A novel mutation in the STK11 gene causes heritable Peutz-Jeghers syndrome - a case report

The Chinese Han kindred with PJS was from Zhanjiang, Guangdong, China. Subjects in the kindred were diagnosed with or without PJS in Guangzhou Women and Children’s Medical Center. The proband III-6 received polyp surgery in the hospital mentioned above and the other patients did in hospitals in Zhanjiang (Fig. 1a). All of the unrelated Han controls were from Guangdong, not younger than 40 years, and diagnosed without PJS symptoms or family history in the First Affiliated Hospital of Guangdong Medical University.

Five patients distributed over three generations exhibited an autosomal dominant mode (Fig. 1a). The age of onset for mucocutaneous pigmentation was as early as at birth in III6, II7, III4, and at two years in II3, (Table 1, Fig. 1b and c). III6 was diagnosed with PJ polyps because of blood in stool and received intestinal surgery at the first year (Table 1). The PJ polyps relapsed and three hamartomatous polyps were excised from cavity of the descending colon at the age of 21 months (Fig. 1b). The largest polyp was 2.7 × 1.8 cm, looked smooth on the surface (Fig. 1b); its pedicle was 0.8 × 0.6 cm (data not shown). As shown in hematoxylin-eosin stained tissue slices, smooth muscle fibers from the muscularis mucosa extended into the inter-cryptas (Fig. 1b). Focal hemorrhage, necrosis and infiltration of a large number of inflammatory cells were observed (Fig. 1b). The proband’s father II3 exhibited pigmentation at both the lips and digits (Fig. 1c). His onset age was not defined but his father said it was before 2 years old. He was diagnosed with PJ polyps in the transverse colon at fifteen years (Table 1). III4, the elder brother of the proband, was diagnosed with PJ polyps in the rectum at two years (Table 1). The polyps extended out of the anus, as his parents described. II7, the uncle of the proband, was diagnosed with PJ polyps because of stomachache at sixteen years (Table 1). A polyp as large as the fist was surgically excised from the stomach, his families said. There is no determined information about the phenotypes of the grandmother I2 who died from neoplasm at forty. Her husband and oldest son told us that she suffered from pigmentation at the lips and digits, but the onset age was unknown. The grandfatherI1 is now eighty-three years old, and the PJS signs are still absent from him.

We sequenced the exons, exon-intron boundaries, 5′ or 3′ untranslated region of the STK11 gene in all of the family members. As a result, a heterozygous two-nucleotide deletion in exon three (c.440_441delGT) co-segregated with the PJS phenotypes (Fig. 2a). The mutation caused a frameshift at site 147 and a premature termination of translation at site 161 (p.Arg147Leufs*15, Fig. 2b). This mutation has not been reported in literatures or recorded in mutation databases. DNA from two hundred of unrelated ethnicity-matched normal individuals were sequenced and as a result the mutation was absent. Human STK11 protein comprises 433 residues, including a kinase catalytic domain (residues 49–309), N- and C-terminal regulatory domains, and a nuclear localization signal near the N-terminal (Fig. 2b). The frameshift led to a deletion of 57% of the catalytic domain and the whole C-terminal regulatory domain. We further studied the affection to mRNA using allele-specific reverse-transcription-polymerase chain reaction assay. As shown in Fig. 2b, all of the patients had a mutation-specific amplicon along with a wild-type-specific one, while two control family members just had the latter.

To study the functional affection of the mutation, we investigated STK11 protein expression in circulating leukocytes, using an antibody that targets residues 332–336 in STK11 and consequently reacts with the wild-type but not affected STK11. As a result, expression of the wild-type STK11 in controls was significantly higher than that in the patients (p = 0.0019, Fig. 2c), suggesting that the disease might result from STK11 haploinsufficiency. Interestingly, we found the wild-type STK11 expressed less in the three PJ polyps from patient III-6 than in circulating leukocytes from the four patients (p = 0.0252, Fig. 2c), suggesting that epigenetic reduction of STK11 expression might be a second contribution to polyps formation. AMPKα has been reported to be phosphorylated at threonine 172 by STK11 [16, 17]. Here we also observed that the phosphorylation levels of AMPKα paralleled with the levels of STK11 expression (Fig. 2c).

PJS onset in most reported patients was within childhood or adolescent years and it was reported that the median time to onset for gastrointestinal symptoms was 13 years of age [2, 15]. In this study, the age of onset for pigmentation in three of the patients was as early as at birth, not firstly but relatively rarely reported. The onset heterogeneity of PJS in this family was not confirmed. There is no determined information about the PJS onset in two of the patients. But it can’t exclude the possibility that the disease onset in the two patients was also as early as at birth. The heterogeneity in polyps location in spite of the same mutation remains to be studied.

As a positive regulation, STK11 is released and shuttles from nucleus to cytoplasm, and interacts with STRADα/β and MO25α/β to stabilize the kinase activity [16, 18]. The entire kinase catalytic domain and amino acids 319–343 in STK11 are necessary for binding to STRAD [18]. So it is self-evident that the mutant identified here is unable to associate with the STRADα/β-MO25α/β complex. Other interacting partners of STK11 have been successively identified and the indispensable motifs in STK11 for interacting with some of them have not yet been determined [19]. So we can’t exclude the possibility that dominant negative effect is a third molecular mechanism by which the mutation results in PJS, if the inactive mutant occupies the partners.