Identification of human D lactate dehydrogenase deficiency

Abstract

Phenotypic and biochemical categorization of humans with detrimental variants can provide valuable information on gene function. We illustrate this with the identification of two different homozygous variants resulting in enzymatic loss-of-function in LDHD, encoding lactate dehydrogenase D, in two unrelated patients with elevated D-lactate urinary excretion and plasma concentrations. We establish the role of LDHD by demonstrating that LDHD loss-of-function in zebrafish results in increased concentrations of D-lactate. D-lactate levels are rescued by wildtype LDHD but not by patients' variant LDHD, confirming these variants' loss-of-function effect. This work provides the first in vivo evidence that LDHD is responsible for human D-lactate metabolism. This broadens the differential diagnosis of D-lactic acidosis, an increasingly recognized complication of short bowel syndrome with unpredictable onset and severity. With the expanding incidence of intestinal resection for disease or obesity, the elucidation of this metabolic pathway may have relevance for those patients with D-lactic acidosis.

Fig. 1

Lactate optical isomers. Mass-spectrometry chromatogram of the separation of L-lactate (purple) and its optical isomer D-lactate. Note the relative position of the hydroxyl group and hydrogen atom. L-lactate is present at approximately 100 times that of D-lactate in the plasma of a healthy control

Fig. 2

Metabolic analysis reveals elevated D-lactate of two patients. The levels of D-lactate in a urine and b plasma are elevated in our patients compared to controls. Increased levels of D-2-hydroxyisovaleric acid (c, d) and D-2-hydroxyisocaproic acid (e, f) are also observed in urine and plasma, respectively. Separate metabolic measurements for Patient 1 and 2 in urine (n = 3; n = 2) and plasma (n = 1; n = 2) are shown. Mean and standard deviation shown

Fig. 3

Identification of human loss-of-function variants in LDHD. a Sanger sequencing of Patient 1 identifies a LDHD homozygous nonsynonymous variant NM_153486.3:c.1388C>T, p.(Thr463Met). Both the mother and the father are heterozygous carriers of the variant. b The variant p.(Thr463Met) encodes for the amino acid methionine (M) instead of the normally present amino acid threonine (T) in a region that is highly conserved across multiple species. c Sanger sequencing of Patient 2 identifies a separate novel, homozygous nonsynonymous variant NM_153486.3:c.1122G>T, p.(Trp374Cys). Both the mother and the father are heterozygous carriers of the variant. d The variant p.(Trp374Cys) is in a region that is conserved across chordates. Sac_c_DLD1 Saccharomyces_cerevisiae_DLD1, Ara_t_AT5G Arabidopsis_thaliana_AT5G06580, Cae_e_F32D8 Caenorhabditis_elegans_F32D8.12, Gal_g_LDHD Gallus_gallus_LDHD, Hom_s_LDHD Homo_sapiens_LDHD, Mus_m_LDHD Mus_musculus_LDHD

Fig. 4

Zebrafish metabolic studies. a Maternal zygotic mutant ldhd^−/− zebrafish larvae (3 dpf, lower panel) show no phenotype differences compared to wildtype zebrafish larvae (upper panel). Scale bar: 250 μm. Levels of D-lactate (b) and L-lactate (c) in response to LDHD activity. Wildtype zebrafish larvae (ldhd WT) and ldhd^−/− zebrafish (ldhd^−/−) at the 1 cell stage were subject to four conditions: uninjected (cntrl), microinjected with human wildtype LDHD RNA (LDHD WT), microinjected with patient variant Thr463Met LDHD RNA (LDHD T463M), or Trp374Cys LDHD RNA (LDHD W374C). Metabolic assays were then performed to detect D-lactate and L-lactate present at 3 dpf. The conditions where LDHD are nonfunctional result in higher levels of D-lactate; in contrast, no effect is seen in the levels of L-lactate. Measurements for each condition were performed on three batches of 10 embryos. Mean and standard deviation shown