.Q112 (boxed) of LRP2 across eight species

Fig. 3

Pedigree of family CS06, clinical evaluations of the proband, and analysis of mutation c.7274A > G (p.D2425G) in LPR2. a Pedigree of family CS06. *Individuals on whom WES was performed, ^§Individuals on whom sanger sequencing was performed. Abbreviation: M2, mutation c.7274A > G. b Ocular positions and movements for patient IV:3. White arrow, esotropia phenotype of right eye. c Sanger sequencing showing heterozygous c.7274A > G mutation in IV:3 (patient) and III:5 (unaffected) respectively. Reference sequences are given at the bottom. Abbreviation: Het., heterozygous; Ref., reference. d Conservation analysis of residue p.D2425 (boxed) of LRP2 across eight species

Fig. 4

Schematic structural of LRP2/megalin and predicted crystal structural models of the wild-type and mutant. a Megalin is composed of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic domain. The extracellular domain harbors four cysteine-rich complement-type ligand binding repeats, which are separated from each other by β-propellers and EGF-like repeats. b-d Crystal structures of wild-type human LRP2 and mutant human LRP2 carrying p.Q112R. c The mutation spot is highlighted red. D, The 112 residue glutamine is replaced by arginine. e–g Crystal structures of wild-type and mutant human LRP2 carrying p.D2425G. e The mutated residue is indicated in green. Amino acids interacted with residue 2425, including Tyr2426, Tyr2434, Phe2473, and Asn2641, are indicated. g The hydrogen bond between residue 2425 and Tyr2434, Tyr2426, Phe2473, as well Asn2641 are eliminated upon the change from wild-type aspartic to mutant glycine