Undiagnosed RASopathies in infertile men

Abstract

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10^-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.

Keywords: RAS/MAPK pathway; cancer; congenital testicular dysgenesis; cryptorchidism; exome sequencing; molecular diagnosis; multidisciplinary management; syndromic male infertility.

Figure 1

Ras/mitogen-activated protein kinase (MAPK) signaling pathway genes with disease-causing variants in idiopathic male infertility patients. (A) Distribution of identified likely pathogenic (LP) and pathogenic (P) variants across genes and clinical phenotypes. Spermatogenic failure (SPGF) was defined as total sperm count of 0–39 million per ejaculate (8). Cryptorchidism (CR) refers to at least one testicle missing in the scrotum at the recruitment or medical history of testicular maldescent. A subfertile normozoospermic man with SOS1 p.I437T presented low-normal sperm count (50 million per ejaculate) and needed infertility management to achieve pregnancy. (B) Prevalent health conditions in eight (of 10 total) idiopathic SPGF patients carrying LP/P variants in Ras/MAPK pathway genes available for the follow-up clinical assessment. (C) Testicular histology of Case 1 carrying PTPN11 c.922A>G (p.N308D) variant showed mixed testicular atrophy and heterogeneous morphology. Most seminiferous tubules (ST) were narrowed due to thickened lamina propria (Lp), and ~30% were completely or partially hyalinized (HT). ST with Sertoli cells (SC) and spermatogonia (SG) represented <10%, and those with Sertoli cells only (SCO) represented 20%. Maturation arrest in the primary (PS) or secondary spermatocyte stage was observed in ~10%. In the rest of ST, all spermatogenic stages were detected, but the number of round spermatids (RS) and elongated spermatids (ES) was reduced. Severe Leydig cell (LC) hyperplasia was noticeable in the stroma surrounding the tubules. Details are provided in Supplementary Table 8. (D) Immunohistochemical staining of PTPN11 in testicular tissue sections in Case 1 with the PTPN11 p.N308D substitution and in a control case with histologically normal active spermatogenesis (38 y, obstructive azoospermia). PTPN11 protein localized in the cytoplasm of spermatogenic cells, LC and SC. Staining intensity in the control case was the strongest in primary spermatocytes, early round spermatids, and LC, whereas in spermatogonia and SC, only faint PTPN11 positivity was detected. In Case 1 tissue, the strongest staining was detected in LC, consistent with the testicular histology (C). In the negative control performed without the PTPN11 antibody, no staining was detected. Brown color indicates chromogen-labeled antibody, and violet color indicates hematoxylin background staining. (E) Testicular mRNA expression of genes with identified LP/P variants in SPGF patients. Normalized log gene expression levels (Y-axis) in distinct testicular cell types were derived from the human infertility single-cell testis atlas (HISTA ver. 2.9.6) (20). The category “immune cells” includes lymphoid T cells and rare B cells as well as myeloid M1/M2 macrophages. The “other cells” category comprises endothelial and myoid cells. Each dot represents data from one cell. Boxes include 25%–75% of the data points, and the line shows median expression. SCT, spermatocytes; RS, round spermatids.

Figure 2

Patients with more than one disease-causing variant and their pedigrees. (A) Pedigree of Case 6 carrying a novel likely pathogenic (LP) variant in Noonan syndrome gene SOS2 c.26A>G (p.E9G) and incidental findings KIF7 c.434A>C (p.Y145S; LP) and CHEK1 c.1036C>T (p.Q346Ter; LP) that are likely co-contributors to the patient’s complex phenotype and comorbidities. (B) Pedigree of Case 9 carrying SPRED1 c.973C>T (p.R325Ter; pathogenic) and an incidental finding TP63 c.1283C>T (p.P428L; LP). Café-au-lait spots, characteristic of Legius syndrome, were reported in three generations. TP63 is linked to several congenital conditions, including hydronephrosis, urethral stenosis (21), and orofacial clefts (22), observed in this family. None of the family members was available for genetic testing. Detailed clinical data on Case 6 and Case 9 are provided in Table 2. d., died; y, years; WT, wild type.

Figure 3

Protein domains of LZTR1 harboring variants linked to dominant or recessive forms of Noonan syndrome. Disease-causing variants in the LZTR1 gene are linked to both autosomal dominant (AD) and recessive (AR) forms of Noonan syndrome (NS), depending on the position of the variant within the encoded protein (24). Heterozygous LP/P variants in the N-terminal Kelch repeat domain are typically linked to AD forms of LZTR1-linked NS, whereas AR forms are mostly caused by biallelic LP/P variants within the C-terminal BTB domain. All identified LP/P findings in the LZTR1 gene were heterozygous, located in the Kelch repeat domain, and previously reported in the ClinVar database as LP/P. Previously, the detected missense substitutions p.R283Q and p.R170Q have been linked with AD form of NS (25) or schwannomatosis (24), respectively. BTB, broad complex, tram track, and bric-a-brac domain.