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Case Reports
, 415 (2), 300-4

Novel Large-Range Mitochondrial DNA Deletions and Fatal Multisystemic Disorder With Prominent Hepatopathy

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Case Reports

Novel Large-Range Mitochondrial DNA Deletions and Fatal Multisystemic Disorder With Prominent Hepatopathy

Marzia Bianchi et al. Biochem Biophys Res Commun.

Abstract

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.

Figures

Supplementary Fig. 1
Supplementary Fig. 1
Radiolabelled 3′-PCR. The wild-type and deleted mtDNA products for patient 1 (A) are 220 and 188 bp, respectively; whereas the wild-type and deleted mtDNA for patient 2 (B) are 225 and 168 bp, respectively. The histograms report the percentage of mtDNA deleted molecules of patient 1 (C) and 2 (D) with respect to control fibroblasts DNA. F: fibroblasts; H: heart; Lv: liver; S: spleen; SM: skeletal muscle; P: pancreas; Lu: lung; K: kidney; B: blood; AG: adrenal gland of patient. Ctrl: control. Data result from three independent experiments.
Supplementary Fig. 2
Supplementary Fig. 2
Quantitative real-time -PCR. The histograms represent the percentage of deleted mtDNA molecules normalized versus media value of three different control fibroblasts. (A) and (B) refers to patient 1 and 2, respectively. Data derived from three independent experiments performed in triplicate. See legend to Supplementary Fig. 1 for details.
Fig. 1
Fig. 1
Histological and electron microscopy in liver tissue of patient 1 (top) and patient 2 (bottom). (A) Masson trichrome, 4×; (B) hematossilin eosin (HE), 40×; (C) electron microscopy (EM), 1500 magnification (Bars, 2500 nm). The Masson thrichrome showed in patient 1 (top-A) disturbed architecture due to fibrotic septa around hepatic nodules. The HE (top-B) evidences ballooned hepatocytes with multinuclei and microvesicular steatosis. Sometimes megamitochondria are present (arrow). With EM (top-C) the mitochondria appeared round with a less electron-dense matrix and markedly reduced cristae. In patient 2 by Masson trichrome (bottom-A) the architecture is preserved; only mild portal fibrosis is evident. The HE (bottom-B) shows oncocytic hepatocytes rich in mitochondria (asterisks). The EM (bottom-C) displays a heavy accumulation of mitochondria throughout the hepatocytes. Again, note the loss of cristae in these mitochondria. Lipids drops are present.
Fig. 2
Fig. 2
Southern blotting of total fibroblasts DNA from patient 1 (A) and patient 2 (B). DNA digestion of patient 1 was performed using PvuII (P) restriction enzyme; for patient 2: PvuII (P), BamHI (B), and SnaBI (S) were used. Total human mtDNA was used as a probe. The bands at 16.6 kb represent the wild-type mtDNA while the smaller ones represent partial deleted mtDNAs. P1 and P2: patient 1 and 2, respectively. C1 and C2: controls 1 and 2, respectively.
Fig. 3
Fig. 3
Western blotting in post-mortem tissues of patient 2. (A) Homogenates from post-mortem tissues were separated by SDS–PAGE, and antibodies against subunits of complex I (NDUFA9, NDUFS3, NDUFB8, NDUFB4), and complex II (SDH 70 kDa) were used; (B) Histograms corresponding to the normalized expression protein. K, Lv, H, SM: kidney, liver, heart and skeletal muscle from patient; LvC, HC, SMC: liver, heart and skeletal muscle from control; (C) SDS–PAGE of patient’s kidney and liver using antibodies against complex II (SDH 70 kDa), complex V (alpha), complex III (core 2), and complex IV (COX II). Complex I subunits content was normalized to the mitochondrial protein SDH70 used as a control for equal loading. All tissues were referred to its own control, except for kidney (control was not available) that was normalized to liver control, considering the similar amount of protein subunits seen in (C). Low content of NDUFB8 and NDUFB4 subunits is evident in liver, kidney, and skeletal muscle, whereas NDUFA9, NDUFS3 as well as complex II, III, and IV subunits are normally expressed.

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