A Novel COL4A5 Pathogenic Variant Joins the Dots in a Family with a Synchronous Diagnosis of Alport Syndrome and Polycystic Kidney Disease.

Alport Syndrome (AS) is the most common genetic glomerular disease, and it is caused by COL4A3, COL4A4, and COL4A5 pathogenic variants. The classic phenotypic spectrum associated with AS ranges from isolated hematuria to chronic kidney disease (CKD) with extrarenal abnormalities. Atypical presentation of the disorder is possible, and it can mislead the diagnosis. Polycystic kidney disease (PKD), which is most frequently associated with Autosomal Dominant PKD (ADPKD) due to PKD1 and PKD2 heterozygous variants, is emerging as a possible clinical manifestation in COL4A3-A5 patients. We describe a COL4A5 novel familial frameshift variant (NM_000495.5: c.1095dup p.(Leu366ValfsTer45)), which was associated with AS and PKD in the hemizygous proband, as well as with PKD, IgA glomerulonephritis and focal segmental glomerulosclerosis (FSGS) in the heterozygous mother. Establishing the diagnosis of AS can sometimes be difficult, especially in the context of misleading family history and atypical phenotypic features. This case study supports the emerging genotypic and phenotypic heterogeneity in COL4A3-A5-associated disorders, as well as the recently described association between PKD and collagen type IV (Col4) defects. We highlight the importance of the accurate phenotyping of all family members and the relevance of next-generation sequencing in the differential diagnosis of hereditary kidney disease.

Long-Term Treatment with the Calcitonin Gene-Related Peptide Receptor Antagonist Erenumab in CADASIL: Two Case Reports.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of cerebral small vessel disease, caused by a mutation in the NOTCH3 gene on chromosome 19. The main clinical features include migraine (often with aura), early onset, recurrent subcortical ischemic strokes, mood disturbances, and cognitive impairment, frequently leading to dementia and disability with a reduction in life expectancy. Cerebral chronic global hypoperfusion, due to impaired cerebrovascular reactivity, seems to play a primary role in CADASIL. Migraine is the most common early feature of the disease, and to date, there are no consensus guidelines for treatment. Given the vasomodulatory influence of many antimigraine drugs, there is concern about their use in this disease. In particular, the calcitonin gene-related peptide (CGRP) system serves as a vasodilatory protective mechanism during cerebral and cardiac ischemia. Blocking this system could exacerbate ischemic events. Herein, we describe two CADASIL patients who were treated with the calcitonin gene-related peptide (CGRP) receptor antagonist erenumab for chronic migraine, reporting a significant reduction in the frequency of attacks and intensity of pain, and an improvement in quality of life without adverse effects.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants.

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Co-Inheritance of Pathogenic Variants in PKD1 and PKD2 Genes Determined by Parental Segregation and De Novo Origin: A Case Report.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, and it is typically caused by PKD1 and PKD2 heterozygous variants. Nonetheless, the extensive phenotypic variability observed among affected individuals, even within the same family, suggests a more complex pattern of inheritance. We describe an ADPKD family in which the proband presented with an earlier and more severe renal phenotype (clinical diagnosis at the age of 14 and end-stage renal disease aged 24), compared to the other affected family members. Next-generation sequencing (NGS)-based analysis of polycystic kidney disease (PKD)-associated genes in the proband revealed the presence of a pathogenic PKD2 variant and a likely pathogenic variant in PKD1, according to the American College of Medical Genetics and Genomics (ACMG) criteria. The PKD2 nonsense p.Arg872Ter variant was segregated from the proband's father, with a mild phenotype. A similar mild disease presentation was found in the proband's aunts and uncle (the father's siblings). The frameshift p.Asp3832ProfsTer128 novel variant within PKD1 carried by the proband in addition to the pathogenic PKD2 variant was not found in either parent. This report highlights that the co-inheritance of two or more PKD genes or alleles may explain the extensive phenotypic variability among affected family members, thus emphasizing the importance of NGS-based techniques in the definition of the prognostic course.

A likely pathogenic ACTG1 variant in a child showing partial phenotypic overlap with Baraitser-Winter syndrome.

Baraitser-Winter syndrome (BRWS) is a rare autosomal dominant disease (AD) caused by heterozygous variants in ACTB (BRWS1) or ACTG1 (BRWS2) genes. BRWS features developmental delay/intellectual disability of variable degree and craniofacial dysmorphisms. Brain abnormalities (especially pachygyria), microcephaly, epilepsy, as well as hearing impairment, cardiovascular and genitourinary abnormalities may be present. We report on a 4-year-old female, who was addressed to our institution because of psychomotor delay associated with microcephaly and dysmorphic features, short stature, mild bilateral sensorineural hearing loss, mild cardiac septal hypertrophy, and abdominal swelling. Clinical exome sequencing detected a c.617G>A p.(Arg206Gln) de novo variant in ACTG1 gene. Such variant has been previously reported in association with a form of AD nonsyndromic sensorineural progressive hearing loss and we classified it as likely pathogenic according to ACMG/AMP criteria, despite our patient's phenotype only partially overlapped BWRS2. Our finding supports the extreme variability of the ACTG1-related disorders, ranging from classical BRWS2 to nuanced clinical expressions not fitting the original description, and occasionally featuring previously undescribed clinical findings.

Disorder of sex development: a rare case of a boy with an XY karyotype and Magnetic Resonance Imaging findings of hermaphroditism.

Disorders of sexual differentiation are rare congenital conditions in which the chromosomal, anatomic or gonadal sex development is atypical. In some of these patients, chromosomal sex is inconsistent with phenotypic sex; in other cases, the phenotype is not classifiable as either male or female, resulting in a condition known as ambiguous genitalia. These are very complex cases in which diagnostic certainty is not always possible. A multidisciplinary team including geneticists, pediatricians, radiologists is certainly needed to approach these patients. We present the case of an 18-year-old boy with an XY karyotype, ambiguous genitalia, uterus and blind-ending vaginal pouch. The patient had not been previously diagnosed with a disorder of sex development. The patient underwent a panel of genetic analyses and diagnostic imaging investigations. Magnetic resonance imaging was decisive for the identification of the internal genital organs, especially the uterus. At the end of investigations, the patient was diagnosed with 46,XY disorder of sex development. Our purpose is to underline the role of imaging in the diagnosis and management of congenital disorders of sex differentiation.

Biallelic PRMT7 pathogenic variants are associated with a recognizable syndromic neurodevelopmental disorder with short stature, obesity, and craniofacial and digital abnormalities.

PURPOSE: Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyzes the methylation of arginine residues on several protein substrates. Biallelic pathogenic PRMT7 variants have previously been associated with a syndromic neurodevelopmental disorder characterized by short stature, brachydactyly, intellectual developmental disability, and seizures. To our knowledge, no comprehensive study describes the detailed clinical characteristics of this syndrome. Thus, we aim to delineate the phenotypic spectrum of PRMT7-related disorder. METHODS: We assembled a cohort of 51 affected individuals from 39 different families, gathering clinical information from 36 newly described affected individuals and reviewing data of 15 individuals from the literature. RESULTS: The main clinical characteristics of the PRMT7-related syndrome are short stature, mild to severe developmental delay/intellectual disability, hypotonia, brachydactyly, and distinct facial morphology, including bifrontal narrowing, prominent supraorbital ridges, sparse eyebrows, short nose with full/broad nasal tip, thin upper lip, full and everted lower lip, and a prominent or squared-off jaw. Additional variable findings include seizures, obesity, nonspecific magnetic resonance imaging abnormalities, eye abnormalities (i.e., strabismus or nystagmus), and hearing loss. CONCLUSION: This study further delineates and expands the molecular, phenotypic spectrum and natural history of PRMT7-related syndrome characterized by a neurodevelopmental disorder with skeletal, growth, and endocrine abnormalities.

Case Report: Crossing a rugged road in a primary immune regulatory disorder.

Over the last decades, Inborn Errors of Immunity (IEI) characterized by an immune dysregulatory picture, isolated or combined with infections, have been increasingly identified and referred as Primary Immune Regulatory Disorders (PIRD). PIRD diagnosis may be difficult due to heterogeneity of time onset, sequence of clinical manifestations and laboratory abnormalities. Moreover, the dissection of a PIRD vs. a secondary immunodeficiency (SID) might be a real challenge since the same indications for immunosuppressant treatments might represent per se a PIRD clinical expression. Here we report a female patient with a history of recurrent respiratory and urinary tract infections since early infancy and a diagnosis of Rheumatoid Arthritis in adulthood. After poor response to several biologicals she was treated with Rituximab and sent to immunology referral for a severe hypogammaglobulinemia. Clinical and immunological features matched a diagnosis of common variable immunodeficiency and when IgG replacement therapy and antibiotic prophylaxis were added a good infectious control was obtained. Next generation sequencing analysis has revealed a novel heterozygous VUS in the IKBKB gene (c.1465A > G; p.Ser489Gly). Functional analysis has shown a reduced capacity of B lymphocytes and CD4 positive T cells in inducing IκBα degradation, with negative impact on NF-kB pathway. Due to recurrent infections attributed to a common condition in childhood and to an exclusive autoimmunity-centered approach in adulthood, both diagnosis and suitable treatment strategies have suffered a significant delay. To reduce the diagnostic delay, pediatricians, general practitioners and specialists should be aware of IEI and the challenges to differentiate them from SID. Furthermore, genetic characterization and functional analysis may contribute to a personalized approach, in a perspective of targeted or semi-targeted therapy.

1p36 Deletion Syndrome and the Aorta: A Report of Three New Patients and a Literature Review.

BACKGROUND: Monosomy 1p36 syndrome is now considered the most common terminal deletion syndrome, with an estimated incidence of 1 in 5000. Cardiac involvement is well described in the literature mainly in terms of congenital heart defects (CHDs) and cardiomyopathies (CMPs). Few data in the literature describe the potential progressive nature of aortic dilatation (root and ascending aorta) in 1p36 deletion syndrome. SKI harboured in the deleted region might play a predisposing factor for this aspect. METHODS: we reviewed the aortic aspect both in the literature and in our cohort, where major attention to the aortic abnormalities was given through dedicated echocardiographic measurements even in previously screened individuals. RESULTS: aortic involvement in 1p36 deletion syndrome was described in the literature three times within the CHD context. We observed three additional patients from our cohort (three out of nine patients) with aortic dilatation. All patients with dilated aorta had SKI haploinsufficiency within the deleted region. CONCLUSIONS: at long-term outcome and with a growing population of this rare disease, this association (1p36 deletion and aortic dilatation) might represent a major concern especially in terms of risk stratification and the potential need for specific management (conservative pharmacologic and eventually surgical) whenever indicated. The present study suggests the need for detailed multicentric studies and indication to periodic echocardiographic screening in addition to baseline tests, especially in individuals with deletions harbouring SKI.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

Mutational spectrum and clinical signatures in 114 families with hereditary multiple osteochondromas: insights into molecular properties of selected exostosin variants.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.

A 23 years follow-up study identifies GLUT1 deficiency syndrome initially diagnosed as complicated hereditary spastic paraplegia.

Glucose transporter 1 (GLUT1) deficiency syndrome (GLUT1DS) was initially described in the early 90s as a sporadic clinical condition, characterized by seizures, motor and intellectual impairment with variable clinical presentation, and without a known genetic cause. Although causative mutations in SLC2A1 were later identified and much more is known about the disease, it still remains largely underdiagnosed. In the current study, a previously described Italian family was re-analyzed using whole exome sequencing and clinically re-evaluated. Affected individuals presented with spastic paraplegia as a predominant symptom, with epilepsy and intellectual disability, inherited as an autosomal dominant trait with variable clinical presentation. While a novel variant of hereditary spastic paraplegia (HSP) was initially hypothesized in this family, previous linkage studies of known HSP genes did not identify the genetic cause. Exome-sequencing study identified a p.Arg126Cys mutation in the SLC2A1 gene, encoding GLUT1, which segregated with the affected members of the family. The diagnosis of GLUT1DS was further confirmed by cerebrospinal fluid analysis, and treatment was started with good initial response. The description of this large family provides further clinical information on this rare disease. It also offers an example of how GLUT1DS can be challenging to diagnose, and emphasizes the importance of lumbar puncture in the workflow of similar syndromes. Finally, it suggests that analysis of SLC2A1 should be considered in the diagnostic work up of HSP, especially if it is associated with epilepsy.

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.

BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent. AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years. RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding. CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.

A spectrum of LMX1B mutations in Nail-Patella syndrome: new point mutations, deletion, and evidence of mosaicism in unaffected parents.

PURPOSE: Nail-Patella syndrome (MIM 161200) is a rare autosomal dominant disorder characterized by hypoplastic or absent patellae, dystrophic nails, dysplasia of the elbows, and iliac horn. In 40% of cases, a glomerular defect is present and, less frequently, ocular damage is observed. Inter- and intrafamilial variable expressivity of the clinical phenotype is a common finding. Mutations in the human LMX1B gene have been demonstrated to be responsible for Nail-Patella syndrome in around 80% of cases. METHODS: Standard polymerase chain reaction and sequencing methods were used for mutation and single nucleotide polymorphism identification and control of cloned sequences. Array-CGH (Agilent, 244A Kit) was used for detection of deletions. Standard cloning techniques and the Snapshot method were used for analysis of mosaicism. RESULTS: In this study, we present the results of LMX1B screening of 20 Nail-Patella syndrome patients. The molecular defect was found in 17 patients. We report five novel mutations and a approximately 2 Mb deletion in chromosome 9q encompassing the entire LMX1B gene in a patient with a complex phenotype. We present evidence of somatic mosaicism in unaffected parents in two cases, which, to our knowledge, are the first reported cases of inheritance of a mutated LMX1B allele in Nail-Patella syndrome patients from a mosaic parent. CONCLUSION: The study of the described case series provides some original observations in an "old" genetic disorder.

Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy.

BACKGROUND: Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations. METHODS: We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity. RESULTS: We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC). CONCLUSION: Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).