Association of Homozygous PROP1 Mutation in a Saudi Family with Combined Pituitary Hormone Deficiency

Background and Objectives: Combined pituitary hormone deficiency (CPHD) is a rare heterogeneous disease. It is characterized by the deficiency of growth hormone (GH) and shortage of at least one or more other hormones of the pituitary gland including thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin. Rare pathogenic variants in nearly 30 genes have been identified as an underlying cause of CPHD pathogenicity. Among these genes, paired-like homeobox 1 (PROP1) has been reported to be the most common cause of CPHD. Materials and Methods: In the present study, we investigated a large family of Saudi origin with three adult sisters suffering from short stature in combination of secondary amenorrhea. Results: Whole-exome sequencing followed by Sanger sequencing shows a homozygous missense variant (NM_006261.5; c.211C > T; p.R71C) in the PROP1 gene segregating with the disease phenotype within the family. In silico analysis studies show that this variant is highly conserved among several orthologues and is predicted as likely pathogenic using various bioinformatics tools. Conclusions: Our finding presents the first Saudi familial case of autosomal recessive form of CPHD caused by the PROP1 variant.


Introduction
Combined pituitary hormone deficiency (CPHD) is a phenotypically variable and genetically heterogeneous disorder. The disease manifestations can occur anywhere between births to puberty. It involves impaired secretion of growth hormone (GH) in association with shortages of other posterior and/or anterior pituitary hormones [1]. Patients with CPHD may exhibit several clinical symptoms such as hypothyroidism, hypocortisolism, short stature, and impaired sexual maturation at the age of puberty. In addition, with or without extra-pituitary phenotypes, some cases might develop hypoplasia of the optic nerve and midline forebrain abnormalities [2,3]. It is reported that 45% of patients with isolated GH deficiency (IGHD), which involves deficiency of GH only, may also develop CPHD over time, particularly a severe type [4].
The frequency of CPHD is estimated to be 1 in 8000 individuals globally [5]. The majority of incidence is caused by non-genetic factors such as brain surgery, infection, irradiation, tumor, chronic poisoning by heavy metals, trauma, and autoimmune diseases. In addition, the condition can be caused by genetic defect in genes associated with the development and maintenance of the anterior part of the pituitary gland [5][6][7][8]. These genetic defects mainly occur spoIt must be radically, however, several familial forms have been reported in different inheritance patterns including autosomal dominant, recessive, and X-linked recessive [9,10]. It is estimated that 5-30% of CPHD cases are familial cases [3,11]. So far, approximately 30 genes have been demonstrated to be implicated in CPHD pathogenesis. Mutations in POU1F1, LHX3, HESX1, LHX4, SOX3, GLI2, and PROP1 are the most frequent causes of CPHD [12]. Most of these genes encode transcription factors involved in normal embryonic pituitary cell differentiation and pituitary organogenesis [13]. PROP1 mutations are the most commonly detected cause of familial CPHD cases, accounting for 30-50%. Nearly 10% of these cases have an affected first-degree relative. Although PROP1 mutations are rare in sporadic CPHD cases, accounting for nearly 6.7% [1,3,14].
The deficiency in Prop1 was initially found in Ames mice leading to dwarf mice and impairment of the level of growth hormones (GH), prolactin (PRL), and/or thyroidstimulating hormone (TSH) [15]. In humans, the clinical features associated with PROP1 gene mutations are extremely variable between individuals from the same family as well as between individuals from different pedigrees in many respects, including the time of disease onset and severity of the disease, function of adrenal gland, onset of puberty signs, and the final height of untreated patients [16].
In the present study, we describe a clinical and genetic characterization of a Saudi family with three sisters exhibiting features of short stature, secondary amenorrhea, and deficiency in pituitary hormones. A homozygous variant in the PROP1 gene was identified in all three affected individuals. This is the first report of a Saudi familial case of an autosomal recessive form of CPHD caused by the PROP1 variant.

Family Recruitment and Ethical Approval
A four-generation family of a Saudi origin including three adult sisters showing symptoms of secondary amenorrhea with short stature were clinically examined at reproductive endocrinology and infertility clinic at Madina Maternity and Children Hospital (MMCH), Medina. History of the disease was obtained from elders of the family and a pedigree was drawn to assess the inheritance of the disorder (Figure 1). The study research was approved by the institutional review board of Taibah University (TUCDREC/27032021) and MMCH (IRB147-2021). Patients signed the written informed and disclosure consent form after receiving an explanation of the study aim to family members in Arabic language. All the experiments in the current research were conducted in accordance with the guidelines of the ethical committee.   A total of 3 mL of peripheral blood samples was obtained from each participant of the  family including three affected (IV-1, IV-5, and IV-6) and three unaffected (IV-2, IV-3, and  IV-4), individuals as well as from both parents (III-1 and III-2) in ethylenediaminetetraacetic acid tubes for genetic analysis. Genomic DNA was extracted using the QiaAmp DNA mini kit (Qiagen cat no.51306) according to manufacturer's protocol. The integrity and purity of DNA was measured using Qubit fluorometer and Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Next-Generation Sequencing
DNA samples of two affected sisters (IV-1 and IV-6) were whole-exome sequenced (WES) and data were analyzed according to the protocol described elsewhere [17]. Briefly, the SureSelect Kit (Agilent, Santa Clara, CA, USA) was used for preparation of DNA libraries and the Illumina Hiseq2000 instrument was used for generating sequence reads (Illumina, San Diego, CA, USA). The online Illumina BaseSpace analysis tool (https:// basespace.illumina.com, accessed on 13 November 2022) was used for standard filtration of variant calling files (VCF) of the two affected participants. Based on family pedigree and consanguineous marriages, it is clear that the disorder depicted an autosomal recessive inheritance pattern. Therefore, only common homozygous and compound heterozygous variants among the two affected participants were filtered. Variants were classified based on the guidelines of ACMG [18].

Sanger Sequencing Validation
After filtration and variant prioritization, specific primers were designed using the Primer 3 tool (http://primer3.ut.ee/ accessed on 15 October 2022) to validate mutated variants. Ensembl genome browser (https://m.ensembl.org/ accessed on 15 October 2022) was used for downloading the reference sequence of genes of interest. Bidirectional Sanger sequencing was performed for all identified variants using basic protocol as previously mentioned. The sequenced reads were aligned with the reference sequence using the BIOEDIT software version 6.0.7 (https://bioedit.software.informer.com, accessed on 13 November 2022) for variant confirmation.

In Silico Analysis
In silico analysis was carried out to predict the pathogenicity effect of identified variants on overall protein structures. Different online tools were used such as Mu-tationTaster (http://www.mutationtaster.org/; accessed on 20 November 2022), Mutation Assessor (http://mutationassessor.org/; accessed on 20 November 2022), VarSome UNIPROT (https://www.uniprot.org (accessed on 28 December 2022)) was used for mapping of the identified mutation to the PROP1 domain. STRING (version 11.5) website (https://string-db.org/; accessed on 1 January 2023) was used for identification interactions network.

Clinical Evaluation
All three affected sisters (IV-1, IV-5, and IV-6) shared similar clinical features of short stature and irregular menstrual cycle. Both parents and the three other healthy siblings had normal growth, normal weight, and normal development of secondary sexual traits, and did not display any abnormal features. The heights of father and mother were 159 cm and 149 cm, respectively. The height range of healthy siblings was between 151 to 163 cm.
Medical history of the three affected sisters showed that they were born after uneventful pregnancies and normal vaginal deliveries, with more than 2.5 kg birth weight. During the early childhood periods, all three patients showed regular developmental milestones with no significant medical complaints.
At the age of 13, the parents observed the growth failure of the first daughter (IV-1) during her admission in middle school. She was referred to an endocrinologist for clinical evaluation. Investigations revealed profound GH deficiency. GH injection and oral contraceptive pills were administered for one year. Follow up showed a 3 cm increase in her height. However, at the age of 16, a menarche with irregular menstrual cycle was observed, but she was, overall, satisfied with the medication. At the age of 34 years, the patient (IV-1) was referred to the endocrinology clinic at MMCH due to secondary amenorrhea with short stature. The hormonal analysis results showed normal follicle-stimulating hormone (FSH) 3. Her affected sibling (IV-5), aged 28 years, complained of an irregular menstrual cycle and short stature. Her height was 137 cm and weight 36.8 kg with normal mental function. She completed her bachelor's degree. At the age of 19, she had spontaneous menarche for two years and at 21 years of age, she developed amenorrhea for one year. The hormonal profile of IV-5 is presented in Table 1. The patient had normal breast development Tanner stage V, and normal pubic and axillary hair. Ultrasonography of the ovaries showed normal echogenicity ovaries with no suspicious cystic or solid lesions. The size of the right ovary was 3.8 cm 3 and the left, 2.2 cm 3 .
The third affected female (IV-6) is currently 23 years old, displaying an irregular menstrual cycle and short stature. Her height and weight is 137 cm and 43.9 kg, respectively. She induced menarche at the age of 23 years of age. The patient had Tanner stage III with normal pubic hair, and she had no axillary hair. The hormonal profile of IV-6 is presented in Table 1.

A Homozygous Variant in the PROP1 Gene Was Discovered
High quality exome sequence reads for individuals (IV-1) and (IV-6) were obtained with more than 100 x coverages. The analysis of the exome data, including annotation of variants, filtration, and prioritization, reveals a homozygous variant C-to-T transition (c.211C > T) in the PROP1 gene ( Figure 2). This homozygous variant in PROP1 was predicted as the most possible pathogenic variant among the family based on the previous studies of the involvement of PROP1 variants in developing CPHD syndrome (Table 2).

A Homozygous Variant in the PROP1 Gene Was Discovered
High quality exome sequence reads for individuals (IV-1) and (IV-6) were obtained with more than 100 x coverages. The analysis of the exome data, including annotation of variants, filtration, and prioritization, reveals a homozygous variant C-to-T transition (c.211C > T) in the PROP1 gene ( Figure 2). This homozygous variant in PROP1 was predicted as the most possible pathogenic variant among the family based on the previous studies of the involvement of PROP1 variants in developing CPHD syndrome (Table 2).
This missense homozygous variant (c.211C > T) in the PROP1 gene is predicted to change amino acid arginine to cysteine (p.R71C) at codon 71 in exon 2 with a CADD score of 24.6. This single nucleotide change is in the N-terminal region of homeodomain. No other pathogenic variants were detected in known CPHD genes.   This missense homozygous variant (c.211C > T) in the PROP1 gene is predicted to change amino acid arginine to cysteine (p.R71C) at codon 71 in exon 2 with a CADD score of 24.6. This single nucleotide change is in the N-terminal region of homeodomain. No other pathogenic variants were detected in known CPHD genes.

Validation of Variants with Sanger Sequencing
The exon 2 of PROP1 gene was sequenced in all available family members by the Sanger approach. The analysis of Sanger reads shows recessively inheritance of variant in a pedigree. Both parents and the three heathy siblings are heterozygous for the mutant allele, whereas all the three affected individuals are homozygous for the variant ( Figure 3A).

Validation of Variants with Sanger Sequencing
The exon 2 of PROP1 gene was sequenced in all available family members by the Sanger approach. The analysis of Sanger reads shows recessively inheritance of variant in a pedigree. Both parents and the three heathy siblings are heterozygous for the mutant allele, whereas all the three affected individuals are homozygous for the variant ( Figure  3A).

Evolutionary Conservation of PROP1 Amino Acid at Position 71
Multiple sequence alignment was performed to identify the evolution of PROP1 protein at position 71 of the amino acid. Sequence alignment shows that this position is highly conserved in various species throughout evolution. The conservation of the arginine acid at this location suggests the importance of this amino acid to the protein structure and function ( Figure 3C).

Protein Network Interaction Analysis of PROP1
Protein-protein interaction (PPI) was used to investigate potential interactions between PROP1 and other associated proteins. Our data in Figure 4 show the network containing 11 nodes and 35 edges with PPI enrichment; p-value 0.00000029. Most notably, PROP1 interacts with different proteins involved in vital biological functions in the pituitary, mammary, and thyroid glands organogenesis including POU1F1, SOX3, POMC, TSHB, PRL, TBX19, GHRHR, and GHRH. Also, the network shows the strong association of PROP1 with other important protein such as furin and catenin beta-1 (CTNNB1).

Evolutionary Conservation of PROP1 Amino Acid at Position 71
Multiple sequence alignment was performed to identify the evolution of PROP1 protein at position 71 of the amino acid. Sequence alignment shows that this position is highly conserved in various species throughout evolution. The conservation of the arginine acid at this location suggests the importance of this amino acid to the protein structure and function ( Figure 3C).

Protein Network Interaction Analysis of PROP1
Protein-protein interaction (PPI) was used to investigate potential interactions between PROP1 and other associated proteins. Our data in Figure 4 show the network containing 11 nodes and 35 edges with PPI enrichment; p-value 0.00000029. Most notably, PROP1 interacts with different proteins involved in vital biological functions in the pituitary, mammary, and thyroid glands organogenesis including POU1F1, SOX3, POMC, TSHB, PRL, TBX19, GHRHR, and GHRH. Also, the network shows the strong association of PROP1 with other important protein such as furin and catenin beta-1 (CTNNB1). . PROP1 protein-protein interaction network analysis using STRING database with confident sore 0.4. Strong association between PROP1 and other pituitary protein including thyroidstimulating hormone POU1F1 and TSHB; short-stature-protein-associated POU1F1, GHRHR, SOX3, and CTNNB1; amenorrhea and infertility POU1F1 and SOX3 proteins.

Discussion
Mutations in transcription factor genes that encode proteins for hypothalamic and pituitary gland function, or differentiation in development can lead to congenital CPHD. CPHD is a rare medical condition that is associated with a wide spectrum of defects including growth abnormality and dysfunction of anterior pituitary causing hyperplasia or hypoplasia [19]. The current study presents phenotypic features and genotypic analysis of a consanguineous Saudi family with CPHD segregating among the family members in an autosomal recessive pattern. A homozygous missense variant c.211C > T: p.(Arg71Cys) in exon 2 (NM_006261.5) of PROP1 was identified for the CPHD phenotype in the family. This variant has been described on the Varsome website as a variant of uncertain significance and had not been previously reported on the ClinVar website. However, an earlier study revealed two novel compound heterozygous mutations in (p.(Arg71Cys)/p.(Arg71His)) of the homeodomain of the first alpha helix of the PROP1 gene similarly affecting the amino acid in two pre-pubertal siblings with CPHD [20].
Familial CPHD due to an inactivating mutation of the PROP1 gene was first reported in 1998. This transcriptional factor was reported previously to play a crucial role in the proper development of somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs [21]. It . PROP1 protein-protein interaction network analysis using STRING database with confident sore 0.4. Strong association between PROP1 and other pituitary protein including thyroid-stimulating hormone POU1F1 and TSHB; short-stature-protein-associated POU1F1, GHRHR, SOX3, and CTNNB1; amenorrhea and infertility POU1F1 and SOX3 proteins.

Discussion
Mutations in transcription factor genes that encode proteins for hypothalamic and pituitary gland function, or differentiation in development can lead to congenital CPHD. CPHD is a rare medical condition that is associated with a wide spectrum of defects including growth abnormality and dysfunction of anterior pituitary causing hyperplasia or hypoplasia [19]. The current study presents phenotypic features and genotypic analysis of a consanguineous Saudi family with CPHD segregating among the family members in an autosomal recessive pattern. A homozygous missense variant c.211C > T: p.(Arg71Cys) in exon 2 (NM_006261.5) of PROP1 was identified for the CPHD phenotype in the family. This variant has been described on the Varsome website as a variant of uncertain significance and had not been previously reported on the ClinVar website. However, an earlier study revealed two novel compound heterozygous mutations in (p.(Arg71Cys)/p.(Arg71His)) of the homeodomain of the first alpha helix of the PROP1 gene similarly affecting the amino acid in two pre-pubertal siblings with CPHD [20].
Familial CPHD due to an inactivating mutation of the PROP1 gene was first reported in 1998. This transcriptional factor was reported previously to play a crucial role in the proper development of somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs [21]. It is composed of three exons that encode 226 amino acids paired-like homeodomains, and it is located in the long arm of chromosome 5q35.3 [22].
In humans, PROP1 deficiency is most frequently associated with defects in GH, FSH, PRL, TSH, and LH; however, deficiency of ACTH is rarely observed. The hormonal profiles of the three affected sisters are different even though they share a similar missense mutation. Interestingly, patient (IV-6) has LH deficiency while the other affected sisters, IV-1 and IV-5, show abnormal TSH results. All three patients have normal FSH and abnormal PRL results. For analysis of free thyroxine (FT4), patients IV-5 and IV-6 have high results of FT4. However, this hormonal test was not performed for patient IV-1. Clinically, two affected sisters, IV-1 and IV-5, are at Tanner stage V with normal pubic and axillary hair, whereas, IV-6 is at Tanner stage III with normal pubic hair and no axillary hair.
This result is consistent with a previous report that individuals with mutations in PROP1 might exhibit different phenotype/genotype correlations. A large study including 73 affected patients with CPHD from 36 families showed high variability in the clinical features and pituitary hormonal (GH, LH, FSH, and TSH) deficiencies that reduced with age progressively, following a distinct pattern and time scale in each CPHD patient [23].
The rate of PROP1 mutations differ significantly between ethnicities. In the previously reported CPHD cases, the most frequent PROP1 mutations among Latin American, Eastern European, and Portuguese patients were c.301_302delAG and c.150delA of exon 2 [12]. These two mutations were found in 90% of CPHD cases. On the other hand, Moalla et al. (2022) recently reported two mutations (p.(Gln114Ter) and p.(Arg73Cys)) in Tunisian families with non-syndromic CPHD [24]. The p.(Arg73Cys) mutation was previously reported as the most common PROP1 mutation among CPHD patients from North Africa [25][26][27]. However, the mutation in p.(Arg71Cys) was described once in a non-consanguineous Italian family as compound heterozygotes with p.(Arg71His) resulting from different clinical features of both affected siblings associated with deficiency in GH and TSH [20]. In this study, we identified no phenotypic effect of heterozygotes mutation p. (Arg71Cys) in PROP1 in either the parents or healthy siblings. PROP1 has both transcriptional activation and DNA-binding ability [1]. Our amino acid alignment result shows p.(Arg71Cys) present in the paired homeo DNA-binding domain, which is highly conserved among other species including bovines, pigs, monkeys, dogs, gorillas, and Gelada baboons. PPI analysis suggests the crucial role of PROP1, which interacts with other families including the Pit-Oct-Unc (POU) transcription factor family; proprotein convertase subtilisin/kexin family; glucagon family; pro-opiomelanocortin; met-enkephalin family, growth hormone family, and the Wnt signaling pathway.
Finally, our patients currently are under estrogen and progestin hormone replacement therapeutics, using ethinylestradiol and norethindrone in combination with norimin to treat the secondary amenorrhea. In addition, patients are using both calcium carbonate and vitamin D3 to prevent low blood calcium and vitamin D deficiency. However, this study has some limitations, including not performing other hormonal analysis such as GH, ACTH, hypercholesterolemia, and insulin-like growth factor 1, as it was not available in the local hospital.
In conclusion, this new likely pathogenic missense mutation (c.211C > T; p.(Arg71Cys)) in the PROP1 gene expands the phenotypic and genetic spectrum of CPHD syndrome. To the best of our knowledge, this is the first report of PROP1 mutation causing CPHD among the Saudi population. The study recommends genetic screening of patients who display clinical phenotypes of short stature with pituitary hormone deficiency to provide early diagnosis of CPHD for adequate follow-up and to avoid any clinical complications in future.

Funding:
The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work; project number (442/127). Also, the authors would like to extend their appreciation to Taibah University for its supervision support.

Institutional Review Board Statement:
The study research is approved by the institutional review board committee of Taibah University (TUCDREC/27032021) and Madina Maternity and Children Hospital (IRB147-2021).
Data Availability Statement: Data available on request from the authors.