Autosomal Recessive Inheritance Research Articles

Spinal Muscular Atrophy: The Past, Present, and Future of Diagnosis and Treatment.

Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance. The first cases of SMA were reported by Werdnig in 1891. Although the phenotypic variation of SMA led to controversy regarding the clinical entity of the disease, the genetic homogeneity of SMA was proved in 1990. Five years later, in 1995, the gene responsible for SMA, SMN1, was identified. Genetic testing of SMN1 has enabled precise epidemiological studies, revealing that SMA occurs in 1 of 10,000 to 20,000 live births and that more than 95% of affected patients are homozygous for SMN1 deletion. In 2016, nusinersen was the first drug approved for treatment of SMA in the United States. Two other drugs were subsequently approved: onasemnogene abeparvovec and risdiplam. Clinical trials with these drugs targeting patients with pre-symptomatic SMA (those who were diagnosed by genetic testing but showed no symptoms) revealed that such patients could achieve the milestones of independent sitting and/or walking. Following the great success of these trials, population-based newborn screening programs for SMA (more precisely, SMN1-deleted SMA) have been increasingly implemented worldwide. Early detection by newborn screening and early treatment with new drugs are expected to soon become the standards in the field of SMA.

Interpretation of SLC3A1 and SLC7A9 variants in cystinuria patients: The significance of the PM3 criterion and protein stability

Cystinuria is a genetic disorder caused by defects in the b0,+ transporter system, which is composed of rBAT and b0,+AT coded by SLC3A1 and SLC7A9, respectively. Variants in SLC3A1 and SLC7A9 follow autosomal recessive inheritance and autosomal dominant inheritance with reduced penetrance, respectively, which complicates the interpretation of cystinuria-related variants. Here, we report seven different SLC3A1 variants and six different SLC7A9 variants. Among these variants were two novel variants previously not reported: SLC3A1 c.223C > T and SLC7A9 c.404A > G. In silico analysis using REVEL correlated well with the functional loss upon SLC7A9 variants with scores of 0.8560–0.9200 and 0.4970–0.5239 for severe and mild decrease in transport activity, respectively. In addition, DynaMut2 was able to predict a decreased protein expression level resulting from the SLC7A9 variant c.313G > A with a ΔΔGStability −2.93 kcal/mol. Our study adds to the literature as additional cases of a variant allow applying the PM3 criterion with higher strength level. In addition, we suggest the clinical utility of REVEL and DynaMut2 in interpreting SLC3A1 and SLC7A9 variants. While a decreased protein expression level is not embraced in the current variant interpretation guidelines, we believe in silico protein stability predicting tools could serve as evidence of protein function loss.

A novel IBA57 variant is associated with mitochondrial iron-sulfur protein deficiency and necrotizing myelopathy in dogs.

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case-control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron-sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57-depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants.

Clinical masks of marble disease

Marble disease, or osteopetrosis (OPT), is rare in the practice of a rheumatologist, internist or pediatrician. This group of hereditary diseases is based on a defect in the formation, development and functioning of osteoclasts (OCL), which leads to disruption of the processes of resorption and remodeling of bone tissue. Disturbance of resorption leads to increased density and changes in the quality of the bones, as a result of which they become more fragile. As a rule, the disease manifests with pathological fractures. In recent decades, 70% of patients with OPT have been found to have mutations in at least 10 genes that lead to impaired functioning of the OCL. Depending on the variant of inheritance, autosomal dominant, autosomal recessive and intermediate types of OPT are distinguished. Autosomal dominant OPT has a benign course that can be asymptomatic or characterized by multiple bone fractures and other spinal anomalies. The disease usually manifests in adulthood or adolescence. Life expectancy in patients of this group does not differ from that in the general population. Malignant, or infantile, OPT is associated with an autosomal recessive inheritance pattern. Its clinical manifestations are observed from the moment of birth, without treatment, patients die within the first decade of life. In such patients, in addition to the skeletal pathology, there is involvement of the hematopoietic system, compression of the cranial nerves and their function disturbance.The article presents a clinical case of autosomal dominant OPT diagnosed in adulthood (at the age of 38), when the patient referred to the doctor for the first time. Differential diagnosis with ankylosing spondylitis and paraneoplastic spondyloarthritis was performed.

Establishment of a novel human induced pluripotent stem cell line (SIPDi001-A) with compound heterozygous mutations in the UBR7 gene from a Li-Campeau syndrome patient

Li-Campeau syndrome (LICAS) is a syndromic neurodevelopmental disorder characterized by autosomal recessive inheritance and global developmental delay. In this study, we reported the generation of a novel induced pluripotent stem cell (iPSC) line derived from peripheral blood mononuclear cells (PBMCs) obtained from a 7-year-old male patient with Li-Campeau syndrome. The patient carries compound heterozygous variants in the UBR7 gene (c.35_54dup, p.S19Rfs*42; c.863 T > C, p.L288P). The iPSC line showed typical cell morphology, robust expression of pluripotent and self-renewal markers, normal karyotype, and trilineage differentiation potential. This iPSC cell line could be valuable for investigating the underlying pathological mechanisms of neurodevelopmental disorders caused by UBR7 mutations.

Case report: Pyruvate kinase deficiency in an infant

Pyruvate kinase deficiency is a rare cause of hemolytic anaemia. We report a case of Non-spherocytic hemolytic anaemia due to a deficiency of the red cell Pyruvate kinase enzyme due to a homozygous type of autosomal recessive inheritance. The child underwent a double volume Exchange Transfusion and received blood transfusions for Rapid Hemolysis on the first day of life. In this report, we stress the need for consideration of Red cell Pyruvate kinase enzyme deficiency as one of the differentials of Non-immune hemolytic anaemia. The study will help human geneticists and paediatricians with early identification or screening.

P-699 A clinical predictive model for live birth in women of advanced age undergoing PGT cycles

Abstract Study question In 37–45-year-old women undergoing preimplantation genetic testing, who will benefit from repeated cycles until birth? Summary answer The chance of delivering after repeated cycles is higher in those with at least one top-quality unaffected embryo in their first preimplantation genetic testing cycle. What is known already PGT for monogenic disease or structural chromosomal rearrangement (PGT-M and PGT-SR) is offered to couples when one or both carry a monogenic mutation or chromosomal rearrangement that put their future offspring at risk of having a genetic disorder so they can select an unaffected embryo for uterine transfer. The tendency to delay childbirth has led to an increase in advanced-aged women seeking PGT treatments. This poses a major challenge to PGT treatments. Study design, size, duration A retrospective cohort study was conducted at a university hospital reproductive center. The computerized database of 158 women aged 37–45 undergoing 753 PGT-M/SR cycles between 2010 and 2021 was analyzed. Participants/materials, setting, methods The reproductive outcomes of women who were 37–45 years of age, starting a PGT-M/SR cycle, were analyzed until the first live birth or until the patient reached the age of 45. Data were analyzed using univariate analysis, multivariable stepwise logistic regression, Kaplan–Meier method, and decision tree analysis. The cumulative live birth rate was calculated in a conservative manner because the assumption that patients who did not return for subsequent cycles had negative results was made. Main results and the role of chance The analysis included 158 women undergoing 753 preimplantation genetic testing cycles. The cumulative live birth rate was 37.342% (59/158). Decision tree analysis revealed that women aged ≤ 40.1 or women > 40.1 with one or more top-quality embryos in their first cycle had the best chance for a live baby (41% and 56%, respectively). Those older than 40.1 without top-quality embryos and seven or fewer dominant follicles had no live births. A Kaplan–Meier curve showed that for autosomal dominant diseases, there was a negligible increase in live birth rate after three cycles, compared to six cycles in autosomal recessive inheritance. Limitations, reasons for caution This study is limited by its retrospective design and the low birth rate in this age group, which reduces its power to detect differences in birth rates between different modes of inheritance. Wider implications of the findings The predictive model for live births in women aged 37-45 developed in this study can be used for clinical decision making and patient consultation. Additional PGT cycles after three in carriers of an autosomal dominant disorder and six in those with an autosomal recessive disorder should be considered prudently. Trial registration number Not applicable

P-047 A novel mutation of DRC1 gene causes multiple morphological abnormalities of the sperm flagella and male infertility in humans

Abstract Study question Can novel mutations of DRC1 gene affect the sperm morphology in humans and how about the outcomes of assisted reproductive therapy of the affected patients? Summary answer An infertile man with multiple morphological abnormalities of the sperm flagella caused by DRC1 biallelic mutations has good outcomes of fertility after intracytoplasmic sperm injection. What is known already The nexin-dynein regulatory complex (N-DRC) functions by linking neighboring doublet microtubules within motile cilia and flagella, stabilizing the axonemal structure and thereby regulating ciliary motility. As a core of N-DRC protein, the disruptions of DRC1 gene have been identified to cause multiple morphological abnormalities of the sperm flagella (MMAF) and male infertility in humans and mice. DRC1 mutations usually resulted in decreased flagellum axoneme stability, thereby causing flagellar structural disorder. Study design, size, duration The 35 patients with MMAF were recruited from the center for reproductive medicine from August 2019 to June 2022. They were diagnosed with primary male infertility caused by abnormal morphologies of sperm flagella and decreased sperm motility. Genetic testing, pedigree analysis, sperm morphological analysis, functional assays, and assisted reproductive therapy were performed in 2022. Participants/materials, setting, methods We identified and confirmed the DRC1 mutation through a 22-gene next-generation sequencing panel and sanger sequencing. Papanicolaou-staining, scanning electronic microscope, and transmission electronic microscope were performed to reveal the changes of sperm morphology and ultrastructure. Immunostaining was conducted to show the effect of DRC1 mutation on the expression and localization on sperm structural proteins. After the ovarian stimulation, a time-lapse monitoring system was applied for the assisted reproductive therapy of DRC1 mutant patient. Main results and the role of chance Biallelic mutations in DRC1 were identified in a 39-years-old proband who suffered from primary infertility for 9 years due to severe asthenoteratozoospermia. Pedigree analysis of the non-consanguineous family manifested an autosomal recessive inheritance pattern. Papanicolaou-staining, scanning and transmission electronic microscopy showed the abnormalities of sperm flagella and severe disorganization of the axoneme in DRC1-deficient male compared to control subject. Immunostaining with sperm-specific markers, TOM20, DNAI1, and DNALI1, showed the composite changes of flagella morphology and molecular components. After intracytoplasmic sperm injection (ICSI), the rate of fertilization was 71.4% (5/7), the rate of embryo cleavage was 100% (5/5), the rate of transferable embryo was 60% (3/5), respectively. Following frozen-thawed embryo transfer, the wife of the proband became pregnant. Limitations, reasons for caution Additional cases are needed to investigate the gene-disease relationship between DRC1 mutations and male infertility owing to abnormal sperm morphology and motility. Wider implications of the findings This study expands the mutant spectrum of DRC1 and describes a good fertility outcome of assisted reproduction therapy with ICSI for the DRC1 mutant patient. Together with the available information regarding male infertility, it provides new information for the genetic diagnosis and counseling of MMAF in the future. Trial registration number Not applicable

A homozygous frameshift variant in SYCP2 caused meiotic arrest and non-obstructive azoospermia.

Genetic causation for the majority of non-obstructive azoospermia (NOA) remains unclear. Mutations in synaptonemal complex (SC)-associated genes could cause meiotic arrest and NOA. Previous studies showed that heterozygous truncating variants in SYCP2 encoding a protein essential for SC formation, are associated with non-obstructive azoospermia and severe oligozoospermia. Herein, we showed a homozygous loss-of-function variant in SYCP2 (c.2689_2690insT) in an NOA-affected patient. And this variant was inherited from heterozygous parental carriers by natural reproduction. HE, IF, and meiotic chromosomal spread analyses demonstrated that spermatogenesis was arrested at the zygotene stage in the proband with NOA. Thus, this study revealed that SYCP2 associated with NOA segregates in an autosomal recessive inheritance pattern, rather than an autosomal dominant pattern. Furthermore, our study expanded the knowledge of variants in SYCP2 and provided new insight into understanding the genetic etiology of NOA.

Whole-exome sequencing identified novel DNAH5 homozygous variants in two consanguineous families with primary ciliary dyskinesia.

Whole-exome sequencing identified novel DNAH5 homozygous variants in two consanguineous families with primary ciliary dyskinesia.

Amyloid goiter secondary to familial Mediterranean fever with E148Q mutation: A unique case.

Amyloid goiter secondary to familial Mediterranean fever with E148Q mutation: A unique case.

Clinical Profile of Congenital Factor XIII Deficiency in Children.

Congenital Factor 13 Deficiency (FXIIID) is a rare bleeding disorder (RBD) of autosomal recessive inheritance, with an incidence of 1 in 3-5million. The clinical symptomatology, diagnosis, and management of FXIIID are described. A retrospective chart review of children with FXIIID was performed from January 2000 through October 2021 at a tertiary care center in Southern India. The diagnosis was performed by the Urea clot solubility test (UCST) and Factor XIII antigen assay. Twenty children (representing 16 families) were included. Male: Female ratio was 1.5:1. The median age of symptom onset was 6 mo, and the median age of diagnosis was 1 y, demonstrating a delay in diagnosis. Consanguinity was present in 15 (75%) with 4 children having affected siblings. Clinical symptomatology ranged from mucosal bleeds to intracranial bleeds and hemarthrosis, with many children having a history of prolonged umbilical bleeding in their neonatal period. Fourteen children were on cryoprecipitate prophylaxis. Four children had breakthrough bleeds due to irregular prophylaxis, including one intracranial bleed due to a delay in cryoprecipitate prophylaxis during the covid pandemic. Congenital FXIIID presents with a wide range of bleeding manifestations. The high prevalence of consanguinity in Southern India can be a cause of FXIIID's high prevalence in this region. There is a propensity for intracranial bleeding with a significant number having this at first presentation. Regular prophylaxis is required and feasible to prevent potentially fatal bleeds.

On the Basics of Pedigree Visualization and Feature Extraction for the Autosomal Recessive Inheritance Pattern

On the Basics of Pedigree Visualization and Feature Extraction for the Autosomal Recessive Inheritance Pattern

Consanguineous couples' experiences and views regarding expanded carrier screening: Barriers and facilitators in the decision-making process.

Expanded carrier screening (ECS) entails a screening offer for multiple recessive disorders at the same time, and allows testing of individuals or couples regardless of ancestry or geographic origin. Children of consanguineous couples have a higher-than-average risk of manifesting autosomal recessive disorders. This study aims to contribute to the responsible implementation of ECS for consanguineous couples. Seven semi-structured interviews were conducted with consanguineous couples who had recently participated in Whole Exome Sequencing (WES)-based ECS at Maastricht University Medical Center (MUMC+), the Netherlands. The test offered at MUMC+ covers a large number of disease-related genes (~2000), including severe, relatively mild, early- and late-onset disorders. Respondents were interviewed about their views on, and experiences with participation in WES-based ECS. Overall, participation was experienced as worthwhile: it enabled respondents to make informed choices with regard to family planning as well as to take on the presumed parental responsibility to deliver their children as healthy as possible. Furthermore, our findings suggest that (1) true consent for having this test requires timely information about the possible implications of a positive test result for specific categories of findings, as well as about the success rates of the available reproductive options; (2) the clinical geneticist can play a pivotal part in informing participants as well as providing clear information about autosomal recessive inheritance; (3) more research is needed to explore what type of genetic risk information is considered 'meaningful' by participants and actually contributes to reproductive decision-making.

Clinical implications of universal germline testing in breast cancer patients in an Arab population: The Jordanian Exploratory Cancer Genetics (Jo-ECAG) study.

e22535 Background: Germline genetic testing (GGT) for breast cancer (BC) has implications for surgical decision-making, treatment selection, clinical follow-up and cascade testing. As uptake of GGT increases globally, it is important to examine its impact on clinician decision-making, particularly in different ethnic groups. Methods: The Jo-ECAG Study consisted of unselected, newly diagnosed cancer patients (pts) from a single center in Jordan. Pts were classified as meeting (in criteria, IC) or not meeting (out of criteria, OOC) National Comprehensive Cancer Network (NCCN) v.1.2020 GGT criteria. Pts underwent a 84 gene test with clinician-reported outcomes collected >2 months post GGT. Analysis was limited to pts with BC. Pts with one PGV in a gene associated with autosomal recessive inheritance (“carriers”) were excluded from overall PGV count. Blank responses were considered uninformative. Descriptive statistics and Fisher’s exact test were employed. Results: 1648 (21 male) Arabic pts with BC, 36% metastatic, with a mean age at diagnosis of 50.5 were tested; 67% were IC. 212 PGVs were identified in 202 (12%) pts, with a significantly higher rate in IC vs. OOC pts (14% vs. 8%, p=0.0002). PGVs were most common in BRCA1/2 (4%), APC I1307K (4%), and other BC-associated genes such as ATM, CHEK2, PALB2 and TP53 (collectively, 3%). 2% of pts were carriers and 57% had uncertain results. Among pts with PGVs and informative responses (Table), 18% had changes to treatment (11% OOC) and 82% to surveillance/follow up (16% OOC) based on GGT results. Genetic counseling and/or GGT was recommended for relatives of >90% of PGV and carrier pts. No changes to treatment or follow-up were made for pts with negative or uncertain results. Conclusions: 1 in 8 Jordanian BC pts had a PGV identified, with a similar breakdown of PGVs observed in other ethnic cohorts, aside from a higher percentage of APC I1307K. Both treatment and surveillance/follow-up decisions were impacted by GGT results. Appropriately treatment or management was not escalated for pts with negative or uncertain results. [Table: see text]

A rare case of isolated IBM — HNRNPA2B1associated myopathy with inclusion bodies

Hereditary inclusion body myopathy (IBM) is a variant of multisystem proteinopathy. It is a generalized progressive disease with autosomal recessive or autosomal dominant inheritance, characterized by the development of a degenerative process in muscle fi bers due to the accumulation of rimmed vacuoles and nuclear intermediate fi laments. This article presents a clinical observation of a rare variant of IBM — HNRNPA2B1-associated myopathy with a phenotypically diverse picture in representatives of one family from diff erent generations.

Investigation of cancer predisposition in Fanconi anemia heterozygotes: A DiscovEHR cohort population study.

10589 Background: Fanconi anemia (FA) is a cancer predisposition syndrome caused by biallelic pathogenic variants in one of 22 genes involved in DNA intrastrand crosslink repair. 20 of these genes have an autosomal recessive inheritance pattern. Of the FA genes, 5 ( BRCA1, BRCA2, BRIP1, PALB2, RAD51C) are known cancer predisposition genes (CPG) when inherited in monoallelic autosomal dominant manner, though the heterozygous predisposition status of the remaining 15 remains unclear. Large population-level exome sequencing projects with linked health records such as the DiscovEHR cohort serve as promising tools to establish sufficient power to investigate all FA genes. Quantifying cancer risk is important for the counseling and surveillance of FA heterozygotes, especially with the ever-increasing use of genetic testing identifying these individuals. Methods: 170,503 individuals enrolled in the DiscovEHR Cohort were analyzed for pathogenic/likely pathogenic variants in 22 FA genes and identified 5834 subjects, the case group. ICD10 phenotype classifications for these subjects were curated to Phecodes and ICD-O-3 Site Recodes for two control arms of analysis. The first control arm included all DiscovEHR subjects excluding the case group and those with a variant of uncertain significance in the genes of interest with a deleterious in-silico score. The second control arm included individuals from the Surveillance, Epidemiology, and End Results (SEER) database. Phecode analysis yielded odds ratios adjusted for age, sex, race, smoking status, and BMI. SEER analysis yielded Standardized Incidence Ratio scores adjusted for age, sex, and birth cohort. When both arms of the analysis were completed Phecodes and SEER analyses were matched and positive signals for cancer risk were defined by statistically significant ratios in both. Results: The 5 known CPGs demonstrated multiple signals for cancer predisposition as expected (p < 0.0001). Results for FANCA (p = 0.4231) and FANCC (p = 0.8142) heterozygotes validated recent results from our group showing no increased risk of cancer in relatives of FA patients with these genotypes. Ten other FA genes also showed no increased risk of cancer. 3 genes suggested a possible increased risk of specific cancers in heterozygotes and are currently being evaluated in a second large population exome cohort to validate these findings. Conclusions: This is the largest study to date on the cancer predisposition risk in Fanconi anemia heterozygotes. We have confirmed the known risk in 5 well-described CPGs and the lack of risk amongst FANCA and FANCC heterozygotes, the most common in population. Further analysis via an additional large population cohort is underway to validate any possible novel cancer predispositions in 3 genes. For the remaining FA genes, this population analysis supports previous literature on known CPGs and the lack of cancer predisposition in most FA genes.

Clinical utility of chromosomal microarray analysis and whole exome sequencing in foetuses with oligohydramnios

Objectives To evaluate the clinical utility of chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in foetuses with oligohydramnios. Methods In this retrospective study, 126 fetuses with oligohydramnios at our centre from 2018 to 2021 were reviewed. The results of CMA and WES were analysed. Results One hundred and twenty-four cases underwent CMA and 32 cases underwent WES. The detection rate of pathogenic/likely pathogenic (P/LP) copy number variant (CNV) by CMA was 1.6% (2/124). WES revealed P/LP variants in 21.8% (7/32) of the foetuses. Six (85.7%, 6/7) foetuses showed an autosomal recessive inheritance pattern. Three (42.9%, 3/7) variants were involved in the renin–angiotensin–aldosterone system (RAAS), which are the known genetic causes of autosomal recessive renal tubular dysgenesis (ARRTD). Conclusion CMA has low diagnostic utility for oligohydramnios, while WES offers obvious advantages in improving the detection rate. WES should be recommended for fetuses with oligohydramnios.

A loss-of-function variant in canine GLRA1 associates with a neurological disorder resembling human hyperekplexia

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon–intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility.

What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility? Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected. The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear. Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2. For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein's cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined. This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients' testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation. Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism. In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility. This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG ('Emmy Noether Programme' project number 464240267). The authors declare no conflict of interest. N/A.