A compound heterozygous Chinese patient with xeroderma pigmentosum variant type caused by novel POLH variants

Xeroderma pigmentosum (XP) is a rare skin disorder that is inherited in an autosomal recessive pattern. XP patients are extremely sensitive to sunlight, and the exposed skin areas are prone to pigmentation, dryness, atrophy, keratosis, ocular abnormality, cancerization at an early age, and infrequent progressive neurologic degeneration.1 Males and females are affected equally. XP was first depicted by dermatologist Moriz Kaposi in 1874, when it was named xeroderma or parchment skin. Previously, because the causative genes are unknown, complementation group analysis of XP was used to categorize affected individuals based on the fusing of their cultured fibroblasts with different XP subtypes and whether they regained unscheduled DNA synthesis. To date, according to the gene that is affected, a total of 8 forms of XP have been identified: XP-A, XP-B, XP-C, XP-D, XP-E, XP-F, XP-G and XP Variant (XP-V). In contrast to XP-V, the other 7 classical types of XPs are all caused by functional defects in genes encoding the nucleotide excision repair (NER) enzyme pathway.1 The gene responsible for XP-V was first discovered in 1999 and named POLH (MIM 603968). POLH encodes DNA polymerase η (pol η), which is a human homologue of the yeast Rad30 protein.2 POLH is located on 6p21.1 and includes 11 exons (NM_006502). Pol η is a 713 amino acid protein with a molecular weight of 78 kDa. The POLH gene product is not considered to be involved in the NER pathway. Instead, it participates in the transcription of UV-damaged DNA because of its relatively low fidelity. All XP patients share similar skin phenotypes. However, the XP-V group differs from other subtypes because it presents rare neurologic symptoms. Herein, we report a 24-year-old male who was diagnosed with XP-V, with no family history of this disease. He presented pigmented freckle-like patches on his skin that were prone to UV exposure at the age of four and gradually progressed all over his face, neck, chest V zone, and extensor side of both upper limbs (Figure 1A,B). Notably, his manifestations were aggravated after sun exposure. He was hospitalized with hyperplasia of the bridge of the nose and had surgery at the age of 20. Histopathological examination of the hyperplasia showed that the proliferative cells were heteromorphic squamous epidermal cells with indistinct boundaries, and parakeratotic cells could be seen (Figure S2). The diagnosis of squamous cell carcinoma was established. There were lymphocytes, histiocytes, and plasma cells infiltrating the stroma, as well as neoplasms in lymphatic vessels. Summary of the patient's and other 9 Chinese XP-V patients' main clinical presentations is listed in Table S1. Laboratory blood tests, including routine blood/urine/stool analysis and tests for hepatic and renal function indicators, electrolyte levels, hepatitis virus, syphilis, HIV, coagulation function indicators, fasting blood glucose, and squamous cell carcinoma-associated antigen, revealed no abnormal findings. Twelve-channel routine electrocardiogram results showed sinus arrhythmia. No remarkable abnormalities were found in the liver, gallbladder, spleen, pancreas, or bilateral kidneys by colour ultrasound. Given the patient's clinical presentation, XP was implicated for diagnosis. Therefore, we performed whole-exome sequencing (WES) on the peripheral blood DNA of the proband to identify the pathogenic variants. Two variants were found in POLH: c.1066C>T (p.Arg356*) and c.2130dupA (p.Leu711Ilefs*34). Sanger sequencing confirmed that p.Arg356* was inherited from his mother, and p.Leu711Ilefs*34 was inherited from his father (Figure 1c–e). p.Arg356* is a known pathogenic variant reported by Broughton et al.3 The allele frequency of the variant c.1066C>T (p.Arg356*) is 0.00002410 within the global population and 0.00005467 within the Asian population, according to the genome aggregation database (gnomAD). The p.Arg356 amino acid residue is highly conserved in mammalian and vertebrate species. According to the research of Broughton et al., the p.Arg356* variant results in the severe truncation of pol η. p.Leu711Ilefs*34, which occurs in the last exon of POLH (exon 11), was discovered for the first time in XP patients and is predicted to extend the sequence to 743 amino acids. The c.2141A>G (p.*714Trpext*8) variant is predicted to cross the stop codon TAG and extend the sequence to 721 amino acids. Opletalova et al. verified that homozygous c.2141A>G (p.*714Trpext*8) combined with c.2074A>G (p.Thr692Ala) results in the absence of pol η expression in skin fibroblasts from XP-V patients, regardless of exposure to long or short UV irradiation.4 To confirm whether the stop-lost variant of POLH can impair function of pol η, we constructed wild-type and mutant POLH (c.2130dupA, c.2141A>G, and c.2074A>G) with fused 3 × FLAG in N-terminal into pIRES2-EGFP vector and transfected them into HEK293T cells, respectively (Figure S1). Western blot using anti-FLAG and anti-POLH antibodies both confirmed that two stop-lost variants leading to mutant POLH protein (p.Leu711Ilefs*34 and p.*714Trpext*8) degradation compared with wild-type POLH (Figure 1F). We demonstrated that c.2074A>G (p.Thr692Ala) alone does not lead to POLH degradation. Non-stop decay is one mechanism of protein quality controls.5 These results suggested that c.2130dupA (p.Leu711Ilefs*34) and c.2141A>G (p.*714Trpext*8) can cause a remarkable decrease of pol η in vitro; therefore, it could be the cause of the XP phenotype. In addition, within the range of WES, we found no suspicious variants of the NER genes of the patient. The XP Incidence is approximately one case per million in the USA and Europe. Japan has a higher frequency of up to 1 in 22 000, and approximately 25% of Japanese XP patients are diagnosed with XP-V. No exact morbidity statistics of XP were made in China. To date, pathogenic XP variants have been identified in less than 50 Chinese patients, and only 9 of them were XP-V patients that harboured biallelic POLH variants6 (Table S1). There is no specific hotspot of variants; they are basically scattered throughout the whole gene. It is worth noting that all POLH variants found in the Chinese population thus far are nonsense or frameshift mutations. A clear relationship has been reported between the type of missense variants and clinical severity;4 it depends on the degree to which the mutation affects the stability and final activity of the pol η. Nevertheless, for patients with an absent or truncated protein, lifestyle, such as the amount of sun exposure, may have a vital influence on the cancer incidence in XP-V patients. Combined with the patient's medical history and WES results, the diagnosis of XP-V was confirmed by the presence of POLH variants. Also, we proved that two stop-lost variants (c.2130dupA and c.2141A>G) of POLH can lead to the obvious downregulation of pol η expression and therefore may cause XP-V. To conclude, we report a new variant of POLH and expand the mutation spectrum for XP. In the study, Xiaerbati Habulieti and Kexin Guo are the co-first authors. Rongrong Wang and Dong-Lai Ma are the co-corresponding authors. Rongrong Wang, Dong-Lai Ma, and Xue Zhang revised the manuscript and supervised the study. Xiaerbati Habulieti, Jiawei Liu, and Dong-Lai Ma cared for the patients. Xiaerbati Habulieti performed the next-generation sequencing. Kexin Guo performed the Sanger sequencing and wrote the initial draft. All authors participated in the design of the study, clinical assessment, and genomics research. All authors take responsibility for acquisition, analysis, and interpretation of the data. All authors have read and approved the final manuscript. We are grateful to the patients and their family members for their participation. This work was financially supported by the National Natural Science Foundation of China (NSFC) [grant number 81788101], the National Key Research and Development Program of China [grant number 2016YFC0905100 and 2016YFC1000504], the CAMS Innovation Fund for Medical Sciences (CIFMS) [grant numbers 2021-I2M-1-018], and the Natural Science Foundation of Beijing [grant numbers 7172167]. The author(s) declare that they have no conflict of interest. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Graphical representation of POLH pIRES2-EGFP vector. Figure S2 Histopathological examination of nasal bridge hyperplasia of the patient. Table S1 Summary of main clinical presentations of 10 Chinese XP-V patients. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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