Mutations in PNKP Cause Recessive Ataxia with Oculomotor Apraxia Type 4

Hereditary autosomal-recessive cerebellar ataxias are a genetically and clinically heterogeneous group of disorders. We used homozygosity mapping and exome sequencing to study a cohort of nine Portuguese families who were identified during a nationwide, population-based, systematic survey as displaying a consistent phenotype of recessive ataxia with oculomotor apraxia (AOA). The integration of data from these analyses led to the identification of the same homozygous PNKP (polynucleotide kinase 3′-phosphatase) mutation, c.1123G>T (p.Gly375Trp), in three of the studied families. When analyzing this particular gene in the exome sequencing data from the remaining cohort, we identified homozygous or compound-heterozygous mutations in five other families. PNKP is a dual-function enzyme with a key role in different pathways of DNA-damage repair. Mutations in this gene have previously been associated with an autosomal-recessive syndrome characterized by microcephaly; early-onset, intractable seizures; and developmental delay (MCSZ). The finding of PNKP mutations associated with recessive AOA extends the phenotype associated with this gene and identifies a fourth locus that causes AOA. These data confirm that MCSZ and some forms of ataxia share etiological features, most likely reflecting the role of PNKP in DNA-repair mechanisms. Hereditary autosomal-recessive cerebellar ataxias are a genetically and clinically heterogeneous group of disorders. We used homozygosity mapping and exome sequencing to study a cohort of nine Portuguese families who were identified during a nationwide, population-based, systematic survey as displaying a consistent phenotype of recessive ataxia with oculomotor apraxia (AOA). The integration of data from these analyses led to the identification of the same homozygous PNKP (polynucleotide kinase 3′-phosphatase) mutation, c.1123G>T (p.Gly375Trp), in three of the studied families. When analyzing this particular gene in the exome sequencing data from the remaining cohort, we identified homozygous or compound-heterozygous mutations in five other families. PNKP is a dual-function enzyme with a key role in different pathways of DNA-damage repair. Mutations in this gene have previously been associated with an autosomal-recessive syndrome characterized by microcephaly; early-onset, intractable seizures; and developmental delay (MCSZ). The finding of PNKP mutations associated with recessive AOA extends the phenotype associated with this gene and identifies a fourth locus that causes AOA. These data confirm that MCSZ and some forms of ataxia share etiological features, most likely reflecting the role of PNKP in DNA-repair mechanisms. Hereditary autosomal-recessive cerebellar ataxias (ARCAs) are rare neurodegenerative disorders that are clinically and genetically very heterogeneous and are characterized by cerebellar ataxia that is frequently associated with peripheral sensorimotor neuropathy. Ataxia with oculomotor apraxia (AOA) is a subgroup involving cerebellar ataxia, sensorimotor axonal neuropathy, oculomotor apraxia, and extrapyramidal features. Chromosomal instability, immunodeficiency, and sensitivity to ionizing radiations, all usually observed in persons with ataxia telangiectasia (AT) and AT-like disorders, are absent in individuals with AOA.1Barbot C. Coutinho P. Chorão R. Ferreira C. Barros J. Fineza I. Dias K. Monteiro J. Guimarães A. Mendonça P. et al.Recessive ataxia with ocular apraxia: review of 22 Portuguese patients.Arch. Neurol. 2001; 58: 201-205Crossref PubMed Scopus (92) Google Scholar Early-onset recessive ataxia (AOA1 or EAOH [MIM 208920]) is a progressive syndrome associated with hypoalbuminemia and elevated levels of cholesterol and is caused by mutations in APTX (aprataxin).1Barbot C. Coutinho P. Chorão R. Ferreira C. Barros J. Fineza I. Dias K. Monteiro J. Guimarães A. Mendonça P. et al.Recessive ataxia with ocular apraxia: review of 22 Portuguese patients.Arch. Neurol. 2001; 58: 201-205Crossref PubMed Scopus (92) Google Scholar, 2Moreira M.C. Barbot C. Tachi N. Kozuka N. Mendonça P. Barros J. Coutinho P. Sequeiros J. Koenig M. Homozygosity mapping of Portuguese and Japanese forms of ataxia-oculomotor apraxia to 9p13, and evidence for genetic heterogeneity.Am. J. Hum. Genet. 2001; 68: 501-508Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 3Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonça P. Costa M. Barros J. Yanagisawa T. et al.The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin.Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (376) Google Scholar Autosomal-recessive spinocerebellar ataxia 1 (AOA2 or SCAR1 [MIM 606002]), a progressive ataxia,4Le Ber I. Bouslam N. Rivaud-Péchoux S. Guimarães J. Benomar A. Chamayou C. Goizet C. Moreira M.C. Klur S. Yahyaoui M. et al.Frequency and phenotypic spectrum of ataxia with oculomotor apraxia 2: a clinical and genetic study in 18 patients.Brain. 2004; 127: 759-767Crossref PubMed Scopus (162) Google Scholar occurring later than AOA1, is characterized by increased alpha-fetoprotein levels and is caused by mutations in SETX (senataxin).5Moreira M.C. Klur S. Watanabe M. Németh A.H. Le Ber I. Moniz J.C. Tranchant C. Aubourg P. Tazir M. Schöls L. et al.Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2.Nat. Genet. 2004; 36: 225-227Crossref PubMed Scopus (394) Google Scholar A third gene, PIK3R5 (phosphoinositide-3-kinase, regulatory subunit 5; AOA3 [MIM 615217]), has been identified in a consanguineous Saudi Arabian family whose affected members have clinical features similar to those of individuals with AOA2.6Al Tassan N. Khalil D. Shinwari J. Al Sharif L. Bavi P. Abduljaleel Z. Abu Dhaim N. Magrashi A. Bobis S. Ahmed H. et al.A missense mutation in PIK3R5 gene in a family with ataxia and oculomotor apraxia.Hum. Mutat. 2012; 33: 351-354Crossref PubMed Scopus (36) Google Scholar Next-generation sequencing technologies have been remarkably useful for the identification of novel genes causing Mendelian diseases. At the same time, these recently developed technologies have allowed the expansion of phenotypes, particularly in various neurological diseases. The association of the same molecular event with a large-spectrum phenotype or very different phenotypes can be explained by several factors, one being the pleiotropism associated with many genes.7Guerreiro R. Brás J. Hardy J. Singleton A. Next generation sequencing techniques in neurological diseases: redefining clinical and molecular associations.Hum. Mol. Genet. 2014; 23: R47-R53Crossref PubMed Scopus (47) Google Scholar We used exome sequencing and homozygosity mapping to study a series of nine Portuguese families affected by early-onset recessive AOA. These families were identified during a nationwide, population-based, systematic survey of hereditary ataxias and spastic paraplegias. Performed in Portugal from 1994 to 2004, this survey used multiple sources of information to identify affected persons in the community and health-care settings. Detailed methods of the survey are described elsewhere.8Coutinho P. Ruano L. Loureiro J.L. Cruz V.T. Barros J. Tuna A. Barbot C. Guimarães J. Alonso I. Silveira I. et al.Hereditary ataxia and spastic paraplegia in Portugal: a population-based prevalence study.JAMA Neurol. 2013; 70: 746-755Crossref PubMed Scopus (87) Google Scholar All individuals studied here presented established clinical criteria for AOA,1Barbot C. Coutinho P. Chorão R. Ferreira C. Barros J. Fineza I. Dias K. Monteiro J. Guimarães A. Mendonça P. et al.Recessive ataxia with ocular apraxia: review of 22 Portuguese patients.Arch. Neurol. 2001; 58: 201-205Crossref PubMed Scopus (92) Google Scholar and samples were collected after receipt of written informed consent from participants. This study used only de-identified, previously collected DNA samples that were stored at the authorized Center for Predictive and Preventive Genetics, Institute for Molecular and Cell Biology biobank and database. After previous exclusion of mutations in the genes known to be directly associated with this phenotype (APTX, SETX, and PIK3R5), we performed whole-genome genotyping in ten individuals from seven families (nine affected and one unaffected). We used Illumina OmniExpress Beadchips, which assay over 700,000 markers, as per the manufacturer’s instructions. We used Illumina GenomeStudio for initial data analysis and quality control, which led to the exclusion of three samples (two from family 2 and one from family 6) from further analysis involving whole-genome genotyping data. Runs of homozygosity (ROH) were identified with PLINK v.1.079Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A. Bender D. Maller J. Sklar P. de Bakker P.I. Daly M.J. Sham P.C. PLINK: a tool set for whole-genome association and population-based linkage analyses.Am. J. Hum. Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (19634) Google Scholar on the basis of having a size greater than 1.5 Mb and a minimum of 50 SNPs per region. We scanned the genome for regions of homozygosity by using a sliding window of 50 SNPs and allowing at most two missing genotypes and one heterozygote call per ROH. We focused the initial homozygosity mapping on pedigrees with reported consanguinity (families 5 and 7). The only homozygous region, a region >1.5 Mb and shared by all affected individuals, was on chromosome 19 (49,506,390–51,400,356) (Figure S1). Upon analysis of the other families, we found that the proband of family 8 also shared a large homozygous tract overlapping this region (chr19: 49,425,838–53,225,722). We performed exome sequencing in 16 individuals from eight families (12 affected and four unaffected). For this analysis, we prepared genomic DNA according to Illumina’s TruSeq Sample Preparation v.3 and performed the exome capture with Illumina’s TruSeq Exome Enrichment according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq2500 with 100-bp paired-end reads. Following quality control procedures, samples yielded between 10.1 and 12.7 Gb of high-quality, aligned data. This amount of data was the result of mean target coverage between 66.39× and 88.5×, 93.5%–94.6% of targets’ being covered at greater than or equal to 10×, and less than 0.2% of targets’ not being covered even once. We performed sequence alignment and variant calling against the reference human genome (UCSC Human Genome Browser hg19) by using the Burrows-Wheeler Aligner10Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar and the Genome Analysis Toolkit.11McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar, 12DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. Philippakis A.A. del Angel G. Rivas M.A. Hanna M. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat. Genet. 2011; 43: 491-498Crossref PubMed Scopus (7101) Google Scholar Prior to variant calling, PCR duplicates were removed with the Picard software. On the basis of the hypothesis that the mutation was rare, we excluded all common SNPs (MAF > 5%) identified in dbSNP v.137 and in our in-house database of sequencing data for other diseases (n > 2,000). Given the apparent autosomal-recessive mode of inheritance in the three families (families 5, 7, and 8) and the fact that two parental pairs were consanguineous, we focused on homozygous variants located in the shared ROH. One PNKP mutation, c.1123G>T (p.Gly375Trp; RefSeq accession number NM_007254.3), was present in homozygosis in all affected individuals and absent in all unaffected ones. Expanding this analysis to the other studied families, we found the same mutation in homozygosis in family 2; probands in families 1, 3, 4, and 6 were compound heterozygotes for different mutations in the same gene. The proband in family 6 was tested by Sanger sequencing, and no causative mutations were found in this gene in family 9. We also used Sanger sequencing to confirm all mutations identified by exome sequencing, establish compound heterozygosity, and verify intrafamilial segregation. We performed PCR amplifications and purified the resulting products with ExoSAP-IT (USB), then performed direct Sanger sequencing of both strands with BigDye Terminator v.3.1 chemistry v.3.1 (Applied Biosystems) and an ABI 3730XL Genetic Analyzer (Applied Biosystems). Sequencing traces were analyzed with Sequencher software v.4.2 (Gene Codes). The main clinical features of the 11 AOA-affected persons (four males and seven females) who were found to have PNKP mutations are presented in Table 1. All of them were either homozygotes or compound heterozygotes for PNKP mutations (Table 2 and Figure 1). Age at onset ranged from 1 to 9 years; the mean was 4.3 ± 2.3 years. Most individuals exhibited dystonia as their first symptom; this was so prominent that many of these individuals underwent diagnostic procedures for extrapyramidal disorders. In all cases, dystonia spontaneously attenuated during the course of the disease. The second most common symptom was ataxia, followed by oculomotor apraxia. All individuals had signs of polyneuropathy with early, generalized areflexia. Distal-muscle wasting and weakness led to tetraplegia and short atrophic hands and feet. Loss of the ability to walk occurred 7–21 years after disease onset, and most individuals were wheelchair bound by adolescence. Cognitive impairment was present in seven persons, two of whom were severely demented. Alpha-fetoprotein, albumin, and cholesterol levels were highly variable in this cohort; alpha-fetoprotein levels were increased in five individuals, albumin was decreased in six, and cholesterol was elevated in five cases, all of which was determined at later stages of disease progression. In all 11 individuals, brain MRIs revealed cerebellar atrophy.Table 1Clinical, Biochemical, and Imaging Features of the Individuals Carrying PNKP MutationsFamily 1Family 2Family 3Family 4Family 5Family 6Family 7Family 8P1P2P3P4P5P6P7P8P9P10P11Consanguinity−yes−−yesyesyes−GendermalefemalefemalefemalemalefemalefemalefemalemalefemalemaleAge at onset (years)59631344723First signdystoniadystoniadystoniaataxiadystoniaataxiaataxiaOMAOMAdystoniadystoniaMore prominent signneurop.neurop.neurop.neurop.neurop.neurop.neurop.neurop.neurop.neurop.neurop.OMA+++++++++++++++++++++++++Age in wheelchair (years)18221820NA152515142015Dystonia+++++++−−−++++Cognitive impairment+++++NA−−−++++Motor deficit++++++++++++++++++++++++++++++++Decreased vibration sense+++NA++NA+++++++++NA++Pyramidal signs−−+−−−−−−+−Obesity−−++−−+−−++−MRI findingsCACACACACACACACACACACAOther featuresBSABSAdem.NANANANANANAdem.BSAα-fetoprotein levels1.5 N1.5 N1.5 NNN1.5 N4 NNNNNAlbumin levelsNN↓↓↓↓NNANA↓↓Cholesterol levelsNNN↑↑N↑NANA↑↑Abbreviations and symbols are as follows: P1–P11, individuals 1–11, respectively; +, present; ++, moderate; +++, severe; −, absent; ↑, increased level; ↓, decreased level; N, normal level; NA, not available; CA, cerebellar atrophy; BSA, brainstem atrophy; OMA, oculomotor apraxia; neurop., neuropathy; dem., dementia. Open table in a new tab Table 2PNKP Mutations that Caused AOA in Affected Members of the Eight Portuguese Families in Whom Mutations Were FoundFamilycDNA (RefSeq NM_007254.3)Protein (RefSeq NP_009185.2)1c.[1123G>T];[1253_1269dupGGGTCGCCATCGACAAC]p.[(Gly375Trp)];[(Thr424Glyfs∗49)]2c.[1123G>T];[1123G>T]p.[(Gly375Trp)];[(Gly375Trp)]3c.[1221_1223del];[1549_1550insTGTACTGC]p.[(Thr408del)];[(Gln517Leufs∗24)]4c.[1221_1223del];[1315_1329delinsGGGT]p.[(Thr408del)];[(Arg439Glyfs∗51)]aOne of the allelic mutations in F4 is formed by a complex allele that was initially not properly identified. Figure S3 shows the detailed view of the alignment at that position, clearly displaying the complex One of the allelic mutations in F4 is formed by a complex allele that was initially not properly identified. Figure S3 shows the detailed view of the alignment at that position, clearly displaying the complex Open table in a new tab and symbols are as follows: P1–P11, individuals 1–11, respectively; +, present; ++, moderate; +++, severe; −, absent; ↑, increased level; ↓, decreased level; N, normal level; NA, not available; CA, cerebellar atrophy; BSA, brainstem atrophy; OMA, oculomotor apraxia; neurop., neuropathy; dem., dementia. Mutations in PNKP were found to [MIM characterized by and developmental J. M. A. B. D. K. et in PNKP and in DNA Genet. 2010; PubMed Scopus Google Scholar this gene was associated with cerebellar in two who had progressive and C. R. D. J. R. H. M.A. de P. A. cerebellar and the spectrum of PNKP 2013; PubMed Scopus Google Scholar ocular signs were described in those of the individuals we studied had or impairment was observed in most not all individuals to in two These families were identified a population-based survey for hereditary to the of our the We can that is the most of AOA in the Portuguese of the families and the most recessive ataxia in this after P. Ruano L. Loureiro J.L. Cruz V.T. Barros J. Tuna A. Barbot C. Guimarães J. Alonso I. Silveira I. et al.Hereditary ataxia and spastic paraplegia in Portugal: a population-based prevalence study.JAMA Neurol. 2013; 70: 746-755Crossref PubMed Scopus (87) Google Scholar at onset for individuals with is to that of individuals with ± than to that of those with ± is than Ber I. Moreira M.C. Rivaud-Péchoux S. Chamayou C. T. M. G. G. et ataxia with oculomotor apraxia clinical and genetic PubMed Scopus Google Scholar, M. B. M. P. Barbot C. M. M. L. M. et with oculomotor apraxia 2: and study of a cohort of patients.Brain. 2009; PubMed Scopus Google Scholar with extrapyramidal and also that of than that of in individuals with albumin be or normal and cholesterol levels are normal or a similar to is in individuals with after some years of progression. On the other was elevated in some individuals with which to be the in those with The different phenotypes so associated with mutations in PNKP not to to either the or the of the mutation (Figure 1). PNKP is a protein with three an which with the and a DNA and a DNA kinase D. R. S. et al.The molecular of the DNA Full Text Full Text PDF PubMed Scopus Google Scholar The same homozygous located in the kinase of the has been found in individuals with progressive cerebellar and in individuals with MCSZ autosomal-recessive syndrome characterized by onset, intractable and developmental J. M. A. B. D. K. et in PNKP and in DNA Genet. 2010; PubMed Scopus Google Scholar, C. R. D. J. R. H. M.A. de P. A. cerebellar and the spectrum of PNKP 2013; PubMed Scopus Google Scholar We have identified this same variant in compound with in family 1. in the and of the protein have only been associated with developmental In most affected individuals, MCSZ was found to be caused by the variant in J. M. A. B. D. K. et in PNKP and in DNA Genet. 2010; PubMed Scopus Google Scholar In the present variants found to be associated with AOA were all located or the kinase region of the The of the most in this cohort is highly (Figure and is located in a the kinase region. several software this variant as E. a database of human and their and Mutat. 2013; PubMed Scopus Google Scholar A of the of PNKP mutations that the studied variants the and the of some DNA kinase and all studied variants of PNKP mutations associated with and developmental delay on enzyme and DNA Res. 2012; PubMed Scopus Google Scholar The of the protein is by the of the kinase to the between the five and the and D. R. S. et al.The molecular of the DNA Full Text Full Text PDF PubMed Scopus Google Scholar, of PNKP mutations associated with and developmental delay on enzyme and DNA Res. 2012; PubMed Scopus Google Scholar The found in family 3, to the kinase region of the these and has for protein and is that the different phenotypes associated with the different mutations result from in the or kinase of the protein or from protein that to or is also that the different phenotypes are the result of an M. I. the role of in DNA 2011; 36: Full Text Full Text PDF PubMed Scopus Google Scholar PNKP has in multiple pathways in DNA-damage and C. R. D. J. R. H. M.A. de P. A. cerebellar and the spectrum of PNKP 2013; PubMed Scopus Google Scholar, M. I. the role of in DNA 2011; 36: Full Text Full Text PDF PubMed Scopus Google Scholar, and genetic Genet. PubMed Scopus Google Scholar The to these a complex and the key of this is the protein kinase which has been to target H. G. K. S. T. M.C. et of is for DNA 2011; PubMed Scopus Google Scholar, of in and DNA 2010; Full Text Full Text PDF PubMed Scopus Google Scholar Mutations in are known to autosomal-recessive Mutations in other as associated with spinocerebellar ataxia with axonal [MIM and also result in in repair. In this the molecular of PNKP in these different and the by also to the phenotypic associated with mutations in this and genetic Genet. PubMed Scopus Google Scholar A analysis this that PNKP with in neurodegenerative diseases and C. R. D. J. R. H. M.A. de P. A. cerebellar and the spectrum of PNKP 2013; PubMed Scopus Google Scholar The phenotypes associated with PNKP mutations also be by has been recently that and of and can both the and kinase of J.R. and both DNA of 2010; PubMed Scopus Google Scholar In this we identified homozygous or compound-heterozygous PNKP mutations in eight of the nine Portuguese families we studied a of 11 affected in mutations in PNKP are the most of AOA. are to this is also the in other also the phenotype associated with mutations in this gene. PNKP and with other this phenotypic This was in by an the in to the are from the Institute of the of and the at the of a from the to and a from to with The for data presented are as Human Genome

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