STK11 Causative Variants and Copy Number Variations Identified in Thai Patients With Peutz-Jeghers Syndrome

Introduction Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder caused by germline mutations in the serine-threonine kinase 11 (STK11) tumor suppressor gene. This syndrome is characterized by hamartomatous gastrointestinal polyps, mucocutaneous melanin pigmentation, and a higher risk of developing various cancers. Methods We summarized the clinical and molecular characteristics of five unrelated Thai patients with PJS. Denaturing high-performance liquid chromatography (DHPLC) screening, coupled with direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA), were applied for the molecular analysis of STK11. Results A total of four STK11 pathogenic changeswere identified in the five PJS patients, including two frameshift variants (a novel c.199dup, p.Leu67ProfsTer96 and a known c.834_835del, p.Cys278TrpfsTer6) and two types of copy number variations (CNV), exon 1 deletion and exons 2-3 deletion. Among reported STK11 exonic deletions, exon 1 and exons 2-3 deletions were found to be the two most commonly deleted exons. Conclusion All identified STK11 mutations were null mutations that were associated with more severe PJS phenotypes and cancers. This study broadens the phenotypic and mutational spectrum of STK11 in PJS.


Introduction
Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant polyposis syndrome characterized by hamartomatous gastrointestinal polyps associated with mucocutaneous melanin pigmentations [1,2]. The estimated incidence of PJS is reported to be between 1 in 8,300 and 1 in 280,000 live births [3,4]. Patients with PJS have a 15-fold relative risk of developing various gastrointestinal and extra-gastrointestinal cancers compared to the general population, which leads to increased mortality [5]. Colorectal and breast cancers are the most common cancers reported in PJS [6,7]. In addition, complications resulting from the polyps, including intussusception, intestinal obstruction, and bleeding, are also common causes of death [3,8]. Germline mutations in the serine-threonine kinase 11 (STK11) tumor suppressor gene, detected in approximately 30-80% of PJS cases, have been demonstrated as the major pathogenesis [9][10][11]. This gene, located on chromosomal region 19p13.3, contains nine coding exons, which are translated into a 433amino-acid protein [9,10]. STK11 protein functions as a master upstream kinase of AMP-activated protein kinase (AMPK) signaling, which is responsible for the regulation of cell polarity, cell proliferation, and cell metabolism [12][13][14][15]. Defective STK11/AMPK activity contributes to phenotypic manifestations of PJS, including polyp formation and cancer susceptibility metabolism [12][13][14][15].
More than 500 mutations of STK11 have been identified, and most of them result in abnormal protein truncation and loss of kinase activity [16][17][18]. The majority of pathogenic mutations in STK11 are point mutations, which consist of missense/nonsense mutations (42.7%), small insertions or deletions (31.1%), and splicing mutations (7.7%) [17]. The copy number variations (CNV) associated with large genomic

DNA analysis for CNV in STK11
Semi-quantitative multiplex PCR/DHPLC was performed to determine STK11 copy number alterations, including deletions and duplications, in the samples showing negative DHPLC-screening results. Two sets of multiplex PCRs were established. The first set included exons 2-6 of STK11. The second set contained exons 7-9 and exon 1 of STK11. Insulin-like growth factor 1 (IGF1) gene was used as an internal control for autosomal chromosomes, while acyl-CoA thioesterase 9 (ACOT9) or dystrophin (DMD) (primer sequences available on request) represented an internal control for X-chromosomes. The multiplex PCR reactions were performed in a total volume of 25 µL containing 100 ng of genomic DNA, 1× Immo buffer, 2 mM of MgCl2, 400 µM of dNTPs, 1× Q solution, 0.75 units of Immolase™ DNA polymerase (Bioline USA, Inc., Kenilworth, NJ, USA) and optimal concentrations of each primer set (

TABLE 2: Two sets of multiplex PCR
Non-denaturing mode of DHPLC was applied for the separation of multiple DNA fragments from multiplex PCR reactions depending on their sizes. The aliquots (10 µL) of multiplex PCR products were injected into a DHPLC system, which maintained the column temperature at 50 ºC. A normal control sample of the same gender was always injected in the same run to compare the results. The DHPLC peak heights reflect the quantity of PCR products in each DNA fragment.
The samples showing abnormal peak heights were also verified for copy number alterations using MLPA. The MLPA reactions were performed using SALSA® MLPA® probemix P101-B3 STK11 (MRC-Holland, Amsterdam, The Netherlands) following the manufacturer's recommendations and analyzed using an Applied Biosystems® 3130xl Genetic Analyzer (Thermo Fisher Scientific) and Coffalyser v.140721.1958 software (MRC-Holland).

Results
A mutation analysis was performed in five unrelated PJS patients. All patients presented with mucocutaneous pigmentation, gastrointestinal polyposis, and polyp-related complications ( Table 3). Three of five patients had a documented family history of PJS. None of the gastrointestinal cancers have been observed, but two gynecological cancers, including sex cord tumorlets with annular tubules (SCTAT), cervical mucinous adenocarcinoma, and papillary breast cancer, were reported in three female patients.     The pathogenicity interpretation of the identified STK11 variants and CNV was performed (as shown in Table 4) according to the 2015-standards and guidelines recommended by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) [19], as well as the 2019 technical standards for the CNV interpretation recommended by the ACMG and the Clinical Genome Resource (ClinGen) [20], respectively.
The samples showing no heteroduplex formation in DHPLC screening results were subsequently determined for STK11 copy number alterations using semi-quantitative multiplex PCR/DHPLC. The exon 1 deletion was identified in PJS01 and PJS02. Meanwhile, DHPLC peaks dropped in all exons of STK11 were found in PJS04 ( Figure 3). MLPA analysis was used to confirm previously detected exon deletions. The exon 1 deletion was found in PJS01 and PJS02 and agrees with DHPLC analysis, but the exons 2-3 deletion was identified in PJS04, and the result is inconsistent with the whole gene deletion in DHPLC analysis (Figure 4). Although the DHPLC results demonstrated the whole deletion of STK11 in PJS04, they were incompatible when analyzed with the sequencing results. Two heterozygous single base changes were identified in intron 7 (c.920+7G>C) and intron 9 (c.*16+45T>A) of STK11 in PJS04 (data not shown). Moreover, the incorporation of another unrelated internal control gene, proline-rich transmembrane protein 2 (PRRT2), with a large amplicon size of 640 bps in multiplex PCR revealed the reduction of DHPLC peaks, which indicated the effect of DNA degradation in PJS04 (data not shown). Using all mutation analysis together, the mutation of PJS04 was therefore concluded to be exons 2-3 deletion of STK11.

Discussion
PJS is a rare inherited disorder in which STK11 is known as the causative gene [9][10][11]. Although mutation studies of PJS have been conducted in many countries, there are very few studies of PJS in Thailand, and the genetics of this syndrome remain obscure. This study attempts to investigate the clinical and molecular characteristics of Thai PJS patients and apply the versatile DHPLC method to perform the mutation analysis of STK11.
Using DHPLC-based heteroduplex screening and direct DNA sequencing, two STK11 point mutations were identified. A 1-bp frameshift duplication (c.199dup, p.Leu67ProfsTer96) was found in PJS03 ( Figure 1). This mutation has never been reported in PJS; however, the early termination caused by this mutation (p.L67Pfs*96) was similar to the effect of a previously reported adjacent mutation (c.198dup), which clearly cosegregated with PJS in the original family of Peutz's study [21]. Both the PJS03 patient and affected members of the original PJS family presented with gastrointestinal polyps, mucocutaneous pigmentation, and cancer development. Nonetheless, there were different manifestations of polyp location and cancer types. SCTAT, a rare ovarian cancer associated with PJS [22], was identified in PJS03. On the other hand, gastrointestinal cancers, colon and gastric cancers, and breast cancer were found in the original PJS family [21]. There were unusual nasal polyps that developed in 6 out of 22 PJS patients of the original PJS family while being absent in PJS04 [21]. Another point mutation is a 2-bp frameshift deletion (c.834_835del, p.C278Wfs*6) found in PJS05 (Figure 2). This mutation has previously been noted in one case of PJS [9], but there was no reported clinical manifestation related to the mutation.
Semi-quantitative multiplex PCR/DHPLC and MLPA analysis revealed two exonic deletions of STK11. The exon 1 deletion was identified in PJS01 and PJS02. While the exons 2-3 deletion was confirmed in PJS04, the different results between DHPLC and MLPA were observed. All DHPLC peaks of the STK11 amplicons decreased, but MLPA indicated exons 2-3 deletion. Two heterozygous variants found in intron 7 (c.920+7G>C) and intron 9 (c.*16+45T>A) of STK11 in PJS04 indicated that the whole gene deletion might not be possible and the DHPLC peak reduction might be caused by other reasons. It was noticed that decreased DHPLC peaks were also found in large-sized PCR amplicons of unrelated PRRT2 genes in PJS04, suggesting the effect of poor DNA quality. Degraded DNA might disrupt semi-quantitative multiplex PCR/DHPLC analysis to complete the DNA profile or produce an inaccurate result. On the other hand, MLPA probes are able to hybridize small target sequences (50-70 nucleotides), providing better tolerance for the analysis of fragmented DNA. These ambiguous results suggested that MLPA, the present standard method to evaluate copy number alterations in STK11, remains needed to prove the correctness of semi-quantitative multiplex PCR/DHPLC results.
Large genomic deletions are common STK11 mutations in PJS patients apart from point mutations.
According to the published reports, the deletions often involve the whole gene or are distributed in the 5' upstream region of STK11. Noticeably, exon 1 and exons 2-3 represent the most frequently deleted exons that correlate to the exonic deletions found in our patients ( Table 5). An overrepresentation of the Alu repetitive sequence has been proposed as a potential mechanism that mediates homologous recombination, leading to recurrent genomic rearrangements and copy number alterations in STK11 [23,24]. Specifically, Alu elements were highly detected in intron 1 and breakpoint characterization of PJS patients harboring STK11 exons 2-3 deletion indicated that Alu elements located within intron 1 and 3 serve as recombination hotspots [23,24]. Other Alu elements were also predicted to mediate exon 2 and exons 1-3 deletions [24]. These data suggested that exon 1 deletion might occur via the same mechanism.   The genotype-phenotype correlation between STK11 mutations and PJS has been investigated in several studies. The STK11 frameshift mutations in PJS03 and PJS05 led to premature termination, resulting in a truncated protein with a disrupted catalytic kinase domain and a complete absence of the C-terminal regulatory region. The STK11 exonic deletions, including exon 1 in PJS01 and PJS02 and exons 2-3 in PJS04, also affected the kinase domain, resulting in the inactivation of STK11 kinase activity. Null STK11 mutations, including both truncating mutations and large deletions, have found to be associated with more severe phenotypes of PJS. Patients with null STK11 mutations tend to have early-onset, frequent gastrointestinal symptoms including polyps, intussusception, and intestinal obstruction compared to patients with missense mutations [11,[25][26][27]. While previous studies showed the median age of diagnosis and first gastrointestinal symptom onset in patients with null STK11 mutations to be 17 and 10 years, respectively [25,26], the median diagnostic age of our patients was 18 years, with intussusception as the most frequent first symptom. In terms of cancer development, there was no difference in cancer risks between null mutations and missense mutations; however, breast cancer was associated with truncating mutations [7,26,28]. Three of the five patients in our study developed cancer, and one patient who carried a truncating mutation, PJS05, also presented with papillary breast cancer. Two rare PJS-related gynecological cancers were diagnosed in the other two patients, including SCTAT in PJS03 and cervical mucinous adenocarcinoma in PJS04. No obvious correlation between these two rare cancers and germline STK11 mutations in PJS has been described; nevertheless, somatic STK11 mutations were detected in approximately 20% of cervical cancers, and half of the harbored mutations were large monoallelic/biallelic deletions [29].

Conclusions
In summary, we report the clinical characteristics and pathogenic STK11 variants, or CNV, of five Thai patients with PJS. Four null mutations, including two STK11 frameshift variants (a novel c.199dup, p.Leu67ProfsTer96, and a known c.834_835del, p.Cys278TrpfsTer6), and two common STK11 exonic deletions, exon 1 deletion and exons 2-3 deletion, were identified. These STK11 mutations were associated with more severe manifestations of PJS. The molecular analysis of the STK11 gene using the rapid and costeffective DHPLC method for mutation screening combined with standard direct DNA sequencing and MLPA is well optimized and demonstrated. This study expands knowledge on the molecular genetics of STK11 mutations, which is beneficial for clinical diagnosis and genetic counseling.