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

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. hh 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 I kappa B alpha 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

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

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%)

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

Chromatin conformation capture in the clinic: 4C-seq/HiC distinguishes pathogenic from neutral duplications at the GPR101 locus.

peer reviewed[en] BACKGROUND: X-linked acrogigantism (X-LAG; MIM: 300942) is a severe form of pituitary gigantism caused by chromosome Xq26.3 duplications involving GPR101. X-LAG-associated duplications disrupt the integrity of the topologically associating domain (TAD) containing GPR101 and lead to the formation of a neo-TAD that drives pituitary GPR101 misexpression and gigantism. As X-LAG is fully penetrant and heritable, duplications involving GPR101 identified on prenatal screening studies, like amniocentesis, can pose an interpretation challenge for medical geneticists and raise important concerns for patients and families. Therefore, providing robust information on the functional genomic impact of such duplications has important research and clinical value with respect to gene regulation and triplosensitivity traits. METHODS: We employed 4C/HiC-seq as a clinical tool to determine the functional impact of incidentally discovered GPR101 duplications on TAD integrity in three families. After defining duplications and breakpoints around GPR101 by clinical-grade and high-density aCGH, we constructed 4C/HiC chromatin contact maps for our study population and compared them with normal and active (X-LAG) controls. RESULTS: We showed that duplications involving GPR101 that preserved the centromeric invariant TAD boundary did not generate a pathogenic neo-TAD and that ectopic enhancers were not adopted. This allowed us to discount presumptive/suspected X-LAG diagnoses and GPR101 misexpression, obviating the need for intensive clinical follow-up. CONCLUSIONS: This study highlights the importance of TAD boundaries and chromatin interactions in determining the functional impact of copy number variants and provides proof-of-concept for using 4C/HiC-seq as a clinical tool to acquire crucial information for genetic counseling and to support clinical decision-making in cases of suspected TADopathies

Co-Inheritance of Pathogenic Variants in <i>PKD1</i> and <i>PKD2</i> 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 Novel <i>COL4A5</i> 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

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&gt;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