Correction: Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes.

[This corrects the article DOI: 10.1371/journal.pone.0015687.].

Molecular characterization of CPS1 deletions by array CGH.

CPSI deficiency usually results in severe hyperammonemia presenting in the first days of life warranting prompt diagnosis. Most CPS1 defects are non-recurrent, private mutations, including point mutation, small insertions and deletions. In this study, we report the detection of large deletions varying from 1.4 kb to >130 kb in the CPS1 gene of 4 unrelated patients by targeted array CGH. These results underscore the importance of analysis of large deletions when only one mutation or no mutations are identified in cases where CPSI deficiency is strongly indicated.

Application of oligonucleotide array CGH in the detection of a large intragenic deletion in POLG associated with Alpers Syndrome.

Mutations in the polymerase γ (POLG) gene are among the most common causes of mitochondrial disease and more than 160 POLG mutations have been reported. However, a large proportion of patients suspected of having POLG mutations only have one (heterozygous) definitive pathogenic mutation identified. Using oligonucleotide array CGH, we identified a compound heterozygous large intragenic deletion encompassing exons 15-21 of this gene in a child with Alpers syndrome due to mtDNA depletion. This is the first large POLG deletion reported and the findings show the clinical utility of using array CGH in cases where a single heterozygous mutation has been identified in POLG.

Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes.

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.

HERV-mediated genomic rearrangement of EYA1 in an individual with branchio-oto-renal syndrome.

Branchio-oto-renal syndrome is characterized by branchial defects, hearing loss, preauricular pits, and renal anomalies. Mutations in EYA1 are the most common cause of branchio-oto-renal and branchio-otic syndromes. Large chromosomal aberrations of 8q13, including complex rearrangements occur in about 20% of these individuals. However, submicroscopic deletions and the molecular characterization of genomic rearrangements involving the EYA1 gene have rarely been reported. Using the array-comparative genomic hybridization, we identified non-recurrent genomic deletions including the EYA1 gene in three patients with branchio-oto-renal syndrome, short stature, and developmental delay. One of these deletions was mediated by two human endogenous retroviral sequence blocks, analogous to the AZFa microdeletion on Yq11, responsible for male infertility. This report describes the expanded phenotype of individuals, resulting from contiguous gene deletion involving the EYA1 gene and provides a molecular description of the genomic rearrangements involving this gene in branchio-oto-renal syndrome.

Cornelia de Lange syndrome case due to genomic rearrangements including NIPBL.

Cornelia de Lange syndrome (CdLS) is a rare multisystem congenital anomaly disorder characterized by growth and developmental delay, distinctive facial dysmorphism, limb malformations and multiple organ defects. Approximately 60-65% of the CdLS subjects have mutation in one of three cohesin proteins, a main regulator of cohesin-associated protein, NIPBL, and two components of the cohesin ring structure SMC1A and SMC3. A prominent role for cohesin is to control chromosome segregation during cell divisions. We have performed MLPA analysis in a group of 11 children with the CdLS but without identifiable point mutations in the NIPBL and SMC1A genes. In a single patient, we identified a large deletion encompassing exons 35 to 47 of the NIPBL gene. Our finding was validated by aCGH and further characterized by long-range PCR and DNA sequencing of the breakpoint junction.

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

Application of dual-genome oligonucleotide array-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes.

PURPOSE: Mitochondrial disorders constitute a group of clinically and genetically heterogeneous diseases for which molecular diagnosis has been a challenge. The current procedures for diagnosis of mitochondrial DNA deletion and depletion syndromes based on Southern analysis and quantitative polymerase chain reaction are particularly inefficient for determining important parameters of deletion endpoints and percent heteroplasmy. We have developed an improved approach for routine analyses of these disorders in a clinical laboratory. METHODS: A custom-designed oligonucleotide array-based comparative genomic hybridization platform was developed to provide both tiled coverage of the entire 16.6-kb mitochondrial genome and high-density coverage of nuclear genes involved in mitochondrial biogenesis and function, for quick evaluation of mitochondrial DNA deletion and depletion. RESULTS: For initial validation, the performance of this array was characterized in 20 samples with known mitochondrial DNA deletions and 12 with apparent depletions. All previously known deletions were clearly detected and the break points were correctly identified by the oligonucleotide array-based comparative genomic hybridization, within the limits of resolution of the array. The extent of mitochondrial DNA depletion and the percentage of deletion heteroplasmy were estimated using an automated computational approach that gave results comparable to previous methods. Conclusions from subsequent application of this approach with >300 new clinical samples have been in 100% concordance with those from standard methods. Finally, for one sample, we were able to identify an intragenic deletion in a nuclear gene that was responsible for the observed mitochondrial DNA depletion. CONCLUSION: We conclude that this custom array is capable of reliably detecting mitochondrial DNA deletion with elucidation of the deletion break points and the percentage of heteroplasmy. In addition, simultaneous detection of the copy number changes in both nuclear and mitochondrial genomes makes this dual genome array of tremendous value in the diagnoses of mitochondrial DNA depletion syndromes.

Molecular mechanisms for subtelomeric rearrangements associated with the 9q34.3 microdeletion syndrome.

We characterized at the molecular level the genomic rearrangements in 28 unrelated patients with 9q34.3 subtelomeric deletions. Four distinct categories were delineated: terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements; each results in haploinsufficiency of the EHMT1 gene and a characteristic phenotype. Interestingly, 25% of our patients had de novo interstitial deletions, 25% were found with derivative chromosomes and complex rearrangements and only 50% were bona fide terminal deletions. In contrast to genomic disorders that are often associated with recurrent rearrangements, breakpoints involving the 9q34.3 subtelomere region are highly variable. Molecular studies identified three regions of breakpoint grouping. Interspersed repetitive elements such as Alu, LINE, long-terminal repeats and simple tandem repeats are frequently observed at the breakpoints. Such repetitive elements may play an important role by providing substrates with a specific DNA secondary structure that stabilizes broken chromosomes or assist in either DNA double-strand break repair or repair of single double-strand DNA ends generated by collapsed forks. Sequence analyses of the breakpoint junctions suggest that subtelomeric deletions can be stabilized by both homologous and nonhomologous recombination mechanisms, through a telomere-capture event, by de novo telomere synthesis, or multistep breakage-fusion-bridge cycles.

A novel chromosome 19p13.12 deletion in a child with multiple congenital anomalies.

We describe a patient with multiple congenital anomalies including deafness, lacrimal duct stenosis, strabismus, bilateral cervical sinuses, congenital cardiac defects, hypoplasia of the corpus callosum, and hypoplasia of the cerebellar vermis. Mutation analysis of EYA1, SIX1, and SIX5, genes that underlie otofaciocervical and/or branchio-oto-renal syndrome, was negative. Pathologic diagnosis of the excised cervical sinus tracts was revised on re-examination to heterotopic salivary gland tissue. Using high resolution chromosomal microarray analysis, we identified a novel 2.52 Mb deletion at 19p13.12, which was confirmed by fluorescent in situ hybridization and demonstrated to be a de novo mutation by testing of the parents. Overall, deletions of chromosome 19p13 are rare.

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

BACKGROUND: direct DNA sequencing is the primary clinical technique for identifying mutations in human disease, but sequencing often does not detect intragenic or whole-gene deletions. Oligonucleotide array-based comparative genomic hybridization (CGH) is currently in clinical use to detect major changes in chromosomal copy number. METHODS: a custom oligonucleotide-based microarray was constructed to provide high-density coverage of an initial set of 130 nuclear genes involved in the pathogenesis of metabolic and mitochondrial disorders. Standard array CGH procedures were used to test patient DNA samples for regions of copy number change. Sequencing of regions of predicted breakpoints in genomic DNA and PCR analysis were used to confirm oligonucleotide array CGH data. RESULTS: oligonucleotide array CGH identified intragenic exonic deletions in 2 cases: a heterozygous single-exon deletion of 4.5 kb in the SLC25A13 gene [solute carrier family 25, member 13 (citrin)] in an individual with citrin deficiency and a homozygous 10.5-kb deletion of exons 13-17 in the ABCB11 gene [PFIC2, ATP-binding cassette, sub-family B (MDR/TAP), member 11] in a patient with progressive familial intrahepatic cholestasis. In 2 females with OTC deficiency, we also found 2 large heterozygous deletions of approximately 7.4 Mb and 9 Mb on the short arm of the X chromosome extending from sequences telomeric to the DMD gene [dystrophin (muscular dystrophy, Duchenne and Becker types)] to sequences within or centromeric to the OTC gene (ornithine carbamoyltransferase). CONCLUSIONS: these examples illustrate the successful use of custom oligonucleotide arrays to detect either whole-gene deletions or intragenic exonic deletions. This technology may be particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing.