Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.

Czech dysplasia (OMIM 609162) is a recently delineated COL2A1 disorder characterized by early-onset progressive pseudorheumatoid arthritis, platyspondyly, short third and fourth metatarsals, normal height, and the absence of ophthalmological problems or cleft palate. Czech dysplasia is caused by a specific missense mutation (R275C, c.823C > T) in the triple helical domain of the COL2A1 gene. We report on a large family with 11 patients with typical Czech dysplasia and sensorineural hearing loss. Hearing loss has hitherto not been considered as a major manifestation of Czech dysplasia. Mutation analysis documented the COL2A1 c.823C > T (R275C) mutation in all affected individuals. Thus, Czech dysplasia is possibly caused exclusively by the R275C mutation, which is a unique situation among the COL2A1 disorders. The family provides further evidence for the remarkably uniform manifestation of the clinical and radiological abnormalities and adds hearing loss to the list of major anomalies of Czech dysplasia.

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STUDY QUESTION Do bi-allelic variants in the genes encoding the MSH4/MSH5 heterodimer cause male infertility? SUMMARY ANSWER We detected biallelic, (likely) pathogenic variants in MSH5 (4 men) and MSH4 (3 men) in six azoospermic men, demonstrating that genetic variants in these genes are a relevant cause of male infertility. WHAT IS KNOWN ALREADY MSH4 and MSH5 form a heterodimer, which is required for prophase of meiosis I. One variant in MSH5 and two variants in MSH4 have been described as causal for premature ovarian insufficiency (POI) in a total of five women, resulting in infertility. Recently, pathogenic variants in MSH4 have been reported in infertile men. So far, no pathogenic variants in MSH5 had been described in males. STUDY DESIGN, SIZE, DURATION We utilized exome data from 1305 men included in the Male Reproductive Genomics (MERGE) study, including 90 males with meiotic arrest (MeiA). Independently, exome sequencing was performed in a man with MeiA from a large consanguineous family. PARTICIPANTS/MATERIALS, SETTING, METHODS Assuming an autosomal-recessive mode of inheritance, we screened the exome data for rare, biallelic coding variants in MSH4 and MSH5. If possible, segregation analysis in the patients’ families was performed. The functional consequences of identified loss-of-function (LoF) variants in MSH5 were studied using heterologous expression of the MSH5 protein in HEK293T cells. The point of arrest during meiosis was determined by γH2AX staining. MAIN RESULTS AND THE ROLE OF CHANCE We report for the first time (likely) pathogenic, homozygous variants in MSH5 causing infertility in 2 out of 90 men with MeiA and overall in 4 out of 902 azoospermic men. Additionally, we detected biallelic variants in MSH4 in two men with MeiA and in the sister of one proband with POI. γH2AX staining revealed an arrest in early prophase of meiosis I in individuals with pathogenic MSH4 or MSH5 variants. Heterologous in vitro expression of the detected LoF variants in MSH5 showed that the variant p.(Ala620GlnTer9) resulted in MSH5 protein truncation and the variant p.(Ser26GlnfsTer42) resulted in a complete loss of MSH5. LARGE SCALE DATA All variants have been submitted to ClinVar (SCV001468891–SCV001468896 and SCV001591030) and can also be accessed in the Male Fertility Gene Atlas (MFGA). LIMITATIONS, REASONS FOR CAUTION By selecting for variants in MSH4 and MSH5, we were able to determine the cause of infertility in six men and one woman, leaving most of the examined individuals without a causal diagnosis. WIDER IMPLICATIONS OF THE FINDINGS Our findings have diagnostic value by increasing the number of genes associated with non-obstructive azoospermia with high clinical validity. The analysis of such genes has prognostic consequences for assessing whether men with azoospermia would benefit from a testicular biopsy. We also provide further evidence that MeiA in men and POI in women share the same genetic causes. STUDY FUNDING/COMPETING INTEREST(S) This study was carried out within the frame of the German Research Foundation sponsored Clinical Research Unit ‘Male Germ Cells: from Genes to Function’ (DFG, CRU326), and supported by institutional funding of the Research Institute Amsterdam Reproduction and Development and funds from the LucaBella Foundation. The authors declare no conflict of interest.