Cross-Sectional Study on Autosomal Recessive Congenital Ichthyoses: Association of Genotype with Disease Severity, Phenotypic, and Ultrastructural Features in 74 Italian Patients.

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCIs) are a clinically heterogeneous group of keratinization disorders characterized by generalized skin scaling due to mutations in at least 12 genes. The aim of our study was to assess disease severity, phenotypic, and ultrastructural features and to evaluate their association with genetic findings in ARCI patients. METHODS: Clinical signs and symptoms, and disease severity were scored in a single-center series of patients with a genetic diagnosis of ARCI. Skin ultrastructural findings were reviewed. RESULTS: Seventy-four consecutive patients (mean age 11.0 years, range 0.1-48.8) affected with lamellar ichthyosis (50/74, 67.5%), congenital ichthyosiform erythroderma (18/74, 24.3%), harlequin ichthyosis (two/74, 2.7%), and other minor ARCI subtypes (four/74, 5.4%) were enrolled. Mutated genes were as follows: TGM1 in 18/74 (24.3%) patients, ALOX12B in 18/74 (24.3%), CYP4F22 in 12/74 (16.2%), ABCA12 in nine/74 (12.2%), ALOXE3 in seven/74 (9.5%), NIPAL4 in seven/74 (9.5%), and CERS3, PNPLA1, and SDR9C7 in 1 patient each (1.4%). Twenty-five previously undescribed mutations in the different ARCI causative genes, as well as two microduplications in TGM1, and two microdeletions in CYP4F22 and NIPAL4 were identified. The mean ichthyosis severity score in TGM1- and ABCA12-mutated patients was significantly higher than in all other mutated genes, while the lowest score was observed in CYP4F22-mutated patients. Alopecia, ectropion, and eclabium were significantly associated with TGM1 and ABCA12 mutations, and large, thick, and brownish scales with TGM1 mutations. Among specific phenotypic features, psoriasis-like lesions as well as a trunk reticulate scale pattern and striated keratoderma were present in NIPAL4-mutated patients. Ultrastructural data available for 56 patients showed a 100% specificity of cholesterol clefts for TGM1-mutated cases and revealed abnormal lamellar bodies in SDR9C7 and CERS3 patients. CONCLUSION: Our study expands the phenotypic and genetic characterization of ARCI by the description of statistically significant associations between disease severity, specific clinical signs, and different mutated genes. Finally, we highlighted the presence of psoriasis-like lesions in NIPAL4-ARCI patients as a novel phenotypic feature with diagnostic and possible therapeutic implications.

Prenatal ultrasound detection of collodion membrane in association with an autosomal recessive congenital ichthyosis due to transglutaminase 1 deficiency.

We describe a case of collodion baby diagnosed prenatally by ultrasound. Classic signs (ectropion, flattened nose, and eclabion) were detected on routine ultrasound at 21 weeks of gestation. At birth, the presence of collodion membrane was confirmed and subsequently, the diagnosis of an autosomal recessive congenital ichthyosis due to compound heterozygosity of the TGM1 gene was made.

TRAPPC9-CDG: A novel congenital disorder of glycosylation with dysmorphic features and intellectual disability.

PURPOSE: TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities. METHODS: Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics. RESULTS: All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites. CONCLUSION: We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.

KBG syndrome: Common and uncommon clinical features based on 31 new patients.

KBG syndrome (MIM #148050) is an autosomal dominant disorder characterized by developmental delay, intellectual disability, distinct craniofacial anomalies, macrodontia of permanent upper central incisors, skeletal abnormalities, and short stature. This study describes clinical features of 28 patients, confirmed by molecular testing of ANKRD11 gene, and three patients with 16q24 deletion encompassing ANKRD11 gene, diagnosed in a single center. Common clinical features are reported, together with uncommon findings, clinical expression in the first years of age, distinctive associations, and familial recurrences. Unusual manifestations emerging from present series include juvenile idiopathic arthritis, dysfunctional dysphonia, multiple dental agenesis, idiopathic precocious telarche, oral frenula, motor tics, and lipoma of corpus callosum, pilomatrixoma, and endothelial corneal polymorphic dystrophy. Facial clinical markers suggesting KBG syndrome before 6 years of age include ocular and mouth conformation, wide eyebrows, synophrys, long black eyelashes, long philtrum, thin upper lip. General clinical symptoms leading to early genetic evaluation include developmental delay, congenital malformations, hearing anomalies, and feeding difficulties. It is likely that atypical clinical presentation and overlapping features in patients with multiple variants are responsible for underdiagnosis in KBG syndrome. Improved knowledge of common and atypical features of this disorder improves clinical management.

Hoarse cry in a newborn with epidermolysis bullosa simplex, generalized severe.

Hoarse cry and respiratory stridor are the signs of potentially life-threatening laryngeal involvement in selected severe and frequently early lethal subtypes of inherited epidermolysis bullosa (EB). We present a newborn with generalized skin blistering and onychodystrophy who developed a hoarse cry and inspiratory stridor. Ultrastructural skin examination revealed tonofilament clumping in basal keratinocytes and genetic testing identified the de novo missense mutation p.Arg125Cys in the KRT14 gene, consistent with EB simplex generalized severe, which is characterized by major morbidity in infancy but a favorable long-term prognosis. The present case underlines the importance to consider EB simplex generalized severe in the differential diagnosis of EB infants presenting hoarseness and stridor.

First Case of KRT2 Epidermolytic Nevus and Novel Clinical and Genetic Findings in 26 Italian Patients with Keratinopathic Ichthyoses.

Keratinopathic ichthyoses (KI) are a clinically heterogeneous group of keratinization disorders due to mutations in KRT1, KTR10, or KRT2 genes encoding keratins of suprabasal epidermis. Characteristic clinical features include superficial blisters and erosions in infancy and progressive development of hyperkeratosis. Histopathology shows epidermolytic hyperkeratosis. We describe the clinical, histopathological, and molecular findings of a series of 26 Italian patients from 19 unrelated families affected with (i) epidermolytic ichthyosis due to KRT1 or KRT10 mutations (7 and 9 cases, respectively); (ii) KTR10-mutated ichthyosis with confetti (2 cases); (iii) KRT2-mutated superficial epidermolytic ichthyosis (5 cases); and (iv) KRT10-mutated epidermolytic nevus (2 cases). Of note, molecular genetic testing in a third case of extensive epidermolytic nevus revealed a somatic missense mutation (p.Asn186Asp) in the KRT2 gene, detected in DNA from lesional skin at an allelic frequency of 25% and, at very low frequency (1.5%), also in blood. Finally, we report three novel dominant mutations, including a frameshift mutation altering the C-terminal V2 domain of keratin 1 in three familiar cases presenting a mild phenotype. Overall, our findings expand the phenotypic and molecular spectrum of KI and show for the first time that epidermolytic nevus can be due to somatic KRT2 mutation.

X-linked ichthyosis: Clinical and molecular findings in 35 Italian patients.

Recessive X-linked ichthyosis (XLI), the second most common ichthyosis, is caused by mutations in the STS gene encoding the steroid sulfatase enzyme. A complete deletion of the STS gene is found in 85%-90% of cases. Rarely, larger deletions involving contiguous genes are detected in syndromic patients. We report the clinical and molecular genetic findings in a series of 35 consecutive Italian male patients. All patients underwent molecular testing by MLPA or aCGH, followed, in case of negative results, by next-generation sequencing analysis. Neuropsychiatric, ophthalmological and paediatric evaluations were also performed. Our survey showed a frequent presence of disease manifestations at birth (42.8%). Fold and palmoplantar surfaces were involved in 18 (51%) and 7 (20%) patients, respectively. Fourteen patients (42%) presented neuropsychiatric symptoms, including attention-deficit hyperactivity disorder and motor disabilities. In addition, two patients with mental retardation were shown to be affected by a contiguous gene syndrome. Twenty-seven patients had a complete STS deletion, one a partial deletion and 7 carried missense mutations, two of which previously unreported. In addition, a de novo STS deletion was identified in a sporadic case. The frequent presence of palmoplantar and fold involvement in XLI should be taken into account when considering the differential diagnosis with ichthyosis vulgaris. Our findings also underline the relevance of involving the neuropsychiatrist in the multidisciplinary management of XLI. Finally, we report for the first time a de novo mutation which shows that STS deletion can also occur in oogenesis.

Keppen-Lubinsky syndrome is caused by mutations in the inwardly rectifying K+ channel encoded by KCNJ6.

Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K(+) channels.

Interstitial 10q21.1q23.31 Duplication due to Meiotic Recombination of a Paternal Balanced Complex Rearrangement: Cytogenetic and Molecular Characterization.

Complex chromosomal rearrangements (CCRs) are structural aberrations involving more than 2 chromosomal breakpoints. They are associated with different outcomes depending on the deletion/duplication of genomic material, gene disruption, or position effects. Balanced CCRs can also undergo missegregation during meiotic division, leading to unbalanced derivative chromosomes and, in some cases, to affected offspring. We report on a patient presenting with developmental and speech delay, growth retardation, microcephaly, hypospadias, and dysmorphic features, harboring an interstitial 10q21.1q23.31 duplication, due to recombination of a paternal CCR. Application of several cytogenetic and molecular techniques allowed determining the biological bases of the rearrangement, understanding the underlying chromosomal mechanism, and assessing the reproductive risk.

Congenital heart defects in molecularly proven Kabuki syndrome patients.

The prevalence of congenital heart defects (CHD) in Kabuki syndrome ranges from 28% to 80%. Between January 2012 and December 2015, 28 patients had a molecularly proven diagnosis of Kabuki syndrome. Pathogenic variants in KMT2D (MLL2) were detected in 27 patients, and in KDM6A gene in one. CHD was diagnosed in 19/27 (70%) patients with KMT2D (MLL2) variant, while the single patient with KDM6A change had a normal heart. The anatomic types among patients with CHD included aortic coarctation (4/19 = 21%) alone or associated with an additional CHD, bicuspid aortic valve (4/19 = 21%) alone or associated with an additional CHD, perimembranous subaortic ventricular septal defect (3/19 = 16%), atrial septal defect ostium secundum type (3/19 = 16%), conotruncal heart defects (3/19 = 16%). Additional CHDs diagnosed in single patients included aortic dilatation with mitral anomaly and hypoplastic left heart syndrome. We also reviewed CHDs in patients with a molecular diagnosis of Kabuki syndrome reported in the literature. In conclusion, a CHD is detected in 70% of patients with KMT2D (MLL2) pathogenic variants, most commonly left-sided obstructive lesions, including multiple left-sided obstructions similar to those observed in the spectrum of the Shone complex, and septal defects. Clinical management of Kabuki syndrome should include echocardiogram at the time of diagnosis, with particular attention to left-sided obstructive lesions and mitral anomalies, and annual monitoring for aortic arch dilatation.

CHARGE syndrome due to deletion of region upstream of CHD7 gene START codon.

BACKGROUND: CHARGE syndrome is an autosomal dominant disorder, characterized by ocular Coloboma, congenital Heart defects, choanal Atresia, Retardation, Genital anomalies and Ear anomalies. Over 90 % of typical CHARGE patients are mutated in the CHD7 gene, 65 %-70 % of the cases for all typical and suspected cases combined. The gene encoding for a protein involved in chromatin organization. The mutational spectrum include nonsense, frameshift, splice site, and missense mutations. Large deletions and genomic rearrangements are rare. CASE PRESENTATION: We report here on a 5.9 years old male of Moroccan origin displaying classic clinical features of CHARGE syndrome. Using CGH array and NGS analysis we detected a microdeletion (184 kb) involving the promoter region and exon 1 of CHD7 gene and the flanking RAB2 gene. CONCLUSION: The present observation suggests that deletion limited to the regulatory region of CHD7 is sufficient to cause the full blown CHARGE phenotype. Different size of deletions can result in different phenotypes, ranging from a milder to severe CHARGE syndrome; this is based on a combination of major and minor diagnostic characteristics, therefore to a more variable clinical features, likely due to the additive effect of other genetic imbalances. MLPA and CGH techniques should be considered in the diagnostic protocol of individuals with a clinical suspect of CHARGE syndrome.

Diagnosis of Noonan syndrome and related disorders using target next generation sequencing.

BACKGROUND: Noonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies. METHODS: In this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL. RESULTS: All expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing. CONCLUSION: Here we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.

Prenatal identification of a pathogenic maternal FGFR1 variant in two consecutive pregnancies with fetal forebrain malformations.

OBJECTIVE: Holoprosencephaly (HPE) is the most common aberration of forebrain development, and it leads to a wide spectrum of developmental and craniofacial anomalies. HPE etiology is highly heterogeneous and includes both chromosomal abnormalities and single-gene defects. METHODS: Here, we report an FGFR1 heterozygous variant detected by prenatal exome sequencing and inherited from the asymptomatic mother, in association with recurrent neurological abnormalities in the HPE spectrum in two consecutive pregnancies. RESULTS: Individuals with germline pathogenic variants in FGFR1 (MIM: 136350) show extensive phenotypic variability, which ranges from asymptomatic carriers to hypogonadotropic hypogonadism, arhinencephaly, Kallmann's syndrome with associated features such as cleft lip and palate, skeletal anomalies, isolated HPE, and Hartsfield syndrome. CONCLUSION: The presented case supports the role of exome sequencing in prenatal diagnosis when fetal midline structural anomalies are suggestive of a genetic etiology, as early as the first trimester of gestation. The profound heterogeneity of FGFR1 allelic disorders needs to be considered when planning prenatal screening even in asymptomatic carriers.

DNA methylation profiling in Kabuki syndrome: reclassification of germline KMT2D VUS and sensitivity in validating postzygotic mosaicism.

Autosomal dominant Kabuki syndrome (KS) is a rare multiple congenital anomalies/neurodevelopmental disorder caused by heterozygous inactivating variants or structural rearrangements of the lysine-specific methyltransferase 2D (KMT2D) gene. While it is often recognizable due to a distinctive gestalt, the disorder is clinically variable, and a phenotypic scoring system has been introduced to help clinicians to reach a clinical diagnosis. The phenotype, however, can be less pronounced in some patients, including those carrying postzygotic mutations. The full spectrum of pathogenic variation in KMT2D has not fully been characterized, which may hamper the clinical classification of a portion of these variants. DNA methylation (DNAm) profiling has successfully been used as a tool to classify variants in genes associated with several neurodevelopmental disorders, including KS. In this work, we applied a KS-specific DNAm signature in a cohort of 13 individuals with KMT2D VUS and clinical features suggestive or overlapping with KS. We succeeded in correctly classifying all the tested individuals, confirming diagnosis for three subjects and rejecting the pathogenic role of 10 VUS in the context of KS. In the latter group, exome sequencing allowed to identify the genetic cause underlying the disorder in three subjects. By testing five individuals with postzygotic pathogenic KMT2D variants, we also provide evidence that DNAm profiling has power to recognize pathogenic variants at different levels of mosaicism, identifying 15% as the minimum threshold for which DNAm profiling can be applied as an informative diagnostic tool in KS mosaics.

Assessment of gene-disease associations and recommendations for genetic testing for somatic variants in vascular anomalies by VASCERN-VASCA.

BACKGROUND: Vascular anomalies caused by somatic (postzygotic) variants are clinically and genetically heterogeneous diseases with overlapping or distinct entities. The genetic knowledge in this field is rapidly growing, and genetic testing is now part of the diagnostic workup alongside the clinical, radiological and histopathological data. Nonetheless, access to genetic testing is still limited, and there is significant heterogeneity across the approaches used by the diagnostic laboratories, with direct consequences on test sensitivity and accuracy. The clinical utility of genetic testing is expected to increase progressively with improved theragnostics, which will be based on information about the efficacy and safety of the emerging drugs and future molecules. The aim of this study was to make recommendations for optimising and guiding the diagnostic genetic testing for somatic variants in patients with vascular malformations. RESULTS: Physicians and lab specialists from 11 multidisciplinary European centres for vascular anomalies reviewed the genes identified to date as being involved in non-hereditary vascular malformations, evaluated gene-disease associations, and made recommendations about the technical aspects for identification of low-level mosaicism and variant interpretation. A core list of 24 genes were selected based on the current practices in the participating laboratories, the ISSVA classification and the literature. In total 45 gene-phenotype associations were evaluated: 16 were considered definitive, 16 strong, 3 moderate, 7 limited and 3 with no evidence. CONCLUSIONS: This work provides a detailed evidence-based view of the gene-disease associations in the field of vascular malformations caused by somatic variants. Knowing both the gene-phenotype relationships and the strength of the associations greatly help laboratories in data interpretation and eventually in the clinical diagnosis. This study reflects the state of knowledge as of mid-2023 and will be regularly updated on the VASCERN-VASCA website (VASCERN-VASCA, https://vascern.eu/groupe/vascular-anomalies/ ).

A New Case of Autosomal-Dominant POLR3B-Related Disorder: Widening Genotypic and Phenotypic Spectrum.

POLR3B encodes the RPC2 subunit of RNA polymerase III. Pathogenic variants are associated with biallelic hypomyelinating leukodystrophy belonging to the POLR-related disorders. Recently, the association with dominant demyelinating neuropathy, classified as Charcot-Marie-Tooth syndrome type 1I (CMT1I), has been reported as well. Here we report on an additional patient presenting with developmental delay and generalized epilepsy, followed by the onset of mild pyramidal and cerebellar signs, vertical gaze palsy and subclinical demyelinating polyneuropathy. A new heterozygous de novo missense variant, c.1297C > G, p.Arg433Gly, in POLR3B was disclosed via trio-exome sequencing. In silico analysis confirms the hypothesis on the variant pathogenicity. Our research broadens both the genotypic and phenotypic spectrum of the autosomal-dominant POLR3B-related condition.