According to the family history inquiry, among the 5 individuals who have passed away in the 1st and 2nd generation, only 1 individual (II-5) died of brain stroke with obesity, hypertension, and gout at the age of 54, while the remaining 4 individuals died of multiple metastases of finger cSCC. II-7 was a tailor. He had several years' history of dorsal digital skin ulcer but never sought for any medical treatment. He finally visited the hospital due to the severe deterioration of the ulcer (finger bone exposing). He took a skin biopsy and was diagnosed as cSCC. He was hospitalized for a finger amputation in Zhuhai People's Hospital in 2010. Although underwent standard chemotherapy, 2 years later, he died of multiple metastasis including lung metastasis and spinal metastasis at 51 years old. II-3, born in 1959, was a blue-collar worker. He was admitted to Tongji Hospital for a nonhealing ulcer of the dorsal finger in 2012. A pathological diagnosis of cSCC was made after the skin biopsy. Axillary metastasis emerged soon after surgical resection of the tumor. Finally, he died of uncontrollable bleeding from axillary metastatic tumor at 57 years old. Two other family members (I-1 and II-1, an accountant and a blue-collar worker) had similar experience and died due to massive bleeding of axillary metastatic tumor at the ages 36 and 45, respectively.
To identify the underlying pathological gene, we performed WES for the proband (IV-6). A heterozygous mutation in the SMARCAD1 gene NM_001254949.2: c.-10+5G>A (if counting from the beginning of the noncoding exon 1, this mutation would be named as c.378+5G>A) was identified. The WES result was further confirmed by Sanger sequencing (Figure 4(a)).
Sanger sequencing was also performed for III-2, III-6, III-10, III-11, IV-1, and IV-4. As shown in the pedigree chart (Figure 2), (+) indicates the mutation has been confirmed by Sanger sequencing. (-) indicates no mutation was detected by Sanger sequencing. III-2, III-6, III-10, and IV-4 were clinically diagnosed with Basan syndrome. They were also confirmed carrying the same mutation by Sanger sequencing.  Figure 4(b) illustrates the mutation of the proband's father (III-10). III-11 and IV-1 did not carry this mutation, while no Basan syndrome was diagnosed.  Figure 4(c) illustrates that the mutation was not discovered in the proband's mother (III-11). This revealed that the mutation cosegregated with Basan syndrome in this pedigree. Bioinformatics analysis showed that this mutation was not detected in the 1000 genome database, ExAC databases, or gnomAD database.
One of the 7 collected blood samples (III-2) was contaminated due to misoperation before performing WES. Therefore, WES was performed for only 6 individuals.  Table 1 summarizes the results. All of the 6 samples had a coverage higher than 98% at 4X. The average depth was 160.22 for III-6, 193.60 for III-10, 136.44 for III-11, 176.87 for IV-1, 157.72 for IV-4, and 136.29 for IV-6. In summary, the sequencing data had reached the required quality for next step analysis.
Based on the WES data, we performed target analysis for the SMARCAD1 gene to further confirm the association between SMARCAD1 and Basan syndrome. As shown in Table 2, all Basan syndrome patients carried the mutation SMARCAD1, chr4: 94253676G>A. Specifically, III-6 had 63 reads mapped to SMARCAD1 variant with a frequency of 0.406; III-10 had 60 reads mapped with a frequency of 0.400; IV-4 had 66 reads mapped with a frequency of 0.485; and IV-6 had 54 reads mapped with a frequency of 0.519. Other family member who was not diagnosed with Basan syndrome did not carry this mutation.
As described above, the family history revealed 4 affected members with cSCC in the 1st and 2nd generations. This family exhibited high cooccurrence rate between Basan syndrome and cSCC. To discover the potential highly related gene mutations, we further investigated WES results in the ClinVar database and COSMIC database, aiming for cancer-related gene mutations. Due to the limited samples available, we set out to apply strict filtering criteria for finding possible associated gene mutations. The criteria only kept mutations occurring in the four individuals with Basan syndrome (III-6, III-10, IV-4, and IV-6) but not in other two non-Basan individuals (III-11 and IV-1). The results from the filtering criteria retained 3434 mutations. Out of these 3434 mutations, only one mutation, rs751141, was mapped to the ClinVar database. The annotation of rs751141 shows that it is a risk factor related to "hypercholesterolemia, familial."
The above-mentioned 3434 mutations were then mapped in the COSMIC database. In order to find more significance related to the FAMILIAL cancer, we only retained the exonic area and presented as "nonsynonymous" or "frameshift." After filtering, three genetic variants appeared in individuals with Basan syndrome but not in non-Basan individuals: rs3085861 (SETBP1), rs663651 (SETBP1), and rs2454206 (TET2). Both SETBP1 gene and TET2 gene have been reported associating with skin cancers, including cSCC. However, according to dbSNP, all of the three genetic variants observed in this study are common variants.
Minigene was preformed to further analyze the SMARCAD1 pre-mRNA splicing. We constructed a Minigene containing genomic sequence from intron 1, pseudoexon 1, pseudoexon 2, and exon 2. Our data indicated that the mutation in the SMARCAD1 gene site can lead to splicing variations: pseudoexon 1, pseudoexon 2, and exon 2 were all detected in the wild type, but pseudoexon 2 was skipped in the mutation type (Figure 5). This mutation was located in the intron 1. No other potential splicing site was found. The Minigene result demonstrated that this mutation in intron 1 may cause the gene-splicing variation. Contrarily, no mutation in the intron (wild-type) exhibited normal expression of pseudoexon 1, pseudoexon 2, or exon 2.