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A homoplasmic mitochondrial transfer Ribonucleic Acid mutation as a cause of maternally inherited hypertrophic cardiomyopathy

Robert W. Taylor, PhD^*, Carla Giordano, MD, Mercy M. Davidson, PhD, Giulia d’Amati, MD, PhD, Hugh Bain, MD, Christine M. Hayes, BSc^*, Helen Leonard, MD, Martin J. Barron, PhD^*, Carlo Casali, MD, PhD^||, Filippo M. Santorelli, MD^¶, Michio Hirano, MD, Robert N. Lightowlers, PhD^*, Salvatore DiMauro, MD and Douglass M. Turnbull, MD, PhD^*,*

^* Department of Neurology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
Department of Neurology, Columbia University College of Physicians and Surgeons, New York, New York, USA
Department of Paediatric Cardiology, Freeman Hospital, Newcastle upon Tyne, United Kingdom
Dipartimento di Medicina Sperimentale e Patologia, Newcastle upon Tyne, United Kingdom
^|| Istituto di Clinica delle Malattie Nervose e Mentali, Newcastle upon Tyne, United Kingdom
^¶ Molecular Medicine, Children’s Hospital "Bambino Gesù," Università di Roma-La Sapienza, Rome, Italy

View larger version (18K): [in a new window]   Figure 1 Pedigrees of family 1 (A) and family 2 (B). An arrow indicates the probands in each family. Solid symbols indicate clinically affected individuals, and those tested for the A4300G mitochondrial deoxyribonucleic acid mutation have an asterisk. In family 1, there is a less extensive family tree, and from the pedigree, either an X-linked or autosomal-recessive pattern of inheritance is possible. However, there was no clinical or pathologic evidence of Fabry disease in either family.

View larger version (57K): [in a new window]   Figure 2 Histochemical analysis of skeletal muscle and cardiac tissue. (A) Age-matched control heart sample dual-stained for cytochrome c oxidase (COX) and succinate dehydrogense (SDH) activity. (B) Right ventricular sample from Patient no. IV-03 (family 1) dual-stained for COX and SDH activity, highlighting the distribution of COX-negative (blue) cells. (C) Left ventricular sample from Patient no. IV-03 (family 1) dual-stained for COX and SDH activity. (D) Left ventricular sample from Patient no. IV-01 (family 2) dual-stained for COX and SDH activity. (E) A transverse section of skeletal muscle from the proband in family 1 (IV-03) reacted for COX activity, showing a normal staining pattern. The scale bar represents 200 µm.

View larger version (38K): [in a new window]   Figure 3 Quantification and distribution of the A4300G mutation in family 1. (A) Scheme of the restriction fragment length polymorphism (RFLP) analysis performed. A single HinfI site cuts the wild-type polymerase chain reaction-amplified product of 239 bp into two fragments of 180 and 59 bp. In the presence of the A4300G mutation, a mismatched reverse primer creates a new HinfI recognition site that cuts the 180-bp fragment into two smaller products of 158 and 22 bp. (B) Labeled products generated by the RFLP analysis were separated through a 12% nondenaturing polyacrylamide gel. Fragment sizes (bp) are shown on the right. mtDNA = mitochondrial deoxyribonucleic acid.

View larger version (33K): [in a new window]   Figure 4 Quantification and distribution of the A4300G mutation in family 2. (A) Labeled products generated by the same restriction fragment length polymorphism (RFLP) analysis described in Figure 3, showing the homoplasmic mutation in the heart deoxyribonucleic acid of both probands (IV-03 in family 1 and IV-01 in family 2) and in the fibroblasts of the proband (IV-01) from family 2. Fragment sizes (bp) are shown on the right. (B) Scheme of the alternative RFLP analysis first described by Casali et al. (14). In combination with the A4300G mutation, a mismatched forward primer creates a new HphI recognition site that cuts the 263-bp polymerase chain reaction-amplified product into two smaller products of 235 and 28 bp. In this assay, wild-type mitochondrial deoxyribonucleic acid (mtDNA) remains uncut. (C) Representative gel of samples from three family members showing labeled products generated by the RFLP analysis described earlier. A total of 23 maternal relatives were screened by this assay and shown to be homoplasmic for the A4300G mutation. Fragment sizes (bp) are shown on the right.

View larger version (60K): [in a new window]   Figure 5 Determination of steady-state mitochondrial transfer ribonucleic acid gene for isoleucine (mt-tRNAIle) levels in solid tissues and cultured cells using high-resolution Northern blots. Small ribonucleic acids (1 µg) were separated through a 13%, 8 mol/l urea denaturing polyacrylamide gel, electroblotted onto membranes and hybridized with radiolabeled probes specific for mt-tRNAIle and mt-tRNALeu(UUR) transcripts as described in Methods: High-resolution Northern blot analysis. Together with the patient samples, each panel shows three appropriate control samples prepared from the identical tissue or cell type. (A) Left ventricle of Patient no. IV-03 (family 1). (B) Skeletal muscle of Patient no. IV-03 (family 1). (C) Left ventricle of Patient no. IV-01 (family 2). (D) Cultured myoblasts of Patient no. IV-03 (family 1). (E) Cultured skin fibroblasts of patient no. IV-03 (family 1; lane 1), Patient no. IV-01 (family 2; lane 2), and Patient no. IV-03 (family 2; lane 3).