Phenotypic expansion of POFUT1 loss of function mutations in a disorder featuring segmental dyspigmentation with eczematous and folliculo-centric lesions

Abstract

Appearance of mosaic disorders in thin Blaschko lines suggests that somatic mutations in keratinocyte precursors underlie their pathogenesis. Germline heterozygous mutations in POFUT1 gene cause Dowling-Degos disease (DDD), a skin disease that features flexural reticulated hyperpigmentation and follicular-based lesions. POFUT1 mosaicism has not been described to date. Here we describe a 9-year old female with segmental hyper- and hypopigmented patches with overlying eczematous plaques and follicular papules. Employing paired whole exome sequencing of saliva and keratinocytes isolated from affected skin, we found a novel germline heterozygous POFUT1 deletion causing frameshift and premature codon termination and somatic copy-neutral loss of heterozygosity on chromosome 20 encompassing POFUT1. Expression levels of POFUT1 as well as other key-regulators of the notch signaling pathway—NOTCH1, NOTCH2 and HES1— were reduced in affected keratinocytes compared to normal keratinocytes. Our findings provide the first evidence of POFUT1 post-zygotic mutation and a phenotypic expansion of POFUT1 loss of function mutations. We show that a recessive loss of function mutation in POFUT1 produces a distinct clinical presentation with features (e.g. dermatitis) that are absent in the generalized form of DDD. This study demonstrates how analysis of mosaic disorders can reveal unexpected phenotypes for known genes.

INTRODUCTION

Cutaneous mosaic disorders can present in clinically recognized patterns; among them are lines of Blaschko representing the pathways of epidermal precursor cells dorsoventral migration during embryogenesis [Moss et al., 1993]. Pigmentary mosaicism presents in flag-like, checkerboard, broad or fine Blaschkolinear patterns. While flag-like and checkerboard patterns suggest that the somatic mutation occurred in melanocyte precursors, a broad Blaschkolinear pattern could result from a melanocyte or keratinocyte derived mosaicism and a fine Blaschkolinear pattern suggests that the affected cells are keratinocytes [Kinsler and Larue, 2018]. Moreover, the presence of epidermal changes, even if subtle, together with pigmentary changes in a Blaschkoid pigmentary mosaicism, suggests the disorder is primarily keratinocyte precursor derived.

Several inherited skin diseases can present with epidermal changes together with pigmentary changes including Dowling-Degos disease (DDD) which is a rare autosomal dominant skin disorder presenting with progressive evolution of small hyperpigmented macules in a reticulated distribution with predilection to flexural regions. Other features include hypopigmented macules, small pigmented papules and follicular-based-like lesions such as comedones and acneiform scars [Zhang et al., 2017]. DDD has been shown to result from mutations in at least 3 genes: KRT5 (MIM 148040), POFUT1 (MIM 607491) and POGLUT1 (MIM 615618) [Basmanav et al., 2014; Betz et al., 2006; Li et al., 2013]. An overlap with the inflammatory disease hidradenitis suppurativa (HS) had been described in patients with PSENEN (MIM 607632) and POFUT1 mutations [Pavlovsky et al.; González-Villanueva et al., 2018].

We studied a 9-year-old female with segmental dyspigmentation, eczematous plaques and follicular-based papules using paired whole exome sequencing (WES) of saliva and keratinocytes cultured from affected skin.

METHODS

The study was approved by the Yale Human Investigation Committee and complied with the declaration of Helsinki principles. Individual consent and parental permission were obtained in writing. For detailed methods see Supplementary Material.

RESULTS

A nine-year-old female presented with congenital Blaschkolinear distributed thin and thick hypo- and hyperpigmented patches over her left upper extremity and trunk. At the age of 7, she developed additional hypopigmented streaks with overlying folliculo-centric papules or itchy eczematous plaques (Fig.1a–d). The eczematous streaks involving her left thumb and index finger were associated with mild nail dystrophy and tapering of both digits (Figure 1c–d). While the hypopigmented streaks were completely covered with papules or scale, the hyperpigmented patch had irregular borders, was interspersed with hypopigmented streaks and only partially covered with folliculo-centric papules. Family history was significant for psoriasis and DDD in her mother (Fig.1e–h). The combination of thin Blaschkolinear distribution, folliculo-centric lesions and eczematous components suggested that the hypopigmented component of the mosaic disorder originates in keratinocyte precursor cell [Kinsler and Larue, 2018]. A biopsy obtained from a hypopigmented patch with folliculo-centric papules demonstrated features resembling DDD (Figure 1i), while a biopsy from the palmar eczematous hypopigmented plaque showed ortho- and parakeratosis, acanthosis and mild perivascular inflammation (Fig.1j). MITF immunostaining demonstrated a normal number of melanocytes (Fig.1k). We therefore performed a paired whole exome sequencing (WES) of genomic DNA isolated from keratinocytes cultured from a Blaschkolinear hypopigmented patch with folliculo-centric papules and from saliva with no somatic mutations identified (Tables S1–2). Plotting B-allele frequency across the genome demonstrated a segment of loss of heterozygosity (LOH) on chromosome 20 extending from 29.52 megabases (Mbs) to the telomere at 62.89 Mbs (Fig.2a). Analysis of germline variants within the region of LOH revealed a novel heterozygous POFUT1 c.889_890del, p.W297Gfs*14 frameshift (Tables S1–S2). Sanger sequencing confirmed heterozygosity for POFUT1 c.889_890del in saliva as well as in fibroblasts cultured from affected skin and confirmed loss of the wild-type allele in the affected keratinocytes (Fig.2b). Copy number variation analysis of WES data suggested copy-neutral LOH which was confirmed by comparative qPCR (Table S3, Fig.2c–d). The same germline mutation was found in the mother. To determine whether the mutant allele is expressed in affected skin, we isolated RNA from keratinocytes cultured from a hypopigmented patch with folliculo-centric papules. After cDNA synthesis and PCR with POFUT1 specific primers, we sequenced the resultant PCR product and found that the mutant allele is expressed exclusively.

Figure 1. Clinical and histologic features of POFUT1 mosaicism.

The patient presented with Blaschkolinear hyper and hypopigmentation with overlying folliculo-centric tiny papules with a goose bump appearance (a,b). Over the left first and second digits there are linear hypopigmented scaly plaques associated with mild nail dystrophy and tapering of both digits (c,d). Pedigree of kindred demonstrating autosomal dominant inheritance of Dowling- Degos trait. Black symbols represent Dowling-Degos disease, patterned symbol represents segmental Dowling-Degos disease (e) Reticulate hyperpigmentation in the sub- mammary folds (f) as well as sides of the neck in the patient’s mother. A biopsy obtained from the linear hypopigmented patch on the arm showed mild epidermal hyperplasia with finger like elongation of the rete ridges and hyperpigmentation of their lower part as well as follicular plugging (g). A skin biopsy obtained from the eczematous plaque on the left palm revealed acanthosis with hyperkeratosis and mild prevascular inflammation (h). MITF immunostaining demonstrated normal number and distribution of melanocytes.

Figure 2.. Mosaic biallelic POFUT1 frameshift deletion underlies Blaschkolinear dyspigmentation with epidermal changes and results in aberrant expression of Notch signaling pathway genes.

B-allele frequency differences between keratinocytes isolated from blaschko-linear hypopigmented patch with folliculo-centric papules and saliva are plotted across the genome by chromosome and physical position. Dashed vertical lines separate individual chromosomes. Somatic loss of heterozygosity (LOH) on chromosome 20q extends from 29.52 megabases (Mbs) to the telomere at 62.89 Mbs and contains the POFUT1 gene (a). Sanger sequencing was employed to confirm the heterozygous germline POFUT1 c.889_890del, p.W297Gfs*14 frameshift. This mutation is enriched in keratinocytes isolated from the linear hypopigmented patch , and no wild-type allele could be identified (b). CoNIFER was employed to detect copy number variations on chromosome 20. Singular value decomposition of standardized z-scores of reads per thousand bases, per million reads (STD-ZRPMK) is plotted against positions on chromosome 20 and shows no evidence for copy number variation (c). This is further corroborated by comparative qPCR assessing the POFUT1 locus. The fold change of the copy number of genomic POFUT1 from affected skin is calculated using relative quantification (RQ) RQ= 0.98, range 0.84–1.15 (d). RNA expression levels of POFUT1, NOTCH1, NOTCH2, HES1, HEY1 and KRT5 were determined by quantitative real-time PCR in keratinocytes cultured from affected keratinocytes and normal keratinocytes. POFUT1 c.889_890del, p.W297Gfs*14 frameshift resulted in decreased expression on POFUT1, NOTCH1, NOTCH2 and HES1. Results are presented as RQ +/− SD (e).

To further confirm the pathogenicity of the POFUT1 mutation, we used qRT-PCR to determine the expression levels of POFUT1 transcript in keratinocytes isolated from hypopigmented patchwith folliculo-centric papules and normal primary keratinocytes as control and found that POFUT1 expression was diminished (Fig.2e). We also applied qRT-PCR to quantify the relative expression of other genes known to be associated with DDD and/or play a critical role in the Notch signaling pathway and found an aberrant expression of NOTCH1, NOTCH2 and HES1 in affected keratinocytes compared to normal controls (Fig.2e).

DISCUSSION

POFUT1 is part of the Notch signaling pathway, an evolutionarily conserved pathway that is important in cell fate determination in almost all tissues and organs during embryogenesis and homeostasis [Nowell and Radtke, 2013]. In the skin, Notch activity is associated with differentiation of the interfollicular epithelium and the hair follicle [Nowell and Radtke, 2013]. Notch signaling also contributes to the regulation of melanocyte homeostasis [Kumano et al., 2008]. One important modification that modulates Notch signal transduction is O-fucosylation of the EGF-like repeats of Notch receptors. POFUT1 adds O-linked fucose to the EGF-like repeats of Notch receptors, after which Notch ligands bind Notch receptors and induce the proteolytic release of the intra-cellular domain of the receptor which translocates into the nucleus in order to induce target gene expression [Basmanav et al., 2014; Betz et al., 2006; Li et al., 2013; Pavlovsky et al.]. DDD has been linked to heterozygous germline mutations in POFUT1. Our findings provide a novel phenotype for post-zygotic POFUT1 mosaicism. While we show that within hypopigmented streaks keratinocytes carry a “second-hit,” we cannot exclude that an earlier event occurred in a multipotent progenitor cell giving rise to the hypopigmented streaks (mutated keratinocytes) and hyperpigmented streaks (mutated melanocytes) as previously proven in other mosaic disorders such as phacomatosis pigmentokeratotica [Groesser et al., 2013].

Our findings also provide a phenotypic expansion of POFUT1 loss of function mutations, since the clinical and histologic features were only partially compatible with DDD. The patient presented with streaks of confluent dyspigmentation rather than streaks of reticulated pigmentation, papules that were tiny with a goose-bump appearance and an eczematous component over the palmar area with nail dystrophy. While heterozygous POFUT1 mutations in DDD result in haploinsufficiency of POFUT1 [Li et al., 2013], in the present case there is recessive loss of function, leading to further attenuated Notch signaling and a distinct clinical presentation. This is further supported in murine models of Notch signaling ablation by either RBP-J deletion or combined loss of Notch1 and Notch2 that resulted in overexpression of pro-inflammatory cytokines causing a chronic inflammation resembling atopic dermatitis [Nowell and Radtke, 2013].

Mosaicism manifests in skin diseases via two primary mechanisms. In the first, a dominant heterozygous post-zygotic somatic mutation is confined to a sub-population of precursor cells in an otherwise wild-type individual. Such a dominant mutation could be constitutionally lethal, and therefore permitted to manifest in the skin by mosaicism, or constitutionally viable. In the second mechanism, an independent somatic mutation arises in a subpopulation of precursor cells in an individual already carrying a germline heterozygous mutation. This could arise via a two-hit mechanism. For example, an inherited heterozygous loss-of-function mutation may be paired with a somatic mutation of the remaining wild type allele, generating segmental disease. Clinically, the combination of a germline heterozygous mutation and a post-zygotic mutation usually manifestas segments of affected skin arising upon otherwise normal-appearing skin [Atzmony et al., 2019] or as generalized skin disease with more severely affected segments [Poblete-Gutiérrez et al., 2004]. The present study demonstrates that second-hit post-zygotic mutations can manifest not only as a phenotypic augmentation, but also as a different phenotype.

Li et al. showed that POFUT1 haploinsufficiency results in small size melanocytes with pale cytoplasm and a decreased number of melanosomes. In a zebrafish model of POFUT1 knockdown there was abnormal distribution and decreased production of melanin, suggesting that dyspigmentation in POFUT1 DDD results from impairment of the ability to synthesize melanin in melanocytes with POFUT1 loss of function [Li et al., 2013]. Since melanosome uptake into keratinocytes could be influenced by the proliferation and differentiation of keratinocytes [Choi et al., 2014], and the present case demonstrates dyspigmentation in streaks with second-hit in keratinocytes, we can assume that abnormal Notch signaling could influence melanosome uptake as was demonstrated in DDD due to KRT5 mutations [Betz et al., 2006].

Notably, Notch positively regulates retinoic acid signaling in the skin, and retinoic acid signaling is active in the hair follicle and inter-follicular epithelium where it induces differentiation [Collins and Watt, 2008]. Thus far, treatment options for DDD are mainly ablative lasers for the papular component and intense pulse dye for the hyperpigmentation [Kumano et al., 2008]. One case report demonstrated good response to topical adapalene [Altomare et al., 1999]. In an attempt to tailor a pathogenesis-directed therapy, treatment with retinoids should be considered in generalized as well as mosaic forms of DDD.

In conclusion, our findings identify a novel clinical phenotype arising from POFUT1 mosaicism and extend the phenotypic spectrum of POFUT1 loss of function mutations. This study demonstrates how analysis of mosaic disorders can reveal unexpected phenotypes for known genes.