MCM9 is associated with germline predisposition to early-onset cancer—clinical evidence

Mutated MCM9 has been associated with primary ovarian insufficiency. Although MCM9 plays a role in genome maintenance and has been reported as a candidate gene in a few patients with inherited colorectal cancer (CRC), it has not been clearly established as a cancer predisposition gene. We re-evaluated family members with MCM9-associated fertility problems. The heterozygote parents had a few colonic polys. Three siblings had early-onset cancer: one had metastatic cervical cancer and two had early-onset CRC. Moreover, a review of the literature on MCM9 carriers revealed that of nine bi-allelic carriers reported, eight had early-onset cancer. We provide clinical evidence for MCM9 as a cancer germline predisposition gene associated with early-onset cancer and polyposis, mainly in a recessive inheritance pattern. These observations, coupled with the phenotype in knockout mice, suggest that diagnostic testing for polyposis, CRC, and infertility should include MCM9 analysis. Early screening protocols may be beneficial for carriers.


INTRODUCTION
Identification of high-risk polyposis and colorectal cancer (CRC) predisposition genes is imperative in order to prevent CRC in carriers and their relatives. The estimated heritability of CRC ranges from 16 to 35% 1 . Genetic predisposition to CRC has been classically associated with germline mutations or, in nonpolyposis cases, epimutations in the DNA mismatch repair (MMR) genes. and NTHL1 (NAP MIM #616415) and the MMR genes (CMMRD MIM #276300). Recently, APC mosaicism was added as a relatively common cause of polyposis in isolated cases 2 . However, only 4-8% of all patients with CRC are presently found to carry germline pathogenic variants in one of the known highpenetrance genes 3 , and overall, a considerable proportion of familial aggregation of CRC remains unexplained 3 . Notably, most cancer predisposition conditions described today are nonsyndromic.
The minichromosome maintenance 9 homologous recombination repair factor gene MCM9 (MIM #610098) functions together with MCM8 in a helicase hexameric complex involved in genome maintenance, meiotic recombination, and repair of double-strand breaks via homologous recombination 4 . The MCM8/9 complex is required for proper localization of the MRN (Mre11-Rad50-Nbs1) complex to DNA double-strand breaks and for DNA resection to enable homologous recombination 5,6 . Cells lacking MCM8/9 are viable but highly sensitive to inter-strand cross-linking-inducing agents, and they exhibit more chromosome aberrations in the presence of mitomycin C than wild-type cells 7 . MCM9 is apparently also required for mammalian MMR 8 as it co-immunoprecipitates with MCM8, MSH2, and MLH1, and its deficiency has been linked to microsatellite instability (MSI) and MMR alterations 8 .
MCM9 knockout mice are viable but undergo embryonic germ cell depletion with production of a reduced quantity of spermatozoa due to defective stem-cell renewal. MCM9 impairment induces meiotic recombination defects and oocyte degeneration 4 . Female mice are sterile, with ovaries that are devoid of oocytes. MCM9 knockout mice were found to be at high risk of hepatocellular carcinomas and ovarian tumors 4,9 . We and others have described several families in which bi-allelic mutations in MCM8 or MCM9 were associated with primary ovarian insufficiency (POI) 7,10-15 . Early-onset polyposis and CRC were reported in only one of them 10 (Table 1). Very recently, Golubicki et al. 16 reported two patients with CRC who had biallelic MCM9 mutations; one also had POI (Table 1). Nevertheless, MCM9 has not been defined as a cancer predisposition gene 17,18 and is not tested in targeted panels.
Prompted by these findings, we re-evaluated a family with an MCM9 pathogenic variant, previously evaluated at our medical center for POI, and revealed that three siblings had early-onset cancer.

RESULTS AND DISCUSSION
The clinical and pathological data of the family described in the present study are shown in Table 1 and Fig. 1.
The parents (III-2, III-3) are first paternal cousins of Middle Eastern Arabic origin. They have five children, three daughters (IV-1, IV-2, IV-4) and two sons (IV-3, IV-5). Two daughters were diagnosed with POI around age 15 years. Karyotype was normal (46, XX), and there was no premutation in the FMR1 gene. Both were infertile. One of the two sons was diagnosed with severe oligoteratoasthenozoospermia (OTA) at age 35 years. Whole-exome sequencing detected the c.1483G > T [p.E495*] variant in the MCM9 gene 14 .  Re-evaluation of the family for the present study revealed that three of the siblings had cancer and the parents had a few colonic polyps. Current clinical and pathological data are shown in Fig. 1 and Table 1.
Patient IV-1, homozygous for the MCM9 variant, is currently 40 years old. She was diagnosed at age 15 years with primary amenorrhea secondary to hypogonadism, and was treated with hormonal replacement. At age 31 years, colonoscopy performed to evaluate rectal bleeding revealed a villous adenoma with highgrade dysplasia and foci of well-differentiated adenocarcinoma.
Patient IV-2, homozygous for the MCM9 variant, was, like her sister, diagnosed with primary amenorrhea around age 15 years. At age 37 years, she was found to have metastatic clear cell carcinoma of the cervix. PET-CT scan showed massive involvement. She died 2 months after diagnosis.
Patient IV-3, heterozygous brother, was diagnosed with OTA. Karyotype, Y micro-deletions, and CFTR gene testing were normal. At age 35 years, he presented with rectal bleeding and weight loss and was ultimately diagnosed with moderately differentiated colon adenocarcinoma with liver involvement. Findings on immunohistochemistry for the four MMR proteins were normal. Surveillance revealed recurrent polyps.
Patient IV-4, heterozygous for the MCM9 variant, is healthy at age 42 years. She has four healthy children and did not report any fertility problems. She has not undergone colonoscopy.
Patient IV-5 has never gone for genetic counseling but has recurrent polyps.
Both parents (III-2 and III-3) are cancer free at age 69 and 74 years. They did not report fertility problems. Colonoscopy performed in the mother at age 68 years for evaluation of rectal bleeding revealed three polyps. The father had one tubular adenoma at age 66 years and one tubulo-villous adenoma with low-grade dysplasia and one hyperplastic polyp at age 68 years. The family history was remarkable for early cancer in three family members, one with suspected medulloblastoma and one reported to have died of an "abdominal tumor" (Fig. 1).

Updated review of published cases
Our review of previous publications on MCM9 carriers from families with cancer (Table 1) yielded 11 additional patients. Of the nine bi-allelic carriers, eight reported fertility problems and eight had early-onset cancer, including six with CRC (ages 31-55 years; mean 41). Three of the four tumors analyzed for MSI were stable. Multiple polyps were documented in four bi-allelic carriers, and gynecologic cancer was reported in two bi-allelic women (ages 14 and 37 years). Among the seven mono-allelic carriers, two (ages 35 and 83 years) had CRC and six (ages 39-83 years) had a few colonic polyps.
This report complements our previous description 14 and, together with the work of Golubicki et al. 16 , supports the recognition of MCM9 as a cancer predisposition gene associated with early-onset cancer, mainly CRC and gynecologic tumors, in addition to infertility. The mode of inheritance is autosomal recessive, with some evidence of a milder mono-allelic, phenotype. The co-occurrence of infertility and early-onset cancer, both conditions associated with mutated MCM9, in the heterozygote brother (Patient IV-3) is intriguing because it may indicate substantial malfunction of MCM9 also in the mono-allelic state. A heterozygous variant in MCM9 leading to Y511* was reported in ClinVar in a patient with POI (ClinVar 156586), and in three MCM heterozygotes with POI 15 . Moreover, MCM8-9 heterozygous variants were identified in 16/131 Dutch cases of polyposis and familial cancer 16 , and CRC and a few polyps at a later age were reported in heterozygote parents 10 . Thus, mono-allelic carriers may be at increased risk, with low penetrance or late onset. The effect of MCM9 mutations might be dosage-dependent whereas bi-allelic variations lead to more severe defects.
However, given the relative high frequency of both infertility and cancer in the general population, incidental irrelevant MCM9 heterozygous findings could be argued.
Of the four tumors analyzed for MSI, three were stable. MCM9 is required for mammalian MMR 18 . MCM8/9 deficiency impairs HRmediated DNA repair, and deficient cells are particularly sensitive to DNA cross-linking agents and inhibition of poly-ADP ribose polymerase 19,20 . This might vindicate both MSI signatures.
To conclude, we provide clinical supporting evidence for MCM9 as a cancer germline predisposition gene associated with earlyonset CRC and polyposis, mainly in a recessive inheritance pattern. The new data presented here, by itself not sufficient to conclude that MCM9 is causal for either polyposis or CRC, however, these observations, coupled with the phenotype in knockout mice 10,16 , suggest that early screening protocols may be beneficial for carriers. Diagnostic testing for polyposis, CRC, and infertility should include analysis of MCM9. Additional studies in other cohorts including patients affected with familial cancer, colonic polyposis, and/or fertility problems could help to further define the role of MCM9 in predisposing to these pathologies.

METHODS Background
In 2016, a report from the HaEmek Medical Center in Israel, by Fauchereau et al. 14 described the genetic findings in a consanguineous family with POI. Whole-exome sequencing of two patients and a non-affected sister yielded a homozygous causal variant in the MCM9 gene, c.1483G > T [p.E495*], apparently leading to loss of a functional protein.

Patient re-evaluation
For the present study, to investigate the possible association among mutant MCM9, infertility, and a predisposition to cancer, we retrieved the clinical and pathological data from the files of the patients evaluated by Fauchereau et al. 14 at our medical center 5 years previously. We searched for additional morbidities and assessed the clinical, oncologic, and pathologic findings. All patients described in this manuscript provided written informed consent. The study was approved by the HaEmek Medical Center Research Ethics Committee.

Literature review
Public resources such as Pubmed and ClinVar were searched for publications on MCM9 carriers using the keyword "MCM9". Data on cancer and colonic polyps among MCM9 carriers and/or family members were retrieved and analyzed.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.