Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on November 10, 2004
Human Molecular Genetics 2005 14(1):95-102; doi:10.1093/hmg/ddi009

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Human Molecular Genetics, Vol. 14, No. 1 © Oxford University Press 2005; all rights reserved

Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB

Jie Liu, Julie G. Nealon and Lee S. Weinstein^*

Metabolic Diseases Branch, National Institute of Diabetes, Digestive and Kidney iseases, National Institutes of Health, Bethesda, MD 20892, USA

^* To whom correspondence should be addressed. Tel: +1 3014022923; Fax: +1 3014020374; Email: leew{at}amb.niddk.nih.gov

Received September 14, 2004; Revised October 13, 2004; Accepted October 25, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Pseudohypoparathyroidism type IB (PHPIB) is associated with abnormal imprinting of GNAS, the gene encoding the heterotrimeric G protein G_s and other alternative products. The gene contains three differentially methylated regions (DMRs) located upstream of the G_s promoter (from upstream to downstream): the paternally methylated NESP55 promoter region, the maternally methylated NESP antisense (NESPAS)/XLs promoter region and the maternally methylated exon 1A region located just upstream of the G_s promoter. We have now performed a detailed analysis of the GNAS methylation profile in 20 unrelated PHPIB probands. Consistent with prior results, all have loss of exon 1A imprinting (a paternal epigenotype on both alleles). All five probands with familial disease had a deletion mutation within the closely linked STX16 gene and a GNAS imprinting defect involving only the exon 1A region. In contrast, the STX16 mutation was absent in all sporadic cases. The majority of these patients had abnormal imprinting of the more upstream regions in addition to the exon 1A imprinting defect, with eight of 15 having a paternal epigenotype on both alleles throughout the GNAS locus. In virtually all cases, the imprinting status of the NESP55 and NESPAS/XLs promoters is concordant, suggesting that their imprinting is co-regulated, whereas the imprinting of the NESPAS/XLs promoter region and XLs first exon is not always concordant even though they are closely linked and lie within the same DMR. Familial and sporadic forms of PHPIB have distinct GNAS imprinting patterns that occur through different defects in the imprinting mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Pseudohypoparathyroidism type IB (PHPIB) is defined by renal resistance to parathyroid hormone (PTH) in the absence of other physical or endocrine abnormalities (except borderline thyrotropin resistance in some cases). The related disorder PHPIA [multihormone resistance with Albright hereditary osteodystrophy (AHO)] is caused by heterozygous inactivating mutations within the coding exons of G_s, the heterotrimeric G protein -subunit required for hormone-stimulated cyclic AMP formation (1). PHPIA only occurs when G_s mutations are inherited maternally, whereas paternally inherited mutations lead to AHO without hormone resistance (a condition also referred to as pseudopseudohypoparathyroidism). On the basis of this observation, it was proposed that G_s is paternally imprinted in a tissue-specific manner (2,3). More recent studies in both mice (4) and humans (5–8) have confirmed that G_s is imprinted in some hormonally responsive tissues.

GNAS, the gene encoding G_s at 20q13, is a complex locus that generates multiple gene products through the use of four alternative first exons that splice onto a common set of downstream exons (exons 2–13, Fig. 1) (1). The most downstream promoter (G_s exon 1) is not methylated but is more transcriptionally active on the maternal allele in some tissues (4–8). In mice, tissue-specific G_s imprinting is also associated with allele-specific differences in histone methylation (9). Alternative promoters located 35 and 47 kb upstream of exon 1 produce transcripts for the G_s isoform XLs and the chromogranin-like protein NESP55, respectively (10–12). The XLs and NESP55 promoters are oppositely imprinted. XLs is expressed only from the paternal allele and its promoter is methylated on the maternal allele, whereas NESP55 is expressed only from the maternal allele and its promoter is methylated on the paternal allele. More recently, the XLs promoter has been shown to generate paternally expressed antisense transcripts that traverse the NESP55 promoter from the opposite direction (NESP antisense, NESPAS) (13–16). Another promoter and first exon (exon 1A) located 2.5 kb upstream of G_s exon 1 is methylated on the maternal allele and generates untranslated transcripts only from the paternal allele (17,18). Studies in mice suggest that this region is a primary imprint mark which regulates G_s imprinting (18,19).

View larger version (12K): [in this window] [in a new window]   Figure 1. General organization and imprinting of the GNAS locus. The epigenotypes of the maternal (Mat, above) and paternal (Pat, below) alleles of GNAS are depicted with regions of methylation shown above (METH) and splicing patterns shown below. Transcriptionally active promoters are indicated by horizontal arrows in the direction of transcription. The alternative first exons and common exon 2 shown are depicted as rectangles with exon 1 being the first coding exon for Gs. Common downstream exons 3–13 as well as the downstream exons of the antisense transcript (NESPAS) are not shown. Exon 1 is paternally imprinted in some tissues, indicated by the dashed arrow. The diagram is not drawn to scale.

 
We and others have shown that PHPIB is consistently associated with loss of imprinting of the exon 1A region and is less often associated with abnormal imprinting of the other more upstream differentially methylated regions (DMRs) of GNAS (17,20,21). We have proposed that the exon 1A region contains a negative regulatory element for the G_s promoter that is both tissue-specific and methylation-sensitive, and that loss of maternal-specific methylation of exon 1A in PHPIB leads to PTH resistance by tissue-specific loss of G_s expression (1,17). Although most PHPIB cases are sporadic, in some cases, the disease is familial and the phenotype occurs when the trait is maternally inherited (22). The familial form of the disease has been shown to be associated with a heterozygous 3 kb deletion mutation within the closely linked STX16 gene (23).

In the present study, we have examined the imprinting status of GNAS in 20 PHPIB probands, and have now more specifically examined the methylation status of both the NESPAS/XLs promoter region and the XLs first exon, which are closely linked within the same DMR. Our results confirm that exon 1A imprinting is consistently lost in PHPIB. In our cohort, familial PHPIB is always associated with the STX16 mutation and a GNAS imprinting defect involving only the exon 1A DMR. In contrast, sporadic PHPIB is not associated with the STX16 mutation and is usually associated with a GNAS imprinting defect that also involves the more upstream DMRs. In almost all cases, the imprinting of the NESP55 and NESPAS/XLs promoters is concordant, suggesting that imprinting of these regions are co-regulated. Imprinting of the NESPAS/XLs promoter region and XLs first exon is not always concordant even though they are closely linked within the same DMR. Familial and sporadic forms of PHPIB have distinct GNAS imprinting defects.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PHPIB is consistently associated with an exon 1A DMR imprinting defect
In our original cohort of 13 PHPIB patients, we found that all had loss of exon 1A DMR imprinting (17). In the present study, we have re-examined the GNAS imprinting status of 10 patients from the original study, plus 10 additional previously unreported patients. Exon 1A methylation was determined by hybridization of an exon 1A-specific probe to genomic DNA that was digested with either PstI alone, or PstI plus either of the methylation-sensitive enzymes AscI or NgoMIV (Fig. 2A). In normal subjects, the double digestions produce both a 2.8 kb band representing the intact PstI fragment and smaller bands resulting from digestion by the methylation-sensitive enzymes, consistent with the presence of one methylated and one unmethylated allele (17). In contrast, the 2.8 kb fragment was completely digested by both enzymes in 19 of 20 PHPIB patients, consistent with total loss of the maternal-specific methylation of exon 1A (Figs 2 and 3). In one patient (P18, Figs 3 and 4), the 2.8 kb band was faintly present in both double digested samples, indicating that methylation of exon 1A in this patient was not totally absent. There was no evidence that this residual methylation was associated with a milder form of PTH resistance in patient P18. These results, as well as our prior results (17), show that exon 1A DMR imprinting is lost in all 23 PHPIB patients that we have examined to date, indicating that this is a consistent finding in PHPIB.

View larger version (32K): [in this window] [in a new window]   Figure 2. Methylation analysis of GNAS DMRs. (A) The restriction maps of the NESP55, NESPAS/XLs and 1A DMRs are shown with each first exon represented as a black box and the position of the probes used for Southern analysis shown below. The 5' extent of the NESPAS and XLs first exons are based on GenBank accession no. AJ251760; the XLs exon likely extends farther upstream than shown (32). For the NESPAS/XLs DMR, the two EcoRI sites (RI) at each end are 17.5 kb apart. (B) Results of Southern analysis of genomic DNA from a normal and three PHPIB patients. Analysis of the NESPAS/XLs promoter and XLs first exon are performed using probes AS2 and XL1, respectively. All patients lacked methylation of exon 1A (data not shown). Patient P15 had abnormal methylation at all sites (both alleles methylated at NESP55, both alleles unmethylated at NESPAS/XLs promoter and XLs). Patient P3 maintained normal methylation at all three of these sites. Patient P7 had abnormal methylation at NESP55 and the NESPAS/XLs promoter, but maintained normal methylation within the XLs first exon. Bg, BglII; S, SacII; F, FspI; N, NgoMIV; A, AscI; Bs, BssHII; E, EagI; X, XmnI; M, MluI; Sa, SacI; P, PstI.

 

View larger version (29K): [in this window] [in a new window]   Figure 3. Summary of GNAS methylation analysis in 20 PHPIB patients. The methylation status [methylated (CH3), unmethylated (–), trace methylation (Tr)] of each allele is shown as maternal/paternal. For the NESP55 (left) and exon 1A (right) DMRs, methylation at all restrictions sites was consistent so their methylation status is summarized with a single notation. For the NESPAS/XLs DMR (center), the results at each restriction site are shown individually. The transcriptional start sites for NESPAS and XLs (arrows) are based on GenBank accession no. AJ251760, although it is likely that the XLs start site is located further upstream than shown (32). The presence or absence of familial disease and the STX16 mutation, respectively, in each patient is indicated to the left. In patient P18, the methylation of NESP55 is not complete (Fig. 4). The normal methylation patterns at each site are shown at the bottom. Initial characterization of the first 10 patients has been previously reported (17).

 

View larger version (52K): [in this window] [in a new window]   Figure 4. GNAS methylation analysis of PHPIB patients P10 and P18. Southern analysis of the NESP55, NESPAS/XLs promoter, XLs and exon 1A regions were performed using the same probes and restriction enzymes as in Figure 2. Patient P10 has no methylation of exon 1A, normal methylation of NESP55 and XLs, but has only trace methylation of the NESPAS/XLs promoter region. In patient P18, the maternal-specific methylation of exon 1A is partially lost, the methylation of the NESPAS/XLs DMR is completely lost and the NESP55 region is mostly methylated, although not completely.

 
Familial PHPIB is associated with an STX16 mutation and a GNAS imprinting defect involving only the exon 1A DMR
A 3 kb deletion mutation within the closely linked STX16 gene has been recently identified in familial PHPIB (23). All five probands within our cohort with a positive family history for PHPIB had the identical STX16 mutation, whereas the mutation was absent in all 15 patients without a positive family history for PHPIB (Fig. 3). Consistent with earlier observations (17,23), all familial cases with the STX16 mutation had loss of exon 1A methylation but had normal methylation of the upstream NESP55 and NESPAS/XLs DMRs (Fig. 3). The clinically affected brother of patient P3 and clinically affected mother of patient P4 both harbored the STX16 mutation.

Patient P16 (II-1, Fig. 5A) has two affected half-brothers (II-2, II-3) from the same mother (I-1), who herself is unaffected. These two half-brothers had the STX16 mutation and the same GNAS imprinting pattern as the affected proband. The clinically affected sister of patient P19 (IV-2, Fig. 5B) has the STX16 mutation and the exon 1A DMR imprinting defect, whereas their clinically unaffected mother (III-5) has the STX16 mutation but not the exon 1A DMR imprinting defect. The mother's paternal aunt (II-1), who herself is clinically unaffected, has three daughters with PHPIB. Therefore, she and her brother (II-2) are most likely clinically silent carriers of the STX16 mutation which was inherited from their father (I-2). The paternally inherited STX16 mutation failed to result in PTH resistance in III-5. In contrast, maternal transmission of the mutation from either II-1 or III-5 resulted in PHPIB in affected offspring. Therefore, renal PTH resistance is associated with loss of exon 1A imprinting, rather than the STX16 mutation, and both the imprinting defect and the clinical phenotype result from maternal transmission of the STX16 mutation.

View larger version (13K): [in this window] [in a new window]   Figure 5. PHPIB inheritance patterns in two familial PHPIB kindreds. Partial pedigrees of kindreds for patients (A) P16 and (B) P19 are shown with males as boxes and females as circles. Probands are indicated with an arrow. Clinically, unaffected family members who did not undergo genetic testing are indicated with open boxes. PHPIB patients who did not undergo genetic testing are partially filled on the upper half. PHPIB patients shown to have both the STX16 mutation and the exon 1A DMR imprinting defect are completely filled. In (B), patient III-5 is a silent carrier who has the STX16 mutation but not the exon 1A DMR methylation defect (indicated with gray in the lower right quadrant). Asterisks indicate patients who were not tested but who are presumed to carry the STX16 mutation.

 
Sporadic PHPIB is associated with more global GNAS imprinting defects
In 15 of 20 patients in the cohort, the STX16 mutation was absent and the family history was negative for PHPIB, consistent with these being sporadic cases. In contrast to the familial cases, most of these sporadic cases had GNAS imprinting defects involving the upstream NESP55 and NESPAS/XLs DMRs as well as the exon 1A DMR. In our initial study (17), we examined the methylation status of NESP55, the XLs first exon and exon 1A regions but did not specifically examine the upstream portion of the NESPAS/XLs DMR including the promoter region. To better define the imprinting patterns observed in PHPIB, we have now examined a larger group of patients and have examined the methylation throughout the NESPAS/XLs DMR, including the NESPAS/XLs promoter region, by Southern analysis using multiple probes and methylation-sensitive restriction enzymes (Figs 2A and 3). The methylation of NESP55 was determined using previously published probes and restriction enzymes (17) (Fig. 2A).

Analysis of these regions identified three general patterns among the 15 sporadic PHPIB patients. In eight of these patients, the methylation of NESP55, the NESPAS/XLs promoter region and the XLs first exon was abnormal, with the NESP55 being methylated on both alleles and the NESPAS/XLs DMR being unmethylated on both alleles (Figs 2B and 3). Combined with the exon 1A imprinting defect, this group of patients has a paternal epigenotype in both alleles throughout the GNAS locus. We had previously ruled out a paternal uniparental disomy of this region in one of these patients (patient P1) (17), and none of these patients has other manifestations that were previously reported in a case of paternal uniparental disomy of 20q (24). In one patient from this subgroup (P18, Figs 3 and 4), the NESP55 methylation abnormality appears to be incomplete, as a small proportion of the genomic DNA is digested by the methylation-sensitive enzymes within this region. This patient also showed a small amount of residual methylation in exon 1A (discussed earlier), although the NESPAS/XLs DMR appears to be completely unmethylated in this patient.

Two of the 15 sporadic PHPIB patients (P8 and P9) had a GNAS imprinting pattern similar to that seen in familial PHPIB, namely loss of exon 1A DMR imprinting but no abnormality of the NESP55 or NESPAS/XLs imprinting (Fig. 3). To rule out the possibility that these two patients had an STX16 deletion that would not be identified by our PCR strategy, we looked for heterozygosity at seven polymorphic sites within the STX16 deleted region located between positions 3959 and 4712 (23). Patient P9 was heterozygous at both 3959 and 4712 positions, confirming that this region was present on both copies of chromosome 20 in this patient. Patient P8 was homozygous at these sites, and therefore we could not rule out the possibility of a small STX16 deletion in this patient. Patients 6, 10 and 11 from our original study (17) appear to also have this GNAS imprinting pattern, but were not included in the present study because we did not have enough material to do detailed analysis of the NESPAS/XLs promoter region.

Interestingly, in four patients, the NESP55 and NESPAS/XLs promoter regions were abnormally imprinted (biallelic methylation of NESP55, no methylation of the NESPAS/XLs promoter region), whereas the XLs first exon remained partially methylated (Figs 2B and 3). Southern analysis of the same DNA samples using all four methylation-sensitive restriction enzymes simultaneously or the frequently cutting methylation-sensitive enzyme HpaII showed these patients to have a pattern similar to that of normal subjects with the presence of a completely methylated fragment, confirming the results obtained were due to the presence of one methylated and one unmethylated allele rather than partial methylation of both alleles (data not shown). Therefore, these patients have concordant imprinting of the NESP55 and NESPAS/XLs promoter regions, with a paternal epigenotype on both alleles, but discordant imprinting of the NESPAS/XLs promoter region and XLs first exon, even though these two regions are closely linked within the same imprinted GC-rich region. In one patient (P10), the methylation of NESP55 and the XLs first exon was normal, whereas much of the maternal-specific methylation of the NESPAS/XLs promoter region was lost (Figs 3 and 4). There were no patients with normal imprinting of the NESP55 and NESPAS/XLs promoters and abnormal imprinting of the XLs first exon.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We and others had previously shown that the PHPIB phenotype is consistently associated with an imprinting defect involving the exon 1A region (17,20,21). In these patients, maternal-specific methylation of exon 1A is lost, resulting in a paternal epigenotype on both parental alleles and biallelic expression of exon 1A-specific transcripts. Our present study describes 10 more PHPIB patients who have the same epigenetic defect, providing further evidence that the PHPIB phenotype is consistently associated with the exon 1A imprinting defect. The importance of this epigenetic defect in the development of PHPIB is underscored by the results of this and other studies (21,23) showing that in familial PHPIB kindreds, clinical affected family members always have the exon 1A imprinting defect, whereas clinically unaffected family members do not have the exon 1A imprinting defect, even when some of these unaffected members have or are presumed to have an underlying genetic mutation. Although in one of our patients (P18) the methylation defect was not fully complete, this was not associated with a less severe phenotype.

We have proposed that the exon 1A DMR has a negative regulatory element for the G_s promoter that is both tissue-specific and methylation-sensitive (3,17). This model would predict that a tissue-specific factor expressed in renal proximal tubules cells, such as a repressor or insulator protein, binds to a site within the exon 1A DMR and suppresses the G_s promoter on the paternal allele, but is unable to bind to the maternal allele because its site is methylated. In PHPIB, the maternal allele is also unmethylated, allowing the factor to bind to both alleles, leading to loss of G_s expression in renal proximal tubules and PTH resistance. Support for this model comes from the fact that mice with paternal, but not maternal, deletion of the exon 1A DMR have increased G_s expression in renal proximal tubules (J. Liu and L.S. Weinstein, submitted for publication) and lower circulating PTH levels (19) (J. Liu and L.S. Weinstein, submitted for publication). Although it is possible that the exon 1A-specific transcripts themselves could be important for tissue-specific G_s imprinting, this seems unlikely due to the fact that in mice these transcripts are expressed ubiquitously, whereas G_s imprinting is highly tissue-specific (18).

Of the five patients in our cohort known to have familial disease, all had both a methylation defect limited to the exon 1A DMR and a previously reported deletion mutation in the closely linked gene STX16 which is located upstream of GNAS (23). This is consistent with other reports showing that familial PHPIB is almost always associated with this methylation pattern (17,20,21). In several of our kindreds, we show that those who maternally inherit STX16 mutation have the exon 1A DMR methylation defect and develop PHPIB, whereas those who paternally inherit the mutation are clinically silent carriers who lack the exon 1A methylation defect. This is consistent with prior reports showing that the development of disease in familial PHPIB kindreds requires maternal inheritance (22,23,25). It appears likely that the region deleted in the STX16 mutation contains one or more cis-acting elements that are required to establish imprinting of the exon 1A DMR, which we have shown in mice to occur during oogenesis (18).

Interestingly, although the STX16 deletion prevents exon 1A DMR imprinting, it has no effect on imprinting of the intervening NESP55 and NESPAS/XLs DMRs, suggesting that imprinting of these regions is normally established by mechanisms that are independent of those required for exon 1A DMR imprinting. Consistent with this, exon 1A deletions in mice have no effect on imprinting of these upstream regions in Gnas (19) (J. Liu and L.S. Weinstein, submitted for publication). Recently, a second primary imprint mark has been identified within the NESPAS/XLs DMR in mice (26), providing further support that NESP55 and NESPAS/XLs imprinting is regulated by a second imprinting control region.

When compared with other published PHPIB cohorts (20,21), our cohort has a much greater frequency of imprinting defects involving the NESP55 and NESPAS/XLs regions, reflecting the fact that our cohort is mostly composed of sporadic cases which are frequently associated with a more global GNAS imprinting defect. It is unclear whether these more global defects result from other underlying genetic mutations or a failure to imprint which may rarely occur by a stochastic process. In eight of our 15 sporadic cases, there was a paternal epigenotype throughout the GNAS locus. Although such a pattern has been observed in a patient with paternal uniparental disomy of 20q (24), this patient had other significant phenotypic abnormalities. We have shown that this global GNAS imprinting defect can occur in a PHPIB patient in the absence of paternal uniparental disomy (17). Two of our sporadic cases had an imprinting defect involving exon 1A alone, even in the absence of the STX16 mutation. Therefore, this abnormal imprinting pattern can occasionally occur in PHPIB by alternative mechanisms.

With the possible exception of one patient, the imprinting status of the NESP55 and the NESPAS/XLs promoter regions were concordant, with loss of maternal-specific NESPAS/XLs promoter methylation always being associated with a gain of methylation of NESP55 on the maternal allele. These results suggest that these two promoter regions are coordinately regulated. We have shown that the NESP55 imprinting is not established until after implantation in mice, and is therefore dependent on the presence of another primary imprint mark (18). Biallelic methylation of NESP55 with loss of maternal NESPAS/XLs promoter methylation is also seen in biparental complete hydatidiform moles (27) and a mouse model (28) (J. Liu and L.S. Weinstein, submitted for publication), which are both unable to establish maternal primary imprints marks, raising the possibility that methylation of an imprinting control region within the NESPAS/XLs promoter (26) is required for the NESP55 promoter to remain unmethylated and transcriptional active. The apparent reciprocal relationship between the NESP55 and NESPAS/XLs promoters is reminiscent of other reciprocally imprinted promoters for overlapping sense and untranslated antisense transcripts such as Air/Igf2r (29) and Xist/Tsix (30). It is possible that inactivation of the NESP55 promoter is not mediated by the NESPAS transcript in cis, because the NESP55 imprinting does not always co-occur with NESPAS expression in different tissues (15,31). Partial loss of maternal-specific methylation of the NESPAS/XLs promoter with normal methylation of NESP55 in patient P10 could reflect the fact that we did not specifically examine the CpG sites in the NESPAS/XLs promoter that are most critical for the NESP55 imprinting or that maintenance of the NESP55 imprinting does not depend upon NESPAS/XLs promoter methylation once it is established.

Another novel observation in our study is the fact that the imprinting of the NESPAS/XLs promoter region and XLs first exon is differentially affected in some PHPIB patients, even though these regions are closely linked within the same DMR. In these cases, normal imprinting of the XLs first exon is maintained even in the presence of an imprinting defect involving NESP55, the NESPAS/XLs promoter and the exon 1A DMR. Although we did not observe any patients with normal methylation of NESP55 and abnormal methylation of XLs, it is possible that this is the methylation pattern in one previously published case based upon the reported allele-specific expression patterns of their respective transcripts (21). Biparental complete hydatidiform moles with a maternal methylation defect also show differences in imprinting between the NESPAS/XLs promoter and the XLs first exon, although in this setting rather than the XLs first exon having normal imprinting, this region had partial methylation of both parental alleles which was variably distributed among its CpG sites (27). The NESPAS/XLs DMR has a complicated methylation pattern in mouse blastocysts that is not uniform throughout the DMR (18,26), indicating that different portions of the DMR are methylated independent of one another and at different stages of development. Further studies in humans and mice will provide a better understanding of how the various imprinted domains of GNAS interact to generate the fully developed paternal and maternal epigenotypes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Patients
The diagnosis of PHPIB was made in all patients reported in this study, based upon the presence of renal PTH resistance (hypocalcemia, hyperphosphatemia, elevated serum PTH levels) in the absence of any features of AHO. Vitamin D deficiency was ruled out in all patients based on the measurement of 25 hydroxyvitamin D levels. Patients P1–P10 in the present study were previously reported as patients 1–5, 7–9, and 11 and 12 in our prior study (17), respectively. This study was approved by the NIDDK/NIAMS Institutional Review Board and informed consent was obtained from all subjects.

Methylation analysis
Genomic DNA was isolated from patient blood samples using the Blood and Cell Culture DNA Maxi kit (QIAGEN, Valencia, CA, USA). Southern analysis was performed using NESP55 and exon 1A-specific probes as previously described (17) (Fig. 2). Methylation within the NESPAS/XLs DMR was determined by Southern analysis using four different probes (Fig. 2). The XLs-specific probe XL2 was the same probe used in our prior study (17). The other probes were generated by PCR using the following pairs of upstream and downstream oligonucleotide primers, respectively: AS1, 5'-GACCCATAGAGCTAAAGC-3' and 5'-CGGATCTCAAGCCCCTAGTC-3'; AS2, 5'-CGACTACCCTAAGATACCCAG-3' and 5'-TGGGCTCGTTGTGAGGCGGTTC-3'; XL1, 5'-TGCTTCAGCCTCAGTCTAGGG-3' and 5'-TCCCAAGTACAAGTACACAGTCGGC-3'.

Genotyping
All DNA samples were screened for the presence of the 3 kb deletion mutation within STX16 by PCR and Southern analysis as previously described (23). In selected patients, we determined the genotype at seven polymorphic sites between positions 3959 and 4712 within the STX16 deleted region as previously described (23).

	ACKNOWLEDGEMENTS

 
This study was supported by the Intramural Research Program of the National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services.

	FOOTNOTES

 
Present address: The Bullis School, Potomac, MD 20854, USA.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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