Appendix e-1:
Data supporting the pathogeneity of R722H and V1044A changes
METHODS
Carrier frequency determination. The frequency of the p.R722H and p.V1044A changes in Finnish control alleles (n= 370 individuals) was determined by PCR and solid-phase minisequencinge1 using sequence-specific primers.
Search of p.R722H change among adult ataxia patients. We screened 224 DNA samples of adult ataxia patients, whose DNA samples were referred to the medical genetics laboratory of Turku University for spinocerebellar ataxia (SCA) diagnostics, and remained without definite diagnosis after SCA mutation screening (screened for mutations of SCA1, 2, 3, 6, 7, 8, 10, 12 and 17; DRPLA, and FRDA). We analyzed the presence of the p. R722H change by sequencing exon 13 as described.4 The complete POLG1 coding sequence was determined of those patients, who were found to carry the p.R722H change.
Structure-functional analysis of R722H within the POLG structure. POLG 3D structure7 with bound DNA template and mapped R722H and V1044A mutations was analyzed as in.e2
RESULTS
Analysis of adult ataxia patients. One adult ataxia patient was homozygote for R722H, and two were heterozygotes, who did not carry a second POLG variant. The homozygote patient manifested at 59 years with exercise intolerance and gait disturbance. Thereafter, the disease progressed rapidly: the patient became wheelchair-bound within a year with severe ataxia, tremor, dysarthria, dysphagia, and diplopia. She had no apparent neuropathy. Brain MRI showed cortical and central atrophy, mild cerebellar atrophy, and an old infarct lesion in the left frontoparietal region. Muscle histology showed mild unspecific changes.
Mapping of R722H and V1044A within the POLG structure. In the 3D structure of POLG7 the arginine (R) 722 locates in the junction between spacer subdomains and accessory subunit, in close vicinity of a common Alpers-disease associated pathogenic site A467 (figure e-1). V1044 locates within the helix partially involved in binding of the single-stranded DNA template.
DISCUSSION
We previously considered R722H to be a polymorphic variant, found in heterozygosity in two Finnish patients with Parkinson’s disease and in 1/268 control individuals.e3 However, here we found it in a patient with Alpers-like phenotype, in compound heterozygosity with a new variant V1044A. Therefore, the pathogenic role of R722H required new consideration. Pathogenic role is supported by a) symptoms typical for POLG-disease, with severe encephalopathy, intractable epilepsy, athetoid-ataxic movement disturbance and rapid psychomotor regression; b) compound heterozygosity of R722H with a novel variant V1044A, the latter not found in 740 ethnically matched control chromosomes; c) combination of a spacer domain with a polymerase domain variant , quite typical for Alpers syndrome; d) previous report of homozygous patients with R722H or compound heterozygous with W748S in two families with non-specified neurological phenotypes,e4 as well as in a patient with severe myoclonic epilepsy of infancy (SMEI), and suggested digenic effect of POLG1 and SCN1A mutations;e5 e) no homozygous healthy controls found. However, neither R722 nor V1044 are well conserved in species (Figure 1 B), which is unusual for pathogenic POLG variants. We screened 370 Finnish control individuals and found the carrier frequency of R722H change to be 1:95, being more common than W748S change (1:125). Screening of 224 samples, from patients suspected for SCA, led us to find one homozygote for R722H, and no compound heterozygous patients, suggesting that if R722H is pathogenic, it is not a major ataxia-causing mutation. The homozygous patient presented at 59 years with rapidly progressive ataxia accompanied by dysarthria, dysphagia and cranial nerve involvement. Although her disease is not typical MIRAS, it mimics POLG-disorders. It remains, however, puzzling that in our diagnostic workup, with altogether over 700 patients screened for POLG1, the patients described here are the only ones found with R722H in compound heterozygous or homozygous form. In conclusion, clinical and DNA data support the role of R722H to be a recessive mutation. We consider it likely, but not proven, that R722H and V1044A are pathogenic in the described patients.
We utilized the recently published POLG1 crystal structure to analyse the structural consequences of the R722H and V1044A changes. The location of R722 in 3D structure is in the close proximity to A467, the major mutation site of Alpers syndrome. Therefore, similar to A467T, R722H has potential to decrease polymerase activity and processivity, and affect interaction with the accessory subunit.e6 V1044 locates within the helix involved in binding of the single-stranded DNA template close to R1047, another mutation site related to Alpers syndrome, and suggests that both these variants could indirectly affect DNA binding and thus decrease DNA polymerase activity. The structural analysis supports R722H to be deleterious, but is less supportive for V1044A, which is also less conserved in species. This case illustrates well the challenges of POLG1 in routine diagnosis: as POLG1 allows considerable normal variation in its amino acid sequence, the pathogenic roles of novel variants are challenging to assess. However, the crystal structure information is a useful new tool to study consequences of the mutations in this protein and to consider their potential pathogenic role.
e-References
e1. Suomalainen A, Syvänen AC. Quantitative analysis of human DNA sequences by PCR and solid-phase minisequencing. Mol Biotechnol 2000;15:123-131.
e2. Palin EJ, Lesonen A, Farr CL, Euro L, Suomalainen A, Kaguni LS. Functional analysis of H. sapiens DNA polymerase gamma spacer mutation W748S with and without common variant E1143G. Biochim Biophys Acta 2010;1802:545-551.
e3. Luoma PT, Eerola J, Ahola S, et al. Mitochondrial DNA polymerase gamma variants in idiopathic sporadic Parkinson disease. Neurology 2007;69:1152-1159.
e4. Komulainen T, Hinttala R, Karppa M, et al. POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype. BMC Neurol 2010;10:29.
e5. Bolszak M, Anttonen AK, Komulainen T, et al. Digenic mutations in severe myoclonic epilepsy of infancy. Epilepsy Res 2009;85:300-304.
e6. Luoma PT, Luo N, Loscher WN, et al. Functional defects due to spacer-region mutations of human mitochondrial DNA polymerase in a family with an ataxia-myopathy syndrome. Hum Mol Genet 2005;14:1907-1920.