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High prevalence of SURF1 c.845_846delCT mutation in Polish Leigh patients
european journal of paediatric neurology 13 (2009) 146–153
Official Journal of the European Paediatric Neurology Society
Original article
High prevalence of SURF1 c.845_846delCT mutation in Polish Leigh patients Dorota Piekutowska-Abramczuka,*, Ewa Popowskaa, Maciej Pronickib, Elzbieta Karczmarewiczc, Dorota Tylek-Lemanskae, Jolanta Sykut-Cegielskad, Tamara Szymanska-Dembinskab, Liliana Bieleckac, Malgorzata Krajewska-Walaseka, Ewa Pronickad a
Department of Medical Genetics, Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland Department of Pathology, Children’s Memorial Health Institute, Warsaw, Poland c Department of Biochemistry and Experimental Medicine, Children’s Memorial Health Institute, Warsaw, Poland d Department of Metabolic Diseases, Endocrinology and Diabetology, Children’s Memorial Health Institute, Warsaw, Poland e Department of Genetics, Medical Faculty of Jagiellonian University, Krakow, Poland b
article info
abstract
Article history:
Leigh syndrome is a neuropathological disorder with typical morphological changes in
Received 6 December 2007
brain, appearing regardless of diverse molecular background. One of the most common
Received in revised form
enzymatic defects in Leigh patients is cytochrome c oxidase deficiency associated with
12 March 2008
recessive mutations in the SURF1 gene. To assess the SURF1 mutation profile among Polish
Accepted 17 March 2008
patients we studied 41 affected children from 34 unrelated families by PCR-SSCP and sequencing. Four novel mutations, c.39delG, c.752-1G>C, c.800_801insT, c.821A>G, and
Keywords:
five described pathogenic changes, c.311_312insAT312_321del10, c.688C>T, c.704T>C,
Leigh syndrome
c.756_757delCA, c.845_846delCT, were identified in 85.3% of analysed probands. One
SURF1 gene
mutation, c.845_846delCT, was identified in 77.6% of SURF1 alleles. Up to now, it has
c.845_846delCT mutation
been reported only in 9% of alleles in other parts of the world. The deletion was used as
Frequency of disease
LSSURF1 marker in population studies. Eight heterozygous carriers of the mutation were found in a cohort of 2890 samples. The estimated c.845_846delCT allele frequency is 1:357 (0.28 0.2%), and the lowest predicted LSSURF1 frequency in Poland 1:126,736.births. Relatively high frequency of LSSURF1 in Poland with remarkable c.845_846delCT mutation dominance allows one to start the differential diagnosis of LS in each patient of Polish (and probably Slavonic) origin from the direct search for c.845_846delCT SURF1 mutation. ª 2008 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ48 22 815 1075; fax: þ48 22 815 7457. E-mail addresses: [email protected], [email protected] (D. Piekutowska-Abramczuk). 1090-3798/$ – see front matter ª 2008 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2008.03.009
european journal of paediatric neurology 13 (2009) 146–153
1.
Introduction
Leigh syndrome (LS) (MIM 256000) is a neurodegenerative disorder with typical morphological changes characterized by progressive symmetrical vascular proliferation and necrotic areas localized in basal ganglia, brainstem, spine, and sometimes other specific structures of the brain.1 Association of the condition with a dysfunction of various steps of mitochondrial respiratory chain and energetic metabolism was revealed many years after the first pathomorphologic description of the syndrome. It was maintained that one of the most common enzymatic defects found in Leigh patients is the severe and generalized cytochrome c oxidase (COX, complex IV) deficiency associated with recessive mutations in the SURF1 gene.2,3 The SURF1 gene is located on chromosome 9q34 in a highly conserved cluster of housekeeping genes and is ubiquitously expressed.4,5 The SURF1 gene consists of nine exons, spanning almost 5000 bp and codes for 300 amino acid protein of w30 kDa weight. The SURF1 protein is embedded in the inner mitochondrial membrane with two predicted transmembrane domains (amino acid positions 61–79 and 275–293) and its central loop facing the intermembrane space.5 The SURF1 protein has been shown to be required for correct assembly of complex IV, but it is not a constituent of this enzyme. It is suggested that SURF1 protein plays an important role at early stages of COX assembly and it may promote the association of subunit MTCO2 with subassembly consisted of MTCO1–COX4– COX5 proteins, which is the crucial step activating cascade incorporation of remaining subunits.6 A new report indicates a possible role of the SURF1 protein in formation of hem a3–CuB centre.7 It was discovered that patients with SURF1 mutations showed accumulation of early COX assembly intermediates and decreased amounts of complete holoenzyme.6,8 In most cases significant reduction of COX activity to 10–25% of the lowest control values was observed in all tissues.9–12 To date, about 60 different mutations in the SURF1 gene have been identified in approximately 90 LS patients.9–42 Among many various deleterious mutations occurring in single families, there are two recurrent changes found in unrelated patients from different populations, c.845_846delCT and c.311_312insAT312_321del10. The last one is invariably associated with two polymorphisms, c.280T>C and c.573C>G.11,12 The main objective of our study was to analyse a large group of children with Leigh syndrome for the presence of mutations in the SURF1 gene and to evaluate the carrier frequency of the condition in Polish population.
2.
Methods
2.1.
Patients and controls
Sixty LS patients were diagnosed in 1981–2006 in the countrywide reference metabolic centre of the Children’s Memorial Heath Institute in Warsaw (Poland). The diagnosis was established depending on similar unchanged criteria. Brain imaging at the progressive stage and/or at autopsy showed typical LS changes. Biochemical analysis of muscle bioptate and/or fibroblast culture (performed in 21 probands) revealed remarkable
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COX deficiency (in muscle 4.61HSP!HSP!3.92, control values 26.4HSP!HSP!7.4 nmol/min/mg protein).10,12 The patients died due to respiratory arrest, usually before 4 years. Molecular analysis was performed in 41 patients coming from 34 families, due to restricted availability of biological material. Thirty-four parents and other relatives were enrolled in the study. The molecular data were partially reported in other papers.10,12,30 The control panel included 40 unrelated healthy subjects. Parental or legal guardian informed consent for clinical examination and DNA analysis was obtained for each subject.
2.2.
Epidemiological studies
Two thousand eight hundred and ninety anonymous newborns’ blood samples collected on Guthrie cards drawn from the newborn screening programme were included in the population screening. Taking into account that the frequency of the SURF1 mutation heterozygotes may be different in various regions of Poland, we evaluated the relative frequencies of carriers from four large districts containing representative amounts of samples. The study protocols were approved by the Bioethics Commission of the Children’s Memorial Health Institute.
2.3.
Molecular analysis
Total DNA of whole blood leukocytes, cultured fibroblasts and skeletal muscles were isolated with standard proteinase K digestion and phenol/chloroform extraction. All nine exons and intron–exon boundaries of the SURF1 gene were amplified with specific primers described by Tiranti et al.,2 assessed on agarose gels and submitted to SSCP analysis on 8% polyacrylamide gel. Shifted DNA fragments were purified by QIAquick PCR Purification Kit (Qiagen) and sequenced in an automated ABI PRISM 310 sequencer using the Dye-Terminator Kit (Applied Biosystems). As a routine both strands were sequenced. DNA samples of both parents were also examined, if available, to confirm the segregation of identified mutations. Sequences of analysed fragments were compared to the SURF1 cDNA sequence (GenBank RefSeq: NM_003172). Numbering of revealed nucleotide changes was based on the reference sequence; position þ1 corresponded to the A of the ATG translation initiation codon. DNA samples tested in population studies were obtained from dried blood spots. A small piece (approximately 2 mm in diameter) was cut from each spot and used in an amplification reaction with specific primers flanking analysed fragment of SURF1 exon 9. PCR reaction was carried out in optimal conditions with initial denaturation stage in 95 C elongated to 50 min. The further analysis was performed in the same way as for patient’s samples.
3.
Results
3.1.
Identification of SURF1 mutations in LS families
Molecular analysis revealed the presence of 9 different SURF1 gene mutations in 36 diagnosed LS patients (Fig. 1A). The
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european journal of paediatric neurology 13 (2009) 146–153
Fig. 1 – Mutations in the SURF1 gene in Leigh patients. (A) Mutations and polymorphisms identified in Polish population. (B) Two common mutations present in Polish (red bars), another Slavonic (Czech Republic, Slovakia, Croatia) and other published (Western Europe, Australia, Northern America, Japan and China) LSSURF1L cases.
majority of identified mutations (7/9) are localized in a region spanning exons 7–9. Five of detected mutations (c.39delGnovel, c.311_312insAT312_321del10, c.756_757delCA, c.800_ 801insT- novel, c.845_846delCT) are frameshift changes. The dinucleotide deletion, c.845_846delCT in exon 9, was found in 28 families in 45 alleles (77.6% of all mutated alleles), in homozygous form in 20 children (17 families) and in heterozygous form in 15 patients (11 families) (Fig. 2). In five families the rearrangement, c.311_312insAT312_321del10 in exon 4, was detected. In one patient it was found in homozygous form, in four other cases in heterozygous form. Both mentioned mutations make 87.9% of all identified pathogenic mutations in Polish LSSURF1 patients. The remaining frameshift mutations, c.39delG, c.756_757delCA, c.800_801insT, were present in individual families. From among three identified nucleotide substitutions two are known (c.688C>T, c.704T>C) and one is a novel transition (c.821A>G). One novel splice site mutation in intron 7 (c.752-1G>C) was detected in heterozygous form in a single family. To confirm the pathogenic character of novel mutations detected in the SURF1 gene, the control group of 40 healthy persons was screened. No changes were found in indicated fragments of the gene. Three recurrent polymorphisms, c.280T>C in exon 4, c.573C>G in exon 6 and 3–5 copies of the tandem repeat TGCGGGG in intron 1, were found in eight LS patients. Besides, one unpublished substitution c.114C>G in exon 3 was detected in two persons from the control group. Mutations in the SURF1 gene were revealed in 36 patients from 29 families (85.3% of all LS families enrolled to our study). In the remaining five families mtDNA mutations in ATPase6
(m.8993T>C in two cases, m.8993T>G in two cases) and in tRNALys gene (m.8363G>A in one proband) were identified.43,44 In 13 families molecular analysis of the SURF1 gene was performed in parents and siblings of the affected patients.
Fig. 2 – Identification of c.845_846delCT mutation in the SURF1 exon 9. (A) Wild type sequence with the underlined region prone to be deleted. (B) Heterozygote for c.845_846delCT mutation. (C) Homozygote for c.845_846delCT mutation.
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european journal of paediatric neurology 13 (2009) 146–153
Family-specific mutations were detected in heterozygous form on parental alleles. Four heterozygous carriers of mutations were identified in healthy siblings of affected children.
3.2.
Clinical characteristics
Clinical signs and symptoms typical for a classical Leigh syndrome were analysed in three genetically defined subgroups of our patients: one with c.845_846delCT homozygous LSSURF1, the second with c.845_846delCT compound heterozygous LSSURF1, and the third with mtDNA (mainly T9883G/ C) mutations. Some differences in the clinical features are emphasized in the Table 1. A similarity in the age of onset (4–18 months) and the age of death below 4 years, first symptoms resembling a gastrointestinal disease (vomiting, sudden decrease in weight gain), and the patient’s special appearance (thin skin, hirsutism, eye movement dissociation) were among remarkable findings in the c.845_846delCT homozygotes. In this group of patients hyperventilation episodes with respiratory alkalosis (pH >7.40) and decreased partial CO2 pressure (pCO2 <30 mmHg) at the beginning of the disease were always found (low HCO3 represents a compensation phenomenon). In two other genetic subgroups a longer survival, development of extrapyramidal signs and dystonia (as well as other features of non-classical LS variants) were seen more frequently.
3.3.
Population studies
Two thousand eight hundred and ninety randomly chosen samples were screened for the presence of the common c.845_846delCT deletion. Eight heterozygous carriers for the mutation were found (Fig. 3). No homozygous case was identified in the analysed group. The frequency of mutated allele was calculated as 0.0014 (1:714). Using the Hardy–Weinberg principle the frequency of c.845_846delCT deletion carriers was estimated as 0.0028 (0.28%, 1:357) with a 0.95 confidence interval ( p 0.05) of 0.002 (0.2%). It was possible to estimate the overall carrier frequency for all mutations in LSSURF1. Taking into account that c.845_846delCT make up approximately 78% of mutated SURF1 alleles in Polish patients, the result is 0.36%, range 0.16–0.56% (1:279, range 1:625 to 1:178; 0.95 confidence interval). The incidence of SURF1 deficient Leigh syndrome in Poland was estimated as 1:311,364 (range 1:1,562,500 to 1:126,736; 0.95 confidence interval).
4.
Discussion
Since 1998 mutations in the SURF1 gene have been demonstrated as a main cause of Leigh syndrome associated with severe cytochrome c oxidase deficiency. The discovery was an essential breakthrough in diagnostic procedure in a set of Polish LSCOX patients. Thirty-six affected children with
Table 1 – Clinical characteristics of Polish LS children in relation to the genotype. Parameters Number of patients Age of onset between 4 and 18 months Normal psychomotor development up to age of 4–6 months Starting gait at time First words at time Infection or other stress at the onset Acute failure to thrive, marked weight decrease, vomiting Increased ventilation at the onset Skin thin, pale, hypervascularity, hirsutism Emotional/mental/development preserved Remarkable muscle hypotonia Tremor, trembling Dissociation of eye movements (nystagmus, squint) Dystonic movements, ataxia, extrapyramidal syndrome Bulbal symptoms (swallow difficulties) Death or respiratory failure <4 years of age Survival >7 years Seizures Dysmorphic features Heart, liver, kidney or endocrine system involvement Acid base characteristics (pH >7.40, pCO2 <30 mmHg, low HCO3, high O2 saturation) Increased lactate concentration (blood, urine, CSF) Typical LS brain CT/MRI/autopsy in patient or siblings
c.845_846delCT SURF1 homozygotes
c.845_846delCT SURF1 heterozygotes
mtDNA mutations
20 19/20 (95%)a 19/20 (95%)
16 8/14 (57%) 12/16 (75%)
5 1/5 (20%) 3/5 (60%)
17/20 14/14 6/20 18/20
(85%) (100%) (30%) (90%)
10/12 9/9 2/16 6/14
(83%) (100%) (13%) (43%)
3/5 3/5 2/5 2/5
11/16 16/20 20/20 18/20 12/20 13/20
(69%) (80%) (100%) (90%) (58%) (65%)
4/12 10/16 14/16 12/13 3/16 9/13
(33%) (63%) (88%) (92%) (20%) (69%)
2/5 (40%) 0 2/5 (40%) 3/5 (60%) 2/5 (40%) 0
6/13 (46%)
1/5 (20%)
4/11 6/12 3/15 2/15 1/15 2/15 4/16
0 2/5 (40) 2/5 (40) 1/5 (20) 0 0 1/5 (20%)
7/20 (35%) 1/20 (5%) 16/18 (89%) 0 1/20 (5%) 3/20 (15%) 3/20 (15%) 16/16 (100%) 16/20 (80%) 20/20 (100%)
a Presence of clinical feature (in percentage) in patients with available data.
(36%) (50%) (20%) (13%) (7%) (13%) (25%)
11/14 (89%) 16/16 (100%)
(60%) (60%) (40%) (40%)
5/5 (100%) 4/5 (80%)
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Fig. 3 – Distribution of the SURF1 c.845_846delCT mutation in different parts of Poland. The numbers represent amount of heterozygous carriers of the mutation identified in studied anonymous samples. The circles indicate LSSURF1L patients matched by place of origin diagnosed in 1981–2006 in Poland, Black circles, homozygotes; grey circles, heterozygotes.
confirmed pathogenic mutation in the SURF1 gene are the largest group from all published cases to date. The SURF1 mutations were identified in 85% of analysed Polish patients with clinical diagnosis of Leigh syndrome. The incidence of detected SURF1 mutations in our group of children is higher than results obtained by other teams, e.g. 20% reported by Zhang et al.,28 26% found by Sue et al.,31 (with correction to 28%22), 57% identified by Pequignot et al.9 and 75% described by Tiranti et al.32 Published results indicate great differences in population genetics of Leigh disease. Thus the high proportion of the SURF1 mutations identified in our group of patients points to genotype homogeneity and accurate selection of patients based on uniform own criteria. Biochemical and histochemical analyses, in all available cases, showed severe and generalized mitochondrial complex IV deficiency.10,12 The assessment of the clinical picture of our patients revealed some specific phenotype characteristics for c.845_846delCT homozygous LSSURF1 probands (Table 1). This group of patients present nearly identical clinical features and a similar disease course. According to our experience, a LSSURF1 suspicion may be undertaken immediately by a paediatrician who is familiar with the disease, even before typical LS changes become confirmed by brain MRI and other Rahman criteria. Intensive weight decrease, vomiting and increased respiratory effort (usually evoked by an upper respiratory tract infection, fever or vaccination) are very characteristic. The child is hypotrophic, with thin pale skin, hirsutism and ‘‘frightened eyes’’. Compulsory hyperventilation, respiratory alkalosis, hypocapnia and lactate level elevation in plasma, urine and/or cerebrospinal fluid are present.29
Subsequent episodes of neurological deterioration (tremor, motoric regression, eye movement dissociation, bulbar signs) appear and progress. Cognition is preserved. Ventilation failure develops and leads to death. The screening for SURF1 gene mutation(s) is often positive in such cases, confirming the initial strassen diagnose of LS. Remarkably, to date most of the reported mutations predict loss of function of the SURF1 protein caused by frameshift, nonsense or splice site changes in the SURF1 gene. This is consistent with the results of our study. Five from nine identified mutations (c.39delG, c.311_312insAT312_321del10, c.756_757delCA, c.800_801insT, c.845_846delCT) introduce a frameshift in an open reading frame and create a premature stop codon. Two missense mutations (c.704T>C, c.821A>G) are predicted to change amino acids (respectively p.Met235Thr, p.Tyr274Cys) in the SURF1 protein and one mutation (c.688C>T) results in stop codon (p.Arg230X) creation. One mutation (c.752-1G>C) probably alters acceptor splice site in intron 7 and prevents its normal splicing like most splicing changes reported to date.13 In summary, 91% of identified mutations in Polish patients lead to premature termination codons, resulting either in synthesis of truncated proteins or nonsense-mediated mRNA decay (NMD).14 The residual activity of complex IV was observed in our patients. We speculate that thisis the result of the presence of some amount of incorrect protein with restricted assembly function to form active cytochrome c oxidase. It is also possible that another protein can complement the lack of the SURF1 protein. The missense mutations in yeast gene MSS51 can compensate for the loss of Shy1p, the homologue of human SURF1.45 We have no information on the human homologue of MSS51. In the group of Polish patients three different polymorphisms were detected. The presence of c.280T>C and c.573C>G substitution, linked with c.311_312insAT312_321del10 mutation, was confirmed in all our patients and family members harbouring this mutation. The third polymorphism (tandem repeat TGCGGGG in intron 1), which is reported in the literature as the most frequent,13 was found in five copies in only two Polish families. We tried to compare the distribution of the SURF1 mutations of Polish patients with different populations (Fig. 4). In literature, information on the ethnic origin of the majority of LSSURF1 cases was lacking for most described cases. However, for some patients data concerning their origin were presented. They were mostly Slavs.14,16,18,25 In our comparative study LS patients were divided into three groups: Polish patients, other Slavonic patients and the rest of the published cases. Mutational analysis of all cases confirmed the marked dominance of two changesdc.311_312insAT312_321del10 and c.845_846delCTd but their distribution was quite different (Fig. 1B). In Western populations the most frequent detected mutation was a complex insertion/deletion in exon 4. It was found on 25% of alleles,2,3,9,11,13,20–22,31–37 while in Slavonic patients (including Polish) it was revealed only in 8–10% of alleles.12,16,this study The second recurrent mutation, c.845_846delCT, occurred particularly frequently in children with Slavonic origin. It was present in 78% of alleles in Polish LS group and in 46% of alleles in other Slavonic patients.10,16,this study Recently, this mutation was reported in one allele in a Bulgarian proband25 and in two
european journal of paediatric neurology 13 (2009) 146–153
151
Fig. 4 – Distribution of alleles with c.845_846delCT mutation in different regions of the world. The numbers represent amount of mutated alleles found in the certain area. Results for Western European cases are presented together. Red circle indicates Poland, green oval indicates three Slavonic countries (Czech Republic, Slovakia and Croatia).
alleles in a patient from Croatia.18 In western populations this deletion was observed in 9% of mutated SURF1 alleles. Thus numerous occurrence of both mutations in several populations dispersed throughout the world may suggest their ancient origin. We cannot exclude that patients harbouring the same mutation are descendants of emigrants from the single ethnic group. Taking into account the presence of many c.845_846delCT mutation cases in Poland (and neighbouring Czech Republic and Slovakia) with the homozygote accumulation in the central and south-eastern part of our country (Fig. 3), it is likely that c.845_846delCT originated from this region. The distinct dominance of only one (or at the most two) mutation in various recessive genes, like HADHA (P.-A.D., unpublished data), NBS146 and DHCR747 unequivocally distinguish the Polish population from other nations, indicating its homogeneity and stability. High frequency of c.845_846delCT mutation allowed us to use the deletion as a marker of SURF1 deficient Leigh syndrome in Poland aimed to evaluate the frequency of the disease. As we know, this is the first such report; there are only few communications of LS frequency in the world estimated on the basis of clinically recognized cases. According to Rahman data, comprising Australian patients,48 the incidence of LS is 1:77,000. Taking into account the fact that in this group only 18% of probands displayed COX deficiency, the LSCOX frequency is 1:427,350. Darin et al.49 estimated the LS frequency in western Sweden as 1:34,000, and compiled LSCOX frequency as 1:69,000. The incidence of LSSURF1 in Poland was evaluated as 1:311,364 and the carrier frequency as 1:279 (0.36 0.2%). LS
associated with SURF1 mutations appears to be a rather rare condition in Poland in comparison with the frequencies of other diseases, e.g. cystic fibrosis (4% according to Bal et al.50) or Smith–Lemli–Opitz syndrome (3.2% according to Ciara et al.47). The majority of LSSURF1 carriers were detected in the central and south-eastern areas of the country. Our finding correlates with a great amount of patients recognized in these regions. We identified no carriers in northern Poland and only one carrier in western Poland. This may be connected with migration of the Polish population at the beginning of the 20thcentury and the resettlements just after the Second World War, concerning especially the western and northern parts of the country. A similar correlation was also observed by Tylki-Szymanska et al.51 in a large group of patients with lysosomal diseases. It seems interesting that the frequency of Leigh syndrome estimated in this study (1:311,364) is similar to that calculated from the number of molecularly confirmed LSSURF1 patients (1:375,000). Probably almost all LS children are referred to our metabolic centre due to long-lasting, alarming clinical features and diagnostic difficulties. Taking into consideration the total number of LS patients (60) diagnosed in our institute in 1981–2006, the frequency of LS in Poland is 1:218,384 births. Recently, as a result of the present study, a search for c.845_846delCT SURF1 gene mutation has been included as a first step of differential diagnosis of LS in Poland (Fig. 5). We suppose that our finding of c.845_846delCT SURF1 gene mutation predominance would be useful not only in Poland and other Slavonic countries but also would have global
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8.
9.
10.
11.
12.
13.
14.
Fig. 5
15.
significance due to the fact that nearly 20 million people of Polish origin are dispersed throughout the world.
16.
Acknowledgements We thank the patients and families who participated in this study and all the physicians who referred the subjects. This work was supported partly by grants from the Polish Ministry of Science and Information No. 2P05E05729 (to P.E.) and Polish MITONet.pl.
17.
18.
19.
20.
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protein SURF1p leads to loss of the active site heme. J Biol Chem 2005;280(18):17652–6. Hanson BJ, Carrozzo R, Piemonte F, et al. Cytochrome c oxidase-deficient patients have distinct subunit assembly profiles. J Biol Chem 2001;276(19):16296–301. Pequignot MO, Desguerre I, Dey R, et al. New splicing-site mutations in the SURF1 gene in Leigh syndrome patients. J Biol Chem 2001;276(18):15326–9. Piekutowska-Abramczuk D, Popowska E, Pronicka E, et al. SURF1 gene mutations in Polish patients with COX deficient Leigh syndrome. J Appl Genet 2001;42:103–8. Head RA, Brown RM, Brown GK. Diagnostic difficulties with common SURF1 mutations in patients with cytochrome oxidase-deficient Leigh syndrome. J Inherit Metab Dis 2004;27: 57–65. Bohm M, Pronicka E, Karczmarewicz E, et al. Retrospective, multicentric survey in 180 children with cytochrome c oxidase deficiency. Pediatr Res 2006;59(1):21–6. Pequignot MO, Dey R, Zeviani M, et al. Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome c oxidase deficiency. Hum Mut 2001;17:374–81. Williams SL, Taanman JW, Hansikova H, et al. A novel mutation in SURF1 causes skipping of exon 8 in a patient with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. Mol Genet Metab 2001;73:340–3. Bruno C, Biancheri R, Garavaglia B, et al. A novel mutation in the SURF1 gene in a child with Leigh disease, peripheral neuropathy, and cytochrome c oxidase deficiency. J Child Neurol 2002;17:233–6. Capkova M, Hansikova H, Godinot C, et al. A new missense mutation of 547C>T in the SURF1 genedbiochemical and molecular genetic study in seven children with Leigh syndrome. Cas Lek Ces 2002;141:636–41. Ogawa Y, Naito E, Ito M, et al. Three novel SURF-1 mutations in Japanese patients with Leigh syndrome. Pediatr Neurol 2002; 26(3):196–200. Cvitanovic LJ, Van Coster R, Seneca S, et al. Leukoencephalopathy associated with respiratory chain complex deficiency. J Inherit Metab Dis 2003;26(Suppl. 2):109. Rossi A, Biancheri R, Bruno C, et al. Leigh syndrome with COX deficiency and SURF1 gene mutations: MR imaging findings. Am J Neuroradiol 2003;24:1188–91. Head RA, Brown RM, Rahman S, Boubriak II , Brown GK. A novel splicing mutation in the SURF1 gene. J Inherit Metab Dis 2003;26(Suppl. 2):100. Moslemi A-R, Tulinius M, Darin N, et al. SURF1 gene mutations in three cases with Leigh syndrome and cytochrome c oxidase deficiency. Neurology 2003;61:991–3. Sacconi S, Salviati L, Sue CS, et al. Mutation screening in patients with isolated cytochrome c oxidase deficiency. Pediatr Res 2003;53(2):224–30. Van Riesen A, Antonicka H, Kramer E, et al. Long-surviving Leigh syndrome patients with COX deficiency and SURF1 mutations. Biochim Biophys Acta 2004;27. 6th Eur Meeting on Mitochondrial Pathology, Nijmegen, The Netherlands. Monnot S, Chabrol B, Cano A, et al. Cytochrome c oxidasedeficient Leigh syndrome with homozygous mutation in SURF1 gene. Arch Pediatr 2005;12(5):568–71. Ostergaard E, Bradinova I, Ravn SH, et al. Hypertrichosis in patients with SURF1 mutations. Am J Med Genet 2005;138(4): 384–8. Tay SKH, Sacconi S, Akman O, et al. Unusual clinical presentations in four cases of Leigh disease, cytochrome c oxidase deficiency, and SURF1 gene mutations. J Child Neurol 2005;20:670–4. Zakharova EY, Tsygankova PG, Fedonyuk ID, Ilina ES. A novel mutation in SURF1 gene in Russian patient with Leigh syndrome. Eur J Hum Genet 2007;15(Suppl. 1):69.
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28. Zhang Y, Yang YL, Sun F, et al. Clinical and molecular survey in 124 Chinese patients with Leigh or Leigh-like syndrome. J Inherit Metab Dis 2007;30(2):265. 29. Pronicka E, Piekutowska-Abramczuk D, Popowska E, et al. Compulsory hyperventilation and hypocapnia of patients with Leigh syndrome associated with SURF-1 gene mutations as a cause of low serum bicarbonates. J Inherit Metab Dis 2001; 24:707–14. 30. Pecina P, Gnaiger E, Zeman J, Pronicka E, Houstek J. Decreased affinity to oxygen of cytochrome c oxidase in patients with Leigh syndrome caused by SURF1 gene mutations. Am J Physiol Cell Physiol 2004;287:C1384–8. 31. Sue CM, Karadimas C, Checcarelli N, et al. Differential features of patients with mutations in two COX assembly genes, SURF-1 and SCO 2. Ann Neurol 2000;47:589–95. 32. Tiranti V, Jaksch M, Hofmann S, et al. Loss-of-function mutations of SURF-1 are specifically associated with cytochrome c oxidase deficiency. Ann Neurol 1999;45:787–90. 33. Thorburn DR, Hutchison WM, Reed KJ, et al. Cytochrome c oxidase deficiency caused by SURF1 mutations shows relative sparing of cognitive function. J Inherit Metab Dis 1999;22 (Suppl. 1):13. 34. Poyau A, Buchet K, Bouzidi MF, et al. Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients. Hum Genet 2000;106:194–205. 35. Coenen MJH, Van Den Heuvel LWJ, Nijtmans LGJ, et al. Surfeit1 gene analysis and two dimensional blue native gel electrophoresis in cytochrome c oxidase deficiency. Biochem Biophys Res Commun 1999;265(2):339–44. 36. Coenen MJ, Smeitink JA, Farhoud MH, et al. The first patient diagnosed with cytochrome c oxidase deficient Leigh syndrome: progress report. J Inherit Metab Dis 2006;29(1):212–3. 37. Coenen MJ, Smeitink JA, Pots JM, et al. Sequence analysis of the structural nuclear encoded subunits and assembly genes of cytochrome c oxidase in a cohort of 10 isolated complex IVdeficient patients revealed five mutations. J Child Neurol 2006; 21(6):508–11. 38. Teraoka M, Yokoyama Y, Ninomiya S, et al. Two novel mutations of SURF1 in Leigh syndrome with cytochrome c oxidase deficiency. Hum Genet 1999;105:560–3. 39. Santoro L, Carrozzo R, Malandrini A, et al. A novel SURF1 mutation results in Leigh syndrome with peripheral neuropathy caused by cytochrome c oxidase deficiency. Neuromusc Disord 2000;10:450–3.
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40. Rahman S, Brown RM, Chong WK, Wilson CJ, Brown GK. A SURF1 gene mutation presenting as isolated leukodystrophy. Ann Neurol 2001;49:797–800. 41. Salviati L, Freehauf C, Sacconi S, et al. Novel SURF1 mutation in a child with subacute encephalopathy and without the radiological features of Leigh syndrome. Am J Med Genet 2004; 128A:195–8. 42. Yuksel A, Seven M, Cetincelik U, Yesil G, Koksal V. Facial dysmorphism in Leigh syndrome with SURF-1 mutation and COX deficiency. Pediatr Neurol 2006;34:486–9. 43. Pronicki M, Sykut-Cegielska J, Matyja E, et al. G8363A mitochondrial DNA mutation is not a rare cause of Leigh syndromedclinical, biochemical, and pathological study of affected child. Folia Neuropathol 2007;45(4):187–91. 44. Pronicki M, Matyja E, Piekutowska-Abramczuk D, et al. Light and electron microscopy characteristics of the muscle of patients with SURF1 gene mutations associated with Leigh syndrome. J Clin Pathol 2008;61(4):460–6. 45. Perez-Martinez X, Broadley SA, Fox TD. Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J 2003;22(21):5951–61. 46. Varon R, Seemanova E, Chrzanowska K, et al. Clinical ascertainment of Nijmegen breakage syndrome (NBS) and prevalence of the major mutation, 657del5, in three Slav populations. Eur J Hum Genet 2000;8:900–2. 47. Ciara E, Popowska E, Piekutowska-Abramczuk D, et al. SLOS carrier frequency in Poland as determined by screening for Trp151X and Val326Leu DHCR7 mutations. Eur J Med Genet 2006;49:499–504. 48. Rahman S, Blok RB, Dahl HHM, et al. Leigh syndrome: clinical features and biochemical and DNA abnormalities. Ann Neurol 1996;39:343–51. 49. Darin N, Oldfors A, Ar Moslemi, Holme E, Tulinius M. The incidence of mitochondrial encephalomyopathies in childhood: clinical features and morphological, biochemical, and DNA abnormalities. Ann Neurol 2001;49:377–83. 50. Bal J, Maciejko D, Mazurczak T. The type and frequency of mutations in CFTR gene occurrence in patients with cystic fibrosis in Poland–implication of results obtained from genetic counseling and diagnostic screening. Pediatr Pol 1995; 70:627–32. 51. Tylki-Szymanska A, Czartoryska B, Lugowska A, Gorska D. The prevalence and diagnosis of lysosomal storage diseases in Poland. Eur J Pediatr 2001;160:261.
Official Journal of the European Paediatric Neurology Society
Original article
High prevalence of SURF1 c.845_846delCT mutation in Polish Leigh patients Dorota Piekutowska-Abramczuka,*, Ewa Popowskaa, Maciej Pronickib, Elzbieta Karczmarewiczc, Dorota Tylek-Lemanskae, Jolanta Sykut-Cegielskad, Tamara Szymanska-Dembinskab, Liliana Bieleckac, Malgorzata Krajewska-Walaseka, Ewa Pronickad a
Department of Medical Genetics, Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland Department of Pathology, Children’s Memorial Health Institute, Warsaw, Poland c Department of Biochemistry and Experimental Medicine, Children’s Memorial Health Institute, Warsaw, Poland d Department of Metabolic Diseases, Endocrinology and Diabetology, Children’s Memorial Health Institute, Warsaw, Poland e Department of Genetics, Medical Faculty of Jagiellonian University, Krakow, Poland b
article info
abstract
Article history:
Leigh syndrome is a neuropathological disorder with typical morphological changes in
Received 6 December 2007
brain, appearing regardless of diverse molecular background. One of the most common
Received in revised form
enzymatic defects in Leigh patients is cytochrome c oxidase deficiency associated with
12 March 2008
recessive mutations in the SURF1 gene. To assess the SURF1 mutation profile among Polish
Accepted 17 March 2008
patients we studied 41 affected children from 34 unrelated families by PCR-SSCP and sequencing. Four novel mutations, c.39delG, c.752-1G>C, c.800_801insT, c.821A>G, and
Keywords:
five described pathogenic changes, c.311_312insAT312_321del10, c.688C>T, c.704T>C,
Leigh syndrome
c.756_757delCA, c.845_846delCT, were identified in 85.3% of analysed probands. One
SURF1 gene
mutation, c.845_846delCT, was identified in 77.6% of SURF1 alleles. Up to now, it has
c.845_846delCT mutation
been reported only in 9% of alleles in other parts of the world. The deletion was used as
Frequency of disease
LSSURF1 marker in population studies. Eight heterozygous carriers of the mutation were found in a cohort of 2890 samples. The estimated c.845_846delCT allele frequency is 1:357 (0.28 0.2%), and the lowest predicted LSSURF1 frequency in Poland 1:126,736.births. Relatively high frequency of LSSURF1 in Poland with remarkable c.845_846delCT mutation dominance allows one to start the differential diagnosis of LS in each patient of Polish (and probably Slavonic) origin from the direct search for c.845_846delCT SURF1 mutation. ª 2008 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ48 22 815 1075; fax: þ48 22 815 7457. E-mail addresses: [email protected], [email protected] (D. Piekutowska-Abramczuk). 1090-3798/$ – see front matter ª 2008 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2008.03.009
european journal of paediatric neurology 13 (2009) 146–153
1.
Introduction
Leigh syndrome (LS) (MIM 256000) is a neurodegenerative disorder with typical morphological changes characterized by progressive symmetrical vascular proliferation and necrotic areas localized in basal ganglia, brainstem, spine, and sometimes other specific structures of the brain.1 Association of the condition with a dysfunction of various steps of mitochondrial respiratory chain and energetic metabolism was revealed many years after the first pathomorphologic description of the syndrome. It was maintained that one of the most common enzymatic defects found in Leigh patients is the severe and generalized cytochrome c oxidase (COX, complex IV) deficiency associated with recessive mutations in the SURF1 gene.2,3 The SURF1 gene is located on chromosome 9q34 in a highly conserved cluster of housekeeping genes and is ubiquitously expressed.4,5 The SURF1 gene consists of nine exons, spanning almost 5000 bp and codes for 300 amino acid protein of w30 kDa weight. The SURF1 protein is embedded in the inner mitochondrial membrane with two predicted transmembrane domains (amino acid positions 61–79 and 275–293) and its central loop facing the intermembrane space.5 The SURF1 protein has been shown to be required for correct assembly of complex IV, but it is not a constituent of this enzyme. It is suggested that SURF1 protein plays an important role at early stages of COX assembly and it may promote the association of subunit MTCO2 with subassembly consisted of MTCO1–COX4– COX5 proteins, which is the crucial step activating cascade incorporation of remaining subunits.6 A new report indicates a possible role of the SURF1 protein in formation of hem a3–CuB centre.7 It was discovered that patients with SURF1 mutations showed accumulation of early COX assembly intermediates and decreased amounts of complete holoenzyme.6,8 In most cases significant reduction of COX activity to 10–25% of the lowest control values was observed in all tissues.9–12 To date, about 60 different mutations in the SURF1 gene have been identified in approximately 90 LS patients.9–42 Among many various deleterious mutations occurring in single families, there are two recurrent changes found in unrelated patients from different populations, c.845_846delCT and c.311_312insAT312_321del10. The last one is invariably associated with two polymorphisms, c.280T>C and c.573C>G.11,12 The main objective of our study was to analyse a large group of children with Leigh syndrome for the presence of mutations in the SURF1 gene and to evaluate the carrier frequency of the condition in Polish population.
2.
Methods
2.1.
Patients and controls
Sixty LS patients were diagnosed in 1981–2006 in the countrywide reference metabolic centre of the Children’s Memorial Heath Institute in Warsaw (Poland). The diagnosis was established depending on similar unchanged criteria. Brain imaging at the progressive stage and/or at autopsy showed typical LS changes. Biochemical analysis of muscle bioptate and/or fibroblast culture (performed in 21 probands) revealed remarkable
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COX deficiency (in muscle 4.61HSP!HSP!3.92, control values 26.4HSP!HSP!7.4 nmol/min/mg protein).10,12 The patients died due to respiratory arrest, usually before 4 years. Molecular analysis was performed in 41 patients coming from 34 families, due to restricted availability of biological material. Thirty-four parents and other relatives were enrolled in the study. The molecular data were partially reported in other papers.10,12,30 The control panel included 40 unrelated healthy subjects. Parental or legal guardian informed consent for clinical examination and DNA analysis was obtained for each subject.
2.2.
Epidemiological studies
Two thousand eight hundred and ninety anonymous newborns’ blood samples collected on Guthrie cards drawn from the newborn screening programme were included in the population screening. Taking into account that the frequency of the SURF1 mutation heterozygotes may be different in various regions of Poland, we evaluated the relative frequencies of carriers from four large districts containing representative amounts of samples. The study protocols were approved by the Bioethics Commission of the Children’s Memorial Health Institute.
2.3.
Molecular analysis
Total DNA of whole blood leukocytes, cultured fibroblasts and skeletal muscles were isolated with standard proteinase K digestion and phenol/chloroform extraction. All nine exons and intron–exon boundaries of the SURF1 gene were amplified with specific primers described by Tiranti et al.,2 assessed on agarose gels and submitted to SSCP analysis on 8% polyacrylamide gel. Shifted DNA fragments were purified by QIAquick PCR Purification Kit (Qiagen) and sequenced in an automated ABI PRISM 310 sequencer using the Dye-Terminator Kit (Applied Biosystems). As a routine both strands were sequenced. DNA samples of both parents were also examined, if available, to confirm the segregation of identified mutations. Sequences of analysed fragments were compared to the SURF1 cDNA sequence (GenBank RefSeq: NM_003172). Numbering of revealed nucleotide changes was based on the reference sequence; position þ1 corresponded to the A of the ATG translation initiation codon. DNA samples tested in population studies were obtained from dried blood spots. A small piece (approximately 2 mm in diameter) was cut from each spot and used in an amplification reaction with specific primers flanking analysed fragment of SURF1 exon 9. PCR reaction was carried out in optimal conditions with initial denaturation stage in 95 C elongated to 50 min. The further analysis was performed in the same way as for patient’s samples.
3.
Results
3.1.
Identification of SURF1 mutations in LS families
Molecular analysis revealed the presence of 9 different SURF1 gene mutations in 36 diagnosed LS patients (Fig. 1A). The
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european journal of paediatric neurology 13 (2009) 146–153
Fig. 1 – Mutations in the SURF1 gene in Leigh patients. (A) Mutations and polymorphisms identified in Polish population. (B) Two common mutations present in Polish (red bars), another Slavonic (Czech Republic, Slovakia, Croatia) and other published (Western Europe, Australia, Northern America, Japan and China) LSSURF1L cases.
majority of identified mutations (7/9) are localized in a region spanning exons 7–9. Five of detected mutations (c.39delGnovel, c.311_312insAT312_321del10, c.756_757delCA, c.800_ 801insT- novel, c.845_846delCT) are frameshift changes. The dinucleotide deletion, c.845_846delCT in exon 9, was found in 28 families in 45 alleles (77.6% of all mutated alleles), in homozygous form in 20 children (17 families) and in heterozygous form in 15 patients (11 families) (Fig. 2). In five families the rearrangement, c.311_312insAT312_321del10 in exon 4, was detected. In one patient it was found in homozygous form, in four other cases in heterozygous form. Both mentioned mutations make 87.9% of all identified pathogenic mutations in Polish LSSURF1 patients. The remaining frameshift mutations, c.39delG, c.756_757delCA, c.800_801insT, were present in individual families. From among three identified nucleotide substitutions two are known (c.688C>T, c.704T>C) and one is a novel transition (c.821A>G). One novel splice site mutation in intron 7 (c.752-1G>C) was detected in heterozygous form in a single family. To confirm the pathogenic character of novel mutations detected in the SURF1 gene, the control group of 40 healthy persons was screened. No changes were found in indicated fragments of the gene. Three recurrent polymorphisms, c.280T>C in exon 4, c.573C>G in exon 6 and 3–5 copies of the tandem repeat TGCGGGG in intron 1, were found in eight LS patients. Besides, one unpublished substitution c.114C>G in exon 3 was detected in two persons from the control group. Mutations in the SURF1 gene were revealed in 36 patients from 29 families (85.3% of all LS families enrolled to our study). In the remaining five families mtDNA mutations in ATPase6
(m.8993T>C in two cases, m.8993T>G in two cases) and in tRNALys gene (m.8363G>A in one proband) were identified.43,44 In 13 families molecular analysis of the SURF1 gene was performed in parents and siblings of the affected patients.
Fig. 2 – Identification of c.845_846delCT mutation in the SURF1 exon 9. (A) Wild type sequence with the underlined region prone to be deleted. (B) Heterozygote for c.845_846delCT mutation. (C) Homozygote for c.845_846delCT mutation.
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Family-specific mutations were detected in heterozygous form on parental alleles. Four heterozygous carriers of mutations were identified in healthy siblings of affected children.
3.2.
Clinical characteristics
Clinical signs and symptoms typical for a classical Leigh syndrome were analysed in three genetically defined subgroups of our patients: one with c.845_846delCT homozygous LSSURF1, the second with c.845_846delCT compound heterozygous LSSURF1, and the third with mtDNA (mainly T9883G/ C) mutations. Some differences in the clinical features are emphasized in the Table 1. A similarity in the age of onset (4–18 months) and the age of death below 4 years, first symptoms resembling a gastrointestinal disease (vomiting, sudden decrease in weight gain), and the patient’s special appearance (thin skin, hirsutism, eye movement dissociation) were among remarkable findings in the c.845_846delCT homozygotes. In this group of patients hyperventilation episodes with respiratory alkalosis (pH >7.40) and decreased partial CO2 pressure (pCO2 <30 mmHg) at the beginning of the disease were always found (low HCO3 represents a compensation phenomenon). In two other genetic subgroups a longer survival, development of extrapyramidal signs and dystonia (as well as other features of non-classical LS variants) were seen more frequently.
3.3.
Population studies
Two thousand eight hundred and ninety randomly chosen samples were screened for the presence of the common c.845_846delCT deletion. Eight heterozygous carriers for the mutation were found (Fig. 3). No homozygous case was identified in the analysed group. The frequency of mutated allele was calculated as 0.0014 (1:714). Using the Hardy–Weinberg principle the frequency of c.845_846delCT deletion carriers was estimated as 0.0028 (0.28%, 1:357) with a 0.95 confidence interval ( p 0.05) of 0.002 (0.2%). It was possible to estimate the overall carrier frequency for all mutations in LSSURF1. Taking into account that c.845_846delCT make up approximately 78% of mutated SURF1 alleles in Polish patients, the result is 0.36%, range 0.16–0.56% (1:279, range 1:625 to 1:178; 0.95 confidence interval). The incidence of SURF1 deficient Leigh syndrome in Poland was estimated as 1:311,364 (range 1:1,562,500 to 1:126,736; 0.95 confidence interval).
4.
Discussion
Since 1998 mutations in the SURF1 gene have been demonstrated as a main cause of Leigh syndrome associated with severe cytochrome c oxidase deficiency. The discovery was an essential breakthrough in diagnostic procedure in a set of Polish LSCOX patients. Thirty-six affected children with
Table 1 – Clinical characteristics of Polish LS children in relation to the genotype. Parameters Number of patients Age of onset between 4 and 18 months Normal psychomotor development up to age of 4–6 months Starting gait at time First words at time Infection or other stress at the onset Acute failure to thrive, marked weight decrease, vomiting Increased ventilation at the onset Skin thin, pale, hypervascularity, hirsutism Emotional/mental/development preserved Remarkable muscle hypotonia Tremor, trembling Dissociation of eye movements (nystagmus, squint) Dystonic movements, ataxia, extrapyramidal syndrome Bulbal symptoms (swallow difficulties) Death or respiratory failure <4 years of age Survival >7 years Seizures Dysmorphic features Heart, liver, kidney or endocrine system involvement Acid base characteristics (pH >7.40, pCO2 <30 mmHg, low HCO3, high O2 saturation) Increased lactate concentration (blood, urine, CSF) Typical LS brain CT/MRI/autopsy in patient or siblings
c.845_846delCT SURF1 homozygotes
c.845_846delCT SURF1 heterozygotes
mtDNA mutations
20 19/20 (95%)a 19/20 (95%)
16 8/14 (57%) 12/16 (75%)
5 1/5 (20%) 3/5 (60%)
17/20 14/14 6/20 18/20
(85%) (100%) (30%) (90%)
10/12 9/9 2/16 6/14
(83%) (100%) (13%) (43%)
3/5 3/5 2/5 2/5
11/16 16/20 20/20 18/20 12/20 13/20
(69%) (80%) (100%) (90%) (58%) (65%)
4/12 10/16 14/16 12/13 3/16 9/13
(33%) (63%) (88%) (92%) (20%) (69%)
2/5 (40%) 0 2/5 (40%) 3/5 (60%) 2/5 (40%) 0
6/13 (46%)
1/5 (20%)
4/11 6/12 3/15 2/15 1/15 2/15 4/16
0 2/5 (40) 2/5 (40) 1/5 (20) 0 0 1/5 (20%)
7/20 (35%) 1/20 (5%) 16/18 (89%) 0 1/20 (5%) 3/20 (15%) 3/20 (15%) 16/16 (100%) 16/20 (80%) 20/20 (100%)
a Presence of clinical feature (in percentage) in patients with available data.
(36%) (50%) (20%) (13%) (7%) (13%) (25%)
11/14 (89%) 16/16 (100%)
(60%) (60%) (40%) (40%)
5/5 (100%) 4/5 (80%)
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Fig. 3 – Distribution of the SURF1 c.845_846delCT mutation in different parts of Poland. The numbers represent amount of heterozygous carriers of the mutation identified in studied anonymous samples. The circles indicate LSSURF1L patients matched by place of origin diagnosed in 1981–2006 in Poland, Black circles, homozygotes; grey circles, heterozygotes.
confirmed pathogenic mutation in the SURF1 gene are the largest group from all published cases to date. The SURF1 mutations were identified in 85% of analysed Polish patients with clinical diagnosis of Leigh syndrome. The incidence of detected SURF1 mutations in our group of children is higher than results obtained by other teams, e.g. 20% reported by Zhang et al.,28 26% found by Sue et al.,31 (with correction to 28%22), 57% identified by Pequignot et al.9 and 75% described by Tiranti et al.32 Published results indicate great differences in population genetics of Leigh disease. Thus the high proportion of the SURF1 mutations identified in our group of patients points to genotype homogeneity and accurate selection of patients based on uniform own criteria. Biochemical and histochemical analyses, in all available cases, showed severe and generalized mitochondrial complex IV deficiency.10,12 The assessment of the clinical picture of our patients revealed some specific phenotype characteristics for c.845_846delCT homozygous LSSURF1 probands (Table 1). This group of patients present nearly identical clinical features and a similar disease course. According to our experience, a LSSURF1 suspicion may be undertaken immediately by a paediatrician who is familiar with the disease, even before typical LS changes become confirmed by brain MRI and other Rahman criteria. Intensive weight decrease, vomiting and increased respiratory effort (usually evoked by an upper respiratory tract infection, fever or vaccination) are very characteristic. The child is hypotrophic, with thin pale skin, hirsutism and ‘‘frightened eyes’’. Compulsory hyperventilation, respiratory alkalosis, hypocapnia and lactate level elevation in plasma, urine and/or cerebrospinal fluid are present.29
Subsequent episodes of neurological deterioration (tremor, motoric regression, eye movement dissociation, bulbar signs) appear and progress. Cognition is preserved. Ventilation failure develops and leads to death. The screening for SURF1 gene mutation(s) is often positive in such cases, confirming the initial strassen diagnose of LS. Remarkably, to date most of the reported mutations predict loss of function of the SURF1 protein caused by frameshift, nonsense or splice site changes in the SURF1 gene. This is consistent with the results of our study. Five from nine identified mutations (c.39delG, c.311_312insAT312_321del10, c.756_757delCA, c.800_801insT, c.845_846delCT) introduce a frameshift in an open reading frame and create a premature stop codon. Two missense mutations (c.704T>C, c.821A>G) are predicted to change amino acids (respectively p.Met235Thr, p.Tyr274Cys) in the SURF1 protein and one mutation (c.688C>T) results in stop codon (p.Arg230X) creation. One mutation (c.752-1G>C) probably alters acceptor splice site in intron 7 and prevents its normal splicing like most splicing changes reported to date.13 In summary, 91% of identified mutations in Polish patients lead to premature termination codons, resulting either in synthesis of truncated proteins or nonsense-mediated mRNA decay (NMD).14 The residual activity of complex IV was observed in our patients. We speculate that thisis the result of the presence of some amount of incorrect protein with restricted assembly function to form active cytochrome c oxidase. It is also possible that another protein can complement the lack of the SURF1 protein. The missense mutations in yeast gene MSS51 can compensate for the loss of Shy1p, the homologue of human SURF1.45 We have no information on the human homologue of MSS51. In the group of Polish patients three different polymorphisms were detected. The presence of c.280T>C and c.573C>G substitution, linked with c.311_312insAT312_321del10 mutation, was confirmed in all our patients and family members harbouring this mutation. The third polymorphism (tandem repeat TGCGGGG in intron 1), which is reported in the literature as the most frequent,13 was found in five copies in only two Polish families. We tried to compare the distribution of the SURF1 mutations of Polish patients with different populations (Fig. 4). In literature, information on the ethnic origin of the majority of LSSURF1 cases was lacking for most described cases. However, for some patients data concerning their origin were presented. They were mostly Slavs.14,16,18,25 In our comparative study LS patients were divided into three groups: Polish patients, other Slavonic patients and the rest of the published cases. Mutational analysis of all cases confirmed the marked dominance of two changesdc.311_312insAT312_321del10 and c.845_846delCTd but their distribution was quite different (Fig. 1B). In Western populations the most frequent detected mutation was a complex insertion/deletion in exon 4. It was found on 25% of alleles,2,3,9,11,13,20–22,31–37 while in Slavonic patients (including Polish) it was revealed only in 8–10% of alleles.12,16,this study The second recurrent mutation, c.845_846delCT, occurred particularly frequently in children with Slavonic origin. It was present in 78% of alleles in Polish LS group and in 46% of alleles in other Slavonic patients.10,16,this study Recently, this mutation was reported in one allele in a Bulgarian proband25 and in two
european journal of paediatric neurology 13 (2009) 146–153
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Fig. 4 – Distribution of alleles with c.845_846delCT mutation in different regions of the world. The numbers represent amount of mutated alleles found in the certain area. Results for Western European cases are presented together. Red circle indicates Poland, green oval indicates three Slavonic countries (Czech Republic, Slovakia and Croatia).
alleles in a patient from Croatia.18 In western populations this deletion was observed in 9% of mutated SURF1 alleles. Thus numerous occurrence of both mutations in several populations dispersed throughout the world may suggest their ancient origin. We cannot exclude that patients harbouring the same mutation are descendants of emigrants from the single ethnic group. Taking into account the presence of many c.845_846delCT mutation cases in Poland (and neighbouring Czech Republic and Slovakia) with the homozygote accumulation in the central and south-eastern part of our country (Fig. 3), it is likely that c.845_846delCT originated from this region. The distinct dominance of only one (or at the most two) mutation in various recessive genes, like HADHA (P.-A.D., unpublished data), NBS146 and DHCR747 unequivocally distinguish the Polish population from other nations, indicating its homogeneity and stability. High frequency of c.845_846delCT mutation allowed us to use the deletion as a marker of SURF1 deficient Leigh syndrome in Poland aimed to evaluate the frequency of the disease. As we know, this is the first such report; there are only few communications of LS frequency in the world estimated on the basis of clinically recognized cases. According to Rahman data, comprising Australian patients,48 the incidence of LS is 1:77,000. Taking into account the fact that in this group only 18% of probands displayed COX deficiency, the LSCOX frequency is 1:427,350. Darin et al.49 estimated the LS frequency in western Sweden as 1:34,000, and compiled LSCOX frequency as 1:69,000. The incidence of LSSURF1 in Poland was evaluated as 1:311,364 and the carrier frequency as 1:279 (0.36 0.2%). LS
associated with SURF1 mutations appears to be a rather rare condition in Poland in comparison with the frequencies of other diseases, e.g. cystic fibrosis (4% according to Bal et al.50) or Smith–Lemli–Opitz syndrome (3.2% according to Ciara et al.47). The majority of LSSURF1 carriers were detected in the central and south-eastern areas of the country. Our finding correlates with a great amount of patients recognized in these regions. We identified no carriers in northern Poland and only one carrier in western Poland. This may be connected with migration of the Polish population at the beginning of the 20thcentury and the resettlements just after the Second World War, concerning especially the western and northern parts of the country. A similar correlation was also observed by Tylki-Szymanska et al.51 in a large group of patients with lysosomal diseases. It seems interesting that the frequency of Leigh syndrome estimated in this study (1:311,364) is similar to that calculated from the number of molecularly confirmed LSSURF1 patients (1:375,000). Probably almost all LS children are referred to our metabolic centre due to long-lasting, alarming clinical features and diagnostic difficulties. Taking into consideration the total number of LS patients (60) diagnosed in our institute in 1981–2006, the frequency of LS in Poland is 1:218,384 births. Recently, as a result of the present study, a search for c.845_846delCT SURF1 gene mutation has been included as a first step of differential diagnosis of LS in Poland (Fig. 5). We suppose that our finding of c.845_846delCT SURF1 gene mutation predominance would be useful not only in Poland and other Slavonic countries but also would have global
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significance due to the fact that nearly 20 million people of Polish origin are dispersed throughout the world.
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Acknowledgements We thank the patients and families who participated in this study and all the physicians who referred the subjects. This work was supported partly by grants from the Polish Ministry of Science and Information No. 2P05E05729 (to P.E.) and Polish MITONet.pl.
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