X-Linked Recessive Combined Pituitary Hormone Deficiency Is Mapped To

Genomics 98 (2011) 440–444

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Genomics

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X-linked recessive combined pituitary hormone deﬁciency is mapped to Xp22.3–Xp11 in a Chinese family ☆

Chun Lin Wang a, Li Liang a,⁎, Zheng Shen b, Chao Chun Zou a, Jun Fen Fu a, Guan Ping Dong a a Department of Endocrinology, Children's Hospital of Zhejiang University School of Medicine, Hangzhou 310003, China b Children's Hospital of Zhejiang University School of Medicine and Zhejiang Key Laboratory for Diagnosis and Therapy of Neonatal Diseases, Laboratory, Hangzhou, 310003, China article info abstract

Article history: Genetic mutations have been identified in a modest proportion of patients with combined pituitary hormone Received 11 April 2011 deficiency (CPHD). We reported a 3-generation family consisting of 18 members, including 5 affected males Accepted 21 September 2011 (the proband, his 2 brothers, his cousin, and his maternal uncle; III1–III4, II8) suffered with CPHD. MRI of the Available online 6 October 2011 pituitary gland showed hypoplasia of the pituitary gland in affected members. By 19 STR markers and linkage analysis, we found that the disease gene localized between the DXS987 and DXS1226 markers (LOD Keywords: score=2.408, θ=0). All affected male patients inherited the same haplotype from the female carrier (I4). Growth hormone Hypothyroidism The proband's mother (II4) and her sister (II3, II6) were obligate female carriers. However, the unaffected fi X-linked males (II7,II9) in the family did not have this haplotype. These observations con rm a new X-linked recessive Genetics inherited disease in a Chinese family with CPHD and the pathogenic gene is mapped to Xp22.1–Xp11. © 2011 Elsevier Inc. All rights reserved.

1. Introduction 2. Results

Combined pituitary hormone deficiency (CPHD) is associated with The proband (III1), his 2 brothers (III2 and III3), and his cousin deficiencies of growth hormone (GH) and at least 1 of the other 5 an- (III4) were all confirmed to have CPHD (hypothyroidism and GH defi- terior pituitary hormones. Genetic mutations, including mutations in ciency). The proband's maternal uncle (II8) had GH deficiency (GHD). several autosomal genes (POU1F1, HESX1, PROP1)[1–3], have been The subjects had no distinctive facial characteristics and exhibited identified in a modest proportion of patients with CPHD. Over the normal adrenal function. The MRI scans of the pituitary gland past decade, many cases of X-linked recessive familial CPHD have revealed hypoplasia of the anterior pituitary gland. The pituitary also been reported. X-linked combined pituitary hormone deficiency stalk was thin and difficult to be visualized in subjects III2 and III3 has been previously linked to specific loci. Duplications at locus (Fig. 1). The proband (III1) and his brother (III2) exhibited mild men- Xq22–q27.2 result in panhypopituitarism or mental retardation with tal retardation (IQ of 55 and 68, respectively, Table 1). isolated growth hormone deficiency [4–6], while duplications at the The STR markers distributed along the X chromosome were used locus Xq21.3–q22 (BTK gene) result in hypogammaglobulinemia for analysis. We noted that the highest LOD scores (2.408 at θ=0) with isolated growth hormone deficiency.[7] Due to the additional were obtained for the DXS987 and DXS1226 markers. Four of the molecular heterogeneity, the inherited basis of many cases of familial affected male subjects (III1–III4) inherited the same haplotype CPHD remains unclear[8]. Here, we report a new X-linked recessive (DXS987-G and DXS1226-G) from their maternal grandmother (I4). inherited disease in a Chinese family with CPHD and the location of The other affected male (II8) inherited this haplotype (DXS987-G the candidate gene domain. and DXS1226-G) from his mother (I4). However, unaffected males (II7,II9) in the family did not have this haplotype. Subject II4 and her sisters (II3,II6) were obligate female carriers; one of their two chromosomes contained the affected haplotypes (DXS987-G and DXS1226-G) (Figs. 2, 3). So the linkage analysis showed that DXS987 and DXS1226 loci had a strong linkage sign, the candidate gene mostly located from fi ☆ This work was supported by the Zhejiang Health Bureau Fund (2010QNA013) and DXS987 (Xp22.1) to DXS1226 (Xp21.3) and con rmed map between Project 81100552 supported by NSFC. We all confirm that neither the manuscript nor DXS8051 (Xp22.3) and DXS1068 (Xp11). any part of it has been published or is being considered for publication elsewhere. The authors participated sufficiently in the work to take public responsibility for its 3. Discussion content and declare there is not a conflict of interest. ⁎ Corresponding author at: 57 Zhugan Xiang, Hangzhou 310003, China. Fax: +86 571 87033296. In this study, we described the clinical characteristics of an X- E-mail address: [email protected] (L. Liang). linked form of CPHD (thyroid hormone and growth hormone

(II8) only had GHD, and III1, III2 also showed mild mental retardation. MRI analyses of their pituitary glands showed hypoplasia of the anterior pituitary gland, and the pituitary stalk was thin and difficult to be visualized. Preliminary linkage analysis (LOD score=2.408) revealed that the disease gene was mapped to Xp22.3–Xp11. To our knowledge, this is the first time to find CPHD or GHD with recessive X-linkage in a family associated with genes on Xp22.3–Xp11. The various phenotypes of this family maybe due to gene mutations on Xp22.3–Xp11

resulting in hypoplasia of the anterior pituitary. The proband (III1) and his brother (III2) had only mild mental retardation without other hypothyroidism symptoms and his uncle (II8) only had GHD. Therefore, we presume it is heterogeneity that affects pituitary cell S secreting growth hormone more than thyroid stimulating hormone AP (TSH). Further studies should be carried out on identiﬁcation of the genes on Xp22.3–Xp11 and its products, which will advance our un- derstanding of this disabling heritable disease. By advancing the un- derstanding, treatment or prevention strategies are possible to be formulated for CPHD associated with these genetic diseases in future.

4. Subject and methods

Fig. 1. MRI scan representative of X-linked recessive combined pituitary hormone de- 4.1. Clinical studies

ficiency in subject III3. Sagittal MRI scans of subject III3 showed hypoplasia of the anterior pituitary gland (AP). The pituitary stalk (S) was thin and difficult to visualize due The studied 3-generation family is consisted of 18 members, in- to its severe attenuation. cluding 5 affected males. The pedigree was consistent with an X- linked recessive condition (Fig. 1). No distinctive facial characteristics were apparent in the affected males. The pituitary gland was studied deficiency) in a Chinese family. It should be noted that an LOD score by magnetic resonance imaging (MRI). of 2.408 is of significance for an X-linked trait. However, it is not def- Subject III , i.e., the proband, was diagnosed with secondly hypo- initely significant because less than 3.0. Therefore these results are 1 thyroidism at the age of 7 years and received levothyroxine sodium preliminary. Further families or expansion of this pedigree are re- as a thyroid hormone substitute. He presented with short stature quired to confirm this finding. with a height of 92 cm and a height standard deviation score (SDS) CPHD is characterized by deficiencies in GH and at least one more of −5.6. His bone age was only 4.8 years. Peak GH levels during pituitary hormone. The molecular basis of pituitary development is the insulin tolerance and arginine stimulation tests were 0.1 ng/ml still not clear. A complex genetic cascade dictates organ commitment, (normalN10 ng/ml), which determined by ELISA. The 2-year growth cell differentiation, and cell proliferation within the anterior pituitary. rate was 2 cm/year (b3rd percentile). The patient received recombi- Mutations in these genes, including HESX1, LHX3, LHX4, PROP1,and nant human GH (rhGH) as a GH substitute. After 2 years of therapy, POU1F1, have been implicated in the etiology of hypopituitarism and his height reached 112 cm (−2.2 SDS), and his growth rate increased exhibiting autosomal dominant and/or recessive inheritance [8,9]. To to 10 cm/year. our knowledge, no gene mutations between Xp22.3 and Xp11 had Subject III , the proband's brother, was diagnosed with hypothy- been reported to be the cause of CPHD or GHD. In the past several de- 2 roidism at 6 years of age. His height was 78 cm (−8.5 SDS), and his cades, many studies of linkage families found that GHD was associated bone age was only 1 year. The growth rate of III was 2 cm/year for with X-linked recessive inheritance. Conley ME et al. relied on cytoge- 2 2 years. He was treated with levothyroxine sodium, and normal thy- netics and linkage analyses to study two unrelated families in which af- roid function was restored soon thereafter. This subject underwent 2 fected males presented isolated growth hormone deficiency; however, different GH provocative tests at the age of 6.5 years, and the findings the immunologic findings were indistinguishable from those of typical of the 2 tests revealed GHD. After 6 months of rhGH therapy, his X-linked agammaglobulinemia (XLA) revealing that the mid-portion height increased to 84 cm (−7 SDS), and his growth rate increased of the long arm of the X chromosome between DXS3 and DXS94 in- to 12 cm/year. When subject III , the proband's second brother, was volved the XLA gene and caused the GHD in combination with XLA 3 diagnosed with hypothyroidism at 1.5 years of age, the family under- [10]. Recently, Stewart et al. reported that the MEF gene (also referred went medical investigation. Subject III presented with a height of to as E74-LIKE FACTOR, ELF4, or gene map locus: Xq26) mutation may 3 be responsible for X-linked hypogammaglobulinemia combined with isolated GHD [7,11]. The SHOX gene (location: Xp22.33; Yp11.3) be- longs to the paired homebox family and is located in the pseudoautoso- Table 1 mal region 1 (PAR1) of the X and Y chromosomes. Defects in this gene Summary of clinical data obtained from 5 males affected with X-linked recessive combined pituitary hormone deficiency. are associated with idiopathic short stature and Leri–Weill syndrome [12–14]. Gene SOX3 (location: Xq27.1) encodes a number of transcrip- Subject Chronological Height GH Thyroid Gonadal IQ a tion factors of the SOX family; mutations in this gene have been associ- age (years) SDS deficiency hormone function score ated with X-linked mental retardation with GHD [4,5,15–17]. An III1 7.4 −5.6 + Low Pre-puberty 55 − expansion of a polyalanine tract (by 11 alanines) within the transcrip- III2 6 8.5 + Low Pre-puberty 68 − tion factor SOX3 (Xq27.1) has been reported in patients with GHD III3 1.5 4.8 + Low Pre-puberty U III 2.5 −6.7 + Low Pre-puberty U and variable learning difficulties [17]. Over- and under-dosage of 4 II8 21 −9.1 + Normal Puberty U SOX3 as well as gene copy number variation are all associated with hy- U: IQ was not evaluated. popituitarism or CPHD [18,19]. All subjects had GH deficiency and their serum ACTH, cortisol, and prolactin concentrations In our study, the 3-generation family contained four males affected were normal. a with GHD combined with hypothyroidism (III1, III2, III3, III4), one male All subjects had normal serum TSH. 442 C.L. Wang et al. / Genomics 98 (2011) 440–444 C.L. Wang et al. / Genomics 98 (2011) 440–444 443

DXS DXS DXS 987 1226 1214

DXS 1068

DXS 8051

Fig. 3. Linkage analysis: parametric analysis, model: recessive_model. The highest LOD scores, 2.408 at θ=0, were obtained for the DXS987 and DXS1226 markers. LOD scores of DXS8051 and DXS1068 were −13.592 and −1.848. So DXS987 and DXS1226 loci had a strong linkage sign, the candidate gene mostly located from Xp22.1 to Xp21.3 and conﬁrmed map from DXS8051 (Xp22.3) to DXS1068 (Xp11).

66 cm (−4.8 SDS). After levothyroxine sodium therapy restored nor- DXS8091, and DXS1073 (Table 2). Blood samples were assayed for STR mal thyroid function, GHD was conﬁrmed at the age of 2 years. Subject markers and divided into 4 panels. Primers with a 5′ FAM ﬂuorescent

III4, the proband's cousin, was diagnosed with hypothyroidism and label were designed online by using Primer3 software (http://frodo.wi. GHD at 2.5 years of age. His height was 68 cm (−6.7 SDS). In the mit.edu/cgi-bin/primer3). Polymerase chain reaction of the ﬂuorescent familial medical investigation, subject II8, the proband's maternal ampliﬁed length polymorphism was performed according to the follow- uncle, was diagnosed with GHD without hypothyroidism at 21 years ing protocol. Each panel contains a 20-μl reaction mixture, including of age. His presenting height was 114.5 cm (−9.1 SDS) and bone age 1×HotStarTaq buffer, 3 mM Mg2+, 0.3 mM dNTP 1 U HotStarTaq poly-

13 years, and he exhibited G2 stage sex development. Subjects III1 merase (Qiagen Inc.), 1 μl of template DNA and a mixture of primers and III2 were evaluated for verbal and performance intelligence (the primers of the STR markers see in supplementary material): Panel quotient (IQ) and for socioadaptive behavior by the Wechsler Intelli- 1: 0.1 μM DXS1001F/R, 0.1 μM DXS1205F/R, 0.1 μM DXS990F/R, 0.1 μM gence Scale for Children—Revised (WISC-R). The serum ACTH, cortisol, DXS1073F/R, 0.25 μMofDXS1106F/R;Panel2:0.1μM DXS1211F/R, and prolactin concentrations were normal in the 5 affected males. 0.1 μM DXS1047F/R, 0.1 μM DXS1068F/R, 0.1 μM DXS1062F/R, 0.1 μM Written informed consent was obtained from each participant. DXS993F/R, 0.1 μM DXS8091F/R; Panel 3: 0.1 μM DXS991F/R, 0.1 μM The study was approved by the Ethics Committee of the Children's DXS1060F/R, 0.1 μM DXS984F/R, 0.3 μMDXS8051F/R;Panel4:0.1μM Hospital of Zhejiang University School of Medicine. of DXS1226F/R, 0.1 μM DXS986F/R, 0.1 μM DXS1214F/R, 0.25 μMof DXS987F/R. The cycling program was 95 °C for 15 min; 11 cycles of 4.2. Linkage study 94 °C for 20 s, 62 °C per cycle for 40 s with a reduction of 0.5 °C after eachcycle,and68°Cfor2min;24cyclesof20sat94°C,40sat56°C, Genomic DNA was extracted from the venous blood collected from and 2 min at 68 °C; and ﬁnal elongation for 1 h at 60 °C. PCR products the 18 family members (I1–I4,II1–II9,III1–III5). We performed analyses were diluted 1:20 and then genotyped with an ABI3130xl Genetic Ana- with a total of 19 short tandem repeat (STR) markers distributed over lyzer. Two-point linkage and haplotypes of the X chromosome and STR the entire X chromosome, including DXS1060, DXS8051, DXS987, markers were calculated using the MLINK computer program (http:// DXS1226, DXS1214, DXS1068, DXS993, DXS991, DXS986, DXS990, www.sph.umich.edu/csg/abecasis/Merlin/index.html). Analyses were DXS1106, DXS1001, DXS1047, DXS1062, DXS1211, DXS984, DXS1205, performed under the assumption that disease allele frequency was

Fig. 2. (A) Pedigree of the family with combined pituitary hormone deﬁciency located in Xp22.3–Xp11. Affected individuals display GH deﬁciency and hypothyroidism. Haplotypes were constructed by using STR markers distributed over the entire X chromosome. The haplotype segregation is indicated by A, C, D, E, G, H, I, and K. Markers mapping to the region are shown as G. (B) The proband's family. III1, III2, III3 were diagnosed with GHD and hypothyroidism; no distinctive or apparent characteristics were observed except for short stature. 444 C.L. Wang et al. / Genomics 98 (2011) 440–444

Table 2 References STR information. [1] S. Radovick, et al., A mutation in the POU-homeodomain of Pit-1 responsible for STR no. Gene position Genetic D Genetic Repeat unit Panel combined pituitary hormone deficiency, Science 257 (1992) 1115–1118. DECODE Marshfield [2] P.Q. Thomas, et al., Heterozygous HESX1 mutations associated with isolated congenital pituitary hypoplasia and septo-optic dysplasia, Hum. Mol. Genet. 10 DXS1060 Xp22.3 12.71 15.12 (tg)25 3 (2001) 39–45. DXS8051 Xp22.3 – 17.29 (gt)24 3 [3] D. Kelberman, et al., Molecular analysis of novel PROP1 mutations associated with – DXS987 Xp22.1 p21.3 28.2 22.18 (tg)27 4 combined pituitary hormone deficiency (CPHD), Clin. Endocrinol. 70 (2009) DXS1226 Xp22 38.7 27.59 (gt)27 4 96–103. DXS1214 Xp21.2 46.21 33.54 (tg)17 4 [4] M. Lagerstrom-Fermer, et al., X-linked recessive panhypopituitarism associated DXS1068 Xp11 60.25 37.33 (ca)19 2 with a regional duplication in Xq25–q26, Am. J. Hum. Genet. 60 (1997) 910–916. DXS993 Xp11.4 – 42.21 (ac)19 2 [5] M. Raynaud, et al., X-linked mental retardation with isolated growth hormone DXS991 Xp11.21 79.2 52.5 (ac)23 3 deficiency is mapped to Xq22–Xq27.2 in one family, Am. J. Med. Genet. 76 DXS986 Xq21.1 86.84 57.37 (tg)15+(ta)14 4 (1998) 255–261. DXS990 Xq21.32 94.92 60.62 (tg)19 1 [6] N.M. Solomon, et al., Increased gene dosage at Xq26–q27 is associated with X- – DXS1106 Xq22 – 66.58 (ca)20 1 linked hypopituitarism, Genomics 79 (2002) 553 559. DXS1001 Xq24 120.35 75.79 (ca)22 1 [7] D.M. Stewart, L. Tian, L.D. Notarangelo, D.L. Nelson, X-linked hypogammaglobulinemia and isolated growth hormone deficiency: an update, Immunol. Res. 38 (2007) DXS1047 Xq26–q27 – 82.07 (ctat)12+(tg)20 2 391–399. DXS1062 Xq24–qter 141.15 82.84 (tg)20 2 [8] M.T. Dattani, Growth hormone deficiency and combined pituitary hormone defi- DXS1211 Xq26.2 142.03 83.92 (ac)17 2 ciency: does the genotype matter? Clin. Endocrinol. (Oxf) 63 (2005) 121–130. DXS984 Xq26.3 145.8 85.55 (ac)19 3 [9] D. Kelberman, M.T. Dattani, Hypopituitarism oddities: congenital causes, Horm. – DXS1205 Xq23 q27 147.46 87.56 (ac)17 1 Res. 68 (Suppl 5) (2007) 138–144. DXS8091 Xq28 167.3 96.14 (ca)18 2 [10] M.E. Conley, A.W. Burks, H.G. Herrod, J.M. Puck, Molecular analysis of X-linked DXS1073 Xq27 188.22 102.35 (tg)20 1 agammaglobulinemia with growth hormone deficiency, J. Pediatr. 119 (1991) 392–397. [11] D.M. Stewart, L. Tian, L.D. Notarangelo, D.L. Nelson, Update on X-linked hypogammaglobulinemia with isolated growth hormone deficiency, Curr. Opin. Allergy Clin. Immunol. 5 (2005) 510–512. 0.00001 and the model of inheritance was X-linked recessive with a [12] E. Rao, et al., Pseudoautosomal deletions encompassing a novel homeobox gene 100% penetrance for males. cause growth failure in idiopathic short stature and Turner syndrome, Nat. Genet. 16 (1997) 54–63. [13] T. Ogata, et al., SHOX nullizygosity and haploinsufficiency in a Japanese family: implication for the development of Turner skeletal features, J. Clin. Endocrinol. Acknowledgments Metab. 87 (2002) 1390–1394. [14] G. Rappold, et al., Genotypes and phenotypes in children with short stature: clinical indicators of SHOX haploinsufficiency, J. Med. Genet. 44 (2007) 306–313. The authors thank the patients for their cooperation with this work. [15] B.C. Hamel, et al., Familial X-linked mental retardation and isolated growth hor- We also thank Center for Human Genetics Research, Shanghai Genesky mone deficiency: clinical and molecular findings, Am. J. Med. Genet. 64 (1996) Bio-Tech CO., Ltd for supporting the genetic research. This work was 35–41. [16] F.A. Hol, et al., Identification and characterization of an Xq26–q27 duplication in a supported by the Zhejiang Health Bureau Fund (2010QNA013) and Pro- family with spina bifida and panhypopituitarism suggests the involvement of two ject 81100552 supported by National Natural Science Foundation of distinct genes, Genomics 69 (2000) 174–181. China. [17] F. Laumonnier, et al., Transcription factor SOX3 is involved in X-linked mental retardation with growth hormone deficiency, Am. J. Hum. Genet. 71 (2002) 1450–1455. [18] K.S. Woods, et al., Over- and underdosage of SOX3 is associated with infundibular Appendix A. Supplementary data hypoplasia and hypopituitarism, Am. J. Hum. Genet. 76 (2005) 833–849. [19] S. Dateki, et al., Mutation and gene copy number analyses of six pituitary transcription factor genes in 71 patients with combined pituitary hormone deficiency: Supplementary data to this article can be found online at doi:10. identification of a single patient with LHX4 deletion, J. Clin. Endocrinol. Metab. 95 1016/j.ygeno.2011.09.002. (2010) 4043–4047.