Unique near-complete deletion of GLI2 in a patient with combined pituitary hormone deficiency and post-axial polydactyly

Growth Hormone & IGF Research 50 (2020) 35–41

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Unique near-complete deletion of GLI2 in a patient with combined pituitary hormone deficiency and post-axial polydactyly

T

⁎

Melitza S.M. Elizabethb, ,1, Annemieke J.M.H. Verkerke,1, Anita C.S. Hokken-Koelegac,f, Joost A.M. Verlouwe, Jesús Argenteg,i, Roland Pfaeffleh, Theo J. Visserd,2, Robin P. Peetersa,d, Laura C.G. De Graaffa,c a

Erasmus MC Rotterdam, Department of Internal Medicine, Subdiv. Endocrinology, the Netherlands Dutch Growth Research Foundation, Rotterdam, the Netherlands c Erasmus MC Rotterdam, Academic Center for Rare Growth Disorders, the Netherlands d Erasmus MC Rotterdam, Academic Center for Thyroid Diseases, the Netherlands e Erasmus MC Rotterdam, Dept of Internal Medicine, Genetic laboratory, the Netherlands f Erasmus MC Rotterdam, Dept of Pediatrics, Subdiv. Endocrinology, the Netherlands g Hospital Infantil Universitario Niño Jesús, Department of Endocrinology, Instituto de Investigación La Princesa, Universidad Autónoma de Madrid, Department of Pediatrics, Madrid, Spain h Hospital for Children and Adolescents, University of Leipzig, Pediatrics, Germany i CIBER de Fisiopatologia de la Obesidad y Nutriciόn (CIBEROBN), Instituto de Salud Carlos III, IMDEA Food Institute, CEIUAM+CSIC, Madrid, Spain b

A R T I C LE I N FO

A B S T R A C T

Keywords: Pituitary gland [MeSH] Transcription factors [MeSH] Gene deletion [MeSH] Polydactyly [MeSH] Brain [MeSH]

Introduction: Combined pituitary hormone deficiency (CPHD) can cause a broad spectrum of health problems, ranging from short stature only, to convulsions or even death. In the majority of patients, the cause is unknown. Methods: The idex case had unexplained CPHD, pituitary anomalies on MRI and polydactyly. In the patients and her unaffected parents, we performed SNP array analysis and Whole Exome Sequencing, after candidate gene analysis turned out negative. Results: We found a unique de novo heterozygous 229.9 kb deletion in the index case on chr. 2q14.2. This deletion covered 12 out of the 13 coding exons of the GLI2 gene, a transcription factor involved in midline formation and previously associated with CPHD. As reported GLI2 deletions and mutations show a large phenotypic variability, we performed a genotype-phenotype analysis. This revealed that GLI2 missense mutations usually present with a ‘ppp-only’ phenotype (pituitary anomalies ± postaxial polydactyly without brain phenotype), whereas the ‘ppp-plus’ phenotype (with major brain malformations and/or intellectual disabilities) is more frequent in patients with larger deletions, and those with frameshift mutations/point mutations or splice variants resulting in a stop codon (p < .001). Conclusion: The present case shows that a deletion of the GLI2 gene only (not affecting any of the adjacent genes) causes pituitary anomalies without brain phenotype. This suggests that brain phenotype only occurs when additional genes adjacent to GLI2 are deleted, or when mutations result in truncated GLI2 mRNA/protein. However, due to the lack of functional data for many GLI2 mutations and based on the available information regarding variable penetrance, phenotype-genotype correlations need to be made with caution.

1. Introduction Pituitary hormone deficiency (PHD) refers to the diminished secretion or absence of one or more of the anterior pituitary hormones. It

can be either acquired (due to head trauma, radiation, brain tumors or surgery) or congenital, and it can be either isolated or a combination of deficiencies (Combined Pituitary Hormone Deficiency or CPHD) [1,2]. CPHD is a rare condition and its prevalence is estimated to be 1 in

⁎ Corresponding author at: Dept. of Internal Medicine, Rg526, Erasmus MC, University Medical Center, Dr Molenwaterplein 40, 3015 GD Rotterdam, the Netherlands. E-mail address: [email protected] (M.S.M. Elizabeth). 1 These authors contributed equally to this manuscript. 2 In honour of Theo J. Visser, who deceased during the preparation of this manuscript.

https://doi.org/10.1016/j.ghir.2019.10.002 Received 3 July 2019; Received in revised form 26 September 2019; Accepted 4 October 2019 Available online 17 October 2019 1096-6374/ © 2019 Published by Elsevier Ltd.

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TruSeq DNA Library preparation (Illumina, Inc., San Diego, CA) was performed on a Caliper Sciclone NGS workstation (Caliper Life Sciences, Hopkinton, MA), followed by exome capture using the Nimblegen SeqCap EZ V2 kit (Roche Nimblegen, Inc., Madison, WI). This capture targets 44 Mb of exonic regions covering 30,246 coding genes, 329,028 exons and 710 miRNAs. Paired-end 2 × 100 bp sequencing was performed at 6 samples per lane on Illumina HiSeq2000 sequencer using Illumina TruSeq V3, resulting in 6Gb of sequencing data.

8000 individuals worldwide [3]. Patients with CPHD show a wide variation in phenotype, depending on the amount and combination of hormones that are deficient. Symptoms include neonatal hypoglycaemia, prolonged neonatal jaundice, micropenis in boys (due to prenatal lack of gonadotropins), short stature, truncal obesity, delayed puberty, and, if the pituitary-adrenal axis is affected, it can even lead to death [4]. Understanding the etiology of CPHD is important for anticipation of clinical problems, for genetic counselling and for the development of future strategies to prevent or treat the disease. Previous genetic studies have reported 30 genes involved in the pathogenesis of CPHD [5]. However, only a minority of the cases of CPHD can be explained by mutations in genes like PROP1 (MIM 601538), HESX1 (MIM 601802), POU1F1 (MIM 173110), LHX3 (MIM 600577), LHX4 (MIM 602146), OTX2 (MIM 600037), GLI2 (MIM 165230), SHH (MIM 600725) and HHIP (MIM 606178). In most cases of CPHD, mutation screening of candidate genes remains negative [6–10]. Glioma-associated oncogene family zinc finger 2 (GLI2) belongs to the GLI-Krupel family of zinc-finger transcription factors. GLI2 is located on chromosome 2q14.2 and consists of a 6.8 kb coding sequence that extends 13 exons and encodes a 1586 amino acid protein [11]. GLI2 is the primary mediator in the Sonic Hedgehog (Shh) signaling pathway, which is crucial for the formation of central nervous system midline and pituitary development [12]. GLI2 is a large and polymorphic gene [11]. Various deletions and mutations have been reported in GLI2, with a large phenotypic variability. To date, it is not clear whether this large phenotypic variability is due to the location of the mutations, the size of the deletion and/or the involvement of adjacent genes. We performed SNP array analysis and Whole Exome Seguencing (WES) in a patient with unexplained CPHD, postaxial polydactyly and pituitary anomalies on MRI. We found a unique near-complete deletion of GLI2, which did not affect any adjacent genes. This deletion sheds a new light on the phenotypic spectrum associated with GLI2 defects.

3.3. Copy number variant analysis by SNP array DNA was hybridized to Illumina Human CytoSNP850K SNP arrays according to standard protocol. Copy number analysis was performed using Nexus 8.0 from BioDiscovery. 3.4. Determining DNA copy number by calculating WES read count ratios Read counts from WES data were evaluated per exon for GLI2 and 2 adjacent genes, IHNBB, upstream and TFCP2L1, downstream of GLI2. As controls, four non-related unaffected individuals, (two males and two females) were used without DNA abnormalities. Readcounts were normalized by total sample yield in Gb. Read counts for the four normal control samples were averaged and used to calculate read count ratios for the patient compared to these controls, and for the separate parents compared to these controls. A ratio approaching 1 indicates presence of both alleles, a ratio approaching 0.5 corresponds with a heterozygous deletion. In the capture kit that we used for WES, no probes were present for the first, non coding exon (exon1) because it is considered as not functionally relevant. 3.5. Genotype-phenotype analysis

3. Material and methods

In order to perform genotype-phenotype analysis, we summarized previously reported GLI2 mutations and large deletions including GLI2. We made an overview of polydactyly and brain phenotype for both deletions and mutations. Patients were classified as having ‘brain phenotype’ when they had intellectual disability (developmental delay), or when one or more of the following brain imaging anomalies was reported (apart from pituitary defects): complete or partial agenesis of the corpus callosum, corpus callosum hypoplasia, myelinisation disturbance, ventriculomegaly, white matter anomalies, cavum septum pellucidum, absent septum pellucidum, hypoplastic septum, microcephaly, holoprosencephaly, asymmetric brain hemispheres, widening of temporal horn, pontocerebellar hypoplasia, temporobasal dysgyria and/or schizencephaly. Chiari I malformation (CM) is a common incidental finding on brain imaging and often asymptomatic [13]. Therefore, Chiari type 1 malformations are not considered ‘brain phenotype’. Ppp-only phenotype was defined as the presence of pituitary anomalies (with or without postaxial. polydactyly), without brain phenotype. Ppp-plus phenotype was defined as the presence of pituitary anomalies (with or without postaxial. polydactyly) with accompanying brain phenotype.

3.1. DNA isolation

3.6. Statistical analysis

Genomic DNA of the patient and her parents was extracted from peripheral whole blood samples according to standard procedures.

The relation between genotype and phenotype was analysed by Chisquared test using SPSS 22.0.

3.2. Whole exome sequening

4. Results

Genomic DNA was fragmented into 200–400 base pairs (bp) fragments using Covaris Adaptive Focused Acoustics shearing according to the manufacturer's instructions (Covaris, Inc., Woburn, MA). Illumina

DNA samples of the patient as well as of her parents were analysed by both SNP array and whole exome sequencing. SNP array analysis revealed a de novo heterozygous 229,9 kb deletion in the index case on

2. Patient The index case was a girl of Danish origin, with unexplained CPHD. She had characteristics associated with GH deficiency, like a prominent forehead and truncal obesity, as well as postaxial polydactyly in both hands. Cognitive functioning was normal. The father of the patient had a height of 184 cm and had no signs of hypopituitarism. The mother had a height of 161.8 cm and was also healthy. Although the original laboratory values and MRI images of the girl were no longer available, the medical file reported that all pituitary hormone levels were low and magnetic resonance imaging (MRI) revealed infundibular hypoplasia. At the age of 3 years, the patient had short stature with a height of 78.5 cm (−4.2 SDS) and low growth velocity. GH values during stimulation test were very low (GH peak 1.9 mUI/L, reference value > 20 mIU/L), after which GH treatment was started. She also received pituitary hormone replacement therapy for all other pituitary axes. Growth velocity improved drastically after the initiation of GH treatment and, at the age of 15 years, the patient had reached a normal adult height of 164.1 cm (+0.3 SDS).

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Fig. 1. Deleted area on chromosome 2 (A) Summery overview of Chr.2q14.2-2q14.3 region in father, mother and index. Deleted area shown in red. (B) Zoomed in overview of Chr.2q14.2-2q14.3 of index (deleted region shown in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

genes INHBB and TFCP2L1 also indicated presence of both alleles. This means that only exon 3–14 of GLI2 were deleted. Detailed read counts are shown in Supplementary Table 1. In order to view our data in the light of the existing literature, we summarized all large GLI2 deletions (Fig. 3a) and mutations (Fig. 3b) published to date [12,14–28]. An overview of polydactyly and brain phenotypes (major brain malformations and/or intellectual disability) is shown in Table 1 for deletions and in Fig. 3b for mutations.

chr. 2q14.2, which affected exon 3–14 of the GLI2 gene (MIM 165230; NP_005261), but none of the adjacent genes (Fig. 1). The largest transcript of GLI2 (Ensembl ENST0000452319.1) contains 14 exons. The first exon is 30 bp only (chr2:121.549.985–121.550.014, build 37) and non-coding. The other 13 exons (exons 2–14) are protein coding. Exon 2, the first coding exon (chr2: 121.554.867–121.555.044, build 37) contains the ATG protein translation initiation site. The deletion of the index case spans from 121.557.260 to 121.787.132, which means it starts after the first coding exon (exon 2) and ends downstream of GLI2. As a result, all coding exons except exon 2, are deleted. Normalized read counts from WES data of GLI2 and two adjacent genes IHNBB and TFCP2L1 confirmed the (near-complete) deletion of GLI2 (Fig. 2). The read count ratios of coding exons 3–14 showed an average ratio of 0.5 (indicating a heterozygous deletion) and read count ratio of the first GLI2 coding exon (exon 2) was 0.87 (indicating the presence of both alleles). The average read count ratios of the adjacent

5. Discussion We performed Whole Exome Sequencing (WES) and SNP array analysis in a female patient with unexplained CPHD, postaxial polydactyly, pituitary stalk hypoplasia and normal cognition, and her unaffected parents. With the SNP array we found a de novo 229 Kb heterozygous deletion at 2q14.2 (Fig. 1). The deletion included 12 out of 13 coding exons of the GLI2 gene, but deleted none of the neighbouring 37

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Fig. 2. Normalized read count ratios of separate exons of genes INHBB (green), GLI2 (red) and TFCP2L1 (blue) of the index patient, compared to four averaged normal controls. A ratio of 1 indicates presence of both alleles, a ratio of 0.5 corresponds to a heterozygous deletion. Probes for the non-coding first exon (exon 1) of GLI2 are not present in the capture kit used for WES. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

better understand the relation between GLI2 genotype and corresponding phenotypes, we visualized the previously reported GLI2 deletions and mutations. Our summary on genotype-phenotype relations resulted in five remarkable findings. First, the ppp-only phenotype (pituitary anomalies ± postaxial polydactyly but no brain anomalies) was most prevalent among patients with (missense) point mutations. Second, the ppp-plus phenotype (pituitary anomalies ± postaxial polydactyly, with major brain malformations or intellectual disabilities) was most prevalent in patients with large deletions. Third, the vast majority (all but two) of patients with brain malformations or intellectual disability had polydactyly, whereas polydactyly was far less prevalent among patients without brain phenotype. The fourth remarkable finding was that frameshift mutations and point mutations, which result in a stop codon, were strongly associated with the ppp-plus-phenotype. Bear et al. reported that individuals with truncating mutations were more likely to have only pituitary anomalies and polydactyly [21]. However, our summary shows that half of the frameshift mutations (6/12) and point mutations (4/7) resulting in a stop codon, were associated with brain anomalies. Considering the mechanism underlying the apparently aggravating effect of truncating mutations, one could speculate that this could be caused by interference at cellular level. Normally, abnormal mRNAs containing premature stop codons (Premature Termination Codons or PTCs) are degraded by the nonsense-mediated mRNA decay (NMD) system [36,37]. However, some PTC-containing mRNAs can escape from the NMD system (NMD-escape) [38,39]. In that case, the escaping abnormal mRNA would result in a truncated GLI2 protein, which might interfere with action of the wild-type protein. However, this is still a hypothetical mechanism which should be subject of future studies.

genes. WES Read count analysis showed hemizygozity for coding exons 3–14 (Fig. 2), which confirmed that only one out of 13 coding exons was present in the normal, homozygous state. The current finding of the near-complete deletion of GLI2 gene, but none of the neighbouring genes, sheds a new light on the phenotypic spectrum associated with GLI2 defects. Hovinga et al. 2018 showed that the phenotype caused by a same GLI2 mutation within the same family, can vary drastically. This phenotypic variability makes it difficult to predict the exact impact of each individual GLI2 mutation. Initially, mutations in GLI2 were thought to be strictly related to holoprosencephaly (HPE) [29–33]. GLI2 mutations were first identified in patients with HPE and patients with manifestations suggestive of midline neurodevelopmental anomalies [20,21,24]. Recent findings, however, suggest that only 2% of mutations within GLI2 are truly related to HPE [28]. Other GLI2 mutations correspond to a phenotype of pituitary insufficiency without HPE, which can coincide with polydactyly and/or facial variants [34,35]. Several point mutations in the GLI2 gene have been identified in patients with hypopituitarism, but deletions affecting the GLI2 region are rare. The few deletions reported were either small intra-exonic deletions or large deletions affecting several Mb. The smallest reported deletion affecting the entire GLI2 gene, is a 1.3 Mb heterozygous deletion, containing GLI2 and five neighbouring genes: EPB4.1 L5 (MIM 611730), RALB (MIM 179551) TMEM185B, INHBB ((MIM147390) and TFCP2L1 (MIM 6097850) [21]. Bear et al. reported a smaller 118 kb deletion, but this deletion affected only part of GLI2 [29]. Various authors provided tables with clinical findings available for patients with GLI2 mutations and deletions [16,19,22,23]. In order to 38

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Fig. 3. GLI2 deletions (3a) and mutations (3b) reported to date, with the corresponding data regarding pituitary anomaly, polydactyly, major brain malformations and/or intellectual disability. Figure adapted from Bear et al. (2014), Gregory et al. (2015) and Goumy et al. (2016) [16,19,22].

eukaryotic genomes (from yeasts to humans), where they help fine-tune the expression levels of target genes leading to phenotypic diversity [40]. Knowing this, we hypothesize that mutations changing a Glutamine (Q) into a stop codon, or those changing a non-Q into a Q, may affect GLI2 transcriptional activation and have a direct or indirect effect on phenotype. Future research is needed to investigate the exact pathophysiological mechanism by which glutamine residues in the GLI2 activator domain affect phenotype. In conclusion, we found a de novo heterozygous 229,9 kb deletion in the index case on chr. 2q14.2 deleting 12 of the 13 coding exons of the GLI2 gene, but none of the adjacent genes. Our subsequent

The last interesting finding was that, within the activator domain, 60% of all mutations in which a Glutamine (Q) residue was involved, were associated with ppp-plus phenotype (3/5). This was remarkable, because only 28% (11/40) of the missense mutations affecting other (non-Q) residues displayed the ppp-plus phenotype. Thus, mutations changing a Glutamine (Q) into a stop codon, or those changing a non-Q into a Q, appeared to be associated with brain anomalies more often than non-Q related mutations. However, the number of mutations available was small and did not reach statistical significance (p = 0,17). It is known that glutamine residues can affect transcription regulation; glutamine-rich repeats are enriched in transcriptional regulators in 39

Deleted region

Size of deletion

40

aCGH/FISH aCGH Cytogentics/SNP aray aCGH SNP array/MLPA aCGH

Maternal NA Maternal NA

de novo

de novo de novo de novo maternal paternal paternal

de novo NA

NA de novo paternal

de novo de novo

Origin

Small ectopic pituitary, Chiari I malformation Normal brain unknown Normal brain

pituitary infundibular hypoplasia

Ventriculomegaly Small AP with normal PP and stalk. White matter anomalies, cavum septum pellucidum NA Normal transcranial ultrasound − Partial ACC Temporal myelinisation disturbance Pituitary anomaly

ACC ACC, Dandy Walker malformation NA

ACC −

Brain features

+ − + −

+

+ NA − NA NA −

NA −

NA NA −

NA NA

GHD

+ + + NA

+

+ + − − − −

multiple (mild) multiple

single incisor multiple

Midfacial hypoplasia Midfacial hypoplasia normal cleft lip and palate

+, multiple

+, +, − +, +, +

+ hypertelorism, megalocornea

− NA

− − −

+, Cleft lip and palate +, multiple including cranial synostosis +, multiple + +

Facial dysmorphisms

+ −

PD

aCGH = array comparative genomic hybridization, ACC = agenesis of corpus callosum, GHD = growth hormone deficiency, PD = post-axial polydactyly or post-axial skin tags, AP = anterior pituitary, PP = posterior pituitary, NA = not available.

sequencing sequencing sequencing sequencing

DNA DNA DNA DNA

20 Mb 19 Mb 6.6 Mb 5.8 Mb 4,3 Mb 1,3 Mb SNP array/WES array/WES

2q14.2–22.1 2q14.1–q22.1 2q14.1-q14.3 2q14.1q14.3 2q14.2-2q14.3 2q14.2

Gustavson et al. [21] Greally et al. [22] Niida et al.[2017] Goumy et al. [23] Kordaß et al. [24] Kevelam et al. [25]

chr band 26.6 Mb

karyotyping karyotyping karyotyping, FISH, microsatellite analysis aCGH DNA sequencing

Deletions affecting smaller part OF GLI2 Bear et al. [26] 2q14.2 118.3 kb Bear et al. [26] 2q14.2 118.3 kb Bear et al. [26] 2q14.2 118.0 kb Bear et al. [26] 2q14.2 151 kb

2q14.2–21.3 2q12.3-q21.3

Peng et al. [19] Gregory et al. [20]

chr band chr band chr band

karyotying karyotyping

Method

Deletion affecting almost complete GLI2 Present case [2018] 2q14.2 229 kb

2q14-q21 2q13-q21 2q14.1-q21

Frydman et al. [1989] Davis et al. [16] Baker et al. [18]

Complete GLI2 deletion as part of larger deletion Antich et al. [14] 2q12q14 chr band Lucas et al. [15] 2q14q21 chr band

Publication

Table 1 Overview of the major clinical findings available for patients with large 2q14 deletions including GLI2 (adapted from Kevelam et al. and Kordaß et al. [19,20]).

M.S.M. Elizabeth, et al.

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M.S.M. Elizabeth, et al.

genotype-phenotype analysis of reported GLI2 defects showed that GLI2 missense mutations usually present with a ‘ppp-only’ phenotype (pituitary anomalies ± postaxial polydactyly without brain phenotype). In contrast, patients with larger deletions, and those with frameshift mutations/point mutations or splice variants resulting in a stop codon, usually have major brain malformations or intellectual disabilities (‘ppp-plus’ phenotype)(p < 0,001). The deletion found in our index case shows that deletion of the GLI2 gene only (not affecting any of the adjacent genes), also presents with the ppp-only phenotype. This suggests that brain problems predominantly occur when additional genes adjacent to GLI2 are deleted, or in case of (yet hypothetical) interference of truncated GLI2 mRNA/protein with wild-type. However, due to the lack of functional data for many GLI2 mutations and based on the available information regarding variable penetrance, phenotype-genotype correlations need to be made with caution. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ghir.2019.10.002.

[11] [12]

[13]

[14]

[15]

[16] [17] [18]

Funding

[19]

For this project, we received financial support from Pfizer.

[20]

Author disclosure summary [21]

ME, AHK, AV, JA, JV, RPf, an RP have nothing to declare. LdG received an Investigator Initiated Research Grant from Pfizer. [22]

Ethical approval

[23]

Informed consent was obtained from all individuals participating in this study and their parents if they were < 18 years old. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. We obtained approval from the medical ethics committees of all participating hospitals.

[24]

[25]

[26] [27]

Acknoledgements

[28]

We thank the patient and her parents for their kind cooperation. Whole Exome Sequencing and SNP array analysis was supported by a grant from Pfizer.

[29]

[30]

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