Genomic deletion size at the epsilon-sarcoglycan locus determines the clinical phenotype
1Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Center of Neurology, University of Tuebingen, Tuebingen, Germany, 2Section of Neurogenetics, Department of Medical Genetics, Panum Institute, University of Copenhagen, Copenhagen, 3Department of Neurology, Aarhus University Hospital, Aarhus, Denmark, 4Institute of Human Genetics, GSF National Research Center for Environment and Health, Munich-Neuherberg, 5Institute of Human Genetics, Technical University, Munich, Germany and 6Department of Paediatrics, University Hospital of Innsbruck, Innsbruck, Austria
Correspondence to: Friedrich Asmus, MD, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Center of Neurology, University of Tuebingen, Otfried-Muller Str. 27, 4th floor, Tuebingen, Germany E-mail: friedrich.asmus{at}uni-tuebingen.de
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Summary |
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Myoclonus–dystonia (M–D, DYT11) is a dystonia plus syndrome characterized by brief myoclonic jerks predominantly of neck and upper limbs in combination with focal or segmental dystonia. It is caused by heterozygous mutations of the epsilon-sarcoglycan (SGCE) gene on chromosome 7q21.3. We present three patients with heterozygous large deletions in the 7q21.13-21.3 region. By quantitative analysis of single nucleotide polymorphism (SNP) oligonucleotide arrays, the deletion size was determined to range from 1.63 to 8.78 Mb. All deletions contained the maternally imprinted SGCE gene and up to 43 additional neighbouring genes. Two of the patients presented with typical M–D, whereas one paediatric patient with split-hand/split-foot malformation and sensorineural hearing loss (SHFM1D, OMIM 220600 [OMIM] ) had not developed M–D at the age of 9 years. This patient had the largest deletion of 8.78 Mb (7q21.13-21.3) containing also SHFM1, DLX6 and DLX5, which had been previously shown to be deleted in SHFM1D. In two patients, the deletions removed the paternal allele of the KRIT1 gene, which is a major cause of cavernous cerebral malformations type 1 (CCM1). Only the adult patient showed asymptomatic cavernous cerebral malformations on cranial MRI, underlining age-dependent penetrance and haploinsufficiency as pivotal features of patients with KRIT1 mutations. All three deletions contained the COL1A2 gene. In contrast to dominant negative point mutations, which cause osteogenesis imperfecta with bone fractures, haploinsufficiency of COL1A2 resulted only in subtle symptoms like recurrent joint subluxation or hypodontia. Assessing copy number variations by SNP arrays is an easy and reliable technique to delineate the size of human interstitial deletions. It will therefore become a standard technique to study patients, in whom heterozygous whole gene deletions are detected and information on neighbouring deleted genes is required for comprehensive genetic counselling and clinical management.
Key Words: 7q21 deletion; myoclonus–dystonia; SCGE; cavernous cerebral malformations; KRIT1
Abbreviations: SNP, single nucleotide polymorphism; ICP, infantile cerebral palsy; CCM, cerebral cavernous malformation
Received April 20, 2007. Revised August 8, 2007. Accepted August 9, 2007.
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Introduction |
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Myoclonus–dystonia (M–D, DYT11, OMIM 159900 [OMIM] ) is a dystonia plus syndrome. In the majority of familial cases, the disorder is caused by a heterozygous mutation in the gene for epsilon-sarcoglycan. Since the cloning of SGCE as causative gene in M–D (Zimprich et al., 2001
), over 20 reports on SGCE point mutations and small deletions have been published [for an overview see (Tezenas du Montcel et al., 2006)]. Maternal imprinting of SGCE has been found to explain a highly reduced penetrance after maternal transmission of heterozygous SGCE mutations (Muller et al., 2002; Grabowski et al., 2003). Whole exon deletions of SGCE were described in two families with typical M–D presenting with action myoclonus of the upper limbs and cervical dystonia as well as writer's cramp (Asmus et al., 2005). Like truncating point mutations, these SGCE exon deletions cause a shift of the translational reading frame and introduce a premature termination codon.
Recently, a case of syndromic M–D in a patient with a paternal deletion of chromosome 7q21 was reported (DeBerardinis et al., 2003). Beside myoclonic jerks, the clinical presentation in this 32-month-old boy included microcephaly, short stature, a dysmorphic facies and a language delay. However, in this report only a deletion interval from 9 to 15 Mb was determined by fluorescence in situ hybridization (FISH) and genotyping of microsatellite markers. The role of neighbouring genes of SGCE in causing additional symptoms remained undetermined.
In the present study, we report on the clinical findings in three additional patients with 7q21 deletions. By delineating the extent of the deletions using quantitative SNP oligonucleotide arrays (SNP arrays) and quantitative PCR assays, specific genotype–phenotype correlations could be detected.
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Clinical assessment
Patients were included in this study, if a deletion of all coding exons of the SGCE gene was detected. Two patients showed clinical features of myoclonus–dystonia according to the published criteria (Asmus and Gasser, 2004
). A previously published paediatric patient with a paternal de novo deletion at the 7q21 band including the SGCE gene was also included (Haberlandt et al., 2001). All patients or their parents agreed to participate in the clinical and genetic part of this study and gave their informed consent. The study had been approved by the local ethics committees. Clinical information was systematically collected including family history, age and distribution of neurological, orthopaedic and dermatological symptoms at onset and at examination, as well as cranial MRI, skull X-rays, electrophysiological findings, response to alcohol and other medications. All patients were assessed by investigators trained in movement disorders and/or paediatrics (F.A., L.H., E.D. and E.H.)
Mutation analysis
Mutational analysis of the SGCE gene
Genomic DNA of all patients was isolated from peripheral blood leukocytes using a modified salting out procedure (Miller et al., 1988). DNA concentrations were measured with the NanoDrop spectrophotometer (ND-1000 V.3.1.2). SGCE point mutations in all patients had been excluded using mutational screening methods previously published (Hjermind et al., 2003).
A quantitative duplex PCR (qPCR) assay for all 11 coding exons and the splice variant exon 11b of the SGCE gene was performed on the LightCycler instrument (Roche Diagnostics) using hybridization probes as described earlier (Asmus et al., 2005). Target and reference amplicons were simultaneously quantified with different labelling of the 5'-hybridization probes for SGCE with LightCycler Red 640 and for beta globin as the internal reference with LightCycler Red 705. The ratios of SGCE/beta globin were calculated based on duplicate quantification results similar to the LC quantification method published for the parkin gene (Hedrich et al., 2001). SGCE/beta-globin ratios between 0.8 and 1.2 were considered as normal and ratios between 0.4 and 0.6 were expected for heterozygous deletions. To assess the parental origin of the deletions we genotyped microsatellite markers D7S2212, D7S820, D7S2410, D7S652 and D7S1513 around the SGCE locus using primers and conditions published in the GDB Human Genome Database (http://gdb.org).
Copy number variation analysis by SNP arrays
Sample preparation and hybridization
The Affymetrix Gene Mapping 100K SNP array set was used for all hybridizations. Each set contained an Xba240 and a Hind240 array covering a total of 116’204 SNPs distributed over the whole human genome with an average spacing of 23 kb. SNPs were genotyped from PCR amplicons of 200–2500 bp in length using either XbaI or HindIII adaptor-ligated genomic DNA. Using a total of 250 ng genomic DNA digestion with XbaI and HindIII, adaptor ligation, purification, fragmentation by DNaseI digestion and biotin labelling were carried out according to the manufacturer's instructions (Affymetrix GeneChip® Mapping 100K manual Rev3, Affymetrix Inc., USA).
Hybridization was performed in the Affymetrix GeneChip Hybridization Oven 640. Arrays were washed and stained in the Affymetrix GeneChip Fluidics Station 450 and scanned with the Affymetrix GeneChip Scanner 3000 7G. Image processing was performed with GCOS 1.4 and genotypes were called with GTYPE 4.0 software using the default call threshold of 0.25.
Copy number analysis
The data analysis of SNP arrays for copy number variations (CNV) has been described in greater detail elsewhere (Wagenstaller et al., in press). Briefly, we used genotype-specific dosage values, which helped to reduce the bias produced by different hybridization characteristics of each allele. Therefore, the raw intensity values at each SNP locus were calculated as mean of the perfect-match probes for both alleles, A and B. The raw intensity values were then median normalized. The log2 ratio of the intensity values were separately calculated for the three genotypes (AA, AB and BB) and the no-calls by dividing the normalized intensity values of the test array by the median values of all arrays for each SNP locus. Mean dosage levels also depend on probe and fragment characteristics like relative GC content and fragment length and were therefore corrected using quadratic regression. We implemented tools to select regions conspicuous for gains and losses of copy number. To select CNVs, we took into account the array-specific SD of the log2 intensity ratios. CNVs were defined on the basis of a minimum of five consecutive SNPs. Further details on the analysis software are available upon request.
Log2 ratios for wild-type SNPs ranged from log2 (0.75) to log2 (1.25). Ratios below these values were regarded as indicative of heterozygous deletions. Deletion sizes were calculated using the physical location of SNPs taken from the dbSNP build 125 (UCSC human genome browser build May 2004). Minimal deletion sizes were determined as physical distance between the outermost SNPs with log2 ratios below 0.75. Maximum deletion sizes were calculated between the SNPs at the deletion border with wild-type log2 ratios. All analysis tools were implemented as R or Perl scripts.
Confirmation qPCR assays of deleted genes
For heterozygous deletions of genes implicated in autosomal dominant human diseases results of CNV analysis were confirmed by additional qPCR experiments. Assessment of gene dosage was performed as described for SGCE with qPCR experiments using hybridization probes (FRET technology) on the LightCycler instrument (for primers and probes of all assays see supplementary material). For the assessment of the telomeric breakpoints in patients 1 and 2, target amplicons for qPCR assays were chosen with a length of 90–130 bp. An assay in exon 1 of the beta-globin gene was used as reference for the quantification with the SYBR green dye on the LightCycler 1.0 instrument. Amplification conditions were chosen as described elsewhere (Tizzano et al., 2005). Ratios of target gene/beta globin were expected to range from 0.4 to 0.6 for heterozygous deletions. All experiments were performed in duplicate and results are given as mean.
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Phenotypes of index patients with heterozygous SGCE deletions
Clinical history and examination of the three index patients from Denmark and Austria revealed the following phenotypes, summarized in Table 1:
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Patient 1 started to have myoclonic jerks of the neck and upper limbs at the age of 18 months without any gross delay of motor milestones. Over the years she had not experienced any progression of her movement disorder. When she was examined at the age of 47 years, she presented with ‘lightning-like’ myoclonic jerks of the neck and arms, which were rarely detectable at rest but which were markedly aggravated by intentional movements like drawing or writing. In addition, torticollis to the right and writers’ cramp of her right hand could be detected. Myoclonus and dystonia improved by about 90% after alcohol intake. She had severe overweight with a BMI of about 35 and was, in contrast to the rest of the family, short of stature. In childhood, she had marked problems with caries. She reported recurrent subluxation of her ankle joints every 2–3 months. Cranial MRI scans at the age of 49 years were unremarkable. Patient 1 refused contacting further pedigree members for a systematic pedigree assessment and genetical testing. According to her report, her first-degree relatives did not show any of her clinical features.
In patient 2, slight involuntary head torsion was observed around the age of 13 months. After an otherwise unremarkable motor development, she developed generalized myoclonic jerks with predominance in the neck and upper limbs around 5 years of age. Her movement disorder almost completely resolved after alcohol ingestion. She had no history of seizures or psychiatric comorbidity. On examination she had blue sclerae and slight ligamentous laxity of her limb joints.
Neither her mother nor her brother had any movement disorders symptoms or orthopaedic problems at examination. Her father died at the age of 82 years of lung cancer. Choreoathetoid movements could only be observed in a cousin of patient 2, who suffered from infantile cerebral palsy (ICP) after complicated delivery. There was no history of acute neurological deficits including cerebral haemorrhage and seizures in any family member.
An MRI scan performed at the age of 59 years showed a total of five hypointense lesions on T2- and gradient echo-weighed images (Fig. 1). These signal alterations were classified as cerebral cavernous malformation (CCM) type IV according to neuroradiological criteria (Zabramski et al., 1994; Labauge et al., 2001).
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Patient 3 has been described in greater detail earlier (Haberlandt et al., 2001
). Based on an ectrodactyly of the right foot and sensorineural hearing impairment with a Mondini dysplasia he was diagnosed with split-hand/split-foot malformation 1D (SHFM1D, OMIM 605617
[OMIM]
). His clinical features included complex facial dysmorphism with microcephaly, a submucous cleft palate, carious primary teeth, hypodontia, sparse, light hair and pale skin. In addition mental and psychomotor developmental delay was noted. At the age of 9 years, neurological examination could not detect any movement disorder. He had been treated with a unilateral cochlear implant and could speak three-word sentences. Cranial MRI at the age of 18 months showed no vascular abnormalities. The examination of the parents and of his four siblings detected no abnormalities.
Results of SGCE qPCR and genotyping
Genomic DNA of all patients and of accessible healthy family members was assessed for deletions of exon 1–11 and the splice variant exon 11b of the SGCE gene. All three index patients showed reductions of the SGCE/beta-globin ratios ranging between 0.42 and 0.60 indicating heterozygous deletions of all coding exons of SGCE (Fig. 2). All other individuals in pedigree 2 showed wild-type ratios (range 0.8–1.2) only, excluding SGCE deletions (Fig. 2). In patient 3, a paternal origin of the de novo deletion had already been shown by genotyping of microsatellite markers in the 7q21 region (Haberlandt et al., 2001). In pedigree 2, DNA was only available of the index patients’ mother, of her healthy brother and of a cousin with ICP. Haplotype reconstruction and loss of heterozygosity for microsatellite markers D7S2410, D7S652 and D7S1513 around the SGCE locus was indicative of a paternal origin of the deleted allele in patient 2. No DNA was available to test for the parental origin of the deletion in patient 1.
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Results of genomic CNV assessment using SNP genotyping arrays
Since the heterozygous deletions in all patients covered all SGCE exons, we aimed at delineating the extent of the genomic defect by using SNP arrays. For each patient genomic DNA was hybridized to SNP arrays in duplicate. Log2 intensity ratios of the target region 7q21-23 were calculated by comparing the intensity values of the test arrays with those of reference arrays (Fig. 3A).
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Log2 signal intensity ratios for patient 1 indicated a heterozygous deletion with a maximum interval of
1.63 Mb from SNP rs7783452 to SNP rs17166512 including 10 genes (for the physical location of the deletions relative to annotated genes see Table 2). In patient 2, the deletion extended from rs7781521 to rs854726 encompassing 30 genes over an interval of 4.99 Mb. The largest heterozygous deletion was detected in patient 3, where the wild-type log2 ratios for rs801856 and rs951987 defined a deletion interval of 8.78 Mb covering 44 genes.
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Confirmation assays with qPCR of annotated genes
The deletions assessed by 100K array sets were validated by qPCR assays. Out of a total of 44 genes in the region, we selected target genes according to their physical annotation, if they were located (1) either directly outside the deletion boundaries suggested by the CNV analysis (FLJ321100, ASNS, PEX1), (2) if heterozygous mutations of the gene had been described in autosomal-dominant human disease (KRIT1, COL1A2, DLX5) or (3) if the loss of the paternal allele had been reported to cause early embryonic lethality in a mouse model (PEG10). In addition we narrowed down the genomic position of the telomeric deletion breakpoints in patients 1 and 2 since both appeared to lie within a maximum interval of 404 kb from SNP rs10499905 to rs854726.
In all index patients, FLJ32110 and ASNS were clearly outside the regions with heterozygous deletions (Fig. 3B). Beside the deletion of the SGCE gene, all patients displayed a heterozygous loss of COL1A2 and PEG10 including the PEG10 promoter region. The deletions in patients 2 and 3 encompassed the PEX1 and KRIT1 genes. DLX5, the most telomeric gene of the gene cluster previously involved in SHFM1D, was only deleted in patient 3.
Assessment of telomeric deletion breakpoints in the distal 7q21.3 region
In patients 1 and 2, 10 additional qPCR assays were designed to cover the region between PEG 10 and rs854726 at intervals of 16–52 kb (see Supplementary Material). Primers were chosen to reside outside repetitive elements to avoid false negative quantification results. In patient 1, the deletion breakpoint was located in a 15.2 kb interval between the qPCR assays at position 94.109 and 94.125 Mb. In the SNP array analysis the outer boundary for this patient was indicated at SNP rs17166512 (position 94.133999 Mb). Likewise, the deletion breakpoint in patient 2 mapped to an interval of 35.7 kb between the qPCR assay at position 94.236 Mb and the outermost SNP rs854726 showing wild type log2 ratios at 94.271893 Mb. Therefore, the minimal distance of the telomeric breakpoints in patients 1 and 2 is about 111 kb.
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Discussion |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialWeb ResourcesReferences |
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We have identified two novel heterozygous genomic deletions in the 7q21.13-21.3 region and refined the size of a previously reported deletion in the same region.
The deletion size ranged from 1.63 to 8.78 Mb and contained between 10 and 44 annotated genes. Our results allowed us to perform a detailed genotype–phenotype analysis. We could identify the following specific phenotypic constellations:
Myoclonus–dystonia and loss of the paternal SGCE allele
Paternally inherited heterozygous deletions of the SGCE gene caused typical Myoclonus–dystonia in patients with a 1.63 Mb and a 4.99 Mb deletion. Both patients had a symptom onset in infancy. The clinical presentation was indistinguishable from patients with SGCE point mutations in respect of age at onset, symptom distribution with predominance in the neck and upper limbs, appearance of action myoclonus as well as alcohol responsiveness. The uniformity of the M–D phenotypes irrespective of whether the patients carry a heterozygous SGCE point mutation or a deletion of the total gene, confirms loss of function as the common mutational mechanism in DYT11. Even for SGCE missense mutations, a loss of epsilon-sarcoglycan function has been suggested by the detection of disturbed endoplasmic reticulum trafficking and proteasome degradation of missense mutated epsilon-sarcoglycan protein in cell culture experiments (Esapa et al., 2007).
Variations in symptom distribution and severity as well as the appearance of psychiatric comorbidity in M–D patients might be attributed to differences in genetic background, epigenetic variations or environmental factors. Myoclonus could only be detected in both adult patients. Because M–D has been shown to have an almost complete penetrance after paternal transmission by the age of 25 years, patient 3 is still at risk of developing M–D in the future (Asmus et al., 2002; Tezenas du Montcel et al., 2006).
In patient 3 and in two previously reported patients (Marinoni et al., 1995; Courtens et al., 2005), de novo deletions of the 7q21 region originated from the paternal allele. The preponderance of a paternal origin has also been shown for large genomic deletions of the APC gene in familial adenomatous polyposis, for partial 18q deletions and for chromosome 5p deletions in Cri-du-chat syndrome (Crow, 2000; Aretz et al., 2004). A paternal disposition for chromosomal rearrangements might be explained by an increased sensitivity of meiotic and post-meiotic stages of spermatogenesis to the induction of large genomic deletions and translocations (Aretz et al., 2004).
KRIT1 and CCM1
In patient 2, the 4.99 Mb deletion also contained the KRIT1 gene, which has been extensively screened in several studies for its association with CCM (Laurans et al., 2003). On cranial MRI, CCM could be diagnosed in patient 2 based on the detection of type IV CCM lesions most pronounced on gradient echo sequences. This finding supports haploinsufficiency of KRIT1 to be the major mutational mechanism in CCM1 (Labauge et al., 2007).
Patient 3 showed no symptoms of CCM at the age of 9 years and had an unremarkable cranial MRI at the age of 18 months. First and most important, this patient has not reached the average age at onset of 30 years described for patients with symptomatic KRIT1 point mutations (Denier et al., 2004). Likewise the CCM load has been reported to be age dependent (Brunereau et al., 2000; Labauge et al., 2001) and penetrance has been shown to be incomplete in CCM1 pedigrees. An adult onset in CCM patients with heterozygous genomic KRIT1 mutations is in agreement with the hypothesis of a need for a second somatic KRIT1 mutation (‘second hit’) to develop CCMs (Labauge et al., 2007).
The detection of KRIT1 mutations in patients with whole SGCE deletions has therefore pivotal consequences on genetic counselling and the need for a follow-up of neuroimaging with gradient echo cranial MRI.
Functional relevance of haploinsufficiency of PEG10 and COL1A2
Mice with a paternal knock-out of the maternally imprinted peg10 gene, the telomeric neighbouring gene of SGCE, show early embryonic lethality by day 10.5 post-coitus due to placenta dysfunction (Ono et al., 2006). Similarly, mice with a maternal uniparental disomy of the same chromosomal region containing peg10 also die at an early embryonic stage (Beechey et al., 2003). In contrast to these mouse models, paternal PEG10 haploinsufficiency is apparently not lethal in humans, since the deleted intervals in all three patients included PEG10, pointing to a different organization of early embryonic placental growth.
All our patients carried a heterozygous deletion COL1A2, the gene encoding the alpha-2 chain of type I collagen located just 154 kb centromeric to SGCE. Clinical findings with blue sclerae in patient 2, hypodontia in patient 3, recurrent subluxation of the ankle joints in patient 1, slight laxity of joints in patients 2 and 3 and short stature in all patients were only subtle. In patient 1, the recurrent subluxation of her ankles might have also been caused by her severe overweight. Without a history of bone fractures after insufficient trauma and with unremarkable skull and spine X-rays, clinical features in all the three patients are compatible with a very mild form of Osteogenesis imperfecta type I due to COL1A2 haploinsufficiency. In contrast to COL1A2 missense point mutations, which in most cases affect a glycine residue in the triple helical domain of the two alpha chains and exert a dominant negative effect on collagen multimerization, haploinsufficiency for COL1A2 can be almost completely compensated.
Split-hand/split-foot malformation with hearing loss
Only in patient 3 with inborn SHFM and sensorineural hearing loss, the paternal 7q21.13-21.3 deletion included the gene cluster SHFM1, DLX6 and DLX5. These genes have been shown to be involved in limb development in mouse models and in human ectrodactyly (Wieland et al., 2004). Only DLX5/6–/– double knockout mice showed a SHFM phenotype in combination with disturbed inner ear development (Robledo et al., 2002; Robledo and Lufkin, 2006). In humans, no point mutations in these genes could be identified (Tackels-Horne et al., 2001). This implicates that the expression pattern of SHFM1, DLX6 and DLX5 is complex, associated and partially redundant. Only haploinsufficiency of all three genes together like in patient 3 is apparently sufficient to cause SHFM1D.
Another gene, pendrin (SLC26A4), which causes a form of dominant and monogenetic Mondini dysplasia, is located over 10 Mb telomeric to the distal deletion breakpoint in patient 3. A dysregulation of pendrin expression by the 7q21.13-21.3 deletion is unlikely because of the large genomic distance.
De Berardinis published a patient (patient D) with a paternal deletion at 7q21.3 including the SGCE gene (DeBerardinis et al., 2003). Patient D showed clear similarities to patient 3, including short stature, facial dysmorphism and delay of cognitive development (Table 1). Recalculating the minimum deletion size in patient D (BAC clones RP11-575G1 to RP11-648L18) resulted in an interval of 12.5 Mb. Out of the 19 additional telomeric genes included in the deletion of patient D (see Supplemenatry Material), Semaphorin 3 E (SEMA3E), when mutated, has been associated with CHARGE syndrome, a complex developmental disorder causing cranial nerve abnormalities (Lalani et al., 2004). Patient D suffered from left-sided facial palsy but showed no other signs indicative of CHARGE syndrome (DeBerardinis et al., 2003).
The telomeric deletion breakpoint in patient D is located between position 94.001 and 95.559 Mb. The breakpoint regions of patients 1 and 2 are located within the same interval. Both reside in an almost contiguous sequence of long and short interspersed nuclear elements (LINE and SINE) of 238 kb length telomeric to PEG 10, but are clearly separated by a minimum interval of 111 kb. Possibly, the breakpoint of patient D is also located within this repetitive sequence.
The role of SNP oligonucleotide genotyping arrays in CNV analysis
The genotype–phenotype correlations shown in this study are based on the delineation of the deletion size by SNP genotyping arrays. To date, all the previous studies on 7q21 deletions used standard techniques like chromosome banding, FISH or microsatellite genotyping. Recently, array comparative genomic hybridization (array CGH) (Engels et al., 2007) and high-density oligonucleotide arrays (SNP arrays) (Redon et al., 2006) have been evaluated for the genome-wide analysis of copy number variations (CNV). Two groups have successfully applied the Affymetrix 100K SNP array set used in the present study for CNV analysis (Hoyer et al., 2007) (Wagenstaller et al., in press).
CNV detection by SNP oligonucleotide arrays provides a much better resolution than G-banded chromosome analyses, but has to cope with noisy log2 intensity ratios by integrating the intensity ratios of adjacent SNPs. Although analyses tools aim to reach a high sensitivity without loosing specificity, the results may contain interspersed SNPs with false negative or false positive log2 intensity ratios. Also the breakpoint determination is not always exact. These limitations of the current SNP array methodology make qPCR assays still indispensable to verify and narrow down the deletion breakpoints in specific cases.
In conclusion, the ease of use of SNP arrays for the assessment of copy number variations, its industrial production and the potential for further increase of resolution may finally outperform the currently favored array CGH technique and will make it a standard tool in the mutational analysis of rare phenotypes.
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Supplementary material |
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Supplementary material is available at Brain online.
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialWeb ResourcesReferences |
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Genomic locations were determined using the ‘May 2004’ Build of the UCSC Genome Browser, Genome Bioinformatics Group, UC Santa Cruz (http://genome.ucsc.edu/cgi-bin/hgGateway) and from the GDB Human Genome Database (http://gdb.org). Disease information was obtained from the Pubmed library, National Library of Medicine and National Institutes of Health, (http://www.pubmed.gov) and from disease reviews on http://www.genetests.org.
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Acknowledgements |
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We thank our patients and their relatives for participating in this study. This work was supported by educational grants of the University of Tuebingen, FORTUENE 1364-0-0, and by the German Network for Hereditary Movement Disorders, GeNeMove (01GM0304), funded by the German Ministry for Education and Research (GMER).
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