Andrew Touati

Recessive mutation in tetraspanin CD151 causes Kindler syndrome-like epidermolysis bullosa with multi-systemic manifestations including nephropathy

Epidermolysis bullosa (EB) is caused by mutations in as many as 19 distinct genes. We have developed a next-generation sequencing (NGS) panel targeting genes known to be mutated in skin fragility disorders, including tetraspanin CD151 expressed in keratinocytes at the dermal-epidermal junction. The NGS panel was applied to a cohort of 92 consanguineous families of unknown subtype of EB. In one family, a homozygous donor splice site mutation in CD151 (NM_139029; c.351+2T>C) at the exon 5/intron 5 border was identified, and RT-PCR and whole transcriptome analysis by RNA-seq confirmed deletion of the entire exon 5 encoding 25 amino acids. Immunofluorescence of probands skin and Western blot of skin proteins with a monoclonal antibody revealed complete absence of CD151. Transmission electron microscopy showed intracellular disruption and cell-cell dysadhesion of keratinocytes in the lower epidermis. Clinical examination of the 33-year old proband, initially diagnosed as Kindler syndrome, revealed widespread blistering, particularly on pretibial areas, poikiloderma, nail dystrophy, loss of teeth, early onset alopecia, and esophageal webbing and strictures. The patient also had history of nephropathy with proteinuria. Collectively, the results suggest that biallelic loss-of-function mutations in CD151 underlie an autosomal recessive mechano-bullous disease with systemic features. Thus, CD151 should be considered as the 20th causative, EB-associated gene.

Multigene Next-Generation Sequencing Panel Identifies Pathogenic Variants in Patients with Unknown Subtype of Epidermolysis Bullosa: Subclassification with Prognostic Implications

Please cite this article as: Vahidnezhad H, Youssefian L, Saeidian AH, Touati A, Sotoudeh S, Abiri M, Barzgar M, Aghazadeh N, Mahmoudi H, Norouz-zadeh S, Hamid M, Zahabiyon M, Bagherian H, Zeinali S, Fortina P, Uitto J, Multigene Next Generation Sequencing Panel Identifies Pathogenic Variants in Patients with Unknown Subtype of Epidermolysis Bullosa: Subclassification with Prognostic Implications, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.07.830.

Genome-wide single nucleotide polymorphism-based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa

Autozygosity mapping (AM) is a technique utilised for mapping homozygous autosomal recessive (AR) traits and facilitation of genetic diagnosis. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using both short tandem repeat (STR) and genome‐wide single nucleotide polymorphism (SNP) array‐based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analysed with an EB‐targeted next‐generation sequencing (NGS) panel. NGS identified mutations in five additional cases, initially undiagnosed due to the presence of compound heterozygosity, deep intronic mutations or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 of 46 cases (95.7%) diagnosed genetically.

Erythrokeratoderma: a manifestation associated with multiple types of ichthyoses with different gene defects

DEAR EDITOR, Erythrokeratoderma refers to a group of closely related disorders of cornification manifesting with hyperkeratotic, often transient and migratory, erythematous figurate plaques with sharply demarcated borders that typically develop in early childhood with or without palmoplantar involvement. Erythrokeratodermas have been historically classified into the two main categories: (i) erythrokeratodermia variabilis et progressiva (also known as erythrokeratoderma figurata variabilis and Mendes da Costa disease), and (ii) progressive symmetric erythrokeratoderma (also known as Gottron syndrome). However, there is debate as to whether these two presentations of erythrokeratoderma are distinct entities, and both manifestations are now included under a single disease entry, erythrokeratoderma, on the OMIM catalogue (www.omim.org). Thus, erythrokeratoderma could be considered as a distinct entity caused by mutations in specific, ichthyosis-related genes. In this context, genetic mutations causing autosomal dominant erythrokeratoderma were initially reported in the gap junction-related genes GJB2, GJB3, GJB4 and GJA1, in addition to in LOR, whereas associations with autosomal recessive erythrokeratoderma have been identified in case reports with mutations in ELOVL4, ABHD5 and KDSR. Here, we provide additional evidence in support of the notion that erythrokeratoderma may be a manifestation associated with multiple types of ichthyoses with different gene defects. Patient 1 is a 3-year-old female born to second-cousin consanguineous parents (Fig. 1a). Cutaneous findings first manifested at 3 months of age with ichthyotic, erythematous, finely scaling plaques with sharply demarcated red-brown borders, occurring initially on the lower extremities in a symmetric pattern. The lesions were protean and displayed a migratory pattern, eventually spreading to all four extremities and the trunk (Fig. 1a). History was negative for collodion membrane at birth, alopecia, palmoplantar keratoderma (PPK), nail dystrophy and dental abnormalities. Significant laboratory findings included elevated values for serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and creatine phosphokinase, suggesting myopathy. Transabdominal ultrasound revealed a mild diffuse hepatic parenchymal hyperechogenicity consistent with fatty liver disease. Patient 2 is an 8-year-old male born to first-cousin consanguineous parents (Fig. 1b). Onset of the clinical manifestations began at 2 months of age as mild generalized ichthyosis along with migratory erythematous thin plaques with welldemarcated red borders localized to the extremities (Fig. 1b). There was no alopecia, collodion membrane, dental abnormalities, PPK or nail involvement. Patient 3 is an 11-year-old female patient born to firstcousin once removed parents (Fig. 1c). Cutaneous manifestations appeared at 6 months of age as well-circumscribed erythematous hyperkeratotic lesions with well-defined brown borders, first occurring on the hands and eventually migrating to the back, neck and extremities following a symmetric and transient pattern. Additionally, large brown ichthyotic plaques developed on the face, reminiscent of typical autosomal recessive congenital ichthyosis (Fig. 1c). The patient also reported progressive bilateral deafness. No alopecia, PPK, nail dystrophy, ectropion or tooth abnormalities were observed. We have recently developed a next-generation sequencing (NGS) array that incorporates 38 different ichthyosis-associated genes: ABCA12, ABHD5, AGPS, ALDH3A2, ALOX12B, ALOXE3, AP1S1, ARSE, CERS3, CLDN1, CYP4F22, EBP, ELOVL4, GJB2, GJB3, GJB4, GJB6, KRT1, KRT10, KRT2, KRT9, LIPN, LOR, NIPAL4, PEX7, PHYH, PNPLA1, PNPLA2, POMP, SLC27A4, SNAP29, SPINK5, ST14, STS, TGM1, TGM5, VPS33B and ZMPSTE24. We applied this sequencing array to DNA from 180 patients with ichthyosis from Iran. The amplicons were designed to cover 99% of the target bases, and the mean coverage from the target region was 493X. The three patients with erythrokeratoderma were found to have distinct homozygous mutations on NGS analysis. Patient 1 harboured a previously unreported homozygous missense mutation in NM_016006(ABHD5): c.977G>A(p.Gly326Glu) (Fig. 1a). Patient 2 had a previously unreported homozygous missense mutation NM_001145717(PNPLA1): c.614C>T (p.Pro205Leu) (Fig. 1b). Patient 3 was found to have a previously reported homozygous deletion mutation NM_001145717 (PNPLA1): c.939_952G>Tdel13(p.Glu313AspfsTer50) (Fig. 1c). All mutations were confirmed by bidirectional Sanger sequencing, and the pathogenic mutations were heterozygous in the parents. Missense mutations were predicted as damaging using the programs PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), FIS mutation assessor (www.mutationassessor.org), GVGD (http://agvgd.hci.utah.edu/), PROVEAN (http://provean.jcvi. org/index.php) and SIFT (http://sift.jcvi.org/). None of these variants were present in the homozygous state in a control

Molecular genetics of a cohort of 635 cases of phenylketonuria in a consanguineous population

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, characterized by intellectual deficit and neuropsychiatric complications in untreated patients with estimated frequency of about one in 10,000 to 15,000 live births. PAH deficiency can be detected by neonatal screening in nearly all cases with hyperphenylalaninemia on a heel prick blood spot. Molecular testing of the PAH gene can then be performed in affected family members. Herein, we report molecular study of 635 patients genetically diagnosed with PKU from all ethnicities in Iran. The disease-causing mutations were found in 611 (96.22%) of cases. To the best of our knowledge, this is the most comprehensive molecular genetics study of PKU in Iran, identifying 100 distinct mutations in the PAH gene, including 15 previously unreported mutations. Interestingly, we found unique cases of PKU with uniparental disomy, germline mosaicism, and coinheritance with another Mendelian single-gene disorder that provides new insights for improving the genetic counseling, prenatal diagnosis (PND), and/or pre-implantation genetic diagnosis (PGD) for the inborn error of metabolism group of disorders.

Next generation sequencing identifies double homozygous mutations in two distinct genes (EXPH5 and COL17A1) in a patient with concomitant simplex and junctional epidermolysis bullosa

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering diseases. We developed a next generation sequencing (NGS) panel covering 21 genes associated with skin fragility disorders, and it was applied to DNA from 91 probands with the diagnosis of EB. In one patient, novel homozygous mutations were disclosed in two different, unlinked EB‐associated genes: EXPH5, chr11 g.108510085G > A; p.Arg1808Ter and COL17A1, chr10 g.104077423delT; p.Thr68LeufsTer106. Consequences of the COL17A1 mutation were examined by RNAseq which revealed a complex splicing pattern predicting synthesis of a truncated polypeptide (85%) or in‐frame deletion of exon 4 (15% of transcripts). Transmission electron microscopy (TEM) and immunostaining revealed findings consistent with EB simplex (EBS) and junctional EB (JEB), and clinical examination revealed a complex phenotype with features of both subtypes. This case illustrates the power of next generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype–phenotype correlations, prenatal testing, and genetic counseling of families at risk for recurrence.

The genetic basis of hyaline fibromatosis syndrome in patients from a consanguineous background: a case series

BackgroundHyaline fibromatosis syndrome (HFS) is a rare heritable multi-systemic disorder with significant dermatologic manifestations. It is caused by mutations in ANTXR2, which encodes a transmembrane receptor involved in collagen VI regulation in the extracellular matrix. Over 40 mutations in the ANTXR2 gene have been associated with cases of HFS. Variable severity of the disorder in different patients has been proposed to be related to the specific mutations in these patients and their location within the gene.Case presentationIn this report, we describe four cases of HFS from consanguineous backgrounds. Genetic analysis identified a novel homozygous frameshift deletion c.969del (p.Ile323Metfs*14) in one case, the previously reported mutation c.134xa0Tu2009>u2009C (p.Leu45Pro) in another case, and the recurrent homozygous frameshift mutation c.1073dup (p.Ala359Cysfs*13) in two cases. The epidemiology of this latter mutation is of particular interest, as it is a candidate for inhibition of nonsense-mediated mRNA decay. Haplotype analysis was performed to determine the origin of this mutation in this consanguineous cohort, which suggested that it may develop sporadically in different populations.ConclusionsThis information provides insights on genotype-phenotype correlations, identifies a previously unreported mutation in ANTXR2, and improves the understanding of a recurrent mutation in HFS.

A novel mutation in ST14 at a functionally significant amino acid residue expands the spectrum of ichthyosis-hypotrichosis syndrome.

BackgroundMutations in the ST14 gene, encoding the serine protease matriptase, have been associated with ichthyosis-hypotrichosis syndrome (IHS), a Mendelian disorder with skin and hair manifestations which include, in addition to ichthyosis and hypotrichosis, hypohidrosis and follicular atrophoderma. However, the understanding of the specific consequences of mutations in ST14 on the development of this syndrome is incomplete.ResultsUsing a targeted next-generation sequencing array of 38 ichthyosis-associated genes on a large cohort of 180 ichthyosis patients from a primarily consanguineous background, a previously unreported homozygous p.Asp482Asn mutation in ST14 was identified in a patient with IHS. This mutation affects an essential site within a ligand-binding domain of matriptase. Comparison with previous reports of IHS allowed further delineation of the phenotype of IHS in correlation with mutations present in these patients. Histological and ultrastructural analysis of skin and hair identified novel features in this disorder.ConclusionsThis study correlates genotypic and phenotypic features of the rare disorder, IHS, expands the spectrum of pathology associated with the disorder, and provides clinical evidence of the importance of the Asp482 amino acid, previously shown to have an essential role in matriptase activation.