Genotype-phenotype correlation in primary carnitine deficiency

Abstract

Primary carnitine deficiency is caused by defective OCTN2 carnitine transporters encoded by the SLC22A5 gene. Lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy. Recently, asymptomatic mothers with primary carnitine deficiency were identified by low carnitine levels in their infant by newborn screening. Here, we evaluate mutations in the SLC22A5 gene and carnitine transport in fibroblasts from symptomatic patients and asymptomatic women. Carnitine transport was significantly reduced in fibroblasts obtained from all patients with primary carnitine deficiency, but was significantly higher in the asymptomatic women's than in the symptomatic patients' fibroblasts (P < 0.01). By contrast, ergothioneine transport (a selective substrate of the OCTN1 transporter, tested here as a control) was similar in cells from controls and patients with carnitine deficiency. DNA sequencing indicated an increased frequency of nonsense mutations in symptomatic patients (P < 0.001). Expression of the missense mutations in Chinese hamster ovary (CHO) cells indicated that many mutations retained residual carnitine transport activity, with no difference in the average activity of missense mutations identified in symptomatic versus asymptomatic patients. These results indicate that cells from asymptomatic women have on average higher levels of residual carnitine transport activity as compared to that of symptomatic patients due to the presence of at least one missense mutation.

Fig. 1

Carnitine transport by fibroblasts obtained from patients with primary carnitine deficiency. Carnitine (0.5 μM) transport was measured for 4 h at 37°C. Nonsaturable transport, measured in the presence of 2 mM cold carnitine, was subtracted from total transport to obtain saturable carnitine transport. Data are means± SE of 6 observations in two separate experiments. Panel A shows data for individual patients, panel B shows the average ± 99% confidence intervals for symptomatic patients and asymptomatic women. Note the logarithmic scale in panel B. *p<0.01 versus controls and mothers; **p<0.01 versus symptomatic patients and controls using 99% confidence intervals.

Fig. 2

Carnitine transport by Chinese Hamster Ovary (CHO) cells expressing normal and mutant carnitine transporters. CHO cells were stably transfected with normal and mutant OCTN2 cDNA cloned in the pEGFP mammalian expression vector. After selection for resistance to G418 (0.8 mg/ml), expression of the trans-gene was verified by fluorescent microscopy. Carnitine (0.5 μM) transport was measured for 1 h (to measure initial entry rates) and corrected for nonsaturable uptake (measured in the presence of 2 mM cold carnitine). Points are averages ± SE of 6 samples. Panel A shows data for individual missense mutations, panel B shows the average for mutations identified in symptomatic patients and mothers. Note the logarithmic scale in panel B. In both groups, carnitine transport was significantly (p<0.01 using ANOVA) reduced as compared to wild type OCTN2, but there was no significant difference between the group of missense mutations identified in symptomatic patients versus asymptomatic women using ANOVA.

Fig. 3

Time-course of ergothioneine transport in normal human fibroblasts. A. Ergothioneine (0.5 μM) transport was measured for the time indicated at 37°C in the absence (open circles) and presence of 2mM cold ergothioneine (nonsaturable transport, filled circles). B. Nonsaturable ergothioneine transport was subtracted from total transport to obtain saturable (net) ergothioneine transport. Data are means ± SE of 3observations. The line in panel B is a linear regression passing for the origin with the parameters indicated.

Fig. 4

Kinetic constants for ergothioneine transport in human fibroblasts. Ergothioneine (0.5–500 μM) uptake was measured for 60 min at 37°C in normal human fibroblasts. Points are averages of triplicates ± SD. A. Total ergothioneine transport was fitted to equation 1 (see methods) for a single saturable transporter with superimposed diffusion. Lines show the best fit to total ergothioneine uptake. B. Ergothioneine transport was corrected for that measured in the presence of 2 mM cold ergothioneine to correct for diffusion. Data were fitted to a Michaelis-Menten equation. Parameters are shown in the figure as averages ± SE. Units for the parameters are: Vmax= nmol/ml cell water/h; Km=μM; Kd=h⁻¹.

Fig. 5

Ergothioneine transport by fibroblasts obtained from patients with primary carnitine deficiency. Ergothioneine (0.5 μM) transport was measured for 4 h at 37°C. Nonsaturable transport, measured in the presence of 2 mM cold ergothioneine, was subtracted from total transport to obtain saturable ergothioneine transport. Data are means ± SE of 6 observations from two separate experiments. Panel A shows data for individual patients, panel B shows the average for symptomatic patients and asymptomatic women. In panel B, there were no significant differences among groups using ANOVA.