Germline NUP98 Variants in Two Siblings with a Rothmund-Thomson-Like Spectrum: Protein Functional Changes Predicted by Molecular Modeling.

Two adult siblings born to first-cousin parents presented a clinical phenotype reminiscent of Rothmund-Thomson syndrome (RTS), implying fragile hair, absent eyelashes/eyebrows, bilateral cataracts, mottled pigmentation, dental decay, hypogonadism, and osteoporosis. As the clinical suspicion was not supported by the sequencing of RECQL4, the RTS2-causative gene, whole exome sequencing was applied and disclosed the homozygous variants c.83G>A (p.Gly28Asp) and c.2624A>C (p.Glu875Ala) in the nucleoporin 98 (NUP98) gene. Though both variants affect highly conserved amino acids, the c.83G>A looked more intriguing due to its higher pathogenicity score and location of the replaced amino acid between phenylalanine-glycine (FG) repeats within the first NUP98 intrinsically disordered region. Molecular modeling studies of the mutated NUP98 FG domain evidenced a dispersion of the intramolecular cohesion elements and a more elongated conformational state compared to the wild type. This different dynamic behavior may affect the NUP98 functions as the minor plasticity of the mutated FG domain undermines its role as a multi-docking station for RNA and proteins, and the impaired folding can lead to the weakening or the loss of specific interactions. The clinical overlap of NUP98-mutated and RTS2/RTS1 patients, accounted by converging dysregulated gene networks, supports this first-described constitutional NUP98 disorder, expanding the well-known role of NUP98 in cancer.

Two unrelated cases with biallelic CHEK2 variants:a novel condition with constitutional chromosomal instability?

Constitutional heterozygous mutations in CHEK2 gene have been associated with hereditary cancer risk. To date, only a few homozygous CHEK2 mutations have been reported in families with cancer susceptibility. Here, we report two unrelated individuals with a personal and familial cancer history in whom biallelic CHEK2 alterations were identified. The first case resulted homozygous for the CHEK2 c.793-1 G > A (p.Asp265Thrfs*10) variant, and the second one was found to be compound heterozygous for the c.1100delC (p.Thr367Metfs*15) and the c.1312 G > T (p.Asp438Tyr) variants. Multiple cytogenetic anomalies were demonstrated on peripheral lymphocytes of both patients. A literature revision showed that a single other CHEK2 homozygous variant was previously associated to a constitutional randomly occurring multi-translocation karyotype from peripheral blood in humans. We hypothesize that, at least some biallelic CHEK2 mutations might be associated with a novel disorder, further expanding the group of chromosome instability syndromes. Additional studies on larger cohorts are needed to confirm if chromosomal instability could represent a marker for CHEK2 constitutionally mutated recessive genotypes, and to investigate the cancer risk and the occurrence of other anomalies typically observed in chromosome instability syndromes.

A Novel Hypothesis on Choroideremia-Manifesting Female Carriers: Could CHM In-Frame Variants Exert a Dominant Negative Effect? A Case Report.

Choroideremia is an X-linked recessive condition presenting in males, with progressive degeneration of retinal and choroidal tissues leading to progressive visual loss. Its pathological mechanism is due to alterations in the CHM gene that encodes for REP1, a protein required for prenylation of Rab by the Rab geranylgeranyl transferase (RGGT). Even though female carriers are predicted to be not affected by the disease, a wide phenotypic spectrum ranging from mild to severe cases has been reported in women. The reason why Choroideremia manifests in female carriers remains elusive. While X chromosome inactivation (XCI) skewing has been proposed as a leading putative mechanism, emerging evidence has shown that CHM could variably escape from XCI. We described a family with an initial clinical suspicion of Retinitis Pigmentosa in which a novel CHM pathogenic splicing variant was found by exome sequencing. The variant, initially found in the 63-year-old female presenting with impaired visual acuity and severe retinal degeneration, segregated in the 31-year-old daughter and the 37-year-old son, both presenting with fundus anomalies. mRNA studies revealed a shorter in-frame CHM isoform lacking exon 10. Molecular modeling of the ternary REP1/Rab/RGGT protein complex predicted significant impairing of REP1/Rab binding without alteration of REP1/RGGT interaction. We suggest that, in our female cases, the biallelic expression of CHM may have led to the production of both the mutant and wild type REP1. The mutant isoform, sequestrating RGGT, could reduce its available amount for Rab prenylation, thus exerting a dominant-negative effect. If confirmed with further studies and in large cohorts of female carriers, the here proposed molecular mechanism could help to explain the complexity of manifestation of Choroideremia in females.

Clinical presentation and molecular characterization of a novel patient with variant POC1A-related syndrome.

Biallelic pathogenic variants in POC1A result in SOFT (Short-stature, Onychodysplasia, Facial-dysmorphism, and hypoTrichosis) and variant POC1A-related (vPOC1A) syndromes. The latter, nowadays described in only two unrelated subjects, is associated with a restricted spectrum of variants falling in exon 10, which is naturally skipped in a specific POC1A mRNA. The synthesis of an amount of a POC1A isoform from this transcript in individuals with vPOC1A syndrome has been believed as the likely explanation for such a genotype-phenotype correlation. Here, we illustrate the clinical and molecular findings in a woman who resulted to be compound heterozygous for a recurrent frameshift variant in exon 10 and a novel variant in exon 9 of POC1A. Phenotypic characteristics of this woman included severe hyperinsulinemic dyslipidemia, acanthosis nigricans, moderate growth restriction, and dysmorphisms. These manifestations overlap the clinical features of the two previously published individuals with vPOC1A syndrome. RT-PCR analysis on peripheral blood and subsequent sequencing of the obtained amplicons demonstrated a variety of POC1A alternative transcripts that resulted to be expressed in the proband, in the healthy mother, and in controls. We illustrate the possible consequences of the two POC1A identified variants in an attempt to explain pleiotropy in vPOC1A syndrome.

A Single Center Retrospective Review of Patients from Central Italy Tested for Melanoma Predisposition Genes.

BACKGROUND: Cutaneous malignant melanoma (CMM) is one of the most common skin cancers worldwide. CMM pathogenesis involves genetic and environmental factors. Recent studies have led to the identification of new genes involved in CMM susceptibility: beyond CDKN2A and CDK4, BAP1, POT1, and MITF were recently identified as potential high-risk melanoma susceptibility genes. OBJECTIVE: This study is aimed to evaluate the genetic predisposition to CMM in patients from central Italy. METHODS: From 1998 to 2017, genetic testing was performed in 888 cases with multiple primary melanoma and/or familial melanoma. Genetic analyses included the sequencing CDKN2A, CDK4, BAP1, POT1, and MITF in 202 cases, and of only CDKN2A and CDK4 codon 24 in 686 patients. By the evaluation of the personal and familial history, patients were divided in two clinical categories: "low significance" and "high significance" cases. RESULTS: 128 patients (72% belonging to the "high significance" category, 28% belonging to the "low significance" category) were found to carry a DNA change defined as pathogenic, likely pathogenic, variant of unknown significance (VUS)-favoring pathogenic or VUS. CONCLUSIONS: It is important to verify the genetic predisposition in CMM patients for an early diagnosis of further melanomas and/or other tumors associated with the characterized genotype.

A novel CDKN2A in-frame deletion associated with pancreatic cancer-melanoma syndrome.

Pancreatic cancer-melanoma syndrome (PCMS) is an inherited condition in which mutation carriers have an increased risk of malignant melanoma and/or pancreatic cancer. About 30% of PCMS cases carry mutations in CDKN2A. This gene encodes several protein isoforms, one of which, known as p16, regulates the cell-cycle by interacting with CDK4/CDK6 kinases and with several non-CDK proteins. Herein, we report on a novel CDKN2A germline in-frame deletion (c.52_57delACGGCC) found in an Italian family with PCMS. By segregation analysis, the c.52_57delACGGCC was proven to segregate in kindred with cutaneous melanoma (CM), in kindred with CM and pancreatic cancer, and in a single case presenting only with pancreatic cancer. In the literature, duplication mapping in the same genic region has been already reported at the germline level in several unrelated CM cases as a variant of unknown clinical significance. A computational approach for studying the effect of mutational changes over p16 protein structure showed that both the deletion and the duplication of the c.52_57 nucleotides result in protein misfolding and loss of interactors' binding. In conclusion, the present results argue that the quantitative alteration of nucleotides c.52_57 has a pathogenic role in p16 function and that the c.52_57delACGGCC is associated with PCMS.

Striking phenotypic overlap between Nicolaides-Baraitser and Coffin-Siris syndromes in monozygotic twins with ARID1B intragenic deletion.

The chromatin remodeling AT-Rich interaction domain containing 1B protein (ARID1B) also known as BAF-associated factor, 250-KD, B (BAF250B) codified by the ARID1B gene (MIM#614556), is a small subunit of the mammalian SWI/SNF or BAF complex, an ATP-dependent protein machinery which is able to activate or repress gene transcription, allowing protein access to histones through DNA relaxed conformation. ARID1B gene mutations have been associated with two hereditary syndromic conditions, namely Coffin-Siris (CSS, MIM#135900) and Nicolaides-Baraitser syndromes (NCBRS, MIM#601358), characterized by neurodevelopment delay, craniofacial dysmorphisms and skeletal anomalies. Furthermore, intellectual impairment and central nervous system (CNS) alterations, comprising abnormal corpus callosum, have been associated with mutations in this gene. Moreover, ARID1B anomalies resulted to be involved in neoplastic events and Hirschprung disease. Here we report on two monozygotic male twins, displaying clinical appearance strikingly resembling NCBRS and CSS phenotype, who resulted carriers of a novel 6q25.3 microdeletion, encompassing only part of the ARID1B gene. The deleted segment was not inherited from the only parent tested and afflicted the first exons of the gene, coding for protein disordered region. We also provide, for the first time, a review of previously published ARID1B mutated patients with NCBRS and CSS phenotype and a computer-assisted dysmorphology analysis of NCBRS and ARID1B related CSS individuals, through the Face2Gene suite, confirming the existence of highly overlapping facial gestalt of both conditions. The present findings indicate that ARID1B could be considered a contributing gene not only in CSS but also in NCBRS phenotype, although the main gene related to this latter condition is the SMARCA2 gene (MIM#600014), another component of the BAF complex. So, ARID1B study should be considered in such individuals.

Autism spectrum disorder in a patient with a genomic rearrangement that only involves the EPHA5 gene.

About one child in 68 is affected by the autism spectrum disorder (ASD), one of the most common neurodevelopmental disorders linked to intellectual disability, especially in males, intellectual disability being diagnosable in about 60-70% of autistic individuals. The biological bases of ASD are not yet fully known, but they are generally considered multifactorial, although many genes and genomic loci have been proposed to be possibly associated with this condition. In this report, we describe the case of a 14-year-old female Italian proband affected by ASD, carrying a novel ~ 270 kb interstitial microduplication, localized at the distal portion of the 4q13.1 region. The rearrangement was inherited from a mild symptomatic father and included a large part of the single EPHA5 gene, a receptor tyrosine kinase involved in the neural development, already indicated to be linked to ASD by previous Genome Wide Association Studies. This imbalance represents, to the best of our knowledge, the smallest duplication identified to date that only impacts the EPHA5 gene. We hypothesize that the duplication of this gene may alter EPHA5 expression and that this may impact the autistic phenotype of the patient.

Sensitivity to asbestos is increased in patients with mesothelioma and pathogenic germline variants in BAP1 or other DNA repair genes.

Pathogenic germline variants in the BAP1 tumor suppressor gene can cause a cancer syndrome called BAP1 tumor predisposition syndrome (BAP1-TPDS), which is characterized by predisposition to mesothelioma, melanoma, renal cell carcinoma, basal cell carcinoma, and other tumors. Other genes that may predispose to mesothelioma are CDKN2A and DNA repair genes. Asbestos exposure has often been reported in patients with malignant pleural mesothelioma (MPM) and germline variants in BAP1, but this exposure has never been quantified. We aimed to search for germline variants in BAP1 among 25 new Italian probands with suspected BAP1-TPDS, summarize the prevalence of these variants in 39 Italian patients with familial MPM and other tumors recruited over a 5-year period, and compare cumulative asbestos exposure in 14 patients with MPM and pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes with that of 67 patients without germline variants in 94 cancer-predisposing genes. We report here a new pathogenic germline variant in BAP1: c.783 + 2 T > C. The prevalence of pathogenic germline variants in BAP1 was 7.7% among patients with familial MPM (3/39). Patients with pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes showed lower cumulative asbestos exposure than patients without germline variants in 94 cancer-predisposing genes (P = .00002). This suggests an interaction between genetic risk factors and asbestos in the development of mesothelioma.

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis.

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as "ferroportin disease", which is due to "loss of function" mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by "gain of function" mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation. In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.

Familial melanoma and multiple primary melanoma.

Cutaneous melanoma (CM) has the highest mortality rates among the most common skin cancers, and its incidence is rising worldwide, thus representing a significant health care burden. CM is considered the most lethal skin cancer if not detected and treated during its early stages. Susceptibility to CM is also associated with an increased presence of atypical nevi and the occurrence of multiple primary melanoma. Personal history of CM increases the risk of developing a second melanoma by 5-8%. A family history of melanoma has also been strongly associated with an increased risk of melanoma. Approximately 5-10% of melanoma cases occur in a familial context. The main genes involved are CDKN2A, CDK4 and MC1R. The recent technological advances have allowed the identification of new genes involved in melanoma susceptibility: breast cancer 1 (BRCA1), BRCA1-associated protein 1 (BAP1), and telomerase reverse transcriptase (TERT).Tests on these genes allow to identify a larger number of high-risk individuals with a potential of developing familial melanoma and primary multiple melanomas. These patients also have a high risk of developing internal organ malignancies, especially pancreatic cancer. It is essential that these individuals receive adequate management along with frequent dermatological examinations, dermoscopic evaluation, genetic counselling and instrumental examinations aimed at the early identification of other tumors associated with CM.

Spectrum of mucocutaneous manifestations in 277 patients with joint hypermobility syndrome/Ehlers-Danlos syndrome, hypermobility type.

Cutaneous manifestations are a diagnostic criterion of Ehlers-Danlos syndrome, hypermobility type (EDS-HT) and joint hypermobility syndrome (JHS). These two conditions, originally considered different disorders, are now accepted as clinically indistinguishable and often segregate as a single-familial trait. EDS-HT and JHS are still exclusion diagnoses not supported by any specific laboratory test. Accuracy of clinical diagnosis is, therefore, crucial for appropriate patients' classification and management, but it is actually hampered by the low consistency of many applied criteria including the cutaneous one. We report on mucocutaneous findings in 277 patients with JHS/EDS-HT with both sexes and various ages. Sixteen objective and five anamnestic items were selected and ascertained in two specialized outpatient clinics. Feature rates were compared by sex and age by a series of statistical tools. Data were also used for a multivariate correspondence analysis with the attempt to identify non-causal associations of features depicting recognizable phenotypic clusters. Our findings identified a few differences between sexes and thus indicated an attenuated sexual dimorphism for mucocutaneous features in JHS/EDS-HT. Ten features showed significantly distinct rates at different ages and this evidence corroborated the concept of an evolving phenotype in JHS/EDS-HT also affecting the skin. Multivariate correspondence analysis identified three relatively discrete phenotypic profiles, which may represent the cutaneous counterparts of the three disease phases previously proposed for JHS/EDS-HT. These findings could be used for revising the cutaneous criterion in a future consensus for the clinical diagnosis of JHS/EDS-HT.

Nosology and inheritance pattern(s) of joint hypermobility syndrome and Ehlers-Danlos syndrome, hypermobility type: a study of intrafamilial and interfamilial variability in 23 Italian pedigrees.

Joint hypermobility syndrome (JHS) and Ehlers-Danlos syndrome, hypermobility type (EDS-HT) are two markedly overlapping heritable connective tissue disorders. The cumulative frequency of JHS and EDS-HT seems high, but their recognition remains an exclusion diagnosis based on different sets of diagnostic criteria. Although proposed by a panel of experts, clinical identity between JHS and EDS-HT is still a matter of debate due to unknown molecular basis. We present 23 families with three or more individuals with a diagnosis of JHS and/or EDS-HT. Rough data from the 82 individuals were used to assess the frequency of major and minor criteria, as well as selected additional features. A series of statistical tools were applied to assess intrafamilial and interfamilial variability, emphasizing intergenerational, and intersex differences. This study demonstrates marked heterogeneity within and between families in terms of agreement of available diagnostic criteria. In 21 pedigrees affected individuals belong to two or three phenotypic sub-categories among JHS, EDS-HT, and JHS + EDS-HT overlap. Intergenerational analysis depicts a progressive shifting, also within the same pedigree, from EDS-HT in childhood, to JHS + EDS-HT in early adulthood and JHS later in life. Female-male ratio is 2.1:1, which results lower than previously observed in unselected patients' cohorts. In these pedigrees, JHS, EDS-HT, and JHS + EDS-HT segregate as a single dominant trait with complete penetrance, variable expressivity, and a markedly evolving phenotype. This study represents a formal demonstration that EDS-HT and JHS contitute the same clinical entity, and likely share the same genetic background, at least, in familial cases.

TFR2-related hereditary hemochromatosis as a frequent cause of primary iron overload in patients from Central-Southern Italy.

OBJECTIVE: Hereditary hemochromatosis (HH) is a common Mendelian disorder of iron metabolism. Eighty percent of northern Europeans descendant HH patients carry the same mutation (p.C282Y) in the HFE gene. Simultaneously, due to a founder effect, its frequency varies considerably between different populations. In Central-Southern Italy the prevalence of p.C282Y mutation is low and in several patients the disease has different causes. Four additional rarer forms have been described. Type 3 HH has been reported in about 50 families and no more than 30 TFR2 pathogenic mutations have been globally identified. The aim of this study is to assess the TFR2 role in non-HFE HH pathogenesis. STUDY DESIGN AND SETTING: TFR2 sequence analysis was performed on 45 Italian patients without HFE mutations. RESULTS: This study revealed TFR2 biallelic pathogenic mutations in 7/45 (15.6%) individuals. Moreover monoallelic TFR2 deleterious defects (18%) or polymorphisms with unclear meaning (36%) were identified. Besides, we recognized 10 novel variants and 9 described changes. CONCLUSION: We believe this to be the largest series of type 3 HH patients described so far. Present findings support the hypothesis of a main role of the TFR2 gene in HH pathogenesis in those regions, such as Central-Southern Italy, where the p.C282Y frequency is low.

Clinical and genetic study of two patients with Zimmermann-Laband syndrome and literature review.

Zimmermann-Laband syndrome (ZLS) is a rare MCA/MR condition mainly characterized by gingival hypertrophy, hypo/aplastic nails and distal phalanges, hypertrichosis and intellectual disability. The molecular basis of ZLS is unknown. Most patients are sporadic, although familial aggregation is also observed with different inheritance patterns. We report on two unrelated children with full-blown characteristics of ZLS. Remarkable variability in expression included severity of neurocognitive involvement and extent of appendicular and facial features. In both, comparative genome hybridization array at a ~ 75 Mb resolution resulted negative, while aminoacid metabolic screening revealed high plasma levels of hypoxanthine and xanthine in one. Literature review identified 50 previously published patients (27 females, 23 males), including 14 familial, clustered in four pedigrees, and 37 sporadic. Tabulation of clinical features confirmed the core phenotype and identified developmental delay as the unique major clinical problem (occurring in 40% of the cases) with a moderately high risk of epilepsy (13%). Segregation analysis in the 20 sporadic patients with available data on healthy sibs and a single pedigree with affected sibs was significantly in contrast with an autosomal recessive mutation. An autosomal dominant mutation with high mutation rate and rare instances of germinal mosaicism seems the most likely inheritance pattern. This work may represent a starting point for future molecular studies aimed at identifying the molecular basis of ZLS.

Elsahy-Waters syndrome: Evidence for autosomal recessive inheritance.

Elsahy-Waters or branchioskeletogenital syndrome is a rare MCA/MR syndrome characterized by moderate mental retardation, hypospadias and characteristic craniofacial morphology, which includes brachycephaly, facial asymmetry, exotropia, hypertelorism/telechantus, broad nose, concave nasal ridge, underdeveloped midface, prognathism, and radicular dentin dysplasia. Here we report on a 44-year-old woman and her 45-year-old brother, born to consanguineous parents, who show a striking resemblance to the earlier described patients. The hitherto reported patients were male and in one pedigree parents were consanguineous. The present report of an affected woman and her brother, born to consanguineous parents, supports autosomal recessive inheritance of this condition. We provide a short review of all previously reported patients with Elsahy-Waters syndrome and related entities.

Palmoplantar keratoderma, pseudo-ainhum, and universal atrichia: A new patient and review of the palmoplantar keratoderma-congenital alopecia syndrome.

Palmoplantar keratoderma (PPK) may concur with congenital alopecia (CA) in various genodermatoses. We report on a 10-year-old girl with generalized atrichia and a severe form of PPK causing pseudo-ainhum, sclerodactyly, and contractures, a phenotype not consistent with any well-defined condition. Non-specific additional findings comprised mild nail dystrophy and widespread keratosis pilaris including ulerythema ophryogenes. Direct sequencing of the GJB2 and LOR coding regions yielded normal results. A review identified two additional sporadic and four familial cases with PPK and CA. Comparison between familial cases suggested the existence of two genetically and phenotypically distinct types of PPK-CA: (i) an autosomal dominant form (Stevanovic type), a variable and benign phenotype without significant hand complications, and (ii) a more complex autosomal recessive variant (Wallis type) with contractures, sclerodactyly, and pseudo-ainhum. Nuclear cataract may represent an additional although not constant finding in the Wallis type PPK-CA. Further reports are required to test this preliminary conclusion.