Genetics of ncHH: from a peculiar inheritance of a novel GNRHR mutation to a comprehensive review of the literature.

BACKGROUND: Normosmic congenital hypogonadotropic hypogonadism (ncHH) is caused by the deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Its typical clinical manifestation is delayed puberty and azoospermia. Homozygous and compound heterozygous mutations in the GNRHR gene (4q13.2) are the most frequent genetic causes of ncHH. OBJECTIVES: (i) Characterization at the molecular level (genetic origin and functional effect) of a unique homozygous mutation (p.Gly99Glu) in a ncHH man; (ii) to provide a comprehensive catalog of GNRHR mutations with genotype-phenotype correlation and comparison of in vitro studies vs. in silico prediction tools. MATERIAL AND METHODS: A ncHH man and his parents, in whom we performed the following: (i) Sanger sequencing, qPCR of the GNRHR gene; (ii) chromosome 4 SNP array; and (iii) competition binding assay and inositol phosphate signaling assay. PubMed and Human Genome Mutation Database (HGMD) search for GNRHR mutations. Bioinformatic analysis of 55 reported variants. RESULTS: qPCR showed two GNRHR copies in the index case. SNP array revealed the inheritance of two homologous chromosomes 4 from the mother (maternal heterodisomy; hUPD) with two loss of heterozygosity regions, one of them containing the mutated gene (maternal isodisomy; iUPD). Functional studies for the p.Gly99Glu mutation demonstrated a right-shifted GnRH-stimulated signaling response. Bioinformatic tools show that commonly used in silico tools are poor predictors of the function of ncHH-associated GNRHR variants. DISCUSSION: Functional analysis of the p.Gly99Glu mutation is consistent with severely decreased GnRH binding affinity (a severe partial loss-of-function mutation). Complete LOF variants are associated with severe and severe/moderate phenotype, whereas partial LOF variants show wide range of clinical manifestations. CONCLUSION: This is the first ncHH patient carrying a novel causative missense mutation of GNRHR with proven 'severe pLOF' due to maternal hUPD/iUPD of chromosome 4. Our literature review shows that functional studies remain essential both for diagnostic and potential therapeutic purposes.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

The Y chromosome-linked copy number variations and male fertility.

Since the first definition of the AZoospermia Factor (AZF) regions, the Y chromosome has become an important target for studies aimed to identify genetic factors involved in male infertility. This chromosome is enriched with genes expressed exclusively or prevalently in the testis and their absence or reduction of their dosage is associated with spermatogenic impairment. Due to its peculiar structure, full of repeated homologous sequences, the Y chromosome is predisposed to structural rearrangements, especially deletions/ duplications. This review discusses what is currently known about clinically relevant Y chromosome structural variations in male fertility, mainly focusing on copy number variations (CNVs). These CNVs include classical AZF deletions, gr/gr deletion and TSPY1 CNV. AZF deletions are in a clear-cut causeeffect relationship with spermatogenic failure and they also have a prognostic value for testis biopsy. gr/gr deletion represents the unique example in andrology of a proven genetic risk factor, providing an eight-fold increased risk for oligozoospermia in the Italian population. Studies on TSPY1 CNV have opened new perspectives on the role of this gene in spermatogenic efficiency. Although studies on the Y chromosome have importantly contributed to the identification of new genetic causes and thus to the improvement of the diagnostic work-up for severe male factor infertility, there is still about 50% of infertile men in whom the etiology remains unknown. While searching for new genetic factors on other chromosomes, our work on the Y chromosome still needs to be completed, with special focus on the biological function of the Y genes.

Molecular analysis of estrogen receptor alpha gene AGATA haplotype and SNP12 in European populations: potential protective effect for cryptorchidism and lack of association with male infertility.

BACKGROUND: A specific haplotype (AGATA) in the estrogen receptor alpha (ER1) gene was recently described as a new risk factor for cryptorchidism in the Japanese population. In this ethnic group, single-nucleotide polymorphism 12 (SNP12) was concluded to be the tag SNP for the AGATA haplotype. MATERIALS AND METHODS: A large group of patients (total number=335) and controls (total number=567) of two Caucasian populations were analysed for the AGATA haplotype and SNP12 to verify whether this genetic variant and its tag SNP were associated with cryptorchidism or with severe spermatogenic failure. RESULTS: We confirm that SNP12 is the tag SNP for the AGATA haplotype also in Caucasians. However, in contrast with the Japanese population we found a protective effect for ESR1 SNP12 on cryptorchidism in the Italian population. No association between SNP12 and severe spermatogenic disturbances was observed. CONCLUSIONS: The observed associations (although with opposite effect) with cryptorchidism encourage future studies on independent cases and controls from different ethnic and geographic origins. On the other hand, in contrast with other ESR1 polymorphisms, SNP12 polymorphism is not associated with severe male factor infertility in two independent European population.

DAZL polymorphisms and susceptibility to spermatogenic failure: an example of remarkable ethnic differences.

Polymorphisms in genes involved in spermatogenesis are considered potential risk factors for male infertility. Recently a polymorphism in the deleted in azoospermia-like (DAZL) gene (T54A) was reported as susceptibility factor to oligo/azoospermia in the Chinese population. DAZL is an autosomal homologue of the Y chromosomal DAZ (deleted in azoospermia) gene cluster and both are considered master regulators of spermatogenesis. The aim of the present study was to screen (i) for mutations of the entire coding sequence of the DAZL gene in patients lacking of the DAZ gene cluster, in order to evaluate if DAZL polymorphisms may influence the AZFc deletion phenotype; (ii) for the two previously described (and eventually newly identified) single nucleotide polymorphisms (SNPs) in a large group of infertile and normospermic men of Italian origin. We failed to detect new mutations. We confirmed previous results showing no evidence for a functional role of the T12A mutation. Surprisingly, the T54A polymorphism, which was present in 7.4% of the Chinese patients was absent in our Caucasian population. This remarkable difference represent an example of how ethnic background is important also for polymorphisms involved in spermatogenesis and contributes to better select clinically relevant tests, specifically based on the ethnic origin of the infertile patients.

The clinical significance of the POLG gene polymorphism in male infertility.

Based on association studies, an increasing number of gene polymorphisms have been proposed as modulators of spermatogenesis. Interestingly, a clear cause-effect relationship between a polymorphism of the POLG gene and oligo(astheno)zoospermia was recently described. The POLG gene contains a polymorphic CAG repeat, and the presence of a homozygous mutant (not10/not10 CAG) genotype was found only in infertile men. In the present study, a large number of infertile patients and normospermic men of Italian origin were studied to define the effect of POLG genotypes on spermatogenic potential and whether the homozygous mutant is specific for spermatogenic disturbances. The mutated genotype was found at the same frequency in both infertile and normospermic men. Mean values of sperm parameters such as sperm count, motility, and morphology did not differ significantly between carriers of the three different genotypes. Our study failed to confirm any influence of the POLG gene polymorphism on the efficiency of the spermatogenesis. More importantly, considering that the homozygous mutant genotype has been found in normospermic fertile men, the analysis of the CAG repeat tract of the POLG gene does not appear to have any clinical diagnostic value.