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Utility of oligonucleotide array--based comparative genomic hybridization for detection of target gene deletions.

Although direct DNA sequencing of specific genes is the primary clinical technique for identifying mutations in human disease, this method detects only point mutations or small deletions. Large intragenic exonic deletions and duplications have been shown to be a frequent cause of many diseases. For example, more than 65% of the mutations in DMD [5] [dystrophin (muscular dystrophy, Duchenne and Becker types)] (which cause Duchenne muscular dystrophy) and approximately 20% of the mutations in GLDC [glycine dehydrogenase (decarboxylating)] (which cause nonketotic hyperglycinemia) are exonic deletions (1, 2). To date, various methods, including multiplex ligation-dependent probe amplification (MLPA) [6] (3), restriction fragment analysis on Southern blots (4), customized fluorescence in situ hybridization (FISH) (5), and quantitative real-time PCR (gPCR) analysis (6), have been used to detect intragenic deletions or duplications. Such testing is currently available, however, only for a small number of specific genes, and the efforts to examine multiple genes simultaneously have been very limited. For example, although multiple exon-deletion MLPA assays have recently been developed for assessing X-linked mental retardation (7), such analyses are restricted to approximately 60 loci.

Oligonucleotide probes on microarrays that correspond to sequences throughout the entire genome have now been shown to produce quantitative hybridization responses under standardized conditions, allowing rapid and relatively inexpensive analysis of chromosomal copy number variation to be performed as a clinical test. Most of the applications of this technology have been designed to detect major changes in chromosomal copy number that affect >50 kb of sequence (8, 9); however, these applications are primarily attributable to the fact that the technology was initially developed as a substitute for karyotyping or fluorescence in situ hybridization analysis. There is no inherent reason why higher-resolution analyses cannot be performed.

We describe the utility of a targeted oligonucleotide array designed to detect both whole-gene deletions and small intragenic deletions in genes involved in mi tochondrial biogenesis or function, or in metabolic disorders such as urea cycle disorders and progressive familial intrahepatic cholestasis [OMIM (Online Mendelian Inheritance in Man) nos. 211600, 602347, and 601847].

Materials and Methods


The clinical details pertaining to case 1 of citrin deficiency have been reported elsewhere (10). In brief, the proband presented with 3 episodes of life-threatening bleeding and significant failure to thrive that responded to a high-protein, low-carbohydrate diet. Western blotting for citrin protein revealed an absence of cross-reactive material. Sequencing analysis revealed a novel heterozygous splice site mutation [c.848 + 3 A>C (IVS8 + 3A>C)] in the SLC25A13 gene, but the second mutant allele was not identified.

The patient in case 2 had progressive intrahepatic cholestasis and presented with intracerebral hemorrhage secondary to a coagulopathy of hepatic origin. A liver biopsy revealed hepatic fibrosis and cholestasis. Immunohistochemistry showed an absence of the bile salt export pump protein, which is encoded by the ABCB11 gene [PFIC2, ATP-binding cassette, subfamily B (MDR/TAP), member 11]. PCR failed to amplify exons 13-17 of the gene; however, sequencing of the remaining ABCB11 exons, as well as the ATPSB1 gene (PFIC1; ATPase, class I, type 8B, member 1), did not detect any mutations.

Case 3 was of a 3-day-old baby girl who presented with poor feeding, encephalopathy, and hyperammonemia (464 [micro]mol/L, reference interval, <40 [micro]mol/L), increased glutamine (3369 [micro]mol/L; reference interval, 238-842 [micro]mol/L), and a significantly increased orotic acid concentration (749 [micro]mol/L per mmol/L creatinine) in the urine. Despite aggressive therapy, the ammonia concentration continued to increase to a peak concentration of 1810 [micro]mol/L. Treatment was discontinued, and the child died on day 4. An enzyme assay of a liver sample revealed reduced ornithine transcarbamylase (OTC) activity; mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) activity was within the reference range. Subsequent sequence analysis of the OTC gene was performed; however, mutations were not detected.

Case 4 was of an 8-year old girl who also presented with hyperammonemia, developmental delay, and an increased glutamine concentration. The CPS1 gene was sequenced, but no mutations were found. Given the possibility of a deletion affecting CPS1 or OTC, we subsequently analyzed the relevant DNA sequence on the oligonucleotide array.

All research testing was carried out with the consent of the families and according to a research protocol approved by the Baylor College of Medicine Institutional Review Board.


A custom 44K array was made using the Agilent oligonucleotide microarray platform (Agilent Technologies). This array included coverage for approximately 130 nuclear genes that are related to metabolic pathways or mitochondrial biogenesis and function, at an average probe spacing of about 250-300 by per oligonucleotide probe.

Total DNA was extracted from peripheral blood leukocytes, liver, or muscle tissue with commercially available DNA-isolation kits according to the manufacturer's protocols (Gents Systems). For each array CGH experiment, 1 [micro]g each of purified patient DNA and sex-matched control DNA was digested with 10 U AluI and 10 U RsaI (Promega) and differentially labeled with cyanine-5 (Cy5) and cyanine-3 (Cy3) fluorescent dyes (PerkinElmer), respectively, by random priming with a Bioprime Array CGH Genomic Labeling Module (Invitrogen). Hybridization was carried out for at least 20 h at 65[degrees]C in a rotating microarray hybridization chamber and then washed according to the manufacturer's protocols (Agilent Technologies). The slide hybridization results were scanned into image files with a GenePix 4000B microarray scanner (Molecular Devices). Agilent's Feature Extraction v9.1 software then located and quantified the array features, and CGH Analytics software analyzed text file outputs for relative changes in copy number. All regions with a minimum of 5 consecutive oligonucleotides indicating significant copy number variation were investigated further; common copy number variants were assessed by position comparison with copy number variation databases and by comparison with parental CGH patterns, when available.


For the intragenic deletions detected with oligonucleotide array CGH, we confirmed the breakpoints by PCR amplification and sequencing of the junction region to determine the exact breakpoints. We confirmed large deletions (>1 Mb) by hybridizing the DNA of the affected individuals and their parents on Agilent Human Genome CGH Microarray 244K whole-genome oligonucleotide arrays to better define the breakpoints before sequence analysis. The GenBank sequences NT_079595 (SLC25A13), NW_921585 (ABCB11), and NC_000023 (OTC), were used as the respective reference sequences. For sequencing, we used sequence-specific oligonucleotide primers linked to M13 universal primers, which were designed to amplify all of the coding exons of the genes described in this report.


In the patient with citrin deficiency (case 1), array CGH showed a heterozygous deletion within the SLC25A13 gene on chromosome 7g21.3 that involved multiple consecutive oligonucleotide probes (Fig. 1A). The hybridization data were consistent with a deletion of approximately 4.6 kb that included parts of introns 2 and 3 as well as all of exon 3. PCR amplification across the putative breakpoints (Fig. 1B) and sequencing of the isolated PCR product identified the breakpoints within the SLC25A13 gene at c.70-862 and c.212 + 3527 (Fig. 1C), which correspond to a 4 532-bp deletion. This heterozygous deletion was also confirmed in the proband's asymptomatic sister and her father by direct DNA sequencing of the junction fragment and by gPCR (data not shown). Previous direct sequencing of the patient's DNA had revealed a novel heterozygous alteration, c.848 + 3 A>C (IVS8 + 3A>C), which SpliceView software (see splice-view.html) had predicted to produce a loss of a splice donor site. We confirmed the aberrant splicing of the c.848 + 3 A>C mutation by cDNA sequencing; in addition, finding these mutations in the proband's mother confirmed the trans configuration of these mutations.

In the patient with progressive intrahepatic cholestasis (case 2), array CGH detected a homozygous deletion at the ABCB11 locus on chromosome 2g31.1 that involved >50 oligonucleotide probes (Fig. 2A). Sequence analysis of the junction region revealed breakpoints consistent with the oligonucleotide array CGH data that indicated deletion of sequence from c.1309+1048 to c.2076-610 (Fig. 2B). This 10.5-kb deletion contains part of intron 12, all of exons 13-17, and part of intron 17 (Fig. 2C). Unfortunately, parental samples for this patient were not available for comparison.

A large deletion of part of one X chromosome (from Xp11.4-21.2) was detected with the gene-based array in the infant girl with hyperammonemia (case 3 ). This approximately 7.4-Mb deletion extended from the telomeric side of DMD to within exon 4 of the OTC gene on the centromeric side (data not shown). Because the oligonucleotide array was targeted only to specific genes of interest, we also analyzed the DNA with an Agilent 244K whole-genome oligonucleotide array to identify the breakpoints (Fig. 3). The deletion breakpoints were determined to be at approximately X:30.7 Mb and X:38.1 Mb. Analysis of DNA from parental blood samples in case 3 (Fig. 3) did not detect any deletions, suggesting germ line mosaicism or a de novo mutation.

We did not detect CPS1 deletions in the patient of case 4, who also presented with hyperammonemia; but instead we found a deletion of the OTC gene. An Agilent 244K array confirmed an approximately 9-Mb deletion encompassing a region of the X chromosome similar to that of patient 3 (Fig. 3 ). The proximal breakpoint was centromeric to the OTC gene at approximately X:38.4 Mb. The distal region of the deletion was more complex, with an approximately 0.2-Mb deleted region followed by a 0.24-Mb nondeleted region and a large 8.84-Mb deletion. We were not able to obtain parental DNA samples for this patient to evaluate the inheritance of these regions; therefore, we do not know whether this pattern is due to a complex rearrangement mechanism or a small polymorphic copy number variant in this region.

Several known genes are in the X-chromosome region between the DMD and OTC genes, including: (a) CYBB [cytochrome b-245, beta polypeptide (chronic granulomatous disease)], which encodes the cytochrome b (3 chain involved in superoxide production and mutations of which cause chronic granulomatous disease; (b) XK [X-linked Kx blood group (McLeod syndrome)], which is the X-linked Kx blood group of the kell precursor and mutations of which have been associated with McLeod syndrome, an X-linked, recessive disorder characterized by abnormalities in the neuromuscular and hematopoietic systems; and (c) RPGR (retinitis pigmentosa GTPase regulator), the X-linked retinitis pigmentosa GTPase regulator isoform B. Deletion of these genes, however, would not explain these patients' clinical presentations with a proximal urea cycle defect.





Oligonucleotide array--based CGH has now been routinely used to detect large chromosomal deletions (9, 11 ). Most of these assays monitor regional changes that may affect multiple genes, although large rearrangements impacting a single gene have been investigated in a few cases. Examples of the latter application include the study of large rearrangements in MECP2 [methyl CpG binding protein 2 (Rett syndrome)], the gene responsible for Rett syndrome and X-linked mental retardation (12), and an investigation of copy number changes in the DMD gene, which are responsible for Duchenne muscular dystrophy (8). We hypothesized that similar approaches could be used to simultaneously screen for intragenic deletions within a group of genes relevant to specific related disorders. We were particularly interested in the genes involved in mitochondrial and metabolism-related disorders to complement the gene-sequencing analyses in our laboratory.

The use of array CGH for gene-based analyses has several advantages over alternative methods, such as MLPA, gPCR, and fluorescence in situ hybridization analysis. In our experience, the initial setups of the latter 3 assays are time consuming, and their performance requires costly validation. In addition, maintaining proficiency with these tests in a low-throughput clinical-testing environment is problematic. Furthermore, these techniques, which are typically designed to analyze 1 or 2 probes at a time, usually require multiple measurements. Such hurdles may have inhibited the rapid introduction of deletion or duplication assays into the clinical-testing sphere as new disease genes have been discovered. The availability of well-characterized oligonucleotides covering the whole human genome from established databases reduces the time to create and validate probes when one compares the time to do the same with MLPA-or gPCR-based assays. Unlike gPCR or MLPA, which evaluates a single region in an exon, this highly multiplexed technology allows us to detect deletions in flanking introns as well as partial exon deletions.

It is advantageous to have oligonucleotide probes on the same array for genes involved in the same pathway or for similar diseases. For example, the patient in case 4 was originally suspected of having a CPS1 deficiency, but a deletion involving the OTC gene was discovered. Similarly, citrin deficiency often presents with an increased citrulline concentration, but it must be distinguished from disease caused by mutations in ASST (argininosuccinate synthetase) and ASL (argininosuccinate lyase). Furthermore, the molecular etiology of intrahepatic cholestasis may involve mutations in ATPSB1 (PFIC1), ABCB11 (PFIC2), or ABCB4 [PFIC3; ATP-binding cassette, sub-family B (MDR/ TAP), member 4]. Thus, it is convenient to be able to analyze a113 of these genes simultaneously.

Large genes may have an inherently higher risk for intragenic deletions. Both the ABCB11 (PFIC2) and SLC25A13 (citrin) genes are large. The ABCB11 gene consists of 27 coding exons spanning 108 kb. The citrin gene consists of 18 exons spanning 201 kb; introns 2 and 3 are particularly large (19.5 kb and 42.2 kb, respectively). A heterozygous 4.6-kb deletion in the citrin gene, consisting of a single exon and flanking intronic sequences, and a homozygous 10-kb deletion in the ABCB11 gene were readily detectable on the oligonucleotide-based microarray assay. On the other hand, OTC is not a large gene. A recent review reported 341 different point mutations (13). The majority of these mutations were private mutations, and, interestingly, mutations were not detected in 80% of patients. Conversely, 6%-20% of OTC mutations have been reported to be large deletions involving all or part of the gene (14-16). Therefore, the possibility of deletions should be considered in clinically and biochemically confirmed OTC-deficient patients. This consideration is particularly important in female patients, because heterozygous intragenic deletions cannot be detected with the traditional sequencing approach. Our results demonstrated that 2 disease-manifesting female patients in fact had large deletions in this region that included all or part of the OTC gene, suggesting that this region on the X chromosome may be particularly prone to deletions.

In summary, our experience suggests that oligonucleotide-based assays offer a valuable tool for clinical analysis of intragenic deletions, duplications, and rearrangements. In particular, we have demonstrated that targeted oligonucleotide arrays can be of great utility when used in conjunction with direct DNA sequencing in the context of autosomal recessive diseases when only one heterozygous mutant allele is detected. We are currently further evaluating extension of this oligonucleotide array CGH approach to the simultaneous analysis of copy number mutations in nuclear genes, mitochondrial DNA depletion, and single deletions in the mitochondrial genome leading to heteroplasmy.

Grant/Funding Support: This study was supported in part by National Institutes of Health fellowship award K12 RR17665 (David Dimmock). The sponsor did not participate in the design, conduct, or interpretation of the data from this study.

Financial Disclosures: The authors have no patents associated with the techniques or products described in this paper. The Department of Molecular and Human Genetics at Baylor College of Medicine offers microarray testing on a fee basis.

Acknowledgments: The authors thank Lin-Ya Tang for technical support.


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Lee-Jun C. Wong, [1], *, ([dagger]) David Dimmock, [1], ([dagger]) Michael T. Geraghty, [2] Richard Quan, [3] Uta Lichter-Konecki, [4] Jing Wang, [1] Ellen K. Brundage, [1] Fernando Scaglia, [1] and A. Craig Chinault [1]

[1] Molecular and Human Genetics, Baylor College of Medicine, Houston, TX;

[2] Genetics, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada;

[3] UC Davis Medical Center, Sacramento, CA; [4] Children's National Medical Center, Washington, DC.

[5] Human genes: DMD, dystrophin (muscular dystrophy, Duchenne and Becker types); GLDC glycine dehydrogenase (decarboxylating); SLC25A13, solute carrier family 25, member 13 (citrin); ARC811, ATP-binding cassette, sub-family B (MDR/TAP), member 11; ATP881, ATPase, class I, type 8B, member 1; OTC, ornithine carbamoyltransferase; CPS1, carbamoyl-phosphate synthetase 1, mitochondrial; CY88, cytochrome b-245, beta polypeptide (chronic granulomatous disease); XK, X-linked Kx blood group (McLeod syndrome); RPGR, retinitis pigmentosa GTPase regulator; MECP2, methyl CpG binding protein 2 (Rett syndrome); A551, argininosuccinate synthetase 1; ASL, argininosuccinate lyase; ARC84, ATP-binding cassette, sub-family B (MDR/TAP), member 4.

[6] Nonstandard abbreviations: MLPA, multiplex ligation-dependent probe amplification; gPCR, quantitative real-time PCR; CGH, comparative genomic hybridization.

* Address correspondence to this author at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, NAB 2015, Houston, TX 77030. Fax 713-798-8937; e-mail

([dagger]) Lee-Jun C. Wong and David Dimmock are co--first authors.

Received January 22, 2008; accepted April 2, 2008.

Previously published online at D01: 10.1373/clinchem.2008.103721
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Title Annotation:Molecular Diagnostics and Genetics
Author:Wong, Lee-Jun C.; Dimmock, David; Geraghty, Michael T.; Quan, Richard; Lichter-Konecki, Uta; Wang, J
Publication:Clinical Chemistry
Date:Jul 1, 2008
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