Hassan Rouba

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Mutations in the protamine locus: association with spermatogenic failure?

The protamine locus consists of a 28.5 kb region with a linear array of the protamine (PRM)1, PRM2, PRM3 and transition nuclear protein (TNP)2 genes. Several studies indicate an abnormal expression pattern of protamine genes associated with male infertility, although the molecular mechanism underlying this observation is unclear. Here, we determined the spectrum of DNA variants present in all four genes in men with unexplained infertility compared with an ancestry-matched fertile/normospermic population. A total of 160 control individuals and at least 125 infertile men with either idiopathic azoospermia or oligozoospermia were sequenced for the open reading frame of PRM1, PRM2, PRM3 and TNP2 genes. All individuals carried an apparently intact Y chromosome. Of the 28 variants identified, 21 were previously described in the literature. The novel variants that were observed only in the infertile cohort included the SNP c.65G>A mutation which resulted in an amino acid change at the codon 22 (p.Ser22Asn) in the PRM1 gene, a mutation in the promoter region of PRM2 (-67C>T) and a nonsense mutation in the PRM3 gene. These data are consistent with that of previous studies which have indicated that mutations in the protamine locus may be an infrequent cause of male infertility.

Association of Spermatogenic Failure with the b2/b3 Partial AZFc Deletion

Infertility affects around 1 in 10 men and in most cases the cause is unknown. The Y chromosome plays an important role in spermatogenesis and specific deletions of this chromosome, the AZF deletions, are associated with spermatogenic failure. Recently partial AZF deletions have been described but their association with spermatogenic failure is unclear. Here we screened a total of 339 men with idiopathic spermatogenic failure, and 256 normozoospermic ancestry-matched men for chromosome microdeletions including AZFa, AZFb, AZFc, and the AZFc partial deletions (gr/gr, b1/b3 and b2/b3). AZFa and AZFc deletions were identified in men with severe spermatogenic failure at similar frequencies to those reported elsewhere. Gr/gr deletions were identified in case and control populations at 5.83% and 6.25% respectively suggesting that these deletions are not associated with spermatogenic failure. However, b2/b3 deletions were detected only in men with spermatogenic failure and not in the normospermic individuals. Combined with our previous data this shows an association of the b2/b3 deletion (p = 0.0318) with spermatogenic failure in some populations. We recommend screening for this deletion in men with unexplained spermatogenic failure.

Y-chromosome AZFc structural architecture and relationship to male fertility

OBJECTIVE To determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure. DESIGN Case-control study. SETTING Infertility clinic (Tenon Hospital, Paris). PATIENT(S) 557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109). INTERVENTION(S) Characterization of 32 partial AZFc deletions. MAIN OUTCOME MEASURE(S) DAZ gene cluster divided into two families (DAZ1/2 and DAZ3/4), CDY1 gene, and Y-chromosome haplogroups. RESULT(S) We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group. CONCLUSION(S) The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a, or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility.

Association of the MTHFR A1298C Variant with Unexplained Severe Male Infertility

The methylenetetrahydrofolate reductase (MTHFR) gene is one of the main regulatory enzymes involved in folate metabolism, DNA synthesis and remethylation reactions. The influence of MTHFR variants on male infertility is not completely understood. The objective of this study was to analyze the distribution of the MTHFR C677T and A1298C variants using PCR-Restriction Fragment Length Polymorphism (RFLP) in a case group consisting of 344 men with unexplained reduced sperm counts compared to 617 ancestry-matched fertile or normozoospermic controls. The Chi square test was used to analyze the genotype distributions of MTHFR polymorphisms. Our data indicated a lack of association of the C677T variant with infertility. However, the homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in severe oligozoospermia group compared with controls (OR = 3.372, 95% confidence interval CI = 1.27–8.238; p = 0.01431). The genotype distribution of the A1298C variants showed significant deviation from the expected Hardy-Weinberg equilibrium, suggesting that purifying selection may be acting on the 1298CC genotype. Further studies are necessary to determine the influence of the environment, especially the consumption of diet folate on sperm counts of men with different MTHFR variants.

No association between T222P/LGR8 mutation and cryptorchidism in the Moroccan population.

Background: Cryptorchidism is the most common genital anomaly in men. The INSL3/LGR8 system is involved in testicular descent via gubernacular development. INSL3 binds with high affinity to its receptor LGR8 and receptor activation is associated with cAMP signaling. Analysis of human INSL3 and LGR8 mutations confirms that some cases of cryptorchidism are caused by mutations in these genes. The T222P mutation is the only one within the LGR8 gene associated with the cryptorchidism phenotype. A strong association of the T222P mutation with cryptorchidism was found in an Italian population. Due to the same mutation being found in patients within the Mediterranean area, a possible founder effect of this mutation is supposed. Methods: We screened 109 patients with cryptorchidism and 250 controls in a Moroccan population. Results: We found that 3 of the 109 patients tested carry the T222P mutation and 4 individuals in the control group also carry the mutation. Conclusions: Our results show in fact that the same mutation is present in the Moroccan population, but an association between cryptorchidism and the T222P mutation was not found.

Association of the C677T polymorphism in the human methylenetetrahydrofolate reductase (MTHFR) gene with the genetic predisposition for type 2 diabetes mellitus in a Moroccan population.

AIMS Type 2 diabetes mellitus (T2DM) is a major public health problem around the world. The C677T and A1298C polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene have been reported to be associated with T2DM and its complications. This study aimed to investigate this association in the Moroccan population. METHODS A case-control study was performed among 282 Moroccan diabetic patients and 232 healthy controls. The MTHFR C677T and A1298C polymorphisms were genotyped by polymerase chain reaction, followed by enzymatic digestion with HinfI and MboII enzymes, respectively. RESULTS There was a significant association between C677T polymorphism and T2DM in both additive and dominant models. In addition, the 677T allele frequency differed significantly between the diabetic and control groups (26.06% vs. 33.20%, respectively). However, no significant association was found between A1298C polymorphism and T2DM. The frequencies of combined genotypes 677CC/1298AA and 677CT/1298AC differed significantly between the diabetic and control groups (32.62% vs. 20.61% and 9.57% vs. 17.55%, respectively). CONCLUSIONS These results show an evident association between the MTHFR C677T polymorphism and T2DM in Moroccan patients but no significant association with the MTHFR A1298C polymorphism.

A Nonsense Mutation in the Hedgehog Receptor CDON Associated With Pituitary Stalk Interruption Syndrome.

BACKGROUND Pituitary stalk interruption syndrome (PSIS) and holoprosencephaly (HPE) are congenital midline defects. Rare mutations in the sonic hedgehog (SHH) signaling gene CDON have recently been reported in patients with HPE. OBJECTIVE To report a unique case of PSIS with a maternally inherited nonsense mutation in the SHH signaling protein CDON. METHOD We performed exome sequencing on a case of PSIS. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) and an ancestry-matched control panel were screened upon identification of CDON mutation. RESULTS We identified a novel heterozygous nonsense mutation (c.2764T>C, Glu922Ter) in a case of PSIS without HPE who presented with neonatal hypoglycemia and cholestasis associated with GH, TSH, and ACTH deficiencies. This mutation was absent in all control databases and from 400 healthy ancestry-matched control subjects. The mutation was inherited from the patients mother, who was operated on in childhood for strabismus. The absence of this variant in control samples suggests that it is likely to be responsible for the phenotype. CONCLUSION We report for the first time a mutation in the CDON gene associated with PSIS.

Prevalence of the mitochondrial A 1555G mutation in Moroccan patients with non-syndromic hearing loss

UNLABELLED Mutations in mitochondrial DNA (mtDNA), especially the A1555G transition in the 12S rRNA gene, are one of the causes of both aminoglycoside-induced and non-syndromic sensorineural hearing loss. OBJECTIVE The aim of this study was to determine the prevalence of the A1555G mitochondrial mutation in Moroccan patients. METHODS We performed molecular characterization by PCR-RFLP and direct sequencing of one hundred and sixty four patients (84 unrelated familial and 80 sporadic cases) with a congenital sensorineural non-syndromic hearing loss and one hundred normal hearing controls for the occurrence of the A1555G mutation. RESULTS Mutational analysis of the mtDNA showed the presence of the homoplasmic A1555G mutation in three families, leading to a frequency of 3.6% similar to that reported for European-populations. No A1555G mutation was detected in sporadic and controls cases. However, we detected in twenty normal hearing controls a novel polymorphism A1557C, which was not found in patient samples. We further evidenced the presence of the A1438G mitochondrial polymorphism in four patients with sensorineural hearing loss and in five controls. CONCLUSION Our results show that the occurrence of the A1555G mutation in hearing impaired patients accounts for 3.6% in a Moroccan patients and those novel mtDNA polymorphisms might contribute to a novel sub-haplogroup specific of the Magrheb.

Mutations in the TSPYL1 gene associated with 46,XY disorder of sex development and male infertility

We screened 100 individuals with anomalies of testicular development or function for mutations in the TSPYL1 gene. A 46,XY female with complete gonadal dysgenesis carried a p.K320R mutation in the highly conserved NAP domain, and a 46,XY male with idiopathic azoospermia harbored a p.R89H mutation, and this data supports the hypothesis that mutations in TSPYL1 may contribute to anomalies of testicular development/function.