Genetics and disorders of sex development

One in every 135 baby boys in Victoria is born with a form of genital abnormality, and the incidence is rising. The US and Europe are also witnessing a similar, alarming trend.

The reason for the rapid increase in the rate of genital abnormalities in male newborns is unknown, but may be due to exposure in utero to endocrine disruptors – molecules that affect the production of androgens at the critical time when the penis is forming.

Disorders of sex development are not uncommon and are part of wide spectrum, but due to the importance that society places on gender identity, the consequences can be traumatic, Professor Andrew Sinclair says.

Sinclair, who heads the early development and disease group at Melbourne’s Murdoch Children’s Research Institute, presented his recent findings on genetic variation underlying disorders of sex development (DSD) at the Lorne Genome conference this week.

As a postdoctoral researcher in London in 1990, Sinclair was the first to identify the human gene that determines maleness, Sex-determining Region, Y-chromosome (SRY).

SRY encodes a transcription factor, whose only apparent function is to activate a related transcription-factor gene, SOX-9.

SOX-9, one of a large family of transcription-factor genes that initiate organogenesis, causes the fetus’ indeterminate gonads to form testis.

If either SRY or SOX-9 are inactivated, the gonads form ovaries – and the chromosomally male fetus develops as a female.

Sinclair has spent the greater part of his research career searching for the genes that co-ordinate male development, and trying to trace the cascade of genetic activity initiated by SRY from its lonely outpost on the Y chromosome.

He is performing whole-genome analyses on a cohort of male and female patients with DSD, using high-density microarrays to identify copy-number variations – micro-deletions and duplications – associated with specific developmental defects, such as failure of the gonads to develop normally.

The approach involves taking a small blood or saliva sample from the patient and extracting genomic DNA. The DNA is placed on an Affymetrix microarray where it hybridises with a subset of nearly two million genetic markers. The chip is then processed at the Australian Genome Research Facility in Parkville.

“Each run yields two million data points – a tsunami of data for each patient that would be unmanageable without algorithms to automatically identify unique regions,” Sinclair says.

In collaboration with Professor Terry Speed at the bioinformatics department at the Walter and Eliza Hall Institute, Sinclair’s team has developed algorithms that rapidly pick out regions that deviate in terms of copy number, and can identify deletions and duplications.

“There is obviously a huge amount of variation in every individual that is not associated with any pathology, so the first thing we do is to subtract out the known normal variation,” he says.

“When we’re looking at a patient’s DNA, we’re focusing on genes involved in gonad development, but there is every possibility that we will see something else, such as a mutation in the BRCA1 tumour suppressor, that puts the patient at increased risk of breast cancer.

“That raises an ethical issue – do we inform the patient? We’ve decided to forward this type of information to an appropriate genetic counselor or medical specialist, who will inform the patient.”

Sinclair says the true frequency of disorders of sex development in the general population is unknown.

“There is also a wide spectrum of disorders, ranging from hypospadias – opening of the shaft of the penis – though ambiguous external genitalia, to complete sex reversal, where a 46XY individual develops as a female.

“We do know that genital abnormalities account for 7.5 per cent of all birth defects. In addition to the rising incidence of defects of penile development (1 in 135 boys), one baby in 3,500 is born with significant ambiguity of the genitalia – data suggests the true incidence is probably higher.”

---PB--- Whole genome analysis

The current state of knowledge around disorders of sex development in physically female individuals with a 46XY male karyotype is that the underlying genetic defect is unknown in 80 per cent of cases, Sinclair says.

Of the rest, 15 per cent involve mutations in SRY, and five per cent involve mutations in SF-1.

SF-1 codes for a steroidogenic factor thought to be essential for normal development of the hypothalamic-pituitary-gonadal signalling system during early embryogenesis.

Sinclair’s team is trying to identify the underlying lesion in the 80 per cent of cases of unknown origin. He says most mutations are spontaneous, but in a small minority of cases, there is a family history of gonad dysgenesis.

“There are two hypotheses. The first is that affected individuals may be explained by mutations in the regulatory and coding regions of known gonad determining genes. The second hypothesis is that mutations in as yet unidentified gonad genes will explain these patients.

“Our whole genome analysis suggests both hypotheses are involved. For example, we have identified deletions in the regulatory region of SOX-9, which is a known key gene in development of the testes but we have also identified some novel gonad genes.”

Sinclair says SOX-9 has a very large, upstream regulatory region incorporating two regulatory elements – one controls the development of the testes, while the other regulates chondrogenesis, or the development of cartilage.

“Our work in humans has identified a regulatory region upstream of SOX-9 that, when deleted, results in development of XY females without skeletal defects, suggesting it is the region that controls SOX-9 expression in the gonad.

“We’ve also identified some new genes involved in gonadal development, so we’re gradually filling in the blanks.”

Sinclair says the research will help to improve clinical management of individuals with DSD. Because of the importance that society attaches to gender identity, the consequences of sex disorders for these patients can be extremely traumatic.

“There are issues around genital surgery and matching it to the gender assigned to the newborn, and there are reproductive consequences: most patients are sterile,” he says.

“There is also the issue of a high risk of cancer for an XY female with undescended testes, which must be removed.

“By identifying these genes, we are able to provide a rapid diagnosis that in turn informs the clinical management required, whether it be surgery or hormone treatment. If we know the gene mutation , and which pathway is affected, the clinical management and outcome is more likely to be significantly improved.

“We’re also feeding important information back to the patient: we can explain that an abnormality in the function of a particular gene is the reason why they have a particular condition. When we can put a label on the problem: patients usually feel more comfortable with who they are.”

---PB--- Developmental cascade

In some cases, the mutation in an XY individual is expressed as complete gonadal failure. In others, the gonads are present but only partially developed, suggesting a mutation in a gene that acts later in the developmental cascade.

Many children present with sexual development problems at puberty. XY females have a fibrous gonadal streak in place of normal testes, despite having a normal Y-chromosome and functional SRY gene, indicating that the problem lies with a gene downstream of SRY.

In the reverse condition, XX males have testes that produce no sperm, lack female internal ducts, and genitalia that range from normal male through to ambiguous.

“About 90 per cent these patients result from the transfer of SRY onto an X-chromosome,” Sinclair says.

When the SRY “maleness” gene was finally located in 1990, it was found just outside pseudoautosomal region on the tip of the Y-chromosome, the only region that undergoes pairing and recombination with a homologous region on the short arm of the X chromosome during meiosis.

In rare cases, recombination captures the SRY gene and transfers it to the corresponding region of the X chromosome, where it deflects development into the male pathway.

But Sinclair says around 10 per cent of XX males have no Y-chromosome sequences attached to the X-chromosome.

“We’re very interested in these cases too. We think the sex reversal involves disruption of ovary-determining genes such as RSPO1, WNT4 and FOXL2.”