A Novel STX16 Deletion in Autosomal Dominant Pseudohypoparathyroidism Type Ib Redefines the Boundaries of a cis-Acting Imprinting Control Element of GNAS

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A Novel STX16 Deletion in Autosomal Dominant Pseudohypoparathyroidism Type Ib Redefines the Boundaries of a cis-Acting Imprinting Control Element of GNAS Agnès Linglart, Robert C. Gensure, Robert C. Olney, Harald Jüppner, Murat Bastepe The American Journal of Human Genetics Volume 76, Issue 5, Pages 804-814 (May 2005) DOI: 10.1086/429932 Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 1 Schematic representation of the human STX16 and GNAS loci (not to scale). Exons are depicted as blackened boxes; arrows indicate direction (sense, antisense) and allelic origin (Mat = maternal [above the line]; Pat = paternal [below the line]) of transcription. Note that Gαs is transcribed from both alleles in most tissues (Campbell et al. 1994; Hayward et al. 1998a, 1998b; Zheng et al. 2001) and that the paternal allele is silenced (dotted arrow) in some tissues, including kidney, adipose tissue, and pituitary (Hayward et al. 1998a, 2001; Yu et al. 1998; Li et al. 2000; Mantovani et al. 2002). Paternal and maternal DMRs are indicated by blackened and unblackened rectangles, respectively. The American Journal of Human Genetics 2005 76, 804-814DOI: (10.1086/429932) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 2 A, Evidence of allelic loss within the STX16 locus in the obligate carriers W-II/5 and W-II/6. All the affected individuals (blackened symbols) as well as the obligate carriers (striped symbols) share the same disease-associated haplotype (gray columns) at 20q13.3; note that not all investigated markers are shown. A recombination event occurred in individual W-II/9 between markers 907-rep2 and 261P9-CA1 and was transmitted to her affected daughter, W-III/12, thus reducing the critical interval likely containing the genetic lesion that causes AD-PHP-Ib. Evidence of allelic loss was found at SNP rs6100157 (bold typeface) in obligate carriers W-II/5 and W-II/6. B, Loss of methylation at GNAS exon A/B in the newborn W-III/12. The methylation status of GNAS exon A/B was assessed by Southern blot analysis of cord blood DNA from patient W-III/12 and of genomic DNA from her unaffected mother (W-II/9), an individual with paternal uniparental isodisomy of chromosome 20q (“UPD”) (Bastepe et al. 2001a), and a healthy control individual (“N”). These samples were digested with a methylation-sensitive (EagI) and a methylation-insensitive (EcoRV) restriction endonuclease, as described elsewhere (Bastepe et al. 2001b). Locations of restriction sites and their methylation status (“+” = methylated; “−” = nonmethylated), the predicted fragments, and the probe (gray bar) are shown in the left panel. Affected individuals (UPD and W-III/12) show loss of methylation at exon A/B, whereas unaffected individuals (N and W-II/9) have one methylated and one nonmethylated allele. Fragment sizes are indicated by arrowheads. Note that the faint hybridizing band below the 6.2-kb band reflects partial methylation (“+/−”) and, hence, incomplete digestion at one of the EagI sites between exon A/B and Gαs exon 1. The American Journal of Human Genetics 2005 76, 804-814DOI: (10.1086/429932) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 3 Delayed onset of PTH resistance in patient W-III/12. Serum calcium, phosphorus, and PTH levels were measured during follow-up visits of the patient—in whom loss of methylation at GNAS exon A/B was identified at birth—and the 4.4-kb deletion was discovered subsequently. Normal ranges are indicated by gray shading. Treatment with an active vitamin D analog (Rocaltrol) was first started at age 21 mo (vertical arrow). However, the parents discontinued the treatment shortly thereafter, and continuous treatment was not resumed until the child was 30 mo old (horizontal arrow). The American Journal of Human Genetics 2005 76, 804-814DOI: (10.1086/429932) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 4 Identification of a novel 4.4-kb deletion in kindred W. A, Simplified map of the 20q13.3 region and the localization of the two known microdeletions. Sequence of the depicted region is included in AL050327 (clone 907D15) and AL139349 (clone 261P9). Exons are depicted as blackened rectangles, and direct repeats are shown as striped arrowheads. The 3-kb and 4.4-kb deleted regions lie between the thin and thick brackets, respectively. The locations of recognition sites for the restriction endonucleases used in the Southern blot analysis are shown under the gene map. The location of the 1.6-kb probe is marked by the gray bar, and primers A, B, C, D, E, and F, which were used for PCR analyses, are indicated by arrowed lines; cen = centromeric; tel = telomeric; N = NESP55; AS = antisense; X = XLαs; A/B = exon A/B. B, Southern blot analysis of genomic DNA from an unaffected (“U”) subject, W-III/4, and an obligate carrier (“OC”), W-II/5, which was digested with three different restriction endonucleases (AflII, BclI, and BgIII). Hybridization with the 1.6-kb 32P-labeled probe (nucleotides 8299–9921 of AL139349) showed the expected wild-type bands of 7.9 kb, 9.8 kb, and 9.2 kb, respectively. Additional bands (arrowheads) in the obligate carrier (6.8 kb, 11.1 kb, and 7 kb, respectively) are compatible with a 4.4-kb deletion removing one AflII site, two BclI sites, and one BglII site—all of which are centromeric of STX16 exon 2. C, Confirmation and exact localization of the deletion by PCR. Amplification across the predicted location of the deletion, performed using one pair of primers (A [forward] and B [reverse]), yielded a 5.9-kb wild-type fragment in the unaffected subject, the affected individual, and the obligate carrier; both the affected individual and the obligate carrier also showed a shorter fragment (of 1.5 kb). Direct nucleotide sequence analysis of the short amplicon revealed the lack of 4,368 bp extending from nucleotide 704 to nucleotide 5072 of AL139349. D, Amplification of the STX16 region with four different primers, located at nucleotides 404 (primer C [forward]), 1020 (primer D [reverse]), 4938 (primer E [forward]), and 5190 (primer F [reverse]) of AL139349. The sizes of expected wild-type PCR products are 616 bp (primers C and D), 252 bp (primers E and F), and 4.8 kb (primers C and F [not shown]). Obligate carriers and affected individuals show an additional 422-bp amplicon (arrow [primers C and F]). Results are shown for each individual in the family (blackened symbols represent affected individuals, striped symbols represent obligate carriers, and unblackened symbols represent unaffected individuals). The American Journal of Human Genetics 2005 76, 804-814DOI: (10.1086/429932) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 5 Biallelic expression of STX16. A, STX16 gives rise to at least three distinct transcripts (left). Isoform A (915 bp) differs from isoform B (965 bp) by a shorter sequence derived from exon 1, whereas isoform C (422 bp) comprises this short exon 1 sequence as well as exons 2–5 and terminates with exon 6a. In addition, exon 4 is alternatively spliced in each isoform, leading to a total of at least six variants. Wild-type STX16 isoform B and the transcripts derived from individuals W-III/3 and F-III/37 (“mut1” and “mut2,” respectively) are depicted (right). The 4.4-kb deletion (thick brackets) removes exons 2–4; the 3-kb deletion (thin brackets) removes exons 4–6. Exons are depicted as blackened boxes, and direct repeats are shown as striped arrowheads. Primers G and H are represented as arrowed lines; cen = centromeric; tel = telomeric. B, RT-PCR analysis of STX16 transcripts in total RNA of immortalized lymphocytes from a healthy control individual (CTRL) and the following individuals with AD-PHP-Ib: W-III/3 (present study), F-III/37 (carrying the 3-kb deletion in STX16 region [Bastepe et al. 2003]), and C-II/1 (carrying a deletion affecting NESP55 but not the STX16 region [Bastepe et al. 2005]). Reactions were performed with (“+”) or without (“−”) reverse transcriptase, as described in the “Subjects and Methods” section. Products derived from the mutant alleles (“mut1” and “mut2”) and from the wild-type alleles (“WT”) are indicated. Note that the wild-type products represent STX16 isoform B with or without sequences derived from exon 4. C, Direct nucleotide sequence of STX16 isoform B amplified from RNA of a healthy control individual shows heterozygosity at the polymorphic nucleotide 527, corresponding to SNP rs2296524. The American Journal of Human Genetics 2005 76, 804-814DOI: (10.1086/429932) Copyright © 2005 The American Society of Human Genetics Terms and Conditions