Human Developmental Genetics

Human Developmental Genetics

The unit is focused on understanding the molecular events governing the formation of the human gonads and the mechanisms of gamete specification and differentiation.


The initial events of mammalian sex determination are genetically determined with SRY as the master regulatory switch that triggers the formation of the testes. Errors in the process of sex-determination are common and can result in a range of phenotypes from complete sex-reversal to minor genital anomalies. Although many of the early cellular and morphological events that occur downstream of SRY action have been characterized (Figure 1), the mechanisms involved in mammalian sex determination is poorly understood. The objective of the unit is to understand reproductive processes by analysing the genetic and epigenetic mechanisms associated with the development of both somatic and germ cell lineages of the mammalian gonad. 

Figure 1: Schematic representation of molecular events during mammalian sex-determination 


Understanding the development of somatic cell lineage of gonad is being achieved by the analysis of patients with DSD (Disorder of Sex Development) phenotypes. The startegy involves comparative genomic hybridization (CGH) to screen for rearrangements associated with DSD and the functional characterisation of pathogenic mutations revealed by Sanger and Next Generation sequencing approaches. This is aimed at both identifying new genetic variants associated with urogenital anomalies and to use these mutations to understand the mechanism of gonad formation and the choice of somatic sex.


1.Mammalian Somatic Sex Determination


We have developed a large collection of biological material from patients with urogenital anomalies, through collaborations with clinical centres worldwide. Much of our research activities on sex-determination (i.e. the somatic cells of the gonad) centre on the use of this unique biological resource. The analyses of these cases have identified unique mutations that offer both a mechanistic insight of gonad development, as well as new clinical diagnostic markers (Figure 2).









Figure 2. (a) Chromatograms representing the mutations in NR5A1 associated with DSD. The position of the mutations on the protein and in-silico prediction by Polyphen are also shown. (b) Nuclear localization of NR5A1. (c) The transcriptional activity and synergism of wild-type and mutated NR5A1 with GATA-4 on human AMH promoter. (d) Interaction between wild-type and mutant NR5A1 proteins with GATA-4.


The unit receives approximately 5 new cases of 46,XY and 46,XX DSD per week from clinical referral centres in France, Europe, India, North Africa and worldwide. These samples are routinely sequenced for mutations in genes known to be involved in the relevant pathology by standard Sanger sequencing approaches.


The specific questions we are asking are


  • What are the effects of the mutations, which we have identified, on the biological activity of the protein?
  • Can these mutations provide an insight into the mechanism(s) of gonad formation and the choice of somatic sex?
  • What are the novel factors/mechanisms associated with anomalies of human gonad development?


1.1.Identification of new variants involved in sex determination by Comparative Genomic Hybridization (CGH):

Human gonadal development is sensitive to gene dosage and errors in human sex determination are often associated with duplication and deletion events in the human genome. This approach forms part of the EuroDSD FP7 project (
We are also funded by the FP7 EuroDSD project to develop a DSD GeneChip, which will sequence 36 genes involved in reproductive processes using an array-based hybridization procedure. The aim of this is to develop a rapid, simple and cost effective method of sequencing many genes.


1.2. The DSD Exome Project:
Defining the genetic basis of DSD cases is a high priority because a failure to adequately understand the cause can lead to premature decisions and irreversible clinical interventions. A lack of clear understanding can have major consequences on the patient and the families concerned and gender identity can be a major concern. The identification of new genetic factors will not only deepen the knowledge on the pathology of these conditions and reveal novel insights into the mechanism of sex determination in the human. We hypothesize that many unexplained cases of DSD carry mutations in coding sequences of genes that have not been recognised to play a key role in determining somatic cell sex. The identification of new genetic factors and the functional analysis of mutant proteins will reveal mechanistic pathways of gonad formation as well as lead to the identification of genetic modifiers influencing the severity of the phenotypes. Exome sequencing has become a highly efficient and cost effective strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare and common Mendelian disorders. The goal of this approach is to identify the functional variant that is responsible for the disease without the high costs associated with either whole-genome sequencing or time-consuming sequencing of many potential candidate genes. It is estimated that the protein coding regions of the human genome constitute about 85% of the disease-causing mutations.
We are currently performing exome sequencing using the SOLiDTM system with an average coverage of x200. The goal of this project is to sequence the entire coding region in the genome of 60 cases of syndromic and non-syndromic DSD cases by 2011. Both sporadic and familial cases are being analysed with half of all cases presenting with some degree of consanguinity. An example of a familial case is shown in figure 3.


 Figure 3. Familial case of DSD. The arrow indicates the proband, a 46,XX SRY-negative male (testicular DSD). Other affected individuals (solid squares) are 46,XY men with unexplained infertility. In collaboration with Dr Inas Mazen, NRC, Cairo, Egypt. 


To date complete exome of 12 cases of DSD has been sequenced. The basic variant statistics of these cases are indicated in Table 1. In three of the 12 cases pathogenic mutations have been identified in genes that have previously not been reported to cause DSD. 


  Individual range
Total number of variants 12510-19494
Heterozygous variants 7009-12523
Homozygous variants 2967-6971
Transition SNPs 9061-14054
Transversion SNPs 3449-5443


Table 1. Summary of variants identified in 12 DSD samples by exome sequencing.

The patient phenotypes that will be screened include – gonadal dysgenesis/agenesis DSD and congenital heart disease, DSD and autism, DSD and multiple congenital anomalies, Müllerian agenesis, 46,XX testicular or ovotesticular DSD (XX male or true hermaphrodite), ovarian cyst formation associated with precocious puberty and primary ovarian insufficiency.


1.3. Specification of Sertoli cells from murine embryonic stem (ES) cells:
Sertoli cells facilitate the progression of germ cells to spermatozoa by providing the niche within the seminiferous tubules as well as by direct contact. The initial embryonal induction and subsequent differentiation and function of Sertoli cells involve SRY, NR5A1 and SOX9. The genetic program and the signal(s) that determine the induction of Sertoli cells are unclear and we are investigating the mechanisms underlying Sertoli cell specification using ES cells as a model system. Besides providing an insight into the mechanisms of testicular morphogenesis, Sertoli cells derived from ES cells may provide a viable alternative to understand the Sertoli cell-germ cell interaction in a co-culture system and improve the derivation of germ cells from ES cells (see below).


1.4. Patient/mutation specific iPSCs:
Recent derivation of individual pluripotent stem cells (iPSCs), provide a practical functional equivalent to human ES cells. To reveal the mechanism of human Sertoli cell development we are developing patient specific iPSC lines from the DSD samples that are currently being screened under the DSD Exome Project. For each of these patients we will have data on the pathogenic mutation(s) and potential genetic modifiers of the phenotype. These iPSCs would then be directed to differentiate into Sertoli/ Sertoli-like cells to study the development and progression of the disease, specific to that individual. The Sertoli cells derived from the patient-specific iPSC lines will have an immediate application to assess the response to drugs and hormones, that would be useful for the management of the DSD in the patient. In the long term, these iPS cell lines may be the most suitable candidates for the cellular replacement therapy in the patient.


2. Germ Cell Development and Maintenance


The specification and differentiation of primordial germs cells (PGCs) into male germ cells is a dynamic process involving a series of synchronised genetic and epigenetic changes that remain ill defined. To understand these events we are using two parallel approaches involving analysis of patients with impaired spermatogenesis and a novel ex-vivo model system.


2.1. Genetic and epigenetic landscape of male germ cells: Our research initially focused on the contribution of Y chromosome microdeletions to spermatogenic failure including a partial deletion of AZFb and various forms of rearrangements within the AZFc region. Recently we reported mutations in the NR5A1 gene associated with severe spermatogenic failure (Figure 4). We continue to search for novel causes of idiopathic male infertility by candidate gene analysis and whole exome sequencing approaches. 


Figure 4. Mutations in the NR5A1 gene associated with male infertility.


A consideration of the causes of human infertility needs to take into account the recent and sharp decline in human male reproductive health. The rapid reduction in semen quality observed in 2 or 3 generations in some countries suggests an environmental aetiology, perhaps in genetically susceptible individuals. Experimental data suggests that exposure of a developing male foetus to a number of environmental factors can negatively influence sexual development and testicular function. Indeed, there is growing evidence associating imprinting disorders in spermatozoa with impaired human spermatogenesis. Our preliminary results indicate that 7% of oligozoospermic patients have imprinting anomalies in their spermatozoa at the H19 and/or MEST loci. We are studying a large cohort of oligozoospermic patients and address the extent of and possible genetic element to these epigenetic modifications.


  • Are methylation changes limited to imprinted loci or genome wide?
  • What is the relationship between methlyation changes and fertility status?
  • Are there genetic changes associated with altered epigenetic states?
  • What are the consequences of methylation anomalies on IVF success?
  • Is there evidence of transgenerational inheritance of epigenetic changes?


These questions are being addressed by epigenetic profiling of sperm DNA using meDIP approaches, detailed genetic analyses of individuals with imprinting anomalies (Y chromosome analysis, expression profiling of the transcripts in sperm-head, exome sequencing) and long term clinical follow-up of men (and their offspring) with these problems.


2.2. Specification of primordial germ cells (PGCs) from murine embryonic stem (ES) cells and their differentiation into male gametes: Role of KIT/SCF signal transduction pathway.


Despite the importance of PGCs in the formation of the germ line, the molecular mechanisms regulating their specification, growth, survival and differentiation remain ill defined. The specification and subsequent differentiation of PGCs is a dynamic process involving stage-specific genetic and epigenetic modifications (Figure 5). Although functional sperm, capable of fertilization have been generated from ES cells, only a small number of highly selected in vitro-derived germ cells were capable of fertilization; the live-born pups survived only a short time and showed imprinting defects. This indicates the need to fully understand the genetic and epigenetic mechanisms underlying the PGC specification, proliferation and differentiation. 



Figure 5: Schematic representation of in vivo and in vitro development of germ cell in mouse. The in vitro scheme of derivation and differentiation of PGCs from mES cells is based on the methodology adopted by us. The known genetic and epigenetic events including genetic profile at each stage of development are shown.


The receptor tyrosine kinase KIT encoded by c-KIT and its ligand stem cell factor (SCF) have been shown to be critical in vivo and in vitro for the appropriate specification, migration, proliferation, survival and differentiation of PGCs. As c-Kit is a pleiotropic gene it is difficult to analyze its role in vivo. ES cells provide an ideal in vitro system to study this developmentally important pathway. But KIT and its ligand SCF are expressed both by the undifferentiated and differentiating ES cells; therefore it is impossible to dissect the role of this receptor-ligand system by ligand starvation followed by the addition of the ligand. We are using a novel strategy that involves the use of ES cells carrying a null (Wlacz) as well as a knock-in (WFKB-kit) allele of Kit that encodes an SCF-independent hybrid KIT receptor which can be activated by the FKBP binding drug, AP20187 (Figure 6a). Briefly, the chimeric c-Kit cDNA, where two FK506 binding domains (FKBP12) are fused to KIT signalling domains, was introduced by homologous recombination into KIT null cells (Figure 6b). The FKB12 domains allow intracellular protein dimerization and reversible activation in response AP20187.
The importance of c-Kit in germ cell development is also supported by our microarray expression data from WlacZ/WFKB-kit cell lines where several genes involved in spermatogenesis show a significant reduction in expression in the KIT null cells as compared to the wild type ES cells. These genes are a part of the Delta SxrB region on mouse Y chromosome that is necessary for spermatogonial proliferation and shows a syntenic homology to a region of the human Y chromosome that is recurrently deleted in patients with infertility. Analysis of KIT null ES cells also show a reduction in the expression of Stella and Blimp-1 and an increase in expression of Hoxa1 suggesting a reduced potential of KIT null ES cells to form PGCs..




Figure 6: (a) Chimeric c-Kit cDNA (b) KIT null mouse ES cell lines.


Preliminary data indicate that the SCF/KIT pathway plasy an important role in the generation and/or differentiation of PGCs from ES cells. We are testing this hypothesis and analyzing the role of KIT/SCF signal transduction pathway in PGC specification and/or differentiation.


  • What are the genetic and epigenetic events associated with PGC specification from ES cells?
  • Does the KIT/SCF signalling pathway play a role in the specification/differentiation of PGCs from ES cells?
  • At what time point during the differentiation process is SCF/KIT signalling required?
  • What is the contribution of mutations in genes regulated by KIT to human cases of infertility?