Diagnosis, treatment, and long-term outcomes of pediatric pemphigus: a retrospective study at tertiary medical centers.

BACKGROUND: Pediatric pemphigus is a rare bullous disease that represents a diagnostic and therapeutic challenge;  evidence on patients' response to various treatments and long-term surveillance data are lacking. We aimed to investigate pediatric pemphigus patients' characteristics, diagnosis, therapeutics, response, and long-term follow-up. METHODS: This is a retrospective study of all pemphigus patients aged <18 years, diagnosed between 2000 and 2023, from three tertiary medical centers in Israel. The diagnosis was confirmed by positive immunofluorescence. RESULTS: Twelve pediatric pemphigus patients were included (mean age 10.7 ± 4.3 years, male:female ratio 1:1). Mean diagnostic delay was 11.1 ± 12.6 months (range 1.8-36 months). Most patients had pemphigus vulgaris with mucosal involvement (58.3%). First-line treatment for all patients included systemic corticosteroids (sCS), with a treatment duration (including tapering down) of 28 ± 18.4 months. Hospitalization did not yield better outcomes. Only three patients achieved sustained complete response with sCS treatment (25.0%), and the rest required additional therapeutics, most commonly rituximab. Rituximab showed a good safety profile and therapeutic response. Follow-up was recorded up to 18.1 years after diagnosis (mean: 5.6 years). Three of five patients with information available more than 5 years after the pemphigus diagnosis still exhibited disease symptoms. CONCLUSIONS: Pediatric pemphigus is associated with a significant diagnostic delay. While sCS can induce remission in most patients as a first-line treatment, long-term disease control requires additional immunomodulators. Long-term follow-up reveals a chronic yet mostly benign disease course in this population and advocates for the use of rituximab in pediatric pemphigus patients.

Persistent Cutaneous Lesions of Darier Disease and Second-Hit Somatic Variants in ATP2A2 Gene.

Importance: Darier disease (DD) is a rare genetic skin disorder caused by heterozygous variants in the ATP2A2 gene. Clinical manifestations include recurrent hyperkeratotic papules and plaques that occur mainly in seborrheic areas. Although some of the lesions wax and wane in response to environmental factors, others are severe and respond poorly to therapy. Objective: To investigate the molecular mechanism underlying the persistency of skin lesions in DD. Design, Setting, and Participants: In this case series, DNA was extracted from unaffected skin, transient and persistent lesional skin, and blood from 9 patients with DD. Genetic analysis was used using paired-whole exome sequencing of affected skin and blood or by deep sequencing of ATP2A2 of affected skin. Chromosomal microarray analysis was used to reveal copy number variants and loss of heterozygosity. All variants were validated by Sanger sequencing or restriction fragment length polymorphism. Interventions or Exposures: Paired whole-exome sequencing and deep sequencing of ATP2A2 gene from blood and skin samples isolated from persistent, transient lesions and unaffected skin in patients with DD. Main Outcomes and Measures: Germline and somatic genomic characteristics of persistent and transient cutaneous lesions in DD. Results: Of 9 patients with DD, all had heterozygous pathogenic germline variants in the ATP2A2 gene, 6 were female. Participant age ranged from 40 to 69 years on enrollment. All 11 persistent skin lesions were associated with second-hit somatic variants in the ATP2A2 gene. The somatic variants were classified as highly deleterious via combined annotation-dependent depletion (CADD) scores or affect splicing, and 3 of them had been previously described in patients with DD and acrokeratosis verruciformis of Hopf. Second-hit variants in the ATP2A2 gene were not identified in the transient lesions (n = 2) or the normal skin (n = 2). Conclusions and Relevance: In this study, persistent DD lesions were associated with the presence of second-hit somatic variants in the ATP2A2 gene. Identification of these second-hit variants offers valuable insight into the underlying mechanisms that contribute to the lasting nature of persistent DD lesions.

Lack of mitochondrial complex I assembly factor NDUFAF2 results in a distinctive infantile-onset brainstem neurodegenerative disease with early lethality.

BACKGROUND: Congenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported. METHODS: Clinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016-2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays. RESULTS: All four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2-4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease. CONCLUSIONS: Biallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage.

Trends of Diagnosis, Disease Course, and Treatment of Atopic Dermatitis 2012-2021: Real-World Data from a Large Healthcare Provider.

In the last decade, new treatments for atopic dermatitis (AD) have emerged. We aimed to describe trends of the diagnosis, disease course, and treatment of AD over a decade (2012-2021) using data from Maccabi Healthcare Services (a 2.7-million-member healthcare provider in Israel). The AD prevalence was stable (4.0% on 31 December 2021 vs. 4.3% on 31 December 2012). The annual AD incidence was also stable (5.8/1000 in 2012 and 5.7/1000 in 2021). AD-related treatment use was highest in the first year post-diagnosis, and it included, among children (n = 87,414) vs. adults (n = 36,865), low-potency topical corticosteroids (TCS) (41.8% vs. 27.1%), mid-potency TCS (30.1% vs. 28.1%), high-potency TCS (34.9% vs. 60.3%), topical calcineurin inhibitor (10.8% vs. 10.1%), phosphodiesterase-4-inhibitor (0.3% vs. 0.7% overall; approved in 2019), phototherapy (0.1% vs. 2.3%), and systemic/biologic treatments (13.0% vs. 13.3%). Among children diagnosed in 2012 and followed through to 2021 (n = 5248), 21.5% had ≥1 AD diagnosis/treatment 10 years later (among 3223 adults: 38.3%). We conclude that the incidence and prevalence rates of AD were comparable to those in similar database studies and remained relatively stable over the past decade. The results underscore the burden of medication use among children and adults, particularly in the first year after AD diagnosis, and the low rate of AD diagnosis among patients originally diagnosed as children 10 years earlier.

Treatment with Methotrexate in Infants and Toddlers with Atopic Dermatitis: A Retrospective Multi-Center Study.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% of children. Methotrexate (MTX) is used off-label as a systemic treatment for AD patients unresponsive to topical therapies, but limited data exist regarding its safety and efficacy in children, especially in those < 4 years old. To further investigate MTX in younger patients, we screened the medical records of three referral centers between 2016 and 2022 and identified 28 infants and toddlers < 4 years old with AD treated with MTX. Mean age upon MTX initiation was 2.7 ± 1.2 years and mean investigator global assessment (IGA) score was 3.78 ± 0.4. Median duration of MTX treatment was five months. Following 12 and 24 weeks of MTX treatment, the response rate was 50% and IGA 0/1 was achieved in 14.2% and 21.4% of patients, respectively. Most treatment cessations were attributed to a lack of efficacy or parental concern. Although adverse events were reported in 57.1% of patients, MTX was discontinued due to such adverse events only in two patients (7.1%). Taken together, MTX demonstrated a high safety profile in AD patients <4 years old. MTX efficacy was moderate and presumably underestimated by parents who opted for premature treatment cessation due to concerns associated with an immunomodulatory drug.

A homozygous variant in CHMP3 is associated with complex hereditary spastic paraplegia.

BACKGROUND: Monogenic neurodegenerative diseases represent a heterogeneous group of disorders caused by mutations in genes involved in various cellular functions including autophagy, which mediates degradation of cytoplasmic contents by their transport into lysosomes. Abnormal autophagy is associated with hereditary ataxia and spastic paraplegia, amyotrophic lateral sclerosis and frontal dementia, characterised by intracellular accumulation of non-degraded proteins. We investigated the genetic basis of complex HSP in a consanguineous family of Arab-Muslim origin, consistent with autosomal recessive inheritance. METHODS: Exome sequencing was followed by variant filtering and Sanger sequencing for validation and familial segregation. Studies for mRNA and protein expression used real-time PCR and immunoblots. Patients' primary fibroblasts were analysed using electron microscopy, immunofluorescence, western blot analysis and ectopic plasmid expression for its impact on autophagy. RESULTS: We identified a homozygous missense variant in CHMP3 (Chr2:86507484 GRCh38 (NM_016079.4): c.518C>T, p.Thr173Ile), which encodes CHMP3 protein. Segregation analysis validated the presence of the homozygous variant in five affected individuals, while healthy family members were found either heterozygous or wild type for this variant. Primary patient's fibroblasts showed significantly reduced levels of CHMP3. Electron microscopy disclosed accumulation of endosomes, autophagosomes and autolysosomes in patient's fibroblasts, which correlated with higher levels of autophagy markers, p62 and LC3-II. Ectopic expression of wild-type CHMP3 in primary patient fibroblasts led to reduction of the p62 particles accumulation and number of endosomes and autophagosomes compared with control. CONCLUSIONS: Reduced level of CHMP3 is associated with complex spastic paraplegia phenotype, through aberrant autophagy mechanisms.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Potential di-genic contribution to guttate leukoderma as the predominant feature of epidermolysis bullosa simplex.

Inherited epidermolysis bullosa (EB) simplex is a heterogeneous group of skin fragility disorders caused by mutations in genes encoding cell-cell or cell-matrix adhesion proteins. A recently identified, rare subtype of EB simplex is due to bi-allelic mutations in the EXPH5 gene, which encodes exophilin5, an effector protein of the Rab27B GTPase involved in intracellular vesicle trafficking and exosome secretion. The EXPH5 EB subtype is characterized by early-onset skin blisters and scars, mainly on extremities, and varying degrees of pigmentary alterations. Here, we present a 31-year-old female with diffuse guttate hypopigmentation on the trunk and extremities since early childhood, with no apparent blisters or scars. We employed whole exome sequencing of germline DNA extracted from the patient's leukocytes to determine the genetic aetiology of the phenotype. A novel homozygous variant in EXPH5, c.1153C>T causing a premature stop codon at amino acid Glutamine 385, was identified. Histologic examination after skin pricking disclosed focal keratinocyte detachment typical to EB. Additionally, we identified a deleterious-predicted variant in ENPP1, a gene associated with disturbed transfer of melanosomes to keratinocytes in Cole disease. Our report expands the clinical spectrum of inherited EB simplex with a possible di-genic synergism contributing to co-presentation with guttate leukoderma.

Characteristics of Atopic Dermatitis Patients Treated with Crisaborole: Real-World Data from a Large Healthcare Provider Database in Israel.

Background: In recent years, new treatments dedicated to atopic dermatitis (AD) have become available in Israel, including crisaborole, a small molecule with unique benzoxaborole chemistry. Objective: To describe baseline characteristics, history of AD therapies, and use of health-care services of early crisaborole users in real-world settings. Methods: A retrospective cohort study was performed using the data of a large health provider in Israel. AD patients treated with crisaborole since it became commercially available in Israel in July 2019 through end of Sep 2020, were included. Baseline demographics and clinical characteristics, prior AD-related treatments and healthcare resource utilization were collected. Results: A total of 441 patients were included (57.8% females, median age = 21.1y; interquartile range = 10.5-40.8). In 62.1%, a dermatologist prescribed the first dispensed crisaborole. Median time from AD diagnosis to crisaborole treatment was 6.6 years. Up to 12 months prior to crisaborole treatment, low-, mid- and high-potency TCS were used by 30.8%, 31.1% and 55.8% of patients, respectively. Treatments related to moderate-to-severe AD were dispensed to 38.5% of patients in the prior 5 years. Asthma and allergic rhinitis were documented among 22.2% and 37.2%, respectively. In the past year, patients had a median of 9 visits to primary care physicians, 84.6% visited a dermatologist (≥5 visits: 12.9%). Conclusion: While crisaborole is indicated for mild-to-moderate disease, results suggest that a significant proportion of patients had history of advanced AD therapies suggestive of moderate-to-severe AD.

Acral peeling in Nagashima type palmo-plantar keratosis patients reveals the role of serine protease inhibitor B 7 in keratinocyte adhesion.

Acral peeling skin syndrome (APSS) is a heterogenous group of genodermatoses, manifested by peeling of palmo-plantar skin and occasionally associated with erythema and epidermal thickening. A subset of APSS is caused by mutations in protease inhibitor encoding genes, resulting in unopposed protease activity and desmosomal degradation and/or mis-localization, leading to enhanced epidermal desquamation. We investigated two Arab-Muslim siblings with mild keratoderma and prominent APSS since infancy. Genetic analysis disclosed a homozygous mutation in SERPINB7, c.796C > T, which is the founder mutation in Nagashima type palmo-plantar keratosis (NPPK). Although not previously formally reported, APSS was found in other patients with NPPK. We hypothesized that loss of SERPINB7 function might contribute to the peeling phenotype through impairment of keratinocyte adhesion, similar to other protease inhibitor mutations that cause APSS. Mis-localization of desmosomal components was observed in a patient plantar biopsy compared with a biopsy from an age- and gender-matched healthy control. Silencing of SERPINB7 in normal human epidermal keratinocytes led to increased cell sheet fragmentation upon mechanical stress. Immunostaining showed reduced expression of desmoglein 1 and desmocollin 1. This study shows that in addition to stratum corneum perturbation, loss of SERPINB7 disrupts desmosomal components, which could lead to desquamation, manifested by skin peeling.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Parental mosaic cutaneous-gonadal GJB2 mutation: From epidermal nevus to inherited ichthyosis-deafness syndrome.

Ichthyosis and deafness syndrome is a group of devastating genodermatoses caused by heterozygous mutations in GJB2, encoding the gap junction protein connexin 26. These syndromes are characterized by severe skin disease, hearing loss, recurrent infections, and cutaneous neoplasms. Cutaneous somatic mutations in the same gene are associated with porokeratotic eccrine ostial dermal duct nevus. Here we report a family in which a parent presented with localized epidermal nevus and his child suffered with hystrix-like ichthyosis with deafness. Histologic examination of the parent's cutaneous lesion revealed verrucous epidermal nevus without features of porokeratotic eccrine ostial dermal duct nevus. Genetic analysis identified the same pathogenic variant, GJB2 c.148G>A (p.D50N), in DNA extracted from the parent's cutaneous lesion and the child's leukocytes, but not in the parent's leukocytes. This study expands the phenotypic heterogeneity of GJB2 mosaic variants in addition to porokeratotic eccrine ostial dermal duct nevus, and emphasizes the importance of molecular diagnosis of mosaic skin diseases considering the risk of severe inherited diseases in the offspring.

Concomitant variants in NF1, LZTR1 and GNAZ genes probably contribute to the aggressiveness of plexiform neurofibroma and warrant treatment with MEK inhibitor.

Neurofibromatosis 1 (NF1) is caused by germline mutations in the NF1 gene and manifests as proliferation of various tissues, including plexiform neurofibromas. The plexiform neurofibroma phenotype varies from indolent to locally aggressive, suggesting contributions of other modifiers in addition to somatic loss of NF1. In this study, we investigated a life-threatening plexiform neurofibroma in a 9-month-old female infant with NF1. Germline mutations in two RASopathy-associated genes were identified using whole-exome sequencing-a de novo pathogenic variant in the NF1 gene, and a known pathogenic variant in the LZTR1 gene. Somatic analysis of the plexiform neurofibroma revealed NF1 loss of heterozygosity and a variant in GNAZ, a gene encoding a G protein-coupled receptor. Cells expressing mutant GNAZ exhibited increased ERK 1/2 activation compared to those expressing wild-type GNAZ. Taken together, we suggest the variants in NF1, LZRT1 and GNAZ act synergistically in our patient, leading to MAPK pathway activation and contributing to the severity of the patient's plexiform neurofibromatosis. After treatment with the MEK inhibitor, trametinib, a prominent clinical improvement was observed in this patient. This case study contributes to the knowledge of germline and somatic non-NF1 variants affecting the NF1 clinical phenotype and supports use of personalized, targeted therapy.

An Update on the Cutaneous Manifestations of Darier Disease.

BACKGROUND: Knowledge about the clinical features of Darier disease, an orphan autosomal-dominant genetic disorder, is sparse and has been evaluated only in few studies. OBJECTIVES: To investigate the clinical features of a large group of patients with Darier disease, and to explore for associations between disease characteristics and severity of the disease. METHODS: Seventy-six individuals with Darier disease were evaluated utilizing a structured questionnaire-based interview, a physical examination, and a retrospective assessment of their medical records. RESULTS: The most frequent locations of lesions were hands (99%) and fingernails (93%). Wart-like lesions on the hands were more visible after soaking them in water for 5 minutes, we therefore named this phenomenon the "wet hand sign". Oral involvement was found in 43% of patients, while 48% of women and 16% of men showed genital lesions. Patients with severe Darier disease had a tenfold greater risk of developing genital lesions than those with mild disease (P = .01). Most patients (88%) in our study exhibited a combination of the four types of the disease patterns of distribution (flexural, seborrheic, nevoid, and acral). CONCLUSIONS: Documentation of disease on the hands and fingernails provides a highly sensitive means to aid in the diagnosis of Darier disease. It is important to evaluate mucosal lesions including genital and oral mucosa.

Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.

Autosomal recessive congenital ichthyosis (ARCI) is a rare and heterogeneous skin cornification disorder presenting with generalized scaling and varying degrees of erythema. Clinical manifestations range from lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) through the most severe form of ARCI, Harlequin ichthyosis (HI). We used homozygosity mapping, whole-exome and direct sequencing to delineate the relative distribution of pathogenic variants as well as identify genotype-phenotype correlations in a cohort of 62 Middle Eastern families with ARCI of various ethnic backgrounds. Pathogenic variants were identified in most ARCI-associated genes including TGM1 (21%), CYP4F22 (18%), ALOX12B (14%), ABCA12 (10%), ALOXE3 (6%), NIPAL4 (5%), PNPLA1 (3%), LIPN (2%) and SDR9C7 (2%). In 19% of cases, no mutation was identified. Our cohort revealed a higher prevalence of CYP4F22 and ABCA12 pathogenic variants and a lower prevalence of TGM1 and NIPAL4 variants, as compared to data obtained in other regions of the world. Most variants (89%) in ALOX12B were associated with CIE and were the most common cause of ARCI among patients of Muslim origin (26%). Palmoplantar keratoderma associated with fissures was exclusively a result of pathogenic variants in TGM1. To our knowledge, this is the largest cohort study of ARCI in the Middle-Eastern population reported to date. Our data demonstrate the importance of population-tailored mutation screening strategies and shed light upon specific genotype-phenotype correlations.

Homozygote loss-of-function variants in the human COCH gene underlie hearing loss.

Since 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While additional recessive variants in the COCH gene (DFNB110) have recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data have a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ.

Biochemical methods can reveal stable protein-protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein-protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay® (PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein-protein interactions, but also can reveal post-translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin-embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma-Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Proximity ligation assay Support Protocol 1: Antigen retrieval method for formalin-fixed, paraffin-embedded tissues Support Protocol 2: Creation of custom PLA probes using the Duolink™ In Situ Probemaker Kit when commercially available probes are not suitable Basic Protocol 2: Imaging, quantification, and analysis of PLA signals.