Available online 22 March 2024, 112212

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Discovery of diverse causes of amenorrhea in an ethnically restricted cohort.

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Expansion of the genotypic spectrum associated with amenorrhea.

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Novel causal variants in SYCP2L, FANCM and GNRHR.

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Importance of functional validation before assigning variant pathogenicity.

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Potential of exome sequencing as a first-line diagnostic test for amenorrhea.

AbstractResearch question

Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1–3.7% women. Genetic factors explain 20–30% of POI cases, but most causes remain unknown despite genomic advancements.

Design

We used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity.

Results

Despite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea – the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO.

Conclusions

This study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.

Section snippetsKey message

Genetic cause of POI, characterised by amenorrhea and elevated gonadotropins in women under 40 years, largely eludes identification. Despite ethnic homogeneity, whole exome sequencing reveals diverse genetic causes of amenorrhea. Our study highlights the complexity of amenorrhea's genetic landscape, suggesting genomic sequencing as a universal diagnostic tool for infertility conditions.

Participants and ethical adherence

Six amenorrheic patients from four Iranian families were included in this study – five diagnosed with POI with elevated FSH (>20 mIU/ml) and one diagnosed with hypogonadotropic hypogonadism. Table 3 includes the available family and medical history. No patient reported a history of autoimmune disease, although ovarian autoantibodies were not assessed. All procedures were approved by the Human Research Ethics Committee of the Royal Children's Hospital, Melbourne (HREC# 22,073). Written informed

A homozygous nonsense SYCP2L variant: c.1528C  >  T p. (Gln510Ter)

Patient 1 was born to consanguineous parents (first cousins) of Iranian descent. At age 23, she was diagnosed with POI after presenting with secondary amenorrhea. FSH was elevated at 117.8 IU/I, and AMH was low at 0.01 ng/ml. Ultrasound identified ovaries that were smaller than normal. Patient 1 had a similarly affected sister (Patient 2) who was diagnosed with POI at age 19 (Fig. 1a) (Table 4).

WES of the two affected siblings and parents identified a homozygous stop-gain SYCP2L variant:

Discussion

In the present study we have used WES to investigate the genetic cause of amenorrhea in six patients from a small cohort of Iranian descent. Interestingly, WES revealed five different genetic causes of amenorrhea including novel gene variants (SYCP2L, FANCM, GNRHR) and cytogenetic causes (46, XY sex reversal, Turners syndrome).

Conclusion

Technological advances have made WES more accessible and affordable while detecting a wider range of genetic aberrations. Despite the challenges associated with next-generation sequencing, including the identification of variants with unknown clinical significance, the potential benefits of this powerful tool for cytogenetic and molecular diagnoses of POI are evident. This study highlights the various factors contributing to amenorrhea within a limited sample of individuals from a single ethnic

Ethics approval

All procedures were in accordance with the Human Research Ethics Committee of the Royal Children's Hospital, Melbourne.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

CRediT authorship contribution statement

Shabnam Bakhshalizadeh: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation. Fateme Afkhami: Writing – review & editing, Resources, Methodology, Investigation. Katrina M. Bell: Writing – review & editing, Investigation, Formal analysis, Data curation. Gorjana Robevska: Writing – review & editing, Project administration, Methodology. Jocelyn van den Bergen: Writing – review & editing, Project

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments/Funding

The research conducted at the Murdoch Children's Research Institute was supported by the Victorian government's operational infrastructure support program. This work also was supported by Cancer Research UK (DRCRPG-May23/100002 and C20724/A26752 to C.S., and C29637/A20183 and DRCNPG-Nov21/100001, with support from the Engineering and Physical Sciences Research Council, to E.W.T.), the BBSRC (BB/T01508X/1 to E.W.T. and C.S.), and UKRI (UKRI Postdoc Guarantee EP/X02749X/1 to Z.X. and E.W.T.).

This

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