Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages

Article Outline

• Abstract
• Introduction
• Materials and methods
  • Patient selection
  • Polymerase chain reaction, sequencing and restriction reaction
• Results
• Discussion
• Acknowledgements
• Appendix A. Supplementary data
• References
• Copyright

Abstract 

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

Our primary aim was to gain more insight into maturation arrest of spermatogenesis (MA). Patients with MA are infertile due to the absence of sperm cells in their ejaculates. A biopsy of the testis of these patients showed that the germ cells stopped developing at a level called meiosis. During meiosis the chromosomes are divided into two daughter cells. We looked for the presence of alterations in genes essential for meiosis. We also investigated the possibility that modifications in human meiosis genes are causing a milder effect and that in such cases meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution. These abnormal germ cells might cause recurrent miscarriages after fertilization. Several changes in three investigated genes were detected. Most of these alterations were also observed in a control group consisting of men with normal semen parameters or men who fathered at least one child. Therefore, these modifications are most likely not the cause of the problems in the patients. However, one change present in an evolutionary important functional domain of the SYCP3 gene was detected in the male partner of a couple suffering from recurrent miscarriages and was absent in >200 controls. Despite our small patient groups, these preliminary results confirm our assumption that abnormalities in meiosis genes might be involved in the formation of gametes with abnormal chromosomal constitution. Yet, further research is necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

Keywords: DNMT3L, maturation arrest, MSH4, mutations, recurrent miscarriages, SYCP3

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Introduction 

Non-obstructive azoospermia is characterized by the complete absence of germ cells in the patients’ ejaculate due to absent or incomplete spermatogenesis. One of the potential phenotypes observed at the testicular level is maturation arrest of spermatogenesis. In this case, spermatogenic cells stop maturing, mostly at the level of primary spermatocytes, which are undergoing meiosis. It is therefore speculated that a defect in one of the genes involved in meiosis might be the underlying cause of the fertility problems of these patients. Knock-out mouse models have indeed shown that deletions of genes essential for meiosis, such as SYCP3, are causing a complete block of spermatogenesis (Yuan et al., 2000). However, until today, studies in humans were often not successful in showing such a relationship. Defects in meiosis genes at the DNA level have been explored by looking at the presence of mutations in these genes (for SYCP3: Martínez et al., 2007, Miyamoto et al., 2003, Stouffs et al., 2005). Mostly these studies were disappointing. The lack of a relationship between the genes analysed and maturation arrest of spermatogenesis can be explained by several hypotheses. First of all, the number of meiosis genes investigated so far remains small. Yet, other still uncharacterized genes might be essential for initiation, progression or completion of meiosis. Finally, mutations in meiosis genes might be rare and thus large patient groups need to be analysed. In order to overcome these pitfalls, this study analysed a larger group of patients with maturation arrest of spermatogenesis and investigated three genes that are involved in meiosis.

On the other hand, mutations in human meiosis genes are potentially causing a milder phenotype than predicted from mouse studies. Most mutations detected in human meiosis genes were heterozygous changes, whereas mouse constructs were always homozygous changes (deletions). Potentially, these heterozygous alterations are either not, or only mildly, affecting meiosis. Alternatively, two or more heterozygous mutations, potentially in different genes, might act together to cause the phenotype. Furthermore, single nucleotide substitutions (SNP) might cause a less severe phenotype than expected from mouse studies in which mostly the major part of the gene was removed. This study postulated that, in such cases, meiosis might be only slightly disturbed and thus could potentially be completed with the production of mature germ cells. However, the chromosomal constitution of these germ cells might be abnormal. After fertilization by these germ cells, non-implantation or miscarriages might be the consequence. So far, this hypothesis remains largely unstudied. For these reasons, a second part of the study was aimed at identifying mutations in meiosis genes in couples with recurrent miscarriages. This study searched for mutations in three genes, SYCP3, MSH4 and DNMT3L. All genes were selected based on observations from mice studies.

SYCP3^−/− male mice were found to be completely sterile due to an arrest of spermatogenesis at the zygotene stage (Yuan et al., 2000). The authors noticed that SYCP3 is required for the assembly of the synaptonemal complex and the pairing of homologous chromosomes. Miyamoto et al. (2003) showed that also in humans, defects in SYCP3 are a frequent cause of male infertility due to a maturation arrest of spermatogenesis. However, others could not confirm this observation (Martínez et al., 2007, Stouffs et al., 2005). Female knock-out mice were still fertile, although a severe reduction in litter size was noticed (Yuan et al., 2000, Yuan et al., 2002). The offspring died in utero due to chromosomal aneuploidy (Wang and Höög, 2006, Yuan et al., 2002). While this paper was being prepared, a first study was published reporting on the presence of mutations in the SYCP3 gene of two out of 26 women with recurrent miscarriages (Bolor et al., 2009). This study also predicts a relationship between meiosis, aneuploidy and recurrent miscarriages.

MSH4 (MutS homologue 4) is a member of the mammalian mismatch repair gene family. These genes were found to be involved in the repair of DNA mismatches and in the control of meiotic recombination. The MSH4 gene was first identified in yeast, where it appeared to be involved in meiosis only. Disruption of the MSH4 gene in yeasts reduces crossover frequency and increases non-disjunction of homologous chromosomes (Ross-Macdonald and Roeder, 1994). In mice too, Msh4 was found to be important for meiosis as it is co-localized with the synaptonemal complex. Homozygous mutant male mice are infertile due to non-obstructive azoospermia and show a severe depletion of spermatocytes. Furthermore, chromosome pairing was found to be abnormal (Kneitz et al., 2000). Also in female Msh4^−/− mice, a loss of germ cells was observed soon after birth (Kneitz et al., 2000). Similarly to males, female gonocytes failed to undergo complete chromosome pairing, causing the loss of oocytes.

The human DNA-cytosine 5-methyltransferase 3-like gene (DNMT3L) was first characterized by Aapola et al. (2000). DNMT3L is homologous to DNMT3A and DNMT3B, two proteins involved in de-novo DNA methyltransferase, but lacks the ability to transfer methyl groups to DNA (Aapola et al., 2000, Aapola et al., 2002). In mice, expression of Dnmt3l was highest in gonocytes, but was also detected in postnatal cells (Bourc’his and Bestor, 2004, La Salle et al., 2007). Several knock-out mouse models have been examined; all show meiotic defects during spermatogenesis and a decrease in the number of germ cells (Hata et al., 2006, Jia et al., 2007, Webster et al., 2005). During meiosis asynapsis or incomplete synapsis was observed (Bourc’his and Bestor, 2004, Webster et al., 2005). During the pachytene checkpoint, the asynapsis is a known trigger for germ cell arrest and apoptosis, explaining the observed phenotype. This asynapsis might be explained by changes in the chromatin structure. Indeed, Webster et al. (2005) observed changes in the acetylation status of histone H4 and histone H3 acetylated at lysine 9 in Dnmt3l^−/− mice compared with wild-type mice. It had also been shown before that DNMT3L acts as a transcriptional regulator through interaction with histone deacetylase HDAC1 and that DNMT3L is involved in methylation of the paternal genome (Aapola et al., 2002, Deplus et al., 2002, La Salle et al., 2007, Webster et al., 2005).

In this study, these three genes were investigated in azoospermic patients with a maturation arrest of spermatogenesis as well as in couples with recurrent miscarriages.

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Materials and methods 

Patient selection 

DNA from a total of 40 azoospermic patients presenting for infertility treatment and showing maturation arrest of spermatogenesis was analysed for the presence of mutations in the selected genes. For all patients, testicular sperm extraction (TESE) was performed, during which a testicular biopsy was also taken for histological examination within the frame of their fertility work-up (Tournaye et al., 1997). The description of the testicular phenotype reported in this study is based on the most advanced stage of spermatogenesis observed in the histological sample, taking into account the absence or presence of spermatozoa in testicular tissues as seen in wet preparations of multiple specimens. When, for example, the histology showed tubules with Sertoli cell-only syndrome as well as with maturation arrest, the histology of this sample was defined as maturation arrest. Moreover, if spermatozoa were found at TESE and the histology showed maturation arrest of spermatogenesis, these samples were referred to as incomplete maturation arrest.

In order to avoid bias caused by differences in ethnicity between patients and controls, the current study further subdivided all patients into two groups: 27 Caucasian patients and 13 Arabic patients were included. Other ethnic groups were excluded from this study. Taking these criteria into consideration, patients were classified in four groups: Caucasian patients with a complete maturation arrest (n=17); Caucasian patients with an incomplete maturation arrest (n=10); Arabic patients with a complete maturation arrest (n=8) and Arabic patients with an incomplete maturation arrest (n=5). Based on the testicular histology, two Caucasian men had a maturation arrest at the level of spermatogonia (one complete and one incomplete). For all other patients, spermatogenesis was disturbed during meiosis. Three of the patients had a history of cryptorchidism. Patients with a known genetic cause of male infertility, such as a karyotype abnormality or a Yq microdeletion were excluded from this study.

Twenty-eight of the 40 patients analysed for the presence of mutations in SYCP3 have already been reported before (Stouffs et al., 2005).

For alterations detected in the patients’ DNA that also predict a change of the corresponding protein, control samples were analysed. For this control group, DNA samples from men with normozoospermia, defined by routine sperm analysis, were used. The number of control samples analysed was dependent on the change observed. At least 30 samples were investigated. If the change appeared to be present in equal frequencies in patients and controls, no more controls were investigated. Otherwise, more control samples were analysed. The selection of the controls analysed was also dependent on the ethnicity of the patients. Controls were always from the same ethnic origin as the patients for whom an alteration was observed. The number of controls analysed for each alteration is shown in Table 1, Table 2.

Table 1. Frequency of the alterations in patients with maturation arrest of spermatogenesis and controls.

Values are number/total (%) or %.

NA=not assessed.

Table 2. Frequency of the alterations in patients with recurrent miscarriages and controls.

Values are number/total (%) or %.

NA=not assessed.

In a second part of the study, the male and female partners of 23 couples with recurrent miscarriages were analysed. These couples were of mixed ethnic origin. This was an unselected patient group with at least two consecutive miscarriages and for whom the karyotypes were normal. Patients with two recurrent miscarriages were included as this is the limit for referral to the Centre for Medical Genetics for genetic investigation. No further selection criteria were used. The control group used was of the same origin as the samples of interest. This control group consisted of men with proven fertility (at least one child) or normozoospermic semen parameters.

Polymerase chain reaction, sequencing and restriction reaction 

DNA was isolated from leukocytes following standard procedures. Primers were designed in order to amplify and sequence the entire coding region and parts of the flanking introns of the three genes (Supplementary Tables 1 and 2, available online only) and were synthesized by Eurogentec (Belgium).

The sequences from GenBank (NM_002440 for MSH4; AF492003 for SYCP3; NM_013369 for DNMT3L) and Ensembl (www.ensembl.org) were used as reference sequences. The A of the ATG translation initiation signal was numbered +1. Intronic alterations located up to 20 base pairs outside the splice site region were examined in this study.

The primers, PCR and sequencing reactions used for SYCP3 were as described before, with the exception of the amount of DNA used (125ng) (Miyamoto et al., 2003, Stouffs et al., 2005). Primers for PCR and sequencing reactions for MSH4 and DNMT3L are described in Supplementary Tables 1 and 2. PCRs for MSH4 and DNMT3L were performed in a 50μl mix containing 250ng of DNA, 1× PCR Buffer II (Applied Biosystems, The Netherlands), 2mmol/l of MgCl₂ (Applied Biosystems), 0.2mmol/l of each dNTP (Amersham Pharmacia Biotech, The Netherlands), 1μmol/l of each primer and 1.25units of Taq polymerase (Applied Biosystems). Thermocycling conditions consisted of an initial denaturation of 5min at 94°C, 30 or 35 cycles of 1min at 94°C, 1min at a variable annealing temperature (Supplementary Table 1) and 2min at 72°C and a final extension of 7min at 72°C. PCR products were analysed on a 2% agarose gel. For exon 10 of the DNMT3L gene, a GC-rich kit (Roche, belgium) was used according to the instructions of the manufacturer. After purification, all samples were sequenced with primers used for amplification. All samples were run on the ABI3130xl Genetic Analyzer (Applied Biosystems).

In order to analyse control samples for the modification observed in exon 20 of the MSH4 gene, a restriction reaction was set up: samples were amplified after which a restriction reaction was performed with DraI. Similarly, after amplification of exon 7 of the SYCP3 gene, a restriction reaction was performed with Hpy166II, after which the results were analysed by agarose gel electrophoresis.

Single nucleotide changes were analysed through Alamut (Interactive Biosoftware) and using Panther (www.pantherdb.org).

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Results 

In the first part of the study, patients with maturation arrest of spermatogenesis were investigated. A total of 11 alterations were detected, of which five were predicted to alter the amino acid sequences (Table 1).

Besides the change c.754A>G reported before, no new changes were found in the SYCP3 gene. This alteration was located after the stop codon, and will therefore probably not alter the amino acid sequence.

Seven sequence alterations of the MSH4 gene were detected, of which four also altered the amino acid sequence (Table 1). The first alteration changing the amino acid sequence was located in exon 2: c.289G>A and causes p.A97T. This alteration was already described in GenBank (www.ncbi.nlm.nih.gov/snp) as rs5745325. In the Caucasian patient group, four patients were homozygous for c.289G>A, while 13 patients were heterozygous. In the Caucasian control group, five men were homozygous AA and 19 men were heterozygous. These results were not statistically different. Also when comparing the patient data to frequencies published in GenBank (HapMap-CEU; www.ncbi.nlm.nih.gov/projects/SNP/snp_viewTable.cgi?pop=1409), no differences were observed. In the patient group, two patients with the homozygous alteration (AA) had a complete maturation arrest and nine patients with a heterozygous alteration (AG) had a complete maturation arrest. Also when comparing the frequencies of this alteration in patients with complete and incomplete maturation arrest of spermatogenesis, no differences were observed. In the Arabic patient group, five patients were heterozygous for c.289G>A. Two of them had a complete maturation arrest.

Alteration c.1766A>G was located in exon 13 and causes the change of a tyrosine into a cysteine (p.Y589C). This change was detected in one Caucasian patient with complete maturation arrest of spermatogenesis and in one out of 61 Caucasian controls. Both patient and control were heterozygous for this alteration. This SNP was reported in GenBank as rs5745459.

In exon 18, two alterations were detected that were always found concurrently: c.2463A>C and c.2520G>C. The latter causes an alteration of the amino acid sequence (p.E840D). These alterations were observed in three patients (two Caucasians and one Arabic) of which two patients were having a complete maturation arrest of spermatogenesis. The same two alterations were detected in one of the 66 Caucasian control samples analysed.

Alteration c.2741G>A was detected as a heterozygous change in two Arabic patients and two Arabic controls. One of the patients was having a complete maturation arrest and the other patient an incomplete maturation arrest of spermatogenesis. This alteration, causing p.S914N, was absent in the Caucasian patients. In GenBank, this SNP was reported as rs5745549.

Two more changes were observed in exon 12 without changing the amino acid sequence. Four known intronic polymorphisms were detected that have been previously reported in GenBank: c.428–12A>T (rs5745326), c.588+11A>G (rs3737574), c.1163–20C>T (rs2047435) and c.1540+8A>C (rs1493367).

In DNMT3L, a single alteration was detected that also predicts a change at the amino acid level: c.832A>G causing p.R278G. The heterozygous change (AG) was detected in 10 Caucasian and four Arabic patients and 12 out of 33 Caucasian controls. The homozygous (GG) variation was detected in two patients and three controls of Caucasian origin (Table 1). This alteration was detected in equal frequencies in patients and controls, and therefore most likely represents a polymorphism. Two more changes were detected at the DNA level that presumably have no influence on the amino acid sequence (Table 1). One intronic SNP was already mentioned in GenBank: c.–7–4C>A (rs2070565).

In the second part of the study, 23 couples (or 46 patients) with recurrent miscarriages were investigated for the presence of changes in these three genes. The results are described in Table 2. A total of 10 alterations were observed in the exons, of which six were predicted to change the amino acid sequence. Three alterations were not detected in the first part of the study. A first change in DNMT3L (c.453C>T) is (most probably) a polymorphism since the amino acid sequence is not altered. A second alteration (c.268G>A) in the MSH4 gene does change the protein sequence: p.A90T. However, since this change was also detected in two men with normozoospermia, this alteration is likely a polymorphism. A last change was detected in the SYCP3 gene: c.548T>C. This SNP also changes the amino acid sequence: p.I183T, changing a non-polar isoleucine into a polar threonine. Although located near a splice site, the intron/exon boundary is probably not affected. This heterozygous change was detected in a single man. The couple of whom the man had this change was sent for genetic counselling after five miscarriages. No karyotypic abnormalities were detected. In order to determine whether the observed change is a polymorphism, men known to have fathered at least one child (n=90) and men with normozoospermic sperm values (n=119) were investigated. The change was not detected in these control groups. Furthermore, this change was located in a Cor1/Xlr/Xmr conserved region, which is the coiled-coil domain of the gene (Alamut; Baier et al., 2007). Analysis through Panther showed a P_deleterious of 0.78116, indicating a likelihood of 78% that the change has a deleterious effect on the protein function.

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Discussion 

For the present study, the primary aim was to gain more insight into genetic causes of maturation arrest of spermatogenesis. Presumably, defects during meiosis might be the underlying cause of the stop in the development of male gametes. Therefore, three genes (SYCP3, MSH4 and DNMT3L) were selected based on their function and phenotype in mice knocked out for these genes. SYCP3 and MSH4 are essential for meiotic recombination, while DNMT3L is involved in the regulation of the methylation of the paternal genome. Only SYCP3 had already been investigated in view of male infertility (Martínez et al., 2007, Miyamoto et al., 2003, Stouffs et al., 2005). Recently, another two changes in the SYCP3 gene have been described in relationship with recurrent miscarriages. These heterozygous changes were predicting an alternatively spliced mRNA sequence and therefore are causing truncated proteins (Bolor et al., 2009).

For the first part of the study, a total of 40 patients with a maturation arrest of spermatogenesis were investigated for the presence of mutations in these genes. Five changes were found in these genes that are predicted to change the amino acid sequence: four changes in MSH4 and one in DNMT3L. Three of the four alterations in MSH4 were already reported to be SNPs in the database of GenBank. Only c.2520G>C causing p.E840D has not been described before. This sequence variation was always found together with another alteration in exon 18 (c.2463A>C). Both SNPs were detected in Caucasian and Arabic patients. Two of these patients were also heterozygotes for the c.1569T>C substitution located in exon 12, while one patient was homozygous for this SNP. One man from the control group with the alteration c.2520G>C was detected. The presence of c.1569T>C was confirmed in exon 12 and c.2463A>C in exon 18. Yet, more patients were found with the SNP in exon 12 alone. Thus it remains unclear whether the SNP in exon 18 segregates with the SNP in exon 12. The observed alteration p.A97T is common in Caucasian men: the HapMap project reported that 43% of men are heterozygous AG and 10% are homozygous AA. These data correspond to the frequencies observed in the azoospermic patients and controls in the present study. Also, none of the analysed patients or controls were homozygous for the SNP in exon 13 (c.1766A>G). However, in the HapMap project 17% of analysed individuals were homozygous GG. This is statistically higher than the frequency observed in current samples analysed. Yet, this SNP is probably not involved in male infertility since the current patient and control group showed no difference. The present study detected the variation p.S914N in Arabic men only. According to the frequencies of HapMap, 8% of European entities are heterozygotes, while no homozygotes are reported. Yet, the number of Caucasian men analysed in this study, is limited.

In DNMT3L, only a single change was detected that is expected to change the amino acid sequence. This alteration has not yet been reported in GenBank. From the current data, it can be concluded that this change is present in equal frequencies in patients and controls.

In conclusion, all five alterations in patients with a maturation arrest of spermatogenesis were also observed in men with normal sperm values in about equal frequencies and, therefore, are most likely polymorphisms. Also previous studies aiming to identify mutations in patients with maturation arrest of spermatogenesis mostly failed to detect disease causing mutations. Rather than analysing more genes, this study decided to broaden the patient group. This decision was supported by the link between recurrent miscarriages and sperm aneuploidy and between chromosomal abnormalities and meiotic defects (Bernardini et al., 2004, Ferguson et al., 2007, Martin, 2008, Sun et al., 2008). For the second part of the study, 23 couples with recurrent miscarriages were selected. As this is a pilot study, patients were only selected based on their karyotype, which was normal. In this part of the study, three additional changes were detected of which one does not influence the amino acid sequence. One change in the MSH4 gene (c.268G>A) was also present in controls and therefore most likely represents a SNP. A last change, however, was located in a conserved region of the SYCP3 gene. This alteration was present in only a single Caucasian man out of the 23 couples suffering from recurrent miscarriages, and was absent in controls.

For analysing the impact of alterations found in patients with recurrent miscarriages, we have used the control groups that are generally used in male infertility studies: men with normozoospermia and men with proven fertility. We are well aware that a control group consisting of men with normozoospermia is not the best group to use. However, based on the large group of controls tested in total (>200) and the absence of description of this SNP in GenBank, we can conclude that this change is not a common polymorphism in Caucasians. The alteration is located in a functional domain and is predicted to alter the function of the gene. Therefore, this change might be the underlying cause of the problems of the couple. However, larger and more appropriate control groups should be analysed. Together with functional studies this will prove the relationship between the observed change and recurrent miscarriages. Furthermore, fluorescent in-situ hybridization analysis performed on spermatozoa from this man might show if the chromosomal constitution is indeed abnormal. Unfortunately, semen was not available for analysis.

Multiple studies have been reported in which infertile patients have been analysed for the presence of mutations in SYCP3 (Martínez et al., 2007, Miyamoto et al., 2003, Stouffs et al., 2005). Except for one study in which two azoospermic patients were reported with a deletion of a single base pair causing a frame shift, no other mutations have been described so far (Miyamoto et al., 2003). This study, together with the study of Bolor et al. (2009), showed that mutations in the SYCP3 gene might also be related with recurrent miscarriages. Problems during meiosis of male as well as female germ cells might cause difficulty in maintaining pregnancies. Despite the fact that no changes were detected in MSH4 and DNMT3L, the current data together with the study of Bolor et al. (2009) encourages the study of more meiosis genes in view of recurrent miscarriages. Also better characterized patient groups, preferentially with data on chromosomal constitution of one or more fetuses, will be useful to understand the relationship between the mutations in meiosis genes and their consequences. Furthermore, data on the frequency of sperm aneuploidy, in case of a mutation in men, will help to understand the impact of the alterations.

In conclusion, no new mutations that can explain the fertility problems of the patients analysed were found. One potential mutation was detected in one male partner of a couple facing recurrent miscarriages. Therefore more work needs to be carried out in terms of numbers of patients and genes investigated as it is clear that emerging research is identifying mutations that may explain recurrent miscarriage/sperm maturation arrest. Further work will determine whether mutation screening may be warranted as part of a fertility work up or genetic counselling.

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Acknowledgements 

K Stouffs is a postdoctoral fellow of the Fund for Scientific Research Flanders, Belgium (FWO-Vlaanderen). The authors wish to thank the laboratory, clinical and paramedical staff of the Centre for Medical Genetics, the Centre for Reproductive Medicine and the Department of Pathology of the UZ Brussel for their assistance. The work was supported by grants from the Fund for Scientific Research Flanders (Belgium), and from the Research Council of the Universitair Ziekenhuis Brussel (UZOR) and Vrije Universiteit Brussel.

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Appendix A. Supplementary data 

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