Comprehensive Molecular Analysis of Disease-Related Genes as First-Tier Test for Early Diagnosis, Classification, and Management of Patients Affected by Nonsyndromic Ichthyosis.

Inherited ichthyoses are a group of clinically and genetically heterogeneous rare disorders of skin keratinization with overlapping phenotypes. The clinical picture and family history are crucial to formulating the diagnostic hypothesis, but only the identification of the genetic defect allows the correct classification. In the attempt to molecularly classify 17 unrelated Italian patients referred with congenital nonsyndromic ichthyosis, we performed massively parallel sequencing of over 50 ichthyosis-related genes. Genetic data of 300 Italian unaffected subjects were also analyzed to evaluate frequencies of putative disease-causing alleles in our population. For all patients, we identified the molecular cause of the disease. Eight patients were affected by autosomal recessive congenital ichthyosis associated with ALOX12B, NIPAL4, and TGM1 mutations. Three patients had biallelic loss-of-function variants in FLG, whereas 6/11 males were affected by X-linked ichthyosis. Among the 24 different disease-causing alleles we identified, 8 carried novel variants, including a synonymous TGM1 variant that resulted in a splicing defect. Moreover, we generated a priority list of the ichthyosis-related genes that showed a significant number of rare and novel variants in our population. In conclusion, our comprehensive molecular analysis resulted in an effective first-tier test for the early classification of ichthyosis patients. It also expands the genetic, mutational, and phenotypic spectra of inherited ichthyosis and provides new insight into the current understanding of etiologies and epidemiology of this group of rare disorders.

Specifications and validation of the ACMG/AMP criteria for clinical interpretation of sequence variants in collagen genes associated with joint hypermobility.

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.

De Novo Mutation in KMT2C Manifesting as Kleefstra Syndrome 2: Case Report and Literature Review.

Diagnosis of pediatric intellectual disability (ID) can be difficult because it is due to a vast number of established and novel causes. Here, we described a full-term female infant affected by Kleefstra syndrome-2 presenting with neurodevelopmental disorder, a history of hypotonia and minor face anomalies. A systematic literature review was also performed. The patient was a 6-year-old Caucasian female. In the family history there was no intellectual disability or genetic conditions. Auxological parameters at birth were adequate for gestational age. Clinical evaluation at 6 months revealed hypotonia and, successively, delay in the acquisition of the stages of psychomotor development. Auditory, visual, somatosensory, and motor-evoked potentials were normal. A brain MRI, performed at 9 months, showed minimal gliotic changes in bilateral occipital periventricular white matter. Neuropsychiatric control, performed at 5 years, established a definitive diagnosis of childhood autism and developmental delay. Molecular analysis of the exome revealed a novel KMT2C missense variant: c.9244C > T (p.Pro3082Ser) at a heterozygous state, giving her a diagnosis of Kleefstra syndrome 2. Parents did not show the variant. Literature review (four retrieved eligible studies, 10 patients) showed that all individuals had mild, moderate, or severe ID; language and motor delay; and autism. Short stature, microcephaly, childhood hypotonia and plagiocephaly were also present. Conclusion. Kleefstra syndrome 2 is a difficult diagnosis of a rare condition with a high clinical phenotypic heterogeneity. This study suggests that it must be taken in account in the work-up of an orphan diagnosis of intellectual disability and/or autism spectrum disorder.

Clinical heterogeneity of Kabuki syndrome in a cohort of Italian patients and review of the literature.

Kabuki syndrome (KS) is a well-recognized disorder characterized by postnatal growth deficiency, dysmorphic facial features, skeletal anomalies, and intellectual disability. The syndrome is caused by KMT2D gene mutations or less frequently KDM6A gene mutations or deletions. We report a systematic evaluation of KS patients from Campania region of Italy; data were also compared with literature ones. We collected data of 15 subjects (8 males and 7 females with age range 10-26 years; mean age 16.9 years) with confirmed diagnosis of KS, representing the entire cohort of patients from Campania Region. Each patient performed biochemical testing and instrumental investigation. Neuro-intellectual development, cranio-facial dysmorphisms, and multisystem involvement data were collected retrospectively. For each category, type of defects and frequency of the anomalies were analyzed. Our observation shows that KS patients from Campania region have some particular and previously underscored, neurological and immunological findings. We found high prevalence of EEG's abnormalities (43%) and MRI brain abnormalities (60%). Microcephaly resulted more common in our series (33%), if compared with major cohorts described in literature. Biochemical features of immunodeficiency and autoimmune diseases including thyroid autoimmunity, polyserositis, and vitiligo were observed with high prevalence (54.5%). Low immunoglobulins levels were a frequent finding. Lymphocyte class investigation showed significantly reduced CD8 levels in one patient.Conclusions: These data confirm great heterogeneity of clinical manifestations in KS and suggest to introduce further clinical diagnostic criteria in order to perform a correct and precocious diagnosis. What is Known • Kabuki syndrome is characterized by growth deficiency, dysmorphic facial features, skeletal anomalies, and intellectual disability • Immune dysfunction is a common finding but autoimmune diseases are rarely seen • Neurological features are common What is New • Some particular facial features could help gestalt diagnosis (hypertelorism, broad nasal bridge, micrognathia, tooth agenesis, cutaneous haemangiomas and strabismus) • Higher prevalence of autoimmune disorders than previously reported • Particular neurological features are present in this cohort (EEG and MRI brain abnormalities).

Pathobiologic Mechanisms of Neurodegeneration in Osteopetrosis Derived From Structural and Functional Analysis of 14 ClC-7 Mutants.

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).

A novel GLA mutation in a Fabry family with glucose-6-phosphate dehydrogenase deficiency.

Fabry disease is an X-linked lysosomal disease caused by mutations of the alpha-galactosidase A (GLA) gene at chromosome subband Xq22.1. To date, more than 600 genetic mutations have been identified to determine the nature and frequency of the molecular lesions causing the classical and milder variant phenotypes and for precise carrier detection. We report here a Fabry family (mother, son and daughter) where the alpha-galactosidase A defect was associated with a glucose-6-phosphate dehydrogenase (G6PD) deficiency. Mutation analysis revealed for the GLA gene the presence of a new mutation, i.e., a small deletion (c.452delA) on exon 3 and for the G6PD gene the presence of 2 mutations, p.V68M (G6PD Asahi, G6PD A+) and p.N126D (G6PD A+) on exon 3 and exon 4, respectively.

Two founder mutations in the SEC23B gene account for the relatively high frequency of CDA II in the Italian population.

Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non-Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non-Italian unrelated patients. Two mutations, E109K and R14W, account for over one-half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ≈2,200 years; whereas for the R14W mutation it is ≈3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy.