Genetic testing and new variants in diagnosis of congenital ichthyoses

Abstract

Background

The aim of this study was to evaluate how diagnostic practice in congenital ichthyoses has evolved during the years 2000–2020 and what kind of gene variants of congenital ichthyosis have been found.

Methods

The study cohort of this register‐based research consisted of a total of 88 patients, whose diagnostic testing was conducted, and ichthyosis diagnoses set at the Department of Dermatology and the Department of Clinical Genetics at Tampere University Hospital during the years 2000–2020.

Results

Diagnosis of ichthyosis was confirmed with genetic testing in 33 cases, and with conventional diagnostic methods, such as clinical findings, skin biopsy and family history of ichthyoses, in 55 cases. We observed four novel variants in patients with the clinical diagnoses of congenital ichthyoses.

Conclusion

When genetic testing became available, it was offered primarily to patients with severe forms of ichthyosis. During the study period next‐generation sequencing became the genetic testing method of choice providing new opportunities in diagnostics.

The aim of this study was to evaluate how diagnostic practice in congenital ichthyoses has evolved during the years 2000–2020 and what kind of gene variants of congenital ichthyosis have been found. We observed four novel variants in patients with the clinical diagnoses of congenital ichthyoses. During the study period, next‐generation sequencing became the genetic testing method of choice providing new opportunities in diagnostics BioRender.com.

1. INTRODUCTION

Hereditary ichthyoses comprise a genetically and clinically heterogenous group of congenital cornification disorders associated with defects in the epidermal barrier (Oji & Traupe, 2009; Schmuth et al., 2013; Vahlquist & Törmä, 2020). Most types of ichthyoses are monogenic disorders and mutations in over 50 genes have so far been found to cause ichthyosis (Fischer & Bourrat, 2020). The known variants involved in ichthyoses affect proteins of structural importance for corneocyte formation, cell–cell junction proteins and enzymes required for the proteolysis of cell junctions, for lipid metabolism and for DNA repair (Schmuth et al., 2013). Characteristic clinical manifestations are scaling and/or hyperkeratosis of the skin, often seen with erythema, erosions and xerosis (Oji & Traupe, 2009; Vahlquist et al., 2018). The severity of the symptoms varies considerably, depending on the specific form of ichthyosis (Vahlquist et al., 2018). There is no curative treatment for ichthyosis and often lifelong treatment of symptoms is required. Accordingly, ichthyosis has been reported to have a negative effect on patients' quality of life (QoL) (Troiano & Lazzeri, 2020).

Due to phenotype variability and heterogeneous aetiology, diagnosing ichthyosis and distinguishing between different forms of ichthyoses is challenging and has previously been based on clinical signs and skin symptoms, the patient's family history, skin biopsies and specific analyses, such as electron microscopy (Schmuth et al., 2013). Diagnosing syndromic ichthyoses entails assessing extracutaneous symptoms (Yoneda, 2016). However, in the last 10 years, genetic testing has provided an opportunity to confirm the diagnosis accurately and rapidly and made it possible to select the most suitable therapeutic options, to clarify the prognosis and to initiate genetic counselling (Schmuth et al., 2013; Vahlquist et al., 2018). In particular, massive parallel sequencing technologies, also known as next‐generation sequencing (NGS), have become the most important genetic testing methods (Fischer & Bourrat, 2020; Takeichi & Akiyama, 2016; Uitto et al., 2020), providing a significant improvement in the application of molecular genetics in the study period. The aim of the study was to evaluate how diagnostic practice in congenital ichthyoses has evolved over the last two decades, and report what variants of congenital ichthyosis were found in a cohort of ichthyosis patients at a university hospital.

2. MATERIALS AND METHODS

2.1. Ethical compliance

The regional Ethics Committee of Tampere University Hospital approved the study protocol (R20128R), and the Declaration of Helsinki was followed.

2.2. Patients and study protocol

The study cohort consisted of patients whose diagnostic testing was conducted, and ichthyosis diagnoses set at the Department of Dermatology or the Department of Clinical Genetics at Tampere University Hospital between 2000 and 2020, where the diagnostics and treatment of genodermatoses are centralised. The patients were sent for a clinical evaluation to a dermatologist, or a clinical geneticist and the genetic testing was conducted in a clinical setting. For this study, the data were collected from the patient records at Tampere University Hospital using International Classification of Diseases Version 10, with the diagnostic codes Q80, Q80.0, Q80.1, Q80.2, Q80.3, Q80.4, Q80.8 and Q80.9 for ichthyosis. The search yielded a total of 122 patients. In addition, one pregnant woman was diagnosed as having a harlequin‐foetus. Patients were excluded from our study if the precise ichthyosis diagnosis had been confirmed prior to the year 2000 (n = 25), if the confirmed diagnosis was not ichthyosis (n = 9), and if the diagnosis was confirmed at another regional hospital during the study period (n = 1).

The medical records of the patients with ichthyosis were evaluated. The medical histories and information on genetic testing were collected from the patient records and the archives of the Department of Clinical Genetics. This is a register study that complies with data protection and information security regulations. Results are reported in such way that individual patients are not identifiable.

2.3. Genetic testing

After clinical assessment at Tampere University Hospital, genetic testing had been conducted in accredited laboratories in Europe and the United States as part of the diagnostic process and clinical patient care. Informed consent to genetic testing was obtained from all patients after genetic counselling. Genomic DNA was extracted from venous blood samples using standard procedures. For targeted single gene sequencing and gene panels that included analysis of 9–39 genes, most commonly 19 genes, Sanger sequencing or NGS were used. Copy number analysis was used when needed, using microarrays, MLPA (Multiplex Ligation‐dependent Probe Amplification) or applying NGS methods. Variant classification and interpretation were performed in the laboratories according to existing guidelines. From 2015, the guidelines modified from those of the American College of Medical Genetics and Genomics in 2015 were followed (Richards et al., 2015). When needed, segregation analysis of the variants was carried out in the family to verify that the compound heterozygous variants were on different chromosomes (in trans).

3. RESULTS

After the implementation of the exclusion criteria, a total of 88 patients, including 57 (65%) males, were diagnosed as having congenital ichthyosis. The most common ichthyosis was ichthyosis vulgaris (n = 24), followed by recessive X‐linked ichthyosis (n = 23) and lamellar ichthyosis (n = 12). In total, genetic testing was performed on 41 out of 88 patients (47%), and in 33 of these (80%) variants were identified (Table 1). When any variants were identified, they were identified in the first analysis in 25 (76%) cases and for eight patients (24%), several genetic tests were required until the variants were identified. The majority of the genetic tests conducted were single gene tests, which were interpreted as confirming the diagnosis in 27 out of 33 patients (82%), whereas a gene panel confirmed the diagnosis of congenital ichthyosis in six out of 33 patients (18%).

TABLE 1.

Patients (n = 33) with molecule genetic diagnosis of congenital ichthyoses.

Phenotype (OMIM number)	Number of patients	Gene	Variant type	Genomic coordinate (GRCh38 unless otherwise specified)	HGVS nucleotide	Predicted effect on protein	CADD score	Zygosity	Inheritance model	Variant classification a	Diagnostic genetic testing method
X‐linked ichthyosis (308100)	5	STS	Whole gene deletion	chrX				Hemi	XLR	P	Sanger and MLPA, or NGS
ARCI type 1 (LI) (242300)	1	TGM1 TGM1	Missense variant Missense variant	chr14:24261776 chr14:24259099	NM_000359.3: c.427C>T NM_000359.3: c.1135G>C	p.(Arg143Cys) p.(Val379Leu)	25.1 23.4	Het Het	AR	P P	Sanger
ARCI type 1 (LI) (242300)	1	TGM1 TGM1	Missense variant Missense variant	chr14:24258646 chr14:24261776	NM_000359.3: c.1187G>T NM_000359.3: c.427C>T	p.(Arg396Leu) p.(Arg143Cys)	28.9 25.1	Het Het	AR	P P	NGS
ARCI type 1 (242300)	1	TGM1 TGM1	Missense variant Missense variant	chr14:24261776 chr14:24259769	NM_000359.3: c.427C>T NM_000359.3: c.919C>G	p.(Arg143Cys) p.(Arg307Gly)	25.1 24.3	Het Het	AR	P P	NGS
ARCI type 1 (LI) (242300)	1	TGM1 TGM1	Missense variant Missense variant	chr14:24259099 chr14:24260013	NM_000359.2: c.1135G>C NM_000359.2: c.803T>A	p.(Val379Leu) p.(Val268Asp)	23.4 29.0	Het Het	AR	P VUS	Sanger
ARCI type 2 (242100)	1	ALOX12B ALOX12B	Missense variant Missense variant	chr17:8076731 chr17:8075687	NM_001139.2: c.1288C>T NM_001139.2: c.1562A>G	p.(His430Tyr) p.(Tyr521Cys)	28.9 28.4	Het Het	AR	VUS P	Sanger
ARCI type 4A (LI) (601277)	1	ABCA12 ABCA12	Nonsense variant Missense variant	chr2:215064205 chr2:215000928	NM_173076.3: c.178C>T NM_173076.3: c.2956C>T	p.(Arg60*) p.(Arg986Trp)	‐ 29.2	Het Het	AR	LP LP	Sanger
ARCI type 4A (LI) (601277)	1	ABCA12	Missense variant	chr2:214990856	NM_173076.3: c.3470C>T	p.(Ser1157Leu)	25.0	Hom	AR	LP	Sanger
ARCI type 4B (harlequin ichthyosis) (242500)	1	ABCA12	Missense variant	chr2:215000928	NM_173076.3: c.2956C>T	p.(Arg986Trp)	29.2	Hom	AR	LP	Sanger
ARCI type 5 (604777)	1	CYP4F22 CYP4F22	Missense variant Missense variant	chr19:15537579 chr19:15550689	NM_173483.4: c.466C>T NM_173483.4: c.1351C>T	p.(Arg156Cys) p.(Arg451Cys)	29.2 31.0	Het Het	AR	P LP	NGS
ARCI type 3 (606545)	1	ALOXE3 ALOXE3	Missense variant Missense variant	chr17:8014843 (GRCh37) chr17:8007518 (GRCh37)	NM_001165960.1: c.1187T>A NM_001165960.1: c.2096T>C	p.Val396Asp p.Leu699Pro	25.3 25.8	Het Het	AR	VUS VUS	NGS
Ichthyosis prematurity syndrome (608649)	1	SLC27A4	Nonsense variant	chr9:128345497	NM_005094.4: c.504C>A	p.(Cys168*)	‐	Hom	AR	P	Sanger
Type 1 annular epidermolytic ichthyosis (607602)	1	KRT10	Missense variant	chr17:40822124	NM_000421.5: c.462T>A	p.(Asn154Lys)	22.5	Het	AD	LP	Sanger
Type 1 annular epidermolytic ichthyosis (607602)	1	KRT10	Missense variant	chr17:40822120	NM_000421.5: c.466C>A	p.(Arg156Ser)	27.2	Het	AD	P	Sanger
Type 1 annular epidermolytic ichthyosis (607602)	1	KRT10	Missense variant	chr17:40822104	NM_000421.5: c.482T>C	p.(Leu161Ser)	28.2	Het	AD	P	Sanger
Type 1 annular epidermolytic ichthyosis (607602)	1	KRT10	Missense variant	chr17:40819532	NM_000421.5: c.1358T>C	p.(Leu453Pro)	32.0	Het	AD	P	Sanger
Congenital reticular ichthyosiform erythroderma, (ichthyosis with confetti) (609165)	1	KRT10	Nonsense variant	chr17:40819522–40,819,523	NM_000421.5: c.1367_1368delAG	p.(Glu456Glyfs*124)	‐	Het	AD	LP	Sanger
Type 2 annular epidermolytic ichthyosis (620148)	1	KRT1	Missense variant	chr12:52679790	NM_006121.4: c.559C>T	p.(Leu187Phe)	26.7	Het	AD	P	Sanger
Superficial epidermolytic ichthyosis (ichthyosis bullosa of Siemens) (146800)	2	KRT2	Missense variant	chr12:52646774	NM_000423.3: c.1435 A>C	p.(Thr479Pro)	29.7	Het	AD	P	Sanger
Type 2 peeling skin syndrome (609796)	1	TGM5	Missense variant	chr15:43260151	NM_201631.4: c.337G>T	p.(Gly113Cys)	26.4	Hom	AR	P	NGS
Type 2 X‐linked dominant chondrodysplasia punctata (302960)	1	EBP	Nonsense variant	chrX:48527015	NM_006579.3: c.328C>T	p.(Arg110*)	‐	Het	XLD	P	Sanger
Netherton syndrome (256500)	5	SPINK5	Nonsense variant	chr5:148091214	NM_006846.4: c.652C>T	p.(Arg218*)	‐	Hom	AR	P	Sanger
Netherton syndrome (256500)	1	SPINK5	Nonsense variant Splicing variant	chr5:148091214 chr5:148100582	NM_006846.4: c.652C>T NM_006846.4: c.1220+1G>C	p.(Arg218*) ‐	‐ ‐	Het Het	AR	P LP	Sanger
Netherton syndrome (256500)	1	SPINK5	Nonsense variant	chr5:148111847	NM_006846.4: c.1772delT	p.(Leu591Glnfs*124)	‐	Hom	AR	LP	Sanger

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; ARCI, autosomal recessive congenital ichthyosis; Hemi, hemizygous; Het, heterozygous; Hom, homozygous; LI, lamellar ichthyosis; LP, likely pathogenic variant; MLPA, multiplex ligation‐dependent probe amplification; NGS, next‐generation sequencing; P, pathogenic variant; Sanger, Sanger sequencing; VUS, variant of uncertain significance; XLD, X‐linked dominant; XLR, X‐linked recessive.

^a According to the American College of Medical Genetics and Genomics variant classification standards.

At the time, genetic testing was not offered to the patients in the diagnostics of mild forms of ichthyoses, such as ichthyosis vulgaris. Diagnosis of ichthyosis vulgaris was confirmed with skin biopsy or with clinical picture and family history in all cases. These patients had typically xerosis, fine scaling, pruritus, and eczema since the first years of life. Late onset of symptoms was quite common also in recessive X‐linked ichthyosis, which was mainly diagnosed with typical clinical findings, such as dark‐brown scaling on extensor surfaces, and confirmed by detection of steroid sulphatase deficiency in skin biopsy, whereas genetic testing was used in five out of 23 (22%) cases. Half of the patients with lamellar ichthyosis were diagnosed with genetic testing. Patients with lamellar ichthyoses had severe symptoms at birth, like collodion, extropium and eclabium, and they developed erythroderma and generalised coarse brown scaling thereafter.

Besides 24 different pathogenic and likely pathogenic variants, we observed four novel variants of uncertain significance (VUS). The first patient had, in a single gene test, a heterozygous TGM1 (transglutaminase 1) VUS c.803T>A, p.(Val268Asp), and also a heterozygous pathogenic variant c.1135G>C, p.(Val379Leu). The patient was born as a collodium baby with ectropium and later developed generalised coarse and large scaling, palmoplantar hyperkeratosis and alopecia, clinically presenting lamellar ichthyosis. The patient was treated with systemic retinoids. One of the parents carried the pathogenic variant c.1135G>C, and the other parent carried the VUS c. 803T>A. Known pathogenic variants of TGM1 cause type 1 autosomal recessive congenital ichthyosis.

The second patient was born as a collodium baby, but no ectropium was observed. The skin was erythrodermic and in some areas, such as the abdomen, there was coarse scaling. The dermatopathologist observed hyperkeratosis in stratum corneum in the skin biopsy, consistent with ichthyosis. At the age of 1 year, only mild ichthyosis was observed with scaling on the scalp and on the body. In a gene panel, the baby was found to be heterozygous for a ALOXE3 (arachidonate lipoxygenase 3) VUS c.1187T>A, p.Val396Asp and also a heterozygous VUS c.2096T>C, p.Leu699Pro. One parent was heterozygous for the VUS c.1187>C and the other heterozygous for the VUS c.2096T>A. ALOXE3 pathogenic variants cause type 3 autosomal recessive congenital ichthyosis.

The third case with a VUS, a heterozygous ALOX12B (arachidonate 12‐lipoxygenase, 12R type) c.1288C>T, p.(His430Tyr) was observed with a heterozygous pathogenic variant c.1562A>G, p.(Tyr521Cys) in a single gene test of a patient, who was born as a collodium baby and developed mild lamellar ichthyoses, mostly manifesting thereafter with dry skin. One parent carried the VUS c.1288C>T and the other parent the pathogenic variant c.1562A>G. Pathogenic variants of ALOX12B are a known cause of type 2 autosomal recessive congenital ichthyosis.

NGS was initially used in the study population in 2016. Thereafter, during the period 2016–2020, when causative mutation for ichthyosis was identified, the method used was NGS in all cases. When using NGS, mutation was always identified in the first analysis (n = 8) or not identified at all (n = 3). Most first‐time genetic testing was performed in the years 2012–2013.

Median age at confirmed genetic diagnosis was 12.7 (range 0–56) years. Median age at molecular diagnosis varied according to the type of ichthyoses and was 19.8 years among those diagnosed as having X‐chromosomal ichthyosis, 15.8 years among those with autosomal recessive congenital ichthyoses, 22.2 years among those with keratinopathic ichthyoses and 0.5 years among those with ichthyosis syndrome. When diagnosis was confirmed with Sanger sequencing, median age at diagnosis was 17.0 (0–51) years and with NGS 6.0 (0–55) years. The success rate for finding a causative variant was 80% when using Sanger sequencing and 73% when using NGS.

Altogether eight patients were diagnosed as having syndromic ichthyosis, seven of them had Netherton's syndrome and one patient had chondrodysplasia punctata (Table 1). Cases of extremely rare ichthyosis were also diagnosed: one patient with congenital reticular ichthyosiform erythroderma and one Harlequin foetus.

4. DISCUSSION

This study including a well‐defined cohort of congenital ichthyoses patients shows that the diagnostics of these cornification disorders has become more specific in recent years. In addition to 24 known pathogenic or likely pathogenic variants, we also identified four novel variants in patients with the clinical diagnoses of congenital ichthyoses. To the best of our knowledge, these VUS have not so far been reported in the literature either as disease causing variants or as benign polymorphisms. These are rare variants in general population and were classified as being VUS based on the types and the locations of the variants (Richards et al., 2015). In silico and segregation analyses in the families also support their role in the pathogenesis of the patients' disease. It is possible that the variants explain the patients' phenotype, but we still cannot rule out the possibility that they are benign variants. Since commercial genetic tests are nowadays widely used as the main diagnostic method on patients with severe forms of ichthyosis, data from novel variants will accumulate faster and thus the interpretation of the results will become easier.

The major strengths of the present study were the long observation period and the consecutively gathered ichthyoses patients. However, only few genetic tests were used during the study period in the diagnosis of the most common types of ichthyoses, such as ichthyosis vulgaris and recessive X‐linked ichthyosis, which also explains the relatively modest total number of molecular genetic diagnoses in the study. However, it has been reported that mild phenotypes of X‐linked ichthyosis can sometimes be difficult to distinguish from ichthyosis vulgaris, especially when there is no family history of ichthyoses (Vahlquist et al., 2018). Nonetheless, X‐linked ichthyosis is more often familial and has also been associated with neurodevelopmental disorders, where early diagnosis is crucial (Hand et al., 2015; Rodrigo‐Nicolás et al., 2018). Moreover, large X‐chromosomal deletions may affect neighbouring genes, leading to contiguous gene deletion syndromes (Fischer & Bourrat, 2020). Finding the familial gene variant enables genetic counselling, carrier testing and family planning according to the X‐chromosomal inheritance model. Also, mild ichthyoses may cause considerable harm and further decrease in quality of life caused by persistent scaling, erythema and pruritus (Troiano & Lazzeri, 2020), hence, exact diagnosis of mild types of ichthyoses is also important for the patients and their families. Therefore, we nowadays recommend genetic testing in all forms of inheritable ichthyoses.

The relative proportions of recessive X‐linked ichthyosis, lamellar ichthyosis and keratopathic ichthyosis in this study are equivalent to the estimated prevalences of these types of ichthyoses (Dreyfus et al., 2014; Hernández‐Martín et al., 2012; Oji et al., 2010). Therefore, it can be assumed that our study accurately reflects these types of ichthyoses in the study region. The relatively high number of Netherton's syndrome diagnoses in our study probably reflects the discovery of the Finnish founder mutation of SPINK5 in 2016 (Hannula‐Jouppi et al., 2016). Instead, the prevalence of ichthyosis vulgaris in this study remained significantly lower than the estimated prevalence of ichthyosis vulgaris, probably due to the associated mild symptoms that do not always require university hospital evaluation (Brown et al., 2009). Diagnosing the foetus with Harlequin ichthyosis highlights the importance of identification of the genotype to be able to offer families genetic counselling and guidance. Since the study design was retrospective and register based, it was not possible to determine the exact age at the appearance of the first symptoms of ichthyoses. Furthermore, a longer observation period would probably have been needed to show if genetic testing, and particularly NGS, accelerates the diagnostic process.

We have described in this study, how diagnostics of hereditary ichthyoses has evolved from clinical diagnosis and use of single gene test to use of gene panels. As the genes included in gene panels varied during the study period, this may have affected the identification of mutations in patients with congenital ichthyoses. Therefore, patients should be offered a new opportunity for a gene test in the event that the mutation has not been found. Accurate diagnosis is important as early as possible in all types of congenital ichthyoses, including mild types of ichthyoses, to offer families genetic counselling, clinical management and guidance (Salik et al., 2023). It has been reported that with NGS, approximately 80%–90% of causative variants can currently be identified (Cheng et al., 2020; Fischer & Bourrat, 2020). In this study, too, 20% of patients did not get a molecular diagnosis despite genetic testing. Nowadays, diagnostics of ichthyosis in our hospital is done in cooperation with dermatologist and genetics at the same visit, and every patient suspected of having hereditary ichthyosis is offered the option of a genetic test, primarily an ichthyosis gene panel. In future, a recall of the patients and conducting whole‐exome sequencing or whole genome sequencing and identifying complex aetiologies may enable more patients to receive an accurate molecular diagnosis and benefit from further development of pathogenesis‐based therapies for ichthyoses.

AUTHOR CONTRIBUTIONS

Conceptualization: T.K., T.S.; Data curation: M.S., T.K., H.H., T.S.; Formal analysis: M.S., T.K., H.H., T.S.; Funding acquisition: T.K.; Investigation: M.S., T.K., H.H., T.S.; Methodology: T.K., H.H., T.S.; Project administration: T.K., T.S.; Resources: T.K., H.H.; Software: H.H.; Supervision: T.K., T.S.; Validation: T.K., H.H., T.S.; Visualization: T.K., T.S.; Writing‐original draft: M.S., T.K., H.H., T.S.; Writing‐review and editing: M.S, T.K., H.H., T.S.

FUNDING INFORMATION

The study was financially supported by a grant from the Finnish Dermatological Society.

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest to declare.

Supporting information

Table S1.

Salo, M. , Kimpimäki, T. , Huhtala, H. , & Saarela, T. (2024). Genetic testing and new variants in diagnosis of congenital ichthyoses. Molecular Genetics & Genomic Medicine, 12, e70000. 10.1002/mgg3.70000

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.