Identification Of A Novel Nuclear Modifier In A Large Chinese LHON Family

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease that generally affects young adults with rapid, painless, severe visual impairment or loss. Mutations of mitochondrial DNA (mtDNA) are the molecular bases for this disorder. The ND1 G3460A, ND4 G11778A and ND6 T14484C mutations, causing a single amino acid exchange, accounts for more than 90% of LHON pedigrees in some countries. Typical features of LHON are incomplete penetrance and male bias among the affected subjects, suggesting other modifier factors including environmental and genetic factors should modulate the phenotypic manifestation of visual impairment.The Shandong LHON family (WZ162 family) carrying G11778A mutation only exhibited very high penetrance, variable severity and age-of-onset in visual impair-ment in matrilineal relatives. Our previous investigation revealed none of other mtDNA variants was implicated to have functional significance and nuclear modifier genes may play an important role in the phenotypic expression of the G11778A mutation in this Chinese family.To explore the nuclear modifier genes, one unaffected (Ⅱ-10) and three affected (Ⅱ-1,Ⅰ-3, Ⅲ-6) individuals from WZ162 family were studied using Whole-exon Sequencing. We identified a mutation in PRICKLE3 gene (PRICKLE3 c.157 C>T) in this family from 33 missense mutations,9 insertion-deletions,4 splice-site mutations. PRICKLE3 c.157C>T mutation was only detected in patients of this family and not detected in all 436 normal subjects and other 179 matrilineal relatives from 55 Chinese LHON families, nor any other mutation in PRICKLE3 exons were. The mutation shows an X-linked dominance genetic model.PRICKLE3 is located in the mitochondria partly, suggesting it may have close relationship with mitochondria. It is widely expressed in various cells and organs such as brain, liver, kidney, lung, placenta, pancreas, and may have important function. Here, to elucidate the effect of PRICKLE3 c.157 C>T mutation on mitochondria, we estiblished lymphoblastic cell lines derived from four affected individuals(G11778A, c.157C>T, short for patient), three genetically unrelated unaffected individuals (G11778A only, short for carrier), four control individuals (short for control).The results of biochemical assays as follows, (1)The PRICKLE3 protein level was analyzed by western blot, and showed no significant change in crude isolated mitochondria and total cell (P>0.05).(2) We analyzed the mitochondrial respiratory chain subunits protein level and found ATP6 protein level was reduced by 35%,44% compared to that of the control group and △mtDNA groups,changing significantly(P<0.05). But ND4, ND5, ND6, CYTB and CO2 protein level showed no significant change (P>0.05). (3) We used Seahorse XF 96 to quantify the rate of O2 consumption (OCR) in intact lymphoblastic cell lines and found the basal OCR, mitochondrial OCR, maximal OCR of patient group was decreased by 52%,59%,52%(P<0.01), compared to that of control group.The mitochondrial OCR of patient group was lower than that of carrier group by 24%(P<0.05).(4) Furthermore,the mitochondrial ATP production of patient group was reduced by 63% (P<0.01)and that of carrier group was reduced by 43%(P=0.05),compared to that of control group. The mitochondrial ATP production of patient groups was less than that of carrier group by 20%(P<0.01). (5) The mitochondrial membrane potential (△ψm) detection showed the percentage of green-fluorescent cells of control group, patient group, carrier group was 23.8%,38%,39%.The greater the percentage is, the lower the △ψm.The Aψm of patient group and carrier group was lower than that of control group (P<0.05), but there is no significant change between patient group and carrier group (P>0.05). (6) To delineate the reaction upon increasing level of ROS under oxidative-stress, the ratio of mean intensity between unstimuLated and stimuLated with H2O2 in each cell line. The average ratios of controls, patient group and carriergroup were 1.68,2.12, and 2.55. The ratio of patient group and carrier group was more than that of control group (P<0.05), but there is no significant change between patient groups and carrier group (P>0.05).The PRICKLE3 c.157C>T is a novel nuclear gene mutation in WZ162 pedigree. This mutation belongs to X-linked dominant inheritance, and it can reduce the mitochondrial ATP production, and may be responsible for penetrance in this Chinese pedigree. But how PRICKLE3 c.157C>T can reduce the mitochondrial ATP production and the correlation between LHON and PRICKLE3 c.157C>T, still need further study.