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Chromosomal Mutation In Isolated Hypospadias Patients Using High-Resolution Melting Curve (HRM)

Darmawati A. Indraswari1*, Ahmad Z. Juniarto1, Andrew H. Sinclair2 and Sultana M. Faradz3
  • 1 Diponegoro University, Indonesia
  • 2 Murdoch Childrens Research Institute, Australia
  • 3 Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Indonesia

INTRODUCTION Hypospadias, one of the most common disorders of male genitalia is thought to have an increased trend worldwide. A defect in the urethral tube in which the urethra opens ectopically on the ventral side of the penis, between the glans and the perineum, ranges from 7.84 to 33.8 per 10,000 births. This condition is included in the mild spectrum of 46, XY disorder of sex development (DSD).(1–4) Hypospadias is the most common congenital penile anomaly (68.3%), followed by chordee (8.6%) and hypospadias plus chordee (5%), and 14% are reported as unspecified penile anomalies. The severity of hypospadias can range from a slightly offset urethral meatus to complete failure of urethral tube formation, which can result in ambiguous genitalia.(5) The increased frequency in the last 20 years has been thought to be associated with environmental changes especially in the increased risk of exposure to chemical compounds.(2,3,6–8) Factors playing roles in the development of CPA are genetics and environmental factors, particularly exposures to environmental endocrine disrupting chemicals (EDCs).(9–12) One of the important genes involved in the development of male external genitalia is WNT5A.(13–15) Those genes play roles in the early development that is androgen independent thus its examination is important. chr METHODS DNA extraction is performed in Molecular and Cytogenetic laboratory of Cebior Faculty of Medicine Diponegoro University Semarang. Mutation analysis of the samples using HRM is performed at MCRI in Melbourne, Australia. The samples are DNAs from 44 individuals who were male (46, XY) with a confirmed clinical diagnosis of isolated hypospadias, no other anomalies or syndromes by urologist and/or clinical geneticist who gave their consent and no confirmed mutations for any of hypospadias candidate genes in previous studies. Salting-out method is used for the DNA extraction. The quality and quantity of DNA is determined using a NanoDrop 1000 spectrophotometer. Curve analysis was done for HRM result. The LightCycler® 480 Gene Scanning Software (Roche, Mannheim, Germany) was employed. The method of DNA sequencing was Sanger sequencing method. The mutations analyses were done by using the Abi Sequence Scanner V1.0 software (Applied Biosystems, California, USA). Sequencing result was compared to reference. Obtained fragment analysis traces were analyzed using the GeneMarker (v 1.97) software. A summary of the results are be presented in tables and graphs. RESULTS HRM analysis of WNT5A exon 5B reveals a difference plot curve shown in Figure 1. The thermal cycle data was shown in Table 1. The melting curves are aberrant with different melting curve. These two samples (2.15 and 1.4) with aberrant patterns from the HRM analysis on WNT5A exon 5B were then sequenced. Sanger sequencing showed that translocations were found in WNT5A exon 5B. Using the Blat Tool in the USCS web browser, the sample showed a translocation in two samples. The translocations are in chromosome 8: 143517007-143517080 (81 nt) in sample 2.15 and chromosome 8: 143516989-143517080 (92 nt) in sample 1.4. The WNT5A is located in chromosome 3 but the sequencing shows a fragment of chromosome 8. The translocated fragment of chromosome 8 is then using BLAT in the UCSC and they reveal no coding gene. This is similar to a deletion in an exon, giving missing expression of certain protein that should be coded by WNT5A. A finding is found in two samples as translocation of a fragment of non-coding region in chromosome 8 into chromosome 3 inside the WNT5A exon 5B. DISCUSSION The HRM technique is an increasingly popular device to diagnose genetic diseases. The study is the first to screen variants of WNT5A in a cohort of patients with hypospadias. An unexpected finding was found in a sample as translocation of chromosome 8 in chromosome 3. A translocation may develop hypospadias through certain mechanisms. One of the most possible mechanisms is that the fragment of chromosome 8 that is translocated in the WNT5A remove the gene that is responsible for the normal development of male external genitalia. Several methods have been used for detection of mutations in hypospadias candidate genes. The development of male external genitalia is determined by two distinct phases, the early androgen-independent phase and the late androgen-dependent phase. Recent studies on WNT5A, the gene that plays an important role in both phases showed that no single mutation was found that may cause hypospadias. Genetic counseling in the family plays an important role in the management of hypospadias. The most common phenotype for hypospadias in Indonesia is penile hypospadias. Important information for the parents and family during the counseling range from confirmed biological gender, the clinical management, the risk of having children with the same condition, to the prevention for the next pregnancy.(18) The study has some limitations. Future studies should involve more samples using a more convenient method. Other advanced techniques such as next generation sequencing should be considered. More studies are needed to confirm the utilization of HRM for mutation screening in hypospadias. Studies to confirm the effect of the mutations and genetic examinations in unaffected individuals are needed. CONCLUSION Chromosomal mutations were found using Sanger sequencing in aberrant pattern from the HRM analysis in two samples that is translocation in chromosome 8: 143516989-143517080 (92 nt) and 143516981-143517080 (100 nt), consecutively. These result in the absence of a part of WNT5A exon 5B, suggesting a possible deletion of WNT5A exon 5B that warrants a functional study to confirm.

Figure 1
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Acknowledgements

Katie Ayers, Stefanie Eggers, Jocelyn Bergen, and Sonja Gustin of Molecular Development in Murdoch Childrens Research Institute Victoria Australia, and the Ministry of Research Technology and Higher Education, Indonesia.

References

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Keywords: Hypospadias, HRM, High-resolution melting curve, Wnt5a, Chromosomal mutation

Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019.

Presentation Type: Poster Presentation

Topic: Miscellaneous

Citation: Indraswari DA, Juniarto AZ, Sinclair AH and Faradz SM (2019). Chromosomal Mutation In Isolated Hypospadias Patients Using High-Resolution Melting Curve (HRM). Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2019.63.00021

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Received: 03 Dec 2018; Published Online: 17 Jan 2019.

* Correspondence: MD. Darmawati A Indraswari, Diponegoro University, Semarang, Indonesia, darmawatiayu@gmail.com