Expanding the Phenotype of Congenital Glucocorticoid Deficiency: An Iranian Patient with Cholestasis due to Pathogenic Variants in the MC2R Gene

Familial glucocorticoid deficiency is caused by variants in the MC2R and MRAP genes. We report an Iranian patient with congenital glucocorticoid deficiency and cholestasis due to pathogenic variants in the MC2R gene. This is the first documented case of a patient with conditions. Clinical evaluations and lab assessments were conducted on a six-month-old male infant. Next-generation sequencing identified the genetic causes of the disease, and Sanger sequencing confirmed the variants through segregation analysis. The clinical presentation included prolonged jaundice, progressive skin hyperpigmentation, seizures, fever, and a large umbilical hernia. Two variants in the MC2R gene, c.560delT and c.676G > C, were detected and classified as pathogenic and likely pathogenic, respectively. The cooccurrence of cholestasis and glucocorticoid deficiency illustrates the clinical heterogeneity caused by MC2R variants. The prevalence of c.560delT and c.676G > C between Iranian populations suggests these variants may be common. The high frequency of c.560delT could be attributed to a founder effect.

Tere is also a report that severe cortisol defciency in early infancy causes cholestatic hepatitis [6].Moreover, evidence suggests a connection between neonatal liver failure and congenital adrenal hyporesponsiveness, as well as the occurrence of addisonian-like crises in congenital hypopituitarism and cholestatic jaundice [8,9].Conversely, alterations in bile fow have also been observed in adrenalectomized mice [2,10].Based on the aforementioned documents, it is prudent to incorporate noninvasive hormonal tests into the initial assessment before considering a liver biopsy in neonates and infants with cholestasis and hypoglycemia.Tis study documents a case of neonatal cholestasis and hepatosplenomegaly accompanied by seizure and hypoglycemia attributed to severe cortisol defciency.
While some authors have reported an association between cortisol defciency and cholestasis, as well as the successful treatment of cholestasis with hydrocortisone replacement therapy, it is essential to underscore the importance of initially measuring cortisol levels in the evaluation of cholestasis, a condition rarely associated with adrenal insufciency [4][5][6][7].

Materials and Methods
2.1.Genetic Investigations.Peripheral blood was taken for DNA extraction.WES was performed by an Illumina HiSeq 4000 platform with the mean read depth of 150X.Te wholeexome sequencing raw data (FASTQ fle) were analyzed using a core i7 CPU (central processor unit), 16 GB RAM in the Linux Ubuntu 20.04 operation system; briefy, FastQC tool (version 0.11.9) was applied for quality control of the reads.Te adapters were removed using the Trimmomatic software (version 0.36).

Review
Literature.We conducted a comprehensive analysis of individual data, integrating clinical characteristics and genetic analyses to gain insight into the disease caused by two variants.Our investigation into the genetics of familial glucocorticoid defciency (FGD) involved an extensive search of databases including PubMed, ScienceDirect, and John Wiley and Springer.Tese databases yielded relevant information.We employed keywords related to FGD, such as "variant," "c.560delT," "c.676G > C," "FGD," "p.Val187Alafs * 29," "p.Gly226Arg," and "MC2R," along with the term "phenotypic variability" to ensure inclusivity.Te collected data encompassed patient demographics, including numbers, age of onset, gender distribution, clinical symptoms, variant types, genotypic profles, and clinical test outcomes.To identify the most prevalent variants, we applied criteria from relevant studies to select those with the highest frequency.Subsequently, we calculated the frequencies of deletion and missense mutations across diverse patient cohorts and geographical regions.

Clinical Features.
A six-month-old male infant, who had been admitted twice due to prolonged jaundice and progressive skin hyperpigmentation since birth, was brought to the hospital after experiencing a seizure with a fever on the frst day of life.He was the frst child of unrelated parents, was formula-fed, and displayed average height and weight (7.800 kg and 67 cm).During the physical examination, generalized jaundice and hyperpigmentation were noted.Te liver and spleen were palpable 4 cm and 3 cm below the costal margin, respectively.Additionally, he presented with a sizable umbilical hernia measuring 3 cm (Figure 1).Routine chest examination, including heart assessment, was conducted.Generalized hyperpigmentation was observed along with a penile stretch length of 2.5 cm and palpable testes in the scrotum.Te eye examination yielded normal results.

Results
Te patient's laboratory fndings are detailed in Table 1.Tyroid hormone levels were within normal range.Te laboratory results of the patient are detailed in Table 1.Tyroid hormone levels were within normal range.Comprehensive metabolic screening for inherited metabolic disorders, which included tandem mass spectrometry (MS), urinary organic acid profle, NH3 levels, and long-chain panel levels, all yielded normal results.Tests for hepatitis, toxoplasmosis, rubella, cytomegalovirus, and herpes simplex returned negative results.Despite elevated ACTH levels in the absence of mineralocorticoid defciency, low plasma cortisol levels indicated a diagnosis of isolated glucocorticoid defciency.Tis led to the initiation of hydrocortisone replacement therapy.After one month, bilirubin levels normalized; after four months, all liver function tests returned to normal.Troughout the eight-month follow-up period, ACTH levels remained low despite hydrocortisone replacement therapy being administered and normal mineralocorticoid function observed.No hypoglycemic episodes were reported during this monitoring period.

Discussion
Familial glucocorticoid defciency (FGD) results from a resistance to the action of adrenocorticotropic hormone (ACTH) on the adrenal cortex, leading to an isolated defciency in glucocorticoids.Tis condition manifests as an isolated defciency in glucocorticoids.FGD is a rare autosomal recessive disorder that typically presents in infancy or early childhood.Clinical features include hyperpigmentation, recurrent infections, failure to thrive, hypoglycemic episodes, and seizures, which can progress to coma or death.Diagnostic evaluation reveals decreased levels of cortisol and androgens along with elevated ACTH levels, while the reninaldosterone axis remains unafected.[6].Tis genetically heterogeneous disorder usually results from variants in the MC2R gene (FGD type 1) and MRAP gene (FGD type 2), though WES has identifed variants in other genes causing other types of FGD [12,17].Tis study details the clinical manifestations of 23 patients (from 21 distinct families) harboring mutations c.560delT (p.Val187Alafs * 29) and c.676G > C (p.Gly226Arg) and analyzed the geographical prevalence of these variants.Furthermore, a novel case is presented involving a family with cholestasis resulting from these deleterious mutations in the MC2R gene is presented, contributing to the broadening of the phenotypic spectrum associated with congenital glucocorticoid defciency.
To the best of our knowledge, only a 32-day-old male infant has been reported with infantile cholestasis due to a homozygous variant c.763_764delAT (p.Met255Valfs * 17) in the MC2R gene [18].In this study, we present a novel Iranian case exhibiting unique clinical manifestations of FGD1.Te patient was found to be compound heterozygous for two previously reported variants in the MC2R gene: c.560delT (p.Val187Alafs * 29) [19] as a pathogenic variant and c.676G > C (p.Gly226Arg) [12] classifed as a likely pathogenic variant.Tis compound heterozygosity resulted in a distinct phenotype previously unreported in the literature.
MC2R expressed in the zona fasciculata encodes the ACTH receptor, and the MRAP gene encodes an accessory protein of MC2R (melanocortin−2 receptor accessory protein).Pathogenic variants of the MC2R gene that produce  International Journal of Endocrinology

Vomiting
Normal adrenal [12] International Journal of Endocrinology  6 International Journal of Endocrinology an aberrant protein can downregulate the cortisol synthesis pathway and abolish ACTH stimulation; defects in this pathway could lead to various clinical manifestations of FGD, including hyperpigmentation, hypoglycemia, eczema, failure to thrive, jaundice, and increased susceptibility to infection [20][21][22].
In addition to high levels of ACTH, 79% of MC2Rrelated patients have hyperpigmentation; hypoglycemia is also observed in 77% of patients, and seizure, jaundice, and vomiting are documented in 20%, 30%, and 14% of the afected individuals, respectively [12].Respiratory stress and recurrent infection are also seen around 10% of patients [12]; almost all these clinical manifestations were noted.Malnutrition has also been reported in some cases which was not present in our patient.An accidental fnding in our case, an umbilical hernia was documented which has not been reported to the best of our knowledge [23].In our study, 18 patients had a homozygous mutation, while one patient had a compound heterozygous genotype.Te mean age of onset was identifed to be 3.8 months.Hyperpigmentation and hypoglycemia are the most common symptoms associated with this variant (Figure 2(a)).
It is important to consider that the frequency of certain variants may be attributed to a founder efect, while others may arise as mutational hotspots [24].Additionally, some variants may exhibit ethnicity-specifc distributions due to founder efects.For instance, specifc variants in genes such as GJB2 variants and CYP21A2 have been found to have elevated frequencies in particular populations [25,26]; for example, the c.560delT is variant is prevalent among Turkish patients [12].Tis variant may have a wider distribution in this country, and it is common likely due to migration from the east to west of Turkey.Notably, the c.560delT mutation was initially identifed in an Iranian family with Northern Iranian ancestry, suggesting a potential founder efect within the Iranian population [11].Furthermore, the c.560delT and c.676G > C variants have been reported in Turkish populations, indicating a shared genetic landscape between these populations [12,14].
In conclusion, when evaluating FGD patients, an initial measurement of cortisol levels is recommended for the initial assessment of cholestasis.

Figure 1 :
Figure 1: (a) Skin hyperpigmentation and umbilical hernia.(b) Electropherogram of the patient and his parents.

Figure 2 :
Figure 2: (a).Te number of afected people according to the phenotypes they have shown.(b) Percentage of patients with MC2R in diferent countries.(c) Te number of study cases separated based on their gender.

Figure 3 :
Figure 3: Geographical distribution of c.560delT and c.676C > G mutations identifed in this study.Te frst report of the c.560delT mutation was in a family from Iran who was in a family with two siblings who originated from Northern Iran, close to the Turkish border.Te c.560delT variant showed a wider distribution most likely refecting migration from the East to the West of Turkey and has been reported in previous article.
Patients with c.560delT.Twenty-one cases were identifed to carry the c.560delT mutation, with 18 patients exhibiting a homozygous mutation one patient displaying a compound heterozygous genotype.Te age of symptom onset was recorded for a total 3.2.Clinical Features of Patients with c.676G> C.Among a group of 23 patients, 2 individuals exhibited homozygosity, while an additional two displayed compound heterozygosity for the c.676C > G variant.Tese patients presented with similar clinical manifestations, including hypoglycemia, hypothyroidism, and reduced levels of sodium (NA), for 16%.Te distribution of these variants was graphically illustrated (see Figure2(b)).Te c.560delT mutation predominantly manifested in patients from Turkey and one family from Northern Iran, with eighteen families hailing from Turkey (Figure

Table 1 :
Laboratory results of the studied patient.

Table 2 :
Information and data related to all patients who carried c.560delT and c.676C > G variants in all articles and in the study.