Pustular diseases and keratinocyte-myeloid synergy.

Pustules are among the most common lesions produced in human skin. Infections by pathogens and drug-induced reactions are frequent causes of pustule formation. In recent years immune-mediated pustular diseases have drawn attention. It is proposed to classify pustular diseases according to the initiating events and sites: purely epidermal pustules, follicular pustules or pustules noted in autoinflammatory syndromes. The unifying pathology in all of the three categories is a microinvasion of activated neutrophils into epidermal or adnexal epithelia. Formation of pustules involves established IL-17 / IL-23, IL-36 / IL-36RN driven pathology, or IL-1 /caspase-activated autoinflammation. Pathophysiology demonstrates an intriguing synergy of keratinocytes with neutrophils. This is called keratinocyte-myeloid synergy (KMS). Non-infectious pustules are formed by IFNα controlling the production of chemoattractants (IL-8, LTB4) or induced by IL-1-regulated inflammasomes and caspase/ IFNß-induced chemotaxins. The presence of physical barriers, for example, cornified cell layers (str. corneum), is instrumental in establishing chemotactic gradients and blocking migrating neutrophils. In follicular KMS-driven pustular disorders, in contrast to epidermal pustules, neutrophil-mediated toxicity propagates lasting and expanding ulcerating diseases with increased levels of circulating immunoglobulin A (IgA). Complexed IgA is suggested to propagate ongoing pustular diseases. These are prerequisites essential for developing pustules in burdensome human skin diseases.

Novel SBF2 mutations and clinical spectrum of Charcot-Marie-Tooth neuropathy type 4B2.

Biallelic SBF2 mutations cause Charcot-Marie-Tooth disease type 4B2 (CMT4B2), a sensorimotor neuropathy with autosomal recessive inheritance and association with glaucoma. Since the discovery of the gene mutation, only few additional patients have been reported. We identified seven CMT4B2 families with nine different SBF2 mutations. Revisiting genetic and clinical data from our cohort and the literature, SBF2 variants were private mutations, including exon-deletion and de novo variants. The neuropathy typically started in the first decade after normal early motor development, was predominantly motor and had a rather moderate course. Electrophysiology and nerve biopsies indicated demyelination and excess myelin outfoldings constituted a characteristic feature. While neuropathy was >90% penetrant at age 10 years, glaucoma was absent in ~40% of cases but sometimes developed with age. Consequently, SBF2 mutation analysis should not be restricted to individuals with coincident neuropathy and glaucoma, and CMT4B2 patients without glaucoma should be followed for increased intraocular pressure. The presence of exon-deletion and de novo mutations demands comprehensive mutation scanning and family studies to ensure appropriate diagnostic approaches and genetic counseling.

Saliva induces expression of antimicrobial peptides and promotes intracellular killing of bacteria in keratinocytes by epidermal growth factor receptor transactivation.

BACKGROUND: Wounds in the oral cavity, constantly exposed to both saliva and bacteria, heal quickly without infection. Furthermore, during licking of skin wounds, saliva promotes wound healing and plays a role in keeping the wound free of infection. OBJECTIVES: To investigate whether saliva induces expression of antimicrobial peptides (AMPs) in human epidermal keratinocytes and whether saliva promotes clearance of intracellular bacteria in these cells. METHODS: Expression of AMPs was investigated in the oral mucosa and ex vivo injured skin by immunohistochemistry. Human beta-defensin-3 expression was investigated in epidermal keratinocytes after saliva stimulation, using real-time polymerase chain reaction and immunofluorescence. RESULTS: We found higher expression of AMPs in the oral mucosa than in the epidermis. Saliva accelerated the injury-induced expression of AMPs in human skin ex vivo and was a potent inducer of the expression of AMPs in epidermal keratinocytes. The expression of AMPs was induced by metalloproteinase-dependent epidermal growth factor receptor (EGFR) transactivation mediated by a salivary lipid. Saliva increased the intracellular clearance of Staphylococcus aureus in keratinocytes through EGFR activation. CONCLUSIONS: These findings suggest a previously unreported role of saliva in innate immunity and demonstrate for the first time that saliva induces gene expression in epidermal keratinocytes.

Novel TPM3 mutation in a family with cap myopathy and review of the literature.

Cap myopathy is a rare congenital myopathy characterized by the presence of caps within muscle fibres and caused by mutations in ACTA1, TPM2 or TPM3. Thus far, only three cases with TPM3-related cap myopathy have been described. Here, we report on the first autosomal dominant family with cap myopathy in three-generations, caused by a novel heterozygous mutation in the alpha-tropomyosin-slow-encoding gene (TPM3; exon 4; c.445C>A; p.Leu149Ile). The three patients experienced first symptoms of muscle weakness in childhood and followed a slowly progressive course. They presented generalized hypotrophy and mild muscle weakness, elongated face, high arched palate, micrognathia, scoliosis and respiratory involvement. Intrafamilial variability of skeletal deformities, respiratory involvement and mild cardiac abnormalities was noted. Muscle MRI revealed a recognizable pattern of fatty muscle infiltration and masseter muscle hypertrophy. Subsarcolemmal caps were present in 6-10% of the fibres and immunoreactive with anti-tropomyosin antibodies. We conclude that the MRI-pattern of muscle involvement and the presence of masseter muscle hypertrophy in cap myopathy may guide molecular genetic diagnosis towards a mutation in TPM3. Regular respiratory examinations are important, even if patients have no anamnestic clues. We compare our findings to all cases of cap myopathy with identified mutations (n=11), thus far reported in the literature.

Immunohistological pointers to a possible role for excessive cathelicidin (LL-37) expression by apocrine sweat glands in the pathogenesis of hidradenitis suppurativa/acne inversa.

BACKGROUND: The cause of follicular occlusion, a key early event in the pathogenesis of hidradenitis suppurativa (HS), also known as acne inversa, remains unknown. OBJECTIVES: To identify changes, if any, in the antimicrobial peptide (AMP) and cytokine expression profile of HS affected human skin. METHODS: Quantitative immunohistomorphometry was used to compare the in situ protein expression of selected AMPs and cytokines in lesional HS skin from 18 patients with that in healthy skin (n = 12). The lesional skin from patients with HS was histologically subclassified based on the predominance of inflammation vs. scarring. RESULTS: Compared with healthy controls, significantly increased immunoreactivity for cathelicidin (LL-37) was noted in the apocrine sweat gland and distal outer root sheath (ORS) of the hair follicle (HF) epithelium in lesional HS skin. Immunoreactivity for LL-37, psoriasin, human ß-defensin 3 (hBD3), α-melanocyte stimulating hormone (α-MSH), macrophage migration inhibitory factor (MIF), tumour necrosis factor (TNF)-α and interleukin (IL)-8 was significantly increased in HS epidermis. LL-37 and TNF-α immunoreactivity was also increased in the dermis of lesional HS skin. In contrast, lysozyme expression was decreased in the epidermis of lesional HS skin, while that of TNF-α and IL-8 was decreased in the proximal ORS of HFs in HS lesions. These differences were most pronounced in HS with predominant inflammation. CONCLUSIONS: Our observations raise the question as to whether excessive secretion of AMPs by the skin, in particular by the apocrine sweat glands, distal HF epithelium, and epidermis, may attract inflammation and thus facilitate or promote HS development.

Oculopharyngeal muscle dystrophy: fine structure and mRNA expression levels of PABPN1.

OBJECTIVE: The underlying molecular mechanism leading to the OPMD causing mutation in the PABPN1 gene has not been elucidated so far. Two models are under consideration: the first model is the polymerase slippage mechanism. The second model is unequal crossing over. The aim of the present study is to correlate clinical, fine structural, and molecular genetic data. MATERIAL AND METHODS: In 6 cases of OPMD, confirmed by electron microscopy, we analyzed mutations in exon 1 of the polyadenine binding protein nuclear1 (PABPN1) gene on chromosome 14q11.1 using DNA isolated from biopsied muscle tissue. Furthermore, the corresponding mRNA from frozen biopsies was analyzed. RESULTS: In addition to the usual expansion of the (GCG)6 sequence to the well known (GCG)8-13 trinucleotide repeats in 5 of the patients, we detected a novel (GCA)2(GCG) insertion in one patient. This mutation favors a pathomechanism of "unequal crossing over" instead of a "polymerase slippage" model. Tubulofilamentous (8.5 nm) nuclear inclusions were especially prominent in an isolated nucleus of a nuclear clump in a severely atrophic muscle fiber. However, no correlation was found between muscle weakness, the frequency of repeats, and the frequency and size of nuclear inclusions. CONCLUSIONS: Muscle weakness was not obviously correlated to the number of repeats, but it is suggested that it might be linked to an increase of the transcription rate representing the ratio between mutated versus normal RT-PCR products.

Dermal fibroblasts from acute inflamed atopic dermatitis lesions display increased eotaxin/CCL11 responsiveness to interleukin-4 stimulation.

BACKGROUND: The presence of eosinophils and/or eosinophil-derived products in the dermis is characteristic for involved skin of patients with atopic dermatitis and contributes to the observed tissue injury. CCL11 is a potent chemoattractant and activator of human eosinophils and interleukin (IL)-4 is a potent inducer of CCL11 expression in dermal fibroblasts. OBJECTIVES: As increased fibroblast CCL11 expression may explain eosinophilic infiltration of involved skin areas in atopic dermatitis, we asked whether dermal fibroblasts from atopic patients differ from fibroblasts of healthy individuals in their ability to express CCL11. METHODS: We compared IL-4-induced CCL11 mRNA expression using reverse transcription-polymerase chain reaction from cultured dermal fibroblasts derived from biopsies of chronic lesional and acute lesional atopic skin as well as from skin biopsies derived from normal skin of healthy donors. RESULTS: Considerable variability in IL-4-induced relative CCL11 mRNA expression was detected in fibroblasts derived from biopsies of different individuals. The lowest median IL-4 concentration inducing half maximal CCL11 mRNA expression (EC(50)) was found in fibroblasts derived from acute inflamed atopic lesions. CONCLUSIONS: Inducibility of CCL11 in dermal fibroblasts upon stimulation with Th2 cytokines explains the tissue eosinophilia observed in the presence of Th2 cytokines and the localization of eosinophils to the dermis. Decreased EC(50) values of IL-4-induced CCL11 expression in fibroblasts from acute inflamed atopic skin lesions indicates increased IL-4 responsiveness in these lesions and further substantiates the special role for IL-4-induced dermal fibroblast CCL11 expression in acute lesions. Variable CCL11 expression in fibroblasts from different patients with atopic dermatitis indicates heterogeneity of factors determining atopic phenotype in atopic dermatitis.

Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract.

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

Incontinetia pigmenti-related myopathy or unsolved "double trouble"?

Incontinentia pigmenti is an X-linked dominant or sporadic multisystemic disorder with involvement of skin, eyes and central nervous system which results from mutations in the gene for NF-kappaB essential modulator (NEMO). We report on a patient with genetically confirmed Bloch-Sulzberger syndrome, who presented with a progressive myopathy and cardiomyopathy. Genetic analyses revealed an intragenic deletion (Intron3 and Exon10) of the NEMO/IKKgamma/IKKAP/IKBKG gene. Further complete sequencing of genes encoding for desmin, lamin A/C, emerin, and FHL1 showed no evidence of pathogenic mutations. A pathological expansion of CCTG repeats of the ZNF9 gene (PROMM) was ruled out by PCR amplification analysis. MLPA-analysis showed no evidence for duplications or deletions of the dystrophin gene. This report highlights the unusual combination of a genetically confirmed incontinentia pigmenti and a proximal myopathy and dilatative cardiomyopathy of unknown origin. We discuss that the striated muscle involvement (i) might be based on the observed intragenic deletion of the NEMO gene, or (ii) on an additional gene defect leading to an adult onset myopathy. Further studies on neuromuscular involvement in patients with incontinentia pigmenti are needed to clarify this issue.

Increased expression of human beta-defensin 3 in mollusca contagiosum.

The human beta defensins (hBDs)-2 and -3 are inducible antimicrobial peptides present in human skin. Besides an important role in fighting bacteria, they also have an antiviral function. Molluscum contagiosum (MC) is a cutaneous viral disease caused by the MC virus. Lesions show a tendency towards spontaneous regression, which might be caused by antiviral proteins such as defensins. We report a marked increase in hBD-3 immunoreactivity in MC lesions in contrast to hBD-2, which was only marginally increased. We suggest a role for the hBD-3 peptide in MC pathogenesis.

Human gammadelta T cells produce the protease inhibitor and antimicrobial peptide elafin.

Human gammadelta T cells rapidly secrete pro-inflammatory cytokines in response to T cell receptor-dependent recognition of pyrophosphates produced by many bacteria and parasites. In further support of an important role of gammadelta T cells in the immune defence against infection, human gammadelta T cells have been shown to produce the antimicrobial peptide LL37/cathelicidin. In this study, we have investigated whether gammadelta T cells can produce additional antimicrobial peptides. To this end, we have screened human gammadelta T cell clones by RT-PCR for mRNA expression of a broad range of antimicrobial peptides. While alpha-defensins were absent and beta-defensins (HBD1) present only in rare gammadelta T cell clones, elafin mRNA was induced by supernatant of Pseudomonas aeruginosa grown under static conditions. Elafin is a protease inhibitor that also displays antimicrobial activity. Constitutive intracellular expression of elafin was demonstrated by flow cytometry and Western blot analysis. Furthermore, trappin-2 (pre-elafin) could be immunoprecipitated in cell lysates but also in the supernatant of gammadelta T cells stimulated by Ps. aeruginosa supernatant. Taken together, our studies reveal a novel effector function of gammadelta T cells which might be important for local immune defence.

Human hair follicle epithelium has an antimicrobial defence system that includes the inducible antimicrobial peptide psoriasin (S100A7) and RNase 7.

BACKGROUND: Hair follicle (HF) ostia represent a potential port of microbial entry into the skin. However, they rarely show clinical signs of infection. This suggests the presence of local, efficient, antimicrobial defence systems, which may include antimicrobial peptides (AMPs). OBJECTIVES: We determined the presence and distribution of the major AMPs, RNase 7 and psoriasin (S100A7), in human scalp HFs. We investigated whether HF production of these AMPs was induced by prototypic microbial products and proinflammatory cytokines, i.e. interferon (IFN)-gamma. Finally, we examined whether the classical pathways for AMP induction, such as toll-like receptor (TLR)4 and TLR5 expression, are present in human HFs and up-regulated after stimulation with bacterium-associated ligands. METHODS: Cryosections from fresh or organ-cultured full-thickness normal human scalp skin treated with lipopolysaccharide (LPS), flagellin, protein A, lipoteichoic acid (LTA) or IFN-gamma were stained for psoriasin and RNase 7 immunoreactivity (IR) as well as for TLR4 and TLR5. In addition, outer root sheath cell culture and semiquantitative analysis of mRNA expression levels of RNase 7 and psoriasin were performed. RESULTS: Specific RNase 7 IR was present throughout the entire HF outer root sheath in situ and in cell culture, whereas psoriasin IR was present only in the most distal compartment and not detectable in cultured ORS cells. Upon treatment with Gram-positive (LTA, protein A) or Gram-negative bacterial (LPS, flagellin) cell wall components, or with the cytokine IFN-gamma, the IR of both psoriasin and RNase 7 was modified. TLR4 and TLR5 IR was detected in the normal HF epithelium and were upregulated after treatment with their respective ligand. The mRNA analysis confirmed the immunohistochemistry results. CONCLUSIONS: This pilot study suggests that normal human scalp HF epithelium possesses a functional antimicrobial defence system, which includes the AMPs RNase 7 and psoriasin, and TLRs, and that these are induced by classical microbial products.

Psoriasin (S100A7) is a principal antimicrobial peptide of the human tongue.

The human tongue is particularly resistant to bacterial infections although the mouth is continuously exposed to a complex and abundant ensemble of microbes, such as the common intestinal bacterium Escherichia coli. We show that lingual epithelia produce and release, as a primary E. coli-killing compound, the S100 protein psoriasin. No significant reduction in psoriasin release could be achieved through repeated rinsing of the epithelial surface of the tongue. Psoriasin is produced in the upper layers of the lingual epithelia but is lacking in the most superficial and basal cells. It displays a gradient pattern of expression with decreasing expression from the anterior one-third to the posterior portion of the tongue. Thus, psoriasin may be the key to the resistance of the human tongue toward E. coli.

Expression of human beta-defensin-2 and -3 in verrucae vulgares and condylomata acuminata.

The human beta defensins (hBDs) 2 and 3 contribute to the inducible antimicrobial peptides in human skin. Besides an important role in fighting bacteria, they may also exert antiviral function. Verrucae vulgares and condylomata acuminata are typical cutaneous viral diseases caused by different subtypes of the human papillomavirus. Their tendency for spontaneous regression could be caused by antiviral proteins like defensins. In a retoperspective study, we investigated lesions of verrucae vulgares and condylomata acuminata for the presence of hBD-2 and hBD-3 by immunohistochemistry. All of the specimens of verrucae vulgares (n = 20) were positive for hBD-2 and hBD-3. The specimens of condylomata acuminata (n = 15) exhibited expression for hBD-2 and hBD-3, with the exception of three and two samples, respectively. The expression levels in condyloma acuminata were lower for both defensins compared with verrucae vulgares. Normal skin tissue exhibited no staining of both hBD-2 and hBD-3 with the exception of two cases. Taken together, this is the first report of an increased expression of the hBD-2 and hBD-3 in cutaneous papillomavirus infections.

Who is really in control of skin immunity under physiological circumstances - lymphocytes, dendritic cells or keratinocytes?

Our views of the skin immunity theatre are undergoing constant change. These not only reflect paradigm shifts in general immunology and skin biology, but also have profound clinical implications, which call for strategic changes in dermatological therapy. Nowhere can this be witnessed at a greater level of instructiveness and fascination than when addressing the question posed by this new Controversies feature. Thus, after a very long period of dominance by T cells and Langerhans cells as 'lead actors' on the skin immunity stage, the lowly keratinocyte has recently made an astounding theatrical appearance as a key protagonist of the innate skin immunity system, which may control even acquired skin immune responses. Further enhancing dramatic complexity and tension, the mast cell has entered as an additional actor claiming centre stage, and the epidermal Langerhans cell has slipped in a surprise appearance as the chief agent of immunotolerance. May you, esteemed reader, enjoy the spectacle offered here by selected immunodermatology authorities who double as 'stage managers' pushing their respective favourite actors into the limelight. You get everything you may expect from a good performance - complete with the impresario's overture that lures you into the theatre and sets the stage, competing divas, recently discovered new talents and even the critic's digest while the performance is still ongoing. By the time the curtain drops, you will have reached your own, independent conclusions on how to answer the title question of this play - at least for the time being...

Antimicrobial skin peptides and proteins.

Human skin is permanently exposed to microorganisms, but rarely infected. One reason for this natural resistance might be the existence of a 'chemical barrier' consisting in constitutively and inducibly produced antimicrobial peptides and proteins (AMPs). Many of these AMPs can be induced in vitro by proinflammatory cytokines or bacteria. Apart from being expressed in vivo in inflammatory lesions, some AMPs are also focally expressed in skin in the absence of inflammation. This suggests that non-inflammatory stimuli of endogenous and/or exogenous origin can also stimulate AMP synthesis without inflammation. Such mediators might be ideal 'immune stimulants' to induce only the innate antimicrobial skin effector molecules without causing inflammation.

Peripheral nerve and skeletal muscle involvement in CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by degeneration of vascular smooth muscle cells (VSMC) of nearly all tissues studied so far. The clinical phenotype of CADASIL shows great variability. The disease is caused by mutations of the Notch3 gene encoding the transmembrane receptor Notch3, which is expressed predominantly in VSMC. In some patients, neuromuscular symptoms have been described. To investigate the fine structural features of peripheral nerve and muscle biopsy specimens in more cases and greater detail, seven electron microscopically confirmed CADASIL patients showing a variable amount of granular osmiophilic material on the surface of VSMC were included in this study. Pathogenic mutations within the cluster region (exon 3 and 4) of the Notch3 gene were identified in six cases. Degeneration and regeneration of nerve fibers in the sural nerves, studied in four cases, was present, although moderate, in all nerve biopsy specimens, whereas an intramuscular nerve fascicle showed more severe changes. Enlarged mitochondria with needle-like calcium precipitates were repeatedly seen. In muscle biopsy specimens, some degree of neurogenic atrophy was apparent in addition to myopathic changes, including occasional ragged red fibers with abnormally large mitochondria, focal tubular aggregates, abnormal terminal cisternae, and myofibrillary abnormalities. Automated sequence analysis of the whole mitochondrial DNA performed in one patient revealed several nucleotide polymorphisms, which were not considered pathogenic. The findings suggest that in CADASIL degeneration of small blood vessels is initiated by defects of the surface membrane of VSMC. Dysfunction of these blood vessels may cause low-grade chronic ischemia with secondary hypoxidosis and a large variety of structural changes noted in skeletal muscle and peripheral nerves, although a primary influence of the underlying genetic defect can not be excluded.

Primary carnitine deficiency: adult onset lipid storage myopathy with a mild clinical course.

We studied two adult patients with myalgia and muscular fatigability during prolonged physical exercise. Serum creatine kinase was increased and muscle biopsy revealed a lipid storage myopathy affecting predominantly the type I fibres. Skeletal muscle carnitine content was reduced to 15% and 21% of the normal mean values, while serum carnitine levels were either normal or decreased. Four months of oral therapy with L-carnitine (3 g per day) resolved the clinical symptoms completely in both patients, and a subsequent muscle biopsy confirmed a marked reduction of lipid storage, along with increased muscle carnitine levels. The analysis of renal carnitine excretion and the exclusion of possible secondary carnitine deficiencies in both patients are compatible with mild defects of the carnitine transporter in one patient and of carnitine biosynthesis in the other. Since myalgia and muscular fatigue are frequent but unspecified complaints of otherwise clinically unremarkable adult patients, it is important to identify myopathies associated with primary carnitine deficiency because they may be amenable to treatment.

NOD2 (CARD15) mutations in Crohn's disease are associated with diminished mucosal alpha-defensin expression.

BACKGROUND: Mutations in NOD2, a putative intracellular receptor for bacterial peptidoglycans, are associated with a subset of Crohn's disease but the molecular mechanism linking this protein with the disease pathogenesis remains unclear. Human alpha defensins (HD-5 and HD-6) are antibiotic effector molecules predominantly expressed in Paneth cells of the ileum. Paneth cells also express NOD2. To address the hypothesis that the function of NOD2 may affect expression of Paneth cell defensins, we compared their expression levels with respect to NOD2 mutations in Crohn's disease. METHODS: Forty five Crohn's disease patients (24 with NOD2 mutations, 21 with wild-type NOD2) and 12 controls were studied. Real time reverse transcription-polymerase chain reaction was performed with mucosal mRNA for HD-5, HD-6, lysozyme, secretory phospholipase A2 (sPLA2), tumour necrosis factor alpha, interleukin 8, and human hypoxanthine phosphoribosyltransferase (housekeeping gene). Immunohistochemistry with anti-HD-5 and histological Paneth cell staining were performed in 10 patients with NOD2 mutations or wild-type genotypes. RESULTS: Ileal expression of HD-5 and HD-6, but not sPLA2 or lysozyme, were diminished in affected ileum, and the decrease was significantly more pronounced in patients with NOD2 mutations. In the colon, HD-5, HD-6, and sPLA2 were increased during inflammation in wild-type but not in NOD2 mutated patients. In both the colon and ileum, proinflammatory cytokines and lysozyme were unaffected by NOD2 status. Immunohistochemistry identified Paneth cells as the sole source of HD-5. CONCLUSION: As alpha defensins are important in the mucosal antibacterial barrier, their diminished expression may explain, in part, the bacterial induced mucosal inflammation and ileal involvement of Crohn's disease, especially in the case of NOD2 mutations.