# Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency

Elena J. Tucker, Michael F. Sharp, Anna Lokchine, Katrina M Bell, Catherine

S. Palmer, Brianna L Kline, Gorjana Robevska, Jocelyn van den Bergen,

Jérôme Dulon, Diana Stojanovski, et al.

► To cite this version:

Elena J. Tucker, Michael F. Sharp, Anna Lokchine, Katrina M Bell, Catherine S. Palmer, et al. Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency. Clinical Genetics, 2024, 106 (3), pp.321-335. 10.1111/cge.14543. hal-04605092

HAL Id: hal-04605092

https://hal.science/hal-04605092v1

Submitted on 16 Jan 2026

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# Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency

Elena J. Tucker<fnref n="1" />,<fnref n="2" /> | Michael F. Sharp<fnref n="3" />,<fnref n="4" /> | Anna Lokchine<fnref n="5" />,<fnref n="6" /> | Katrina M. Bell<fnref n="1" />,<fnref n="7" /> |

Catherine S. Palmer<fnref n="8" /> | Brianna L. Kline<fnref n="1" /> | Gorjana Robevska<fnref n="1" /> |

Jocelyn van den Bergen<fnref n="1" /> | Jérôme Dulon<fnref n="9" /> | Diana Stojanovski<fnref n="8" /> |

Katie L. Ayers<fnref n="1" />,<fnref n="2" /> | Philippe Touraine<fnref n="9" /> | Wayne Crismani<fnref n="3" /> | Sylvie Jaillard<fnref n="5" />,<fnref n="6" /> |

Andrew H. Sinclair<fnref n="1" />,<fnref n="2" /> ID

## Correspondence

Elena J. Tucker, Reproductive Development, Murdoch Children's Research Institute, Melbourne, VIC, Australia.

Email: elena.tucker@mcri.edu.au

## Funding information

National Health and Medical Research Council, Grant/Award Numbers: 1074258, 1054432, 1062854; State Government of Victoria; CHU Rennes

## Abstract

Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, *Fanconi anemia complementation group A (FANCA)*, is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous *FANCA* variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic *FANCA* variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel *FANCA* variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in *FANCA*, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.

## KEYWORDS

DNA damage repair, FANCA, Fanconi anaemia, functional genomics, infertility, premature ovarian insufficiency

<footnote n="1">¹Reproductive Development, Murdoch Children's Research Institute, Melbourne, Victoria, Australia</footnote>

<footnote n="2">²Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia</footnote>

<footnote n="3">³DNA Repair and Recombination Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia</footnote>

<footnote n="4">⁴The Faculty of Medicine, Dentistry and Health Science, University of Melbourne, Victoria, Australia</footnote>

<footnote n="5">⁵CHU Rennes, INSERM, EHESP, IRSET (Institut de recherche en santé, environnement et travail) - UMR_S1085, Univ Rennes, Rennes, France</footnote>

<footnote n="6">⁶Service de Cytogénétique et Biologie Cellulaire, CHU Rennes, Rennes, France</footnote>

<footnote n="7">⁷Bioinformatics, Murdoch Children's Research Institute, Victoria, Australia</footnote>

<footnote n="8">⁸Department of Biochemistry and Pharmacology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia</footnote>

<footnote n="9">⁹Department of Endocrinology and Reproductive Medicine, AP-HP, Sorbonne University, Paris, France</footnote>
# 1 | INTRODUCTION

Premature ovarian insufficiency is a leading cause of infertility affecting up to 4% of women.¹ It is characterised by loss or lack of ovarian activity before the age of 40, manifesting as amenorrhea and elevated follicle stimulating hormone (FSH). Current clinical guidelines² recommend cytogenetic analysis and investigation of a single causative gene, *Fragile X Messenger Ribonucleoprotein 1 (FMR1)*, which are responsible for ~15% of cases collectively. Growing evidence, however, demonstrates the benefits of genomic sequencing that can identify likely cause in more than a quarter of remaining patients.³,⁴ A genetic diagnosis has multiple benefits such as enabling screening for gene-specific co-morbidities, appropriate patient counselling and cascade testing for family members who could benefit from early interventions.

The genetic cause of POI is highly diverse, with over 80 causative genes that have varied roles in processes such as ovarian development, meiosis, DNA damage repair, mitochondrial function, immune regulation, folliculogenesis, hormone signalling and more.⁵,⁶ POI can be the first clinical manifestation of many genetic syndromes and the underlying genetic cause can have broad health implications.⁷,⁸ For example, POI caused by genetic disruption of DNA damage repair can also lead to elevated cancer risk.⁹

Cellular division and cell cycle control play a critical role in the development and maintenance of oocytes. During fetal development oocytes arrest at prophase I and remain dormant until meiotic resumption after menarche. The genomic integrity of oocytes must be maintained throughout this long process to ensure fertility preservation. Furthermore, during meiosis, oocytes undergo homologous recombination which involves DNA double strand breaks. Disruption to meiosis and DNA damage repair is known to lead to oocyte depletion and premature loss of ovarian function. In fact, over 30 genes involved in these processes have been found to harbour POI-associated variants¹⁰ (Table 1).

The ubiquitous role of DNA damage repair genes means that their disruption is often associated with syndromic POI. For example, variants in *nibrin* (NBN), which is involved in initiating DNA double strand break repair, are associated with Nijmegen Breakage Syndrome characterised by POI, intellectual disability, immunodeficiency and cancer predisposition.³⁶ Variants in *minichromosome maintenance 8 homologous recombination repair factor 8 (MCM8)* or *MCM9*, which encode helicases required for double strand break repair, can be associated with cancer predisposition and POI.⁵²,⁵³ Interestingly, there is a phenotypic spectrum associated with many of these genes, with some individuals presenting with only isolated POI,⁷ although the reason for the varied presentation is poorly understood.

One genetic syndrome which predisposes to POI is Fanconi Anaemia (FA).⁵⁴ Affected individuals are also predisposed to progressive bone marrow failure, myelodysplastic syndrome and acute myeloid leukaemia. Other features can include limb, kidney, eye and skin abnormalities.⁵⁵ Endocrine dysfunction, genitourinary abnormalities and infertility are also frequent features of FA.¹⁹,⁵⁶,⁵⁷ FA can be caused by variants in at least 22 genes, all of which act in a common

genetic and biochemical pathway. The inheritance pattern of FA is autosomal recessive, with the exception of variants in *Fanconi anemia complementation group B (FANCB)* which are X-linked, and a rare dominant variant in *RAD51 recombinase (RAD51)*. Variants in *Fanconi anemia complementation group A (FANCA)* are the most common cause of FA and are responsible for ~60%-70% of cases.⁵⁴,⁵⁵,⁵⁸ This gene encodes a subunit of the FA core complex, which is responsible for recruiting DNA repair machinery to areas of DNA damage. Sanger sequencing of the FANCA gene, however, only detects ~60% of causative variants due to limitations in detecting gene rearrangements/deletions that are frequent at this locus.⁵⁹

Although the FANCA gene is a well-established cause of classical FA, it has also been proposed that heterozygous variants in this gene are responsible for isolated POI¹⁸,⁶⁰ although whether these variants are truly causal is controversial.⁶¹ It is important to establish whether heterozygous variants in FANCA can cause POI as this will have significant impact on future variant curation and the diagnosis and potential outcomes for affected individuals.

Here we identify compound heterozygous FANCA variants in French sisters presenting with isolated POI. Functional studies were not able to confirm sensitivity to DNA damage, potentially explaining the non-syndromic nature of the phenotype. Our cases highlight the potential involvement of FANCA deletions to POI and the challenges in their detection using WES.

Our data adds evidence that monoallelic loss of FANCA is an unlikely cause of POI and highlights the challenge and importance of functional validation before variants can be upgraded from uncertain significance to pathogenic for clinical management.

# 2 | RESULTS

## 2.1 | Sisters presented with isolated POI

The proband (P1), of Congolese descent, presented with primary amenorrhea and pubertal delay at 16 years of age (Figure 1A). FSH was elevated at 33 IU/L and anti-Mullerian hormone (AMH) was low at 0.2 ng/mL (Table 1). Ovaries could be detected by ultrasound but were small (surface area right: 0.8 cm², left: 0.5 cm²) and had no active follicles. No additional family history was noted, specifically, there were no *café au lait* spots, reports of recurrent infection or family history of cancer. Her mother died shortly after her birth so age of maternal menarche and menopause could not be recalled. There was no notable facial dysmorphia and she was of normal stature (1.58 m). There were no skeletal abnormalities of the hands or wrists. Bone density examination was within normal limits.

The proband's sister (P2) presented at age 23 with secondary amenorrhea after normal puberty and menarche. FSH was elevated at 67 IU/L and AMH was low at 0.2 ng/mL. Ovaries were visualised by ultrasound with a surface area of 1.4 cm² on the right and 2.0 cm² on the left and one active follicle. Like her sister, she was of normal stature (1.65 m), had no skeletal abnormalities of the hands or wrists and no other family or medical history was identified or reported.
<table><caption>TABLE 1 List of genes in which variants have been associated with human POI.</caption><thead><tr><td>Gene</td><td>Name</td><td>Role</td><td>Mode of inheritance</td><td>Phenotype</td><td>References</td></tr></thead><tbody><tr><td>ANKRD31</td><td>Ankyrin repeat domain 31</td><td>Stabilises and regulates the binding of DSB formation factors</td><td>AD</td><td>POI</td><td>11</td></tr><tr><td>ATM</td><td>ATM serine/threonine kinase</td><td>Role in cellular responses to genomic damage</td><td>AR</td><td>Ataxia telangiectasia + POI</td><td>12</td></tr><tr><td>BLM</td><td>Bloom syndrome, RecQ helicase-like</td><td>DNA helicase, regulates homologous recombination</td><td>AR</td><td>Bloom syndrome + POI</td><td>13</td></tr><tr><td>BUB1B</td><td>BUB1B, mitotic checkpoint serine/threonine kinase</td><td>Spindle assembly checkpoint component</td><td>AD</td><td>POI</td><td>14</td></tr><tr><td>CCDC155</td><td>Coiled-coil domain containing 155</td><td>Modulates chromosome movement for DSB repair</td><td>AR</td><td>POI</td><td>15</td></tr><tr><td>DMC1</td><td>DNA meiotic recombinase 1</td><td>Required for meiotic homologous recombination</td><td>AR</td><td>POI (azoospermia in males)</td><td>16</td></tr><tr><td>ERCC6</td><td>Excision repair cross-complementation group 6</td><td>Transcription coupled nucleotide excision repair</td><td>AD</td><td>POI</td><td>17</td></tr><tr><td>FANCA</td><td>Fanconi anaemia complementation group A</td><td>Fanconi anaemia core complex</td><td>AR, AD?</td><td>Fanconi anaemia + POI (azoospermia in males)</td><td>18–20</td></tr><tr><td>FANCC</td><td>Fanconi anaemia complementation group C</td><td>Fanconi anaemia core complex</td><td>AR</td><td>Fanconi anaemia + POI</td><td>19,21</td></tr><tr><td>FANCG</td><td>Fanconi anaemia complementation group G</td><td>Fanconi anaemia core complex</td><td>AR</td><td>Fanconi anaemia + POI</td><td>19,20</td></tr><tr><td>FANCM</td><td>Fanconi anaemia complementation group M</td><td>Interacts with the FA core complex in DNA damage repair</td><td>AR</td><td>POI (azoospermia in males)</td><td>22,23</td></tr><tr><td>FANCD1/ BRCA2</td><td>BRCA2 DNA repair associated</td><td>Homologous recombination and fork stabilisation</td><td>AR</td><td>POI, cancer predisposition</td><td>24–26</td></tr><tr><td>HELQ</td><td>Helicase, POLQ-like</td><td>Double strand break repair enzyme</td><td>AR</td><td>POI</td><td>3</td></tr><tr><td>HFM1</td><td>HFM1, ATP-dependent DNA helicase homologue</td><td>Required for synapsis between homologous chromosomes</td><td>AR</td><td>POI</td><td>27</td></tr><tr><td>HROB</td><td>Homologous recombination factor with OB-fold</td><td>Involved in DNA damage repair – recruits MCM8 and MCM9 helicases</td><td>AR</td><td>POI</td><td>28</td></tr><tr><td>HSF2BP</td><td>Heat shock transcription factor 2 binding protein</td><td>Localises at recombination nodules to mediate cross over events in meiosis</td><td>AR</td><td>POI</td><td>29</td></tr><tr><td>MCM8</td><td>Minichromosome maintenance 8 homologous recombination repair factor</td><td>Required for homologous recombination-mediated repair of double-strand breaks</td><td>AR</td><td>POI, cancer predisposition</td><td>30</td></tr><tr><td>MCM9</td><td>Minichromosome maintenance 9 homologous recombination repair factor</td><td>Required for homologous recombination-mediated repair of double-strand breaks</td><td>AR</td><td>POI, cancer predisposition</td><td>31</td></tr><tr><td>MRE11A</td><td>MRE11 homologue, double strand break repair nuclease</td><td>Required for double strand break repair</td><td>AR</td><td>Ataxia-telangiectasia-like disorder + POI</td><td>32,33</td></tr><tr><td>MSH4</td><td>MutS homologue 4</td><td>Meiotic crossover</td><td>AR</td><td>POI</td><td>34</td></tr><tr><td>MSH5</td><td>MutS homologue 5</td><td>Meiotic crossover</td><td>AR</td><td>POI</td><td>35</td></tr><tr><td>NBN</td><td>Nibrin</td><td>Required for the repair of double strand breaks</td><td>AR</td><td>Nijmegen breakage syndrome + POI, infertility</td><td>7,36</td></tr><tr><td>PRDM9</td><td>PR domain containing 9</td><td>Meiosis-specific histone H3 methyltransferase and a major determinant of meiotic recombination hotspots</td><td>AD</td><td>POI</td><td>11</td></tr><tr><td>RAD51B</td><td>RAD51 paralog B</td><td>RAD51 paralog required for homologous recombination</td><td>AR</td><td>POI</td><td>37</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><td>Gene</td><td>Name</td><td>Role</td><td>Mode of inheritance</td><td>Phenotype</td><td>References</td></tr></thead><tbody><tr><td>REC8</td><td>REC8 meiotic recombination protein</td><td>Meiosis-specific component of the cohesin complex</td><td>AR</td><td>POI</td><td>28</td></tr><tr><td>RECQL4</td><td>RecQ helicase-like 4</td><td>DNA helicase that unwinds double-stranded DNA, involved in DNA repair</td><td>AR</td><td>Rothmund-Thomson syndrome + POI</td><td>38</td></tr><tr><td>SGO2</td><td>Shugoshin 2</td><td>Maintains the integrity of the cohesion complex, required for meiosis</td><td>AR</td><td>POI</td><td>39</td></tr><tr><td>SPIDR</td><td>Scaffolding protein involved in DNA repair</td><td>Homologous recombination repair gene</td><td>AR</td><td>POI</td><td>40</td></tr><tr><td>SPO16</td><td>Synaptonemal complex reinforcing element</td><td>Involved in meiotic synapsis and crossover maturation</td><td>AR</td><td>POI</td><td>41</td></tr><tr><td>STAG3</td><td>Stromal antigen 3</td><td>Subunit of cohesin, required for proper pairing and segregation of chromosomes during meiosis</td><td>AR</td><td>POI</td><td>42</td></tr><tr><td>SWI5</td><td>SWI5 homologous recombination repair protein</td><td>Regulates homologous recombination through the RAD51 pathway</td><td>AR</td><td>POI</td><td>3</td></tr><tr><td>SYCE1</td><td>Synaptonemal complex central element protein 1</td><td>Part of the synaptonemal complex that physically links homologous chromosomes</td><td>AR</td><td>POI</td><td>43</td></tr><tr><td>SYCP2L</td><td>Synaptonemal complex protein 2 like</td><td>Component of the synaptonemal complex that physically links homologous chromosomes</td><td>AR</td><td>POI</td><td>44</td></tr><tr><td>TP63</td><td>Tumour protein p63</td><td>Transcription factor involved in development and surveillance of oocyte integrity</td><td>AD</td><td>Syndromic and isolated POI</td><td>45,46</td></tr><tr><td>WRN</td><td>Werner syndrome, RecQ helicase-like</td><td>DNA helicase that unwinds double-stranded DNA, involved in DNA repair</td><td>AR</td><td>Werner syndrome + POI</td><td>47</td></tr><tr><td>XRCC2</td><td>x-ray repair complementing defective repair in Chinese hamster cells 2</td><td>Repair of DNA double strand breaks by homologous recombination</td><td>AR</td><td>Fanconi anaemia + POI</td><td>48,49</td></tr><tr><td>XRCC4</td><td>x-ray repair cross complementing 4</td><td>Non-homologous end-joining DNA repair</td><td>AR</td><td>Dwarfism, metabolic syndrome + POI</td><td>50</td></tr><tr><td>ZSWIM7</td><td>Zinc finger, SWIM-type containing 7</td><td>Required for homologous recombination during prophase I of meiosis</td><td>AR</td><td>POI</td><td>51</td></tr></tbody></table>

Abbreviations: AD, autosomal dominant; AR, autosomal recessive.

## 2.2 | WES identified a candidate variant of uncertain significance (VUS) in FANCA

WES was performed on each sister and filtered for moderate-high priority rare variants in candidate POI genes, potentially biallelic variants and likely LOF variants (Supplementary Table 1). This revealed 15 heterozygous variants in candidate POI genes shared by the sisters, four genes of which have been conclusively associated with autosomal recessive POI in humans: homologous recombination factor with OB-fold (HROB), prolyl endopeptidase like (PREPL), twinkle mtDNA helicase (TWNK) and FANCA. The sisters also shared variants in 13 different genes with potential biallelic inheritance, though none of these genes have previously been associated with POI. Lastly, they shared a

predicted LOF variant in one gene, *iodothyronine deiodinase 1* (DIO1), however, again this gene had no known association with POI.

This initial data analysis failed to identify the genetic cause of POI in the sisters. We further considered the heterozygous variants in genes previously associated with human POI (Supplementary Table 2). Variants in PREPL, TWNK and FANCA have previously been described in syndromic POI involving myopathy, Perrault syndrome and FA respectively. In contrast, the sisters of this study presented with isolated POI. Although the fourth gene, HROB, has previously been associated with isolated POI,²⁸ the HROB variant was consistently predicted likely benign by online algorithms and monoallelic variants in HROB are unlikely causative. In contrast, the variant in FANCA had conflicting pathogenicity predictions with a deleterious prediction by
Polyphen, CADD and SIFT, but not MutationTaster. The FANCA variant was validated by Sanger sequencing (Figure 1B). The altered residue was modestly conserved (Figure 1C). Interestingly, this variant has been identified previously in an individual with FA according to ClinVar (Accession ID: VCV000997461.8) however, further detail of the patient phenotype is not available publicly. Modelling of the Fanconi Anaemia complex reveals that the altered residue is positioned at the FANCA dimer interface with FANCG (Figure 1D).

## 2.3 | Further interrogation of WES data identified an intragenic deletion in FANCA

Given there is controversy about the causal role of heterozygous FANCA variants in POI, with substantial evidence suggesting heterozygous variants are tolerated, we manually inspected WES data to determine whether a copy number variant (CNV) might have escaped detection. Visual inspection of WES data using control samples from each WES batch, identified a likely intragenic deletion affecting Exons 28-31 in both sisters with ~50% coverage compared to controls when normalised to flanking exon coverage (Figure 2A,B). In contrast, the other three genes with heterozygous variants did not have discernible differences in coverage compared to controls (Supplementary Figure S1). We confirmed the FANCA deletion using recently developed bioinformatic tools to detect CNVs from WES data,⁶² with the deletion detected by two of three incorporated callers (Figure 2C). Finally, we confirmed heterozygous intragenic deletion of Exons 28-31 via clinical microarray, which revealed a heterozygous ~13.6 kb FANCA deletion (arr[GRCh38] 16q24.3 (89752083_89765675)x1) in each sister. This deletion has not previously been reported in the general population according to DGV, although the region has been involved in other CNVs deleting varying exons.

## 2.4 | The intragenic deletion breakpoints co-localise with SINE repetitive elements

Based on chromosomal microarray, the boundary of the genomic deletion was uncertain. The intragenic deletion was refined by PCR amplification and sequencing (Figure 2D). Sequencing revealed a SINE repeat element at each breakpoint and 15 bp overlapping sequence (Figure 2E), determining the genomic variant as a 14.38kB deletion, Chr16: 89751663_89766042del. At the cDNA level, this can be defined as NC_000016.10(NM_000135.4):c.2602-976_3066 + 475del encoding a predicted protein with an internal deletion of 155 residues, NC_000016.10(NP_000126.2):p.(Phe868_Gln1022del).

## 2.5 | The heterozygous missense variant and intragenic deletion are in *trans*

If the intragenic deletion and missense variant are causing autosomal recessive POI, the two variants must be inherited on alternative

alleles. We generated patient lymphoblastoid cell lines from which RNA could be extracted and analysed. Analysis of patient cDNA using primers flanking the deleted region confirmed that the intragenic deletion caused a stable transcript lacking exons 28-31 (Figure 3A,B). In contrast, RT-PCR with allele-specific primers that only amplify the non-deleted allele (Figure 3A,C) revealed that all full length FANCA transcript harboured the c.4177G > A, p.Val1393Met missense variant (Figure 3C). This confirmed that the deletion and the missense variants are in *trans* and the patients have inherited biallelic variants in FANCA.

## 2.6 | The intragenic deletion encodes stable FANCA with an internal deletion

The deletion spanning 4 exons of FANCA is predicted to encode an in-frame protein lacking 155 amino acids, p.(Phe868_Gln1022del). Many of these residues are highly conserved (Figure 4B), suggesting they are likely required for FANCA structure/function. Some are altered by known pathogenic missense variants (Figure 4A) supporting the requirement for this region to be intact. To investigate whether loss of these residues renders FANCA unstable, we performed western blot of patient lymphoblasts. This revealed two stable FANCA species in patient lymphoblasts corresponding to full length FANCA and FANCA with an 18 kDa internal deletion (Figure 4C).

## 2.7 | No DNA damage sensitivity was detected in patient cells

Given biallelic pathogenic variants in FANCA are usually associated with syndromic POI because of defects in DNA damage repair, patient PBMCs were analysed for hypersensitivity to DNA crosslinking agents. We analysed 50 metaphasic cells treated with mitomycin C (MMC) and these did not indicate chromosomal instability. In contrast, we have previously demonstrated DNA damage sensitivity using this chromosomal breakage assay in a patient with POI due to pathogenic FANCM variants.⁶³

We hypothesised that the variants may be milder than typical FANCA variants, leading to a mild deficiency of DNA damage repair that could be sufficient to induce ovarian damage, but not cause the full clinical diagnosis of FA. To further investigate whether patient cells may have a subtle defect in DNA damage repair, we analysed patient lymphoblasts for their ability to mono-ubiquitinate FA complementation group D2 (FANCD2) in response to exposure to DNA damaging agents. Lymphoblasts from the sisters in this study were able to adequately ubiquitinate FANCD2 suggesting the DNA damage repair pathway is intact (Figure 5A). Furthermore, there was no excess in γH2AX expression in response to hydroxyurea (Figure 5B), nor was there altered survival in response to varying concentrations of MMC (Figure 5C). Collectively, these assays demonstrate a preserved DNA damage response in patient lymphoblasts.
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Heterozygous missense FANCA variant in sisters with premature ovarian insufficiency. (A) Family pedigree showing two affected sisters, (B) Sanger validation of the heterozygous c.4177G > A, p.Val1393Met missense variant. (C) Conservation track demonstrates moderate conservation of p.Val1393, (D) Modelling of the p.Val1393Met variant with its position in one protein of the FANCA dimer at the outside of the complex and its position in the other protein of the dimer at the interface between FANCA and FANCG.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** Detection of a FANCA intragenic deletion that escaped detection by initial WES analysis. (A, B) Manual inspection of WES coverage data indicated comparative reduction in coverage of four exons of FANCA compared to controls in P1 (top) and P2 (bottom). P1 and P2 were run in different WES batches so batch effects are controlled for by comparison to different controls. (A) Full FANCA genomic locus. (B) Zoomed in on exonic deletion). (C) CxGO (recently designed for prediction of CNVs from WES data) detected the exonic deletion using two of three callers. (D) Long-range PCR of genomic DNA amplified a product in sisters harbouring an intragenic deletion. (E) Sange sequencing PCR products allowed break point mapping and indicated the involvement of SINE repetitive elements.</figcaption></figure>
<figure><img src="image_4.png" /></figure>

**FIGURE 3** The FANCA intragenic deletion and missense variants are in trans. (A) Diagram shows RT-PCR design with primers that span the deleted region (F1 + R1) and allele-specific primers (F2 + R2) with a forward primer within the intragenic deletion that will amplify only full-length FANCA cDNA. (B) Gel electrophoresis of RT-PCR products using F1 + R1 demonstrated two PCR products consistent with the expression of full-length mRNA and mRNA with the internal deletion. Skipping of exons 28–31 was confirmed by sequencing, as shown (right). (C) Gel electrophoresis of RT-PCR products using allele specific F2 + R2 primers confirmed expression of full-length transcript. Sequencing (right) confirmed the wildtype exonic junction (Exon 31/32) and presence of the c.4177G > A, p.Val1393Met missense variant confirming the deletion and missense variants are compound heterozygous.

## 2.8 | Patient lymphoblasts do not have altered dynamin-related protein 1 (DRP1) expression

There is some recent evidence suggesting a possible role of FANCA in the maintenance of mitochondrial integrity and function,⁶⁴⁻⁶⁶ though the mechanism for this is yet to be fully elucidated. For example, loss of FANCA has been associated with elevated mitochondrial fission protein, DRP1.⁶⁴ Defects in mitochondrial function, and in particular mitochondrial dynamics via balance of fusion and fission, have been known to lead to POI in humans or mice.⁶⁷,⁶⁸ We therefore examined DRP1 expression in patient lymphoblasts by western blot. This revealed normal levels of mitochondrial DRP1 in comparison to control lymphoblasts (Figure 5D). Mitochondrial dynamics in patient lymphoblasts was assessed by microscopy and a normal mitochondrial network morphology was observed in lymphoblasts from both patients compared to control (Figure 5E).

# 3 | DISCUSSION

## 3.1 | The evolving phenotypic spectrum of DNA damage sensitivity

Biallelic variants in FANCA are the primary cause of Fanconi anaemia, accounting for approximately 60%-80% of FA cases. Variants in some

components of the FA pathway can manifest clinically as isolated infertility, including POI or azoospermia.²⁰,²²,²⁶,³⁷,⁴⁹ Frequently, the first reports of genotype-phenotype associations are biased towards more severe presentations, but as genomic sequencing approaches become more broadly used, there is increased awareness of mild clinical manifestations of disorders that were traditionally viewed as severe paediatric conditions. For example, variants in NBN are a well-established cause of the chromosomal instability syndrome, Nijmegen breakage syndrome, but have more recently been identified as a cause of apparently isolated POI/infertility.⁷ Variants in *mitochondrial transcription factor A (TFAM)* were first associated with a lethal infantile multisystem disorder but have since been associated with a much milder syndromic POI phenotype.⁶⁹⁻⁷¹ Conversely, cancer is often a late-onset condition and sometimes predisposition to cancer is not known at the time of publication of new disease genes. For example, the first reports of *MCM8* and *MCM9* variants suggested these genes were associated with isolated POI, however further cases have since demonstrated an association with syndromic POI.³⁰,³¹,⁵²,⁵³ As more cases are identified and long-term follow-up of affected patients is performed, we will get a better understanding of the genetic basis of POI and its associated conditions, such as cancer. The current study highlights the potential for biallelic FANCA variants to be associated with isolated POI without evidence of DNA damage sensitivity. A caveat to our study, however, is that DNA damage sensitivity was measured in patient lymphoblasts. A negative result, therefore, cannot
<figure><img src="image_5.png" /><figcaption>FIGURE 4 Legend on next page.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**FIGURE 5** Patient lymphoblasts had no discernible defect in DNA damage repair or mitochondrial dynamics. (A) Lymphoblasts from Sisters 1 and 2 were cultured for 16 h with 0, 30 and 300 nM of MMC before whole cell lysates were analysed by SDS-PAGE and western blotting. Revert Total Protein Stain is used as a loading control. FANCD2 ubiquitination is observed in both patients as well as control in response to MMC treatment. (B) Lymphoblasts from Sisters 1 and 2 were cultured for 16 h with 0, 0.2 and 2 mM of Hydroxyurea before whole cell lysates were analysed by SDS-PAGE and western blotting. Revert Total Protein Stain is used as a loading control. γH2A.X is observed in both patients as well as control in response to hydroxyurea treatment, there is no increase observed compared to the control. (C) Lymphoblasts were treated with MMC for 96 h in a dose response, in triplicate. Survival was calculated using flow cytometry with the dead cells stained with propidium iodide. The surviving fraction was normalised to untreated cells for each cell line. Error bars represent standard error of the mean. (D) Whole cell lysates of lymphoblasts from Sisters 1 and 2 were analysed by SDS-PAGE and western blotting. Sisters 1 and 2 had normal levels of DRP1 in comparison to controls. (E) Morphological analysis of patient lymphoblasts demonstrated normal mitochondrial morphology. Red indicates solute carrier family 25 member 6 (SLC25A6) antibody staining, highlighting mitochondrial networks. Dashed box denotes zoom region, scale bar = 5 µm.</figcaption></figure>

detectable by careful and targeted manual visualisation of WES coverage, by two of three CNV callers applied to WES data and by chromosomal microarray. This highlights the importance of CNV analysis to complement SNV analysis of exome data. This is particularly relevant for loci such as FANCA, which are prone to large deletions due to large numbers of repetitive elements.⁵⁸,⁷⁶ The original report by Yang et al.¹⁸ describing heterozygous variants in FANCA in POI patients, failed to address the potential for copy number variants. It is possible that the patients harbouring heterozygous deleterious FANCA variants also harbour gene rearrangements that have escaped detection. The investigation of POI pathogenesis should include analysis for potential CNVs, whether this be achieved by whole genome sequencing, analysis of exome data or microarray.

## 4 | CONCLUSION

Here we identify biallelic variants in FANCA in sisters with isolated premature ovarian insufficiency. One variant is a likely loss-of-function allele, whereas the other is a missense that remains a variant of

uncertain significance. No defect in DNA damage sensitivity could be detected in patient lymphoblasts. Our case demonstrates the potential involvement of biallelic FANCA variants in isolated POI pathogenesis, although further functional validation is required before the missense variant can be considered causal. We also highlight the challenges of FANCA variant detection, the potential for misinterpretation of heterozygous variants and the need for caution when drawing conclusions in such cases.

## 5 | MATERIALS AND METHODS

### 5.1 | Participants

Available family and medical history are included in Table 2. All patients had POI, defined by menstrual disturbance and elevated FSH (>20 mIU/mL) measured twice at least 1 month apart as per the ESHR guidelines,² had no history of ovarian surgery or gonadotoxic therapy, had normal 46,XX chromosomal complement, and were negative for FMR1 premutation.
<table><caption>TABLE 2 Summary of phenotype of sisters, P1 and P2.</caption><thead><tr><th>Patient</th><th>Age at diagnosis</th><th>Karyotype</th><th>Amenorrhea</th><th>Secondary sex characteristic</th><th>US</th><th>FSH IU/l (Ref &lt;20)</th><th>LH IU/l (Ref &lt;60)</th><th>Estradiol pg/mL (Ref &gt;30)</th><th>AMH (ng/mL) (Ref &gt;1.5)</th><th>Other</th></tr></thead><tbody><tr><td>1</td><td>16</td><td>46,XX</td><td>Primary</td><td>Delayed</td><td>Small ovaries:<br/>Right 0.8 cm²<br/>Left 0.5 cm²<br/>No follicles</td><td>33</td><td>ND</td><td>5</td><td>0.2</td><td>Normal bone density<br/>No café au lait spots/<br/>skin hyperpigmentation<br/>No facial dysmorphia<br/>No cancer history<br/>No upper limb<br/>abnormalities</td></tr><tr><td>2</td><td>23</td><td>46,XX</td><td>Secondary</td><td>Normal</td><td>Normal ovaries:<br/>Right 1.4 cm²<br/>Left 2.0 cm²<br/>1 follicle</td><td>67</td><td>22</td><td>43</td><td>0.2</td><td>Normal bone density<br/>No café au lait spots/<br/>skin hyperpigmentation<br/>No facial dysmorphia<br/>No cancer history<br/>No upper limb<br/>abnormalities</td></tr></tbody></table>

## 5.2 | Ethics

Written informed consent was obtained from each participant. All procedures were approved by the Ethics Committee of Rennes University Hospital and the French law (CCTIRS Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé) and/or the Human Research Ethics Committee of the Royal Children's Hospital, Melbourne (HREC/22073).

## 5.3 | General molecular techniques

Genomic DNA was extracted from EDTA-blood and was assessed by NanoDropTM 1000 spectrophotometer and Qubit dsDNA BR Assay (Thermo Fisher Scientific). Selected variants were validated by Sanger sequencing using BigDye v3.1 Terminators (Applied Biosystems) and ABI3130X. Primer sequences are available on request.

## 5.4 | Whole-exome sequencing (WES)

DNA underwent WES with SureSelect Human All Exon V6 (Agilent) capture and sequencing on the NovaSeq 6000 (Illumina). WES data were processed using Cpipe<sup>77</sup> and analysed as previously described<sup>46</sup> (Supplementary Material).

## 5.5 | Clinical microarray

For validation of CNVs detected from WES data, 500 ng of DNA from each patient was used for Illumina Infinium Omni2.5 high density microarrays, performed by the Victorian Clinical Genetics Service. Data were processed and analysed using Illumina GenomeStudio 2.0 and Illumina KaryoStudio v1.4.

## 5.6 | Human cell culture

Whole blood samples were layered onto Ficoll-Plaque Plus (GE Health) and PBMCs were separated by density gradient centrifugation. Freshly isolated lymphocytes were transformed with Epstein Barr Virus in the presence of phytohemagglutinin (Life Technologies). Transformed cells were maintained in RPMI 1640 + 20% FBS.

## 5.7 | DNA damage sensitivity assay

72 h blood culture was performed with phytohemagglutinine, without treatment and after exposure to Mitomycin C (Sigma-Alrich, Saint-Louis, MI, USA). Metaphase spreads were coloured by Giemsa. Baseline and DNA-induced chromosome breakage and rearrangements were scored on 50 metaphases in the proband, her sister and in a normal control. We have used this protocol previously to demonstrate DNA damage sensitivity in POI patients.<sup>63</sup>

## 5.8 | FANCD2 ubiquitination and DNA damage assay

Patient lymphoblasts were cultured for 16 h with 0, 30 and 300 nM of MMC or 0, 0.2 and 2 mM of hydroxyurea. Cells were harvested and lysed by sonication in RIPA buffer. Lysates were run on 3%-8% tris-acetate NuPage polyacrylamide gels (Invitrogen) in tris-acetate running buffer and NuPage antioxidant (Invitrogen). Proteins were transferred to PVDF-fl membrane (Merck) using wet transfer (Bio-Rad) Revert total protein staining (Li-cor) was performed on the membrane for normalisation. Antibodies used were: anti-FANCD2 (ab108928, abcam), anti-rabbit IgG DyLight™ 800 (SA5-10036, Invitrogen), anti-phospho-H2A. X ser139 (05-636, Merck) and anti-mouse IgG DyLight™ 800 (SA5-10176, Invitrogen), Membrane was imaged using Li-Cor Odyssey CLx.
5.9 | Western blot

Patient lymphoblasts were harvested and lysed by sonication in RIPA buffer. Lysates were run on 4%-12% Bis-Tris Bolt polyacrylamide gels (Invitrogen) in MOPS running buffer (Invitrogen). Proteins were transferred to PVDF-fl membrane (Merck) using wet transfer (Bio-Rad). Revert total protein staining (Li-cor) was performed on the membrane for normalisation. Antibodies used were: anti-FANCA (A9529, abclonal), anti-rabbit IgG DyLight™ 800 (SA5-10036, Invitrogen), anti-DRP1 (BD Biosciences, 611 113), Anti-Tim29 (Sigma HPA041858-100UL), Anti-Tom20 (Santa Cruz, sc-17 762). Membranes were imaged using Li-Cor Odyssey CLx.

5.10 | Mitomycin C survival assay

Lymphoblasts were seeded in a 96 well plate and treated a dilution series of MMC (ranging from 7.8 to 500 nM) in triplicate. Cells were left at 37°C at 5% CO₂ for 96 h. Cells were stained with Propidium Iodide (0.5 µM) and counted on BD Fortessa flow cytometer. Propidium iodide stain cells were counted as dead cells and non-stained cells were counted as live cells. Percentage survival was calculated (live cells/(live cells + dead cells) × 100) and normalised to no-treatment control for each cell line.

5.11 | RNA analysis

RNA was extracted using the ReliaPrep RNA Cell Miniprep System (Promega). cDNA was synthesised using the GoScript reverse transcriptase system (Promega). RT-PCR was performed using primers available upon request.

5.12 | Mitochondrial morphology analysis

Lymphoblasts were seeded on to coverslips coated with 0.01% Poly-L-Lysine (Merck) and grown overnight in RPMI 1640 (Gibco) with 20% FBS (Cell Sera) at 37°C, 5% CO₂. The following day, cells were fixed with 4% Paraformaldehyde (Proscitech) in PBS containing 5% [w/v] Sucrose (Sigma). Following fixation, cells were washed and permeabilised with 0.1% [v/v] Triton-X 100 (Sigma). Antibodies used were: SLC25A6 (Abcam ab154007) and goat anti-rabbit AlexaFluor 568 (Invitrogen). Cells were stained with Hoechst 33258 (10 µg/mL, Sigma) and mounted on slides with mounting media (0.1 M Tris-Cl pH 8.0, 90% glycerol, 0.2 M 1,4-Diazabicyclo[2.2.2]octane (Sigma)). Imaging of lymphoblasts was performed using a Leica SP8 confocal microscope equipped with a 552 nm 20 mW solid state laser and a Direct Modulation laser diode (DMOD) 405 nm 50 mW laser. Samples were mounted on a 63X HC PL APO CS2 oil lens and images were captured with the Leica LAS X SP8 software. Laser intensity and gain were optimised for each sample. Z-stacks were acquired as the cells are very rounded and Z-projections are presented to

visualise the cell volume using Fiji image analysis software (https://imagej.nih.gov/ij/).

AUTHOR CONTRIBUTIONS

EJT, SJ and AHS conceived the study. EJT, MFS, AL, CSP, BLK, GR, JvdB, JD, DS, KLA, WC, SJ acquired the data. EJT, MFS, AL, KMB, CSP, BLK, GR, JvdB, JD, DS, KLA, WC, SJ, AHS analysed and interpreted the data. KMB, EJT, SJ performed the bioinformatic and genomic analyses. PT and SJ recruited patients, liaised with families and collected and reviewed clinical data. EJT wrote the manuscript, with input from all authors. All authors reviewed and approved the manuscript.

ACKNOWLEDGEMENTS

This work was supported by a National Health and Medical Research Council (NHMRC) program grant (1074258 to AHS), NHMRC fellowships (1054432 to EJT, 1062854 to AHS), a Suzi Carp postdoctoral scholarship (to EJT), a Norman Beischer Scietnific Fellowship (to EJT), a CHU Rennes grant (Appel à Projets Innovations 2019 to SJ). The research conducted at the MCRI was supported by the Victorian government's operational infrastructure support program. Open access publishing facilitated by The University of Melbourne, as part of the Wiley - The University of Melbourne agreement via the Council of Australian University Librarians.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ORCID

Andrew H. Sinclair https://orcid.org/0000-0003-2741-7992

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Tucker EJ, Sharp MF, Lokchine A, et al. Biallelic FANCA variants detected in sisters with isolated premature ovarian insufficiency. *Clinical Genetics*. 2024;106(3):321-335. doi:10.1111/cge.14543