FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice.

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.

Monogenic etiologies of persistent human papillomavirus infections: A comprehensive systematic review.

PURPOSE: Persistent human papillomavirus infection (PHPVI) causes cutaneous, anogenital, and mucosal warts. Cutaneous warts include common warts, Treeman syndrome, and epidermodysplasia verruciformis, among others. Although more reports of monogenic predisposition to PHPVI have been published with the development of genomic technologies, genetic testing is rarely incorporated into clinical assessments. To encourage broader molecular testing, we compiled a list of the various monogenic etiologies of PHPVI. METHODS: We conducted a systematic literature review to determine the genetic, immunological, and clinical characteristics of patients with PHPVI. RESULTS: The inclusion criteria were met by 261 of 40,687 articles. In 842 patients, 83 PHPVI-associated genes were identified, including 42, 6, and 35 genes with strong, moderate, and weak evidence for causality, respectively. Autosomal recessive inheritance predominated (69%). PHPVI onset age was 10.8 ± 8.6 years, with an interquartile range of 5 to 14 years. GATA2,IL2RG,DOCK8, CXCR4, TMC6, TMC8, and CIB1 are the most frequently reported PHPVI-associated genes with strong causality. Most genes (74 out of 83) belong to a catalog of 485 inborn errors of immunity-related genes, and 40 genes (54%) are represented in the nonsyndromic and syndromic combined immunodeficiency categories. CONCLUSION: PHPVI has at least 83 monogenic etiologies and a genetic diagnosis is essential for effective management.

Secondary ACMG and non-ACMG genetic findings in a multiethnic cohort of 16,713 pediatric participants.

PURPOSE: Clinical next-generation sequencing is an effective approach for identifying pathogenic sequence variants that are medically actionable for participants and families but are not associated with the participant's primary diagnosis. These variants are called secondary findings (SFs). According to the literature, there is no report of the types and frequencies of SFs in a large pediatric cohort that includes substantial African-American participants. We sought to investigate the types (including American College of Medical Genetics and Genomics [ACMG] and non-ACMG-recommended gene lists), frequencies, and rates of SFs, as well as the effects of SF disclosure on the participants and families of a large pediatric cohort at the Center for Applied Genomics at The Children's Hospital of Philadelphia. METHODS: We systematically identified pathogenic (P) and likely pathogenic (LP) variants in established disease-causing genes, adhering to ACMG v3.2 secondary finding guidelines and beyond. For non-ACMG SFs, akin to incidental findings in clinical settings, we utilized a set of criteria focusing on pediatric onset, high penetrance, moderate to severe phenotypes, and the clinical actionability of the variants. This criteria-based approach was applied rather than using a fixed gene list to ensure that the variants identified are likely to affect participant health significantly. To identify and categorize these variants, we used a clinical-grade variant classification standard per ACMG/AMP recommendations; additionally, we conducted a detailed literature search to ensure a comprehensive exploration of potential SFs relevant to pediatric participants. RESULTS: We report a distinctive distribution of 1464 P/LP SF variants in 16,713 participants. There were 427 unique variants in ACMG genes and 265 in non-ACMG genes. The most frequently mutated genes among the ACMG and non-ACMG gene lists were TTR(41.6%) and CHEK2 (7.16%), respectively. Overall, variants of possible medical importance were found in 8.76% of participants in both ACMG (5.81%) and non-ACMG (2.95%) genes. CONCLUSION: Our study revealed that 8.76% of a large, multiethnic pediatric cohort carried actionable secondary genetic findings, with 5.81% in ACMG genes and 2.95% in non-ACMG genes. These findings emphasize the importance of including diverse populations in genetic research to ensure that all groups benefit from early identification of disease risks. Our results provide a foundation for expanding the ACMG gene list and improving clinical care through early interventions.

Interpretation of genomic sequence variants in heritable skin diseases: A primer for clinicians.

Over 1000 heritable disorders have cutaneous manifestations, some of which are syndromicin association with extracutaneous manifestations, whereas others are limited to the skin. The genetic basis of many of these conditions has been deciphered, and mutation analyses using next-generation sequencing approaches, including whole-exome sequencing, whole-genome sequencing, and whole-transcriptome analysis, are now increasingly becoming part of the diagnostic process. Besides confirming the diagnosis, information on the specific mutations can be used for subclassification with prognostication and identification of the carriers, leading to accurate genetic counseling. It also forms a basis for prenatal testing and preimplantation genetic diagnosis. Furthermore, the ongoing therapeutics developments for heritable skin diseases are often allele-specific, necessitating the knowledge of the specific genes and mutations. Although practicing clinicians increasingly employ molecular diagnostics for heritable skin diseases, they often lack the sufficient knowledge required to interpret the implications of the mutations with precision. The purpose of this primer is to provide an overview of mutation-detection strategies and how to interpret genetic information for improved diagnostics and the management of such patients.

Inherited ichthyosis as a paradigm of rare skin disorders: Genomic medicine, pathogenesis, and management.

Great advances have been made in the field of heritable skin disorders using next-generation sequencing (NGS) technologies (ie, whole-genome sequencing, whole-exome sequencing, whole-transcriptome sequencing, and disease-targeted multigene panels). When NGS first became available, the cost and lack of access to these technologies were limiting factors; however, with decreasing sequencing costs and the expanding knowledge base of genetic skin diseases, fundamental awareness of NGS has become prudent. The heritable ichthyoses comprise a genotypically and phenotypically heterogeneous group of monogenic keratinization disorders characterized by persistent scaling, with at least 55 distinct genes currently implicated in causing nonsyndromic and syndromic forms of the disease. By providing a simplified overview of available NGS techniques and applying them in the context of ichthyosis, one of the most common genodermatoses, we hope to encourage dermatologists to offer, when appropriate, genetic testing earlier in patients with unsolved presentations. With the aid of NGS, dermatologists can provide diagnostic certainty in cases of suspected genodermatoses and offer potentially life-changing genome-guided and targeted therapies as they become available.

Acquired ichthyosis, asteatotic dermatitis or xerosis? An update on pathoetiology and drug-induced associations.

Acquired ichthyosis (AI) is a relatively rare cutaneous entity characterized by transient, generalized scaling and pruritus in the absence of family history of ichthyosis or atopic disease. The hyperkeratosis in AI can range from the mild, white-to-brown scaling resembling that in ichthyosis vulgaris (IV) to the more prominent dark brown scaling phenotype, similar to that found in lamellar ichthyosis. The disease can wax and wane in relation to endogenous and/or exogenous factors. Histopathology of AI is similar to that found in IV. AI is usually of cosmetic concern to patients but can, in some cases, reflect the presence of more serious conditions, including malignancies, autoimmune diseases or metabolic disorders. In some cases, AI can be an adverse effect of a medication or the cutaneous symptom of a toxic exposure. Other conditions, such as severe xerosis or eczema, can present with clinical findings similar to AI, making diagnosis a challenge. Furthermore, cases of AI are sporadic throughout the literature and have been documented across a wide variety of medical settings distinct from dermatology, which often contribute to misdiagnosis of this disease. Definitive management requires prompt identification and treatment of the inciting factors combined with conservative therapies, which can include topical emollients, keratolytics, retinoids or corticosteroids, and in rare cases, oral retinoids.

Mutation update: The spectra of PLEC sequence variants and related plectinopathies.

Plectin, encoded by PLEC, is a cytoskeletal linker of intermediate filaments expressed in many cell types. Plectin consists of three main domains that determine its functionality: the N-terminal domain, the Rod domain, and the C-terminal domain. Molecular defects of PLEC correlating with the functional aspects lead to a group of rare heritable disorders, plectinopathies. These multisystem disorders include an autosomal dominant form of epidermolysis bullosa simplex (EBS-Ogna), limb-girdle muscular dystrophy (LGMD), aplasia cutis congenita (ACC), and an autosomal recessive form of EBS, which may associate with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and/or congenital myasthenic syndrome (EBS-MyS). In this study, genotyping of over 600 Iranian patients with epidermolysis bullosa by next-generation sequencing identified 15 patients with disease-causing PLEC variants. This mutation update analyzes the clinical spectrum of PLEC in our cohort and in the literature and demonstrates the relationship between PLEC genotype and phenotypic manifestations. This study has integrated our seven novel PLEC variants and phenotypic findings with previously published data totaling 116 variants to provide the most complete overview of pathogenic PLEC variants and related disorders.

Genetic heterogeneity of heritable ectopic mineralization disorders in a large international cohort.

PURPOSE: Heritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization. METHODS: Sequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo. RESULTS: A total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity. CONCLUSION: The results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.

Whole-transcriptome sequencing identifies postzygotic ATP2A2 mutations in a patient misdiagnosed with herpes zoster, confirming the diagnosis of very late-onset segmental Darier disease.

An 82-year-old female patient presented with a recent onset of painful skin lesions in unilateral distribution on the abdominal area following the lines of Blaschko; the initial diagnosis of Varicella-Zoster infection was made. However, because the individual lesions appeared as hyperkeratotic papules and were unresponsive to antiviral therapy, a skin biopsy was performed, which revealed hyperkeratosis, suprabasal acantholysis and dyskeratosis with corps ronds and grains, consistent with acantholytic dyskeratotic acanthoma. Since this entity has been associated with Darier disease, whole-transcriptome sequencing by RNA-Seq was performed on RNA isolated from a lesion and from adjacent normal appearing skin, and a recently developed bioinformatics pipeline that can identify both genomic sequence variants and the presence of any of 926 viruses was applied. Two pathogenic missense mutations in the ATP2A2 gene were identified in the lesional but not in normal appearing skin, and no evidence of Varicella-Zoster infection was obtained. These findings confirm the diagnosis of segmental Darier disease due to postzygotic mutations in the ATP2A2 gene, and attest to the power of a novel single-step application of RNA-Seq in providing correct diagnosis in this rare genodermatosis.

Pathogenic DST sequence variants result in either epidermolysis bullosa simplex (EBS) or hereditary sensory and autonomic neuropathy type 6 (HSAN-VI).

DST encodes bullous pemphigoid antigen-1 (BPAG1), a protein with eight tissue-specific isoforms expressed in the skin, muscle, brain and nerves. Accordingly, mutations in this gene are associated with epidermolysis bullosa simplex (EBS) and hereditary sensory and autonomic neuropathy type 6 (HSAN-VI). The genotypic spectrum is attested to by 19 distinct mutations but genotype-phenotype correlation for both disorders is not well established. In this study, we performed next-generation sequencing (NGS) on two families with different phenotypic presentations, one foetus (P1) with musculoskeletal and neurological malformations established by prenatal ultrasound and family history, and a 15-year-old female patient (P2) with skin blistering. P1 had a novel homozygous nonsense mutation, DST: NM_001144769, c.3805C>T, p.R1269* within a region of genetic homozygosity (ROH). This mutation resides within the plakin domain of BPAG1 and ablates all isoforms of this protein, leading to novel extracutaneous phenotypes consistent with HSAN-VI in P1. P2 had a recurrent homozygous mutation DST: NM_001723.7, c.3370C>T, p.Gln1124* that presented with giant, trauma-induced skin blisters without extracutaneous involvement. This mutation is located within the coiled-coil domain present in the skin isoform of DST, BPGA1-e, associated with EBS. In summary, we report two families with pathogenic DST variants and expand the spectrum of DST genotype and phenotypes.

Losartan treatment improves recessive dystrophic epidermolysis bullosa: A case series.

Recessive dystrophic epidermolysis bullosa (RDEB) manifests with blistering and erosions of the skin and mucous membranes due to mutations in COL7A1. The repetitive wound healing processes lead to extensive cutaneous scarring. The scarring is driven by inflammatory processes, particularly the TGF-ß signaling pathways, resulting in excess synthesis and deposition of the extracellular matrix, especially collagen. There is currently no effective or specific treatment for RDEB. Losartan, an angiotensin II type 1 receptor antagonist, is an inhibitor of TGF-ß activity. Previous preclinical studies with hypomorphic Col7a1 mice recapitulating features of RDEB have suggested that losartan may improve the clinical features of RDEB. In this case series, we assessed the effects of losartan on the clinical and histopathologic features in seven patients with RDEB; three females and four males; aged 18.1 ± 9.1 years. The diagnosis was based on characteristic clinical features and the presence of biallelic loss-of-function mutations in COL7A1. Daily oral administration of losartan (0.7 mg/kg) for six weeks resulted in subjective improvement of the clinical features, as judged by the treating physicians and the patients, and the severity of the disease objectively improved based on Birmingham Epidermolysis Bullosa Severity (BEBS) score (30.1 ± 12.8 versus 23.3 ± 10.4, before and after treatment, p = 0.018), accompanied by improvement of quality of life, as determined by the EB-QoL questionnaire (24.0 ± 8.1 versus 17.7 ± 5.5, p = 0.018). Histopathology of the selected lesions revealed after treatment increased number of mast cells, and enhanced microvasculature in the mid and lower dermis. The width of collagen bundles in dermis was suggested to be decreased in four samples and changed from dense to loose in appearance. In summary, this case series reports beneficial effects of losartan on RDEB as a potentially novel treatment.

Ichthyosis follicularis syndromes in patients with mutations in GJB2.

Ichthyosis follicularis (IF) manifests as generalized spiny follicular projections found in syndromic diseases secondary to SREBF1 and MBTPS2 mutations. We sought the genetic cause of IF in two distinct families from a cohort of 180 patients with ichthyosis. In Family 1, the proband (Patient 1) presented with IF, bilateral sensorineural hearing loss and punctate palmoplantar keratoderma. Using DNA from peripheral blood lymphocytes, two compound heterozygous mutations, c.526A>G and c.35delG, were discovered in GJB2. In Family 2, the proband (Patient 2) presented with a previously unreported IF phenotype in the context of keratitis-ichthyosis-deafness syndrome, and whole-exome sequencing found a de novo heterozygous mutation, c.148G>A in GJB2. Histopathology was consistent with porokeratotic eccrine ostial and dermal duct naevus (PEODDN) and IF in Patients 1 and 2, respectively. Our findings add to the clinical and histopathological spectrum of IF and emphasize the association of PEODDN-like entities with GJB2 variants.

Evaluation of neurodevelopmental symptoms in 10 cases of neonatal ichthyosis and sclerosing cholangitis syndrome.

Neonatal ichthyosis and sclerosing cholangitis (NISCH) syndrome is an extremely rare entity with only 19 patients described in the literature. We report an extended family with the disorder and investigate the association of neurodevelopmental symptoms. Patients with CLDN1 mutations, and specifically « the Moroccan¼ c.200_201delTT deletion, may be an increased risk for neurodevelopmental symptoms such as learning disabilities, mental retardation, and language delay.

Whole-Transcriptome Analysis by RNA Sequencing for Genetic Diagnosis of Mendelian Skin Disorders in the Context of Consanguinity.

BACKGROUND: Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences. METHODS: We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis. RESULTS: We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel. CONCLUSIONS: Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.

Ancestral patterns of recessive dystrophic epidermolysis bullosa mutations in Hispanic populations suggest sephardic ancestry.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genodermatosis caused by mutations in the gene coding for type VII collagen (COL7A1). More than 800 different pathogenic mutations in COL7A1 have been described to date; however, the ancestral origins of many of these mutations have not been precisely identified. In this study, 32 RDEB patient samples from the Southwestern United States, Mexico, Chile, and Colombia carrying common mutations in the COL7A1 gene were investigated to determine the origins of these mutations and the extent to which shared ancestry contributes to disease prevalence. The results demonstrate both shared European and American origins of RDEB mutations in distinct populations in the Americas and suggest the influence of Sephardic ancestry in at least some RDEB mutations of European origins. Knowledge of ancestry and relatedness among RDEB patient populations will be crucial for the development of future clinical trials and the advancement of novel therapeutics.

Genomics-based treatment in a patient with two overlapping heritable skin disorders: Epidermolysis bullosa and acrodermatitis enteropathica.

Next-generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg·kg-1 ·day-1 ) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics-guided treatment.

Increased level of cathelicidin (LL-37) in vitiligo: Possible pathway independent from vitamin D receptor gene polymorphism.

Vitiligo is a multifactorial skin disease with established role of genetics and autoimmunity in its pathogenesis. Vitamin D receptor (VDR) polymorphisms have been suggested to correlate with risk of vitiligo in some ethnic populations. On the other hand, cathelicidin, one of the innate immune system components, has a role in development of some chronic skin diseases and VDR regulates the expression of cathelicidin. We aimed to determine the plasma level of cathelicidin and its association with the VDR gene polymorphisms as well as plasma vitamin D level in patients with vitiligo. Ninety vitiligo patients and 90 non-vitiligo controls participated in this study. Blood levels of 25(OH) vitamin D and cathelicidin were determined with ELISA. Genotyping for VDR polymorphisms (ApaI, TaqI, FokI and BsmI) was done with RFLP-PCR method. Mean blood level of cathelicidin was significantly higher in vitiligo patients as compared to controls (P < .0001). Mean blood level of vitamin D was significantly lower in patients than controls (P = .01). Statistically significant differences were not observed for both genotype and allele frequencies of BsmI, ApaI and TaqI polymorphisms. There was a borderline increased risk of vitiligo in over-dominant model of FokI polymorphism with OR = 1.8 and P = .051. Our findings was suggestive of the potential role of cathelicidin in the pathogenesis of vitiligo; however, future evaluations are needed to determine its precise mechanism. Genetic study of VDR gene polymorphism was suggestive of increased risk of vitiligo in association with a FokI polymorphism in Iranian population.

The matriptase-prostasin proteolytic cascade in dermatologic diseases.

The proper development and function of skin and hair are dependent on proteolytic activities. Specifically, the matriptase-prostasin cascade is a series of proteolytic reactions in the epidermis integral to normal regulation of desquamation. An increasing amount of research describing this pathway has recently become available, and the importance of this pathway is exhibited by the association of genetic defects in this pathway with human diseases of the skin and hair. Given the relevance of this pathway to dermatology, we provide a review of the current understanding of its relevance to distinct clinical entities, including ichthyosis-hypotrichosis and Netherton syndromes.