Cystatin M/E Variant Causes Autosomal Dominant Keratosis Follicularis Spinulosa Decalvans by Dysregulating Cathepsins L and V.

Keratosis follicularis spinulosa decalvans (KFSD) is a rare cornification disorder with an X-linked recessive inheritance in most cases. Pathogenic variants causing X-linked KFSD have been described in MBTPS2, the gene for a membrane-bound zinc metalloprotease that is involved in the cleavage of sterol regulatory element binding proteins important for the control of transcription. Few families have been identified with an autosomal dominant inheritance of KFSD. We present two members of an Austrian family with a phenotype of KFSD, a mother and her son. The disease was not observed in her parents, pointing to a dominant inheritance with a de novo mutation in the index patient. Using whole-exome sequencing, we identified a heterozygous missense variant in CST6 in DNA samples from the index patient and her affected son. In line with family history, the variant was not present in samples from her parents. CST6 codes for cystatin M/E, a cysteine protease inhibitor. Patient keratinocytes showed increased expression of cathepsin genes CTSL and CTSV and reduced expression of transglutaminase genes TGM1 and TGM3. A relative gain of active, cleaved transglutaminases was found in patient keratinocytes compared to control cells. The variant found in CST6 is expected to affect protein targeting and results in marked disruption of the balance between cystatin M/E activity and its target proteases and eventually transglutaminases 1 and 3. This disturbance leads to an impairment of terminal epidermal differentiation and proper hair shaft formation seen in KFSD.

Kohlschütter-Tönz Syndrome With a Novel ROGD1 Variant in 3 Individuals: A Rare Clinical Entity.

Kohlschütter-Tönz syndrome (OMIM 226750) is a rare disorder with autosomal recessive inheritance among epileptic encephalopathy syndromes. To date, only 31 Kohlschütter-Tönz syndrome families have been reported in the literature. Early-onset epilepsy, progressive global developmental delay, and amelogenesis imperfecta are the main components of the syndrome. Mutations in ROGDI (MIM 226750) and SLC13A5 (MIM 615905) are responsible for Kohlschütter-Tönz syndrome. Here, we report on the clinical and molecular characteristics of 3 individuals from 2 families, all harboring the same homozygous novel deleterious variant in ROGD1, along with a long-term follow-up and review of the literature. Although the phenotypic features are almost consistent in Kohlschütter-Tönz syndrome, overlooking dental findings and diverse degrees of variability in clinical findings makes diagnosis challenging occasionally. Because there is a limited number of reported patients, identification of new patients and delineation of clinical and molecular findings will increase the awareness of clinicians and enable establishing genotype-phenotype correlations.

Spektrum der Ichthyosen in einer österreichischen Ichthyosekohorte von 2004-2007.

HINTERGRUND: Ichthyosen sind eine heterogene Gruppe von Krankheiten, deren klinische Klassifizierung schwierig ist. Hier wird die Ichthyosekohorte eines Expertisezentrums für Genodermatosen im Detail beschrieben. PATIENTEN UND METHODIK: Eingeschlossen wurden Patienten mit klinisch oder genetisch bestätigter Ichthyose, die zwischen 2004 und 2017 untersucht und in einer Datenbank aufgenommen wurden. Krankheitsbeginn, Phänotyp, Histologie, Komorbiditäten und Familienanamnese wurden detailliert beschrieben. Bei den genetisch getesteten Patienten wurden Jahr und Methode der genetischen Testung protokolliert und die Prävalenz der unterschiedlichen Autosomal-rezessive-kongenitale Ichthyose (ARCI)-Gene und -Phänotypen, die Prävalenz der syndromalen Ichthyosen und die Genotyp-Phänotyp-Korrelationen analysiert. ERGEBNISSE UND METHODIK: Von den insgesamt 198 eingeschlossenen Patienten wurden 151 genetisch getestet. 81 Patienten hatten eine Ichthyosis vulgaris (IV), 43 eine X-chromosomale Ichthyose (XLI), 38 eine ARCI, 9 eine keratinopathische Ichthyose (KPI) und ein Patient eine Exfoliative Ichthyose. 26 Patienten litten an einer syndromalen Ichthyose. Im Vergleich zu den syndromalen Ichthyosen wurde bei den häufigen Ichthyosen (IV, XLI) und KPI eine gute Phänotyp-Genotyp-Korrelation beobachtet. In 91 % der ARCI-Patienten konnte die exakte Diagnose durch genetische Testung gestellt werden. Lediglich bei 33 % der Patienten mit syndromaler Ichthyose bestand vor der genetischen Testung ein Verdacht auf die tatsächliche Diagnose. In 86 % der Fälle wurde eine kausale Mutation nachgewiesen. SCHLUSSFOLGERUNGEN: Die Arbeit beschreibt das Spektrum der Ichthyosen an einem Expertisezentrum und zeigt, dass für diese Gruppe die genetische Testung von Genodermatosen ein diagnostischer Standard werden sollte.

Spectrum of ichthyoses in an Austrian ichthyosis cohort from 2004 to 2017.

BACKGROUND: Ichthyoses are a heterogeneous disease group, which makes clinical classification challenging. An ichthyosis cohort at a center for genodermatoses is presented in detail. PATIENTS AND METHODS: Patients with clinically and/or genetically confirmed ichthyosis seen from 2004 to 2017 and listed in a database were included. Disease onset, phenotype, histology, comorbidities and family history were described in detail. In genetically tested patients, the prevalence of various ARCI genes, ARCI phenotypes and syndromic ichthyoses, as well as genotype-phenotype correlation and year/method of genetic testing was assessed. RESULTS: Of all 198 patients who were included in the cohort, 151 were genetically tested. 81 had ichthyosis vulgaris, 43 X-linked ichthyosis, 38 autosomal recessive congenital ichthyosis (ARCI), 9 keratinopathic ichthyosis (KPI) and one exfoliative ichthyosis. 26 individuals suffered from syndromic ichthyoses. A good genotype-phenotype correlation was observed for common ichthyoses and KPI; the correlation was less good in syndromic ichthyoses. In 91 % of ARCI patients an accurate diagnosis was obtained by genetic testing. In only 33 % of syndromic ichthyoses was the definitive diagnosis suspected before genetic testing, which revealed a causative mutation in 86 % of cases. CONCLUSION: This study describes the spectrum of ichthyoses in a center of expertise and shows that genetic testing should become a diagnostic standard for this disease group.

Cleft lip/palate and hereditary diffuse gastric cancer: report of a family harboring a CDH1 c.687 + 1G > A germline mutation and review of the literature.

Hereditary diffuse gastric cancer (HDGC) is an autosomal-dominantly inherited cancer syndrome associated with a high risk for diffuse gastric and lobular breast cancer, caused by heterozygous CDH1 germline mutations. Of note, also cleft lip/palate (CLP) has been described in few HDGC families. Here we report on an extensive pedigree presenting with HDGC, CLP and a CDH1 splice site mutation (c.687 + 1G > A) and review the literature for families with CDH1 mutations, HDGC and CLP. Transcript analysis showed that the c.687 + 1G > A mutation leads to loss of the last 42 bp of exon 5 and is consequently predicted to cause loss of 14 amino acids in the first extracellular cadherin repeat (EC) domain. Five mutation carriers developed diffuse gastric cancer and four individuals presented with CLP. Wild type CDH1 expression levels did not differ between CDH1 mutation carriers with CLP compared to those without CLP. Beside this extensive pedigree, we outline another previously unreported HDGC/CLP family with a CDH1 (c.1711 + 1G > C) germline mutation in this study. Review of the literature revealed a significant enrichment of CDH1 mutations within the EC domains in CLP/HDGC families (Fisher's exact test, p = 0.007) in comparison to CDH1 mutations associated with HDGC only. Report of further CLP/HDGC associated mutations is necessary to confirm this observation. This study highlights that CLP represents an important phenotypic feature of CDH1 germline mutation carriers and emphasizes the inclusion of CLP in the HDGC testing criteria. The underlying causes for the appearance of variable phenotypes in CDH1 mutation carriers could include genetic variation, epigenetic changes and environmental factors and should be investigated in future studies.

SYNE1-ataxia: Novel genotypic and phenotypic findings.

INTRODUCTION: SYNE1 encodes nesprin-1, a scaffold protein which is involved in the binding between cytoskeleton, nuclear envelope and other subcellular compartments. In 2007, recessive truncating SYNE1 mutations have been linked to a genetic form of pure cerebellar ataxia with adult onset and mild phenotype. Subsequent reports described a number of patients with SYNE1-ataxia and widespread neurological involvement including features of motor neuron disease. Recently, heterozygote missense SYNE1 mutations have been associated with muscular disorders, such as Emery-Dreifuss muscular dystrophy, arthrogryposis multiplex congenita and dilated cardiomyopathy. METHODS: Herein we describe novel genotypic and phenotypic findings in an independent cohort of 5 patients with SYNE1-ataxia referring to the Department of Neurology of the Innsbruck Medical University and performed a review of the related literature. RESULTS: We report 3 novel mutations and describe for the first time myocardial involvement in a patient with a complicated spastic-ataxic phenotype and C-terminal mutation. In the literature, mutations associated with additional motor neuron signs spanned over the entire gene, but patients with a particularly severe phenotype and premature death bore C-terminal mutations. CONCLUSION: Our findings support a genotype-phenotype correlation in SYNE1-ataxia and suggest the need for a systematic cardiologic evaluation in the setting of complicated spastic-ataxia phenotypes.

Evidence of mild founder LMOD3 mutations causing nemaline myopathy 10 in Germany and Austria.

OBJECTIVE: To expand the clinical and genetic spectrum of nemaline myopathy 10 by a series of Austrian and German patients with a milder disease course and missense mutations in LMOD3. METHODS: We characterized the clinical features and the genetic status of 4 unrelated adolescent or adult patients with nemaline myopathy. RESULTS: The 4 patients showed a relatively mild disease course. They all have survived into adulthood, 3 of 4 have remained ambulatory, and all showed marked facial weakness. Muscle biopsy specimens gave evidence of nemaline bodies. All patients were unrelated but originated from Austria (Tyrol and Upper Austria) and Southern Germany (Bavaria). All patients carried the missense variant c.1648C>T, p.(Leu550Phe) in the LMOD3 gene, either on both alleles or in trans with another missense variant (c.1004A>G, p.Gln335Arg). Both variants were not reported previously. CONCLUSIONS: In 2014, a severe form of congenital nemaline myopathy caused by disrupting mutations in LMOD3 was identified and denoted as NEM10. Unlike the previously reported patients, who had a severe clinical picture with a substantial risk of early death, our patients showed a relatively mild disease course. As the missense variant c.1648C>T is located further downstream compared to all previously published LMOD3 mutations, it might be associated with higher protein expression compared to the reported loss-of-function mutations. The apparent clusters of 2 mild mutations in Germany and Austria in 4 unrelated families may be explained by a founder effect.

High risk of peri-implant disease in periodontal Ehlers-Danlos Syndrome. A case series.

OBJECTIVES: Periodontal Ehlers-Danlos syndrome (pEDS) has recently been delineated as a molecularly defined cause of early severe periodontitis. Here we report that implant treatment failed in three affected individuals from one family. MATERIALS AND METHODS: Longitudinal data before and after implant treatment were examined for three individuals with genetically confirmed pEDS in the course of a large-scale pedigree analysis. RESULTS: Most detailed information was available for individual 1 in whom first periodontal bone loss was diagnosed at age 16 years. Rapid progression resulted in multiple tooth extractions at age 23 years and interforaminal placement of four implants. After primary implant success, peri-implant bone loss accompanied by highly inflamed tissues and receding gums led to explantation five years later. In individual 2, severe periodontitis was diagnosed at age 15 years and resulted in extraction of all mandibular teeth at age 28 years. Four interforaminal implants were placed. Peri-implant bone loss was diagnosed four years later, when up to three implant threads were exposed. Individual 3 showed complete tooth loss at age 29 years. He was restored with ten implants and removable prosthesis. Peri-implant bone loss was diagnosed radiologically eight years later, when seven implant threads were exposed. CONCLUSION: This is the first report on severe peri-implant bone loss in pEDS. Retention of teeth as long as possible is the primary objective in pEDS as satisfying prosthetic solutions are missing. Further evaluation of dental management in individuals with pEDS is needed to develop concise treatment guidelines.

PRUNE1 Deficiency: Expanding the Clinical and Genetic Spectrum.

BACKGROUND: Primary microcephaly and profound global developmental delay have been considered the core clinical phenotype in patients with bi-allelic PRUNE1 mutations. METHODS: Linkage analysis and whole-exome sequencing (WES) in a multiplex family and extraction of further cases from a WES repository containing 571 children with severe developmental disabilities and neurologic symptoms. RESULTS: We identified bi-allelic PRUNE1 mutations in twelve children from six unrelated families. All patients who survived beyond the first 6 months of life had early-onset global developmental delay, bilateral spastic paresis, dysphagia and difficult-to-treat seizures, while congenital or later-evolving microcephaly was not a consistent finding. Brain MRI showed variable anomalies with progressive cerebral and cerebellar atrophies and T2-hyperintense brain stem lesions. Peripheral neuropathy was documented in five cases. Disease course was progressive in all patients and eight children died in the first or early second decade of life. In addition to the previously reported missense mutation p.(Asp106Asn), we observed a novel homozygous missense variant p.(Leu172Pro) and a homozygous contiguous gene deletion encompassing most of the PRUNE1 gene and part of the neighboring BNIPL gene. CONCLUSIONS: PRUNE1 deficiency causes severe early-onset disease affecting the central and peripheral nervous systems. Microcephaly is probably not a universal feature.

Variable expressivity of TCTEX1D2 mutations and a possible pathogenic link of molar-incisor malformation to ciliary dysfunction.

OBJECTIVE: Clarification of the molecular basis of a ciliopathy associated with molar-incisor malformation in a consanguineous Turkish family. DESIGN: Full dental and clinical examinations, histologic analysis, comprehensive genetic analyses including exome sequencing, ciliary function tests and transmission electron microscopy of ciliary biopsies in the surviving patient. RESULTS: Two siblings had situs inversus and complex heart defects suggestive of ciliary dysfunction. The affected girl who died in utero showed severe chest abnormalities compatible with Jeune syndrome which were not present in the affected boy. Dental investigations in the boy showed typical signs of molar-incisor-malformation. Exome sequencing identified a homozygous intragenic deletion in TCTEX1D2 which is predicted to completely remove protein function. Ciliary function tests and electron microscopy showed mild irregularities of motile cilia such as compound cilia and loss of membranes. CONCLUSIONS: Our findings support the suggestion that TCTEX1D2 mutations have variable expressivity and may be associated with disturbances of embryonic development caused by both, ciliary signaling and motile dysfunction. The presence of molar-incisor-malformation in the living patient raises the possibility of a pathogenetic link of this rare dental anomaly to ciliary dysfunction during tooth development at least in some individuals.

SLC13A5 is the second gene associated with Kohlschütter-Tönz syndrome.

BACKGROUND: Kohlschütter-Tönz syndrome (KTZS) is a rare autosomal-recessive disease characterised by epileptic encephalopathy, intellectual disability and amelogenesis imperfecta (AI). It is frequently caused by biallelic mutations in ROGDI. Here, we report on individuals with ROGDI-negative KTZS carrying biallelic SLC13A5 mutations. METHODS: In the present cohort study, nine individuals from four families with the clinical diagnosis of KTZS and absence of ROGDI mutations as well as one patient with unexplained epileptic encephalopathy were investigated by clinical and dental evaluation, parametric linkage analysis (one family), and exome and/or Sanger sequencing. Dental histological investigations were performed on teeth from individuals with SLC13A5-associated and ROGDI-associated KTZS. RESULTS: Biallelic mutations in SLC13A5 were identified in 10 affected individuals. Epileptic encephalopathy usually presents in the neonatal and (less frequently) early infantile period. Yellowish to orange discolouration of both deciduous and permanent teeth, as well as wide interdental spaces and abnormal crown forms are major clinical signs of individuals with biallelic SLC13A5 mutations. Histological dental investigations confirmed the clinical diagnosis of hypoplastic AI. In comparison, the histological evaluation of a molar assessed from an individual with ROGDI-associated KTZS revealed hypocalcified AI. CONCLUSIONS: We conclude that SLC13A5 is the second major gene associated with the clinical diagnosis of KTZS, characterised by neonatal epileptic encephalopathy and hypoplastic AI. Careful clinical and dental delineation provides clues whether ROGDI or SLC13A5 is the causative gene. Hypersensitivity of teeth as well as high caries risk requires individual dental prophylaxis and attentive dental management.

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Clinical, morphological, biochemical, imaging and outcome parameters in 21 individuals with mitochondrial maintenance defect related to FBXL4 mutations.

FBXL4 deficiency is a recently described disorder of mitochondrial maintenance associated with a loss of mitochondrial DNA in cells. To date, the genetic diagnosis of FBXL4 deficiency has been established in 28 individuals. This paper retrospectively reviews proxy-reported clinical and biochemical findings and evaluates brain imaging, morphological and genetic data in 21 of those patients. Neonatal/early-onset severe lactic acidosis, muscular hypotonia, feeding problems and failure to thrive is the characteristic pattern at first presentation. Facial dysmorphic features are present in 67% of cases. Seven children died (mean age 37 months); 11 children were alive (mean age at follow-up 46 months), three children were lost to follow-up. All survivors developed severe psychomotor retardation. Brain imaging was non-specific in neonates but a later-onset, rapidly progressive brain atrophy was noted. Elevated blood lactate and metabolic acidosis were observed in all individuals; creatine kinase was elevated in 45% of measurements. Diagnostic workup in patient tissues and cells revealed a severe combined respiratory chain defect with a general decrease of enzymes associated with mitochondrial energy metabolism and a relative depletion of mitochondrial DNA content. Mutations were detected throughout the FBXL4 gene albeit with no clear delineation of a genotype-phenotype correlation. Treatment with "mitochondrial medications" did not prove effective. In conclusion, a clinical pattern of early-onset encephalopathy, persistent lactic acidosis, profound muscular hypotonia and typical facial dysmorphism should prompt initiation of molecular genetic analysis of FBXL4. Establishment of the diagnosis permits genetic counselling, prevents patients undergoing unhelpful diagnostic procedures and allows for accurate prognosis.

Genotype-based databases for variants causing rare diseases.

Inherited diseases are the result of DNA sequence changes. In recessive diseases, the clinical phenotype results from the combined functional effects of variants in both copies of the gene. In some diseases there is often considerable variability of clinical presentation or disease severity, which may be predicted by the genotype. Additional effects may be triggered by environmental factors, as well as genetic modifiers which could be nucleotide polymorphisms in related genes, e.g. maternal ApoE or ABCA1 genotypes which may have an influence on the phenotype of SLOS individuals. Here we report the establishment of genotype variation databases for various rare diseases which provide individual clinical phenotypes associated with genotypes and include data about possible genetic modifiers. These databases aim to be an easy public access to information on rare and private variants with clinical data, which will facilitate the interpretation of genetic variants. The created databases include ACAD8 (isobutyryl-CoA dehydrogenase deficiency (IBD)), ACADSB (short-chain acyl-CoA dehydrogenase (SCAD) deficiency), AUH (3-methylglutaconic aciduria (3-MGCA)), DHCR7 (Smith-Lemli-Opitz syndrome), HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2 deficiency), HSD17B10 (17-beta-hydroxysteroid dehydrogenase X deficiency), FKBP14 (Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss; EDSKMH) and ROGDI (Kohlschütter-Tönz syndrome). These genes have been selected because of our specific research interests in these rare and metabolic diseases. The aim of the database was to include all identified individuals with variants in these specific genes. Identical genotypes are listed multiple times if they were found in several patients, phenotypic descriptions and biochemical data are included as detailed as possible in view also of validating the proposed pathogenicity of these genotypes. For DHCR7 genetic modifier data (maternal APOE and ABCA1 genotypes) is also included. Databases are available at http://databases.lovd.nl/shared/genes and will be updated based on periodic literature reviews and submitted reports.

Kohlschütter-Tönz syndrome: mutations in ROGDI and evidence of genetic heterogeneity.

Kohlschütter-Tönz syndrome (KTS) is a rare autosomal recessive disorder characterized by amelogenesis imperfecta, psychomotor delay or regression and seizures starting early in childhood. KTS was established as a distinct clinical entity after the first report by Kohlschütter in 1974, and to date, only a total of 20 pedigrees have been reported. The genetic etiology of KTS remained elusive until recently when mutations in ROGDI were independently identified in three unrelated families and in five likely related Druze families. Herein, we report a clinical and genetic study of 10 KTS families. By using a combination of whole exome sequencing, linkage analysis, and Sanger sequencing, we identify novel homozygous or compound heterozygous ROGDI mutations in five families, all presenting with a typical KTS phenotype. The other families, mostly presenting with additional atypical features, were negative for ROGDI mutations, suggesting genetic heterogeneity of atypical forms of the disease.

Inherited ichthyoses/generalized Mendelian disorders of cornification.

Inherited ichthyoses, defined as the generalized form of Mendelian disorders of cornification, are characterized by visible scaling and/or hyperkeratosis of most or all of the skin. This etiologically and phenotypically heterogenous group of conditions is caused by mutations in various different genes important for keratinocyte differentiation and epidermal barrier function. Diagnosing a specific entity is a particular challenge for the nonspecialist presented with the common clinical scaling. For the clinician, this review outlines an algorithmic approach for utilizing diagnostic clues to narrow down the differential diagnosis and to guide further testing and treatment options.

SIMPLEX: cloud-enabled pipeline for the comprehensive analysis of exome sequencing data.

In recent studies, exome sequencing has proven to be a successful screening tool for the identification of candidate genes causing rare genetic diseases. Although underlying targeted sequencing methods are well established, necessary data handling and focused, structured analysis still remain demanding tasks. Here, we present a cloud-enabled autonomous analysis pipeline, which comprises the complete exome analysis workflow. The pipeline combines several in-house developed and published applications to perform the following steps: (a) initial quality control, (b) intelligent data filtering and pre-processing, (c) sequence alignment to a reference genome, (d) SNP and DIP detection, (e) functional annotation of variants using different approaches, and (f) detailed report generation during various stages of the workflow. The pipeline connects the selected analysis steps, exposes all available parameters for customized usage, performs required data handling, and distributes computationally expensive tasks either on a dedicated high-performance computing infrastructure or on the Amazon cloud environment (EC2). The presented application has already been used in several research projects including studies to elucidate the role of rare genetic diseases. The pipeline is continuously tested and is publicly available under the GPL as a VirtualBox or Cloud image at http://simplex.i-med.ac.at; additional supplementary data is provided at http://www.icbi.at/exome.