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Motor chip: a comparative genomic hybridization microarray for copy-number mutations in 245 neuromuscular disorders.

Neuromuscular disorders (NMDs) [9] are a highly heterogeneous group of genetically determined diseases encompassing many conditions that, directly or indirectly, impair muscle function by affecting the muscles and/or their nervous control. In the annually published gene table of NMDs (http://www.musclegenetable.org) (1), 495 clinical entries and 272 distinct causative nuclear genes have been annotated to date. Genetic and clinical redundancy reflects the broad phenotypic variability included under the term "neuromuscular disorders," embracing myopathies, cardiomyopathies, and neuromyopathies. In addition, at least 99 mapped NMD loci await identification of the causative gene (1), which will extend the number of genes involved in neuromuscular phenotypes.

NMDs are either genetically heterogeneous, in that the same disease may be caused by mutations in many different genes (2), or clinically heterogeneous, when the same gene may be mutated in several different clinical conditions (3). Consequently, molecular diagnosis can be very expensive and time-consuming, requiring a multistep methodological approach and sometimes adding frustration because of the high number of genes that need to be analyzed. Exome sequencing based on next-generation sequencing will be helpful, but even with this powerful technology, copy-number mutations can remain undetected.

A wide range of deleterious mutations have been detected in NMD genes. They include single-nucleotide substitutions that may affect gene expression differently, short insertions or deletions of bases, large deletions or duplications, and other complex chromosomal rearrangements.

When we reviewed the freely available mutation databases (4) to determine the impact of different types of mutations in NMD genes, we observed that most of the characterized pathologic alleles correspond to point mutations and that other mutational mechanisms, such as deletions or duplications, are probably underestimated. About 5% to 6% of gene mutations causing inherited disorders are large deletions or duplications (5, 6), although this percentage is dependent on genomic regions and the techniques used.

Array-based comparative genomic hybridization (aCGH) is the most widelyused technology for detecting copy-number variations (CNVs) and for their mapping along chromosomes (7), and its use is based on the resolution level of the specific design and the platform used (8).

aCGH has proved an excellent technique for detecting DNA copy-number changes in the human genome (9). It has been used for the analysis of tumor genomes (10) and is increasingly used for the genetic testing of individuals with unexplained developmental delay or multiple congenital anomalies (11,12).In addition, new high-density technologies (13) and the possibility of designing customized aCGH focused on specific chromosomal locations for increasing the resolution in a targeted genomic region of interest (14, 15) have enhanced the spectrum of molecular-diagnostic applications.

In NMD molecular diagnosis (16-18), aCGH applications initially focused on Duchenne and Becker muscular dystrophies, which are caused predominantly by DMD [10] (dystrophin) deletions (75%) or duplications (10%) on the X chromosome (19). Other aCGH designs have focused on sarcoglycanopathies (20), collagen VI-related myopathies (21), and amyotrophic lateral sclerosis (22). To date, there are either general or gene-specific aCGH designs, but general arrays have low resolution, whereas gene-specific arrays are useful only when the genetic cause is already suspected. This is not the case for the majority of NMDs characterized by high genetic heterogeneity, for which a comprehensive aCGH design targeting all NMDs could be very useful.

We describe gene selection, the design strategy, and experimental validation of a customized exon-specific oligonucleotide CGH array that we named "Motor Chip" (present release NMDv02.15-Hg18). Motor Chip is capable of testing 425 nuclear genes simultaneously, including 245 disease genes already clearly involved in NMDs and 180 selected genes that are possible candidates in novel neuromuscular phenotypes. Using the Motor Chip to investigate a panel of NMD patients with no previous molecular characterization, we identified the first case of limb girdle muscular dystrophy type 2C (LGMD2C) caused by 2 distinct deletions that inactivate both alleles of the SGCG [sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein)] gene.

Materials and Methods

REFERENCE DNA SAMPLES

Anonymous blood samples were collected into EDTA-containing Vacutainer tubes (BD) from healthy and unrelated individuals of the same ethnic origin. Genomic DNA was isolated by phenol/chloroform extraction. Written informed consent was obtained from all participants (patients and controls), and the protocol was approved by the Ethics Committee of the Seconda Universita degli Studi di Napoli (approval no. 862/2008).

After DNA quantification (see Supplemental Materials and Methods in the Data Supplement that accompanies the online version of this article at http:// www.clinchem.org/content/vol57/issue11), 6 sex-matched DNA samples were pooled and used as male or female reference DNA samples in aCGH experiments.

DOUBLE-BLIND DNA SAMPLES

We used DNA samples from patients with deletions or duplications involving genes included in the Motor Chip design that had previouslybeen detected with alternative diagnostic methods (see Table 1 and Supplemental Materials and Methods in the online Data Supplement). In addition, we used DNA samples from patients with NMDs and an incomplete molecular diagnosis, as well as samples from patients with an NMD clinical diagnosis alone (see Table 1 in the online Data Supplement). Additional DNA samples from healthy individuals were analyzed as negative controls. All DNA samples were blind-tested in aCGH experiments.

GENE SELECTION AND aCGH DESIGN STRATEGY

We selected 245 NMD genes reported in the NMD gene table (1) and classified according to 16 different phenotypes (see Table 2 in the online Data Supplement). Because not all disease genes have been identified, we also included an additional 180 genes (see Table 2 in the online Data Supplement) that we considered putative candidate genes according to criteria indicated in the note to Table 2 in the online Data Supplement. We have included 425 genes in the present release of the Motor Chip.

The design was based on 2 principal criteria: the selection of a competitive microarray platform and an affordable cost of the test per sample. We used the Agilent oligonucleotide-based microarray technology (23) and the SurePrint G3 8 x 60K format (59 090 available features), currently the most cost-effective option (see Table 3 in the online Data Supplement).

Probe selection was performed by using the Web-based Agilent eArray database (version 5.0; Agilent Technologies, https://earray.chem.agilent.com/earray/) and choosing from among 24.3 x [10.sup.6] computationally validated oligonucleotide probes (NCBI Build 36.1, Hg18). We selected onlya limited number of probes (2132 features, 4.8%) with the eArray Genomic Tiling option to design oligonucleotide probes spanning specific chromosomal regions inadequately represented in the Agilent database. For the Motor Chip, we designed exon-specific oligonucleotide probes to obtain full coverage of the coding sequence of each gene (24, 25).

The selected 425 genes correspond to 40 362 512 bp of genomic sequence and to 3 383 769 bp of coding sequence covered with 44 379 oligonucleotide probes (99.8% coverage). The number of probes for each group, the percentage of custom probes, and the mean number of probes per gene are summarized in Table 1.

THE DMD GENE

For DMD, in which about 85% of the mutations are deletions or duplications (19), we selected partially overlapping probes (15 bp as the maximum-acceptable overlap) covering all of the exons and 200 bp to the 5' and 3' intronic sides. We also selected intragenic probes spaced 2000 bp apart to cover the entire DMD gene (see Fig. 1A in the online Data Supplement).

OTHER GENES

For all of the other selected genes, we designed partially overlapping probes (15 bp as the maximum-acceptable overlap) that covered only all of the exons and the 5' and 3' untranslated regions (see Fig. 1B in the online Data Supplement). Alternatively spliced isoforms were also considered. To obtain reliable calls, we selected at least 3 probes per exon (26). We also selected probes spaced 250 bp apart in a 2000-bp genomic region at the 5' end of the gene that should include the putative promoter (see Fig. 1C in the online Data Supplement).

To obtain whole-genome coverage, we randomly filled the residual free space on the array (14 711 features, approximately 23.4%) with probes from the commercially available Agilent Human Genome 44K CGH Microarray.

Biological features were randomly distributed on the microarray. The routinely used Human CGH 1K Agilent Normalization Probe Group (1262 features) and the Human CGH 1K Agilent Replicate Probe Group (5000 features) were also included in the design.

Results

VALIDATION STRATEGY

After an overall evaluation of the Motor Chip design (see Supplemental Materials and Methods in the online Data Supplement), we analyzed 56 double-blind DNA samples (see Table 1 in the online Data Supplement). These samples included 26 from patients in whom genomic imbalances involving NMD genes had previously been detected by different methods [PCR, LogPCR, multiplex ligation-dependent probe amplification (MLPA), quantitative PCR, and aCGH], 7 from patients with NMDs and an incomplete molecular diagnosis (autosomal recessive inheritance and a causative point mutation identified in only 1 allele), and 19 from patients with an NMD clinical diagnosis alone. Three samples from affected individuals with deleterious mutations undetectable with the Motor Chip, 1 sample from a healthy carrier individual, and 8 anonymous samples from healthy individuals were also included as negative controls.

The Motor Chip detected no clinically relevant genomic imbalances in any of the 12 negative controls, with the exception of a previously undetected aneuploid karyotype (47,XYY) in sample no. 40, from a dystrophic patient with a DMD nonsense mutation (see Fig. 2 in the online Data Supplement). This result was validated by MLPA, confirming that the genomic backbone of the Motor Chip was also able to detect more complex and unexpected chromosomal imbalances. Annotated CNVs were detected in negative controls and were also present in some patients (see Table 4 in the online Data Supplement).

DOUBLE-BLIND DIAGNOSIS OF KNOWN MUTATIONS

For samples from patients with deleterious genomic imbalances, the results obtained with the Motor Chip were consistent with those obtained by different methods (Table 2; see Table 1 in the online Data Supplement).

Nine samples (nos. 1-3, 5, 7-9, 11, and 12) presented hemizygous or heterozygous copy-number imbalances involving 1, 2, or more DMD exons (see Fig. 3 in the online Data Supplement). In these cases, the Motor Chip was able to define breakpoint boundaries more precisely than previously used methods because of more accurate coverage of DMD intragenic regions (Table 2). Two unrelated samples (nos. 4 and 10) presented an isolated homozygous deletion of exon 7 in SGCG (see Fig. 4A in the online Data Supplement). Interestingly, only 3 probes showing the same direction in copy-number change were sufficient for a reliable aberration call.

Sample 6 presented a heterozygous deletion of exons 2-8 in the CAPN3 [calpain 3, (p94)] gene (see Fig. 4B in the online Data Supplement). Five samples (nos. 13, 14, 17, 19, and 20) showed heterozygous SPAST (spastin) deletions or duplications (see Fig. 5 in the online Data Supplement), and 2 other samples (nos. 18 and 23) presented heterozygous and homozygous deletion, respectively, of exons 31-34 in SPG11 [spastic paraplegia 11 (autosomal recessive)] (see Fig. 6 in the online Data Supplement). Five other samples (nos. 27, 28, 29, 30, and 32) showed a heterozygous deletion on chromosome 13q12.12 spanning about 1.5 Mb and including 7 genes: SGCG, SACS [spastic ataxia of Charlevoix-Saguenay (sacsin)], TNFRSF19 (tumor necrosis factor receptor superfamily, member 19), MIPEP (mitochondrial intermediate peptidase), PCOTH, (prostate collagen triple helix protein) [11]; SPATA13 (spermatogenesis associated 13), and C1QTNF9 (C1q and tumor necrosis factor related protein 9). Two of these genes, SGCG and SACS, are NMD genes (see Fig. 7 in the online Data Supplement). CNVs covering this region have previously been reported (27, 28). Two of these patients (nos. 29 and 32) also presented a SACS point mutation on the second allele that produced the observed recessive neuromuscular phenotype (29, 30). Finally, 2 samples (nos. 38 and 39) presented a wide heterozygous deletion and a duplication, respectively, both of which involved the PMP22 (peripheral myelin protein 22) and other flanking genes (see Fig. 8 in the online Data Supplement).

NOVEL FINDINGS IN PATIENTS WITH A KNOWN MUTATION IN A SINGLE ALLELE

For samples from patients with an incomplete molecular diagnosis, 4 patients (nos. 24, 25, 26, and 33) presented point mutations in SPG11, and 3 patients (nos. 21, 35, and 36) presented mutations in SETX (senataxin), ZFYVE26 (zinc finger, FYVE domain containing 26), and SACS, respectively. When analyzed on the Motor Chip, only sample no. 21 showed a heterozygous deletion of exons 16-24 in SETX, confirming the diagnosis of autosomal recessive ataxia with oculomotor apraxia type 2 (AOA2). This result was confirmed by long-range PCR and direct sequencing of the breakpoint (Fig. 1; see Supplemental Materials and Methods in the online Data Supplement). The deletion (NG_007946.1:g.62507_83156delins25) was perfectly coincident with that previously described in an unrelated AOA2 patient (31), confirming that deleterious copy-number changes are not infrequent occurrences in SETX(32). No clinically relevant deleterious genomic imbalances were detected in the other samples.

NOVEL FINDINGS IN PATIENTS WITHOUT MUTATIONS

Finally, we analyzed 19 samples with an NMD clinical diagnosis alone, 3 of which were from patients with familial spastic paraparesis or ataxia with an autosomal dominant inheritance (sample nos. 16, 34, and 37), whereas the others (sample nos. 41-56) were mainly from patients with a generic diagnosis of LGMD. In 4 of these samples (nos. 42,45, 55, and 56), the Motor Chip detected copy-number changes in 3 different NMD genes.

Sample no. 42 corresponded to a familial case with a severe LGMD phenotype. We detected a wide heterozygous duplication at 2p13.3 that spanned about 735 kb and included 10 genes: CD207 (CD207 molecule, langerin), VAX2 (ventral anterior homeobox 2), ATP6V1B1 (ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunitB1), ANKRD53 (ankyrin repeat domain 53), TEX261 (testis expressed 261), NAGK (N-acetylglucosamine kinase), MCEE (methylmalonyl CoA epimerase), MPHOSPH10 [M-phase phosphoprotein 10 (U3 small nucleolar ribonucleoprotein)], PAIP2B [poly(A) binding protein interacting protein 2B], and ZNF638 (zinc finger protein 638), as well as the first 20 exons of DYSF [dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)], which causes LGMD2B (33). We used real-time PCR to confirm and better define the extension of this duplication (Fig. 2).

Sample no. 45 corresponded to a familial case with an LGMD/Becker muscular dystrophy-like phenotype. We detected an intragenic heterozygous duplication in LAMA2 (laminin, alpha 2) spanning about 48.8 kb and involving exons 5-12. The copy-number change was confirmed by real-time PCR, which also better positioned the 5' and 3' breakpoints to the 129503539-129507201 and 129573766-129593644 intervals, respectively (Fig. 3A). These results were perfectly coincident in 2 affected sisters of the proband, but after direct sequencing of all coding exons, we were unable to identify any causative mutation on the second allele.

We identified another copy-number change in LAMA2 in sample no. 55, a sporadic case of generic myopathy with sarcotubular proliferation. We detected a heterozygous deletion of about 164.4 kb that removed exons 13-37. The breakpoint was precisely mapped by long-range PCR and direct sequencing (NG_008678.1:g.347377_511797del) (Fig. 3B). This rearrangement was maternally inherited, and all attempts to identify the causative mutation on the second allele proved ineffective. Direct sequencing of all coding exons highlighted only 3 single-nucleotide polymorphisms (rs17057184, NM_000426.3:c.6279C/T (p.A2093A), and rs6569606). One (NM_000426.3: c.6279C/T) was located in exon 45, which was not involved in a copy-number change, and was shown to be heterozygous by genomic DNA analysis. An mRNA analysis of the patient's lymphocytes revealed that this single-nucleotide polymorphism was no longer heterozygous, indicating that the other allele was not expressed.

Sample no. 56 corresponded to a familial case with a severe LGMD phenotype. Analysis with the Motor Chip revealed this individual to be compound heterozygous for 2 different wide deletions involving SGCG, which caused LGMD2C (34). One allele presented the same deletion of about 1.5 Mb involving 7 genes (including SGCG and SACS) described above for sample nos. 27, 28, 29, 30, and 32 (Fig. 4A). On the other allele, we detected a shorter deletion of about 114.5 kb that removed the first 6 exons of SGCG (Fig. 4A).

[FIGURE 1 OMITTED]

By real-time PCR and long-range PCR we confirmed both copy-number changes and further refined the breakpoints. The 1.5-Mb microdeletion was coincident with that previously reported by Breckpot et al. (35) caused by nonallelic homologous recombination involving highly homologous segmental duplications in the 5' and 3' breakpoint-containing regions, which we narrowed down to the 22402586-22407180 and 23830032-23832533 intervals, respectively (Fig. 4A). Conversely, the breakpoint of the shorter microdeletion was precisely mapped [chr13: g.22620716_22789085del (NCBI build 36.1)] (Fig. 4B). It is most likely due to homologous recombination mediated by 2 Alu elements with a high sequence similarity (AluSp at the 5' end and AluY at the 3' end).

Discussion

The Motor Chip is an NMD-focused oligonucleotide-based CGH array and represents the first comprehensive approach to analyzing deleterious copy-number changes in a wide set of NMD genes. The release presented here (NMDv02.15-Hg18) includes 425 nuclear genes, 245 of which are already implicated in neuromuscular phenotypes. The exon-specific probe selection strategy used in the Motor Chip design permitted not only full coverage of the coding sequence for all of the selected genes by using only 44 379 features but also a reduction in the costs per test (see Table 3 in the online Data Supplement), an added value for laboratory use. The technical protocol can be carried out in 6 person-hours (see Table 3 in the online Data Supplement), and it is suitable for use in automated liquid-handling systems.

[FIGURE 2 OMITTED]

There is a commercially available MLPA assay (MRC-Holland), which is frequently used as an alternative method for detecting copy-number changes, for only 44 of the 245 NMD genes included in the Motor Chip. The Motor Chip is cheaper and less time-consuming than MLPA analysis, especially when a panel of candidate genes needs to be investigated.

Validation experiments demonstrated the diagnostic sensitivity and specificity of the Motor Chip design, with 100% concordance between Motor Chip results and those generated by other methods. Of all of the double-blinded DNA samples analyzed, 26 with previously detected pathologic chromosomal imbalances were correctly addressed with the Motor Chip. For the other 27 samples with an incomplete or undetermined molecular diagnosis, only 5 showed previously undetected copy-number changes in NMD genes.

By detecting a wide heterozygous intragenic deletion in SETX, we confirmed the clinical diagnosis of AOA2 for patient no. 21. We also provided the molecular diagnosis of LGMD2C for patient no. 56, who was shown to be a compound heterozygote for 2 distinct wide microdeletions involving SGCG. Interestingly, 1 of these alleles corresponded to a previously reported CNV (27,28), which was also detected in other samples analyzed in this study together with SACS point mutations, producing autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) (29, 30, 35). Recently, a combined LGMD2C/ARSACS phenotype in a 10-year-old girl was also reported as being due to a similar homozygous microdeletion (36). The individual we have described is the first LGMD2C case with distinct microdeletions involving both SGCG copies. Exonic deletions are rare events in sarcoglycanopathies; however, our results suggest that screening for exonic deletions/duplications or more-complex copy-number changes should be carried out for patients in whom a mutation has not been identified in both alleles, as well as in apparently homozygous cases in which segregation of the mutations cannot be confirmed in the parents.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Copy-number imbalances involving DYSF or LAMA2 were also detected in another 3 patients (nos. 42, 45, and 55). Their pathogenic significance was unclear, however. In particular, the observed LAMA2 intragenic duplication and deletion were in frame and apparently without causative mutations on the second allele. CNVs resembling these rearrangements have not been annotated in the Database of Genomic Variants (http://projects.tcag.ca/variation/); however, intragenic copy-number changes for LAMA2 have been reported very recently (37).

The Motor Chip was therefore able to provide a molecular diagnosis in 2 of 27 of these cases, a result consistent with the possible role of deleterious copy-number imbalances in NMDs, which have been estimated at 5%-10%.

Compared with the commercially available Agilent Human Genome CGH Microarrays, the Motor Chip design presents further advantages. They include greater efficiency in detecting intragenic deleterious copy-number changes and a reduction in CNV calls of uncertain pathogenic significance. As highlighted in Table 2, the Motor Chip contains more probes than standard Agilent aCGH designs for most of the pathogenic rearrangements that have been identified. Eight intragenic copy-number changes were undetectable with any of the Agilent aCGH designs considered. Compared with the Motor Chip, these copy-number changes showed a detection rate of about 41.9%, 54.8%, 67.7%, and 77.4% for the 60K, 180K, 244K, and 400K designs, respectively. Therefore, for most NMD monogenic disorders, full exon-specific coverage is necessary to detect causative mutations. As very recently proposed (25), this approach may be applied to a wide range of different clinical conditions.

The Motor Chip also included 180 putative NMD genes, which were selected on the basis of their role in neuromuscular functions. It was designed to be a tool for both enhancing molecular diagnosis and discovering novel disease genes. The oligonucleotide probes selected for full exonic coverage of these genes provided satisfactory signal quality for both channels in the validation experiments performed to date. Only by using the Motor Chip in routine laboratory analyses will it be possible to verify the potential involvement of these genes in neuromuscular diseases.

Oligonucleotide-based microarrays can be updated quickly and easily. For the Motor Chip, selected probes were grouped according to the phenotypic classification of genes described in Table 1. Consequently, features covering a specific gene can be easily added or removed to rapidly enhance array design. Importantly, subsets of these experimentally validated probes could be used in a dedicated microarray design for investigating specific NMD genes, which would further reduce the costs per test.

Unlike commonly used microarray design strategies (in which probes are spaced specifically at high density along the chromosomal regions of interest), all of the genes included in the Motor Chip, with the exception of DMD, were covered with exon-specific oligonucleotide probes alone. Although this approach reduces the number of probes required, contains costs, and increases the detection rate, it might make breakpoint boundary characterization more difficult. This feature can be considered a minor criticism. In many cases, breakpoint boundaries can be mapped precisely by long-range PCR with appropriately designed primer pairs. For genes characterized by wide introns, we intend to add other suitably spaced intragenic probes to the next release of the Motor Chip. Very recently, general recommendations for performing aCGH-based diagnostic tests have advised using a uniform genome coverage with a resolution of 5 Mb to 400 kb throughout the genome (12). The backbone of the next version of the Motor Chip will therefore be redesigned with probes spaced approximately 300 kb apart throughout the genome, for an estimated resolution of about 1 Mb. The current release, in which the entire genome is covered by randomly selected probes, is nevertheless able to identify unexpected genomic imbalances, as confirmed by the detection of both annotated CNVs and the aneuploid (47,XYY) karyotype in sample no. 40.

The molecular diagnosis of CNVs in NMDs will be facilitated byuse of the Motor Chip. Present and future releases of the Motor Chip are freely accessible to other laboratories on request via the Web-based eArray interface.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revisingthe article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: V. Nigro, grant no. FP7/2007-2013 under grant agreement no. 223143 (TECHGENE) and Ministero della Salute (RF-MUL-2007-666195).

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Anna Cuomo and Laura Mondrone for sequencing. We thank Aon Benfield Italia S.p.A.--Milan for the generous gift of a robot. We acknowledge the EUROBIOBANK (L. Politano) supported by TREAT-NMD.

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(35.) Breckpot J, Takiyama Y, Thienpont B, Van Vooren S, Vermeesch JR, Ortibus E, Devriendt K. A novel genomic disorder: a deletion of the SACS gene leading to spastic ataxia of Charlevoix-Saguenay. Eur J Hum Genet 2008;16:1050-4.

(36.) McMillan HJ, Carter MT, Jacob PJ, Laffan EE, O'Connor MD, Boycott KM. Homozygous contiguous gene deletion of 13q12 causing LGMD2C and ARSACS in the same patient. Muscle Nerve 2009;39:396-9.

(37.) Cossee M, Bartoli M, Allamand V, Guittard C, Delague V, Krahn M, et al. P1.15 DNA microarrays for revisiting molecular pathology in neuromuscular disorders. Neuromuscul Disord 2010; 20:604.

Giulio Piluso, [1] Manuela Dionisi, [1] Francesca Del Vecchio Blanco, [1] Annalaura Torella, [1] Stefania Aurino, [1,2] Marco Savarese, [1,2] Teresa Giugliano, [1] Enrico Bertini, [3] Alessandra Terracciano, [3] Mariz Vainzof, [4] Chiara Criscuolo, [5] Luisa Politano, [6] Carlo Casali, [7] Filippo Maria Santorelli, [8] and Vincenzo Nigro [1,2] *

[1] Dipartimento di Patologia Generale, Seconda University degli Studi di Napoli, Naples, Italy; [2] Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; [3] Dipartimento di Neuroscienze, Unita di Medicina Molecolare, Ospedale Pediatrico "Bambino GesU," Rome, Italy; [4] The Human Genome Research Center (HGRC), University of Sao Paulo, Sao Paulo, Brazil; [5] Dipartimento di Scienze Neurologiche, University degli Studi "Federico II," Naples, Italy; [6] Servizio di Cardiomiologia e Genetica Medica, Seconda University degli Studi di Napoli, Naples, Italy; [7] Dipartimento di Neurologia e ORL, University di Roma "La Sapienza"--Polo Pontino, Latina, Italy; [8] IRCCS Fondazione "Stella Maris," Pisa, Italy.

* Address correspondence to this author at: Dipartimento di Patologia Generale, Seconda University degli Studi di Napoli, S. Andrea delle Dame, via L. De Crecchio 7, 80138 Napoli, Italy. Fax +39-0815665704; e-mail vincenzo.nigro@unina2.it, nigro@tigem.it.

Received May 19, 2011; accepted August 23, 2011.

Previously published online at DOI: 10.1373/clinchem.2011.168898

[9] Nonstandard abbreviations: NMD, neuromuscular disorder; aCGH, array-based comparative genomic hybridization; CNV, copy-number variation; LGMD2C, limb-girdle muscular dystrophy type 2C; MLPA, multiplex ligation-dependent probe amplification; AOA2, ataxia with oculomotor apraxia type 2; ARSACS, autosomal recessive spastic ataxia of Charlevoix-Saguenay.

[10] Human genes: DMD, dystrophin (Duchenne and Becker muscular dystrophy); SGCG, sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein); CAPN3, calpain 3, (p94); SPAST, spastin; SPG11, spastic paraplegia 11 (autosomal recessive); SACS, spastic ataxia of Charlevoix-Saguenay (sacsin); TNFRSF19, tumor necrosis factor receptor superfamily, member 19; MIPEP, mitochondrial intermediate peptidase; PCOTH, prostate collagen triple helix protein; SPATA13, spermatogenesis associated 13; C1QTNF9, C1q and tumor necrosis factor related protein 9; PMP22, peripheral myelin protein 22; SETX, senataxin; ZFYVE26, zinc finger, FYVE domain containing 26; CD207, CD207 molecule, langerin; VAAX2, ventral anterior homeobox 2; ATP6V1B1, ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B1; ANKRD53, ankyrin repeat domain 53; TEX261, testis expressed 261; NAGK, N-acetylglucosamine kinase; MCEE, methylmalonyl CoA epimerase; MPHOSPH10, M-phase phosphoprotein 10 (U3 small nucleolar ribonucleoprotein); PAIP2B, poly(A) binding protein interacting protein 2B; ZNF638, zinc finger protein 638; DYSF, dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive); LAMA2; laminin, alpha 2.

[11] At this time, the HUGO Gene Nomenclature Committee has not approved this gene symbol or name.
Table 1. Probe selection summary for Motor Chip design
(NMDv02.15-Hg18).

Group Genes, Total Custom
(a) Description n probes, n probes, n

1 Muscular dystrophies 23 7064 66
2 Congenital muscular 8 1013 43
 dystrophies
3 Congenital myopathies 12 1954 394
4 Distal myopathies 2 178 5
5 Other myopathies 10 749 99
6 Myotonic syndromes 5 590 62
7 Ion channel muscle diseases 11 1484 57
9 Metabolic myopathies 19 1092 92
10 Hereditary cardiomyopathies 47 4738 160
11 Congenital myasthenic 10 495 24
 syndromes
12 Spinal muscular atrophies 15 1212 34
13 Hereditary ataxias 22 1646 83
14 Hereditary motor and 26 2316 75
 sensory neuropathies
15 Hereditary paraplegias 18 1789 100
16 Other neuromuscular 17 1224 59
 disorders
17 Genes involved in metabolic 88 7539 357
 pathways and muscle
 organization
18 Putative DYSF interactors 14 1117 105
 identified by
 bioinformatic approach
19 Putative DYSF interactors 20 1763 99
 identified by yeast
 2-hybrid library
 screening
20 Putative TRIM32 (b) 10 426 30
 interactors identified by
 yeast 2-hybrid library
 screening
21 Additional genes highly 48 2628 154
 expressed in muscle
Prom Putative promoter regions 3362 34
 Total 425 44 379 2132

Group Custom Probes/
(a) Description probes, % gene, n

1 Muscular dystrophies 0.93 307
2 Congenital muscular 4.24 127
 dystrophies
3 Congenital myopathies 20.16 163
4 Distal myopathies 2.81 89
5 Other myopathies 13.22 75
6 Myotonic syndromes 10.51 118
7 Ion channel muscle diseases 3.84 135
9 Metabolic myopathies 8.42 57
10 Hereditary cardiomyopathies 3.38 101
11 Congenital myasthenic 4.85 50
 syndromes
12 Spinal muscular atrophies 2.81 81
13 Hereditary ataxias 5.04 75
14 Hereditary motor and 3.24 89
 sensory neuropathies
15 Hereditary paraplegias 5.59 99
16 Other neuromuscular 4.82 72
 disorders
17 Genes involved in metabolic 4.74 86
 pathways and muscle
 organization
18 Putative DYSF interactors 9.40 80
 identified by
 bioinformatic approach
19 Putative DYSF interactors 5.62 88
 identified by yeast
 2-hybrid library
 screening
20 Putative TRIM32 (b) 7.04 43
 interactors identified by
 yeast 2-hybrid library
 screening
21 Additional genes highly 5.86 55
 expressed in muscle
Prom Putative promoter regions 1.01
 Total 4.80 104

(a) Genes in group 8 corresponding to malignant hyperthermia
[Kaplan (1)] are included in groups 1-7.

(b) TRIM32, tripartite motif containing 32.

Table 2. Pathogenic copy-number imbalances detected using Motor Chip
(NMDv02.15-Hg18) VS Agilent Human Genome CGH Microarrays.

 Motor Chip (NMDv02.15-Hg18) results

Patient Pathogenic
no. ID Sex Gene CNV

1 X661 F DMD DEL exon 27
2 TU119 F DMD DEL exon 55
3 TU358 F DMD DUP exons 50-62
4 X029 F SGCG DEL exon 7
5 X282 M DMD DEL exon 55
6 X640 M CAPN3 DEL exons 2-8
7 R073 M DMD DEL exons 47-48
8 R085 M DMD DUP exons 3-4
9 TU263 M DMD DEL exons 42-43
10 1130 F SGCG DEL exon 7
11 X660 F DMD DEL exon 27
12 X669 F DMD DUP exon 54
13 N015 M SPAST DEL exons 2-17 (3' UTR included)
14 N016 M SPAST DEL exon 17 (3' UTR included)
17 N021 M SPAST DEL exons 1-17 (3' UTR included)
18 N009 F SPG11 DEL exons 31-34
19 N012 F SPAST DUP exons 8-9
20 N013 F SPAST DEL exons 8-16
21 ZA F SETX DEL exons 16-24
23 N003 M SPG11 DEL exons 31-34
27 AT04 M SACS Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
28 AT08 M SACS Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
29 AT06 M SACS Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
30 AT07 F SACS Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
32 AT03 F SACS Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
38 RM M PMP22 Wide DEL (CDRT15, (c) HS3ST3B1, and
 PMP22)
39 SA M PMP22 Wide DUP (CDRT15, HS3ST3B1, PMP22,
 TEKT3, CDRT4, and FAM18B3)
42 3484 M DYSF Wide DUP (CD207, VAX2, ATP6V1B1,
 ANKRD53, TEX261, NAGK, MCEE,
 MPHOSPH10, PAIP2B, ZNF638, and DYSF
45 3159 F LAMA2 DUP exons 5-12
55 X730 F LAMA2 DEL exons 13-37
56 X415 F SGCG Wide DEL (SGCG, SACS, TNFRSF19,
 MIPEP, PCOTH, SPATA13, and C1QTNF9)
 SGCG DEL exons 1-6

 Motor Chip (NMDv02.15-Hg18) results

 Min (Max)
Patient Chr aberration
no. ID Sex Zygosity Probes (a) length (bp)

1 X661 F Het 9 X 3314 (4767)
2 TU119 F Het 53 X 41928 (44125)
3 TU358 F Het 578 X 522437 (523840)
4 X029 F Hom 3 13 184 (29128)
5 X282 M Hem 53 X 41928 (44127)
6 X640 M Het 21 15 8284 (34232)
7 R073 M Hem 51 X 40598 (42392)
8 R085 M Hem 57 X 46607 (47748)
9 TU263 M Hem 44 X 39127 (44420)
10 1130 F Hom 3 13 184 (29128)
11 X660 F Het 9 X 3314 (4767)
12 X669 F Het 22 X 17349 (19385)
13 N015 M Het 79 2 70365 (119775)
14 N016 M Het 46 2 29782 (471171)
17 N021 M Het 83 2 93423 (119963)
18 N009 F Het 11 15 4440 (13189)
19 N012 F Het 6 2 1643 (20254)
20 N013 F Het 25 2 20372 (31009)
21 ZA F Het 28 9 16925 (24044)
23 N003 M Hom 11 15 4440 (13189)
27 AT04 M Het 308 13 1323181 (2369225)
28 AT08 M Het 308 13 1323181 (2369225)
29 AT06 M Het 309 13 2194641 (2659348)
30 AT07 F Het 308 13 1323181 (2369225)
32 AT03 F Het 308 13 1323181 (2369225)
38 RM M Het 71 17 1060503 (1396119)
39 SA M Het 69 17 1381145 (1436540)
42 3484 M Het 73 2 734477 (865685)
45 3159 F Het 23 6 48846 (106088)
55 X730 F Het 94 6 143239 (208282)
56 X415 F Het 308 13 1323181 (2369225)
 Het 18 13 114551 (302571)

 Motor Chip (NMDv02.15-Hg18) results

Patient 5' Breakpoint 3' Breakpoint
no. ID Sex boundary boundary

1 X661 F 32374291-32375205 32378519-32379058
2 TU119 F 31529042-31529841 31571769-31573167
3 TU358 F 31236430-31237173 31759610-31760270
4 X029 F 22767637-22792762 22792946-22796765
5 X282 M 31529042-31529841 31571769-31573169
6 X640 M 40439563-40463933 40472217-40473795
7 R073 M 31823808-31824752 31865350-31866200
8 R085 M 32764472-32764931 32811538-32812220
9 TU263 M 32203829-32208623 32247750-32248249
10 1130 F 22767637-22792762 22792946-22796765
11 X660 F 32374291-32375205 32378519-32379058
12 X669 F 31581707-31582989 31600338-31601092
13 N015 M 32142840-32165853 32236218-32262615
14 N016 M 32225840-32232892 32262674-32697011
17 N021 M 32142652-32142795 32236218-32262615
18 N009 F 42650067-42650071 42654511-42663256
19 N012 F 32194826-32205468 32207111-32215080
20 N013 F 32194826-32205468 32225840-32225835
21 ZA F 134136976-134136978 134153903-134161020
23 N003 M 42650067-42650071 42654511-42663256
27 AT04 M 21593561-22464962 23788143-23962786
28 AT08 M 21593561-22464962 23788143-23962786
29 AT06 M 21303438-21593502 23788143-23962786
30 AT07 F 21593561-22464962 23788143-23962786
32 AT03 F 21593561-22464962 23788143-23962786
38 RM M 14036295-14050847 15111350-15432414
39 SA M 14051331-14051328 15432473-15487871
42 3484 M 70768787-70899988 71634465-71634472
45 3159 F 129506877-129506874 129555720-129612965
55 X730 F 129555720-129612876 129756115-129764002
56 X415 F 21593561-22464962 23788143-23962786

 Agilent Human Genome aCGH (b)
Patient
no. ID Sex 60K 180K 244K 400K

1 X661 F 1 (1) 1 (1) 1 (1) 1 (1)
2 TU119 F 1 (1) 4 (4) 5 (5) 8 (8)
3 TU358 F 12 (12) 44 (44) 58 (58) 101 (101)
4 X029 F 0 (0) 0 (2) 0 (4) 0 (7)
5 X282 M 1 (1) 4 (4) 5 (5) 8 (8)
6 X640 M 0 (0) 1 (2) 2 (4) 2 (7)
7 R073 M 1 (1) 2 (2) 5 (5) 10 (10)
8 R085 M 1 (1) 2 (2) 5 (5) 9 (9)
9 TU263 M 1 (1) 2 (2) 4 (4) 7 (8)
10 1130 F 0 (0) 0 (2) 0 (4) 0 (7)
11 X660 F 1 (1) 1 (1) 1 (1) 1 (1)
12 X669 F 1 (1) 2 (2) 2 (2) 3 (4)
13 N015 M 3 (3) 7 (9) 8 (11) 13 (23)
14 N016 M 1 (14) 2 (44) 2 (52) 7 (105)
17 N021 M 3 (3) 8 (9) 10 (11) 17 (23)
18 N009 F 1 (1) 1 (1) 1 (1) 2 (3)
19 N012 F 0 (1) 0 (1) 0 (1) 1 (3)
20 N013 F 1 (1) 2 (2) 3 (3) 5 (6)
21 ZA F 1 (1) 2 (2) 2 (2) 4 (5)
23 N003 M 1 (1) 1 (1) 1 (1) 2 (3)
27 AT04 M 29 (43) 97 (139) 134 (188) 230 (336)
28 AT08 M 29 (43) 97 (139) 134 (188) 230 (336)
29 AT06 M 39 (50) 131 (153) 172 (204) 316 (365)
30 AT07 F 29 (43) 97 (139) 134 (188) 230 (336)
32 AT03 F 29 (43) 97 (139) 134 (188) 230 (336)
38 RM M 14 (21) 48 (69) 57 (81) 119 (170)
39 SA M 21 (22) 68 (72) 80 (83) 168 (173)
42 3484 M 19 (21) 52 (61) 83 (101) 126 (151)
45 3159 F 1 (2) 4 (7) 7 (12) 10 (20)
55 X730 F 3 (4) 10 (13) 18 (23) 28 (39)
56 X415 F 29 (43) 97 (139) 134 (188) 230 (336)
 3 (7) 10 (17) 13 (23) 23 (44)

(a) Chr, chromosome; Min (Max), minimum (maximum); DEL, deletion;
Het, heterozygote; DUP, duplication; Hom, homozygote; Hem,
hemizygote, UTR, untranslated region.

(b) Number of probes in the commercially available Agilent aCGH
designs for Min (Max) aberration length

(c) Human genes not mentioned in text: CDRT15, CMT1A duplicated
region transcript 15; HS3ST3B1, heparan sulfate (glucosamine)
3-O-sulfotransferase 3B1; TEKT3, tektin 3; CDRT4, CMT1A duplicated
region transcript 4; FAM18B3, family with sequence similarity 18,
member B3.
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Title Annotation:Molecular Diagnostic and Genetics
Author:Piluso, Giulio; Dionisi, Manuela; Blanco, Francesca Del Vecchio; Torella, Annalaura; Aurino, Stefani
Publication:Clinical Chemistry
Date:Nov 1, 2011
Words:7249
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