Stimulatory guanine nucleotide binding protein subunit ... - Springer Link

Eur J Pediatr (1999) 158: 200±203

Ó Springer-Verlag 1999

ENDOCRINOLOGY

U. Walden á R. WeissoÈrtel á Z. Corria á D. Yu á L. Weinstein á K. Kruse á H. G. DoÈrr

Stimulatory guanine nucleotide binding protein subunit 1 mutation in two siblings with pseudohypoparathyroidism type 1a and mother with pseudopseudohypoparathyroidism Received: 13 February 1998 / Accepted in revised form: 7 September 1998

Abstract Pseudohypoparathyroidism (PHP) type 1a is characterized by multihormone resistance and a constellation of somatic features referred to as Albright hereditary osteodystrophy. Several mutations in the gene coding for the Gsa subunit (GNAS1) have been described. Clinical symptoms are heterogeneous and initially laboratory parameters may be normal. We identi®ed a 4 base pair deletion within GNAS1 in two a€ected siblings with PHP type 1a and their mother with presumed pseudo PHP. The female proband was diagnosed after an episode of apnoea and seizures. The younger brother was asymptomatic during infancy and had normal plasma parameters. PHP was diagnosed at the age of 4.4 years. Regular check-ups of siblings in families with index cases are therefore important. Molecular genetic analyses or biochemical screening for stimulatory guanine nucleotide binding protein defects should be performed. Conclusion Di€erent symptoms may be seen in patients with the same mutation causing pseudohypoparathyroidism or pseudopseudohypoparathyroidism. Therefore, clinical and biochemical investigations should be performed in all family members with an index patient. Key words Gs protein á GNAS1 deletion á Hot spot mutation á Pseudohypoparathyroidism 1a á Imprinting Abbreviations AHO Albright hereditary osteodystrophy á BMI body mass index á GNAS1 GS asubunit á Gs stimulatory guanine nucleotide binding protein á PHP pseudohypoparathyroidism á PPHP pseudopseudohypoparathyroidism á PTH parathyroid hormone Introduction

Pseudohypoparathyroidism (PHP) is a term for a group of disorders in which parathyroid hormone (PTH) resistance, characterized by hypocalcaemia, hyperphosphataemia and PTH elevation, is a major * Presented in part at the 35th Montpellier, France 1996

annual meeting of the ESPE,

K. Kruse University Children's Hospital of LuÈbeck, LuÈbeck, Germany Z. Corria á D. Yu á L. Weinstein Metabolic Disease Branch, N1H, NIODK, Bethesda, Maryland, USA

feature [9]. Albright hereditary osteodystrophy (AHO) is an autosomal dominant disease characterized by a constellation of somatic features, including short stature, obesity, brachydactyly and subcutaneous calci®cations. In AHO kindreds, some patients have these features in association with resistance to multiple H. G. DoÈrr (&) á U. Walden á R. WeissoÈrtel Division of Paediatric Endocrinology, University Hospital for Children and Adolescents, Loschgestrasse 15, D-91054 Erlangen, Germany, Tel.: +49-9131-853-3732, Fax: +49-9131-853-3113

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hormones which activate stimulatory guanine nucleotide binding protein (Gs)-coupled signalling pathways, such as PTH and TSH while others have only the somatic features without hormone resistance. These two presentations of AHO are termed PHP type 1a and pseudopseudohypoparathyroidism (PPHP), respectively. The observation that the normally brisk rise in urinary or plasma cAMP in response to exogenous PTH is absent in patients with PHP type 1a suggested that the defect is proximal to the generation of cAMP (receptor, Gs or adenylyl cyclase). Since there is resistance to multiple hormones it was unlikely that the disease could be caused by a defect in a single receptor. Most AHO patients (whether with PHP or PPHP) have a 50% de®ciency in Gs bioactivity when measured in membranes from several tissues. A small number of patients with AHO, multihormone resistance and normal Gs activity are classi®ed as PHP type 1c. Gs de®ciency in AHO patients is associated with decreased expression of the Gsa-subunit (GNAS1) mRNA and/or protein. Many heterozygous loss-of-function mutations within the gene encoding the GNAS1 have been identi®ed in AHO patients. The gene contains 13 coding exons and has been mapped to the long arm of chromosome 20 (20q13.2±13.3). Within AHO kindreds, identical GNAS1 mutations are present in patients with PHP and PPHP. Despite the presence of identical GNAS1 mutations, PPHP patients have no clinical evidence of hormone resistance and have a normal cAMP response to exogenous PTH. Except for a 4 bp deletion in exon 7, and a mutation in exon 13, no GNAS1 mutations have been identi®ed in more than one AHO kindred. We report two siblings with PHP type 1a and their mother probably su€ering from PHP who all have the same 4 bp deletion within exon 7 of GNAS1 which has been previously identi®ed in unrelated AHO kindreds [13]. Despite the identi®cation of the index case, diagnosis was delayed in the younger sibling.

Patients and methods Methods Intact PTH was measured by chemiluminescense immunometric assay (Nichols Institute Diagnostics, California, USA). Urinary cAMP (u-cAMP) and cAMP concentrations were measured by radioimmunoassay (Amersham Buchlar), in plasma obtained 5±10 min or urine obtained 60 min after intravenous application of 0.5 lg/kg human PTH1±38. TSH was measured by enzyme immunometric assay, Serono Diagnostika, Freiburg/Germany. The body mass index (BMI) was calculated as the body weight to (body length)2 (kg/m2). Height standard deviation scores were calculated according to German standards [2]. Bone age was assessed according to Greulich and Pyle [4]. Molecular genetic analyses of PCR-ampli®ed GNAS genomic and direct sequencing were performed as previously described [11]. Patients Case 1 (female patient, born 18.06.86) The female patient was delivered at 37 weeks gestational age by Caesarean section for gestosis and fetal distress. Birth weight was 2420 g and birth length 45 cm. At the age of 5 months, the girl was hospitalized for apnoea and hypoglycaemia. Normal plasma calcium and phosphate levels were determined at that time. Postnatal TSH screening was normal. At 3.8 years, the girl was referred to hospital again with an episode of apnoea and seizures. On examination, typical features of AHO, such as short stature, obesity, round face, broad neck, short metacarpals and developmental delay were noticed. Small subcutaneous ossi®cations were palpable in the abdomen and forearm. The girl had Tanner stage 1. Body height was 100.1 cm, weight 22.7 kg, height SDS for chronological age )0.8, bone age 6 years, height SDS for bone age )3.9, and BMI 28.0 kg/m2 (>97th percentile). Serum calcium was low (1.3 mmol/L), phosphate and PTH were both elevated (8.0 mg/dl; 668 pg/ml). TSH was normal at diagnosis (2.7 lU/ml) and increased to 6.0 lU/ml during the course of the disease (Table 1). Case 2 (male patient, born 31.08.90) The younger brother presented to our clinic at the age of 6 months for a routine check up. At this time, the measured laboratory pa-

Table 1 Laboratory ®ndings of the two siblings and their mother. (n.d. = not done) Normal values (children)

Serum Ca (mmol/l) PO4 (mg /dl) PTH (pg/ml) Alkaline Phosphatase (U/L) TSH (lU/ml) hPTH-test (0.5 lg/kg i.v.) 0¢®5±10¢/ 60¢ Plasma cAMP (nmol/L) Urine cAMP (nmol/mg crea) *During course of disease

Sister At time of diagnosis

2.1±2.7 3.4±7.0 10±55

1.3 (¯) 8.0 (­) 668 (­)

130±500 0.3±3.5

469 2.7/6.0*

Increase of >80 Increase of >80

19.8 ® 18.0 17.2 ® 10.0

Brother At 0.5 years 2.5 6.5 34.2 387 n.d. n.d. n.d.

Mother At time of diagnosis 1.8 (¯) 7.0 (­) 395 (­) 1047 (­) 1.9/6.3* 5.3 ® 6.6 6.3 ® 5.7

2.4 3.0 61 122 n.d. n.d. n.d.

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Discussion

GNAS1 mutations can lead to either PHP type 1a or PPHP [5, 6, 7, 9]. It has been observed that maternal transmission of disease leads to o€spring with PHP type 1a while paternal transmission leads to PPHP in o€spring, suggesting that GNAS1 may be an imprinted gene [3]. It has been shown that in mice with a targeted mutation of the Gsa gene, maternal transmission leads to PTH resistance while paternal transmission does not and the mouse gene is imprinted on the paternal allele [14]. PHP and PPHP are never seen in the same generation although they occur in one family [7]. The maternal transmission of PHP type 1a in this family is consistent with this model. The 4 bp deletion identi®ed in this kindred is identical to that in some other unrelated cases, providing further evidence that this is a deletion hot spot [13]. Further mutations of the GNAS1 have been found, but none has so far been identi®ed in more than one kindred. Similar to previously described cases [1], PTH resistance was not connatal and slowly developed over the ®rst years of life. Therefore normal serum levels of calcium, phosphate and PTH at a very young age does not rule out PHP type 1a. Hypothyroidism developed after the diagnosis of PTH resistance in both siblings. TSH elevation occurs in most patients with PHP and may be present at birth [8] or may develop later during the course of disease. Di€erent clinical symptoms may occur in patients with the identical mutation even in one family [5, 8]. The reason for this phenomenon remains unclear. Marguet et al. [5] reviewed the heterogeneity of clinical symptoms in 148 patients with PHP and proposed an extended classi®cation, including six subtypes. The variable onset of both somatic and endocrine features indicates that family members should be followed closely for signs of AHO or hormone resistance.

We found an identical GNAS1 mutation in a mother with PPHP and two o€spring with PHP type 1a. This is consistent with previous observations that identical

References

rameters were normal and he showed no clinical symptoms of PHP. He was monitored by his general practitioner and was referred to our clinic again at the age of 4.4 years. At this time, typical AHO features (round face, obesity, brachydactyly) were noticed. He had developed subcutaneous calci®cations, especially in the forearm. He had Tanner stage 1. Body height was 103.5 cm, weight 24.0 kg, bone age 4.5 years. Height SDS for both chronological and bone age was )0.8, and BMI was 22.4 kg/m2 (>97th percentile). His serum calcium was initially normal (2.5 mmol/L) but low at the time of diagnosis (1.8 mmol/L). His serum phosphate had mildly increased (6.5 mg/dl to 7.0 mg/dl) whereas PTH had increased from normal values of 34.2 pg/ml to 395 pg/ml within 4 years. During the course of disease, both siblings were treated with L-thyroxine (25 lg/day). The mother, a farmer and housewife with normal elementary school education, had mild clinical evidence of AHO with short stature (150 cm; height SDS)3.3), shortening of the hands and feet, short metacarpals and obesity (BMI 40.0 kg/m2). Laboratory results are shown in Table 1. Serum PTH levels were slightly elevated on two occasions. Neither subcutaneous calci®cations nor other endocrine disturbances were present.

Results

Table 1 summarizes the laboratory data. A nondenaturing polyacrylamide gel of a PCR-ampli®ed genomic fragment including exon 7 of GNAS1 showed an extra slower migrating band in samples derived from the proband and her sibling and mother, but not from her father (Fig. 1). Direct sequencing con®rmed the presence of a heterozygous 4 bp deletion in exon 7 in the three a€ected family members as previously reported in other AHO kindreds [7, 10, 11, 12, 13]. This mutation encodes a frameshift with a premature stop codon.

Fig. 1 Nondenaturing acrylamide gel (6% acrylamide gel in TrisBorate-EDTA bu€er) of PCR products including GNAS1 exon 7 ampli®ed from genomic DNA of: (1) negative control (no mutation in exon 7); (2) case 1 (PHP); (3) case 2 (PHP); (4) father (una€ected); (5) mother (probable PPHP); (6) unrelated PHP patient previously shown to have heterozygous 4 bp deletion within exon 7 [13]. Direct DNA sequencing of the PCR product from the proband demonstrated the same heterozygous 4 bp deletion previously described in exon 7 of GNAS1 [7, 11, 12, 13]

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