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Human Molecular Genetics Advance Access originally published online on November 21, 2005
Human Molecular Genetics 2005 14(24):3911-3920; doi:10.1093/hmg/ddi415
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Deletion of the ANKRD15 gene at 9p24.3 causes parent-of-origin-dependent inheritance of familial cerebral palsy

Israela Lerer, Michal Sagi, Vardiella Meiner, Tirza Cohen, Joel Zlotogora and Dvorah Abeliovich*

Department of Human Genetics, Hadassah Hebrew University Hospital and Medical School, Jerusalem, Israel

* To whom correspondence should be addressed at: Department of Human Genetics, Hadassah Hebrew University Hospital, Ein Kerem, Jerusalem 91120, Israel. Tel: +972 26776016; Fax: +972 26777499; Email: dvoraha{at}cc.huji.ac.il

Received August 3, 2005; Accepted November 1, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A four-generation family was studied in which nine children had congenital cerebral palsy (CP), characterized by quadriplegia and mental retardation. All the affected children were born to healthy, related fathers, whereas the children of their healthy female relatives were unaffected. Linkage analysis attributed the condition to chromosome 9p24.3, where a 225 kb deletion was identified. The deletion spans a single gene, ANKRD15 (ankyrin repeat domain 15), which is ubiquitously expressed. In the affected children, the ANKRD15 is not expressed in lymphoblastoid cells, whereas in their healthy fathers, who harbor the same deletion, the expression of ANKRD15 did not deviate from controls. This expression pattern can be interpreted as a maternal imprinted gene that is expressed only from the paternal allele. The expression of ANKRD15 in lymphoblastoid cells from the control group was monoallelic but not imprinted. The monoallelic expression was restricted to the ANKRD15 gene, whereas biallelic expression was found in the DOCK8 gene, which resides at the telomeric side of the deletion. No correlation was found between the expression of the ANKRD15 gene and the pattern of DNA methylation in the CpG islands 5' of the gene. However, differences in methylation pattern were found in the CpG islands flanking the DMRT1 gene, which is located at the 3' side of the ANKRD15 gene. In the affected individuals, as in the control group, the CpG islands were hypo-methylated, whereas in the healthy fathers, the CpG islands were hyper-methylated in cis with the deletion. This unique family demonstrates a phenomenon of a deletion that creates imprinting-like inheritance. The implication of this family to sporadic CP is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cerebral palsy (CP) is a group of non-progressive chronic disorders impairing control of movement and posture that results from an insult to the developing central nervous system (1Go,2Go). The characteristic signs of CP are spasticity, movement disorders, muscle weakness, ataxia and rigidity. The clinical classification of CP is based on the nature of the motor deficit as well as other symptoms accompanying it, such as mental retardation, optic atrophy, hearing loss and speech impediments. CP has been linked to unusual stresses that infants may undergo at birth and the susceptibility of an immature central nervous system to injury from a variety of agents such as cerebral anoxia, traumatic injury to the brain, fetal infection, hyperbilirubinemia, hypoglycemia, hypothyroidism as well as an inborn error in amino acid metabolism (3Go,2Go).

CP is the most common cause of severe physical disability in childhood, occurring in 0.15–0.25% of live births (1Go,4Go). The vast majority of CP patients are sporadic and only 2% are considered to be inherited. Recently, a new analysis of a Swedish CP children database (Goeteborg data) indicated that genetic causes might be the etiology in 40% of the children (5Go). Although familial aggregation of CP is very unusual, it may be of utmost importance in understanding the pathogenesis of idiopathic CP, which has no obvious explanation.

Here, we present a family in which nine children were born with typical CP to related fathers and the mechanism underlying its inheritance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Description of BS family
An Israeli family (BS) of Jewish Moroccan origin was referred to genetic counseling because several children were affected with congenital neurodegenerative disease resembling CP (Fig. 1). The affected children were born to unrelated healthy parents following normal pregnancies and were delivered with no reported complications. Congenital hypotonia appeared at first, and over the first year, it evolved to spastic quadriplegia with accompanying transient nystagmus. All affected individuals had various degrees of mental retardation and most of them were institutionalized in special institutions for severely retarded children. Neuroimaging studies showed brain atrophy and ventriculomegaly. Extensive biochemical laboratory studies did not reveal any possible explanation for the severe phenotype. High-resolution chromosome analysis indicated normal karyotype; subtelomeric FISH analysis was normal.



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  		Figure 1. Pedigree of BS family and the segregation analysis. For each polymorphism, the numbers 1–10 represent the different alleles and (–) indicates hemizygosity of D9S1858. The arrow points to the recombination between D9S288 and D9S1858. Black squares or circles are affected, dark gray ones are carriers, individuals I-1, II-12, III-22 and III-23 are carriers that were found through the linkage study.

 
Nine of the 20 paternal offspring were affected, whereas all 12 maternal offspring were healthy. All the affected children were born to related clinically healthy fathers (II-2, II-10, III-9, III-10; Fig. 1), indicating that the inheritance is dependent on the parental gender ({chi}2=7.51, P<0.01). A possible explanation for this mode of inheritance is a maternal imprinted gene expressed only from the paternal allele.

Linkage study pointed to deletion at 9p24.3
Linkage analysis was performed assuming that the mode of inheritance in the family is autosomal dominant with incomplete penetrance. On the basis of the affected individuals and the obligatory carriers, a candidate region in the distal end of the short arm of chromosome 9p24 was identified, with maximum LOD score values of 3.53 and 3.45 for D9S1779 and D9S917, respectively. All the affected individuals and carriers share the same haplotype, which was inherited from the mother I-1 who transmitted it to four of her healthy children (II-2, II-5, II-10 and II-12). The affected individuals and obligate carriers were hemizygous for the marker D9S1858, indicating a deletion at this locus. Recombination between D9S1858 and D9S288 in individual III-3 narrowed the candidate region to <4 Mb.

On the basis of the linkage analysis and the deletion presence, prenatal diagnosis was offered for at-risk pregnancies. Eight prenatal diagnoses were performed; in four of them, the pregnancies continued and healthy children were born and in the remainder, the pregnancies were terminated.

Characterization of the deletion
Segregation analysis was performed using polymorphic dinucleotide repeats in proximity to D9S1858, retrieved from the sequence of the contig NT_008413 [GenBank] . The polymorphic repeats 587410(ca) on the telomeric side and 821991(gt) on the centromeric side were outside the deletion and provided an estimation of the deletion size from 100 to 240 kb (Fig. 2A). Serial Southern hybridizations detected differences in the intensity of the relevant restriction fragments when compared with control fragments (Fig. 2B) and allowed localization of the breakpoints within 5–10 kb from each side. Primers designed from the boundaries of the deletion generated in a long-range PCR a 7 kb fragment from family members with the deletion (data not shown), thus enabled us to identify the breakpoints at two Alu elements: AluSq (located in position 817 061–817 211) and AluSx (located in position 590 601–590 750), which share 90% of sequence homology, resulting in a deletion of 225 kb. PCR primers were generated to amplify the junction fragment and it was found only in BS family members who have the deletion (Fig. 2C): it was not found among DNA samples of 210 anonymous control individuals, including 130 Jewish individuals of a similar ethnic background.



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  		Figure 2. Characterization of the deletion. (A) Physical map of the deletion region. The deletion encompasses five polymorphic markers: 636066gt, D9S1858, 708946gt, 728581ga and 757874ca (the numbers represent the position of the repeats in the contig NT_008143), the markers 587410ca and 821991gt are not included in the deletion. The probes tel5 and cen3 were used for Southern hybridization. Arrows above Jf-f and Jf-r represent the location of the primers used for the amplification of the junction fragment. (B) Southern hybridization—genomic DNA digested with HindIII (H) or with PvuII (P) was hybridized with the telomeric probe (T-tel5) (Fig. 2B-1) and the centromeric probe (C-cen3) (Fig. 2B-2). GJB6 probe (G) serves as a dosage control (29Go). The intensity of the bands was equal in spouses (II 6, III 8), whereas in family members with deletion, the intensity of the G-band was stronger than the telomeric or centromeric bands, which demonstrate the deletion (IV 3, II 5). (C) The junction PCR fragment (878 bp) obtained from DNA of the affected children (IV 3, IV 6) and their fathers (III 9, III 10), but not from their mothers (III 8, III 11); the control PCR fragment was generated in all the samples. The underlined sequences point to the breakpoints in the Alu elements.

 
The only gene within the deletion is ANKRD15 (ankyrin repeat domain 15). The DOCK8 gene (dedicator of cytokinesis 8) is located telomeric to the ANKRD15 gene and the DMRT1 gene (doublesex and mab-3 related transcription factor 1) lies on its centromeric side. The 5' upstream sequences of the DMRT1 gene might be included in the deletion (Fig. 3A).



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  		Figure 3. Expression studies. (A) Physical map and the order of the known genes in the deletion region. Schematic presentation of the ANKRD15 gene structure; arrows indicating primers (II, III and V) location for generating the transcript variants A and B. (B) Northern hybridization—RNA from fetal tissues probed with exon 3 of the ANKRD15; a band of 6.2 kb is present in all tissues. (C) RT–PCR of variants A and B of the ANKRD15 gene in LCLs derived from fathers (III-9, III-10 and II-10) and the affected children (IV-3, IV-6, III-20); C1 and C2 are normal controls. The 3' end of the NF1 gene served as positive control for the cDNA synthesis. V1573 and V1574 are chorionic villi cells from fetuses with paternal deletion. V1 and V2 are matched controls.

 
Expression study of the ANKRD15 gene
Spanning 275 kb, the ANKRD15 gene is ubiquitously expressed in various tissues (GeneCards). It includes at least 12 exons (Fig. 3A) and two transcripts that code for the same protein (1194 amino acids) but use different promoters and are designated variant A, exons 2–12 (NM_153 186.2), and variant B, exons 1, 3–12 (NM_015 158.1) (6Go). Other alternative transcripts may exist according to the analysis of the identified cDNAs clones and the genomic DNA (AceView, NCBI).

High expression of ANKRD15 was found by northern hybridization of fetal and adult tissues probed with ANKRD15 exon 3. A major band of 6.2 kb is characteristic of the fetal tissues: brain, lung, liver and kidney (Fig. 3B). In the adult, high expression was prominent in the heart, skeletal muscle and kidney, whereas a very faint band was observed in the brain (data not shown).

The expression of the ANKRD15 gene in BS family members was analyzed in lymphoblastoid cell lines (LCLs) established from the affected individuals and their fathers by RT–PCR. In the affected children (IV-3, IV-6, III-20), there was no expression of variant A and very low expression of variant B, whereas in the fathers (III-9, III-10, II-10), the expression was similar to the normal controls (C1, C2), suggesting monoallelic expression of the paternal allele (Fig. 3C). In extra-embryonic tissues (chorionic villi) of fetuses with paternal deletion (V1573, V1574), the expression was the same as that of matched controls (V1 and V2) (Fig. 3C). These results indicate that the gene is not uniformly expressed.

The mode of expression of ANKDR15 in LCL established from control individuals was done utilizing SNPs in the coding region gene (1236 C/G-dbSNP: 912 175 and 1255 G/T-dbSNP: 912 174) by comparing the genomic DNA sequence to the cDNA sequence. Three heterozygous individuals (16-3, 123-4, 189-13) showed monoallelic expression in both transcripts (variants A and B): in two individuals, the expressed allele was paternal and in one it was maternal (Table 1). Individuals 189-13 and 123-4 were normal females, and the pattern of chromosome X inactivation in their LCLs was random, indicating that the LCLs were not monoclonal and that in the majority of the cells, the same allele is expressed. Monoallelic expression was also found for variant B only in human fibroblasts (in Fib SA, the maternal allele was expressed); however, biallelic expression was found in amniotic cells (Table 1). In all cases where monoallelic expression was observed, there was a preference for the expression of the 1236G-1255T haplotype. In individuals homozygous to the alternative haplotype (CG), there was normal expression, and in the individuals with maternal deletion, their paternal allele was normally expressed regardless of the haplotype, thus excluded the preferential expression of the GT haplotype as a consequence of PCR failure.


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  		Table 1. Expression of ANKRD1 5 in individuals without deletion
 
The expression of DOCK8 and other genes, such as BRCA1, BRCA2 and NF1, was biallelic in the control individuals as well as in all BS family members. The expression of DMRT1 is restricted in adults to the testis and, therefore, it could not be assessed.

Methylation pattern in the deletion segment
In order to examine whether the mode of ANKRD15 expression is attributed to differential methylation, four CpG islands within the deleted segment and in its flanking regions were chosen for methylation analysis (Fig. 4). Regions b (688 221–688 685) and c (692 515–694 200) were within the deletion, whereas regions a (485 219–486 988) and d (822 641–824 211) were outside. In the regions a, b and c, no differences were found in methylation pattern of affected and healthy individuals: regions a and b were hypo-methylated, whereas region c was hyper-methylated [analyzed by PCR amplification after HpaII digestion of genomic DNA (Fig. 4B) or sequencing after bisulfite modification (Fig. 4C)].



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  		Figure 4. Methylation status of CpGs islands. (A) Physical map of the CpG islands; regions a, b and c were within the ANKRD15 gene and region d is located in the promoter sequence of the DMRT1 gene. (B) Methylation status of regions a (634 bp), b (221 bp) and c (493 bp) according to PCR products after DNA digestion with HpaII (+) or without the enzyme (–). Regions a and b are hypo-methylated, whereas region c is hyper-methylated. No significant difference was noted between the affected children and the healthy fathers. (C) Methylation status of region c according to bisulfite modification and sequencing. Black and white circles denote methylated and non-methylated cytosines, respectively.

 
Region d, which encompasses CpG islands flanking the DMRT1 gene, was found to be hypo-methylated in normal control individuals. In this region, a different methylation pattern was found within BS family members: in the affected individuals, region d was hypo-methylated, whereas in the healthy deletion carriers, this region was hyper-methylated (Fig. 5). Using heterozygosity to the SNP (T/A dbSNP: 3 739 583), we were able to demonstrate in the carrier brothers (III-9 and III-10) that the methylation occurred on their maternal chromosome, the chromosome bearing the deletion (Fig. 5A). To confirm this result, we used the polymorphic gt-repeat at nucleotide 821 991 (Fig. 2A) that is located in the proximity of the CpG islands. Following HpaII digestion, a 847 bp fragment containing both the repeat and the CpG islands was generated only in carriers of the maternally derived deletion (Fig. 5B). This PCR fragment served as a template in nested PCR for the repeat polymorphism. In all individuals (III-9, III-10, II-5, II-2, II-10, II-12, III-22 and III-23), the 138 bp allele (maternal) was co-amplified with the hyper-methylated CpG, indicating that the methylation occurred on the maternal allele in cis with the deletion (Table 2). The same allele is hypo-methylated through paternal transmission (IV-3, IV-6, III-3, III-7, III-19 and III-20). These results confirmed that hyper-methylation occurred only through maternal transmission of the deletion.



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  		Figure 5. Methylation status in the CpG islands of DMRT1 gene. (A) The DMRT1 CpG island sequence (region d) after bisulfite modification. Black and white circles represent methylated and non-methylated cytosines, respectively. Hypo-methylation was found in individuals without deletion (16-3, 197) as in the affected (IV-3, IV-6 and III-19) and hyper-methylation in some clones was found in the fathers (III-9, III-10 and II-10). The polymorphism T/A (dbSNP, 3739583) was informative in III-9 and III-10 and shows that the hyper-methylation occurred on the maternal chromosome. (B) Methylation status of region d according to PCR performed after DNA digested with HpaII. PCR product (847 bp) was generated only in the healthy family members that have the deletion.

 

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  		Table 2. The parental origin of the hyper-methylated allele in the DMRT 1 gene
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we present a four-generation family with unique features: (a) the phenotype of the affected individuals is indistinguishable from CP, which is usually non-genetic; (b) the trait is inherited through carrier fathers, whereas the offspring of carrier mothers are healthy. At face value, the mode of inheritance implies an imprinted gene that is expressed from the paternal allele. Linkage analysis pointed at 9p24.3 as the gene location in which a 225 kb deletion spanning the ANKRD15 gene was identified. This deletion was not found among 210 individuals of a control group.

The ANKRD15 that contains ankyrin repeats in its C-terminus is evolutionary conserved; its orthologs are found among multi-cellular organisms. It was first obtained from human cDNA library and termed as KIAA0172 (7Go), and later it was characterized as a tumor suppressor gene (KANK) in renal cell carcinoma (6Go). ANKRD15 is ubiquitously expressed in various tissues during fetal development and adult life (including in LCL). In BS family, the ANKRD15 gene is expressed in healthy individuals who carry the deletion on the maternal allele and a normal paternal allele, whereas in the affected individuals, who are carriers of the paternal deletion and a normal maternal allele, the ANKRD15 gene is repressed.

Recently, VAB-19, the nematode ortholog of ANKRD15, was characterized, and in the presence of a mutated gene, an interesting phenotype of arrest in the 2-fold epidermal elongation period was created (8Go). Shortly after muscle contraction begins, muscle detaches from the epidermis, the mutant embryos twitch normally but never roll; these were essentially paralyzed after the 2-fold stage. In this study (8Go), it was shown that the VAB-19 gene product is essential for a defined time during embryo development and is important in attaching muscles to the epidermis via intermediate filament junctions. Sarkar et al. (6Go) have also shown that in the kidney tumor cell line (G-402), there is a difference in the ß-actin distribution, depending on whether the cells express the ANKRD15 (KANK) gene. This supports the view that the ANKRD15 protein is important for the cytoskeleton structure and may be involved in crucial adhesion complexes.

The ANKRD15 gene is expressed in the fetal brain; if the gene product is indeed essential in a specific threshold and in a precise time window during the development of the fetal brain, then its absence may explain the severe phenotype in the affected children.

The characterized deletion may be rare, but other factors can influence the level of ANKRD15 gene expression and lead to its association with sporadic CP. As there are several binding sites, GCGTG, for hypoxia-induced factor 1 at the upstream sequences of the ANKRD15 gene (position 451 268–451 648), the expression of the gene may be oxygen dependent. Hypoxia and low birth weight (fewer cells in the fetal brain during critical developmental processes) are associated with the etiology of CP (9Go,10Go) and may potentially affect the level of ANKRD15 expression. Thus, combinations of factors that may affect the level of ANKRD15 expression possibly explain the sporadic nature of CP.

The parent-of-origin-dependent inheritance in BS family raises the possibility that ANKRD15 is imprinted and expressed from the paternal allele; this possibility was also raised by Sarkar et al. (6Go). Normal control LCLs and fibroblasts showed monoallelic expression rather than the parent-of-origin effect-dependent expression. Monoallelic and/or variable expression has been documented for other genes and may even be common (11Go–13Go). The expression study identified two haplotypes based on the SNPs 1236G/C and 1255T/G; the haplotype 1236G-1255T was preferentially expressed in heterozygous cases, suggesting that the monoallelic expression is dependent on crucial cis elements.

An exception to the monoallelic expression was in embryonic fetal tissues: in normal amniotic cells, the expression was biallelic and in the chorionic villi of fetuses with paternal deletion, there was expression of the maternal allele. The mode of the ANKRD15 expression supports the view of a complex locus with more than one promoter and several different transcripts that may code for different proteins.

In order to better understand the control mechanism governing the monoallelic expression, we analyzed the methylation pattern of the CpG islands in the promoter region of the ANKRD15 gene. Surprisingly, there were no differences between the affected individuals, their fathers and the control group. However, differences in the methylation pattern were found in the promoter region of the DMRT1 gene. Hyper-methylation occurred only on the deleted chromosome when it was transmitted through the mother, but upon paternal transmission, this region was hypo-methylated, as in individuals without deletion. It is reasonable to assume that hypo-methylation of the promoter region of the DMRT1 gene is essential for normal spermatogenesis (14Go); however, the connection between the hypo-methylated DMRT1 promoter and the repression of ANKRD15 expression has not yet been resolved. An intriguing possibility lies in the existence of an antisense transcript, which, if it normally exists, can be connected to the monoallelic expression and to the tissue specificity expression pattern of the ANKRD15 gene (15Go). In the presence of the deletion, the hypo-methylation of the DMRT1 promoter enables creation of a longer antisense that overlaps the sequences of the ANKRD15 gene and represses its expression from the normal allele. Trans-interaction mechanisms between the two alleles (i.e. transvection and paramutation), although rare among mammals, may provide an alternative explanation for these results (16Go).

Although there is no direct evidence for imprinted genes of 9p (17Go,18Go), apparently the deletion itself creates an imprinting effect. This may exemplify a mechanism in which gene order alterations, resulting in novel juxtaposition of DNA sequences, create or abolish situations of imprinting (19Go–21Go). Terminal deletions of 9p are associated with a wide range of abnormal phenotypes characterized by mental retardation and craniofacial dysmorphic features (22Go), gonadal anomalies and sex reversal phenotype (23Go) or normal phenotype that was interpreted as a benign polymorphism (24Go,25Go). The size of the deletion, the location of the breakpoints, the mode of expression of the relevant genes and possibly the parental origin are factors that will influence the variable phenotypes of carriers with 9p deletions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patients and other family members signed an informed consent form, and the study was approved by the hospital Ethics Committee.

Cell cultures and DNA samples
LCLs were established from several affected individuals and their fathers by transformation of lymphocytes with EBV. Fibroblast cultures and LCLs from control individuals and amniocyte cultures were obtained from the Human Genetics Department at Hadassah Hospital. Control DNA samples were taken from the genetic screening samples, which were stored anonymously (except for ethnic origin). DNA was extracted using the salting-out procedure.

Linkage study
Polymorphic DNA marker sequences were retrieved from the Genome Data Base. PCR was done using 0.5 µM primers (MWG Biotech AG), with 0.5 units of Taq polymerase (Bioline), in the presence of 0.2 mM DNTPs (Bioline) and 1.5 mM MgCl2. The annealing temperature range was 55–58°C. PCR products were separated on denatured polyacrylamide gel (8%) and detected with silver staining (26Go). The LOD score values were calculated with LIPED (J. Ott version 1988).

Characterization of the deletion
The deletion length was estimated by segregation analysis of dinucleotide repeats, which were retrieved from the published sequences of chromosome 9 (AL136979 [GenBank] , AL136365 [GenBank] , AL390279) and analyzed as in the linkage study (primers 1–6, Table 3). For Southern hybridization, 10 µg genomic DNA was digested with restriction enzymes (New England Biolabs) according to the manufacturer's recommendations, run on 0.8% agarose gel and blotted to Sure Blot CHEMI membrane (Intergen). Probes, generated by PCR using primers 7–8 (Table 3), were labeled with 11dUTP dig (Roche Diagnostics). Hybridization was performed at 42°C for 18 h in the presence of 50% formamide. The last wash was done with 0.1x SSC—1% SDS for 15 min at 65°C. Detection was carried out using the Sure Blot CHEMI detection kit (Intergen). X-OMAT films (Kodak) were exposed for 30–120 min at room temperature.


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  		Table 3. PCR primers used in the study
 
Long-range PCR was done with the Expand Long system (Roche Diagnostics) using designed primers no. 9 (Table 3). The PCR fragment of ~7 kb was mapped by restriction enzyme analysis and the junction fragment was sequenced with the BigDye terminator kit using the ABI 310 sequencer (Applied Biosystems). On the basis of the sequence of the junction fragment, primers were designed to amplify a shorter fragment and a pair of primers was used as positive control (nos 10 and 11, Table 3).

Expression study
Northern hybridization was done on RNA blots from fetal and adult tissues (MTN Blot, Clontech). Exon 3 of the ANKRD15 gene was labeled with 11dUTPdig (Roche Diagnostics) through PCR (using the primers no. 12; Table 3) and probed for 1 h at 68°C. Last wash was performed with 0.1x SSC—0.1% SDS for 30 min at the same temperature.

RT–PCR first-strand synthesis was carried out on 1 µg of total RNA (extracted with RNAeasy kit, Qiagen). The reaction mixture included oligo dT (1 µg), random primers (2 µg), 100 µM dNTPs (Roche Diagnostics) and 100 units of SuperScript reverse transcriptase (Invitrogen); the mixture was incubated for 2 h at 42°C. PCR on the first strand was performed with the Expand Long Enzyme system (Roche Diagnostics) according to the manufacturer's instructions and included specific primers (nos 13 for variant A, 14 for variant B and 15 for the 3' of the NF1 gene which serves as a control, Table 3). PCR products were run on 2% agarose gel and visualized under UV illumination.

The expression pattern in samples without the deletion was evaluated by comparing genomic (primers 16) and cDNA (primers 13 and 14) sequences in the ANKRD15 utilizing the following SNPs: dbSNP, 912 175 and dbSNP, 912 174. Primers nos 17 and 18 (Table 3) were used for dbSNP, 2 297 079 and dbSNP, 1 887 957 in the DOCK8 gene. The mode of BRCA1, BRCA2 and NF1 expressions was analyzed, respectively, utilizing the following SNPs: dbSNP, 16 942; dbSNP, 1 801 406 and dbSNP, 1 801 052.

X-inactivation test
Genomic DNA cut with HpaII served as a template for PCR using primers from both sides of the trinucleotide repeats in the androgen receptor (no. 26, Table 3). PCR products were analyzed on ABI 3100 with GeneScan software (Applied Biosystems) and compared to the alleles obtained without HpaII digestion (27Go).

DNA methylation study
Bisulfite modification was performed with the CpGenome DNA modification kit (INTERGEN). PCR primers were designed for the modified strand (reviewed in 28Go). Region c was amplified using primers no. 21 and then nested PCR was performed with primers no. 22. A segment of region d was amplified with primers no. 24 (Table 3). The PCR products were cloned with the TA cloning kit (Invitrogen) and sequenced with M13 primers by the ABI 310 sequencer.

Methylation restriction analysis was performed by digestion of 100 ng genomic DNA with 20 units of HpaII for 18 h at 37°C. The restriction enzyme was inactivated at 95°C for 10 min and then the different regions (a, b, c, d) were amplified with the relevant primers (nos 19, 20, 23, 25; Table 3). PCR products were run on 2% agarose gel and visualized under UV illumination.

Databases
Sequence viewer, Ace View, Locus Link http://www.ncbi.nlm.nih.gov, Gene Cards http://bioinfo.weizmann. ac.il


   ACKNOWLEDGEMENTS
 
We would like to thank all the family members for their cooperation, Mrs Susan Mendelson for expert assistance with sequencing and Mrs Aviva Yechezkel for assistance with the LCLs. This work was supported by a grant from the Israeli Academy of Science.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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