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. 2015 Apr 13:6:123.
doi: 10.3389/fgene.2015.00123. eCollection 2015.

Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency

Uwe Ahting  1 , Johannes A Mayr  2 , Arnaud V Vanlander  3 , Steven A Hardy  4 , Saikat Santra  5 , Christine Makowski  6 , Charlotte L Alston  4 , Franz A Zimmermann  2 , Lucia Abela  7 , Barbara Plecko  7 , Marianne Rohrbach  8 , Stephanie Spranger  9 , Sara Seneca  10 , Boris Rolinski  11 , Angela Hagendorff  12 , Maja Hempel  13 , Wolfgang Sperl  2 , Thomas Meitinger  14 , Joél Smet  3 , Robert W Taylor  4 , Rudy Van Coster  3 , Peter Freisinger  15 , Holger Prokisch  14 , Tobias B Haack  14
Affiliations

Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency

Uwe Ahting et al. Front Genet. .

Abstract

Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.

Keywords: NFU1; iron–sulfur cluster; lipoic acid; mitochondrial respiratory chain; pulmonary hypertension.

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Figures

FIGURE 1
FIGURE 1
Simplified schematic view of iron–sulfur cluster assembly machinery in mitochondria of human cells. Known protein components are inticated in green, described according to its encoding gene. Disease-associated components are indicated in bold. Yellow spots represent sulfur molecules and red spots iron molecules. The molecules are assembled in [2Fe–2S]-cluster and [4Fe–4S]-clusters by the ISC machinery.
FIGURE 2
FIGURE 2
Brain MRI studies in NFU1-mutant patients 2 [T2-weighted axial scans at age 1 2/12 years (A,B) and 2 2/12 years (C,D)], 3 [axial T2-weighted (E) and diffusion tensor image (F) at age 3 months], and 5 [T2-weighted coronar scans (G,H) and MR spectroscopy (I) at age 3 months]. A marked difference is seen in the extent and localization of the lesions ranging from progressive symmetric white matter lesions with necrotic regions in patient 2 (C,D) to reduced volume of supratentorial white matter (E) and diffusion restriction on DTI (F) and alterations in brainstem (G) and upper spinal cord (H).
FIGURE 3
FIGURE 3
Gene structure of NFU1 and position of the identified pathogenic variants (reference cDNA sequence NM_001002755.2 and genomic DNA of chromosome 2 NC_000002.11). Bold script indicates newly identified pathogenic variants. Introns are not drawn to scale.
FIGURE 4
FIGURE 4
Genetic analysis of patient 1. (A) Electropherogram of the strand of cDNA of NFU1. The box indicates the position of pathogenic variant c.622G>T. (B) Break point analysis of the deletion on the non-expressed allele of NFU1.The box above the electropherogram indicates the breakpoint; below illustrates the position of the deletion in the chromosome.
FIGURE 5
FIGURE 5
Genetic analysis of patient 3. (A) Electropherogram of the strand of cDNA of NFU1. The box indicates the position of the pathogenic c.544C>T variant. (B) Conservation of the amino acid residue at position 182 affected by the pathogenic c.544C>T, p.Arg182Trp variant in the indicated species. The box marks the changed position.
FIGURE 6
FIGURE 6
Immunofluorescence studies of cultured skin fibroblast cells from patient 3 and a control showing reduced lipoic acid content and reduced amount of SDHA in the patient cells as compared to controls. (A,B) Staining on lipoic acid; A = control cells, B = patient cells. Blue = DAPI-stain (nuclei), green = porin, red = lipoic acid. (C,D) Staining on SDHA; C = control cells, D = patient cells. Blue = DAPI-stain (nuclei), red = porin, green = SDHA. In all (A–D) the lower-right picture shows the merge of the other three pictures.
FIGURE 7
FIGURE 7
Lipoic acid loading and OXPHOS complex expression in patient 7. (A) Western blot showing expression levels of lipoic acid residues of subunit E2 of PDH (PDH E2) and subunit E2 of αKGDH (αKGDH-E2) in skeletal muscle, liver, and cultured skin fibroblast for patient 7 (p) compared to a control sample (c). The mitochondrial protein VDAC1 (porin) was used as loading control. Together with both skeletal muscle samples of control and patient, a patient with a pathogenic mutation in IBA57 (cIBA) was loaded in parallel to illustrate the similarity of defective lipoylation. (B) Western blot showing expression levels of different subunits of all five OXPHOS complexes (NDUFB8 for complex I, Ip for complex II, core2 for complex III, COXII for complex IV, and Valpha for complex V) in skeletal muscle, liver, and cultured skin fibroblasts in patient 7 (p) and a control sample (c). There is absence of CRM-signal for complex II subunit in skeletal muscle and cultured skin fibroblasts. In liver the CRM-signal for complex II is almost equal to the control loaded at 25% (c25%). CRM-signal for complex I is undetectable (skin fibroblasts) or lower than c25% (skeletal muscle and liver). For skeletal muscle a patient with a pathogenic mutation in IBA57 (cIBA) was loaded in parallel to illustrate the similarity of lowered OXPHOS subunit expression. (C) Blue native-PAGE with in-gel activity staining is shown for liver (left side) and skeletal muscle (right side) in patient 7 (p) and a control sample (c) illustrating severely decreased complex I in liver and complex II activity in both tissues. Complex I activity in skeletal muscle is partially decreased. Samples are loaded in duplo and one gel is used for in-gel activity staining of complex I, III and IV (left panel) while the other is used for complexes V and II (right panel).
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