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Detection and analysis of common pathogenic germline mutations in Peutz-Jeghers syndrome

BACKGROUND Different types of pathogenic mutations may produce different clinical phenotypes, but a correlation between Peutz-Jeghers syndrome (PJS) genotype and clinical phenotype has not been found. Not all patients with PJS have detectable mutations of the STK11/LKB1 gene, what is the genetic basis of clinical phenotypic heterogeneity of PJS? Do PJS cases without STK11/LKB1 mutations have other pathogenic genes? Those are clinical problems that perplex doctors. AIM The aim was to investigate the specific gene mutation of PJS, and the correlation between the genotype and clinical phenotype of PJS. METHODS A total of 24 patients with PJS admitted to the Air Force Medical Center, PLA (formerly the Air Force General Hospital, PLA) from November 1994 to January 2020 were randomly selected for inclusion in the study. One hundred thirty-nine common hereditary tumor-related genes including STK11/LKB1 were screened and analyzed for pathogenic germline mutations by high-throughput next-generation sequencing (NGS). The mutation status of the genes and their relationship with clinical phenotypes of PJS were explored. RESULTS Twenty of the 24 PJS patients in this group (83.3%) had STK11/LKB1 gene mutations, 90% of which were pathogenic mutations, and ten had new mutation sites. Pathogenic mutations in exon 7 of STK11/LKB1 gene were significantly lower than in other exons. Truncation mutations are more common in exons 1 and 4 of STK11/LKB1, and their pathogenicity was significantly higher than that of missense mutations. We also found SLX4 gene mutations in PJS patients. CONCLUSION PJS has a relatively complicated genetic background. Changes in the sites responsible for coding functional proteins in exon 1 and exon 4 of STK11/LKB1 may be one of the main causes of PJS. Mutation of the SLX4 gene may be a cause of genetic heterogeneity in PJS.


INTRODUCTION
It is currently believed that Peutz-Jeghers syndrome (PJS) is an autosomal dominant genetic disease predominantly caused by germline mutations in the STK11/LKB1 gene. PJS is characterized by multiple hamartoma polyps in the gastrointestinal tract, pigmentation at specific sites, and hereditary tumors [1][2][3][4]. Pathogenic mutations of STK11/LKB1 lead to inactivation of its expression product and loss of inhibition of mammalian target of rapamycin (mTOR) activity, which leads to abnormal activation of the LKB1/mTOR signal pathway and the occurrence of black spots on the skin and gastrointestinal hamartoma polyps [5]. More than 400 different pathogenic STK11/LKB1 gene mutations are included in the Human Gene Mutation Database (HGMD), most of which are microminiature. Different types of pathogenic mutations may produce different clinical phenotypes, but no correlations of PJS genotype and clinical phenotype has been found so far [6], Not all patients with PJS have detectable mutations in the STK11/LKB1 gene. What is the genetic basis of clinical phenotypic heterogeneity in PJS? Do PJS patients without STK11/LKB1 mutations have other pathogenic genes? These are clinical problems that perplex doctors [7,8]. We enrolled 24 patients treated for PJS. Peripheral venous blood and normal tissue adjacent to polyps were collected for high-throughput next-generation sequencing (NGS) of 139 hereditary colorectal tumor-related genes including STK11/LKB1 to study the correlation between genotype and clinical phenotype of PJS and explore the internal molecular mechanism of the clinical phenotypes. October 21, 2021 Volume 27 Issue 39

Study participants
Patients with PJS, from 18-70 years of age, met the clinical diagnostic criteria of PJs, had complete clinicopathological data, well preserved specimens, were eligible for inclusion. All participants gave their signed informed consent. Patients who could not provide experimental specimens or did not agree to participate in the study were excluded. Twenty-four PJS patients admitted to the Air Force Medical Center (formerly the Air Force General Hospital) from November 1994 to January 2020 met the above criteria and were enrolled. Their clinical information is shown in Table 1. Twenty-three were inpatients, one was an outpatient, 11 had family histories, and 12 had early onset pigment spots that had appeared when they were younger than 3 years of age. All patients met the PJS diagnostic criteria recommended by the National Comprehensive Cancer Network (NCCN) [9]. The experimental samples included 5 mL peripheral venous blood samples collected from 19 patients into tubes containing EDTA-2Na, and paraffin-embedded normal tissue surgically removed from areas adjacent to polyps in five patients. other population databases were searched for the mutation frequency of this gene. The location of genes with a mutation frequency < 0.01 in the HGMD database (HGMD-PUBLIC version 20152) were used for pathogenicity analysis.
SPSS 24.0 was used for statistical analysis of the acquired data. Qualitative results were reported as numbers and percentages. The chi-square test or Fisher's exact probability method was used for between-group comparisons. P < 0.05 was considered

STK11/LKB1 gene detection results and pathogenicity analysis
Twenty of the 24 PJS patients (83.3%) in this group had STK11/LKB1 gene mutations ( Table 3). All were heterozygous and ten were newly discovered mutation sites not included in the dbSNP database. There were eight frameshift mutations, five splicesite mutations, four missense mutations and three nonsense mutations. The mutations occurred in eight of the ten exons in the STK11/LKB1 gene, mutations in exons 1 and 4 and 4 each in exon 7, two in each exons 5 and 8, and one in exons 2, 3, and 6. Frameshift mutations, splice-site mutations, and nonsense mutations were all related to pathogenicity. Frameshift mutations accounted for 62.5% (5/8) that were clearly pathogenic, and 37.5% (3/8) that might cause disease. Splice-site mutations accounted for 40% (2/5) that are clearly pathogenic, and 60% (3/5) that might cause disease. All three nonsense mutations were clearly pathogenic, and the missense mutations were related to and might cause disease. Sites of unclear clinical significance accounted for 50% (2/4); of the 11 truncated mutations, eight cases were clearly pathogenic and three were likely to cause disease. The pathogenicity of STK11 gene mutations in exon 7 was significantly lower than that of other exons (P = 0.000). Truncation mutations were significantly more pathogenic than missense mutations (P = 0.012). The prediction results of bioinformatics tools for missense mutations are shown in Table 4, and the relevant database records and the pathogenicity judgment of all mutations are shown in Table 5 Considering that the type of specimen may impact on the detection rate of STK11/LKB1 gene mutations, we analyzed the paraffin-embedded tissue and blood samples separately. The detection rate of STK11/LKB1 mutations in 60 patients with paraffin samples was 60% (3/5), slightly less than the 89.4% (17/19) of the blood samples from 19 patients. The difference in mutation detection rate of this gene in the two types of sample was not statistically different (P = 0.116).

SLX4 gene detection results and pathogenicity analysis
SLX4 gene mutation (Table 6) was detected in 5 PJS patient samples in this group, with a total detection rate of 20.83% (5/24), all of which were heterozygous mutations. The mutation occurred in 4 of 15 exons of SLX4 gene. Mutation types include: 3 missense mutations, one splice-site mutation, and one non-frameshift mutation. No truncation mutation was found. The SLX4 gene is a tumor suppressor gene, and there are three newly discovered mutation sites. The prediction results of three cases of missense mutations by bioinformatics tools (Table 7), the collection of relevant databases and the judgment of the pathogenicity of all mutations (Table 8) are as follows.

Other gene detection results and pathogenicity analysis
A total of 55 mutations of 46 genes other than STK11/LKB1 and SLX4 were detected in 21 cases (Table 9), f a detection rate of 87.5% (21/24). Twenty-three of the genes were related to cancer suppression and had 32 different mutation sites. Two mismatch repair MMR genes were detected, MSH2, MSH6. Except for a frameshift mutation (frameshift deletion) in the BRIP1 gene detected in one patient (No. 18), the rest were missense mutations (Table 10). October

STK11/LKB1 genotype-phenotype correlation analysis
Investigation of the relationship between genotype and family history found that the proportion of patients with truncated mutations was slightly higher in those with a family history than in those without a history (60% vs 50%). The proportion of splicesite mutations was lower in those with a family history (20% vs 30%), and the proportion of nonsense mutations was higher in patients with a family history (20.0% vs 11.1%). The proportions of missense mutations were the same (20% vs 20%), and the proportion of frameshift mutations were also equal (40% vs 10%). There were no significant difference between-group differences in P truncation mutation = 0.653, P splice site mutation = 0.606, P nonsense mutation = 0.371, P missense mutation = 1.000, and P frameshift mutation = 1.000. Evaluation of the relationship between genotype and early onset/late onset found that the proportion of truncated mutations in patients with early onset was higher than that in patients with late onset (72.7% vs 33.3%). In patients with early onset, the October    percentages of frameshift mutations (54.5% vs 22.2%) and sense mutations (18.2% vs 11.1%) were higher than those in late onset patients. The percentages of splice-site mutations (9% vs 44.4%) and missense mutations were lower (18.2% vs 22.2%). There were no significant between-group differences in P truncation mutation = 0.078, P frameshift mutation = 0.142, P nonsense mutation = 0.660, P splice site mutation = 0.069, P missense mutation = 0.822.

DISCUSSION
The STK11/LKB1 gene located on chromosome 19p13.3 is considered to be a tumor October   suppressor gene [14] and is widely expressed in human tissues. Pathogenic mutation of STK11 can inactivate its expressed product, which results in the loss of its inhibitory effect on the activity of mammalian target of rapamycin (mTOR), leading to the occurrence of skin and mucous membrane black spots and gastrointestinal polyps [5]. Research on whether there is a correlation between the PJS genotype and clinical phenotype is ongoing. Although the correlation is currently unclear [6,17], some studies have reported positive results. For example, Forcet et al [18] reported that patients often present with only black spots and without gastrointestinal polyps when heterozygous mutations occur in exon 8 of the STK11 gene. Amos et al [19] found that PJS patients with missense mutations had a first episode of polypectomy and appearance of other symptoms significantly later than those with truncated mutations or no detectable mutations. In a study including 116 PJS patients in 52 families, Wang et al [20] found that nearly 30% of the mutations occurred in exon 7, and some of those mutations affected the protein Kinase domain XI region, which is associated with 90% of cases with gastrointestinal polyp dysplasia. An analysis of the start region of the STK11/LKB1 coding sequence by Hearle et al [21] found that a change in promoter sequence was unlikely to be the cause of PJS. In this study the time that dark spots first appeared, which is a relatively objective indicator, was the basis of clinical classification, and was used to determine whether there was a correlation between the appearance of the spots and any of the genotypes. Spots that appear in early childhood will be noticed. On the other hand, unless there are obvious clinical symptoms, it is extremely difficult to know about gastrointestinal polyps that appear in early childhood. Also, PJS is an autosomal dominant genetic disease and does not completely follow Mendelian inheritance [6]. In clinical practice, it is often found that neither parent has a family history but their child has the disease. This is difficult to fully explain if the disease is caused by a single gene. Therefore, whether the patient has a family history was also included in the basis of clinical classification.
This study did not found that patients with different clinical phenotypes (early onset/late onset and with or without a family history) had statistically significant differences in their STK11/LKB1 gene mutations and loci. However, we found that the October 21, 2021 Volume 27 Issue 39 most truncation mutations of the STK11/LKB1 gene mostly occurred in exons 1 and 4, most missense mutations occurred in exon 7, and that truncation mutations were significantly more pathogenic than missense mutations. The results indicate that changes in the sites encoding functional proteins in exon regions 1 and 4 may be among the main causes of PJS. Also, the percentage of STK11/LKB1 truncation mutations in patients with early onset PJS was higher than that in patients with late onset PJS, and the between-group difference in the percentage of missense mutations was not significant. Because the evidence of a correlation with missense mutations was not strong, it suggests that early onset PJS is more likely to be caused by pathogenic mutations in STK11/LKB1, while late onset disease is likely to be clinically heterogeneous. The study results also suggest that analysis of the age of appearance of dark spots in a large sample of PJS patients would yield some interesting findings. For the first time, we detected more concentrated mutations in the SLX4 gene in PJS patients. The SLX4 (FANCP) gene is a tumor suppressor gene located on chromosome 16p13.3 [21]. It serves as a key scaffold element for the assembly of multiprotein complexes containing enzymes involved in DNA maintenance and repair [22] and has low to moderate expression in all adult and fetal tissues and specific adult brain regions [23]. It has been reported that[24] truncated mutations in the SLX4 gene were detected in families with Fanconi anemia, and it was determined that SLX4 mutations are clearly related to one of the subtypes of the disease. Fanconi anemia is a rare autosomal recessive genetic disease [25]. In addition to blood system-related manifestations, the clinical manifestations of FA include multiple congenital malformations, brown pigmentation of the skin, and tumor susceptibility [26]. There are many similarities with PJS, mutations in the SLX4 gene have been detected in patients with PJS in previous studies, the first of which was found in this group. SLX4 is considered October 21, 2021 Volume 27 Issue 39 . At present, it is believed that[27-29] the loss of DNA MMR genes causes the accumulation of mismatches in the process of DNA replication, resulting in the occurrence of microsatellite instability and partial junctions. Colorectal cancer has obvious genetic characteristics. We also detected mutations in some MMR genes ( MSH2 and MSH6) in PJS, and the role of SLX4 gene is highly similar to that. Perhaps the mutation of the SLX4 gene may explain the genetic heterogeneity of PJS to some extent.

CONCLUSION
In conclusion, we discovered a series of new gene mutation sites, analyzed their pathogenicity, and enriched the mutation spectrum of PJS pathogenic genes. And through the summary of the clinical phenotypes with different STK11 genotypes, to explore whether they are related, and get some tendentious research results. The detection of SLX4 gene mutations in patients with PJS was reported for the first time.
The relationship between SLX4 gene mutations and the occurrence of PJS is still unclear, but may help to explain the genetic heterogeneity of PJS.

Research background
Different types of pathogenic mutations may produce different clinical phenotypes, but no exact correlation between Peutz-Jeghers syndrome (PJS) genotype and clinical phenotype has been found so far. So it is necessary to study the correlation between genotype and clinical phenotype of PJS, and explore the internal molecular mechanism of different clinical phenotypes.

Research motivation
The authors included 24 cases of treated PJS cases as study participants, collected peripheral venous blood or normal tissue adjacent to polyps for high-throughput nextgeneration sequencing (NGS) of 139 hereditary colorectal tumor-related genes including STK11/LKB1 to study the correlation between genotype and clinical phenotype of PJS.