Rare deleterious BUB1B variants induce premature ovarian insufficiency and early

menopause Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Qing Chen1,2,3, Hanni Ke4,5,6, Xuezhen Luo1,2, Lingbo Wang1,2,7, Yanhua Wu1,2, Shuyan

Tang1,2,3, Jinsong Li7, Li Jin1, Feng Zhang1,2,3,*, Yingying Qin4,5,6,* and Xiaojun Chen1,2,*

*To whom correspondence should be addresses at: Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China.MANUSCRIPT Tel: +86-21-31246783; Email: [email protected] (F.Z.); Email: [email protected] (Y.Q.); Email:

[email protected] (X.C.)

UNCORRECTED MANUSCRIPT © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

1

1

Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

(Shanghai Institute of Planned Parenthood Research), State Key Laboratory of Genetic

Engineering at School of Life Sciences, Fudan University, Shanghai 200011, China

2 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases,

Shanghai 200011, China

3 State Key Laboratory of Reproductive Medicine, Center for Global Health, School of

Public Health, Nanjing Medical University, Nanjing 211166, China

4 Center for Reproductive Medicine, Shandong University, Jinan 250021, China

5 National Research Center for Assisted ReproductiveMANUSCRIPT Technology and Reproductive

Genetics, Jinan 250021, China

6 The Key Laboratory of Reproductive Endocrinology, Shandong University, Ministry of

Education, Jinan 250021, China

7 State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular

Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute

of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of

Chinese Academy of Sciences, Shanghai 200031, China UNCORRECTED MANUSCRIPT

2

Abstract

Losing of ovarian functions prior to natural menopause age causes female infertility and Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

early menopause. Premature ovarian insufficiency (POI) is defined as the loss of ovarian

activity before 40 years of age. Known genetic causes account for 25% – 30% of POI

cases, demonstrating the high genetic heterogeneity of POI and the necessity for further

genetic explorations. Here we conducted genetic analyses using whole-exome sequencing

in a Chinese non-syndromic POI family with the affected mother and at least four

affected daughters. Intriguingly, a rare missense variant of BUB1B c.273A>T

(p.Gln91His) was shared by all the cases in this family. Furthermore, our replication study using targeted sequencing revealed a novel stop-gainMANUSCRIPT variant of BUB1B c.1509T>A (p.Cys503*) in one of 200 sporadic POI cases. Both heterozygous BUB1B variants were

evaluated to be deleterious by multiple in silico tools. BUB1B encodes BUBR1, a crucial

spindle assembly checkpoint component involved in cell division. BUBR1 insufficiency

may induce vulnerability to oxidative stress. Therefore, we generated a mouse model

with a loss-of-function mutant of Bub1b, and also employed D-galactose-induced aging

assays for functional investigations. Notably, Bub1b+/− female mice presented late-onset

subfertility, and they were more sensitive to oxidative stress than wild-type female UNCORRECTEDcontrols, mimicking the clinical phenotypes of POI cases MANUSCRIPT affected by deleterious BUB1B variants. Our findings in human cases and mouse models consistently suggest, for the

3

first time, that heterozygous deleterious variants of BUB1B are involved in late-onset POI

and related disorders. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Introduction

Premature ovarian insufficiency (POI), previously referred as premature ovarian failure or premature menopause, is defined as loss of ovarianMANUSCRIPT activity before the age of 40 years (1). Clinical diagnostic criteria of POI include amenorrhea/oligomenorrhea for over 4

months, and elevated serum follicle-stimulating hormone (FSH) levels (> 25 IU/L) twice

at least 4 weeks apart (2). Women with POI are often perplexed by series of associated

comorbidities like hot flush, night sweats, emotional instability and osteoporosis, which

seriously affect their well-beings (3). The prevalence of POI is approximately 1% − 2%

of women (4). Nowadays, an increasing number of women tend to have their first child

after the age of 30 years, while over 48.5 million couples worldwidely troubled by UNCORRECTEDinfertility during reproductive age (5). MANUSCRIPT POI can be induced by chemotherapy, radiotherapy, surgery, toxin, infection and

other environmental factors. However, previous evidence supported a strong genetic 4

contribution to the pathogenesis of idiopathic POI (6). With the wide use of whole-exome

sequencing (WES) and other genomic technologies, multiple pathogenic genetic variants Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

or genomic loci have been identified in POI (7). To date, approximately 25% − 30% of

POI cases have been explained by genetic alterations (8). Firthermore, previous studies

on the patients presenting relatively mild fecundity impairment (secondary amenorrhea)

in non-syndromic POI families suggested complex pathogenic mechanisms (9-11).

Herein, we conducted WES in a non-syndromic POI family and identified a

heterozygous deleterious missense variant of BUB1B (Budding Uninhibited By

Benzimidazoles 1 Homolog Beta) in the affected mother and daughters. Our subsequent replication study revealed a heterozygous stop-gainMANUSCRIPT BUB1B variant in one of 200 sporadic POI cases. To evaluate the contributions of heterozygous deleterious BUB1B

variants to POI pathogenesis, we generated a Bub1b+/− mouse model, the female mice of

which exhibited late-onset subfertile. Moreover, Bub1b+/− female mice with daily

D-galactose (D-gal) administration mimicked the phenotypes of POI cases in this study.

Our findings revealed the genetic contributions of BUB1B to human POI, and suggest

that heterozygous deleterious variants in BUB1B increase the risk of POI.

UNCORRECTEDResults MANUSCRIPT Identification of a rare deleterious variant of BUB1B in a Chinese POI family

5

In a Chinese non-syndromic POI family (family1, Fig. 1A), the affected females

presented normal menarche and puberty. However, they all presented with variable Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

severities of POI around 40 years of age. The proband (IV-3) experienced menopause in

2015 when she was 40 years old after two years of menstrual disturbance, and her

younger sister IV-4 was diagnosed with POI in 2016. Additionally, two elder sisters and

mother of the proband also suffered from menopause around age of 40 (family members

and their ages at menopause: III-1, 41; IV-1, 39; IV-2, 37). The youngest sister IV-5, who

is now in her 39 years, has been representing signs of POI such as oligomenorrhea and

intermittent menopause for two years. Trans-vaginal ultrasonography of the examined cases in this study showed normal uterus and smallMANUSCRIPT ovaries with no obvious follicles (Table 1).

The POI-affected cases of this family have normal 46, XX karyotypes. The

mitochondrial genome was also amplified in these cases, and no rare deleterious variant

was found. Additionally, -wide array-based comparative genomic

hybridization (CGH) did not identify any causative copy number variation (CNV) in the

affected cases (data not shown).

Further genetic analysis was performed using WES with a 106 × mean coverage UNCORRECTEDdepth. Given that the POI or early menopause in MANUSCRIPT this family was inherited in an autosomal-dominant manner, we analyzed rare heterozygous protein-altering (e.g.,

stop-gain, frameshift, essential splicing-site and missense) variants shared by all the 6

affected cases in this family. The variants with minor allele frequencies (MAF) < 0.1% in

the 1000 Genomes Project (http://www.1000genomes.org) or the Genome Aggregation Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Database (gnomAD; http://gnomad-old.broadinstitute.org) were retained for further

analyses. Preliminary predictions of deleterious variants were analyzed by four

bioinformatic tools: SIFT (http://sift.jcvi.org), PolyPhen-2

(http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org),

and DANN (12) before further evaluation by the American College of Medical Genetics

and Genomics (ACMG) classification (13).

Intriguingly, a rare missense variant c.273A>T (p.Gln91His) in BUB1B (GRCh37: Chr15:40462771; GenBank: NM_001211.5) was MANUSCRIPT identified to be shared by all the POI-affected cases in family 1 (Fig. 1A). Sanger sequencing was used for variant

verification (Fig. 1B). This variant has an extremely low frequency in human population:

0.02% in East Asians of the gnomAD database, and absent in the PGG.Han database that

archives deep-sequencing data (∼30–80×, 319 individuals) and low-pass sequencing data

(∼1.7×, 11670 individuals; and ∼4×, 208 individuals) of Han Chinese (14). Additionally,

the variant BUB1B c.273A>T was predicted to be deleterious by all four bioinformatic

tools (Table 2). UNCORRECTEDBUB1B encodes BUBR1, a spindle assembly checkpoint MANUSCRIPT (SAC) protein essential for normal mitotic progress as it monitors the activities of to prevent cells from

prematurely entering (15). Interestingly, a previous study on Drosophila 7

revealed that BubR1 is vital to maintain sister-chromatid cohesion during meiotic

progression and normal maintenance of synaptonemal complex in females (16). The Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

human BUB1B variant c.273A>T alters a residue (p.Gln91) locating in the important

tetratricopeptide repeat (TPR) domain (Fig. 1E), which is evolutionarily highly conserved

from mammals to yeast and worms. All these findings strongly indicate that BUB1B

variant c.273A>T might be involved in the etiology of POI in family 1.

Replication study in sporadic POI cases revealed a novel BUB1B stop-gain variant

To further investigate the genetic contribution of BUB1B to POI, we conducted a replication study on 200 Chinese sporadic cases MANUSCRIPT with POI using targeted sequencing towards the entire 5′ UTR region, all exons, splicing regions and the entire 3′ UTR region

of BUB1B. Interestingly, a BUB1B stop-gain variant c.1509T>A (p.Cys503*), located at

chr15:40492552 (GRCh37), was identified in one case (P0013, Fig. 1C). This variant has

not been reported in any public databases of human control populations, i.e., it is a novel

BUB1B variant. Sanger sequencing confirmed the paternal origin of this BUB1B

stop-gain variant (Fig. 1C and D). We also conducted WES analysis in subject P0013 and

no candidate pathogenic variants were found in any known POI-causative . UNCORRECTEDIntriguingly, the sporadic POI case P0013 also showed MANUSCRIPT a late-onset POI symptom (Table 1), which is similar to those of the cases in family 1.

8

BUB1B c.1509T>A variant locates in exon 11 of BUB1B and may lead to a

truncation of the entire C-terminal region and part of the middle region of BUBR1 (Fig. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

1E). According to previous studies, the middle region of BUBR1 is involved in

promoting stable kinetochore-microtubule attachments (17), and the C-terminal region

contains the kinase domain that is essential for the transition and organization of

microtubules into stable midzone arrays (18). These previous findings support the

pathogenic role of BUBR1 stop-gain variant p.Cys503* in case P0013.

Shortened reproductive lifespan in Bub1b+/− female mice Previous animal models provided evidence for theMANUSCRIPT potential involvement of BUBR1 in female fertility and aging. For example, complete loss of BubR1 caused embryonic

lethality in mice (19). Mouse hypomorphic Bub1b mutants with approximately 10% of

normal expression levels exhibited premature aging and infertility (20). Additionally, the

Bub1b+/GTTA mice carrying a heterozygous stop-gain mutation showed a reduced lifespan

and developed series of age-related features; however, female reproductive phenotypes

and ovarian functions were not investigated in this model (21).

To further explore the relationship between heterozygous deleterious BUB1B UNCORRECTEDvariants and the late-onset POI phenotypes observed MANUSCRIPT in our cases, we generated a Bub1b+/− mouse model through CRISPR-Cas9 technology. The mutated mice carried a

heterozygous Bub1b frameshift variant (c.255del, p.Glu89Argfs*7; GenBank: 9

NM_009773.3), which shifted the reading frame and produced a premature stop codon

(Supplementary Material, Fig. S1A and B). The genotype of Bub1b+/− mice was verified Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

by Sanger sequencing (Supplementary Material, Fig. S1C). Quantitative RT-PCR further

demonstrated that the mRNA levels of Bub1b in the Bub1b+/− mouse ovaries reduced to

approximately 55% of that in wide-type controls (Supplementary Material, Fig. S1D),

which might be induced by nonsense-mediated mRNA decay (22).

For the investigations on fertility of Bub1b+/− female mice, we mated both Bub1b+/−

and wild-type female mice to Bub1b+/− male mice when they were 8 weeks old. Their

pregnancy rates and litter sizes were continuously recorded. The mean number of live births in wild-type and Bub1b+/− female mice was nearlyMANUSCRIPT equal during the first 6 months, and the offsprings were born with a conventional Mendelian pattern of inheritance (Table

3). However, it was surprising to observe that Bub1b+/− female mice were no longer

pregnant from the 7th month, while the wild-type female controls were still fertile.

To further explore this late-onset phenotype, we compared the ovarian functions

between Bub1b+/− and wild-type female mice of 16 and 38 weeks old, respectively.

Firstly, we measured the expression levels of aging marker p21 and ovarian functional

index Amh (23, 24). Comparing to 16-week-old female mice, significantly increased p21 UNCORRECTEDexpression and decreased Amh expression were observed MANUSCRIPT in 38-week-old female mice, while notably, for which the ovarian failure degree of Bub1b+/− female mice was more

severe than that of wild-type female controls (Supplementary Material, Fig. S2A and B). 10

Consistently, the ovary sizes were obviously smaller in 38-week-old Bub1b+/− female

mice than those in wild-type female mice (Supplementary Material, Fig. S3). Zoomed-in Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

views of the ovaries showed that the primordial follicles were almost absent in the ovary

of 38-week-old Bub1b+/− female mice (Supplementary Material, Fig. S3F). All the above

experimental observations in mouse models indicated that the reproductive lifespan of

Bub1b+/− female mice was shortened when compared with the wild-type female controls,

mimicking the late-onset phenotypes of POI and early menopause in our cases with

BUB1B deleterious variants.

Sensitivity to oxidative stress in the ovary of Bub1bMANUSCRIPT+/− mice BubR1 is a potential target of reactive oxygen species (ROS), and the mice with BubR1

insufficiency showed hypersensitivity to oxidative stress (25). However, it is still

unknown whether ROS influences ovarian functions when BubR1 is insufficient.

Recently, ROS-induced pathologic damage and accumulation of advanced glycation end

(AGE) products have been widely used to investigate aging processes and ovarian

functions (26, 27), and D-gal has been used to induce POI in mouse models (28).

Therefore, we subcutaneously injected D-gal (400 mg/kg/day) daily for 8 weeks in UNCORRECTEDfemale mice. At the end of the injections, the growth MANUSCRIPT rates were obviously reduced in D-gal-treated Bub1b+/− female mice (Bub1b+/− + D-gal) when compared with

D-gal-treated wild-type female mice (Bub1b+/+ + D-gal), although no significant 11

differences was observed among groups on the day of pre-treatment or post-treatment

(Table 4). As an aging marker, the mRNA level of p21 was higher in the ovaries of D-gal Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

group than that of saline group (Fig. 2A). Instruigingly, the levels of p21 mRNA

expression and serum AGE in Bub1b+/− + D-gal group were significantly higher than

those in Bub1b+/+ + D-gal group (Fig. 2A and B). All these experimental observations in

D-gal-treated mouse models suggested that Bub1b+/− female mice were remarkably

sensitive to the stimulation of D-gal.

To evaluate the consequence of D-gal on the ovarian function of Bub1b+/− female

mice, we first examined the level of serum FSH. As shown in Fig. 2C, Bub1b+/− + D-gal group showed a significantly increased serum FSH MANUSCRIPTlevel when compared with Bub1b+/+ + D-gal group. Then we examined the mRNA level of Amh in mouse ovarian tissues.

Interestingly, Bub1b+/− + D-gal group showed a significantly lower mRNA level of Amh

than that in Bub1b+/+ + D-gal group (Fig. 2D), indicating the reduction in ovarian reserve.

To further visualize this influence, we performed follicle counting after

hematoxylin-eosin staining. As shown in Fig. 2E, although there was no obvious change

in the ovary size between two groups (i and ii), Bub1b+/− + D-gal group lost most of

primordial follicles in ovarian cortexes, and exhibited more severe necroptosis (29) in UNCORRECTEDovarian medullas than the controls (iii to vi). Consistently, MANUSCRIPT the number of developing follicles was obviously reduced in Bub1b+/− + D-gal group (Fig. 2F). Taken together,

these findings conformed that the mouse ovaries with Bub1b insufficiency were sensitive 12

to environmental stress such as ROS. Therefore, BUB1B deleterious variants and

oxidative stress may jointly lead to a late-onset subfertility in women. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Discussion

BUB1B-encoded BUBR1 is a critical SAC protein that helps maintain high fidelity of

segregation during metaphase-anaphase transition in mitotic cells (30).

Human BUBR1 is a multi-domain protein kinase, mainly containing the Lys-Glu-Asn

box (KEN), TPR, Gle2-binding sequence (GLEBS), Cdc20 binding site (IC20BD),

destruction box (D-BOX), kinetochore attachment regulatory domain (KARD) and kinase domains (Fig. 1E) (31). The BUB1B variant c.273A>TMANUSCRIPT (p.Gln91His), which was identified in our Chinese POI family, affected an evolutionarily conserved residue of

BUBR1 throughout vertebrates (Supplementary Material, Fig. S4A). This variant also

affected a connective site between two 훼-helices of TPR (Supplementary Material, Fig.

S4B). The TPR domain is essential for BUBR1 to promote and stabilize proper

-microtube attachment during chromosome segregation (32). Notably,

BUBR1 residue Gln91 plays an important role in interacting with Blinkin, a central

component for the establishment of proper kinetochore-microtubule attachments (33). UNCORRECTEDThe other BUB1B variant p.Cys503* identified inMANUSCRIPT this study is predicted to truncate more than half of BUBR1, including D-BOX, KARD and kinase domains (Fig. 1E).

Particularly, the KARD domain in BUBR1 is crucial in both mitotic and meiotic 13

processes; deletion of this domain or prevention of its phosphorylation abolishes

formation of kinetochore microtubules (17, 34). Additionally, the kinase domain of Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

BUBR1 has capacity to catalyze CENP-E phosphorylation that is important in

maintaining proper microtubule capture at kinetochores and assembling of the central

spindle during mitotic process (18, 35). Mutations in the kinase domain reduced

conformational stability of BUBR1 in humans (36). Therefore, BUB1B variant

p.Cys503* can affect the normal function of BUBR1 in ensuring high-fidelity

chromosome segregation.

BubR1/BUBR1 is also required for diverse meiotic functions, including persisting spindle assembly checkpoint activity, establishing MANUSCRIPTkinetochore-microtubule attachments, and mastering the time of meiosis I throughout yeast to mammals (37, 38). Additionally,

an early study revealed that loss-of-function mutation in Drosophila Bub1b caused

chromosome mis-division in females, probably as a consequence of the failure to

segregate properly in meiosis II (16). Furthermore, BubR1 depletion in mouse oocytes

led to a reduced capacity of sustaining in prophase I and failure of polar body extrusion

(39). Therefore, BubR1 might be essential in female reproduction through acting on both

mitosis and meiosis. Our experimental observations in this study revealed, for the first UNCORRECTEDtime, the contributions of human BUB1B variants to ovarian MANUSCRIPT disorders. Defective BUBR1 protein in the spindle checkpoint generates aneuploid daughter

cells during cell division in humans in a remarkably dosage-dependent pattern (40). 14

Clinical and genetic analyses have shown that bi-allelic mutations of BUB1B lead to

mosaic variegated aneuploidy syndrome (MIM 257300), a disease mostly associated with Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

severe intrauterine growth retardation and microcephaly (41). Mono-allelic BUB1B

mutations can lead to premature chromatid separation (PCS) (MIM 176430), a cellular

phenotype observed as separate and splayed chromatids with discernible centromeres

during metaphase (42). Notably, although people with PCS are usually innocuous, they

has been revealed in subfertile families (43, 44). Therefore, non-syndromic POI may be

associated with heterozygous BUB1B deleterious variants in humans, which has not been

reported before. A previous study showed that Bub1b+/GTTA miceMANUSCRIPT presented reduced lifespans with series of age-related features and were fertile (21). Consistently, our animal models also

revealed that Bub1b+/− female mice has normal pregnant cycles and litter sizes, but

presented shorten reproductive lifespans when compared with the wild-type female mice

under normal conditions (Table 3). Intriguingly, low expressions of BubR1 in mice led to

a reduced tolerance to oxidative stress (25), and down-regulation of BUBR1 activity in

human aortic smooth muscle cells increased the level of ROS production (45). These

previous reports indicate the possibility that human subjects with BUB1B mutations are UNCORRECTEDlikely to be susceptible to environmental factors. MANUSCRIPT Consistently, our cases carrying heterozygous BUB1B deleterious variants all

presented with late-onset non-syndromic POI. In this study, we treated the Bub1b+/− 15

mouse model with D-gal to accelerate aging by increasing oxygen free radicals.

Intriguingly, this assay revealed that Bub1b+/− female mice were more prior to aging than Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

wild-type female mice under the treatment with D-gal (Fig. 2A and B), and the ovary of

Bub1b+/− female mice appeared to be more vulnerable to the stimulation of ROS than the

wild-type controls (Fig. 2C−F). Therefore, the BUB1B-associated female subfertility

could be potentially, partially explained by the influence of environment factors, such as

oxidative stress.

BUB1B expression was reduced in the abnormally fertilized zygotes from women

with fertilization failure (46). Moreover, during natural aging process, BUBR1/BubR1 levels are remarkably decreased in the oocytes fromMANUSCRIPT women around 40 years of age and aged mice (66 weeks of age) (47, 48). Therefore, the down-regulation of human BUBR1

in ovaries caused by heterozygous deleterious variants of BUB1B is likely to induce

late-onset subfertility in women around age 40, while the perturbation in young women is

insufficient to present clinical abnormalities.

In conclusion, our findings in human cases and mouse models suggest that

heterozygous deleterious variants in BUB1B could reduce reproductive lifespans of

female carriers possibly via the interaction with environmental factors, which in turn UNCORRECTEDincrease the risk of late-onset POI. MANUSCRIPT

16

Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020 Materials and Methods

Study subjects

The Chinese family with non-syndromic POI cases was enrolled at the Obstetrics and

Gynecology Hospital of Fudan University. In this family, five daughters and their

affected mother were all of normal height (family member, height: III-1, 155cm; IV-1,

160cm; IV-2, 150cm; IV-3, 155cm; IV-4, 156cm; IV-5, 160cm). A cohort of 200 Chinese

sporadic POI patients were obtained from the Center for Reproductive Medicine of Shandong University for replication study. AllMANUSCRIPT the subjects had normal 46, XX karyotypes, and the CGG repeats in 3′ UTR of FMR1 were revealed to be within the

normal range of 27 to 31 by a FrafilEaseTM PCR Reagent Kit. Subjects with interventions

of ovarian surgery or radiotherapy or chemotherapy were excluded. This study was

approved by the institutional review boards at Fudan University and the Center for

Reproductive Medicine of Shandong University.

MtDNA sequencing UNCORRECTEDGenomic DNA samples were extracted from peripheral MANUSCRIPT blood using a Puregene Blood Core Kit B (QIAGEN, Hilden, Germany) according to standard laboratory practice. The

mitochondrial genome was amplified in all POI cases in family 1 using the primers 17

shown in Supplementary Material, Table S1. Sequencing data were aligned to the human

reference mitochondrial genome (GenBank: NC_012920) through UCSC Genome Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Browser (human genome assembly hg19). Haplogroups were assigned with Haplogrep 2

(https://haplogrep.i-med.ac.at), and variants were annotated based on the human genome

database MITOMAP (http://www.mitomap.org).

CNV analysis

CNV analysis was performed on the proband and her affected mother in family 1 using

Agilent SurePrint G3 Human 1 × 1M CGH microarray. Experimental details of the array-based CGH assay have been previously MANUSCRIPT described (49). DNA processing, microarray handling, and data processing were conducted according to the Agilent

oligonucleotide CGH protocol (version 6.3). Genomic CNVs were analyzed with Agilent

Genomic Workbench (version 7.0).

WES and targeted sequencing

For each affected case, 1.5 μg of genomic DNA was used to prepare a captured library

using a Agilent SureSelectXT Human All Exon V6 kit (Santa Clara, USA), and then UNCORRECTEDsequenced on a HiSeq X Ten platform (Illumina, SanMANUSCRIPT Diego, USA). Raw data were aligned to the human reference genome sequence (UCSC Genome Browser hg19) with

the Burrows–Wheeler Alignment tool. Variant calling was accomplished using the 18

Genome Analysis Toolkit (50), and ANNOVAR software were further used to annotate

all variants. The target sequencing towards all exons, the entire 3′ and 5′ UTR regions of Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

BUB1B (NM_001211.5; H. sapiens, GRCh37/hg19, February 2009), and approximately

150-bp flanking regions of these DNA fragments was carried out in 200 sporadic POI

patients.

Bub1b+/− mouse model

Bub1b mutated mouse model was generated through CRISPR-Cas9 technology. Briefly,

two-month-old B6D2F1 (C57BL/6 × DBA2) female mice were superovulated and mated with C57BL/6 male mice to collect zygotes. Cas9MANUSCRIPT and sgRNA were prepared as previously described (51). After Cas9/sgRNA injection, the zygotes were further cultured

in KSOM medium (Millipore, Darmstadt, Germany) at 37°C under 5% CO2 to reach the

2-cell stage, followed by embryo transfer into oviducts of pseudopregnant ICR females at

0.5 day postcoitum as previously described (51). The founders were genotyped and mated

with C57BL/6 for several generations before phenotyping of female mice. Primers for

mouse genotyping are shown in Supplementary Material, Table S1.

UNCORRECTEDD-gal-induced aging mouse model MANUSCRIPT Mutated and wild-type female mice (8 weeks old) were injected subcutaneously with

D-gal (400 mg/kg/day; G0750, Sigma-Aldrich, St. Louis, USA) or same amount of saline 19

daily for 8 weeks. The body weight and the estrous cycle were recorded every day. At the

end of treatment, all mice were sacrificed when they were in diestrus. Blood was Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

collected from the hepatic portal vein and the left ovary, left kidney, part of liver, and

uteri were immediately excised and stored at −80°C for biochemical analysis and the

right ovary was fixed in 10% neutral formalin fix solution for histological studies. All

animal procedures were performed under the ethical guidelines of the Animal Care and

Use Committee of Fudan University and Shanghai Institute of Biochemistry and Cell

Biology, Chinese Academy of Sciences.

Measurement of serum AGE and FSH levels MANUSCRIPT All blood was placed at 4°C overnight with an angle of 45 − 60 degrees. After

centrifugation at 3000 rpm at 4°C for 5 min, sera were separated and stored at −80°C

before analysis. The level of serum AGE was detected using Mouse AGEs ELISA Kit

(Zhen Ke Biotechnology, Shanghai, China) and the level of serum FSH was detected

using Mouse FSH ELISA Kit (Ji Ning Biotechnology, Shanghai, China).

mRNA expression analysis UNCORRECTEDFive left ovaries of mice in each group were collected MANUSCRIPT to extract DNA, mRNA and protein using the All Prep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany).

Total RNAs of 1 μg were immediately converted into cDNAs with HiScript II QRT 20

SuperMix (Vazyme). The cDNAs were individually diluted to 10 ng as templates for the

subsequent real-time fluorescence quantitative PCR with AceQ qPCR SYBR Green Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Master Mix (Vazyme). Mouse Gapdh was used as an internal control. Expressions of the

target genes were normalized to Gapdh, and mRNA levels were quantified according to

the 2−∆∆Ct method. The primers used for expression analysis were shown in

Supplementary Material, Table S1.

Hematoxylin-eosin staining and quantification of ovarian follicle

For histologic analysis, mouse right ovaries were fixed at room temperature for 24 h in 10% neutral formalin fix solution (Sangon Biotech, Shanghai,MANUSCRIPT China) before being dehydrated and embedded in paraffin. For microscopic analysis, consecutive sections were cut into 5

µm thick and stained with hematoxylin and eosin. Every 20th and the next sections were

chosen to calculate the total number of follicles per ovary. Ovarian sections were

photographed using Olympus IX 83 (Japan). The criteria for classifying developmental

stages of ovarian follicles were based on the standard established by Pedersen and Peters

(52). Follicles were counted if they sharply appeared in one section but not the next

section, and the total numbers of follicles at any particular developmental stage were UNCORRECTEDcalculated as previously reported (53). MANUSCRIPT

21

Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Statistical analysis

All data were plotted as a mean ± standard error (n = 5), and statistical analysis was

performed using GraphPad Prism version 6.02 (La Jolla, USA). Between-group

differences were assessed by an unpaired two-tailed Student's t-test. A P value under 0.05

was considered statistically significant.

MANUSCRIPT

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22

Supplementary Material

Supplementary Material is available at HMG online. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Acknowledgments

We gratefully acknowledge the patients for participating and supporting this study. This

work was supported by National Key Research and Development Program of China

(2017YFC1001100), National Natural Science Foundation of China (31625015 and

31521003), Excellent Medical Personnel Training Project of Shanghai Municipal Health

Commission (2017BR035), Shanghai Medical Center of Key Programs for Female Reproductive Diseases (2017ZZ01016), ShanghaiMANUSCRIPT Municipal Science and Technology Major Project (2017SHZDZX01), and Science and Technology Major Project of Inner

Mongolia Autonomous Region of China (zdzx2018065) to the State Key Laboratory of

Reproductive Regulation and Breeding of Grassland Livestock.

Conflict of Interest statement. None declared.

UNCORRECTED MANUSCRIPT

23

References

1. Anasti, J.N. (1998) Premature ovarian failure: an update. Fertil. Steril., 70, 1-15. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

2. The ESHRE Guideline Group on POI, Webber, L., Davies, M., Anderson, R., Bartlett,

J., Braat, D., Cartwright, B., Cifkova, R., de Muinck Keizer-Schrama, S., Hogervorst,

E. et al. (2016) ESHRE Guideline: management of women with premature ovarian

insufficiency. Hum. Reprod., 31, 926-937.

3. Webber, L., Anderson, R.A., Davies, M., Janse, F. and Vermeulen, N. (2017) HRT for

women with premature ovarian insufficiency: a comprehensive review. Hum. Reprod.

Open, 2017, hox007. 4. Luborsky, J.L., Meyer, P., Sowers, M.F., GoldMANUSCRIPT, E.B. and Santoro, N. (2003) Premature menopause in a multi-ethnic population study of the menopause transition.

Hum. Reprod., 18, 199-206.

5. Christou-Kent, M., Dhellemmes, M., Lambert, E., Ray, P.F. and Arnoult, C. (2020)

Diversity of RNA-Binding Proteins Modulating Post-Transcriptional Regulation of

Protein Expression in the Maturing Mammalian Oocyte. Cells, 9, 662.

6. Qin, Y., Jiao, X., Simpson, J.L. and Chen, Z.J. (2015) Genetics of primary ovarian

insufficiency: new developments and opportunities. Hum. Reprod. Update, 21, UNCORRECTED787-808. MANUSCRIPT 7. Jiao, X., Ke, H., Qin, Y. and Chen, Z.J. (2018) Molecular Genetics of Premature

Ovarian Insufficiency. Trends Endocrinol. Metab., 29, 795-807. 24

8. Rossetti, R., Ferrari, I., Bonomi, M. and Persani, L. (2017) Genetics of primary

ovarian insufficiency. Clin. Genet., 91, 183-198. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

9. Qin, Y., Guo, T., Li, G., Tang, T.S., Zhao, S., Jiao, X., Gong, J., Gao, F., Guo, C.,

Simpson, J.L. et al. (2015) CSB-PGBD3 Mutations Cause Premature Ovarian Failure.

PLoS Genet., 11, e1005419.

10. Zhang, D., Liu, Y., Zhang, Z., Lv, P., Liu, Y., Li, J., Wu, Y., Zhang, R., Huang, Y., Xu,

G. et al. (2018) Basonuclin 1 deficiency is a cause of primary ovarian insufficiency.

Hum. Mol. Genet., 27, 3787-3800.

11. Delcour, C., Amazit, L., Patino, L.C., Magnin, F., Fagart, J., Delemer, B., Young, J., Laissue, P., Binart, N. and Beau, I. (2019) MANUSCRIPT ATG7 and ATG9A loss-of-function variants trigger autophagy impairment and ovarian failure. Genet. Med., 21, 930-938.

12. Quang, D., Chen, Y. and Xie, X. (2015) DANN: a deep learning approach for

annotating the pathogenicity of genetic variants. Bioinformatics, 31, 761-763.

13. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W.,

Hegde, M., Lyon, E., Spector, E. et al. (2015) Standards and guidelines for the

interpretation of sequence variants: a joint consensus recommendation of the

American College of Medical Genetics and Genomics and the Association for UNCORRECTEDMolecular Pathology. Genet. Med., 17, 405-424. MANUSCRIPT 14. Gao, Y., Zhang, C., Yuan, L., Ling, Y., Wang, X., Liu, C., Pan, Y., Zhang, X., Ma, X.,

Wang, Y. et al. (2020) PGG.Han: the Han Chinese genome database and analysis 25

platform. Nucleic Acids Res., 48, D971-D976.

15. Chan, G.K., Jablonski, S.A., Sudakin, V., Hittle, J.C. and Yen, T.J. (1999) Human Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at

kinetochores and binds the cyclosome/APC. J. Cell Biol., 146, 941-954.

16. Malmanche, N., Owen, S., Gegick, S., Steffensen, S., Tomkiel, J.E. and Sunkel, C.E.

(2007) Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and

maintenance of synaptonemal complex. Curr. Biol., 17, 1489-1497.

17. Suijkerbuijk, S.J., Vleugel, M., Teixeira, A. and Kops, G.J. (2012) Integration of

kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments. Dev. CellMANUSCRIPT, 23, 745-755. 18. Huang, Y., Lin, L., Liu, X., Ye, S., Yao, P.Y., Wang, W., Yang, F., Gao, X., Li, J.,

Zhang, Y. et al. (2019) BubR1 phosphorylates CENP-E as a switch enabling the

transition from lateral association to end-on capture of spindle microtubules. Cell

Res., 29, 562-578.

19. Wang, Q., Liu, T., Fang, Y., Xie, S., Huang, X., Mahmood, R., Ramaswamy, G.,

Sakamoto, K.M., Darzynkiewicz, Z., Xu, M. et al. (2004) BUBR1 deficiency results

in abnormal megakaryopoiesis. Blood, 103, 1278-1285. UNCORRECTED20. Baker, D.J., Jeganathan, K.B., Cameron, J.D., Thompson, MANUSCRIPT M., Juneja, S., Kopecka, A., Kumar, R., Jenkins, R.B., de Groen, P.C., Roche, P. et al. (2004) BubR1 insufficiency

causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genet., 26

36, 744-749.

21. Wijshake, T., Malureanu, L.A., Baker, D.J., Jeganathan, K.B., van de Sluis, B. and Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

van Deursen, J.M. (2012) Reduced life- and healthspan in mice carrying a

mono-allelic BubR1 MVA mutation. PLoS Genet., 8, e1003138.

22. Baker, K.E. and Parker, R. (2004) Nonsense-mediated mRNA decay: terminating

erroneous expression. Curr. Opin. Cell Biol., 16, 293-299.

23. Tran, N.D., Cedars, M.I. and Rosen, M.P. (2011) The role of anti-mullerian hormone

(AMH) in assessing ovarian reserve. J. Clin. Endocrinol. Metab., 96, 3609-3614.

24. Aravinthan, A. (2015) Cellular senescence: a hitchhiker's guide. Hum. Cell, 28, 51-64. MANUSCRIPT 25. Matsuda, D., Matsumoto, T., Honma, K., Ikawa-Yoshida, A., Onimaru, M., Furuyama,

T., Nakatsu, Y., Tsuzuki, T. and Maehara, Y. (2016) BUBR1 Insufficiency in Mice

Increases their Sensitivity to Oxidative Stress. In Vivo, 30, 769-776.

26. Zhao, H., Li, J., Zhao, J., Chen, Y., Ren, C. and Chen, Y. (2018) Antioxidant effects

of compound walnut oil capsule in mice aging model induced by D-galactose. Food

Nutr. Res., 62.

27. Wang, J.L., Liu, B., Zhang, C., Wang, X.M., Zhen, D., Huang, X.M., Chen, W. and UNCORRECTEDGao, J.M. (2019) Effects of icariin on ovarian function MANUSCRIPT in d-galactose-induced aging mice. Theriogenology, 125, 157-167.

28. Yan, Z., Dai, Y., Fu, H., Zheng, Y., Bao, D., Yin, Y., Chen, Q., Nie, X., Hao, Q., Hou, 27

D. et al. (2018) Curcumin exerts a protective effect against premature ovarian failure

in mice. J. Mol. Endocrinol., 60, 261-271. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

29. Chaudhary, G.R., Yadav, P.K., Yadav, A.K., Tiwari, M., Gupta, A., Sharma, A.,

Pandey, A.N., Pandey, A.K. and Chaube, S.K. (2019) Necroptosis in stressed ovary. J.

Biomed. Sci., 26, 11.

30. Cleveland, D.W., Mao, Y.H. and Sullivan, K.F. (2003) Centromeres and kinetochores:

From epigenetics to mitotic checkpoint signaling. Cell, 112, 407-421.

31. Sewart, K. and Hauf, S. (2017) Different Functionality of Cdc20 Binding Sites within

the Mitotic Checkpoint Complex. Curr. Biol., 27, 1213-1220. 32. Alfieri, C., Chang, L., Zhang, Z., Yang, J., Maslen,MANUSCRIPT S., Skehel, M. and Barford, D. (2016) Molecular basis of APC/C regulation by the spindle assembly checkpoint.

Nature, 536, 431-436.

33. Bolanos-Garcia, V.M., Lischetti, T., Matak-Vinkovic, D., Cota, E., Simpson, P.J.,

Chirgadze, D.Y., Spring, D.R., Robinson, C.V., Nilsson, J. and Blundell, T.L. (2011)

Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for

kinetochore-mitotic checkpoint regulation via an unanticipated binding Site.

Structure, 19, 1691-1700. UNCORRECTED34. Yoshida, S., Kaido, M. and Kitajima, T.S. (2015) MANUSCRIPT Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes. Dev. Cell, 33,

589-602. 28

35. Mao, Y.H., Abrieu, A. and Cleveland, D.W. (2003) Activating and silencing the

mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1. Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Cell, 114, 87-98.

36. Suijkerbuijk, S.J., van Dam, T.J., Karagoz, G.E., von Castelmur, E., Hubner, N.C.,

Duarte, A.M., Vleugel, M., Perrakis, A., Rudiger, S.G., Snel, B. et al. (2012) The

vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase. Dev. Cell,

22, 1321-1329.

37. Touati, S.A., Buffin, E., Cladiere, D., Hached, K., Rachez, C., van Deursen, J.M. and

Wassmann, K. (2015) Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest. Nat. Commun.MANUSCRIPT, 6, 6946. 38. Karess, R.E., Wassmann, K. and Rahmani, Z. (2013) New insights into the role of

BubR1 in mitosis and beyond. Int. Rev. Cell Mol. Biol., 306, 223-273.

39. Homer, H., Gui, L. and Carroll, J. (2009) A spindle assembly checkpoint protein

functions in prophase I arrest and prometaphase progression. Science, 326, 991-994.

40. Bohers, E., Sarafan-Vasseur, N., Drouet, A., Paresy, M., Latouche, J.B., Flaman, J.M.,

Sesboue, R. and Frebourg, T. (2008) Gradual reduction of BUBR1 protein levels

results in premature sister-chromatid separation then in aneuploidy. Hum. Genet., 124, UNCORRECTED473-478. MANUSCRIPT 41. Hanks, S., Coleman, K., Reid, S., Plaja, A., Firth, H., Fitzpatrick, D., Kidd, A.,

Mehes, K., Nash, R., Robin, N. et al. (2004) Constitutional aneuploidy and cancer 29

predisposition caused by biallelic mutations in BUB1B. Nat. Genet., 36, 1159-1161.

42. Garcia-Castillo, H., Vasquez-Velasquez, A.I., Rivera, H. and Barros-Nunez, P. (2008) Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

Clinical and genetic heterogeneity in patients with mosaic variegated aneuploidy:

delineation of clinical subtypes. Am. J. Med. Genet. A, 146A, 1687-1695.

43. Rudd, N.L., Teshima, I.E., Martin, R.H., Sisken, J.E. and Weksberg, R. (1983) A

dominantly inherited cytogenetic anomaly: a possible cell division mutant. Hum.

Genet., 65, 117-121.

44. Gabarron, J., Jimenez, A. and Glover, G. (1986) Premature centromere division

dominantly inherited in a subfertile family. Cytogenet. Cell Genet., 43, 69-71. 45. Guntani, A., Matsumoto, T., Kyuragi, R., Iwasa,MANUSCRIPT K., Onohara, T., Itoh, H., Katusic, Z.S. and Maehara, Y. (2011) Reduced proliferation of aged human vascular smooth

muscle cells--role of oxygen-derived free radicals and BubR1 expression. J. Surg.

Res., 170, 143-149.

46. Suo, L., Zhou, Y.X., Jia, L.L., Wu, H.B., Zheng, J., Lyu, Q.F., Sun, L.H., Sun, H. and

Kuang, Y.P. (2018) Transcriptome profiling of human oocytes experiencing recurrent

total fertilization failure. Sci. Rep., 8, 17890.

47. Pan, H., Ma, P., Zhu, W. and Schultz, R.M. (2008) Age-associated increase in UNCORRECTEDaneuploidy and changes in gene expression in mouse MANUSCRIPT eggs. Dev. Biol., 316, 397-407. 48. Riris, S., Webster, P. and Homer, H. (2014) Digital multiplexed mRNA analysis of

functionally important genes in single human oocytes and correlation of changes in 30

transcript levels with oocyte protein expression. Fertil. Steril., 101, 857-864.

49. Boone, P.M., Bacino, C.A., Shaw, C.A., Eng, P.A., Hixson, P.M., Pursley, A.N., Kang, Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

S.H., Yang, Y., Wiszniewska, J., Nowakowska, B.A. et al. (2010) Detection of

clinically relevant exonic copy-number changes by array CGH. Hum. Mutat., 31,

1326-1342.

50. McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A.,

Garimella, K., Altshuler, D., Gabriel, S., Daly, M. et al. (2010) The Genome Analysis

Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing

data. Genome Res., 20, 1297-1303. 51. Wang, L., Li, M.Y., Qu, C., Miao, W.Y., Yin,MANUSCRIPT Q., Liao, J., Cao, H.T., Huang, M., Wang, K., Zuo, E. et al. (2017) CRISPR-Cas9-mediated genome editing in one

blastomere of two-cell embryos reveals a novel Tet3 function in regulating

neocortical development. Cell Res., 27, 815-829.

52. Pedersen, T. and Peters, H. (1968) Proposal for a classification of oocytes and

follicles in the mouse ovary. J. Reprod. Fertil., 17, 555-557.

53. Myers, M., Britt, K.L., Wreford, N.G., Ebling, F.J. and Kerr, J.B. (2004) Methods for

quantifying follicular numbers within the mouse ovary. Reproduction, 127, 569-580. UNCORRECTED MANUSCRIPT

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Legends to Figures Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

MANUSCRIPT

Figure 1. Identification of rare deleterious BUB1B variants in a Chinese pedigree and a UNCORRECTEDsporadic case with POI. (A) A pedigree (family 1) affected MANUSCRIPT by a rare deleterious variant of BUB1B c.273A>T (p.Gln91His). The proband is indicated by a black arrow. Black

solid circles indicate affected female cases with POI. The grey solid circle indicates a 32

female case with incipient symptoms of POI. The clinical phenotype of subject II-3

(indicated by a question mark) was unavailable. WT, wild-type. (B) Sanger sequencing Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

confirmed the heterozygous BUB1B c.273A>T variant. The mutated position and

nucleotide was indicated by a red arrow and a red box, respectively. (C) A novel

stop-gain variant of BUB1B c.1509T>A (p.Cys503*) was identified in a sporadic female

case P0013. (D) Sanger sequencing confirmed the heterozygous BUB1B c.1509T>A

variant. (E) A schematic diagram depicts the isoforms of BUB1B-encoded BUBR1

protein in mammalians (31). The locations of two deleterious BUB1B variants of this

study are indicated by black arrows. The Lys-Glu-Asn box (KEN), tetratricopeptide repeat (TPR), Gle2-binding sequence (GLEBS),MANUSCRIPT Cdc20 binding site (IC20BD), destruction box (D-BOX), kinetochore attachment regulatory domain (KARD) and kinase

domains are indicated. The chromosomal coordinates of BUB1B c.273A>T and

c.1509T>A in GRCh37 are chr15:40462771 and chr15:40492552, respectively. The

NCBI reference sequence numbers for human BUB1B and BUBR1 are NM_001211.5

and NP_001202.4, respectively.

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Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

MANUSCRIPT

Figure 2. Bub1b+/− female mice exhibited sensitivity to D-gal stimulation. Biochemical UNCORRECTEDmarkers in the serum and pathological changes in the ovary were investigated. (A) UNCORRECTEDEffects of D-gal on AGE in the serum of Bub1b+/− female MANUSCRIPT mice. (B) Effects of D-gal on

p21 expression in the ovary of wild-type and Bub1b+/− female mice. (C) Effects of D-gal

34

on FSH in the serum of Bub1b+/− female mice. (D) Effects of D-gal on Amh expression of

in the ovary of Bub1b+/− female mice. (E) Representative staining of the ovaries in each Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

group (i and ii). The ovarian cortexes are marked with black boxes, and the ovarian

medullas are marked with red boxes. (iii and iv) Zoomed-in views of the ovarian cortexes.

The red arrowhead indicates a primordial follicle. (v and vi) Zoomed-in views of the

ovarian medullas. Black asterisks represent atretic follicles, and black arrows indicate

apoptosis or tissue necrosis in the ovary. (F) Follicle ratio counting was performed in the

wild-type and Bub1b+/− female mice treated with D-gal. Follicle ratio refers to the

percentage of the follicle number at a specific developmental stage to the total follicle number. The NCBI reference sequence number for MANUSCRIPTmouse Bub1b is NM_009773.3. Data were presented as mean ± SD of three independent experiments. Statistical significance:

* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant. AGE, advanced glycation end

products; FSH, follicle-stimulating hormone.

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Table 1. Clinical characteristics of the POI subjects affected by rare BUB1B variants Family 1 Sporadic Case Characteristic Proband (IV-3) Sister (IV-4) P0013 First menses (years old) 14 13 14 Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020 Age of POI (years old) 40 40 36 Weight (kg) 59.0 58.5 54.0 Height (cm) 155 156 157 FSH (IU/L) 68.61 45.08 48.12 LH (IU/L) 31.57 30.40 32.06 PRL (ng/ml) 4.93 7.55 5.54 E2 (pg/ml) 28 47 22 T (ng/ml) 0.44 – – TSH (µIU/ml) 0.34 – – Ultrasound imaging Small ovary Small ovaries – Ovary size (right/left) (mm) – /18*12 13*7/19*14 – Follicles per ovary (right/left) (n) Not obvious Not obvious 0/0 FMR1 CGG repeats (n) 27/31 26/27 30/30 Familial case Yes MANUSCRIPTYes No E2 estradiol, FSH follicle-stimulating hormone, LH luteinizing hormone, PRL prolactin, T testosterone, TSH thyroid stimulating hormone.

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Table 2. Overview of rare deleterious BUB1B variants observed in female subjects with POI Minor allele frequencyb Functional predictionc cDNA Protein Subject gnomAD MutationT changea change 1KGP PGG.Han SIFT PolyPhen-2 ACMG DANN East Asians aster Probably Probably Uncertain Family 1 c.273A>Td p.Gln91His 0 2.00E-04 0 Deleterious 0.996 damaging damaging Significance Disease P0013 c.1509T>Ae p.Cys503* 0 0 0 N/A Damaging Pathogenic 0.992 causing N/A not applicable. aThe GenBank accession number of BUB1B is NM_001211.5. bAllele frequencies were estimated according to the 1000 Genomes Project (1KGP), gnomAD and PGG.Han databases. cMutation assessment using the SIFT, PolyPhen-2, MutationTaster, ACMG and DANN tools. High DANN scores suggest that the variants are likely to have deleterious effects. The DANN cutoff is usually set at 0.93. MANUSCRIPT dThe position of the variant BUB1B c.273A>T in GRCh37 is Chr15: 40462771. eThe position of the variant BUB1B c.1509T>A in GRCh37 is Chr15: 40492552.

37 UNCORRECTED MANUSCRIPT Table 3. Fertility of Bub1b+/− female mice and genotype analysis of live births Genotype of Genotype of Days between Number (%) of live births male mice female mice pregnancies +/+ +/− −/− Bub1b+/− a WT 23 ± 2.3 31 (52) 29 (48) N/A Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020 Bub1b+/− Bub1b+/‒ 24.2 ± 2.6ns 21 (36) 37 (64) 0 (0) b N/A not applicable. ns not significant versus wild-type (WT) female mice. aThe GenBank accession number of Bub1b is NM_009773.3. bBub1b−/− mice are embryogenically lethal.

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Table 4. Body weight changes in wild-type and Bub1b+/− female mice on the first and last day of injection Group Pre-treatment (g) Post-treatment (g) Growth rate (%) Bub1b+/+ + Saline 18.66 ± 0.90 22.54 ± 0.73 20.88 ± 3.29 Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020 Bub1b+/+ + D-gal 18.42 ± 1.28 21.65 ± 1.24 17.66 ± 3.18ns Bub1b+/− + D-gal 18.50 ± 0.83 21.10 ± 0.87 14.10 ± 0.92** The body weight change in C57BL/6 female mice between the day of pre-treatment and post-treatment (mean ± SD). ns not significant (versus Bub1b+/+ + Saline group), ** P < 0.01 (versus Bub1b+/+ + D-gal group), n = 5 in each group.

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Abbreviations

1KGP: 1000 Genomes Project Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

ACMG: American College of Medical Genetics and Genomics

AGE: advanced glycation end

BUB1B: Budding Uninhibited By Benzimidazoles 1 Homolog Beta

CGH: comparative genomic hybridization

CNV: copy number variation

D-BOX: destruction box

D-gal: D-galactose E2: estradiol MANUSCRIPT FSH: follicle-stimulating hormone

GLEBS: Gle2-binding sequence

gnomAD: genome Aggregation Database

IC20BD: Cdc20 binding site

KARD: kinetochore attachment regulatory domain

KEN: Lys-Glu-Asn box

LH: luteinizing hormone UNCORRECTEDMAF: minor allele frequencies MANUSCRIPT PCS: premature chromatid separation

POI: premature ovarian insufficiency 40

PRL: prolactin

ROS: reactive oxygen species Downloaded from https://academic.oup.com/hmg/article-abstract/doi/10.1093/hmg/ddaa153/5873246 by Goteborgs Universitet user on 28 July 2020

SAC: spindle assembly checkpoint

T: testosterone

TPR: tetratricopeptide repeat

TSH: thyroid stimulating hormone

WES: whole-exome sequencing

WT: wild-type

MANUSCRIPT

UNCORRECTED MANUSCRIPT

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