Lynn M. Boyden

Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K+ and H+ excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin–RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na–Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na–Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K+ and pH homeostasis.

Skint1 , the prototype of a newly identified immunoglobulin superfamily gene cluster, positively selects epidermal γδ T cells

B cells, αβ T cells and γδ T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. αβ T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules. The molecules that select γδ T cells are unknown. Vγ5+Vδ1+ cells comprise 90% of mouse epidermal γδ T cells. By mapping and genetic complementation using a strain showing loss of Vγ5+Vδ1+ cells due to a failure of thymic selection, we show that this defect is caused by mutation in Skint1, a newly identified gene expressed in thymus and skin that encodes a protein with immunoglobulin-like and transmembrane domains. Skint1 is the prototypic member of a rapidly evolving family of at least 11 genes in mouse, with greatest similarity to the butyrophilin genes. These findings define a new family of proteins mediating key epithelial-immune interactions.

Skint-1 is a highly specific, unique selecting component for epidermal T cells

αβ T-cell repertoire selection is mediated by peptide–MHC complexes presented by thymic epithelial or myeloid cells, and by lipid–CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1+ dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1+ dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid–CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane–cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.

Adenosine metabolism and murine strain-specific IL-4-induced inflammation, emphysema, and fibrosis.

To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and alpha1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-beta1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A(1), A(2B), and A(3) adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals.

Whole-Exome Sequencing Reveals Somatic Mutations in HRAS and KRAS, which Cause Nevus Sebaceus

ACKNOWLEDGMENTS We thank the patients and their families for taking part in this project. We also thank S Aasi, R Khosla, and P Lorenz for their valuable assistance. Bryan K. Sun, Andrea Saggini, Kavita Y. Sarin, Jinah Kim, Latanya Benjamin, Philip E. LeBoit and Paul A. Khavari Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA; Department of Dermatology, University of California, San Francisco, San Francisco, California, USA and Department of Dermatology, University of Rome Tor Vergata, Rome, Italy E-mail: bryansun@stanford.edu

Dominant de novo mutations in GJA1 cause erythrokeratodermia variabilis et progressiva, without features of oculodentodigital dysplasia

Genetic investigation of inherited skin disorders has informed understanding of skin self-renewal, differentiation, and barrier function. Erythrokeratodermia variabilis et progressiva (EKVP) is a rare, inherited skin disease characterized by transient figurate patches of erythema, localized or generalized scaling, and frequent palmoplantar keratoderma. By employing exome sequencing, we show that de novo missense mutations in GJA1 (gap junction protein alpha 1) cause EKVP. The severe, progressive skin disease in EKVP subjects with GJA1 mutations is distinct from limited cutaneous findings rarely found in the systemic disorder oculodentodigital dysplasia, also caused by dominant GJA1 mutations. GJA1 encodes connexin 43 (Cx43), the most widely expressed gap junction protein. We show that the GJA1 mutations in EKVP subjects lead to disruption of Cx43 membrane localization, and aggregation within the Golgi. These findings reveal a critical role for Cx43 in epidermal homeostasis, and provide evidence of organ-specific pathobiology resulting from different mutations within GJA1.

Mutations in KDSR Cause Recessive Progressive Symmetric Erythrokeratoderma

The discovery of new genetic determinants of inherited skin disorders has been instrumental to the understanding of epidermal function, differentiation, and renewal. Here, we show that mutations in KDSR (3-ketodihydrosphingosine reductase), encoding an enzyme in the ceramide synthesis pathway, lead to a previously undescribed recessive Mendelian disorder in the progressive symmetric erythrokeratoderma spectrum. This disorder is characterized by severe lesions of thick scaly skin on the face and genitals and thickened, red, and scaly skin on the hands and feet. Although exome sequencing revealed several of the KDSR mutations, we employed genome sequencing to discover a pathogenic 346 kb inversion in multiple probands, and cDNA sequencing and a splicing assay established that two mutations, including a recurrent silent third base change, cause exon skipping. Immunohistochemistry and yeast complementation studies demonstrated that the mutations cause defects in KDSR function. Systemic isotretinoin therapy has achieved nearly complete resolution in the two probands in whom it has been applied, consistent with the effects of retinoic acid on alternative pathways for ceramide generation.

Dominant de novo DSP mutations cause erythrokeratodermia-cardiomyopathy syndrome

Disorders of keratinization (DOK) show marked genotypic and phenotypic heterogeneity. In most cases, disease is primarily cutaneous, and further clinical evaluation is therefore rarely pursued. We have identified subjects with a novel DOK featuring erythrokeratodermia and initially-asymptomatic, progressive, potentially fatal cardiomyopathy, a finding not previously associated with erythrokeratodermia. We show that de novo missense mutations clustered tightly within a single spectrin repeat of DSP cause this novel cardio-cutaneous disorder, which we term erythrokeratodermia-cardiomyopathy (EKC) syndrome. We demonstrate that DSP mutations in our EKC syndrome subjects affect localization of desmosomal proteins and connexin 43 in the skin, and result in desmosome aggregation, widening of intercellular spaces, and lipid secretory defects. DSP encodes desmoplakin, a primary component of desmosomes, intercellular adhesion junctions most abundant in the epidermis and heart. Though mutations in DSP are known to cause other disorders, our cohort features the unique clinical finding of severe whole-body erythrokeratodermia, with distinct effects on localization of desmosomal proteins and connexin 43. These findings add a severe, previously undescribed syndrome featuring erythrokeratodermia and cardiomyopathy to the spectrum of disease caused by mutation in DSP, and identify a specific region of the protein critical to the pathobiology of EKC syndrome and to DSP function in the heart and skin.

Phenotypic spectrum of autosomal recessive congenital ichthyosis due to PNPLA1 mutation

DEAR EDITOR, The ichthyoses are rare skin disorders linked by the common finding of scale and concomitant barrier function abnormalities. Recently, mutations in PNPLA1, which encodes patatin-like phospholipase domain containing 1 and plays a role in the formation of the epidermal lipid barrier, have been identified as a rare cause of nonsyndromic autosomal recessive congenital ichthyosis (ARCI). We identified patients with PNPLA1 mutations within our registry of 732 ichthyosis kindreds. DNA was isolated from blood, and either multiplex targeted next-generation sequencing of 42 genes known to cause disorders of keratinization, or whole-exome sequencing was performed (Appendix S1; see Supporting Information). Fourteen unrelated probands with ARCI were found to be compound heterozygous or homozygous for mutations in PNPLA1,which were confirmed with Sanger sequencing. PNPLA1 mutations segregated with disease in all kindreds, five of which were consanguineous (Figs S1–14; see Supporting Information). In total 16 different PNPLA1 mutations were observed, including two that result in early termination, a splice-site mutation and 13 missense substitutions at conserved residues (Fig. 1g and Fig. S15; see Supporting Information). All missense mutations are within the more highly conserved N-terminal half of the protein, and all but two are clustered within the patatin domain. Two patients are homozygous for missense mutations at S53, the nucleophilic serine in the putative lipid hydrolase site, and one is homozygous for a missense mutation at D172, the other critical residue in the catalytic dyad. The phenotypes of all patients with PNPLA1 mutations are described in Table 1, with representative clinical photos provided in Figures S1–14 (see Supporting Information). At birth, seven patients presented with a collodion membrane (one with vernix-like hyperkeratosis), and eight showed generalized erythema and/or scaling. Mature phenotypes included scale that was fine or plate like, and erythema ranging from minimal to severe (Fig. 1a–f). The presence and degree of ectropion and palmoplantar keratoderma are variable, although they are either absent or mild in the majority of patients. Only seven of 19 patients were born with a collodion membrane. Generally, the spectrum of phenotypic severity appears difficult to correlate with specific PNPLA1 genotypes. While some patients with the same genotype exhibit consistent clinical features, there are others with identical or similar mutations who show notable variation in phenotype. For instance, the affected siblings of kindred ICH162 (Fig. S2; see Supporting Information), both of whom are compound heterozygous for the same missense mutations, vary significantly in the severity of their presentations. ICH162-1 was born collodion and developed plate-like scale, palmoplantar keratoderma and severe ectropion, whereas her sister had generalized scale and erythema at birth with no collodion membrane, and now has fine white scale; mild ectropion presented only within the past 2 years at age 80 years. Interestingly, we report two families that appear to display dominant inheritance, but in which sequencing revealed PNPLA1 mutations consistent with recessive inheritance. In kindred ICH201 (Fig. S3; see Supporting Information), which was previously published as having autosomal dominant ichthyosis, there are actually two different recessive PNPLA1 genotypes in affected individuals. The proband (ICH201-1) is compound heterozygous for A34T and S140P, while her two affected children (ICH201-3 and ICH201-4) are both homozygous for S140P, having presumably inherited a second copy of this mutation from their unaffected father. Mutation A34T has previously been observed with homozygous inheritance in a report of a kindred from Galicia, Spain. Kindred ICH201 is also from Galicia, and collection of the extended family history revealed additional family members with ichthyosis (the proband’s deceased sister and a deceased nephew from another sister), despite no known history of consanguinity. These observations suggest that A34T and S140P may be founder mutations present at low frequency in Galicia, a region in which founder mutations in TGM1 have also been reported. Kindred ICH454 (Fig. S7; see Supporting Information) is also notable for an affected parent with two affected children and the resulting appearance of dominant inheritance. In this family, all three are homozygous for the same PNPLA1 mutation as a result of the consanguineous union of the proband and a first cousin who is a heterozygous carrier. Two of the missense mutations we report are distal to the patatin domain (amino acids 16–185, Fig. 1g). Mutation C216R is homozygous in the three affected members of kindred ICH454 (described above), and mutation P235L is homozygous in patient ICH592-1, a child of first cousins of

CARD14-associated papulosquamous eruption: A spectrum including features of psoriasis and pityriasis rubra pilaris

Background: Heterozygous mutations in caspase recruitment domain family member 14 gene (CARD14) have been shown to be associated with psoriasis and familial pityriasis rubra pilaris (PRP). Many subjects with CARD14 mutations display features of both disorders, which can result in diagnostic uncertainty. In addition, these eruptions are often recalcitrant to conventional psoriasis therapies such as methotrexate, oral retinoids, and tumor necrosis factor‐&agr; inhibitors. Objective: We sought to describe the clinical characteristics, family history, and response to therapy in subjects with papulosquamous eruptions due to mutations in CARD14. Methods: Subjects were referred for genetic testing as part of a registry of subjects with inherited disorders of keratinization. DNA was isolated from blood or saliva, and multiplex targeted sequencing or whole exome sequencing was performed. Clinical histories of subjects with CARD14 mutations were reviewed. Results: We identified 15 kindreds with CARD14‐associated papulosquamous eruption (CAPE). Characteristic features of CAPE include early age of onset; prominent involvement of the cheeks, chin, and ears; family history of psoriasis or PRP; minimal response to conventional topical and systemic psoriasis therapies; and improvement with ustekinumab. Limitations: Relatively small sample size. Conclusions: Many subjects with CARD14 mutations display characteristics of both psoriasis and PRP. We propose the term CARD14‐associated papulosquamous eruption to describe this spectrum of disease. Subjects with clinical features suggestive of CAPE should undergo CARD14 sequencing and may benefit from treatment with ustekinumab.