Validation of sensitivity and specificity of triplet-primed PCR (TP-PCR) in the molecular diagnosis of FRAXE

&HFtOLD3LQKHLURGD6LOYD 9DOLGDWLRQRI6HQVLWLYLW\DQG6SHFLILFLW\RI 7ULSOHW3ULPHG3&5733&5LQWKH 0ROHFXODU'LDJQRVLVRI)5$;( RXWXEURGH 80LQKR_ &HFtOLD3LQKHLURGD6LOYD 9DOLGDWLRQRI6HQVLWLYLW\DQG6SHFLILFLW\RI7ULSOHW 3ULPHG3&5733&5LQWKH0ROHFXODU'LDJQRVLVRI)5$;( 8QLYHUVLGDGHGR0LQKR (VFRODGH&LrQFLDV &HFtOLD3LQKHLURGD6LOYD 9DOLGDWLRQRI6HQVLWLYLW\DQG6SHFLILFLW\RI 7ULSOHW3ULPHG3&5733&5LQWKH 0ROHFXODU'LDJQRVLVRI)5$;( *HQpWLFD0ROHFXODU 7UDEDOKRHIHWXDGRVREDRULHQWDomRGD 'LVVHUWDomRGH0HVWUDGR 'RXWRUD$QD3DXOD)HUQDQGHV0RQWHLUR6DPSDLR &DUYDOKR HGD 'RXWRUD3DXOD0DULD9LHLUD-RUJH 8QLYHUVLGDGHGR0LQKR (VFRODGH&LrQFLDV RXWXEURGH I DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ II Agradecimentos No culminar deste longo ano gostaria de exprimir o meu reconhecimento a todos aqueles que, de diversas formas, contribuíram para o sucesso deste trabalho. À minha orientadora Doutora Paula Jorge pela inestimável orientação, pelo conhecimento científico, pelo apoio, por todas as dúvidas e questões respondidas, pela paciência e por me ter acolhido como membro do grupo. Mas, especialmente, por toda a confiança e amizade. À minha co-orientadora Professora Ana Paula Sampaio pela disponibilidade e ajuda nos momentos necessários. Ao Dr. Nuno Maia e Dr. Bárbara Rodrigues pelo apoio, pelos ensinamentos, pela paciência e especialmente pela sincera amizade. À Doutora Isabel Marques pelos ensinamentos, sugestões e críticas construtivas, essenciais para o desenvolvimento deste trabalho. À Doutora Rosário Santos pela oportunidade de pertencer ao grupo de trabalho que permitiu a realização desta dissertação. A todos os elementos do Centro de Genética Médica Doutor Jacinto Magalhães pelo acolhimento e disponibilidade. Aos meus amigos pelo companheirismo e incentivo. Em especial à Daniela e Andreia que me acompanharam durante o mestrado, cujo apoio e amizade foram essenciais. À minha família pelo apoio incondicional, sem o qual, nada disto seria possível. A todos um sincero Muito Obrigada! III STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. ___________________________________ Cecília Pinheiro da Silva IV Resumo Em rotina, a determinação do tamanho da região repetitiva GCC do gene AFF2 inclui análise baseada em PCR e em Southern blot (SB) sendo esta última uma metodologia cara e demorada. Por isso, noutros genes, o SB está a ser substituído por outras alternativas como o Triplet-repeat primed PCR (TP-PCR). Quanto sabemos, este método nunca foi aplicado ao diagnóstico de FRAXE. A síndrome do XE frágil (FRAXE) é uma forma moderada de défice intelectual associada a défice de aprendizagem, hiperatividade e em casos raros, comportamentos autistas. FRAXE, com uma frequência estimada de 1/50000, é uma doença associada a repetição de tripletos causada pela expansão do tripleto GCC em5’UTRdogeneAFF2. O amplo e inespecífico espectro clínico de FRAXE faz com que testes moleculares sejam essenciais para um diagnóstico definitivo. Neste trabalho, um novo TP-PCR foi desenvolvido usando um primer com ligação antes das repetições, um primer (GCC)5 com cauda, que também se liga a uma segunda região em AFF2,e um primer idêntico à cauda inespecífica. O ensaio foi otimizado e validado recorrendo a sete amostras com tamanho de alelos conhecidos. Amostras de ADN de 500 mulheres com um PCR de rotina não informativo também foram testadas. Primeiramente, o ensaio determinou corretamente o tamanho dos alelos em 475 amostras com um genótipo de GCC na faixa normal. Nas restantes 25 amostras originalmente genotipadas como homoalélicas, o ensaio determinou 19 com alelos na faixa normal, quatro alelos intermédios e duas pré-mutações. De entre o grupo de 19, quatro foram incorretamente genotipadas devido a um T>C SNP na região próxima das repetições (validado por sequenciação de Sanger). Este SNP também foi identificado em homozigotia numa amostra. Para verificar o tamanho correto das repetições nas amostras com resultados discrepantes, estas foram analisadas por PCR de rotina e Southern blot sendo que, a presença de alelos expandidos, foi confirmada. Descrevemos então uma ferramenta simples, precisa e específica que pode ser usada para determinar o número de repetições (até 100 repetições) em alelos do gene AFF2. O ensaio identificou as amostras homoalélicas de forma inequívoca evitando a necessidade de uma segunda técnica demorada e representando uma alternativa atrativa para laboratórios de diagnóstico. Além disto, o ensaio identificou seis amostras com alelos expandidos que são putativamente patogénicos e instáveis representando um valor acrescentado no diagnóstico molecular de FRAXE. Palavras-chave: AFF2, diagnóstico, FRAXE, repetição de GCC, TP-PCR V Abstract Routinely, the sizing of the AFF2 gene GCC repetitive region includes PCR-based and Southern blot (SB) analyses, the latter being a very expensive and time-consuming methodology. For that reason, in other genes, SB is being replaced by alternative approaches such as triplet-repeat primed PCR (TP-PCR). To the best of our knowledge, this method has never been applied to the diagnosis of FRAXE. Fragile XE syndrome (FRAXE) is a form of mild to moderate intellectual disability associated with learning deficits, hyperactivity as well as autistic behaviour in rare cases. FRAXE, with an estimated frequency of 1/50000, is a trinucleotide repeat disease mostly caused by a GCC expansion in 5’UTRofthe AFF2 gene. The broad and unspecific spectrum of FRAXE clinical presentation makes molecular testing essential for a definitive diagnosis. Herein, a novel TP-PCR was developed using a primer binding upstream the repeat, a (GCC)5tail primer, also binds to a second region within AFF2, and a primer identical to the unspecific tail. The assay was optimized and validated resorting to seven samples with known allele sizes. DNA samples from 500 unrelated females with a previous uninformative routine PCR testing result were further tested. Firstly, the assay correctly sized 100% of the alleles in 475 samples with a normal-range GCC genotype. In the remaining 25 samples originally genotyped as homoallelic, our assay determined 19 with alleles within the normal range, four intermediate alleles and two premutations. Among the first group of 19, four had been incorrectly genotyped due to a T>C SNP near the repetitive region (validated by Sanger sequencing), this SNP was also identified in homozygosity in one sample. To verify the correct repeat length in the discrepant samples, they were additionally analysed by routine PCR and SB and the presence of the expanded alleles was confirmed. We describe a simple, accurate and specific tool that can be used to determine AFF2 alleles up to 100GCC repeats. The assay unambiguously identified homoallelic samples often obviating the need of a second, usually time-consuming technique and representing an attractive alternative for diagnostic laboratories. Furthermore, in six samples this assay correctly identified a putatively pathogenic and unstable expanded allele which had escaped detection with the previously performed PCR representing an added value in the molecular diagnosis of FRAXE. Keywords: AFF2, diagnosis, FRAXE, GCC repeat, TP-PCR VI Table of contents List of abbreviations and Sigla ..................................................................................... VIII List of Figures ................................................................................................................. IX List of Tables .................................................................................................................. XI List of Graphs ................................................................................................................. XI Introduction ...................................................................................................................... 1 1. Repeat expansions diseases ................................................................................... 1 1.1 X-linked intellectual disability ............................................................................ 1 1.1.1 FRAXE associated gene (AFF2) .................................................................. 2 1.1.1.1 FRAXE GCC alleles ........................................................................... 3 1.1.1.2 Other FRAXE gene variants .................................................................. 5 2. Diagnostic methodologies ..................................................................................... 6 2.1 Cytogenetic techniques ....................................................................................... 6 2.2 Polymerase chain reaction (PCR) ....................................................................... 6 2.2.1 Triplet-repeat primed PCR (TP-PCR) .......................................................... 7 2.2 Southern blot ....................................................................................................... 8 2.3 Sanger sequencing ............................................................................................... 9 2.4 Diagnostic methodologies at CGMJM (Centro de Genética Médica Doutor Jacinto Magalhães) .................................................................................................... 9 3. Objectives /Aims ................................................................................................. 10 Materials and Methods ................................................................................................... 12 1. Samples ................................................................................................................ 12 2. Primers selection and construction ...................................................................... 12 3. Triplet-repeat primed PCR .................................................................................. 13 3.1 PCR amplification conditions ........................................................................... 13 3.2 Fragment size determination ............................................................................. 15 4. Sanger sequencingsymmetric PCR ................................................................... 15 5. Sanger sequencing – asymmetric PCR ................................................................ 16 6. Southern blot – AFF2 repetitive region ............................................................... 17 7. Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MSMLPA) ........................................................................................................................ 18 Results and Discussion ................................................................................................... 19 1. TP-PCR amplification ......................................................................................... 19 2. Optimization of the amplification conditions ...................................................... 20 2.1 Fluorescent labelling ......................................................................................... 21 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 2 AFF2 (AF4/FMR2 family member 2) gene and ARX (aristaless related homeobox) gene mutations are some of the causes of XLID4,5. Non-syndromic X-linked intellectual disability (NS-XLID) is defined as a ‘nonprogressive intellectual impairment segregating in an X-linked manner without consistent somatic or diagnosticfeatures’.SeveralNS-XLID genes have been identified, GD11, oligopherenin 1, PAK3, RPS6KA3, IL1PAPL, TM4SF2 and AFF2, with the latter being the most prevalent form of NS-XLID4. 1.1.1 FRAXE associated gene (AFF2) Following the identification of FRAXA gene in 1991, it became clear that some cytogenetically“fragile”positivefamiliesdidnothavetheFMR1 CGG expansion leading to the identification of a fragile site 600 kb distal to FRAXA. This fragile site named FRAXE is located at Xq286–8. Coincident with the fragile site is AFF2 gene, formerly named FMR2 (Fragile X mental retardation gene 2)6,9 and, similarly to FRAXA, the expansion of a GCC repeat located adjacent to a CpG island upstream of the gene causes its silencing and leads to mental impairment10,11. The AFF2 gene is composed of 22 exons spanning at least 500 kb, it has 6 isoforms with alternative splicing in exons 2, 3, 5, 7 and 21 (scheme in Figure 1)12,13. The FMR2 protein (coded by AFF2 gene) is a member of the FMR2/AF4 protein family localized in nuclear speckles where splicing factors are stored, assembled and modified and in the nucleolus when splicing is blocked. Due to similarity with AF4, it Figure 1 Schematic representation of the AFF2 gene (not at scale). Adapted from Javed et al., 2012. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 3 Figure 2 Schematic representation of FRAXE GCC allelic categories. Adapted from Garber et al., 2008 . N 30 60 200 FM PM I (GCC) n has been considered a putative transcription factor with its N-terminal domain showing transactivation activity and a RNA binding protein probably involved in the regulation of alternative splicing through interaction with G-quartet RNA structure9,14. AFF2 is abundantly expressed in the brain, particularly the amygdala and hippocampus and in the placenta6. A study using AFF2 knockout mice suggested that the gene was important for a normal function of the central nervous system and that its loss caused learning and memory impairment as well as abnormalities in sensory perception. These results support the role of the FMR2 protein in the human disorder15. 1.1.1.1 FRAXE GCC alleles There are few reports on FRAXE GCC allele sizes, herein, both Murray et al., 1996 and Annesi et al., 2004 allele categorizing was considered (Figure 2)16,17. Normal individuals have alleles with 6 to 30 GCC repeats that are stable upon transmission. Alleles with 31 to 60 repeats are considered intermediate or grey-zone and may vary slightly upon transmission. Alleles with repeats ranging from 61 to 200 GCC are considered unstable and designated premutations16,17. When the repeats expand to over 200 copies, full mutation, the gene is silenced due to methylation, causing mental impairment. AFF2 gene methylation is variable with reports showing that alleles with 130GCC can be methylated6. The GCC repeat can either expand or contract and is equally unstable when transmitted through both male and female lines14. 18 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 4 Unlike FRAXA repeats that show AGG interruptions conferring stability upon replication, evidence shows that FRAXE repeats are pure, thus, for FRAXE, repeat length may be the only factor of stability. Therefore, the mutation mechanism is probably similar with that of others triplet diseases without interruptions such as Huntington disease and myotonic dystrophy, where the threshold for instability is approximately 40 repeats19. Murray et al., 1996 detected instability in alleles with 37 and 66 repeats transmitted to male sons. The allele of 37 was transmitted as a mosaicism pattern of 27 and 37 repeats and the allele of 66 expanded to 87 repeats suggesting a potential instability for alleles over the normal range of 30 repeats16. a) Normal Different studies reported a modal FRAXE repeat number of 15/16 (laboratory variation) in New York Caucasian, Finnish and British samples and of 18 in Chinese subjects supporting the existence of ethnic differences in repeat allele distribution as well as a founder effect19. In Europe, the most common alleles range from six to 25 repeats with 15 repeats being the predominant6. b) Full mutation FRAXE full mutation, with a frequency of 1/50000 (estimation), is the most common cause of non-syndromic X-linked intellectual disability. It is characterize by mild (IQ of 50-70) to borderline (IQ 70-85) mental impairment with learning and communication difficulties. Some individuals exhibit attention deficit, hyperactivity and autistic behaviours. Because it is a non-syndromic form of intellectual disability with no typical physical features, individuals with FRAXE are not easily distinguished from the general population, however, there has been reports of some clinical features such as long narrow face, mild facial hypoplasia, high-arched palate, irregular teeth, hair and nasal abnormalities, angiomata, clinodactyly and thick lips6,9. The phenotype associated with FRAXE is variable, there are cases where individuals carrying the expanded and methylated allele have a normal IQ. These cases might be explained by the AFF2 expression in the brain during a developmental time window since it is unclear when the methylation of the expanded allele and the extinction of AFF2 expression occurs20. c) Premutation Using Drosophila as a model, it has been showed that the expression of a GCC premutation allele (GCC 90) can cause neuronal degeneration similar to that found in a “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 5 FRAXA CGG premutation model suggesting that a GCC premutation could contribute to unknown causes of ataxia21. Premature ovarian failure does not seem to be associated with FRAXE premutation as in FRAXA but, an excess of small alleles with fewer than 11 repeats was detected in women with premature ovarian failure by Murray et al.,1998, some small alleles were due to deletions within the GCC repeat region22. d) Intermediate The role of FRAXE intermediate alleles is still poorly understood, with studies showing an excess of intermediate alleles in boys with learning difficulties when compared to a control population suggesting that such alleles may not be as benign as previously supposed. This apparent excess could be explained by somatic expansion in tissues such as the brain, secondary structure at DNA or mRNA with an adverse effect on gene function or other causes16. A study in an Italian population showed a correlation betweenintermediateallelesandParkinson’sdiseasebutnotmentalimpairment which is in concordance to the notion that abnormal mRNA levels have an adverse effect on the gene function as showed to premutation alelles17. However, in another study with Parkinson’s disease patients, no association between intermediate alleles and clinical featuresofParkinson’sdiseasewasfound23. 1.1.1.2 Other FRAXE gene variants Intellectual disability cases due to AFF2 gene by partial or entire gene deletions, partial duplications and translocations have been described. Partial deletions are usually associated with milder phenotypes or autism with severity depending on the location and extent of the deletion while the complete loss of the gene function leads to FRAXE24,25. Stettner et al., 2011 described a case where a small deletion caused mild intellectual disability, speech delay, aggression, impulsivity, attention deficit and autistic features24. A case with a partial duplication with emotional impairment, auditory processing deficit, infantilized speech and macrosomia reinforced the clinical variability associated with AFF2 mutations25. Another study found an excess of missense variants in highly conserved sites of AFF2 gene in males with autism spectrum disorder supporting that this gene harbours susceptible alleles that can contribute to the risk of autism and so implicating the gene role in brain function9. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 6 2. Diagnostic methodologies 2.1 Cytogenetic techniques Fragile X syndrome, as implicit by the name, is associated with a fragile site in the X chromosome that is expressed as an isochromatid gap in metaphase stage. This fragile site is usually observed in less than 50% of metaphase spreads of cell cultures with folate depletion7,8. Among other cytogenetic techniques, fluorescence in situ hybridization (FISH) can identify folate sensitive fragile sites allowing its use as a diagnostic method for Fragile X syndrome (Figure 3)8. However, FISH over-diagnoses “Fragile X syndrome”becauseit shows other fragile sites and non-specific chromosomal breakages. Furthermore, because premutations do not express the fragile site cytogenetically, it would be missed in FISH. Thus, after the identification of the causative gene in 1991, cytogenetic techniques were discontinued and molecular methods started to be developed and routinely used3. 2.2 Polymerase chain reaction (PCR) To determine the size of a repetitive region a PCR technique can be applied. The use of one primer fluorescently labelled allows the amplified product to be analysed by capillary electrophoresis. PCR is a rapid and easy method that usually amplifies small alleles but not large alleles, particularly when using standard conditions. In the presence of females or mosaicism with two alleles varying in size, amplification may favour the smaller allele. The amplification of CG-rich regions also requires special conditions and can be very time-consuming and expensive. These difficulties rise with increasing number the repeats leading to amplification failure26. Figure 3 Location of a folate-sensitive fragile site by Fluorescence In situ hybridisation (in yellow: X centromere). Adapted from Flynn et al., 1993. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 7 Several commercial PCR methods that are able to detect alleles with high GC content without formation of nonspecific products have been developed. A triplet-repeat primed PCR using three different primers or triplet-repeat primed-methyl sensitive PCR is one of those methods27. Another method is methyl sensitive PCR, a technique involving bisulfite treatment that converts cytosine to uracil reducing the CG content and consequently reducing the high melting temperature, hairpins and secondary structures. This method has been described as advantageous for Huntington’s disease diagnosis allowing an amplification with conventional PCR technique and a direct visualization of larger alleles27,28. To the best of our knowledge none of these were applied to FRAXE. 2.2.1 Triplet-repeat primed PCR (TP-PCR) In 1996, Warner et al., developed a simple fluorescent PCR system that could rapidly screen expanded CAG repeat in myotonic dystrophy. In this methodology three different primers were used including a specific flanking fluorescently labelled primer (P1) that dictated the specificity and two primers withacommon5’sequence(P3,P4)29. The P4 primer had the sequence (GCA)5 or (TGC)5 atthe3’terminus, depending on the strand of the CAG repeat to be amplified, linked with a tail artificially produced, containing little or no self-complementarity, no complementarity with (GCA)5 or (TGC)5 and no homology to known human sequences. The P3 primer, with the same artificial tail as P4, binds to the end of the amplifiedproductsduetothestabilisingeffectofthe5’tail sequence. A 10:1 ration for P3 to P4 was used in order to exhaust P4 in the early amplification cycles and, consequently, reduce priming at the (CAG)n sequence in earlier rounds combined with a long extension time to allow complete extension of the larger products29. A scheme showing the TP-PCR methodology principle and expected result is presented in Figure 4. The technique was proved to be robust and reliable in the identification of CAG expanded alleles but not their size, that is, TP-PCR can be used to screen DNA samples and exclude the presence of an expansion. Another advantage relies in the ability to identify expanded alleles in poor quality DNA as opposite to other methods that require large amounts of DNA (e.g. Southern blot)29. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 8 Standardized TP-PCR protocols have been published for several repeat diseases such as Friedreich ataxia, Huntington disease, SCA10, SCA12, SCA2, SCA7, SCA8, SCA3 and also Fragile X syndrome and Fragile X associated primary ovarian insufficiency30,31. LabGenomics® developedacommerciallyavailablekit‘LabGscan™PCRKit®’for the detection of the CGG expansion in the FMR1 gene based on this TP-PCR technique. In this case, besides the TP-PCR pattern, also the total number of repeats is obtained32. 2.2 Southern blot Many laboratories use conventional PCR as a first screening test and use a second methodology when confronted with results such as absence of amplification, uninformative female samples or to exclude mosaicism either in repeat size and/or methylation status. In repeat expansion disorders, this second methodology is commonly Southern blot that can quantify full mutations resistant to PCR amplification and, when combined with a methylation-sensitive endonuclease, allows the determination of its methylation status5,27. Southern blot is a good strategy allowing the detection of large premutations, full mutations and mosaic patterns. This technique resolution is around Figure 4 Schematic representation of the TP-PCR principle. A: Annealing of P4 primer containing a sequence complementary to the repeats. B: Fluorescently labelled P1 recognizes a sequence upstream the repetitive region. C: Selective amplification with P3 primer in later cycles. D: illustration of the electropherogram after capillary electrophoresis separation; each peak represents a fragment with a unique number of repeats. Expanded alleles (right) produce a continuous ladder of peaks out of the normal range. Adapted from Catalli et al., 2010. A B C D Example: 13 repeats Example: >200 repeats “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 9 150bp so an accurate size of FMR1 gene premutations (ex: 70CGG ≈ 210bp) requires the use of PCR. In FMR1 the sizing is crucial to distinguish between large normal, intermediate and premutated alleles and to assess the risk of expansion upon transmission27,33. Briefly, a DNA sample is digested using restriction enzymes and subjected to electrophoresis in horizontal agarose gel. After overnight separation, the fragments are denaturized, neutralized and transferred to a nitrocellulose, polyvinylidene or Nylon membrane through blotting. During this process, the fragments are carried with the buffer and retained in the membrane surface by capillary transfer or using a vacuum blotting device for approximately 4 hours. The nucleic acids are then fixated to the membrane using heat or ultraviolet light and undergo an overnight hybridization step. After several washing steps to remove unhybridized probe, incubation with an antibody and with a substrate, it is subjected to autoradiography with X-ray film or chemiluminescence depending on the probe labelling to visualize the targeted region(s)34–36. 2.3 Sanger sequencing Determining the order of nucleic acids in polynucleotide chains is essential to a variety of research applications. Sequencing is crucial to the identification and interpretation of human sequence variation as it relates to health and disease37,38. Sanger’s ‘chain-termination’ or dideoxy technique uses dideoxynucleotides (ddNTPs) lacking the 3’ hydroxyl group tagged with a specific fluorescent dye. Combining ddNTPs at a fraction of concentration of standard dNTPs, one primer (usually M13-tail) and DNA polymerase results in DNA strands of different lengths. The resulting dye-labelled fragments are submitted to capillary electrophoresis producing fluorescence emissions of four different colours revealing the DNA sequence37,38. This methodology is used for instance to identify missense variants in Fragile X patients when no expansion is observed39. 2.4 Diagnostic methodologies at CGMJM (Centro de Genética Médica Doutor Jacinto Magalhães) The laboratory developed a multiplex PCR technique that simultaneously amplifies the repetitive region of FMR1 and AFF2 genes and the 24bp duplication in ARX gene5. All samples belonging to individuals showing intellectual disability are tested using this “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 10 multiplex strategy and three important mutational hotspot regions can be simultaneously screened5. This routinely used technique amplifies regions that include both FMR1 and AFF2 genes triplet repeat regions using primers flanking those regions and allowing the discrimination of alleles in the normal, intermediate and premutation ranges after capillary electrophoresis separation. Because expanded alleles are usually not amplified, when only one allele in the normal range is obtained in a female sample, two distinct explanations, with different clinical impact, can be formulated, a) it is a normal homoallelic case or b) the second allele is expanded and putatively implicated in the clinical features40. In the case of male patients the routine PCR method can also overlook mosaicism for premutation and full mutation alleles (due to the preferential amplification of smaller alleles and/or competition between the three primer-pairs)26. Expansions causing the silencing of the AFF2 gene are diagnosed through Southern blot, a very time-consuming and expensive methodology that also requires a large amount of DNA41. To avoid these difficulties, there is a need for a screening method that is both easy and fast to perform which is where the triplet-repeat primed PCR technique comes in. TPPCR was proposed by Warner et al., 1996 for other genes/triplet repeats to detect larger alleles in disorders with triplet repeat expansions29. Furthermore, the confirmation of homoallelism by the TP-PCR, would avoid the use of the time-consuming and expensive Southern blot. This methodology is already used to screen FMR1 expansions and to determine the FMR1 AGG interspersion pattern. However, to the best of our knowledge, it was never described in AFF2 gene. 3. Objectives /Aims The main aim of this thesis is the development of a triplet-primed polymerase chain reaction (TP-PCR) assay for the analysis of the AFF2 gene repetitive region. Other goals include testing and validating this new methodology in seven samples with previously known GCC size in terms of: - Primer sequence; - Primer labelling; - PCR amplification conditions; - Primers concentrations; “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 11 - PCR enhancers; - DNA quality. After validation and establishment of the best conditions it will be applied in 500 female samples with a previous “unknown” genetic status (only one size allele was obtained after routine PCR). This study has been approved by DEFI – Departamento de Ensino, Formação e Investigação do Centro Hospitalar Universitário do Porto, CHUP E.P.E., as well as the Hospital’s Ethical Committee - N/ REF.ª 2018.179(154-DEFI/153-CES). All the DNA samples used were anonymously stored at the lab after being tested for Fragile X and signed informed consent. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 18 7. Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MSMLPA) Multiplex ligation dependent probe (MLPA®) was carried out on five samples suspected to have a small deletion. MLPA® was performed using SALSA® MLPA® ME029FMR1/AFF2Probemixandaccordingtomanufacturer’sinstructions.Briefly,the DNA samples are denaturated and undergo a hybridisation reaction during 18 hours. Products are split into two parts, one for probe ligation and the other for ligation and digestion by HhaI of unmethylated DNA, both undergo an equal final PCR reaction.43 MLPA® data was analysed using Coffalyser.Net software (MRC-Holland, Netherlands). “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 19 Results and Discussion 1. TP-PCR amplification As schematized in Figure 5, the TP-PCR amplification is obtained by the combination of a forward primer (green arrow), a primer with five CGGs (complementary to the GCCs) that anneals in the triplet repeats (yellow arrow) and a third equal to the specific primer tail (orange arrow). A distinct fragment is also obtained due to primer ligation further down the sequence (sequence shown in dark yellow). Because of a timine (T) in the exact middle of the primer with five GCC repeats, with optimized conditions, a specific ligation is possible. This allowed the analysis of the repeat region total length simultaneously with the triplet-repeat profile. After amplification and separation by capillary electrophoresis, TP-PCR profiles were obtained (Figure 6). The first peak “contains” five repeats (as the primer binds to five GCCs) and each peak after this corresponds to an increase of one repeat. Figure 6 shows an example of a sample with 15GCCs. Due to some unspecific binding in the CG rich region subsequent to the GCC repeats, some small peaks can be also be observed. The peak at 188.5bp (Figure 6) corresponds to the total allele size. Confirmation of these results was done using samples previously sequenced. Figure 5 Schematic representation of the primers binding sites during the TP-PCR amplification strategy. Sequence of the AFF2 gene (NCBI reference sequence: NG_016313.2) from position c.4952 to c.5156. Due to the existence of an almost perfect (GCC) 5 repeat downstream the polymorphic GCC repeat a second PCR product is obtained. c.4952 c.5156 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 20 2. Optimization of the amplification conditions GC-rich DNA sequences have higher melting temperatures caused by increased hydrogen bonds strength and secondary structures such hairpins, knots and tetraplexes that, combined with hinder denaturation and primer annealing, may lead to polymerase arrest and a premature extension. These sequences are, then, difficult to amplify by PCR and, regularly, result in little or none expected product as well as the amplification of several unspecific products44,45. Small changes in the PCR protocol can have a huge impact on product formation. Thus, an optimization of the assay is required with different approaches such as magnesium chloride concentration, buffer pH, denaturation and annealing time and temperature, cycle number, modified nucleotides, among others44,45. Herein, several parameters were optimized including the primer labelling, cycle number, annealing temperature, annealing and extension times, primers concentration and PCR enhancers (Betaine, DMSO, Q-solution and 7-deaza-2’-deoxyguanosine). Before optimization, a standard amplification program was used (Table 9). Figure 6 TP-PCR result obtained in GeneMapper® software after capillary electrophoresis of the amplified products. Besides the triplet-repeat profile (left) it is also possible to obtain the total size of the fragment (right). 15 7 6 5 9 8 10 11 12 13 14 188.5bp Unspecific binding Relative Fluorescent units (RFU) Basepairs (bp) “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 21 Table 9 Thermocycler program used for the TP-PCR amplification. Step Temperature Time (min) Cycles Initial denaturation 98ºC 5:00 1 Denaturation 98ºC 1:00 45 Annealing 60ºC 1:00 Extension 68ºC 2:00 Final extension 68ºC 10:00 1 2.1 Fluorescent labelling Two different labels were tested in the same primer sequence (NED and HEX). NED (chemical structure not available) is a fluorescent dye used to fluorescently label oligonucleotidesatthe5’-end that can be used in several applications such as real-time PCR, hybridization probes and other fluorescence-based genetic analysis applications. NED has an absorbance maximum of 546nm and an emission maximum of 575nm thus, emitting in yellow at the visible spectrum46. Hexachloro-fluorescein (HEX) is a version of fluorescein used to fluorescently label oligonucleotidesateitherthe5’- or3’-end that can be used in several applications such as real-time PCR, in hybridization probes and for structure-function studies. Oligo primers labelled at the 5’-end can be used to generate fluorescently-labelled PCR or genetic analysis products. HEX has an absorbance maximum of 535nm and an emission maximum of 556nm thus emitting in green at the visible spectrum47. A comparison of the results obtained with the forward primer labelled with NED or HEX is shown in Figure 7. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 22 HEX labelling leads to a more irregular profile and less amount of amplified product (240 RFU) compared to NED (over 400RFU). The latter (NED), althoughwitha‘V’ shaped profile, allows the easy identification of each fragment. In the following experiments and in the development of the methodology, only NED labelling was used. 2.2 Annealing temperature Primers with a high GC content may require higher annealing temperatures (>55ºC) when compared to primers with low GC content (<50%). The use of low annealing temperature can increase the quantity of non-specific products but if the temperature is too high, poor annealing of the primers may occur48,49. In this case, the first test (Table 9 from Section 2.Optimization of amplification conditions) was performed using an annealing temperature of 60ºC for 1min (Figure 8). Figure 7 TP-PCR result obtained using a forward primer labelled with NED (A) or HEX (B). A B 15 15 188.5bp 188.5bp Basepairs (bp) Basepairs (bp) Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 23 At 60ºC, the expected size was obtained, however, with small amplification intensity (RFU). Two different annealing experiments were, then, conducted. In one there was a combination of 15 cycles with an annealing of 57ºC followed by 30 cycles with annealing at 60ºC. In another experiment, 15 cycles with an annealing temperature of 55ºC followed by 30 cycles with annealing at 58ºC. The results of other temperature/ nº cycles combination are shown below in Figure 9. Another example of these tested conditions is shown in Figure 10. Figure 8 TP-PCR result obtained with an annealing temperature of 60ºC. Relative Fluorescent units (RFU) Basepairs (bp) 18 197.1bp Figure 9 TP-PCR result obtained with an annealing temperature of 15x 57ºC/30x 60ºC (A) and 15x 55ºC/30x 58ºC (B). A B Relative Fluorescent units (RFU) Basepairs (bp) Relative Fluorescent units (RFU) Basepairs (bp) 197.1bp 197.1bp 18 18 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 24 Although the profiles obtained with the two different amplifications conditions (A and B) aresimilar,‘cleaner’ peaks are present in the second experiment (B) with 15 cycles at 55ºC followed by 30 cycles with annealing at 58ºC in both samples (Figures 9 and 10). Furthermore (Figure 10), it can be also noted that the fragment that corresponds to the total size of the allele is favoured in the condition B which may be because a lower annealing temperature favours “nonspecific” products48. In this particular experiment, the binding of the primer further down in the sequence is beneficial because a second validation of the total allele size can be obtained. Following these results, the amplification testing protocol was establish as described in Table 3 (Materials and Methods Section 3.1 PCR amplification conditions). 2.3 Primer concentrations A ratio of 10:1 between the tail sequence primer (g.AFF2_TP_PCR_TAIL or Tail) and the repeat sequence primer (g.AFF2_TP_PCR_GCC or GCC) should be used to exhaust the primer binding in the early amplification cycles in order to reduce priming at (GCC)n, produced in earlier rounds29. Figure 10 TP-PCR result obtained with an annealing temperature of 15x 57ºC/30x 60ºC (A) and 15x 55ºC/30x 58ºC (B). A B Relative Fluorescent units (RFU) 39 15 260.5bp 188.5bp 15 260.5bp Basepairs (bp) Relative Fluorescent units (RFU) Basepairs (bp) 39 188.5bp “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 25 In the development of this TP-PCR, a ratio of 2:1 (as in routine experience) was tested with a concentration of 0.2pmol/µL for both the forward (g.AFF2_TP_PCR_F or F) and Tail primers and of 0.1pmol/µL for the GCC primer. Three other different primer concentration combinations were tested, one with the forward primer reduced to half (0.1pmol/µL), the Tail primer reduced to half (0.1pmol/µL) and a third with the GCC primer reduced to a concentration of 0.04pmol/µL with a ratio of 5:1 between the Tail primer (0.2pmol/µL) and the GCC primer (0.04pmol/µL). The results of the four conditions tested in the same DNA sample are shown below in Figure 11. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 26 Analysing these results allow us to conclude that, a decrease in the forward primer concentration gives a weaker signal, hampering the proper repeat number counting. The same occurs with the decrease in the GCC primer concentration where the signal is below 100RFU. A reduction in the tail primer does not seem to change the amplification profile. Following these results, the concentrations of 0.2pmol/µL for primers F and Tail and that of 0.1pmol/µL for primer GCC, with a ratio of 2:1, were used. Figure 11 TP-PCR result obtained after testing different primer concentrations. A: 0.2pmol/µL F and Tail and 0.1pmol/µL GCC. B: 0.1pmol/µL F (reduced to half), 0.2pmol/µL Tail and 0.1pmol/µL GCC. C: 0.2pmol/µL F, 0.1pmol/µL Tail (reduced to half) and 0.1pmol/µL GCC. D: 0.2pmol/µL F and Tail and 0.04pmol/µL for GCC (ratio 5:1). A D C B Basepairs (bp) Basepairs (bp) Basepairs (bp) Basepairs (bp) Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) 188.5bp 188.5bp 188.5bp 188.5bp 15 15 15 15 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 27 2.4 PCR additives Particularly GC-rich DNA sequences can form secondary structures that are difficult to amplify by PCR and, commonly, result in little or absence of expected product as well as the amplification of unspecific products. The inclusion of appropriate PCR additives to the PCR mix can be essential to increase yield, specificity and accuracy of the amplification44. There is a variety of additives and enhancing agents that can be useful to the PCR optimization such as dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (Betaine), formamide, glycerol, non-ionic detergents, bovine serum albumin (BSA), polyethylene glycol and tetramethylammonium chloride44. Both Betaine and DMSO facilitate strand separation of double helix DNA by altering its melting characteristics and are ideal as additives because they are inexpensive, easily obtainable and compatible with other biological agents50. Betaine, an amino acid analog with both positive and negative charges close to neutral pH, equalizes the contribution of GCand ATbase pairing to the stability of the DNA duplex44,50. DMSO acts by disrupting inter and intra strand re-annealing50. Several agents with application in GCrich DNA to facilitate product formation are commercially available but their chemical composition is unknown44,45. Q-solution provided by Qiagen® is one of those cases, it is described as an agent that facilitates amplification of difficult templates by modifying the melting behaviour of DNA, it improves suboptimal PCR due to the presence of secondary structures or GCrich templates but it does not compromise PCR fidelity51. 7-deaza-2’-deoxyguanosine (7-deaza-dGTP) can weaken base to base interactions thus, solving superstructures, making it useful to improve PCR product yield. It can also be helpful in GCrich sequences sequencing, especially in low amounts of template or poor DNA quality52. Besides Betaine, DMSO and 7-deaza-dGTP, also the addition of Q-solution was tested. Figure 12 shows the results of the impact of those additives on the TP-PCR amplification, on the left add component is indicated: 0.5x of Q-solution (A) 0.8M of Betaine (B) and 10% of DMSO (C) compared to their absence (on the right). Results D and E compare the substitution of Betaine and DMSO (left) with 0.2mM 7-deaza-dGTP (right) and G the presence (left) or absence (right) of 7-deaza-dGTP. In another experiment (F), both Betaine and DMSO concentrations were reduced to 0.56M Betaine and 7% DMSO (right). “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 34 3.1 Normal alleles The novel TP-PCR technique detected 15 heterozygous samples with AFF2 alleles within the normal range. In Figures 15 and 16 are some examples of samples that had previously revealed an uninformative PCR results. In the first example, alleles of 14 and 15GCC (Figure 15) were identified. This result shows that the established conditions allow the discrimination of alleles differing in one repeat. In this second example (Figure 16), a difference of 12 repeats (15 and 27 GCC) can be observed. In our opinion the fact that alleles are ~36bp apart in size can explain why the second allele was not observed with the routine PCR. Furthermore, the fact that routine PCR used three primer pairs (multiplexing FMR1, AFF2 and ARX) hampers the amplification of the second allele. This bias towards the smallest alleles seems to be occurring more frequently with AFF2 than FMR1 gene alleles (P. Jorge, personal communication). Figure 15 TP-PCR result obtained from a sample with a 14GCC and 15GCC alleles. 14 15 Relative Fluorescent units (RFU) 185.5bp 188.5bp Basepairs (bp) Figure 16 TP-PCR result obtained from a sample with a 15GCC and 27GCC alleles. 15 27 Relative Fluorescent units (RFU) Basepairs (bp) 188.5bp 224.4bp “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 35 3.2 Intermediate alleles In the 500 tested samples, four carry intermediate alleles (ranging from 31 to 60 repeats) were identified (Figures 17 to 20). Relative Fluorescent units (RFU) Basepairs (bp) 25 32 216.7bp 236.8bp Figure 17 TP-PCR result obtained from a sample with a 25GCC and 32GCC alleles. Relative Fluorescent units (RFU) Basepairs (bp) 19 36 199.5bp 248.9bp Figure 18 TP-PCR result obtained from a sample with a 19GCC and 36GCC alleles. Figure 19 TP-PCR result obtained from a sample with a 16GCC and 47GCC alleles. Relative Fluorescent units (RFU) Basepairs (bp) ~47 16 ~285.7bp 190.8bp Relative Fluorescent units (RFU) Basepairs (bp) 193.6bp ~49 17 288.6bp Figure 20 TP-PCR result obtained from a sample with a 17GCC and 49GCC alleles. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 36 3.3 Premutation alleles The new developed method allowed the identification of two female premutation carriers, one with 15 and 84GCC alleles and the other with 15 and ~107GCC alleles (Figures 21 and 22). Figure 21 TP-PCR result obtained from a sample with a 15GCC and ~84GCC alleles. Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) Basepairs (bp) Basepairs (bp) 15 15 15 188.5bp 188.5bp 188.5bp ~84 ~395.3bp Figure 22 TP-PCR result obtained from a sample with a 15GCC and ~107GCC alleles. Relative Fluorescent units (RFU) Relative Fluorescent units (RFU) Basepairs (bp) Basepairs (bp) 15 188.5bp 188.5bp 188.5bp 15 15 ~107 ~463bp “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 37 In the expanded range (over 100 repeats) it was not possible to accurately quantify the exact size of the allele. However, a clear distinction between true homoallelic and an expansion can be attained and this is the purpose as a screening method. If needed for diagnostic purposes, the exact number of GCC repeats in expanded alleles, should be obtained by Southern blot. 3.4 Confirmatory techniques 3.4.1 Sizing of larger alleles The routine PCR was optimized to allow the amplification of larger alleles. Essentially, only AFF2 locus was amplified by removing the primers recognizing FMR1 and ARX loci, as well as slight changes in the amplification program allowed the quantification of AFF2 second allele up to ~107GCCs. Results are shown in Figures 23 to 25. Figure 23 PCR result obtained from a sample with a 15GCC and 27GCC alleles. Relative Fluorescent units (RFU) Basepairs (bp) 252.5bp (15GCC) 287.8bp (27GCC) Relative Fluorescent units (RFU) Basepairs (bp) 252.5bp (15GCC) ~459.6bp (84GCC) Figure 24 PCR result obtained from a sample with a 15GCC and ~84GCC alleles. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 38 A confirmation of the allele sizes obtained by TP-PCR, even for the large alleles of 84 and 107GCC, was obtained. 3.4.2 Southern blot Southern blot analysis with an AFF2 specific probe (AFF2 AJ31-Dig1 GeneProber™) was performed using, among others, samples with larger alleles. A calibration curve using DIG II and DIG III size markers was used to calculate the approximate number of GCCs (Figure 26). In female samples, a normal size fragment of 2.1kb fragment corresponds to the active X chromosome and the 4.5kb fragment to the methylated inactive X chromosome. A Relative Fluorescent units (RFU) Basepairs (bp) 253.1bp (15GCC) ~529bp (107GCC) Figure 25 PCR result obtained from a sample with a 15GCC and ~107GCC alleles. Figure 26 Southern blot analysis. A: Southern blot result. B: Calibration curve. y = 1,5054e0,2786x 0 1 2 3 4 5 6 7 8 9 10 02468 Molecular weight (kb) Migration distance (cm) ♂ +control 15/107GCC 15/84GCC 17/49GCC 16/47GCC 19/36GCC 25/32GCC 14/80GCC 11/31GCC DIG II+III DIG II+III DIG III DIG II 9416bp 5148bp 3530bp 2027bp 1375bp 1584bp 4361bp 2322bp 1904bp 6557bp 4973bp 2.1kb 4.5kb 1 2 3 4 5 6 7 8 9 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 39 FRAXE full mutation male (>300GCCs) was used as a positive control showing methylated fragments larger than 6kb (Lane 1). The results obtained by the Southern blot technique were concordant with those from the novel TP-PCR. The intermediate size alleles’ fragments migrated slightly above 2.1kb and the premutation alleles between 2.3kb (84GCC) and 2.4kb (107GCC). The methylated premutation alleles are not clearly distinguish because the agarose concentration used is unable to discriminate such small changes. The 5.5kb fragment obtained inthe“methylated”areain lanes 2-6, 8 and 9 correspond to the change c.4022193G>A in AFF2 gene. This single nucleotide polymorphism (SNP), described in Ensembl as rs5980369, occurs within the sequence recognized by AflIII. When the A is present, the restriction enzyme cannot cut and a larger fragment is obtained (Figure 27)57. Furthermore, the observed amount of methylated and unmethylated allele is similar so we can conclude that a random methylation pattern is occurring at AFF2 locus. The two premutated alleles were confirmed (Lanes 2 and 3) and no mosaicism was detected. 3.4.3 Methylation-specific Multiplex ligation-dependent probe (MSMLPA®) MS-MLPA® kit combines the detection of small copy number changes with the methylation status. In this particular case MS-MLPA ME029 FMR1/AFF2 Probemix (MRC-Holland) detects deletions or duplications in the FMR1 and AFF2 genes. Besides, although it cannot measure the repeats length, it can quantify the methylation status particularly those from expanded alleles58. Figure 27 Scheme of the AFF2 gene with restriction sites for several restriction enzymes. NotI and AflIII enzymes (dark box) and FxE-AJ31 probe were used in the Southern blot. *rs5980369 (c .4022193G>A). Adapted from FMR2 CCG triplet repeat non-radioactive Southern blot genotyping protocol. * “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 40 Because partial deletions or duplications can cause intellectual disability24,25 some samples with unexpected results were tested using the MS-MLPA kit. This probemix has 27 probes for FMR1 and AFF2 with seven of FMR1 and five of AFF2 containing a HhalI recognition site and providing information on the methylation status. Reference probes (n=13) are also present including two probes with HhaI recognition site that can be used as controls to confirm HhaI digestion59. Coffalyser software calculates the normalized ratios of HhaI digested to undigested for each of the methylation-specific probes60. No copy number changes were detected as well as no skewing of the methylation pattern (Figure 28). As presented in Figure 28, in the undigested sample, a ratio of approximately 1 is observed in all FMR1 and AFF2 probes which means a normal copy number (2 copies) in both genes43. In the digested sample, the probes with HhaI recognition site, RARB-up [HHA1] (Dig) and EME1-1 [HHA1] (Dig), confirm the correct digestion (ratio of 0). For the FMR1 and AFF2 probes with HhaI recognition site, a ratio of approximately 0.5 is Figure 28 Example of MLPA ® showing calculated ratios for each probe in a female sample with 19GCC and 36GCC. Undigested sample Digested sample “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 41 obtained, corresponding to a random (normal) X-chromosome inactivation pattern in normal female samples43,59. 3.4.4 Sanger sequencing The TP-PCR revealed one case of homozygosity but with a different number of repeats when calculating the total size on the second peak on the right (Figure 29). Also, four cases of heterozygosity with a difference of two repeats was not detected by routine PCR. Curiously, this difference in the number of triplets was also not observed in the total length fragment (Figure 30). In one sample the TP-PCR suggested 19GCC homoallelism while the peak on the right indicated a total size of 17GCC (aproximately 194bp) (Figure 29). In one of the four supposedly heterozygous samples the TP-PCR detected two alleles of 18 and 20GCC but only one fragment migrated at 197bp, which is correspondent to a 18GCC allele (Figure 30). These results were confirmed after several repetitions and also using primers of the routine PCR (Figures 31 and 32). Figure 29 TP-PCR result obtained from a homoallelic sample with 19GCC alleles. Relative Fluorescent units (RFU) Basepairs (bp) 19 193.8bp (17GCC) Figure 30 TP-PCR result obtained from a heterozygous sample with 18GCC and 20GCC alleles. 20 197bp (18GCC) 18 Relative Fluorescent units (RFU) Basepairs (bp) “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 42 In order to further understand the reason why this occurred, the samples were sequenced by Sanger sequencing method (Figures 33 and 34). According to the reference sequence for the AFF2 gene the GCC repeats are followed by a ‘CTGCCGCCCCGGCT’ (NCBI reference sequence: NG_016313.2) Figure 31 PCR result obtained in the same sample showed in Figure 30. Relative Fluorescent units (RFU) Basepairs (bp) 258.4bp (17GCC) Figure 32 PCR result obtained in in the same sample showed in Figure 31. Relative Fluorescent units (RFU) Basepairs (bp) 261.7bp (18GCC) Figure 33 Sanger sequencing result after capillary electrophoresis of the amplified product of the sample with 19GCC detected by TP-PCR. T>C c.5030 c.5104 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 43 In our samples, a T>C change at the 5067 position was observed. This single nucleotide polymorphism (SNP) is described in Ensembl, rs868949662, and has a frequency of C: 0,02161. In the homoallelic sample (19GCC), the C is present in homozygosity (Figure 33) while in the other samples the C is present in heterozygosity (Figure 34). Figure 34 Sanger sequencing result after capillary electrophoresis of the amplified product of the sample with 18GCC and 20GCC detected by TP-PCR. c.5100 c.5027 T>C c.4952 c.5156 Figure 35 Schematic representation of the primers binding sites during the TP-PCR amplification strategy. AFF2 gene (NCBI reference sequence: NG_016313.2) sequence from c.4952 to c.5156 is shown. C in red marks the position of the c.5067T>C change observed in some samples. “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 50 containing and highly GC-rich DNA sequences. Mol. Biotechnol. 54, 1048–1054 (2013). 46. GeneLinkTM. Fluorescent Dyes - NED. Available at: http://genelink.com/newsite/products/MODPDFFILES/26-6445.pdf. (Accessed: October 29th 2019) 47. GeneLinkTM. Fluorescent Dyes - HEX. Available at: http://www.genelink.com/newsite/products/MODPDFFILES/26-6432.pdf. (Accessed: October 29th 2019) 48. Rychlik, W., Spencer, W. J. & Rhoads, R. E. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409–6412 (1990). 49. Kramer, M. F. & Coen, D. M. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Curr. Protoc. Cell Biol. 10, A.3F.1-A.3F.14 (2001). 50. Jensen, M. A., Fukushima, M. & Davis, R. W. DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis. PLoS One 5, 1–5 (2010). 51. Qiagen®. HotStarTaq Plus DNA Polymerase. Available at: https://www.qiagen.com/no/products/discovery-and-translational-research/pcrqpcr/pcr-enzymes-and-kits/end-point-pcr/hotstartaq-plus-dnapolymerase/#productdetails. (Accessed: September 18th 2019) 52. Jung, A., Ruckert, S., Frank, P., Brabletz, T. & Kirchner, T. 7-deaza-2′- deoxyguanosine allows PCR and sequencing reactions from CpG islands. J. Clin. Pathol. - Mol. Pathol. 55, 55–57 (2002). 53. Kalousová, M. et al. Comparison of DNA isolation using salting-out procedure and automated isolation (MagNA system). Prep. Biochem. Biotechnol. 47, 703– 708 (2017). 54. Miller, S. A., Dykes, D. D. & Polesky, H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215 (1988). 55. Qiagen®. EZ1 Advanced XL. Available at: https://www.qiagen.com/br/products/instruments-and-automation/nucleic-acidpurification/ez1-advanced-xl-instrument/#orderinginformation. (Accessed: October 25th 2019) 56. Clark, R. et al. The FRAXA and FRAXE allele repeat size of boys from the Avon Longitudinal Study of Parents and Children (ALSPAC). Wellcome Open Res. 4, 116 (2019). 57. rs5980369 SNP (Ensembl). Available at: https://www.ensembl.org/Homo_sapiens/Variation/Explore?r=X:148497497148498497;v=rs5980369;vdb=variation;vf=142015606. (Accessed: October 22nd 2019) 58. MRC-Holland. SALSA MLPA ME029 FMR1/AFF2 probemix. Available at: http://www.mlpa.com/WebForms/WebFormProductDetails.aspx?Tag=_tz2fAPI “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 51 AupKyMjaDF-E-t9bmuxqlhe_Lgqfk8Hkjuss.&ProductOID=_CV2A4kIQFnY. (Accessed: September 25th 2019) 59. MRC-Holland. SALSA MLPA® ME029 FMR1/AFF2 probemix. Available at: http://www.mlpa.com/WebForms/WebFormProductDetails.aspx?Tag=_tz2fAPI AupKyMjaDF-E-t9bmuxqlhe_Lgqfk8Hkjuss.&ProductOID=_CV2A4kIQFnY. (Accessed: October 21st 2019) 60. Nygren, A. O. H., Lens, S. I. & Carvalho, R. Methylation-specific multiplex ligation-dependent probe amplification enables a rapid and reliable distinction between male FMR1 premutation and full-mutation alleles. J. Mol. Diagn. 10, 496–501 (2008). 61. rs868949662 SNP (Ensembl). Available at: https://www.ensembl.org/Homo_sapiens/Variation/Explore?db=core;r=X:148500 586-148500840;tl=dzIXADhJcnBjlzw6-5311349733897256;v=rs868949662;vdb=variation;vf=183105250. (Accessed: July 5th 2019) “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 52 Annex 1. Summary of data reggarding the 500 tested samples. Sample number FRAXE E1 FRAXE E2 TP-PCR result 65 15 15 16/16 66 14 14 15/15 67 14 14 15/15 68 15 15 16/16 69 18 18 19/19 70 14 14 15/15 71 14 14 15/15 72 13 13 14/14 73 16 16 19/19 SNP 74 14 14 15/15 75 14 14 15/15 76 14 14 15/15 77 14 14 15/15 78 14 14 15/15 79 14 14 15/15 80 14 14 15/15 81 17 17 18/18 82 23 23 23/23 83 15 15 16/16 84 15 15 15/15 85 14 14 15/15 86 18 18 11/18 87 21 21 21/27 88 14 14 15/15 89 15 15 16/18 90 16 16 16/31 91 17 17 18/18 92 14 14 15/15 93 23 23 24/24 94 15 15 15/15 95 15 15 15/15 96 18 18 18/18 97 15 15 15/15 98 15 15 15/15 99 14 14 15/15 100 17 17 18/18 101 20 20 21/21 102 18 18 19/36 103 14 14 15/15 104 14 14 15/15 105 15 15 15/15 106 15 15 15/15 107 11 11 12/12 108 15 15 15/15 109 15 15 16/16 110 14 14 15/15 111 14 14 15/15 112 14 14 15/15 113 17 17 18/20 SNP 114 19 19 20/20 115 14 14 15/15 116 14 14 15/15 117 14 14 15/15 118 17 17 18/18 119 14 14 15/15 120 20 20 21/21 121 14 14 15/15 122 14 14 15/15 123 14 14 15/15 124 14 14 15/15 125 14 14 15/18 126 14 14 15/15 127 14 14 15/15 128 14 14 15/15 Sample number FRAXE E1 FRAXE E2 TP-PCR result 1 14 14 15/15 2 14 14 15/15 3 14 14 15/15 4 14 14 15/15 5 14 14 15/15 6 14 14 15/15 7 14 14 15/15 8 14 14 15/15 9 14 14 15/15 10 19 19 20/20 11 14 14 15/15 12 17 17 18/20 SNP 13 14 14 15/15 14 14 14 15/15 15 14 14 15/15 16 14 14 15/15 17 14 14 15/15 18 14 14 15/15 19 14 14 15/15 20 14 14 15/15 21 13 13 14/14 22 14 14 15/15 23 17 17 18/18 24 19 19 20/27 25 14 14 15/15 26 13 13 14/14 27 15 15 16/16 28 15 15 16/16 29 14 14 15/15 30 14 14 15/15 31 14 14 15/15 32 14 14 15/15 33 14 14 15/15 34 14 14 15/15 35 14 14 15/15 36 14 14 15/15 37 14 14 15/15 38 17 17 18/18 39 14 14 15/15 40 14 14 15/15 41 14 14 15/15 42 14 14 15/15 43 14 14 15/15 44 22 22 23/23 45 14 14 15/15 46 14 14 15/15 47 14 14 15/15 48 20 20 21/21 49 14 14 15/15 50 14 14 15/15 51 15 15 16/16 52 17 17 18/18 53 14 14 15/15 54 17 17 18/18 55 14 14 15/15 56 14 14 15/15 57 14 14 15/15 58 14 14 15/15 59 14 14 15/15 60 15 15 16/16 61 14 14 15/15 62 14 14 15/15 63 14 14 15/27 64 14 14 15/15 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 53 Sample number FRAXE E1 FRAXE E2 TP-PCR result Sample number FRAXE E1 FRAXE E2 TP-PCR result 129 14 14 15/15 199 14 14 15/15 130 17 17 18/18 200 14 14 15/15 131 17 17 18/18 201 14 14 15/15 132 14 14 15/15 202 14 14 15/15 133 15 15 16/16 203 14 14 15/15 134 14 14 15/15 204 26 26 20/26 135 14 14 15/15 205 14 14 15/25 136 14 14 15/15 206 15 15 16/16 137 24 24 25/32 207 15 15 16/16 138 15 15 15/15 208 17 17 18/18 139 14 14 15/19 209 14 14 15/15 140 14 14 15/15 210 15 15 16/16 141 16 16 16/24 211 14 14 15/15 142 15 15 15/15 212 14 14 15/15 143 15 15 15/15 213 20 20 19/21 144 15 15 15/15 214 14 14 15/15 145 15 15 15/17 SNP 215 14 14 15/15 146 15 15 15/15 216 14 14 15/15 147 15 15 15/15 217 17 17 18/18 148 16 16 16/16 218 14 14 15/15 149 15 15 15/15 219 14 14 15/15 150 15 15 15/15 220 14 14 15/15 151 15 15 15/15 221 14 14 15/15 152 15 15 15/15 222 15 15 16/16 153 15 15 15/15 223 14 14 15/15 154 15 15 15/15 224 15 15 16/16 155 15 15 15/15 225 14 14 15/15 156 15 15 15/15 226 14 14 14/15 157 15 15 15/15 227 14 14 15/15 158 15 15 15/15 228 17 17 18/18 159 14 14 15/15 229 15 15 16/47 160 15 15 15/15 230 14 14 15/15 161 15 15 15/15 231 14 14 15/15 162 19 19 19/19 232 14 14 15/15 163 15 15 15/84 233 14 14 15/15 164 15 15 15/15 234 14 14 15/15 165 16 16 16/16 235 14 14 15/15 166 15 15 15/15 236 15 15 16/16 167 15 15 15/15 237 14 14 15/15 168 15 15 15/15 238 14 14 15/15 169 18 18 18/25 239 14 14 15/19 170 15 15 15/15 240 14 14 15/15 171 15 15 15/15 241 14 14 15/15 172 15 15 15/15 242 14 14 15/15 173 15 15 15/15 243 19 19 20/20 174 24 24 24/24 244 17 17 18/18 175 15 15 15/15 245 14 14 15/15 176 15 15 15/26 246 14 14 15/15 177 15 15 15/15 247 14 14 15/15 178 18 18 18/24 248 19 19 20/20 179 15 15 15/15 249 14 14 15/15 180 15 15 15/15 250 24 24 25/25 181 15 15 15/15 251 14 14 15/15 182 15 15 15/15 252 14 14 15/15 183 15 15 15/15 253 15 15 16/16 184 14 14 15/15 254 14 14 15/15 185 18 18 18/18 255 14 14 15/15 186 16 16 17/17 256 17 17 18/18 187 14 14 15/15 257 14 14 15/15 188 17 17 18/18 258 14 14 15/30 189 17 17 18/18 259 14 14 15/15 190 14 14 15/107 260 15 15 16/16 191 14 14 15/15 261 14 14 15/15 192 14 14 15/15 262 15 15 16/16 193 14 14 15/15 263 14 14 15/15 194 14 14 15/15 264 14 14 15/15 195 22 22 23/23 265 14 14 15/15 196 17 17 18/18 266 17 17 18/18 197 14 14 15/15 267 14 14 15/15 198 18 18 19/19 268 14 14 15/15 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 54 Sample number FRAXE E1 FRAXE E2 TP-PCR result Sample number FRAXE E1 FRAXE E2 TP-PCR result 269 14 14 15/15 338 22 22 24/24 270 14 14 16/16 339 17 17 18/18 271 14 14 15/15 340 14 14 15/15 272 14 14 15/15 341 14 14 15/15 273 14 14 15/15 342 14 14 15/15 274 15 15 15/15 343 15 15 15/15 275 16 16 18/18 344 17 17 18/18 276 14 14 15/15 345 15 15 16/16 277 14 14 15/15 346 10 10 11/18 278 14 14 15/15 347 14 14 15/15 279 19 19 20/20 348 14 14 15/15 280 14 14 15/15 349 14 14 15/15 281 14 14 15/15 350 18 18 19/19 282 14 14 15/15 351 17 17 18/18 283 14 14 15/15 352 14 14 15/15 284 15 15 16/16 353 15 15 16/16 285 23 23 25/25 354 14 14 15/15 286 14 14 15/15 355 16 16 17/17 287 14 14 15/15 356 14 14 15/15 288 14 14 15/15 357 14 14 15/15 289 14 14 15/15 358 14 14 15/15 290 15 15 15/15 359 13 13 14/14 291 14 14 15/15 360 14 14 15/18 292 14 14 15/15 361 14 14 15/15 293 14 14 15/15 362 18 18 16/16 294 14 14 15/15 363 17 17 18/18 295 14 14 15/15 364 14 14 15/15 296 14 14 15/15 365 22 22 18/18 297 15 15 16/16 366 15 15 15/15 298 10 10 11/11 367 15 15 16/16 299 14 14 15/15 368 14 14 15/15 300 14 14 15/15 369 14 14 15/15 301 15 15 16/16 370 14 14 15/15 302 14 14 15/15 371 18 18 15/24 303 14 14 15/15 372 14 14 15/26 304 14 14 15/15 373 27 27 17/28 305 23 23 24/24 374 17 17 19/19 306 15 15 16/16 375 14 14 15/15 307 14 14 15/15 376 14 14 15/15 308 14 14 15/15 377 14 14 15/15 309 14 14 15/15 378 13 13 14/15 310 14 14 15/15 379 14 14 15/15 311 19 19 20/20 380 16 16 17/19 312 14 14 15/15 381 14 14 15/15 313 15 15 16/16 382 14 14 15/15 314 13 13 15/15 383 14 14 15/15 315 13 13 15/15 384 14 14 15/15 316 13 13 15/15 385 14 14 15/15 317 13 13 15/15 386 14 14 15/15 318 17 17 18/18 387 14 14 15/15 319 18 18 19/19 388 14 14 15/15 320 14 14 15/15 389 20 20 21/21 321 13 13 15/15 390 14 14 15/15 322 14 14 15/15 391 17 17 18/18 323 14 14 15/15 392 14 14 15/15 324 13 13 15/15 393 14 14 15/15 325 14 14 15/15 394 14 14 15/17 326 13 13 15/15 395 14 14 15/15 327 14 14 15/15 396 14 14 15/15 328 13 13 15/15 397 17 17 18/18 329 13 13 15/15 398 14 14 15/15 330 13 13 15/15 399 14 14 15/15 331 13 13 15/15 400 19 19 20/20 332 13 13 15/15 401 14 14 15/15 333 13 13 15/15 402 16 16 15/15 334 13 13 15/15 403 14 14 15/15 335 13 13 15/15 404 14 14 15/15 336 13 13 15/15 405 16 16 17/49 337 13 13 15/15 406 16 16 17/17 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 55 Sample number FRAXE E1 FRAXE E2 TP-PCR result Sample number FRAXE E1 FRAXE E2 TP-PCR result 407 17 17 15/20 477 14 14 15/15 408 14 14 15/23 478 14 14 15/15 409 17 17 18/18 479 14 14 15/15 410 18 18 18/19 480 14 14 15/15 411 20 20 19/21 481 14 14 15/15 412 13 13 14/21 482 14 14 15/15 413 17 17 18/18 483 15 15 15/15 414 14 14 15/15 484 14 14 15/15 415 12 12 13/15 SNP 485 18 18 20/20 416 15 15 16/20 486 14 14 15/15 417 17 17 18/18 487 14 14 15/15 418 14 14 15/20 488 14 14 15/15 419 15 15 16/16 489 15 15 16/16 420 14 14 15/34 490 14 14 15/15 421 14 14 15/15 491 14 14 15/15 422 14 14 15/15 492 10 10 15/31 423 14 14 15/15 493 14 14 15/15 424 14 14 15/15 494 14 14 15/15 425 16 16 16/17 495 17 17 18/18 426 17 17 18/18 496 15 15 16/16 427 14 14 15/15 497 14 14 15/15 428 14 14 15/15 498 14 14 15/15 429 14 14 15/15 499 19 19 20/20 430 15 15 15/15 500 14 14 15/15 431 14 14 15/15 432 14 14 15/15 433 14 14 15/15 434 14 14 15/15 435 19 19 20/20 436 14 14 15/15 437 14 14 15/20 438 14 14 15/23 439 15 15 15/15 440 15 15 15/15 441 15 15 16/16 442 14 14 15/15 443 14 14 15/15 444 14 14 15/15 445 14 14 15/15 446 14 14 15/15 447 13 13 14/15 448 14 14 15/15 449 13 13 14/29 450 13 13 14/14 451 14 14 15/15 452 16 16 16/17 453 14 14 15/19 454 14 14 15/15 455 14 14 15/15 456 14 14 15/15 457 14 14 15/15 458 15 15 16/16 459 14 14 15/15 460 14 14 15/15 461 14 14 15/16 462 14 14 15/15 463 14 14 15/20 464 13 13 14/14 465 17 17 18/18 466 14 14 15/15 467 14 14 15/15 468 14 14 15/15 469 14 14 15/15 470 14 14 15/15 471 14 14 15/15 472 17 17 18/18 473 14 14 15/15 474 22 22 23/23 475 14 14 15/15 476 14 14 15/15 “ValidationofSensitivityandSpecificityofTripletPrimed PCR (TP-PCR) in the Molecular Diagnosis ofFRAXE” 56