A Next Generation Solution to Disorders of Sex Development

Daniel Shen
Thomas Jefferson High School for Science and Technology

This article was originally included in the 2019 print publication of the Teknos Science Journal

I carefully place a paper-thin membrane onto the petri dish filled with tris-EDTA (TE) buffer with metal forceps. Drawing up the solution of impure genomic DNA, I gently pipette it out until a clear spot dots the membrane, floating in a delicate balancing act. Satisfied with my work, I take a seat by the computer as the machine to my left whirs to life and flashes fluorescent green. At the Vilain Lab in Children’s National Medical Center, I spend a good deal of my time wheeling the mouse around and watching as speckles of fluorescent green, red, and blue dot the computer screen like fireworks. These vibrant colors map out major patterns in sequence across the entire human genome. This very technology has incredible potential to revolutionize the study and treatment of rare diseases.

Disorders of Sex Development (DSD) is a broad category, created in early 2005, that encompasses any abnormal development of chromosomal, gonadal, or anatomic sex, which replaced the outdated “intersex” and “hermaphrodite” terms [6]. Approximately 1 in every 4,500 births is a DSD case [4]. Recognition of anatomic abnormalities at birth, prompts a slew of physical examinations, blood tests, hormone stimulation tests, and genetic tests [3]. Based on the outcome of these tests, pediatricians are challenged with assigning a gender to the baby, determining the cause for the DSD, and managing its symptoms. Parents often must consider surgical options to modify the genitalia and make it consistent with the child’s assigned gender; however, they must also think about the child’s own gender identity. The diagnostic work up and interventions must be conducted efficiently since optimal timing for surgery is between 2-6 months of age [6].

Since 2005, researchers have been searching for potential diagnostic technologies for DSDs. In 2013, scientists developed Next Generation Sequencing (NGS), a technological breakthrough that provided an incredibly fast method of sequencing the entire human genome [2]. Although researchers had previously considered sequencing, it was difficult to accurately assess the large scale mutations on the X and Y chromosomes with existing short range sequencing technology [1]. In the Vilain Lab in D.C. where I intern, the NGS technology has been paired with a multi-million dollar instrument by Bionano Genomics known as Saphyr. It is a mapping device capable of visualizing large scale mutations in the genome such as those in DSD cases. However, Saphyr can only be used with long, high quality DNA samples.

Each week, two to six vacuum-sealed test tubes full of patient blood arrived in the lab. For the next three days, I conducted two slightly different protocols to determine the optimal DNA extraction method. I carefully pipetted cloudy solutions of agarose into rectangular cells and precisely aliquoted pink fluorescent dyes, transforming the sample of blood to genomic DNA. Besides blood, cell cultures and soft tissue are also viable sources of genomic DNA and both require a similar protocol. 

After the DNA was extracted from its original source, it was then mapped by the Saphyr machine and sequenced over the course of several days until the instrument generated a multitude of files containing suspected locations of mutations known as “breakpoints.” Physically checking the validity of these locations using a polymerase chain reaction (PCR) was of paramount importance. Since these breakpoints were correct, the lab was ultimately able to categorize each mutation as benign or pathogenic. It took just a few months from the time of the blood draw for  patients and families to receive the genetic diagnoses [1].  

Researchers including Yanjie Fan, who works at a provincial pediatric hospital in China, have replicated the Vilain Lab’s pioneering DSD work. Fan and other researchers were able to identify 28.1% of pathologic gene sequences associated with DSDs as compared to only 10% with single gene tests that were previously used [4]. However, the cost of this novel technology posed the question of whether it could benefit underprivileged patients. Fan stated that NGS costs approximately the same as 2 single-gene tests and is significantly more effective at diagnosing rare diseases and identifying candidate genes.

DSDs can also pose ethical problems in the field of athletics. Caster Semenya, a South African runner was diagnosed with hyperandrogenism, resulting in elevated levels of testosterone in her body [5]. At the age of 18, Semenya broke the record for the 800- meter dash [5]. However, many questioned the validity of this exceptional achievement, given Semenya’s genetic diagnosis. The medical advisory board, under the leadership of Eric Vilain drew a defining line and established that, for the 2012 Olympics, competitors with over 10 nanomoles of testosterone in their blood would not be permitted to compete in female Olympic events [7]. Bioethicist Silvia Camporesi offers a counter perspective: “Compare Caster Semenya with Bolt,” and she’s not such an outlier. Using hyperandrogenism to define fairness is too narrow” [5]. This controversial topic serves to highlight the political and social implications of DSD research.

Each week as part of its regular lab meeting, the Vilain Lab discussed a DSD case with pediatric facilities across the nation from Children’s hospitals in Michigan, Cincinnati, and China. Often, children and their families were forced into the harsh realities of these  complex disorders and undergo immense psychological turmoil. As an aspiring researcher and physician, I look forward to the implementation of this technology. Next-Generation Sequencing is an incredible tool that can not only help patients with DSDs, but can also provide a scientific basis for setting fair standards among athletes.

References

[1] Barseghyan, H., Délot, E. C., & Vilain, E. (2018). New technologies to uncover the molecular basis of disorders of sex development. Molecular and Cellular Endocrinology, 468. https://doi.org/10.1016/j.mce.2018.04.003

[2] Behjati, S., & Tarpey, P. S. (2013). What is next generation sequencing? Archives of Disease in Childhood - Education and Practice. https://doi.org/10.1136/archdischild-2013-304340

[3] Délot, E. C., & Jeanette, P. C. (2017). Genetics of Disorders of Sex Development The DSD-TRN Experience. Endocrinology and Metabolism Clinics of North America, 46(2). https://doi.org/10.1016/j.ecl.2017.01.015

[4] Fan, Y., Zhang, X., Wang, L., Wang, R., Huang, Z., Sun, Y., . . . Yu, Y. (2017). Diagnostic Application of Targeted Next-Generation Sequencing of 80 Genes Associated with Disorders of Sexual Development. Scientific Reports (Nature). https://doi.org/10.1038/srep44536

[5] Kessel, A. (2018, February 17). The tense debate around Caster Semenya and Dutee Chand demonstrate the intersection between race, gender and medical imperialism. Retrieved January 17, 2019, from The Guardian website: https://www.theguardian.com/world/2018/feb/18/the-unequal-battle-privilege-genes-gender-and-power

[6] Kun, K. S., & Kim, J. (2012). Disorders of Sex Development. Korean Journal of Urology, 1-8. https://doi.org/10.4111/kju.2012.53.1.1

[7] Reardon, S. (2016, May 11). The Spectrum of Sex Development: Eric Vilain and the Intersex Controversy. Retrieved January 29, 2019, from Scientific American website: https://www.scientificamerican.com/article/the-spectrum-of-sex-development-eric-vilain-and-the-intersex-controversy/