Novel Pathogenic Mutations of FERMT1 in two Chinese Kindler Syndrome Families

Background: Kindler syndrome (KNDLRS) is a very rare autosomal recessive disorder characterized by bullous poikiloderma with photosensitivity. Loss-of-function mutations in FERMT1, which located on chromosome 20p12.3, were responsible for KNDLRS. Numerous mutations in FERMT1 have been reported to be associated with KNDLRS. Results: The present study reported two Chinese KNDLRS families, and affected individuals from both families presented with poikiloderma, palmoplantar hyperkeratosis, and diffuse cigarette paper like atrophy on hands. Skin biopsy of the proband from family 2 showed atrophy of epidermis, hyperkeratosis, dilated blood vessels in upper dermis, and microbubbles at the dermis and epidermis junction. Medical Whole Exome Sequencing V4 combined with Sanger sequencing revealed mutations in FERMT1 with affected individuals. Compound heterozygous nonsense mutations (c.193C>T, c.277C>T) were found with family 1, and a homozygous frameshift mutation (c.220delC) was observed in family 2. According to the clinical features and genetic analysis, KNDLRS was diagnosed in two Chinese families. Conclusions: This study revealed two novel pathogenic mutations in FERMT1 that caused KNDLRS and briey summarized all pathogenic mutations in FERMT1 that have been documented via the PubMed.

Background Kindler syndrome (KNDLRS; OMIM #173650) is a very rare autosomal recessive genodermatosis characterized by acral blistering, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility [1]. It was rst reported by Theresa Kindler in a 14-year-old girl with congenital blistering of her hands and feet. By linkage and homozygosity analysis, the pathogenic gene responsible for KNDLRS was mapped to chromosome 20p12.3, and homozygous as well as compound-heterozygous mutations of FERMT1 were identi ed with the KNDLRS patients. Although light microscopy may be helpful, it is best to directly sequence FERMT1 for unequivocal diagnosis. At present, about 91 mutations of FERMT1 have been documented in the literature. Mutation types include missense, splicing, regulatory, small indels, gross deletions/insertions/duplications (HGMD) [2]. Most pathogenic mutations in FERMT1 reported are predicted to lead to premature termination of translation, which results in the loss of the kindlin-1 protein and impairs its function. The clinical and genetic aspects of the disease have recently been reviewed, however no clear genotype-phenotype correlations are established [3,4].
In skin, kindlin-1 is localized within the epidermis and particularly in basal keratocytes but not in epidermal melanocytes and dermal broblasts. How kindlins act is less well-de ned, while diseasecausing mutations show that kindlins are essential for integrin activation, adhesion, cell spreading and signaling [5]. Kiritsi et al has reported that the restoration of kindlin-1 led to structurally normal skin, while loss of kindlin-1 severely impaired keratinocyte proliferation [6]. Loss of kindlin-1 in mouse keratinocytes recapitulates KNDLRS and produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility [7].

Results
Pedigree of two families Pedigree of two families were shown in Fig. 1A and 2A. Both pedigrees presented an autosomal recessive inheritance manner. All parents of the proband are healthy without any clinical phenotype. In addition, family 2 has a consanguineous marriage history.

Patients with Kindler syndrome in two Chinese families
In family 1, physical examination ( Fig. 1B. a-i) revealed an 11-year-old female presented with vulnerable gums, erosion on the gingivitis and skin blisters in the neck. In addition, hypogastric presence blisters and heterochromatism. Moreover, the skin of hands showed atrophy and blisters. The skin pigment of both lower limbs of patient is abnormal, and the skin is atrophic, spreading from the knee to the extremities.
The proband in family 2, a 30-year-old woman had a more severe phenotype ( Fig. 2B. a-j). Both trunk and back of the skin showed diffuse blisters and heterochromatism. As for the limbs of patient, it showed that limb skin atrophy is obvious, accompanied by limb blisters, skin peeling with cracks. Skin biopsy showed atrophy of epidermis, hyperkeratosis, dilated blood vessels in upper dermis, and microbubbles at the dermis and epidermis junction (Fig 2B. k-m).

Gene Variant Distributions in two Families
By using the Medical WES V4 and Sanger sequencing veri cation, we found c.193C>T and c.277C>T compound heterozygous mutations of FERMT1 in proband III-1 from family 1 (Fig. 3C). A c.193C>T mutation inherited from the mother (Fig. 3A), and it had been reported as a pathogenic nonsense mutation previously. A c.277C>T mutation inherited from the father (Fig. 3B) was a novel nonsense mutation, and a bioinformatics analysis showed that it might be a pathogenic mutation.
As for proband IV-1 from family 2, a homozygous mutation (c.220delC (p.H74Tfs*31) in the exon3 of FERMT1 was found to be associated with disease phenotypes. Speci cally speaking, a 1 bp deletion was in the exon3 of FERMT1 (NM_017671; c.220delC (p.H74Tfs*31)) ( Fig. 3F), which resulted in a frameshift and premature termination of FERMT1. It was con rmed that each of her parents was carrying a heterozygous mutation (Fig. 3D-E). According to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines, the mutation was initially identi ed as a likely pathogenic.

Three dimensional structural changes of proteins caused by mutations in two families
To further verify the effect of mutations on FERMT1 coding protein, we use protein structure prediction software (SWISS-model) Review of 91 FERMT1 mutations reported in KNDLRS.
Literatures from 1984 to 2020 as well as the results gained in current study revealed about 91 different mutations in FERMT1. The mutations included missense, frameshift, nonsense, and splice mutations. The locations of FERMT1 mutations spread from exons 1 to 15, introns (1, 7 to 11, 13 to 14), and regulatory regions (Fig. 5). The mutation c.193C > T reported previously was in BLUE, while the other two novel mutations identi ed in this study were highlighted in GREEN. The results demonstrated that most mutations produced a premature termination of FERMT1 and may lead to production of nonfunctional proteins or even absent kindlin-1 protein.

Discussion
KNDLRS is caused by FERMT1 mutations on chromosome 20p12.3 [9]. FERMT1 contains 15 exons and encodes the 677-amino acid protein kindlin-1, which plays an important role in keratinocyte migration, adhesion, and proliferation [1]. The FERMT1 gene product belongs to a family of focal adhesion proteins (kindlin-1, -2, -3) that bind several beta integrin cytoplasmic domains. Ussar S and his collaborators found that deleting kindlin-1 in mice gives rise to skin atrophy [10]. Kindlin-1 play a critical role in integrin activation and that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers [11]. Has et al had reported a novel nonsense mutation E304X in the exon7 of FERMT1, which was predicted to cause loss of the FERM and PH domains and consequently to impair the function of the protein or, more likely, to lead to nonsense-mediated mRNA decay and complete loss of kindlin-1 protein expression [12]. Has C et al reviewed that there was an association of FERMT1 missense and in-frame deletion mutations with milder disease phenotypes, and later onset of complications. It has been reported that a c.1729del (p. Ser577AlafsX14) mutation results in a premature stop codon that deletes most of the FERM 3 domain of kindlin-1 or, more likely, triggers mRNA degradation via nonsense-mediated decay mechanisms [13]. All these loss-of-function FERMT1 mutations demonstrate the importance of kindlin-1 in maintaining epithelial integrity, although the mechanism linking this mutant protein to photosensitivity and poikiloderma remains to be determined. The protein N-terminal was very important for its function.
Harburger et al reported that the in-frame deletion of isoleucine 623 affects the FERM 3 subdomain close to the binding site for β1 integrin [14]. Except for nonsense mutation and small deletion mutation, a novel genomic deletion (about 3.9 kb) was identi ed in Italian KNDLRS patients, and this deletion resulted in the loss of exons 10 and 11 of FERMT1 and leading to a truncated kindlin-1 [15].
To date, 91 different pathogenic FERMT1 mutations have been reported (Fig.5) These mutations were scattered throughout FERMT1, including all the exons, introns (1, 7 to 11, 12 to 13), and regulatory regions, without obvious hotspots identi ed. Most of the mutations were nonsense mutations or frameshift variants, which will result in loss of function and may lead to absent kindlin-1 protein or the production of dysfunctional proteins [16]. As a recessive disorder, most of these mutations in FERMT1 were reported to be homogenous in KNDLRS patients and few were compound heterogeneous mutations.
To date, excluding two milder phenotype related mutations Has et al reported in 2011, no additional homozygous FERMT1 missense and in-frame deletion mutations had been identi ed, therefore it still needs more evidence to clarify the genotype-phenotype correlations with KNDLRS.
Here we reported two patients from different KNDLRS families. With the detailed medical history consultation and clinical examination, they are preliminarily diagnosed as KNDLRS. Validated by the Medical WES V4 and Sanger sequencing, we found the pathogenic variants in FERMT1. The patient III-1 has the compound heterozygous variant, and the patient IV-1 who is from a consanguineous marriage family has the homozygous variant. It was known that the majority of KNDLRS patients are homozygous for their mutations, and are predominantly offspring of consanguineous marriages, or originate from isolated populations [17]. c.193C>T and c.277C>T which are both nonsense mutations will lead to the early termination of protein translation, c.220delC which is a frameshift variant, will lead to the amino change p.H74Tfs*31. To the best of our knowledge, the mutations of c.220delC and c.277C>T have not been reported previously. According to the 2015 ACMG guidelines [18], these mutations were preliminarily determined to be pathogenic. In addition, we used the SIFT, PolyPhen-2, Mutation Taster, GERP++, REVEL to predict the pathogenicity of variants, all results were shown as unknown. We also used the Swissmodel to predict the mutated protein structure, all mutations will lead to the loss of protein functional domain. Although the precise consequence of this mutation remains to be established, the N-terminal region of kindlin's is believed to be important for interaction with binding partners, such as integrins, mis ling, and integrin linked kinase [19,20].

Conclusions
In summary, we have reported two Chinese Kindler syndrome patients, who had been molecularly con rmed to carry loss-of-function FERMT1 mutations (c.193C > T and c.277C > T; c.220delC). These ndings will expand the mutation spectrum of FERMT1 and provide a detailed mutation repertoire of FERMT1 in KNDLRS.

Molecular genetic analysis
For the Medical Whole Exome Sequencing (WES) V4, we prepared 1 to 3 μg of genomic DNA and used the Bioruptorsonicator (Diagenode, NJ, USA) to make about 180bp fragments. By using a DNA sample prep reagent set 1 (NEBNext, MA, USA), the paired-end sequencing libraries were prepared according to Illumina protocols. The ampli ed DNA was captured use GenCap Medical whole exome capture kit (MyGenosticsGenCap Enrichment technologies). The DNA probes were designed to tile along the exon regions of 23000~ genes. The DNA capture was conducted according to manufacturer's protocol. Finally, the high throughput sequencing was performed by Illumina HiSeq X sequencer for paired read 150bp.
After sequencing, we used bioinformatics to screen and analyze the potential pathogenic mutations.
After the suspicious mutation highly related to the disease phenotype is determined, Sanger sequencing was conducted to verify with all the other family members available. Appropriate primer in pairs were designed for mutation sites (Forward-AAGAGTCTACAGGGCACAGG and Reverse-CTAATGCCATCCCAGTCCCT), and the ampli ed product is a 481bp fragment. The sequencing results was compared with FERMT1 (NM_017671) as in GRCh37/hg19 human reference sequence. Clinvar [21], Using SWISS-model, we predicted three-dimensional structure of both wild and mutated kindlin-1 proteins that encoded by FERMT1. Speci c steps according to software requirements [23,24]. In brief, input the protein sequence to be predicted, and click build model rst, then choose the right model. Finally, the predicted model is opened with the visual analysis software Swiss-pdbviewer.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests   Pedigree of family 2 and the clinical features of KNDLRS patient. Pedigree of the family 2 (circle indicated female, square indicated male, two lines indicated marriage between close relatives, black indicated carrier of FERMT1 mutation and blank indicated normal). The arrow represents the proband IV-1 (A). B. The phenotype picture of a 30-year-old female proband in family 2. a-b showed diffuse skin blisters and heterochromatism in trunk and back. c-j showed the four limbs of patient. It showed that limb skin atrophy is obvious, limb blisters, skin peeling with cracks. k-m. Routine hematoxylin and eosin (H&E) stains showed atrophy of epidermis, hyperkeratosis, and dilated blood vessels in upper dermis, and microbubbles at the dermis and epidermis junction.   reported in present study were highlighted in GREEN.