Growth hormone deficiency with advanced bone age: phenotypic interaction between GHRH receptor and CYP21A2 mutations diagnosed by sanger and whole exome sequencing

SUMMARY Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency and, clinically, patients have delayed bone age. High sequence similarity between CYP21A2 gene and CYP21A1P pseudogene poses difficulties for exome sequencing interpretation. A 7.5 year-old boy born to second-degree cousins presented with severe short stature (height SDS −3.7) and bone age of 6 years. Clonidine and combined pituitary stimulation tests revealed GH deficiency. Pituitary MRI was normal. The patient was successfully treated with rGH. Surprisingly, at 10.8 years, his bone age had advanced to 13 years, but physical exam, LH and testosterone levels remained prepubertal. An ACTH stimulation test disclosed a non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency explaining the bone age advancement and, therefore, treatment with cortisone acetate was added. The genetic diagnosis of a homozygous mutation in GHRHR (p.Leu144His), a homozygous CYP21A2 mutation (p.Val282Leu) and CYP21A1P pseudogene duplication was established by Sanger sequencing, MLPA and whole-exome sequencing. We report the unusual clinical presentation of a patient born to consanguineous parents with two recessive endocrine diseases: non-classic congenital adrenal hyperplasia modifying the classical GH deficiency phenotype. We used a method of paired read mapping aided by neighbouring mis-matches to overcome the challenges of exome-sequencing in the presence of a pseudogene.


INTRODUCTION
Isolated growth hormone deficiency is the most common pituitary hormone deficiency; it can be congenital or acquired. Although the most distinctive clinical manifestation is growth failure, there are many other clinical features including a delayed bone age. Amongst the congenital cases, a genetic aetiology can be established in about 10% of patients, with a higher prevalence in familial (34%) compared with sporadic (4%) IGHD. The main genetic causes to date are deleterious mutations in the genes encoding GH (GH1) or the receptor for GHRH (GHRHR) (1).
Congenital adrenal hyperplasia is a genetically heterogeneous disorder, most frequently caused by recessive, loss of function mutations in the 21-hydroxylase enzyme, encoded by CYP21A2, and it can have a wide spectrum of clinical manifestations. The non-classic form can be asymptomatic or associated with signs of postnatal androgen excess: rapid growth, advanced bone age, precocious pubarche, menstrual abnormalities, hirsutism, acne, and/or infertility (2,3). Non-classic CAH due to 21-hydroxylase deficiency is caused mainly by recombination between CYP21A2 and a nearly identical pseudogene, CYP21A1P (4)

CASE REPORT
A boy born at term by vaginal delivery (length 50 cm, weight 3,400 g) to second-degree cousins presented at 7.5 years with severe short stature (102.5 cm, SDS-3.7), high-pitched voice, blue sclera and prominent forehead. Genital examination revealed Tanner stage I, normal penile length (4 cm) and prepubertal testes (length 1.5 cm). Bone age was 6 years according to the Greulich and Pyle method. Clonidine and combined (insulin, TRH, GnRH) pituitary stimulation tests resulted in a peak GH of 0.6 ng/ml indicating GH deficiency, and a peak cortisol of 16.1 mcg/dl, initially interpreted as partial ACTH deficiency. Pituitary MRI was normal: anterior lobe 4.6 mm height (normal for age 4.5±0.6 mm), normal pituitary stalk and topic posterior pituitary. The patient was successfully treated with rGH (33 mcg/kg/day) with a first-year growth velocity of 11.7 cm/year. Surprisingly, at 10.8 years of age he presented with advanced bone age (13 years) ( Figure 1) despite absence of signs of puberty and prepubertal serum LH and testosterone levels. A cosyntropin stimulation test showed respectively, basal and peak levels, cortisol 6.1 and 18.8 mcg/dl, 17 hydroxyprogesterone 9.4 and 52.0 ng/ml and androstenedione 1.2 and 2.0 ng/ml, indicating non-classic 21-hydroxylase deficiency. Cortisone acetate (15mg/m2/day) was added to his treatment. At 12.2 years of age pubic hair was Tanner stage 2 and testicular length had increased to 2.5 cm indicating onset of central puberty. At 19.5 years his adult height was 166.5 cm, below his target height of 178.5 cm (Figure 2).

GENETIC TESTING AND DISCUSSION
At first, we performed Sanger-sequencing. No mutations in GH1, GHRH, or GHRHR were found (5). Using specific primers for the active CYP21A2 gene (6), a homozygous c.
844G>T, p.Val282Leu mutation (previously known as p.Val281Leu) was found (parents were heterozygous) ( Figure 3A); MLPA was also done showing CYP21A1P and C4B duplication. The p.Val282Leu mutation in CYP21A2 is the most commonly found mutation in patients with non-classic CAH (4) and leads to a mild mutant that retains 20-50% of 21hydroxylase activity (7).
In order to establish the unidentified genetic cause of IGHD, whole-exome sequencing (WES) was performed. Briefly, we aligned sequence reads to the 1000 Genomes Phase 1 reference mapped to GRCh37 using BWA v0.5.9 (8) and removed duplicate read pairs using PICARD v1.74. We performed realignment, recalibration and variant calling using GATK v3.3 (9) and applied GATK VQSR filter (10) to remove low-quality variants. Variant annotation was retrieved by using ANNOVAR (11) revealing a homozygous c.431C>T, p.Leu144His mutation in GHRHR. This mutation is indeed detected in the homozygous state by Sanger sequencing, but it was originally overlooked by the first Sanger sequencing (patient 5 of reference (5)). This mutation has been previously described in unrelated patients with IGHD from Sergipe/Brazil, Pakistan, Spain and the United States, and has been reported to lead to reduced cAMP production after GHRH stimulation with normal cellsurface localization of the receptor, suggesting a defect in ligand binding (12). To explore a possible founder effect we analyzed the C/T polymorphisms at positions −261 and −235 of the GHRHR promoter, away 7333 and 7359 bp, respectively, from the c.431C>T mutation.
Our patient was homozygous C at position −235 and homozygous C at position −261. This haplotype is identical to that of the previously reported patients with p.Leu144His from north-eastern Brazil and Spain but different from the patient from north-eastern United States (13). Therefore, the present patient is not related to the previously reported patient from the United States; however, a common ancestor for both families from Brazil and that of Spain, who share the same haplotype, cannot be excluded.
Ectopic posterior pituitary lobe and an interrupted stalk on MRI are increasingly being used for the diagnosis of GHD. However, it should be noted that all patients with GHD reported to date with mutations in GH1 and GHRHR (as well as and in PROP1) have had a normal stalk and topic posterior lobe, as the present case did (5,14,15).
At first, interpretation of WES indicated the CYP21A2 p.Val282Leu mutation in heterozygous state due to the high sequence similarity between CYP21A2 gene and CYP21A1P pseudogene and the copy number duplication at CYP21A1P pseudogene ( Figure 3B). This was in disagreement with the clinical diagnosis and Sanger sequencing. To resolve this gene-pseudogene twist in exome-sequencing, we proposed a method of paired read mapping aided by neighbouring four mis-matches using the exome-sequencing data and manually sorted out the real genotype for the variant of interest at CYP21A2 ( Figure 3C). For the CYP21A2 mutation, there were 44 paired reads supporting the T allele and 0 supporting the G allele. We concluded that the genotype at CYP21A2 c.844 position is T/T. For the corresponding CYP21A1P mutation, the evidence showed 78 paired reads supporting T and 73 supporting G; thus, it should be G/T at corresponding CYP21A1P position.
In conclusion, clinicians must be aware that patients born to consanguineous parents may have more than one genetic disease and should be alert to unusual phenotypes and treatment outcomes. Whole-exome sequencing was able to establish the genetic cause of IGHD but initially presented difficulties in diagnosing the genotype of CYP21A2/CYP21A1P. Our experience reveals the strengths and challenges of each sequencing technology and its applications. Advanced bone age (13 years) in a patient with chronological age of 10.8 years with isolated growth hormone deficiency.