Abstract
Background
The data on Leydig cell hypoplasia (LCH) resulting from biallelic Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) inactivating variants is limited to case series.
Methods
We aim to describe our patients and perform systematic review of the patients with LHCGR inactivating variants in the literature. Detailed phenotype and genotype data of three patients from our centre and 85 (46,XY: 67; 46,XX: 18) patients from 59 families with LHCGR-inactivating variants from literature were described.
Results
Three 46,XY patients (age 6–18 years) from our center, with two reared as females, had two novel variants in LHCGR. Systematic review (including our patients) revealed 72 variants in 88 patients. 46,XY patients (n = 70, 56 raised as females) presented with pubertal delay (n = 41) or atypical genitalia (n = 17). Sinnecker score ≥3 (suggesting antenatal human chorionic gonadotropin (hCG) inaction) was seen in 80% (56/70), and hCG-stimulated testosterone was low (<1.1 ng/mL) in 77.4% (24/31), whereas puberty/postpubertal age, high luteinizing hormone (LH) (97.6%, 41/42) and low (<1.0 ng/mL) basal testosterone (94.9%, 37/39) was observed in most. Follicle stimulating hormone was elevated in 21/51 of these patients. Variants with <10% receptor function were exclusively seen in cohorts with Sinnecker 4/5 (10/15 vs 0/5, P = 0.033). 46,XX patients (n = 18) presented with oligo/amenorrhea and/or anovulatory infertility and had polycystic ovaries (7/9) with median LH of 10 IU/L (1.2–38).
Conclusion
In summary, this study comprehensively characterizes LHCGR variants, revealing genotype-phenotype correlations and informing clinical management of LCH. In 46,XY LCH patients, pubertal LH inaction is uniform with variable severity of antenatal hCG inaction. Few mutant LHCGR have differential actions for LH and hCG.
Introduction
Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is a G-protein coupled receptor that contains 674 amino acids and is a common receptor for both luteinizing hormone (LH) and human chorionic gonadotropin (hCG) (1). In 46,XY fetuses, hCG acts on LHCGR to mediate androgen synthesis and male sexual differentiation, whereas during puberty, LH acts on LHCGR to mediate androgen synthesis and pubertal development (2). Consequently, 46,XY patients with LHCGR variants that severely impair LHCGR activity present as cases of differences of sex development (DSD) with female-like external genitalia, absent puberty, and harbor hypoplastic Leydig cells on testicular histology. These patients have been traditionally classified as Leydig cell hypoplasia (LCH) type-1 to differentiate them from patients with LCH type-2, who have milder LHCGR defects with male external genitalia and present with micropenis and/or delayed puberty (3). As LH mediates ovulation and luteinization of ovarian granulosa cells, 46,XX patients with LHCGR inactivating variants usually present with anovulatory infertility (4, 5).
Since the first description of this disease in 1995 (6, 7), ~50 probands have been published worldwide to date, but none from India. Also, a comprehensive systematic phenotype and genotype analysis of patients with LHCGR inactivating variants has not been done. This study aims to describe patients with LCH managed at our center and also present a detailed individual patient data based (IPD) systematic analysis of the phenotype and genotype of the published patients with LHCGR inactivating variants to enhance the understanding of this rare disorder.
Material and methods
This study includes a detailed phenotypic and genotypic description of three patients with LCH managed at our center. This study was approved by the Institutional Ethics Committee (EC/OA-196/2023) and the waiver of informed consent was obtained considering the retrospective nature of study. Hormonal analysis was performed as described previously (8). Genomic DNA was isolated from peripheral blood leukocytes by standard techniques. Molecular screening of the LHCGR gene was performed as a part of the targeted clinical exome panel using next-generation sequencing and verified by sanger sequencing as described previously. (9, 10)
Systematic review of literature
A Systematic review of literature was performed as per the PRISMA guidelines across PubMed database from inception till December 2022 with following search items: ‘(LHCGR mutation) OR (LH receptor mutation)) OR (luteinizing hormone receptor mutation) OR (Leydig cell hypoplasia)) OR (Leydig cell receptor defect)) OR (luteinizing hormone receptor defect)) OR (Luteinizing hormone/Chorionic gonadotropin receptor mutation)’ restricted to human studies and English language (11). Cross-references of selected articles were used to find additional studies. A total of 1249 publications were initially found, and 48 studies were included for final analysis (Fig. 1). The search and inclusion of the articles was done by two independent reviewers. Joanna Briggs Institute (JBI) critical appraisal tool was used in this systematic review, 40/48 articles were of high quality and remaining eight articles were of moderate quality (12; https://jbi.global/sites/default/files/2019-05/JBI_Critical_Appraisal-Checklist_for_Case_Series2017_0.pdf) (Supplementary Data, see section on supplementary materials given at the end of this article). Per patient demographic details including the geographical region of the origin (https://population.un.org/wpp/DefinitionOfRegions), clinical, hormonal, radiological, and genotypic details were tabulated. Sinnecker scoring was used for description of external genitalia in 46,XY patients (13) and for data analysis, genital phenotypes were categorized into male-like genitalia (Sinnecker score of 1), female-like genitalia (Sinnecker scores of 4 or 5) and atypical genitalia (Sinnecker scores of 2 or 3). Based on the age at presentation, patients with 46,XY were categorized as in minipubertal age (0 to ≤ 0.5 years), pre-pubertal age (> 0.5 to ≤ 9 years), pubertal age (> 9 years to ≤ 14 years) and post-pubertal age (> 14 years). A hCG-stimulated testosterone of <1.1 ng/mL was considered subnormal (14).
PRISMA flow chart for Leydig cell hypoplasia: literature search.
Citation: Endocrine Connections 13, 11; 10.1530/EC-24-0246
The reported variants were tabulated according to the domain of the protein affected and the mechanism of functional defect. For phenotype-genotype correlation, only probands’ data were included. Variants were classified into those with truncating effect on the protein (T), which included deletion, nonsense, and frameshift variants, and those without truncating effect on the protein (NT), which included missense variants, in-frame insertions, and duplications. This analysis excluded four splice-site variants, whose effects on the protein truncation were not clear. Additionally, variants whose functional studies have been reported were classified into ‘severe variants’ (which result in ≤10% of the residual receptor function, 10% being an arbitrary cut-off) and ‘non-severe variants’ (which result in >10% of residual receptor function). Probands with compound heterozygous variants with one T and another NT variant or severe and another non-severe variant were classified into milder categories.
Statistical analysis was performed using SPSS software version 25.0 (IBM). Categorical data were expressed as absolute numbers and percentages, whereas continuous data were expressed as median and ranges as appropriate. Fisher’s exact or chi-square test was used to compare categorical variables, whereas the independent t-test or Mann–Whitney U-test was used to compare continuous variables between two groups. A two-sided P-value <0.05 was considered statistically significant.
Results
Our patients
Patient 1: An 18-year-old boy, born of third-degree consanguineous marriage presented with micropenis and delayed puberty. His arm-span was 7 cm more than his height (168 cm). He had micropenis with stretched penile length of 3.5 cm (−6.1 SDS), Tanner stage 2 pubic hair, testicular volume of 8 cc on each side, and gynecomastia. On hormonal evaluation, patient had high LH, normal follicle stimulating hormone (FSH) and very low basal as well as hCG-stimulated testosterone was low after 3 days of 1500 units of injection hCG. The details of hormonal and radiological evaluation is tabulated in Table 1. Karyotype analysis confirmed 46,XY. Genetic analysis identified a previously described biallelic LHCGR variant, p.Cys131Arg (15). He was initiated on intramuscular testosterone enanthate (150 mg every 3 weeks for 9 months, followed by 200 mg every 2 weeks for 9 months). After 18 months of therapy, his height increased to 173 cm, penile length increased to 6.5 cm (−4.25 SDS), and testicular volume improved to 12 cc on each side. His inhibin B was 131 pg/mL, while semen analysis showed azoospermia. Later, he was initiated on subcutaneous hCG therapy (2000 units thrice weekly for 3 months, followed by 5000 units twice weekly for 3 months) but without further increase in testicular volume whereas serum testosterone levels remained low (0.05 ng/mL). Hence, he was restarted on intramuscular testosterone replacement therapy.
Clinical and hormonal profile of our 46,XY patients with LHCGR inactivation.
| Patient | Sex of rearing, age at evaluation (years) | Presenting symptom | Follicle stimulating hormone (IU/L) | Luteinising hormone (IU/L) | Testosterone (ng/mL) |
Anti-Mullerian hormone (ng/mL) | Imaging findings | LHCGR variant |
|---|---|---|---|---|---|---|---|---|
| 1* | Male, 18 | Delayed puberty | 7.62 | 19.92 | 0.11 | > 24 | Right testis: 2.5 × 1.6cm, left testis: 2.6 × 1.9cm), presence of prostate gland, epididymis, absent mullerian structures | (c.391T > C), p.Cys131Arg, Missense, Homozygous, Exon 5 |
| 2 | Female, 17.8 | Delayed puberty | 4.32 | 15.29 | < 0.025 | > 24 | Gonads noted in bilateral labial folds, no mullerian structures | (c.1799C > T), p.Thr600ile, Missense, Homozygous, Exon 11 |
| 3 | Female, 6.2 | Inguinal masses | 2.49 | 0.45 | 0.025 | > 24 | Bilateral gonads in the labial folds, no mullerian structures | Homozygous, deletion of Exon 1 to 2 ((chr2:g.(487292228_48731226)_(48755671_?)del) |
*Patient 1: hCG stimulated testosterone 0.11ng/ml, Inhibin B 464 pg/mL
Patient 2: A 17-year-8-month-old patient, with no history of consanguinity reared as a girl, presented with primary amenorrhea and absent breast development. There was a history of left inguinal hernia surgery at the age of 9 months. There was no family history of similar complaints. Her height was 168 cm, Tanner stage was B1, P1, with normal female external genitalia (Sinnecker score 5). Her hormonal details are presented in Table 1. Her karyotype was 46,XY and genetic analysis identified a novel exon 11, biallelic LHCGR variant, p.Thr600Ile. This variant has not been reported in 1000 genomes, gnomAD (v3.1), and gnomAD (v2.1), predicted damaging on in-silico tools and the reference codon is conserved across species. Her gender identity was female, and she was initiated on estrogen replacement therapy. Presently patients gonads are in the labial folds; she has been informed about her genetic condition and risk of gonadal malignancy, and he has decided not to go for gonadectomy at present. Hence she has been advised to undergo ultrasonogram of the gonads at 6 monthly intervals.
Patient 3: A 6-year-2-month-old child, reared as a girl presented with bilateral inguinal hernia at infancy. She had Sinnecker 5 external genitalia with palpable bilateral gonads in labia majora. Her hormonal profile is detailed in Table 1. Her karyotype was 46,XY and genetic analysis showed novel biallelic deletion in Exon 1–2 (chr2:g. (48729228_48731226)_ (48755671_?)del) of LHCGR gene. She underwent herniotomy and gonadal biopsy at 6 years of age. The Mullerian structures were absent as per the laparoscopic findings. Histology report showed testicular tubules with Sertoli cells and absent Leydig cells.
Systematic review
As detailed in Fig. 1, this review yielded 48 articles including 85 (46,XY: 67; 46, XX: 18) patients with LHCGR inactivating variants belonging to 59 families. There were 49 probands with 46,XY and 10 probands with 46,XX karyotype. For the IPD analysis, details of three patients from the current report were added to the dataset of published cases. Hence, the final cohort consisted of 88 patients (46,XY: 70; 46,XX: 18) from 62 families.
46,XY patients with LCH
Seventy 46,XY patients from 52 families have been described. Notably, 56 patients were raised as females, and 14 were raised as males.
Phenotype of 46,XY patients with LCH raised as females (n = 56)
Median age of presentation of 46,XY patients who were raised as females was 15 (range: 0–42) years. Majority of patients (n = 39/56) presented in pubertal/post-pubertal ages (> 9 years of age) while 17 patients (17/56) presented in mini/pre-pubertal ages (age ≤ 9 years).
In pubertal/post-pubertal aged cohort, all patients (n = 33, data not available for four patients) presented with delayed puberty/primary amenorrhea. Most patients (22/24) had absent thelarche except for two, who had Tanner stages B4 and B2. Pubic hair stages were commonly P2 (n = 8/21) or P1 (n = 7/21), followed by P4 (n = 4/21) and P3 (n = 2/21). Genital phenotype (n = 37) was predominantly female-type (97.3%, Sinnecker 5:31; Sinnecker 4:5) and one patient had typical male genitalia with Sinnecker score 1. Gonads (n = 34) were commonly present in inguinal region (28, 82.35%), followed by labia majora (n = 2), abdomen (n = 2) and non-visualized on USG/MRI in two patients. Median (range) right and left testicular volumes as measured by sonogram, were 4.69 (1.17–12), and 4.27 (1.08–12) cc, respectively. Pubertal age details about serum hormonal parameters are given in Table 2. Most pubertal/post-pubertal age patients had low basal (28/30, < 1.0 ng/mL, median 0.17 (0.025–4.3)) and hCG-stimulated testosterone (15/17, < 1.1 ng/mL, median 0.22 (0.05–5.1)), except for two siblings. All (n = 33) pubertal/post-pubertal aged patients had high LH (≥ 10 IU/L, median 24.3 (11.2–68)), and 19/32 had high FSH (≥ 10 IU/L, median 12.42 (3.7–59)). Serum AMH (n = 3) was 46, 67.5 and >23 ng/mL and inhibin B (n = 2) was 365.6 and 548 pg/mL.
Serum hormone levels in 46,XY individuals with LHCGR variants, analysed according to their age and sex of rearing.
| Variables | Reared as females (n = 56) | Reared as males (n = 14) | |
|---|---|---|---|
| Median age, range | 15 (0–42) years | 18 (0–51) years | |
| Female type genitalia | 51/56 | 1/14 | |
| Gonadal position | |||
| Labial/scrotal | 5 | 9 | |
| Inguinal | 39 | 5 | |
| Abdominal | 4 | – | |
| Absent | 2 | – | |
| Biochemical characteristics | Normal range | ||
| Luteinizing hormone (IU/L) | |||
| Mini-pubertal age, value, n, elevated in | 0.9–2.64 | < 0.5, n = 1 | N, n = 1 |
| Pre-puberty age | < 0.05–0.42 | 2.66 (0.01–16), n = 10 | 1.0 (0.02–6.3), n = 3 |
| Pubertal age | 0.5–7.9 | 22.71 (12.2–68), n = 6 | 10.9 and 0.73, n = 2 |
| Post Pubertal age | 2.5–10 | 27.1 (14.1–65), n = 26 | 19.9 (15.6–95), n = 5 |
| Follicle stimulating hormone (IU/L) | |||
| Mini-pubertal age, value, n, elevated in | 0.9–2.93 | 1.3, n = 1 | N, n = 1 |
| Pre-puberty age | < 0.06–1.84 | 2.97 (1.1–9.09), n = 10 | 1.30 (0.05–3), n = 3 |
| Pubertal age | 0.4–10.5 | 10.6 (3.7–40), n = 5 | 6.5 and 1.45, n = 2 |
| Post Pubertal age | 2.5–10 | 15.73 (4.32–59), n = 24 | 8.62 (2.26–83), n = 5 |
| Testosterone (ng/mL) | |||
| Mini-pubertal age, value, n, elevated in | 0.52–1.88 | 0 and 0.05, n = 2 | L, n = 1 |
| Pre-puberty age | 0.03–0.43 | 0.085 (0.02–0.72), n = 10 | 0.074 (0–0.1), n = 3 |
| Pubertal age | 0.3–7.6 | 0.14 (0.10–3.9), n = 7 | 0.1 and < 0.12, n = 2 |
| Post Pubertal age | 4–11 | 0.17 (0.03–4.3), n = 22 | 0.52 (0.11–0.94), n = 7 |
| hCG stimulated testosterone (ng/mL) | |||
| Mini-pubertal age, value, n, elevated in | > 1.1 | 0.10 and 0.09, n = 2 | 0.1, n = 1 |
| Pre-puberty age | > 1.1 | 0.12 (0.1–0.9), n = 3 | 0.0 (0–5.9), n = 3 |
| Pubertal age | > 1.1 | 0.18 (0.15–5.10), n = 4 | 0.42, n = 1 |
| Post Pubertal age | > 1.1 | 0.275 (0.05–3.5), n=8 | 2.17 (0.1–10), n = 6 |
| Anti-Mullerian hormone (ng/mL) | |||
| Mini-pubertal age, value, n, elevated in | – | – | – |
| Pre-puberty age | 45–196 | 33 (23–130.31), n = 4 | 144.8, n = 1 |
| Pubertal age | 2–103 | – | 81.1, n = 1 |
| Post Pubertal age | 2–16.5 | 46 (23–67.5) n = 3 | >24, n = 1 |
| Inhibin B (pg/mL) | |||
| Mini-pubertal age, value, n, elevated in | 99–439 | 89.0, n = 1 | – |
| Pre-puberty age | 13–418 | 790 and 108, n = 2 | – |
| Pubertal age | – | – | |
| Post pubertal age | 169–216 | 548 and 365.6, n = 2 | 424 (153–464), n = 3 |
In mini-pubertal/prepubertal aged cohort (n = 17), four were suspected based on positive family history while other probands presented with concern for atypical genitalia (n = 12/17, clitoromegaly or labial fusion) or inguinal masses (n = 1/17). Genital phenotype was as follows: female-type genitalia in 15 patients (Sinnecker score 5 in 12 and 4 in 3) and atypical genitalia in two patients (score of 3 in one and 2 in one patient). Gonads (n = 16) were inguinal in n = 11; labial or scrotal in n = 3 and abdominal in n = 2. In prepubertal age group, 9/10 patients had high LH (≥ 0.3 IU/L, median 2.66 (0.01–16)), and 3/3 patients had low hCG-stimulated testosterone (< 1.1 ng/mL) (Table 2). Serum Basal DHEAS levels in one prepubertal patient (5 years) was 100 ng/mL and median DHEAS in four post pubertal patients was 275 ng/mL (1.8–2996).
Overall, in 46,XY patients who were raised as females, gender incongruence with the sex of rearing has not been reported. Thirty-four patients had undergone bilateral gonadectomy, one had unilateral gonadectomy, and two had a testicular biopsy. Histopathology showed absent Leydig cells in most, though some had a few immature Leydig cells. Seminiferous tubules showed varying maturity of Sertoli cells and a reduced number of germ cells with arrested spermatogenesis. Normal testicular tissue and gonadoblastoma (1 mm foci) were reported in one case each. Most patients were started on estrogen replacement therapy post-surgery.
Phenotype of 46,XY patients with LCH raised as males
Fourteen 46,XY patients were raised as males and presented at median age of 18 (range: 0–51) years. For one patient with genital Sinnecker score of 5 and inguinal gonads, the age at presentation was not reported. Out of the rest of 13 patients, five patients (n = 5/13) presented at birth/early childhood for atypical genitalia (n = 4) or micropenis (n = 1). Their Sinnecker scores were 2 in two patients, 3 in two patients and 1 in one patient and gonadal location were inguinal in 4 and scrotal in 1. Others (n = 9/13) presented at pubertal/postpubertal age with varying concerns of micropenis and cryptorchidism (n = 1), delayed puberty (n = 2), infertility (n = 4), and hypogonadism (n = 1). On examination, all patients had micropenis, with Sinnecker-scores of predominantly 1 (n = 8), and none (n = 7) had gynecomastia. Pubic hair stages were P1 in two, P2 in three, and P3 in one patient. Their gonadal position was scrotal (n = 8) and inguinal region (n = 1) and the median testicular volume by clinical assessment was 14.5 (8–25) cc on right side and 14.5 (8–25) cc left side gonads.
Age-specific serum LH levels were elevated (≥ 0.3 IU/L, median 1 (0.02–6.30) in 6 months to ≤ 9 years; ≥ 10 IU/L, median 17.1 (0.73–95) in > 9 years) in all patients except three, one each in mini-pubertal (reported as normal), prepubertal, and pubertal age groups. Serum FSH was high (≥ 10 IU/L, median 8.62 (2.26–83)) in 4/7 postpubertal patients. Baseline testosterone was uniformly low in all patients, while its response to hCG stimulation was normal (> 1.1 ng/mL) in 5/11 patients (range: 1.2– 0 ng/mL). Median (range) serum AMH (n = 3) and inhibin-B (n = 3) were 81.1 (> 24–144.8 ng/mL) and 424 (153–464 pg/mL), respectively.
Gender incongruence with the sex of rearing has not been reported. All evaluated (n = 4) patients had oligo-azoospermia and infertility. Testicular histopathology (n = 4) showed immature seminiferous tubules with arrested spermatogenesis in all, with absent (n = 3) or immature Leydig cells (n = 1). Treatment details were available for nine patients. In two prepubertal patients (one at birth and another at 1.7 years), testosterone treatment for 3–6 months increased penile size in both and gonadal size in one. Testosterone replacement led to virilization in all patients at pubertal and post-pubertal ages (n = 6). Notably, testosterone treatment led to a remarkable increase in testicular volume (from 8 and 4 cc to 15 cc and 12 cc, respectively) in two patients and sperm count (from azoospermia to 3 million/mL resulting in fertility) in one. hCG-treatment was attempted in two patients. In the first patient, the use of 5000 units twice/week of hCG for 1 year increased serum testosterone to 1.7 ng/mL but failed to improve azoospermia. This patient achieved fertility with the help of micro-testicular sperm extraction (16). In another patient, hCG treatment (5000 units/week for 2 months followed by 3000 units/week) increased serum testosterone levels to normal, testicular volume from 25 to 30 cc, and sperm count from azoospermia to 5.3 million/mL in 4 months (17).
46,XX patients with LHCGR inactivating variants
Median age of presentation of 46,XX patients was 29.5 (range: 8–53) years. Eighteen 46,XX patients from 15 families have been described. In five families (6 patients), the female patients were diagnosed during family screening of affected 46,XY siblings. The others presented for infertility (n = 10) or primary amenorrhea (n = 2). Whenever reported, the onset of thelarche was at a normal age, and at presentation, breast development was normal in all patients. Except for two patients who had menarche at 12, all others had menarche after 16 years of age. After menarche, all patients had oligomenorrhoea. Median (range) serum LH, FSH, estradiol, progesterone, and AMH for post-pubertal age patients (> 13 years, for 46,XX individuals) were 10 IU/L (1.2–38, n = 13), 8.8 IU/L (4.3–26.3, n = 13), 48.0 pg/mL (25–73.54, n = 10), 0.36 ng/mL (0.17–1.57, n = 6) and 1.97 ng/mL (1.12–7.39, n = 4), respectively. The serum estradiol levels were low/low normal in 7/12 patients, while testosterone levels, whenever reported, were normal or low. On sonogram, 3/8 patients had a hypoplastic uterus, while few simple cysts or polycystic morphology was a common ovarian finding (7/9). All patients who attempted conception had anovulatory infertility (n = 13). The response during in-vitro fertilization treatment was uniform in the form of multi-follicular development after induction but retrieval of none (9/11) or only a few oocytes (2/11) with hCG administration.
Genotype of patients with LHCGR inactivating variants
To date (including our patients), 62 families with 88 patients harboring LHCGR inactivating have been described. Parental consanguinity was present in 57.70% (30/52). Seventy-two different variants were described in the homozygous (n = 42 families), compound-heterozygous (n = 17), heterozygous (n = 2), and double homozygous (n = 1) states. The variants were missense (n = 33), nonsense (n = 14), deletions (n = 8), splice-site (n = 6), frameshift (n = 6), insertions (n = 4), and duplication (n = 1). Seven variants (c.161+4A>G, p.Cys131Arg, p.Ala483Asp, p.Ser616Tyr, p.Asn312Ser, pArg479Ter, and pGln18_Pro19ins (n = 33) were recurrent (observed in two or more families) of which two (c.161+4A>G and p.Ala483Asp) were seen in Turkish patients (Supplementary data). There were two patients with heterozygous pathogenic variants Leu17_Gln18insLeuPro, Exon 1 and p.Ile114Phe, Exon 4 causing Leydig cell hypoplasia (18, 19). Functional studies were available for 50% of variants (n = 35).
Phenotype–genotype co-relation analysis
Phenotype–genotype correlation analysis was not considered for the 46,XX cohort due to the small sample size (n = 15) and a fairly uniform phenotype in most patients.
For phenotype-genotype analysis, 46,XY patients (n = 50) were categorized as per their genital phenotype into male-like genitalia (n = 7), atypical genitalia (n = 5), or female-like genitalia (n = 38) (Table 3). Probands with phenotypes male-like genitalia or female-like genitalia presented at later ages (18.2 and 16 years, respectively) with hypogonadism, contrasting those with the atypical genitalia cohort who presented early (0–2.3 years). Gonads were mostly scrotal in the phenotype male-like genitalia cohort but commonly undescended in the other two cohorts. The groups did not differ in serum biochemistry (LH, FSH, basal and hCG-stimulated T). The prevalence of truncating variants (42.8% vs 39.4%) was comparable between cohorts male-like genitalia and female-like genitalia (with Sinnecker-score 1 vs 4/5). However, the prevalence of severe variants as per in vivo functional analysis (< 10% receptor function) was only seen in the cohort with female-like genitalia (Sinnecker-score 4/5), which was statistically significant (66.6% vs 0%, P = 0.033). The distribution of pathogenic variants as per the domains affected were as follows: extracellular (n = 24), transmembrane (n = 15), signal peptide (n = 3), cytoplasmic tail (n = 1) with no statistical difference with respect to serum testosterone and genital phenotype between extracellular vs other domains (Supplementary data). Similarly, there was no statistical difference in serum testosterone and genital phenotype in patients harboring compound heterozygous (CHz) (n = 13) vs homozygous variants (Supplementary Data).
Phenotypic and genetic characteristics in 46,XY probands with LHCGR variants, categorized by the degree of atypical genitalia.
| Male-like external genitalia (Sinnecker score 1) (n = 7) | Atypical genitalia (Sinnecker score 2&3) (n = 5) | Female-like external genitalia (Sinnecker score 4&5) (n = 38) | P value typical male vs female-type | P (typical male+atypical) vs female-type | ||||
|---|---|---|---|---|---|---|---|---|
| Clinical phenotype | ||||||||
| Age of presentation, years | 18.2 (0–34), n = 7 | 0 (0–2.3), n = 5 | 16 (0–37), n = 38 | 0.277 | 0.272 | |||
| Presenting complaints | ||||||||
| Atypical genitalia | 2/7 | 5/5 | 12/34 | 1 | 0.388 | |||
| Hypogonadism | 5/7 | 0/5 | 22/34 | 1 | 0.571 | |||
| Gonadal position | Right | Left | Right | Left | Right | Left | ||
| Scrotal/labial | 6/7 | 6/7 | 0/4 | 1/ 4 | 5/34 | 5/34 | 0.000 | 0.010 |
| Undescended | 1/7 | 1/7 | 4/4 | 3/4 | 29/34 | 29/34 | 0.007 | 0.106 |
| Inguinal | 1/7 | 1/7 | 4/4 | 3/4 | 24/34 | 24/34 | ||
| Abdominal/absent | 0/7 | 0/7 | 0/4 | 0/4 | 5/34 | 5/34 | ||
| Serum biochemistrya | ||||||||
| LH, IU/L | 18.51 (0.73–95), n = 6 | 10.9, n = 1 | 24.25 (12.2–68), n = 24 | 0.554 | 0.122 | |||
| FSH, IU/L | 6.01 (1.45–83), n = 6 | 6.5, n = 1 | 12 (4.7–59), n = 23 | 0.185 | 0.027 | |||
| Testosterone, ng/mL | 0.28 (0.1–0.6), n = 6 | 0.1, n = 1 | 0.17 (0.03–4.3), n = 20 | 0.064 | 0.890 | |||
| hCG stimulated testosterone > 1.1 ng/mL | 3/6 | 0/4 | 0/16 | 0.500 | 1 | |||
| Genotype | ||||||||
| Truncating pathogenic variants | 3/7 (42.8%) | 0/5 | 13/33 (39.40%) | 1 | 0.738 | |||
| Severe pathogenic variants (< 10% receptor function) | 0/5 | 0/5 | 10/15 (66.6%) | 0.140 | 0.033 | |||
Data is expressed as median (range) or n/N as applicable.
a FSH, LH and testosterone values are for patients > 9 years of age.
FSH, follicle stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone.
Discussion
This study is the first report of patients with LCH from India and summarizes the phenotypic and genotypic characteristics of all patients published to date. The three patients from our center depict the wide range of phenotypic variability in the presentation of 46,XY LCH. 46,XY LCH has been previously classified as type-1 (with female external genitalia) and type-2 (with male-like genitalia) based on the clinical details of a few (n = 18) patients (3). Our systematic analysis of published literature reveals that around 10% of the 46,XY LCH patients have atypical genitalia and suggests that the external genital phenotype in LCH is a continuum.
Most (80%) 46,XY LCH patients were raised as females, had female-type external genitalia, and presented with delayed puberty. This common phenotype in 46,XY patients is well explained by the physiology of LHCGR activity in different stages of life. Female sexual differentiation in a 46,XY fetus suggests unresponsiveness of LHCGR to hCG in antenatal life, while similar unresponsiveness to LH at puberty results in poor androgen production and delayed puberty. Accordingly, most patients in this cohort had low serum testosterone levels (< 0.3 ng/mL) except for two patients. Notably, the latter two 46,XY siblings with compound heterozygous variants (p.Ile374Thr/p.Thr392Ile) with female-type external genitalia (suggesting antenatal hCG inaction) had low-normal pubertal basal serum testosterone levels (3.9 ng/mL and 4.3 ng/mL, normal range: 4–12 ng/mL), suggesting some pubertal LH action. These siblings had an undetectable testosterone response to exogenous hCG stimulation (20, 21).
About 20% of 46,XY LCH patients had male external genitalia and were raised as males. Such a degree of genital virilization is indirect evidence of normal/partial hCG action on LHCGR during embryonal life. Surprisingly, the same patients lacked pubertal LH action and had low basal serum testosterone (median of 0.42 ng/mL (0.1–0.94)). Of note, patients from three families with biallelic variants (p.Ile625Lys, exon 10 deletion, and exon 10: c.947_950del) had a testosterone response to hCG-stimulation (17, 22, 23). These observations indicate a differential sensitivity of the mutant LHCGR to LH and hCG. A differential response of the receptor to hCG vs LH has been classically described in a patient with a biallelic exon 10 deletion who had male external genitalia (Sinnecker score 1) and low pubertal baseline testosterone but had a remarkable increase in testicular volume, serum testosterone, and sperm count with hCG therapy. Functional studies have confirmed the preserved response of mutant exon 10 deleted LHCGR to hCG but not to LH (17). While traditionally, LH and hCG have been considered equivalent, at least some ligand-specific differences in the intracellular signaling pathways invoked by LHCGR binding of LH vs hCG have been described in human granulosa-cell and mice Leydig-cell studies (24). Most functional studies on LHCGR defects have been done using hCG. Our observations signify the need for exploring the functional LHCGR defects using LH as well, which we believe may reveal exciting results.
Although the data is limited in minipubertal age, the pubertal age hormonal profile of 46,XY LCH patients reveals interesting patterns. Notably, in the pre-pubertal age, most LCH patients had higher age-matched LH (≥ 0.3 IU/L in 10/12) and FSH (≥ 1.0 IU/L in 11/12), suggesting a likely physiological contribution of LH-mediated testicular steroids to the prepubertal quiescence of the hypothalamic-pituitary-gonadal axis. Similar examples of higher pre-pubertal gonadotropin levels are observed in disorders with poor gonadal sex-steroid production, such as 46,XX aromatase deficiency and 46,XY 17-alpha-hydroxylase deficiency (25, 26, 27). Serum FSH levels were elevated (≥10 IU/mL) in about half of pubertal/post-pubertal LCH patients. The gonadal regulation of FSH involves a composite negative regulation by gonadal steroids and inhibin-B (28, 29). Given that testosterone deficiency is seen in most cases, normal FSH levels in half of the LCH patients indicate adequate inhibin-B production by variably preserved Sertoli cells. Normal/high levels of inhibin-B and AMH in these patients may help to distinguish LCH from gonadal dysgenesis. Adrenarche in this group of patients has not been explicitly described in the literature and needs further studies.
Gonadal germ-cell malignancy is rare in 46,XY LCH patients, with only one report of small gonadoblastoma foci. Greater germ-cell maturation till the spermatid stage in LCH patients may explain the minimal risk of gonadal malignancy in them (30). Most patients with female-like genitalia and male-like genitalia were raised as females and males, respectively, with no report of gender incongruence, which signifies the effect of antenatal androgens on brain imprinting and gender identity (30). Notably, 5/7 patients with atypical genitalia were prepubertal, and longer follow-up data are required to understand the true gender identity in this sub-cohort.
Three of nine patients who were raised as males and treated with testosterone replacement therapy increased their testicular volume. The precise mechanism of this intriguing phenomenon is unknown, but a synergistic action of intact FSH and exogenous testosterone on Sertoli cell proliferation is a plausible explanation. Exogenous testosterone-induced testicular growth has also been observed in juvenile monkeys and 46,XY males with LH β-subunit deficiency (31).
Seventy-two different variants in the LHCGR gene (11 exons) have been described in 62 families to date. While variants are distributed throughout the gene, the maximum variants (n = 25) has been reported in exon 11. Most variants described are unique, but seven of them are recurrent. Two recurrent variants have been described in Turkish families (c.161+4A>G and p.Ala483Asp), suggesting the possibility of a probable founder effect. This would require further studies to confirm (32, 33).
On genotype-phenotype correlation analysis in 46,XY LCH patients, no correlation was observed when variants were classified as per their effect on protein truncation. This can be explained as most non-truncating variants have been shown to cause intracellular retention of receptor protein, causing a severe functional defect in receptor function. When the analysis is limited to variants whose functional studies have been done, the variants resulting in severe functional defects (arbitrarily defined as <10% of wild-type receptor activity) were exclusively seen in patients with female-like genitalia. Further studies are needed to corroborate these findings. Results of functional studies of LHCGR variants need to be interpreted with caution as methodological differences have been shown to cause variability in results (34). Glycoprotein hormone receptors are known to have ligand-independent constitutional activity. However, in this study, similar phenotype was seen for patients with variants that affect the extracellular domain vs other domains (35, 36). In the study analysis, there was no significant difference in phenotype in patients harboring Compound heterozygous (CHz) vs homozygous variants. Notably, a unique combination of a ligand-defective with a signaling-defective mutant has shown at least partial rescue of the receptor in an earlier study (37).
The phenotype of 46,XX with LHCGR inactivating variants was uniform and reflective of LH inaction: delayed menarche, oligo-amenorrhea, anovulatory infertility, and failure of oocyte retrieval with hCG treatment despite multi-follicular development on ovulation induction. 46,XX LHCGR-In patients (n = 18) are less frequently described than 46,XY (n = 70). As it is an autosomal recessive condition, it is expected to be equally prevalent in 46,XX and 46,XY karyotypes. This suggests that most 46,XX patients probably remain undiagnosed. Strong clinical suspicion is required to delineate patients with LHCGR inactivating variants from PCOS due to some biochemical (serum LH) and radiological (polycystic morphology) overlap between the two disorders. We believe that LHCGR inactivating variants should be suspected in patients with NIH phenotype D PCOS who present with oligo-anovulation and have complete absence of clinical or biochemical hyperandrogenism. This would have prognostic implications for fertility and assisted reproduction treatment.
Our study is the first description of Indian LCH patients. The strength of our study is the detailed individual patient data-based analysis of all published patients with LHCGR inactivating variants. The limitation of our analysis includes heterogeneous data as reported in different case reports/series and the inherent limitation of functional studies.
Conclusion
To conclude, we report the first three Indian LCH patients with widely variable phenotypes and two novel variants in LHCGR. LCH is a rare but interesting cause of hypogonadism. While pubertal LH inaction is uniform, the severity of antenatal hCG inaction is variable in 46,XY patients. However, a few mutant LHCGR have differential actions for LH and hCG. The genotype-phenotype correlation analysis confirmed that the estimated severity of LHCGR defect, as per in vivo functional studies, correlated with phenotypic severity. High LH, very low basal and hCG-stimulated testosterone, with normal to high FSH, is the biochemical phenotype that clinches the diagnosis in most 46,XY patients. Such a distinct biochemical phenotype is not present in 46,XX patients, requiring a high index of suspicion for diagnosis.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-24-0246.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Consent to participate
Considering the retrospective nature of the analysis, a waiver for consent was obtained from the Ethics Committee.
Consent for publication
All the co-authors have consented to the publication of the study results.
Availability of data and material
Authors confirm that data supporting the findings of this study are available within the article.
Statement of Ethics
This study was conducted after approval by the Institutional Ethics Committee of Seth GS Medical College and KEM Hospital (EC/OA-196/2023), Mumbai. Individual patient identities were kept confidential and coded prior to analysis.
Author contribution statement
RB and SRJ collected and analyzed the data with the help of SSM. RB and SRJ wrote the manuscript in consultation with ARL, SKB, SSM, VS, MPK, VP, NSS, and TRB. Genetic analysis was done by SA and SCH. The corresponding author (ARL) had full access to all the data in the study and takes responsibility for the decision to submit the article for publication. All authors approved the final version of the manuscript and agree to be accountable for the work and to ensure that any questions relating to the accuracy and integrity of the paper are appropriately investigated and resolved.
Data availability statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.
All parameters are expressed in median (range), except for n<3 where individual values are given. Age stratification is as follows: mini pubertal age (0 to ≤ 0.5 years), pre-pubertal age (> 0.5 to ≤ 9 years), pubertal age (> 9 to ≤ 14 years), post pubertal age (> 14 years). N: exact value not given, and reported as normal. L: exact value not given, and reported as low. The normal range for biochemistry is derived from Fanelli F, Baronio F, Ortolano R, Mezzullo M, Cassio A, Pagotto U & Balsamo A. Normative basal values of hormones and proteins of gonadal and adrenal functions from birth to adulthood. Sexual Development 2018 12 50–94. (https://doi.org/10.1159/000486840)
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