Heterozygous deletion of the NSDHL gene in an Appenzeller Mountain Dog with verrucous epidermal keratinocytic nevi

IF 16.4 1区 化学 Q1 CHEMISTRY, MULTIDISCIPLINARY
Sarah Kiener, Brett Wildermuth, Nadine M. Meertens, Vidhya Jagannathan, Tosso Leeb
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The encoded NAD(P)H steroid dehydrogenase-like protein is a C4 demethylase involved in post-squalene cholesterol biosynthesis. Pathogenic <i>NSDHL</i> variants result in disruption of an essential step in cholesterol biosynthesis with a subsequent aggregation of toxic intermediates and a lack of cholesterol in the skin (Caldas &amp; Herman, <span>2003</span>; König et al., <span>2000</span>). In heterozygous female dogs, this presents as a cornification disorder and is inherited as an X-linked semidominant trait (Bauer et al., <span>2017</span>; Christen et al., <span>2020</span>; Leuthard et al., <span>2019</span>). <i>NSDHL</i>-associated disorders in humans cause a more severe phenotype involving congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD syndrome; König et al., <span>2000</span>). In hemizygous males, such variants have been described as embryonic lethal (Happle et al., <span>1980</span>).</p><p>A 10-month-old female intact Appenzeller Mountain Dog was presented with an 8 month history of severe, progressive hyperkeratosis of the paw pads causing lameness and a primarily left-sided multifocal hyperkeratosis of the haired skin, causing alopecia and mild pruritus. The lesions began on the left inner pinna and hind paw pads and slowly progressed to involve all four paws, although the right front was only mildly affected. Stripes of alopecia with hyperkeratosis were present on the lateral and caudal left thigh, with multiple smaller areas on the left tarsus, left lateral neck and left hip. Complete blood count, serum biochemistry and skin cytology were within normal limits and a fungal culture was negative. Multiple skin punch biopsies were taken under sedation to further pursue a diagnosis. Based on these results the paw pads were treated topically in an attempt to reduce cholesterol precursors in the skin and therefore hyperkeratosis. Two-percent ketoconazole cream and then 2% simvastatin ointment were tried successively, without improvement. The lesions on the haired skin then cleared completely with oral ketoconazole 5 mg/kg once daily but the paw pads remained quite hyperkeratotic, requiring repeated trimming (Figure 1).</p><p>Histopathologically, skin biopsies showed moderate to severe hyperplasia of the epidermis and infundibula of the hair follicles, forming spiked fronds (Figure 2). The infundibula of the hair follicles often showed striking compact parakeratotic hyperkeratosis (with retained nuclei as a sign of delayed maturation of keratinocytes), the epidermis showed orthokeratotic (with loss of nuclei as occurs in normal keratinocyte maturation) to parakeratotic hyperkeratosis. The dermis showed mild perivascular lymphoplasmacytic inflammation. The histopathological alterations resembled the findings described in Labrador Retrievers with <i>NSDHL</i>-related congenital cornification disorder (Bauer et al., <span>2017</span>).</p><p>We performed Sanger sequencing of all exons of the <i>NSDHL</i> gene. The primer sequences are given in Table S1. However, no variant was detected in the coding sequence of <i>NSDHL</i>. We subsequently performed short-read whole-genome sequencing of the affected dog at 20× coverage using Illumina TruSeq PCR-free DNA libraries with ~400 bp insert size. Data processing was performed, with respect to the genome reference assembly UU_Cfam_GSD_1.0, as previously described (Jagannathan et al., <span>2019</span>). Visual inspection of the short-read alignments at the position of the <i>NSDHL</i> gene revealed a large heterozygous deletion of &gt;120 kb comprising the entire <i>NSDHL</i> gene (Figure S1). The deletion was not present in the dam of the affected dog as a deletion-specific amplicon could not be generated on genomic DNA isolated from blood leukocytes of the dam (Table S1). Samples from the father were not available for genotyping. However, assuming that hemizygous <i>NSDHL</i> variants cause lethality, it can be concluded that the variant arose from a <i>de novo</i> mutation event, either in the germline of one of the parents or during the early embryonic development of the affected dog.</p><p>We describe an Appenzeller Mountain Dog with clinical signs suggestive of an <i>NSDHL</i> defect. This differential diagnosis was further supported by the clear therapeutic success of cholesterol precursor reduction. Genetic investigation revealed a large heterozygous <i>de novo</i> deletion spanning the entire <i>NSDHL</i> gene. These results highlight the importance of advanced genomic analysis, such as whole genome sequencing, in identifying structural variants that are easily missed by Sanger sequencing of targeted PCR amplicons.</p><p><b>Sarah Kiener:</b> Conceptualization; investigation; visualization; writing – original draft; writing – review and editing. <b>Brett Wildermuth:</b> Conceptualization; investigation; visualization; writing – original draft; writing – review and editing. <b>Nadine M. 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引用次数: 0

Abstract

Dermal mosaicism can result in skin disorders with specific distribution patterns of the lesions. The most common example is X-chromosomal functional mosaicism, in which the distribution pattern results from random X-chromosome inactivation (Lyonization) (Vreeburg & van Steensel, 2012). Three different skin patterns have been described, namely Blaschko lines, the checkerboard pattern and lateralization (Happle, 2006). Verrucous epidermal keratinocytic nevi (OMIA 002117) caused by variants in the X-chromosomal NSDHL gene may present with any of these patterns. The encoded NAD(P)H steroid dehydrogenase-like protein is a C4 demethylase involved in post-squalene cholesterol biosynthesis. Pathogenic NSDHL variants result in disruption of an essential step in cholesterol biosynthesis with a subsequent aggregation of toxic intermediates and a lack of cholesterol in the skin (Caldas & Herman, 2003; König et al., 2000). In heterozygous female dogs, this presents as a cornification disorder and is inherited as an X-linked semidominant trait (Bauer et al., 2017; Christen et al., 2020; Leuthard et al., 2019). NSDHL-associated disorders in humans cause a more severe phenotype involving congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD syndrome; König et al., 2000). In hemizygous males, such variants have been described as embryonic lethal (Happle et al., 1980).

A 10-month-old female intact Appenzeller Mountain Dog was presented with an 8 month history of severe, progressive hyperkeratosis of the paw pads causing lameness and a primarily left-sided multifocal hyperkeratosis of the haired skin, causing alopecia and mild pruritus. The lesions began on the left inner pinna and hind paw pads and slowly progressed to involve all four paws, although the right front was only mildly affected. Stripes of alopecia with hyperkeratosis were present on the lateral and caudal left thigh, with multiple smaller areas on the left tarsus, left lateral neck and left hip. Complete blood count, serum biochemistry and skin cytology were within normal limits and a fungal culture was negative. Multiple skin punch biopsies were taken under sedation to further pursue a diagnosis. Based on these results the paw pads were treated topically in an attempt to reduce cholesterol precursors in the skin and therefore hyperkeratosis. Two-percent ketoconazole cream and then 2% simvastatin ointment were tried successively, without improvement. The lesions on the haired skin then cleared completely with oral ketoconazole 5 mg/kg once daily but the paw pads remained quite hyperkeratotic, requiring repeated trimming (Figure 1).

Histopathologically, skin biopsies showed moderate to severe hyperplasia of the epidermis and infundibula of the hair follicles, forming spiked fronds (Figure 2). The infundibula of the hair follicles often showed striking compact parakeratotic hyperkeratosis (with retained nuclei as a sign of delayed maturation of keratinocytes), the epidermis showed orthokeratotic (with loss of nuclei as occurs in normal keratinocyte maturation) to parakeratotic hyperkeratosis. The dermis showed mild perivascular lymphoplasmacytic inflammation. The histopathological alterations resembled the findings described in Labrador Retrievers with NSDHL-related congenital cornification disorder (Bauer et al., 2017).

We performed Sanger sequencing of all exons of the NSDHL gene. The primer sequences are given in Table S1. However, no variant was detected in the coding sequence of NSDHL. We subsequently performed short-read whole-genome sequencing of the affected dog at 20× coverage using Illumina TruSeq PCR-free DNA libraries with ~400 bp insert size. Data processing was performed, with respect to the genome reference assembly UU_Cfam_GSD_1.0, as previously described (Jagannathan et al., 2019). Visual inspection of the short-read alignments at the position of the NSDHL gene revealed a large heterozygous deletion of >120 kb comprising the entire NSDHL gene (Figure S1). The deletion was not present in the dam of the affected dog as a deletion-specific amplicon could not be generated on genomic DNA isolated from blood leukocytes of the dam (Table S1). Samples from the father were not available for genotyping. However, assuming that hemizygous NSDHL variants cause lethality, it can be concluded that the variant arose from a de novo mutation event, either in the germline of one of the parents or during the early embryonic development of the affected dog.

We describe an Appenzeller Mountain Dog with clinical signs suggestive of an NSDHL defect. This differential diagnosis was further supported by the clear therapeutic success of cholesterol precursor reduction. Genetic investigation revealed a large heterozygous de novo deletion spanning the entire NSDHL gene. These results highlight the importance of advanced genomic analysis, such as whole genome sequencing, in identifying structural variants that are easily missed by Sanger sequencing of targeted PCR amplicons.

Sarah Kiener: Conceptualization; investigation; visualization; writing – original draft; writing – review and editing. Brett Wildermuth: Conceptualization; investigation; visualization; writing – original draft; writing – review and editing. Nadine M. Meertens: Investigation; visualization; writing – original draft; writing – review and editing. Vidhya Jagannathan: Data curation; writing – review and editing. Tosso Leeb: Conceptualization; funding acquisition; writing – original draft; writing – review and editing.

This study was funded by grant 310030_200354 from the Swiss National Science Foundation.

The authors declare no conflict of interest.

The diagnostic examinations of the affected dog were conducted during the clinical workup, did not constitute an animal experiment and therefore did not require official or institutional ethical approval. The collection of blood samples from control dogs was approved by the Cantonal Committee for Animal Experiments (Canton of Bern; permit BE94/2022).

Consent for the use of samples and data for research purposes was obtained from the owners of the dogs in this study.

Abstract Image

一只患有疣状表皮角化细胞痣的阿彭策尔山地犬的 NSDHL 基因杂合子缺失。
皮肤嵌合可导致具有特定皮损分布模式的皮肤病。最常见的例子是 X 染色体功能性嵌合,其分布模式是随机 X 染色体失活(Lyonization)的结果(Vreeburg &amp; van Steensel, 2012)。目前已描述了三种不同的皮肤模式,即布拉什科线、棋盘格模式和侧化(Happle,2006 年)。由 X 染色体 NSDHL 基因变异引起的疣状表皮角化细胞痣(OMIA 002117)可能表现为上述任何一种模式。所编码的 NAD(P)H 类固醇脱氢酶样蛋白是一种 C4 脱甲基酶,参与蝶呤后胆固醇的生物合成。致病性 NSDHL 变体会导致胆固醇生物合成的一个重要步骤中断,随后有毒中间产物聚集,皮肤中缺乏胆固醇(Caldas &amp; Herman, 2003; König et al.)在杂合子雌性犬中,这种疾病表现为粟粒化障碍,是一种 X 连锁半显性遗传性状(Bauer 等人,2017 年;Christen 等人,2020 年;Leuthard 等人,2019 年)。人类的 NSDHL 相关疾病会导致更严重的表型,包括先天性半身发育不良伴鱼鳞状痣和肢体缺陷(CHILD 综合征;König 等人,2000 年)。一只 10 个月大的雌性完整阿彭策尔山地犬在 8 个月前出现严重的进行性爪垫角化过度,导致跛行,而且主要是左侧多灶性毛发皮肤角化过度,引起脱发和轻度瘙痒。病变始于左侧耳廓内侧和后爪垫,慢慢发展到所有四只爪子,但右前爪仅受到轻微影响。左侧大腿外侧和尾部出现带状脱发和角化过度,左侧跗骨、左侧颈部和左臀部有多个较小的区域。全血细胞计数、血清生化和皮肤细胞学检查均在正常范围内,真菌培养呈阴性。为了进一步确诊,医生在镇静状态下进行了多处皮肤穿刺活检。根据这些结果,对爪垫进行了局部治疗,试图减少皮肤中的胆固醇前体,从而减少过度角化。先后尝试了 2% 酮康唑乳膏和 2% 辛伐他汀软膏,但效果不佳。之后,每天口服一次 5 毫克/千克酮康唑,毛发皮肤上的病变完全消退,但爪垫上的角化过度仍然严重,需要反复修剪(图 1)。毛囊基底层经常出现明显的角化不全角化过度(细胞核保留,这是角质形成细胞成熟延迟的表现),表皮出现角化过度(正常角质形成细胞成熟时细胞核脱落)至角化不全角化过度。真皮出现轻度血管周围淋巴浆细胞炎症。组织病理学改变与患有与NSDHL相关的先天性角化障碍的拉布拉多猎犬的研究结果相似(Bauer等人,2017年)。我们对NSDHL基因的所有外显子进行了桑格测序。我们对 NSDHL 基因的所有外显子进行了 Sanger 测序,引物序列见表 S1。然而,在 NSDHL 的编码序列中没有检测到变异。随后,我们使用Illumina TruSeq不含PCR的DNA文库对患犬进行了20倍覆盖率的短线程全基因组测序,插入片段大小约为400 bp。数据处理是根据之前描述的基因组参考组装 UU_Cfam_GSD_1.0 进行的(Jagannathan 等人,2019 年)。对 NSDHL 基因位置的短读数比对进行目测发现,整个 NSDHL 基因存在一个 120 kb 的大杂合缺失(图 S1)。由于从患病犬母体血液白细胞中分离出的基因组 DNA 无法产生缺失特异性扩增片段(表 S1),因此患病犬母体中不存在该缺失。父亲的样本无法进行基因分型。然而,假设半杂合子 NSDHL 变异会导致致死,那么可以得出结论,该变异是由父母一方的种系或患犬的早期胚胎发育过程中发生的从头突变引起的。我们描述了一只阿彭策尔山地犬,它的临床症状提示其患有 NSDHL 缺陷。胆固醇前体减少剂的明显治疗效果进一步支持了这一鉴别诊断。遗传学调查显示,NSDHL 基因有一个大的杂合性从头缺失。 这些结果凸显了先进的基因组分析(如全基因组测序)在确定结构变异方面的重要性,而这些结构变异很容易被目标 PCR 扩增子的 Sanger 测序所遗漏:构思;调查;可视化;写作--原稿;写作--审阅和编辑。Brett Wildermuth:概念化;调查;可视化;写作--原稿;写作--审阅和编辑。纳丁-M-梅尔滕斯调查;可视化;写作--原稿;写作--审阅和编辑。Vidhya Jagannathan:数据整理;写作--审阅和编辑。本研究得到了瑞士国家科学基金会(Swiss National Science Foundation)310030_200354 号基金的资助。作者声明没有利益冲突。对患病犬的诊断检查是在临床工作中进行的,不构成动物实验,因此不需要官方或机构的伦理批准。对照组犬只血液样本的采集已获得州动物实验委员会(伯尔尼州;许可证号:BE94/2022)的批准。
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来源期刊
Accounts of Chemical Research
Accounts of Chemical Research 化学-化学综合
CiteScore
31.40
自引率
1.10%
发文量
312
审稿时长
2 months
期刊介绍: Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
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