Pseudohypoparathyroidism Type Ib Associated with Novel Duplications in the GNAS Locus

Context: Pseudohypoparathyroidism type 1b (PHP-Ib) is characterized by renal resistance to PTH (and, sometimes, a mild resistance to TSH) and absence of any features of Albright's hereditary osteodystrophy. Patients with PHP-Ib suffer of defects in the methylation pattern of the complex GNAS locus. PHP-Ib can be either sporadic or inherited in an autosomal dominant pattern. Whereas familial PHP-Ib is well characterized at the molecular level, the genetic cause of sporadic PHP-Ib cases remains elusive, although some molecular mechanisms have been associated with this subtype.Objective: The aim of the study was to investigate the molecular and imprinting defects in the GNAS locus in two unrelated patients with PHP-Ib.Design: We have analyzed the GNAS locus by direct sequencing, Methylation-Specific Multiplex Ligation-dependent Probe Amplification, microsatellites, Quantitative Multiplex PCR of Short Fluorescent fragments and array-Comparative Genomic Hybridization studies in order to characterize two unrelated families with clinical features of PHP-Ib.Results: We identified two duplications in the GNAS region in two patients with PHP-Ib: one of them, comprising ~320 kb, occurred ‘de novo’ in the patient, whereas the other one, of ~179 kb in length, was inherited from the maternal allele. In both cases, no other known genetic cause was observed.Conclusion: In this article, we describe the to-our-knowledge biggest duplications reported so far in the GNAS region. Both are associated to PHP-Ib, one of them occurring ‘de novo’ and the other one being maternally inherited.This work was partially supported by Grants IT-795-13 and IT-472-07 from the Basque Department of Education (http://www.hezkuntza.ejgv.euskadi.net/r4​3-2591/es). TV is supported by the FPI Program of the University of Basque Country (UPV-EHU, http://www.ehu.es/p200-home/es)

Dosage and methylation analysis of the <i>GNAS</i> locus.

<p>In all cases, on the X-axis, MS-MLPA probes for copy number determination (P1 to P28) or methylation analysis (P6 to P17) are shown according to their chromosome location (detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117691#pone.0117691.t001" target="_blank">Table 1</a>). On the Y-axis, the final probe ratio is shown. (A) Dosage analysis of the <i>GNAS</i> locus in GS0131 (black bars) and GS0155 (grey bars) compared against the average of four controls (white bars). (B) Methylation analysis of the <i>GNAS</i> locus in GS0131 (black bars) and GS0155 (grey bars) compared against the average of four controls (white bars). GS0131 shows complete loss of methylation at NESPAS, XLαs and exon A/B, and gain of methylation at NESP55, whereas GS0155 shows complete loss of methylation at A/B, partial gain at XLαs and NESPAS and partial loss at NESP55. (C) Dosage analysis of the <i>GNAS</i> locus in GS0131 parents (upper panel) and GS0155 parents (lower panel) compared against the average of four controls. In all cases, black bars represent the father, grey bars represent the mother and white bars represent the controls. Mother of GS0155 presents the same duplication as her daughter, whereas absence of alterations in GS0131 parents demonstrates its ‘de novo’ origin. (D) Methylation analysis of the <i>GNAS</i> locus in GS0131 parents (upper panel) and GS0155 parents (lower panel) compared against the average of four controls. In all cases, black bars represent the father, grey bars represent the mother and white bars represent the controls. There is not any methylation alteration in the parents of GS0131. Mother of GS0155 presents a partial loss of methylation in XLαs (P12-P15) and NESPAS (P9-P11), and a partial gain of methylation in NESP55 (P6-P8), exon A/B not being affected. See text for details.</p

Determination of the parental origin of the duplication in family GS0131.

<p>Electropherogram showing the quantitative multiplex PCR of short fluorescent fragments (QMPSF) of microsatellite D20S459 from family GS0131 members against exon 6 of <i>AIRE</i> (21q22), used as a control amplicon. From up to down, results for the index case, mother and father. Vertical arrows show the control fragment. The horizontal arrow indicates the maternally-inherited duplicated allele in the index case. Figures show the size of the fragments in base pairs (red: weight marker; green: maternally-inherited allele; blue: paternally-inherited allele). Similar data were obtained using other control amplicons: exon 2 of <i>HNF1A</i> (12q24) and exon 3 of <i>HNF1B</i> (17q12). Data of these experiments are not shown.</p

Genomic location of the probes used for the MS-MLPA analysis and the 27 polymorphic markers in chromosome 20q mentioned in the text.

<p>Genomic coordinates (according to GRCh37/hg19 build) of the 28 probes used for the MS-MLPA analysis and the 27 polymorphic markers in chromosome 20q. They are situated in the table according to their chromosomal location. In the case of the MS-MLPA probes, “Probe numbers” indicate the reference used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117691#pone.0117691.g002" target="_blank">Fig. 2</a>. Probes highlighted in bold (P6 to P17) are used to identify methylation aberrations in the locus.</p><p>Genomic location of the probes used for the MS-MLPA analysis and the 27 polymorphic markers in chromosome 20q mentioned in the text.</p

High density aCGH assay results for the 20q13 region of both families.

<p>In both cases, affected genes and regions mentioned in the text are highlighted in red on the X-axis. On the Y-axis, binary logarithm of the sample/control ratio. miR296 location is indicated in bold (corresponds to chr20:57392669–57392748). Red arrows indicate the location of the probes at each breakpoint. (A) Results for GS0131 index case. Won probes correspond to coordinates chr20:57381447–57701869. (B) Results of family GS0155 (upper panel, index case; lower panel, mother). Won probes correspond to coordinates chr20:57281908–57461486.</p

Genotyping results of the 27 polymorphic markers at 20q in both families.

<p>Fluorescent PCR and genotyping results of the 27 polymorphic markers in chromosome 20q, situated in the table according to their chromosomal location. Figures indicate the obtained size of the amplicons, or the number of pentanucleotide repeats, in the case of the A/B pentanucleotide marker. In all cases, first columns indicate paternally inherited alleles; the second columns indicate maternally inherited alleles (in the cases in which alleles are not directly informative, this inheritance has been assigned assuming both paternal alleles are present; see text for details). It was not possible to determine the inheritance of the single nucleotide variation (SNV) c.140–53. Bold figures indicate informative markers.</p><p>Genotyping results of the 27 polymorphic markers at 20q in both families.</p