Expanding the clinical spectrum of biallelic ZNF335 variants.

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in 2 unrelated families. We describe, herein, 2 additional affected individuals with biallelic ZNF335 variants, 1 individual with a homozygous c.1399 T > C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A > G, p.(Glu1333Gly) variants with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra-axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335-associated microcephaly.

A new mutation in the calcium-sensing receptor gene causing hypocalcaemia: case report of a father and two sons.

BACKGROUND: Regulation of calcium is mediated by parathyroid hormone (PTH) and 1.25-dihydroxyvitamine D3. The calcium-sensing receptor (CaSR) regulates PTH release by a negative feedback system. Gain-of-function mutations in the CaSR gene reset the calcium-PTH axis, leading to hypocalcaemia. PATIENTS AND METHODS: We analysed a family with hypocalcaemia. The proband was a 47-year-old man (index, patient I1), who presented with paraesthesias in both limbs. He has two sons (patient II1 a nd I I2). The probands' lab results showed: serum calcium of 1.95 mmol/l, albumin 41 g/l, phosphate 0.81 mmol/l and PTH 6.6 ng/l (normal 15-65 ng/l). Based on this analysis, we suspected a hereditary form of hypocalcaemia and performed genetic testing by polymerase chain reaction and Sanger sequencing of the coding regions and intron boundaries of the CaSR gene. Genetic analysis revealed a new heterozygous mutation: c.2195A>G, p.(Asn732Ser) in exon 7. The lab results of patient II1 showed: serum calcium of 1.93 mmol/l, phosphate 1.31 mmol/l, albumin 41 g/l, and PTH 24.3 ng/l. His genotype revealed the same activating mutation and, like his father, he also lost his scalp hair at an early adolescent age. Patient II2 is asymptomatic, and has neither biochemical abnormalities, nor the familial CaSR gene mutation. He still has all his scalp hair. CONCLUSIONS: 1) The c.2195A>G, p.(Asn732Ser) mutation in exon 7 of the CaSR gene leads to hypocalcaemia, and has not been reported before in the medical literature. 2) Possibly, this mutation is linked to premature baldness.

Are AZFb deletions always incompatible with sperm production?

Deletions on the long arm of the Y chromosome are a well-known cause of male infertility and it is generally accepted that deletions involving the AZFb region are not compatible with sperm production. Here, we report on two patients for whom basic diagnostic tests showed a deletion of the AZFb region. Unexpectedly, both patients had some residual sperm production. Subsequently, extension and additional analyses of the AZFb region disclosed an aberrant deletion pattern. Therefore, these results emphasize the need for a detailed and powerful analysis of cases where first-line Yq deletion tests reveal an AZFb deletion. Moreover, our study clearly demonstrated that only a very careful selection of test markers will avoid the pitfall of a 'no further treatment possible' wrongful conclusion.

Detailed molecular characterization of a novel IDS exonic mutation associated with multiple pseudoexon activation.

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

Array comparative genomic hybridization in male infertility.

BACKGROUND: Male infertility caused by a maturation arrest of spermatogenesis is a condition with an abrupt stop in spermatogenesis, mostly at the level of primary spermatocytes. The etiology remains largely unknown. METHODS: We focused on patients with a complete arrest at the spermatocyte level (n = 9) and used array comparative genomic hybridization to screen for deletions or duplications that might be associated with maturation arrest. Interesting copy number variations (CNVs) were further examined by using quantitative PCR. Where appropriate, the expression pattern was analyzed in multiple human tissues including the testis. RESULTS: A total of 227 CNVs were detected in the patient group. After the elimination of CNVs that were also present in the control group or that were not likely to be involved in male infertility, the remaining 11 regions were investigated more in detail. We first determined the expression pattern of seven genes, for which expression had not been reported to be investigated in testicular tissue, after which one region could be eliminated. Next, all 10 promising candidate regions were analyzed by quantitative PCR in a control population. CONCLUSIONS: Eight deletions/duplications were absent in our control group, and therefore might be linked with the male infertility in our patients. One of these alterations, however, has been detected in a proven fertile father group. Further research is necessary to determine the relationship between the observed genomic alterations and maturation arrest of spermatogenesis. Furthermore, several of the above genes have not been studied at the functional level and consequently, more research is required to determine their role in spermatogenesis.

Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages.

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

[Genetics and male infertility]. / Genetica van de mannelijke onvruchtbaarheid.

Infertility is a problem affecting many couples with a child wish. In about half of these couples a male factor is (co-) responsible for the fertility concern. For part of these patients a genetic factor will be the underlying cause of the problems. This paper gives an overview of the studies performed in the Department of Embryology and Genetics of the Vrije Universiteit Brussel and the Centre for Medical Genetics of UZ Brussel in order to gain more insight into the genetic causes of male infertility. The studies, focusing on men with fertility problems, can be subdivided into three groups: studies on deletions on the long arm of the Y chromosome, studies on X-linked genes and studies on autosomal genes. It is obvious that Yq microdeletions should be considered as a cause of male infertility. Only for patients with a complete AZFc deletion, a small number of spermatozoa can be retrieved. However, even for these patients assisted reproductive technologies are necessary. Complete AZF deletions are found in 4.6% of the patients visiting the centres for Reproductive Medicine and Medical Genetics of the UZ Brussel and for whom no other cause of the fertility problems have been detected. Taken into consideration this low prevalence of Yq microdeletions, it is obvious that also other factors, including genetic factors, must be causing fertility problems. Potentially, gr/gr deletions (partial deletions of the AZFc region) might influence the fertility status of the patients. It remains, however, unclear which of the genes located in the deleted regions are important for the progression of spermatogenesis, in case of partial or complete AZF deletions. In our studies we have also investigated mutations in genes located on the X chromosome. In analogy to the Y chromosome, the X chromosome is interesting in view of studying male infertility since men only have a single copy of the sex chromosomes. As a consequence, mutations in genes crucial for spermatogenesis will have an immediate impact on the sperm production. The genes NXF2, USP26 and TAF7L were investigated for the presence of mutations. All observed single nucleotide changes were also present in control samples, questioning their relationship with male infertility. We also studied five autosomal genes: SYCP3, MSH4, DNMT3L, STRA8 and ETV5. Only for the genes STRA8 and ETV5, changes were detected that were absent in a control population existing of men with normozoospermia. Functional analysis of the changes in ETV5 and the localization of the change observed in STRA8 showed that also these alterations were probably not the cause of the fertility problems in these men. It can be concluded that mutations are rarely detected in men with fertility problems. This low frequency of mutations has also been confirmed in several published studies. Therefore, further research is necessary to determine the impact of genetic causes on male infertility.

The role of the testis-specific gene hTAF7L in the aetiology of male infertility.

The X-linked TAF7L gene is homologous to the autosomal transcription factor TAF7. Together with its testis-specific expression pattern, this might point to an important function in spermatogenesis. In order to analyse the involvement of the hTAF7L gene in the aetiology of male infertility, a total of 25 patients with maturation arrest of spermatogenesis have been analysed for the presence of mutations in this gene. Four alterations of the nucleotide sequence, with concomitant changes in the amino acid sequence, have been observed in 12 patients. All sequence alterations were also found either in a control group consisting of men with proven fertility or in a control group with men with normal spermatogenesis. Therefore, these alterations are probably polymorphisms.

Characterization of the genomic organization, localization and expression of four PRY genes (PRY1, PRY2, PRY3 and PRY4).

PRY (PTP-BL related on the Y chromosome) has been proposed as a candidate spermatogenesis gene. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of the cDNA sequence with the genomic sequence, five exons were identified. Analysis of GenBank-derived clones on the Y chromosome revealed the presence of two full-length copies in azoospermia factor region b (AZFb) (PRY1 and PRY2) and two shorter versions of the PRY gene containing exons 3, 4 and 5 in AZFc (PRY3 and PRY4). A clone containing sequences homologous to exons 3, 4 and 5 is located in area 5L (between AZFa and AZFb), a clone containing a sequence homologous to exon 5 is located in area 5M (in AZFb) and a clone containing a fragment homologous to exon 3 is located in 6F. A repeat structure of exons 1 and 2 is present on the short arm of the Y chromosome as well as on the long arm. PRY1 and PRY2, two gene copies that are located in AZFb, a region often deleted in patients with severe male infertility, were shown to be expressed in the testis. PRY may therefore play an important role in spermatogenesis.

Validation of a simple Yq deletion screening programme in an ICSI candidate population.

This study reports on the validation of a diagnostic screening programme for Yq deletions in a population of infertile men. First, an unselected group of 402 intracytoplasmic sperm injection (ICSI) candidate patients was screened prospectively by means of three polymerase chain reactions (PCR) each with one marker in the region AZFa, AZFb or AZFc. With this screening strategy, eight males (2.2%) were found to carry a deletion in Yq11. Secondly, a subgroup of males were further analysed by multiplex PCR with 27 sequence-tagged sites. In this group of 229 cytogenetically normal males with azoospermia, cryptozoospermia or extreme oligozoospermia, including some patients with varicocele or a history of cryptorchidism, only one additional microdeleted patient was found with the multiplex PCR. Hence we obtained a frequency of 2.2% (9/402) or 4% (9/229) in the unselected and selected patient groups respectively. We conclude that in a diagnostic programme for Yq deletions in ICSI candidates it might be sufficient to use only four markers representing the three AZF regions and a more distal region in AZFc. In this way, it is possible to detect most, if not all, Yq deletions which might be the causal factor in the patient's infertility.