One Hundred Twenty-One Dystrophin Point Mutations Detected from Stored DNA Samples by Combinatorial Denaturing High-Performance Liquid Chromatography

One Hundred Twenty-One Dystrophin Point Mutations Detected from Stored DNA Samples by Combinatorial Denaturing High-Performance Liquid Chromatography

Journal of Molecular Diagnostics, Vol. 12, No. 1, January 2010 Copyright © American Society for Investigative Pathology and the Association for Molecu...

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Journal of Molecular Diagnostics, Vol. 12, No. 1, January 2010 Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology DOI: 10.2353/jmoldx.2010.090074

One Hundred Twenty-One Dystrophin Point Mutations Detected from Stored DNA Samples by Combinatorial Denaturing High-Performance Liquid Chromatography

Annalaura Torella,* Amelia Trimarco,* Francesca Del Vecchio Blanco,* Anna Cuomo,* Stefania Aurino,*† Giulio Piluso,* Carlo Minetti,‡ Luisa Politano,§ and Vincenzo Nigro*† From the Dipartimentos di Patologia Generale,* and Medicina Sperimentale,§ Seconda Universita` degli Studi di Napoli, Naples, the Telethon Institute of Genetics and Medicine,† Naples; and the Universita` degli Studi di Genova,‡ Istituto Giannina Gaslini, Genua, Italy

Duchenne and Becker muscular dystrophies are caused by a large number of different mutations in the dystrophin gene. Outside of the deletion/duplication “hot spots ,” small mutations occur at unpredictable positions. These account for about 15 to 20% of cases , with the major group being premature stop codons. When the affected male is deceased , carrier testing for family members and prenatal diagnosis become difficult and expensive. We tailored a costeffective and reliable strategy to discover point mutations from stored DNA samples in the absence of a muscle biopsy. Samples were amplified in combinatorial pools and tested by denaturing high-performance liquid chromatography analysis. An anomalous elution profile belonging to two different pools univocally addressed the allelic variation to an unambiguous sample. Mutations were then detected by sequencing. We identified 121 mutations of 99 different types. Fifty-six patients show stop codons that represent the 46.3% of all cases. Three nonobvious single amino acid mutations were considered as causative. Our data support combinatorial denaturing high-performance liquid chromatography analysis as a clear-cut strategy for time and cost-effective identification of small mutations when only DNA is available. (J Mol Diagn 2010, 12:65–73; DOI: 10.2353/jmoldx.2010.090074)

on the X chromosome, and the 14-kb transcript encodes a full-length protein (dystrophin) of 427 kd (Dp427m). Both DMD and BMD arise due to mutations at the dystrophin gene locus, which comprises 79 exons and eight tissue-specific promoters. The most common mutations are large intragenic deletions or duplications, encompassing one or more exons, but point mutations are about 15 to 20% of cases, with the major group being premature stop codons.2–9 Patients and their families confer great value to mutation detection for genetic counseling, but also for therapeutic options, since there are claims of novel mutation-targeted treatments.10 –12 Unfortunately, very often muscle biopsies are not possible because the affected family member is deceased. We have tailored a cost-effective and reliable strategy to discover point mutations from DNA samples. Based on the sensitivity of denaturing high-performance liquid chromatography (DHPLC) to detect mutations, especially in A/T-rich sequences, such as the dystrophin gene,6,7 we developed a combinatorial DHPLC approach to screen pooled samples.

Materials and Methods Patients We used archive DNA samples from six different centers: Laboratory of Molecular Biology, Scientific Institute E. Medea, Lecco; Department of Neurological and Psychiatric Sciences, University of Padua; Institute of Neurology, Catholic University, Policlinico Gemelli, Rome; Muscular and Neurodegenerative Disease Unit, Giannina Gaslini Institute, University of Genova; DepartSupported by grants from Telethon-UILDM GUP04008 (2005–2007) and TIGEM-11B and TIGEM-C20B, Ministero dell’Istruzione dell’Universita` e della Ricerca (MIUR: PRIN 2004 and 2006) (to V.N. and C.M.), Ministero della Salute (d.lgs 502/92), Ricerca d’Ateneo (to V.N. and L.P.). A.T. is a fellow of the Luigi Califano Foundation.

Duchenne (DMD [MIM 310200]) and Becker muscular dystrophies (BMD [MIM 300376]) are allelic inherited disorders of muscle. They affect males in ⬎99% of cases, being transmitted as X-linked recessive traits.1 The DMD gene spans 2.2 million bp of genomic DNA

Accepted for publication July 20, 2009. Address reprint requests to Professor Vincenzo Nigro, M.D., Laboratorio di genetica medica, Dipartimento di Patologia Generale, Seconda Universita` degli Studi di Napoli, S. Andrea delle Dame, via L. De Crecchio 7, 80138 Napoli, Italy. E-mail: [email protected] or [email protected]

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Hepes; 25 mmol/L MgSO4 ⫻ 7 H2O; 100 nm KCl; 100 mmol/L (NH4)2 SO4], 0.25 ␮mol/L each dNTP, 0.5 U AmpliTaq Gold (Applied Biosystems, Foster City, CA).

Primer Design

Figure 1. Extraction dates of DNA samples.

ment of Experimental Medicine, Cardiomyology and Medical Genetics, Second University, Naples; and Centro de Estudos do Genoma Humano, Instituto de Biocieˆncias Universidade de Sa˜o Paulo, Brasil. Diagnosis was determined by clinical features consistent with DMD or BMD, along with an X-linked family history. Informed consent was obtained from patients, when possible, according to the guidelines of Eurobiobank or Telethon.

Archive Samples One hundred fifty-three DNA archive samples were stored in Tris-EDTA at 4°C. Fifteen were extracted by phenol-chloroform before 1994, whereas 31 were extracted from 1994 to1999, and 46 from 2000 to 2004 (Figure 1). More recent samples (from 2005 to 2007) were extracted using a FlexiGene DNA kit (Qiagen, Hamburg, Germany). Old samples were often recovered as dry pellets. In this case, we rehydrated the pellet. We evaluated the DNA integrity by 0.6% agarose gel electrophoresis. We did not re-precipitate any of the samples. When required, we performed a preamplification step using the GenomiPhi HY DNA amplification kit (GE Healthcare, Chalfont St. Giles, UK), according to the manufacturer’s instruction. This kit provides microgram quantities of DNA from nanogram amounts of starting material in only a few hours. The limit of polymerase chain reaction (PCR) product size using this archived DNA was about 1000 bp.

Genomic sequence for Dp427m, the main dystrophin isoform found in muscle, was obtained from GenBank (NM 004006.1). Its exon 1 encodes a unique N-terminal MLWWEEVEDCY amino acid sequence and is expressed in the skeletal muscle and heart. For each dystrophin exon and muscular promoter a primers pair was designed using the Primer 3 software package with the following criteria: product size between 200 and 400 bp, primer size between 24 and 28 nucleotides, and melting temperature between 58°C and 62°C (Table 2). Primer pairs were chosen to include flanking-intron sequence. Primer sequences were checked by BLASTn to avoid matching with repeated human sequences or covering single nucleotide polymorphisms in the vicinity of exon sequences. Only in the case of exon 26, we designed two primer pairs that split it into two overlapping fragments. Following these requirements, we created a series of amplicons, all with the same melting characteristics. All were amplified using the same PCR conditions. Primers were synthesized by MWG Biotech AG, Ebersberg, Germany. All PCR share the same conditions (95°C 30 seconds, 60°C 90 seconds, 68°C 90 seconds for 33 cycles).

Amplification of Genomic DNA PCR reactions were set up semiautomatically using an automatic liquid handling Eppendorf epMotion and 384/96-well plates. DNA was amplified in a final reaction volume of 18 ␮l by using 30 ng of genomic DNA for each pool, buffer LB [20 mmol/L Tris; 10 mmol/L Hepes; 2.5 mmol/L MgSO4 ⫻ 7 H2O; 10 nm KCl; 10 mmol/L (NH4)2 SO4], 1.5 mmol/L MgCl2, 0.25 ␮mol/L each dNTP, 0.5 ␮mol/L each primers, 0.5 U AmpliTaq Gold (Applied Biosystems).

Sample Optimization Each DNA sample was diluted to a final concentration of 30 ng/␮l, and 1 ␮l was used in each pool. To control for the possibility of unequal PCR product yield, short tandem repeat (STR) polymorphic markers DXS8015HEX and DXS1204-FAM (Table 1) were amplified from single and pooled DNA templates, in a final reaction volume of 20 ␮l, by using 0.5 ␮mol/L each marker primer, buffer LB 10⫻ [200 mmol/L Tris; 100 mmol/L Table 1.

WAVE System DHPLC Analysis The dystrophin exons and flanking intronic sequences and the muscular promoter were analyzed using highthroughput denaturing high-performance liquid chromatography (HT-DHPLC). PCR products were directly analyzed. Using pooled samples a preliminary annealing step is not required. The system is based on DHPLC. The WAVE DHPLC system is an ion-pair, reverse-phase HPLC

STR Markers DXS1204-FAM

DXS8015-HEX

F Primer: 5⬘-ATGAACCCTTAACTCATTTAGCAGG-3⬘ R Primer: 5⬘-AGCNTGCACCAACATGCC-3⬘ Length: 237–251 bp

F Primer: 5⬘-AGTCTTCTCAGGCCAGAGC-3⬘ R Primer: 5⬘-AGGACCAACTTTCACATGC-3⬘ Length: 174–190 bp

F, forward; R, reverse.

Dystrophin Point Mutations 67 JMD January 2010, Vol. 12, No. 1

Table 2.

Primer Design and DHPLC Conditions Primers

Exon Pm 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26a 26b 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

Forward 5⬘-GGTAGACAGTGGATACATAACAAATGCATG-3⬘ 5⬘-TTTAATTTGGATGCCCCAAACCAG-3⬘ 5⬘-GATAATCGTGAAAATGTATCATTGGA-3⬘ 5⬘-TTGTCGGTCTCCTGCTGGTCAGTG-3⬘ 5⬘-TTGCAACTAGGCATTTGGTCTCTTACC-3⬘ 5⬘-TCTATTTATCACTGAAGATCAAGGAC-3⬘ 5⬘-ACACTCAAGACTTAAGGACTATGGGC-3⬘ 5⬘-ATATAGAAACCAAAAATTGATGTGTAG-3⬘ 5⬘-TTCTACCATGTTGGAAAGTAGTCCT-3⬘ 5⬘-TTGTGCAGCATTGGAAGCTCCTGA-3⬘ 5⬘-ACCACACCGATTTACCTAGAG-3⬘ 5⬘-GATAGTGGGCTTTACTTACATCCTTC-3⬘ 5⬘-GCAGAAATAAATTTCACCATTTGAGAGC-3⬘ 5⬘-GATACTTTGGCAAATTATTCATGCC-3⬘ 5⬘-GTGAGAAACTAGCTGTAAAAGACACG-3⬘ 5⬘-CTATAGTGGTGTATGGAATGCAACC-3⬘ 5⬘-GTCTGACCTCTGTTTCAATACTTCTCAC-3⬘ 5⬘-GTGTCAGGCAGGAGTCTCAGATTGAGA-3⬘ 5⬘-TGAATTACTCATCTTTGCTCTCATGCTG-3⬘ 5⬘-GCTTTCAGATCATTTCTTTCAGTCTG-3⬘ 5⬘-CTTGCCTTACTGCTTTTTAATACCTTC-3⬘ 5⬘-GAGTTTGCTGACAATTTAGGAAAACATGGC-3⬘ 5⬘-GTTTGAATCATATAGATTTCAAGTACAG-3⬘ 5⬘-ACCAGTAATGCCTTATAACGGGTCTCG-3⬘ 5⬘-ATCCAATATGCAATGCCATCAGTTCCC-3⬘ 5⬘-GTCTATGCCAGAAAGGAGGCCTTGA-3⬘ 5⬘-TCTAAGCTTTCTGTTATTTACATACTGATG-3⬘ 5⬘-CTCATTCTAACTGGATGTTGTGAGAAAG-3⬘ 5⬘-CTGTCTGCTGCATTTTGAATTACCTGC-3⬘ 5⬘-TCAGAAGATACTGAGCATTTGCTGATAATCC-3⬘ 5⬘-CAGGATTACAGAAAAGCTATCAAGAGT-3⬘ 5⬘-GTTGTTCTTTGTAGAGCATGCTGACT-3⬘ 5⬘-GACCAGTTATTGTTTGAAAGGCAAA-3⬘ 5⬘-CAAACATGGAATAGCAATTAAGGGGATCTC-3⬘ 5⬘-ACAGAAATATAAAAGTTCCAAATAAGT-3⬘ 5⬘-ACAAGACATTACTTGAAGGTCAATGC-3⬘ 5⬘-CCAATAATGCCATGGTATGTCTCTG-3⬘ 5⬘-CTTCAAGTCCTATCTCTTGCTCATGG-3⬘ 5⬘-GCATGTGATTAGTTTAGCAACAGGAGG-3⬘ 5⬘-TGAAGACTGTACTTGTTGTTTTTGATCAG-3⬘ 5⬘-ATAACTGCAGCCAGAAGTGCACTATAC-3⬘ 5⬘-ATGTGGTTAGCTAACTGCCCTGGGC-3⬘ 5⬘-GGAGGAGGTTTCACTGTTAGGAAGC-3⬘ 5⬘-GCAACACCATTTGCTACCTTTGGGA-3⬘ 5⬘-CTTGATCCATATGCTTTTACCTGCA-3⬘ 5⬘-AGTACAACTGCATGTGGTAGCACACTG-3⬘ 5⬘-ATTGCCATGTTTGTGTCCCAGTTTGC-3⬘ 5⬘-AAAGACAAGGTAGTTGGAATTGTGCTG-3⬘ 5⬘-GCTTATGCCTTGAGAATTATTTACCT-3⬘ 5⬘-TTGCTAACTGTGAAGTTAATCTGCAC-3⬘ 5⬘-CACCAAATGGATTAAGATGTTCATGAAT-3⬘ 5⬘-GAAATTGGCTCTTTAGCTTGTGTTTC-3⬘ 5⬘-GTAAAAGGAATACACAACGCTGAAG-3⬘ 5⬘-TTTAAAATGTCTCCTCCAGACTAGC-3⬘ 5⬘-GACCTGAGGATTCAGAAGCTGTTTACGA-3⬘ 5⬘-TGAGTTCACTAGGTGCACCATTCTGA-3⬘ 5⬘-GCACATATTCTTCTTCCTGCTGTCCTG-3⬘ 5⬘-ACTTCTAGATATTCTGACATGGATCGC-3⬘ 5⬘-GAATGCCACAAGCCTTTCTTAGCACTTC-3⬘ 5⬘-ATGTGGCCTAAAACCTTGTCATATTGCC-3⬘ 5⬘-CCTAAAGAGAATAAGCCCAGGTATC-3⬘ 5⬘-GAGAACATAATTTCTCTCCTTTTCCTCCC-3⬘ 5⬘-TGGAGATTAATGTTGTCTTTCCTGTTTGCGA-3⬘ 5⬘-TCCTGTTTTCTTGACTACTCATGGTAAATGC-3⬘ 5⬘-TATTTCTGATGGAATAACAAATGCTC-3⬘ 5⬘-GAGTCCTAGCTAGGATTCTCAGAGG-3⬘ 5⬘-AGAAGTGTTTACCCTCTAGGAAAGGGTC-3⬘ 5⬘-CCACTACTGTGGAAATACTGGCTACTC-3⬘ 5⬘-GATATACACCTCCTTTGCCATCTTGCC-3⬘ 5⬘-TGGTAGAAGGTTTATTAAAGAGTGTTCTTTGGG-3⬘ 5⬘-CATCCTGTCCTAAATCTGATCTCACC-3⬘ 5⬘-TGCGTGTGTCTCCTTCACCACCTCA-3⬘ 5⬘-CATAACTGTGTGGTGGGTTTTTTCTCCA-3⬘ 5⬘-TTTCAGGAATGTTCGATTAGGTCTTGAA-3⬘ 5⬘-CTGAGTCCCTAACCCCCAAAGCA-3⬘ 5⬘-CCATGGTATATAAAATTTGGTGATGA-3⬘ 5⬘-TAATTCTGTTTTCTTTTGGATGACTTAGCC-3⬘ 5⬘-GCTTGAGGGTTTTCTTTGTTATTTATGAGCAAG-3⬘ 5⬘-TCCCTTTCTGATATCTCTGCCTCTTCC-3⬘ 5⬘-AACAGAGTGATGCTATCTATCTGCACC-3⬘

DHPLC Reverse

5⬘-TTCTCCGAAGGTAATTGCCTCCCAGATCTGAGTCC-3⬘ 5⬘-AATGACACTATGAGAGAAATAAAACGG-3⬘ 5⬘-CAGTTTCTGGTCTGAAATTCTACTAAGTTT-3⬘ 5⬘-CAAAGCCCTCACTCAAACATGAAGC-3⬘ 5⬘-AGATTAATGTTACCCAAAAGGAAACC-3⬘ 5⬘-TGGGGAAAAATATGTCATCAGAGTC-3⬘ 5⬘-TACCATACTAAAAGCAGTGGTAGTCCAG-3⬘ 5⬘-ATGCATATAAAACAGAAAACATCTTG-3⬘ 5⬘-AAGCAGTGTTAGATTATCTTGGAAGC-3⬘ 5⬘-TAGTTTACCTCATGAGTATGAAACTGGTC-3⬘ 5⬘-CACAAGCTTCCAAAACTTGTT-3⬘ 5⬘-GAAAGCACGCAACATAAGATACACCT-3⬘ 5⬘-ACTTCAGCTGATTATGAGTGTGTG-3⬘ 5⬘-CGTGTCTTTTACAGCTAGTTTCTCAC-3⬘ 5⬘-TGGGTTTTTATAAGACCATTGAAAGC-3⬘ 5⬘-TGAGATAGTCTGTAGCATGATAATTGG-3⬘ 5⬘-AAGCTTGAGATGCTCTCACCTTTTCC-3⬘ 5⬘-GCACGGAGTTTACAAGCAGCACAAAATGAG-3⬘ 5⬘-CCCTAAGAAGATTATCTAAATCAACTCGTG-3⬘ 5⬘-CCAAGAAATACCTATTGATTATGCTC-3⬘ 5⬘-TTATTGTTTCATGTTAGTACCTTCTGG-3⬘ 5⬘-GATAAGCGTGCTTTATTGTTTTGAC-3⬘ 5⬘-AACAAGTAAATAAAAATGAGGGTAG-3⬘ 5⬘-ATCCACCCCAGCTGTAAAACACTGATC-3⬘ 5⬘-CTTAGTTAAGTACGTTGAGGCAAGC-3⬘ 5⬘-ACCAGGAAAGAGCAGACTGTATACGAC-3⬘ 5⬘-TTCAACTGCTTTCTGTAATTCATCTGGAG-3⬘ 5⬘-CACTATGCCTCACATATGACCATG-3⬘ 5⬘-TTCTATTTGGTACTTGACCTCTTTTA-3⬘ 5⬘-CTGAGAGCTGTATCTGCTATACATTAATGC-3⬘ 5⬘-AAGAATGGAAGCTGATTCCCAGATGTAC-3⬘ 5⬘-TGCCCAACGAAAACACGTTCCTTAG-3⬘ 5⬘-GTACCTGCGTATTTGCCACCAGAAAT-3⬘ 5⬘-GAAGTGTTTGTGGTCTCAGCATGC-3⬘ 5⬘-ACGTATGTTCAAAATAACCTTCAGTG-3⬘ 5⬘-AAGCTTCTAGCCTTTTCTCTTACC-3⬘ 5⬘-GGACAAAGATGATTGAAGTAACTGGTG-3⬘ 5⬘-CACAAGTTTCCACCTTGGAGTAGATC-3⬘ 5⬘-CAGTTGGAGACTTATCTAAGTTCTTTCC-3⬘ 5⬘-GTTTCTGATGACTAAGAGTCTGAAGCAG-3⬘ 5⬘-GTATAATAAAATCTGGTATTGACATTC-3⬘ 5⬘-CATACGTGGGTTTGCCAGTAACAACTC-3⬘ 5⬘-ATGATCACCTTGTAAAATACGAATG-3⬘ 5⬘-CCTGAAAACAAATCATTTCTGCAAG-3⬘ 5⬘-TCCATCACCCTTCAGAACCTGATCT-3⬘ 5⬘-CATTCCTATTAGATCTGTCGCCCTAC-3⬘ 5⬘-TAACCTAATGGGCAGAAAACCAATG-3⬘ 5⬘-TTAACACATGTGACGGAAGAGATGG-3⬘ 5⬘-TCCTGAATAAAGTCTTCCTTACCACACT-3⬘ 5⬘-TGATTATAAATAGTCCACGTCAATGG-3⬘ 5⬘-TCTCTCTCACCCAGTCATCACTTCATAG-3⬘ 5⬘-GGAGAGTAAAGTGATTGGTGGAAAATC-3⬘ 5⬘-AAATGTGAGGGGGATATATGAACTTAAG-3⬘ 5⬘-GTCTACTGTTCATTTCAGCTTTAACGTG-3⬘ 5⬘-CACCACCCCATTATTACAGCCAACAG-3⬘ 5⬘-CACAAGAGTGCTAAAGCGGAAATGCC-3⬘ 5⬘-GTGGCCTTTTTGCTCCACATCTTTTCC-3⬘ 5⬘-TGTGCTTAACATGTGCAAGGCACGAG-3⬘ 5⬘-TGCTCCGTCACCACTGATCCTTCTATC-3⬘ 5⬘-TTGTGGGAAGATAACACTGCACTCAAG-3⬘ 5⬘-TCCTATCCTCACAAATATTACCATGA-3⬘ 5⬘-CAAGATGCAATAAAGTTAAGTGATAAAAGC-3⬘ 5⬘-TACTCACTTGTGAATATACAGGTTAGTCAC-3⬘ 5⬘-TAACTTGGAGGAAACATGGCCATGTCC-3⬘ 5⬘-TAGTATCAAGATCTTCAAATACTGGCCAATAC-3⬘ 5⬘-CTAAGCCTCCTGTGACAGAGCCC-3⬘ 5⬘-TCCCATCTAGAACTAGGGTAATTAGCCAAC-3⬘ 5⬘-CCTACTGCCTACTGAAGAGCTAATATGAG-3⬘ 5⬘-AACTAACAGCAACTGGCACAGGAGA-3⬘ 5⬘-TGAACTAACTCTCACGTCAGGCTGGCGTC-3⬘ 5⬘-TGGGAGTGAAAGGAGGGTGTTCAGCT-3⬘ 5⬘-GCGAGCGAATGTGTTGGTGGTAGCAGCACCC-3⬘ 5⬘-TATTTGCCTGGCATACAACTAGCCTCA-3⬘ 5⬘-TCCTGTGCTATCCTACCTCTAAATCCCTC-3⬘ 5⬘-GTGCAAGTGTATGCACTCTGCATACC-3⬘ 5⬘-GCACCTATAAAAAGTGCTCTCTGAGG-3⬘ 5⬘-GGCCAAATATTCATGTCCCTGTAATACG-3⬘ 5⬘-TGATCCCAGCAAATCTGAGTCCCAC-3⬘ 5⬘-AGCAGGATGAGACAGACAGAAGCCAT-3⬘ 5⬘-TCTGCTCCTTCTTCATCTGTCATGACTG-3⬘

bp

%A

Melt(°C)

531 347 222 194 193 344 287 280 283 205 292 332 362 227 244 276 225 301 156 360 360 270 357 233 315 274 258 355 356 300 259 203 265 293 299 243 229 237 311 276 261 260 297 331 268 296 336 252 372 243 271 388 265 410 312 288 233 243 225 392 353 154 207 154 157 341 216 391 342 230 262 131 168 226 280 429 230 269 127 159

49% 45% 53% 50% 50% 50% 53% 49% 50% 52% 49% 48% 48% 51% 50% 48% 50% 50% 56% 50% 47% 52% 50% 52% 50% 51% 51% 50% 50% 51% 51% 50% 49% 50% 49% 50% 52% 52% 50% 51% 50% 54% 47% 48% 48% 48% 47% 49% 48% 49% 51% 49% 50% 47% 49% 48% 49% 49% 50% 47% 49% 55% 52% 53% 53% 49% 51% 48% 53% 51% 50% 54% 54% 50% 50% 50% 54% 50% 55% 54%

58.8 53 57.6 59.9 55.3 57.9 59.5 57.3 59.9 58.8 57.4 56.9 54.2 58.4 56.1 57.3 58.3 56.4 60.1 59.5 57.3 60.1 57.6 57.4 58.1 56.2 56.4 58.6 55.8 58.4 59.6 56.3 58.2 57 55 58.3 57.7 60.6 55.7 56.3 56.2 63.2 56.4 54.8 56 58.2 55 57.9 55.6 57.4 59.3 58.8 58.3 54.3 57.2 59.3 58.2 60 57.4 60.9 57.4 58 57.1 56.6 56.9 59.7 56.4 59.6 62.5 58 59.8 58.2 59.6 56.4 59 60.8 61.9 55 56.5 58

%A indicates the starting concentration of buffer A (without acetonitrile) used to load samples. Melt(°C) indicates the preferred temperature of analysis.

68 Torella et al JMD January 2010, Vol. 12, No. 1

Table 3.

Combinatorial Pools

Pool 1

Pool 2

Pool 3

1 2 3 Pool 4

4 5 6 Pool 5

7 8 9 Pool 6

1 4 7

2 5 8

3 6 9

Samples were divided into groups of nine. For each group we created six overlapping pools, each one containing three DNA samples from three different patients, so that each sample was present in a unique combination of two different pools.

method optimized to separate heteroduplex from homoduplex DNA fragments (Transgenomic Inc., Omaha, NE).

Sequence Analysis PCR amplicons were purified using the EXOSAP purification kit (GE Healthcare, Chalfont St. Giles, UK): 2 ␮l of ExoSAP-IT was directly added to 5 ␮l of PCR product and incubated at 37°C for 15 minutes. ExoSAP-IT was inactivated by heating at 80°C for 15 minutes. The sequence reactions were purified

Figure 2. Quality control of PCR yield. A–C: Analysis of each individual sample using the STR DXS8015. D: Analysis of a pool containing three samples.

Figure 3. Examples of aberrant DHPLC profiles. The figure shows different DHPLC profiles with growing complexity from A to D. A: Exon 6 showed a heteroduplex in both pools 1 and 4 sharing the DNA sample TU19, in which a frameshift mutation (c.401 404 delCCAA) was detected. B: Exon 27 heteroduplexes in both pools 2 and 6 sharing the DNA sample TU124, in which a splicing mutation (c.3433-1 A⬎G) was detected. C: Exon 29 heteroduplexes in pools 1, 4, and 5. Pools 1 and 4 shared the DNA sample TU181, pools 1 and 5 shared the DNA sample TU188. The same nonsense mutation (c.3940 C⬎T) was detected in both samples. D: Three different exon 14 heteroduplexes in pools 2 and 6 and 1 and 4, corresponding to combination of a mutation (**) and a known polymorphism (*). Arrow indicates homoduplexes.

Dystrophin Point Mutations 69 JMD January 2010, Vol. 12, No. 1

by Applied Biosystems BigDye XTerminator purification kit to remove unincorporated dye and other contaminants. Samples were analyzed using an ABI3130xL and sequencing analysis software (Applied Biosystems).

Results We screened 153 DNA samples from unrelated DMD or BMD patients. These samples were extracted and studied many years ago without obtaining a genetic diagnosis (Figure 1). We preliminarily excluded deletions or duplications by MLPA and Log-PCR.3,4

Combinatorial Pools To speed up the analysis and improve sensitivity, we pooled DNA samples in 17 units, each comprising samples Table 4.

from nine male patients. For each unit, we assembled six pools, each one containing DNA from three different patients, so that each DNA sample was present in two different pools and thus analyzed in duplicate (Table 3). This enabled the parallel amplification of three DNA samples in one run and allowed us to detect point mutations without the annealing with control DNA. To avoid pooling samples with significantly different PCR yield, we preliminarily genotyped STR markers in each of the DNA samples with the ABI-Prism 3130 xl using Gene Mapper software. We used two different X markers (DXS8015 and DXS1204) for the amplification of separate samples to determine tandem repeat lengths. On the basis of STR analyses, we created the pools by mixing three DNA samples with a different number of repeats (Figure 2). We analyzed the DMD exons, flanking intronic sequences and the muscle-promoter using HT-DHPLC.

Nonsense Mutations Sample

Exon

DNA change

Stop

Protein

New

Disease

3761 TU182-TU294-TU183-TU378 TU139-3443 TU184 TU86 TU180 TU318 TU187-TU189 TU05 G11 TU70 F1 TU01 TU107 TU51-TU185 TU32 TU12 475 TU342 TU181-TU188-TU102 TU218 R46 TU24 TU271 TU190 TU63 TU266 TU194 TU60 TU112-TU178-TU84 TU159 G2-G8-R42 TU186 R88 TU152 3448 TU02-TU157 TU87 TU18 TU208-G13 F4 G3

6 6 7 10 10 11 14 17 19 19 20 20 23 23 24 25 26a 27 28 29 30 33 34 35 35 37 39 39 41 41 42 46 48 57 59 59 60 65 68 70 70 70

c.409 G⬎T c.433 C⬎T c.583 C⬎T c.1062 G⬎A c.1093 C⬎T c.1292 G⬎A c.1652 G⬎A c.2125 C⬎T c.2302 C⬎T c.2380 G⬎T c.2414 C⬎G c.2521 C⬎T c.2956 C⬎T c.3151 C⬎T c.3259 C⬎T c.3409 C⬎T c.3580 C⬎T c.3625 C⬎T c.3843 G⬎A c.3940 C⬎T c.4117 C⬎T c.4600 C⬎T c.4690 C⬎T c.4979 G⬎A c.4996 C⬎T c.5209 C⬎T c.5476 G⬎T c.5530 C⬎T c.5773 G⬎T c.5899 C⬎T c.6023 C⬎A c.6678 G⬎A c.7006 C⬎T c.8422 A⬎T c.8713 C⬎T c.8880 G⬎A c.8944 C⬎T c.9461 T⬎A c.9829 G⬎T c.10108 C⬎T c.10135 A⬎T c.10171 C⬎T

TAA TGA TGA TGA TAA TGA TGA TAA TGA TAG TGA TAA TAA TGA TAG TAG TAG TAA TGA TGA TAG TAG TAA TGA TGA TAA TAA TGA TAG TGA TGA TGA TAG TAG TGA TGA TGA TAG TAA TGA TAA TGA

E137X R145X R195X W354X Q365X W431X W551X Q709X R768X E794X S805X Q841X Q986X R1051X Q1087X Q1137X Q1194X Q1209X W1281X R1314X Q1373X Q1534X Q1564X W1660X R1666X Q1737X E1826X R1844X E1925X R1967X S2008X W2226X Q2336X K2808X R2905X W2960X R2982X L3154X E3277X R3370X K3379X R3391X

Yes No No No No No Yes No No Yes Yes No No No No No No Yes Yes No No No Yes Yes No Yes No No No No Yes Yes No Yes No Yes No No Yes No No No

DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD BMD BMD DMD DMD DMD BMD DMD DMD DMD Carrier DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD BMD BMD/DMD DMD DMD DMD

Resulting TGA stop codons are indicated in bold.

70 Torella et al JMD January 2010, Vol. 12, No. 1

Table 5.

Frameshift Mutations

Sample

Exon

DNA change

Protein

New

Disease

TU326 TU19 3451-3453 TU65 TU55 TU150 TU16 TU07 TU03 TU386 TU23 TU267 TU137 TU115 TU27 TU177 TU44 TU103 G7 TU29-G10 TU08-R49 TU13 R44 TU304 TU06 3488 TU211 TU04 TU57 R37 TU33 TU62 TU151 TU179-G1 TU192-TU193 G12 TU214

5 6 7 8 11 11 11 12 14 16 22 22 25 25 26 26 30 30 30 33 35 36 37 40 40 42 44 48 55 56 58 59 62 65 68 70 73

c.321 delT c.401_404 delCCAA c.593_594 insA c.713_714 delTT c.1188 insT c.1181del G c.1300_1310 delCTCAGGGTAGC c.1482 delG c.delGTA 1603insCT c.1859 delT c.2880 2884 delCAAAC c.2887 del T c.3285 3288 delCAGT c.3420 del C c.3447 delGGlnsTT c.3464 3471 del GTTGGAG c.4100 delA c.4119 delG c.4186 insA c.4565delT (Stop TAA) c.4871_4872 delAG c.5091 delG c.5272 _5280 del TCAGAGCTC ins CCAA c.5606 del G c.5697 dup A c.5973_5974 ins A c.6353 delA c.6980del A c.8081 del G c.8284 ins A c.8597 8598deiTT c.8732 insA c.9204_9207 del CAAA c.9429_9430 del GC c.9926_9929 ins AAGC c.10105 del G c.10386 del T

G109V fs X1 N135V fs X5 H198Q fs X19 L239A fs X7 G397W fs X1 G394A fs X12 L434X K494K fs 7 V535L fs X47 L620R fs X12 K961L fs X5 S963P fs X40 S1096_D1097I fs X9 H1140Q fs X13 K1149N-E1150X G1155E fs X20 Q1367R fs X15 E1374R fs X8 Y1396X fs V1522G fs X2 K1625G fs X27 A1698L fs X22 S1758P fs X13 R1869K fs X4 L1900I fs X5 E1992R fs X11 Q2118R fs X3 K2329S fs X8 F2694S fs X31 I2762N fs X 10 L2866R fs X28 N2912Q fs X2 N3068K fs X20 Q3143H fs X9 H3309Q fs X7 V3369F fs X8 N3462K fs X3

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes No Yes Yes Yes Yes No Yes Yes No Yes Yes Yes No No Yes Yes Yes Yes

carrier DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD BMD/DMD DMD BMD/DMD

Each pool was amplified for all of the 79 dystrophin gene exons and promoter. PCR products were directly analyzed by WAVE system using predetermined temperature and elution buffers concentrations (Table 2). The WAVE system provides rapid, automated scanning Table 6.

for single nucleotide polymorphisms, even when the nature and location of the mutations are unknown. DHPLC analysis of the pools allowed the unambiguous identification of the mutant sample, avoiding the subsequent screening of three single DNA samples.

Putative Splicing Defects

Sample

Position

DNA change

Splice site

New

Disease

TU22-TU77 TU34 TU296 TU219 TU309 2082 TU124 TU164 TU105 TU332 TU379 TU114 TU209 TU133 TU54-1707 TU36-TU97 TU30

Intron 2 Intron 5 Intron 5 Intron 6 Intron 11 Intron 11 Intron 26 Exon 26 Intron 35 Intron 48 Exon 58 Intron 58 Intron 58 Exon 65 Intron 65 Intron 70 Intron 70

c.94⫺1 G⬎A c.358⫺2 A⬎G c.358⫺2 A⬎T c.530⫹1 G⬎A c.1331⫹2 T⬎C c.1332⫺9 A⬎G c.3433⫺1 G⬎A c.3603 G⬎A c.5026⫺6 A⬎G c.7098⫹1 G⬎A c.8668 G⬎A c.8668⫹1 G⬎A c.8668⫹3 A⬎T c.9560 A⬎G c.9563⫹1 G⬎A c.10223⫹1 G⬎A c.10223⫹5 G⬎T

Acceptor Acceptor Acceptor Donor Donor Acceptor Acceptor Donor Acceptor Donor Donor Donor Donor Donor Donor Donor Donor

No No No Yes Yes No No Yes No No No Yes Yes No No No Yes

BMD DMD DMD DMD/BMD DMD/BMD DMD DMD DMD DMD DMD DMD DMD DMD/BMD DMD DMD DMD DMD

Dystrophin Point Mutations 71 JMD January 2010, Vol. 12, No. 1

Table 7.

Functional Mutations

Sample

Exon

DNA change

Protein

New

Disease

TU118 TU42 TU109

3 69 70

c.160_162 del CTC c.10010 G⬎A c.10101_10103 delAGA

L54del C3337Y E3367del

Yes Yes No

DMD DMD BMD/DMD

The presence of a variation within a fragment appears as altered chromatogram shapes of the two different pools sharing the same DNA. This type of scanning unequivocally points out the patient and the fragment for sequence analysis (Figure 3). This approach reduced the turnaround time and was more costeffective.

Sequence Analysis From 153 samples tested, we identified 121 causative mutations of 99 different types. We detected stop codons in the relative majority of patients (56/121 ⫽ 46.3%, Table 4), while we found frameshift mutations in 42 cases (34.7%, Table 5), splice mutations in 20 (16.5%, Table 6), and missense mutation in three patients (2.5%, Table 7). In addition, we detected 36

Table 8.

variations classified as polymorphisms or private variants (Table 8).

Discussion Identification of a pathogenic point mutation in a DMD or BMD patient confirms the clinical diagnosis and allows definitive carrier testing and prenatal diagnosis for family members.8,9 Precise knowledge of the mutation is also required for some of the emerging therapies, such as exon skipping,10 or suppression of premature stop codons.11,12 We found mutations in 121 DMD-BMD patients out of 153 DNA samples tested (Figure 4). In 32 DNA samples (20.9%), no mutation was found. Complete sequence analysis of all exons in these samples con-

Variants and Polymorphisms Variations

Position

New

Number*

c.32-78 G⬎T 94-16 ins T c.832-54 A⬎G c.837 G⬎A T279T c.853 G⬎A G285R 1225 A⬎T T409S c.1603-57 T⬎C 1635 A⬎G R545R 1869 C⬎T L623L c.2176 G⬎T V726F c.2391 T⬎G N797K c.3604-95 delG c.3936 G⬎C L1312F 4234-13A⬎G c.4510 H1504Y c.4675-53 G⬎T c.4878 G⬎T V1626V; 5016 T⬎A N1672K c.5326-54 A⬎C 5234 G⬎A R1745H 5586⫹93insCT c.5723 A⬎T D1908V GAT-⬎GTT c.5795 A⬎G Q1932R c.6118-76 ins TA 6290⫹27 T⬎A c.6443 T⬎C L2148P CTC-⬎CCC 6913-114A⬎T 6913-114A⬎T c.3561 A⬎T 6913-114 c.7200⫹53 C⬎G 8027⫹11 C⬎T c.8571 T⬎C T2857T c.8810 A⬎G Q2937R c.9085-23 C⬎A 9649⫹15 T⬎C c.10789 L3597L; 10554-30_10554-35 del TTTC c.3685*49 c⬎t

Intron 1 Intron 2 Intron 8 Exon 9 Exon 9 Exon 11 Intron 14 Exon 14 Exon 16 Exon 18 Exon 20 Intron 26 Exon 29 Intron 30 Exon 32 Intron 33 Exon 35 Intron 37 Exon 37 Intron 39 Exon 40 Exon 41 Intron 42 Intron 43 Exon 45 Intron 47 Intron 47 Intron 47 Intron 49 Intron 54 Exon 58 Exon 59 Intron 60 Intron 66 Exon 75/intron 74 Intron 79

No Yes No No Yes Yes No No No Yes No Yes Yes Yes Yes No Yes No No Yes No Yes Yes No No No No Yes No No Yes No Yes No Yes Yes

1 5 1 2 1 1 2 7 1 1 1 2 1 2 1 1 2 1 7 1 1 1 2 2 1 4 1 1 2 6 1 1 1 5 1 1

*Number of samples with the same variation.

72 Torella et al JMD January 2010, Vol. 12, No. 1

Figure 4. Distribution of all causative point mutations along the dystrophin cDNA. Segments corresponding to groups of 10 exons are indicated in dark and light gray.

firmed the DHPLC negative results. Considering that 153 DNA samples correspond to 20% of all patients that show no deletions or duplications, about 20% of 20% of patients cannot be diagnosed by DNA analysis alone. This indicates that mRNA13 or CGH array13,14 analyses can be necessary to diagnose about 4% of all DMD/BMD patients. Notably, 22 unrelated patients shared the same mutations (Tables 4 – 6). Among the 56 nonsense mutations, 34 (60.7%) show the TGA termination codon that is considered optimal for readthrough therapy.11 Notably, among the 37 different frameshift mutations, 31 (83.8%) are absent from the Leiden database.2 We identified three putative functional mutations (Table 7). One is the substitution of a highly conserved cysteine at position 3337 within the second half of the dystroglycan-binding domain. A similar mutation (C3340Y) has been associated with Duchenne muscular dystrophy.15 There is the loss of aspartic acid in position 3368 with the substitution of the glutamic acid in position 3367 with aspartic acid. This produces the loss of glutamic acid in position 3367 that is known to be associated with a particular DMD phenotype.16 The first half of the C terminus and the cysteine-rich (D-domain; amino acid residues 3080 –3408) are highly conserved regions of dystrophin. The region is involved in interactions with dystroglycan that mediates attachment of dystrophin to the cytoplasmic surface of

the cell membrane. Deletions or chain-terminating nonsense mutations involving the D-domain usually result in DMD. The third is the c.160_162 CTC deletion in exon 3 in DMD, which resulted in the loss of an evolutionary conserved leucine in position 54 in the actin-binding domain. This is the same amino acid position replaced by an arginine described in a boy with Duchenne muscular dystrophy.17 Interestingly, this missense mutation in position 54 was questioned because it was not completely studied by DNA sequencing. After 16 years, our findings support the causative role of the change. Two unusual mutations were also identified. Premature stop codons in positions 1281 and 1314 associated with BMD and not DMD. This could be explained by the skipping of exon 28 or 29, respectively.18 Our DNA-based mutation screening strategy is suitable for high-throughput applications in patients for which mRNA is unavailable. Sample pooling, together with identical PCR conditions for all fragments, were set up for the simultaneous detection of any mutations type within exons and exon flanking regions. The preliminary analysis of single and pooled samples by STR markers permitted us to confirm the same PCR efficiency in all combinatorial pools. In the protocol, the pooling was conservative, since we only mixed three samples. With the availability of more sensitive methods of DNA detection (ie, multicolor fluorescence) we

Dystrophin Point Mutations 73 JMD January 2010, Vol. 12, No. 1

can foresee possibilities to pool dozens of samples with further impressive reduction of costs.

9.

Acknowledgments

10.

We thank Marina Fanin, Giuliana Galluzzi, Enzo Ricci, Federico Zara, Claudio Bruno, Sara Scapolan, Maria Teresa Bassi, and Mayana Zatz for DNA samples and Alessandra Ferlini for helpful discussion. We acknowledge the SUN-Naples Human Mutation Gene Bank (Cardiomyology and Medical Genetics), which is partner of the Eurobiobank Network.

11. 12. 13.

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