A Clinical Workflow for Cost-Saving High-Rate Diagnosis of Genetic Kidney Diseases.

SIGNIFICANCE STATEMENT: To optimize the diagnosis of genetic kidney disorders in a cost-effective manner, we developed a workflow based on referral criteria for in-person evaluation at a tertiary center, whole-exome sequencing, reverse phenotyping, and multidisciplinary board analysis. This workflow reached a diagnostic rate of 67%, with 48% confirming and 19% modifying the suspected clinical diagnosis. We obtained a genetic diagnosis in 64% of children and 70% of adults. A modeled cost analysis demonstrated that early genetic testing saves 20% of costs per patient. Real cost analysis on a representative sample of 66 patients demonstrated an actual cost reduction of 41%. This workflow demonstrates feasibility, performance, and economic effect for the diagnosis of genetic kidney diseases in a real-world setting. BACKGROUND: Whole-exome sequencing (WES) increases the diagnostic rate of genetic kidney disorders, but accessibility, interpretation of results, and costs limit use in daily practice. METHODS: Univariable analysis of a historical cohort of 392 patients who underwent WES for kidney diseases showed that resistance to treatments, familial history of kidney disease, extrarenal involvement, congenital abnormalities of the kidney and urinary tract and CKD stage ≥G2, two or more cysts per kidney on ultrasound, persistent hyperechoic kidneys or nephrocalcinosis on ultrasound, and persistent metabolic abnormalities were most predictive for genetic diagnosis. We prospectively applied these criteria to select patients in a network of nephrology centers, followed by centralized genetic diagnosis by WES, reverse phenotyping, and multidisciplinary board discussion. RESULTS: We applied this multistep workflow to 476 patients with eight clinical categories (podocytopathies, collagenopathies, CKD of unknown origin, tubulopathies, ciliopathies, congenital anomalies of the kidney and urinary tract, syndromic CKD, metabolic kidney disorders), obtaining genetic diagnosis for 319 of 476 patients (67.0%) (95% in 21 patients with disease onset during the fetal period or at birth, 64% in 298 pediatric patients, and 70% in 156 adult patients). The suspected clinical diagnosis was confirmed in 48% of the 476 patients and modified in 19%. A modeled cost analysis showed that application of this workflow saved 20% of costs per patient when performed at the beginning of the diagnostic process. Real cost analysis of 66 patients randomly selected from all categories showed actual cost reduction of 41%. CONCLUSIONS: A diagnostic workflow for genetic kidney diseases that includes WES is cost-saving, especially if implemented early, and is feasible in a real-world setting.

Clinical and Genetic Characterization of Patients with Bartter and Gitelman Syndrome.

Bartter (BS) and Gitelman (GS) syndrome are autosomal recessive inherited tubulopathies, whose clinical diagnosis can be challenging, due to rarity and phenotypic overlap. Genotype-phenotype correlations have important implications in defining kidney and global outcomes. The aim of our study was to assess the diagnostic rate of whole-exome sequencing (WES) coupled with a bioinformatic analysis of copy number variations in a population of 63 patients with BS and GS from a single institution, and to explore genotype-phenotype correlations. We obtained a diagnostic yield of 86% (54/63 patients), allowing disease reclassification in about 14% of patients. Although some clinical and laboratory features were more commonly reported in patients with BS or GS, a significant overlap does exist, and age at onset, preterm birth, gestational age and nephro-calcinosis are frequently misleading. Finally, chronic kidney disease (CKD) occurs in about 30% of patients with BS or GS, suggesting that the long-term prognosis can be unfavorable. In our cohort the features associated with CKD were lower gestational age at birth and a molecular diagnosis of BS, especially BS type 1. The results of our study demonstrate that WES is useful in dealing with the phenotypic heterogeneity of these disorders, improving differential diagnosis and genotype-phenotype correlation.

Chiari 1 malformation and exome sequencing in 51 trios: the emerging role of rare missense variants in chromatin-remodeling genes.

Type 1 Chiari malformation (C1M) is characterized by cerebellar tonsillar herniation of 3-5 mm or more, the frequency of which is presumably much higher than one in 1000 births, as previously believed. Its etiology remains undefined, although a genetic basis is strongly supported by C1M presence in numerous genetic syndromes associated with different genes. Whole-exome sequencing (WES) in 51 between isolated and syndromic pediatric cases and their relatives was performed after confirmation of the defect by brain magnetic resonance image (MRI). Moreover, in all the cases showing an inherited candidate variant, brain MRI was performed in both parents and not only in the carrier one to investigate whether the defect segregated with the variant. More than half of the variants were Missense and belonged to the same chromatin-remodeling genes whose protein truncation variants are associated with severe neurodevelopmental syndromes. In the remaining cases, variants have been detected in genes with a role in cranial bone sutures, microcephaly, neural tube defects, and RASopathy. This study shows that the frequency of C1M is widely underestimated, in fact many of the variants, in particular those in the chromatin-remodeling genes, were inherited from a parent with C1M, either asymptomatic or with mild symptoms. In addition, C1M is a Mendelian trait, in most cases inherited as dominant. Finally, we demonstrate that modifications of the genes that regulate chromatin architecture can cause localized anatomical alterations, with symptoms of varying degrees.

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis.

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Broadening the ocular phenotypic spectrum of ultra-rare BRPF1 variants: report of two cases.

INTRODUCTION: BRPF1 gene on 3p26-p25 encodes a protein involved in epigenetic regulation, through interaction with histone H3 lysine acetyltransferases KAT6A and KAT6B of the MYST family. Heterozygous pathogenic variants in BRPF1 gene are associated with Intellectual Developmental Disorder with Dysmorphic Facies and Ptosis (IDDDFP), characterized by global developmental delay, intellectual disability, language delay, and dysmorphic facial features. The reported ocular involvement includes strabismus, amblyopia, and refraction errors. This report describes a novel ocular finding in patients affected by variants in the BRPF1 gene. METHODS: We performed exome sequencing and deep ocular phenotyping in two unrelated patients (P1, P2) with mild intellectual disability, ptosis, and typical facies. RESULTS: Interestingly, P1 had a Chiari Malformation type I and a subclinical optic neuropathy, which could not be explained by variations in other genes. Having detected a peculiar ocular phenotype in P1, we suggested optical coherence tomography (OCT) for P2; such an exam also detected bilateral subclinical optic neuropathy in this case. DISCUSSION: To date, only a few patients with BRPF1 variants have been described, and none were reported to have optic neuropathy. Since subclinical optic nerve alterations can go easily undetected, our experience highlights the importance of a more detailed ophthalmologic evaluation in patients with BRPF1 variant.

Prospective genetic germline evaluation in a consecutive group of adult patients aged <60 years with myelodysplastic syndromes.

Relevance of germline (GL) predisposition in myelodysplastic syndromes (MDSs) was stressed in both 2022 WHO and International Consensus classifications, but its incidence is probably underestimated, especially in young adult patients. We selected a cohort of 31 consecutive de novo MDS patients with unusual young age (<60 years). We performed exome sequencing (ES) on DNA extracted from noninvasive sources (peripheral blood and saliva), filtering for a panel of 344 genes specifically tailored for detecting GL variants related to clonal and nonclonal cytopenia. We observed at least one high- or low-confidence GL MDS variant in 7/31 (22.6%) and 9/31 (29.0%) of cases, respectively. Four of 31 patients (12.9%) confirmed having established MDS/AML predisposing disorders. We found heterozygous variants in genes involved in DNA repair/cancer predisposition (ATM, ATR, FANCM, PARN, BRCA1, BRCA2, CHEK2, MSH2) in 9/31 (29.0%) cases and variants affecting ribosome biogenesis (SBDS), hematopoietic stem cell (GATA2), and megakaryocyte (ANKRD26) differentiation in single cases. Two cases had variants in RBBP6, a gene previously described exclusively in familial myeloproliferative neoplasms. Lastly, four cases had variants in genes related to inherited anemias (CUBN and PIEZO1 genes). Our results showed that "young" MDS patients aged 40-60 years carried reported and unreported GL variants with an unexpectedly high proportion, and these events co-occurred with somatic mutations recurrent in myeloid neoplasms. We explored the "no man's land" of the young adult MDS cases adopting a practical and scalable diagnostic tool, capable to detect GL variants avoiding invasive methods.

Multimodal phenotyping of foveal hypoplasia in albinism and albino-like conditions: a pediatric case series with adaptive optics insights.

Aim of the present study is to evaluate the relationship between genetic and phenotypic data in a series of patients affected by grade I and II of foveal hypoplasia with stable fixation and good visual acuity using multimodal imaging techniques. All patients underwent complete clinical and instrumental assessment including structural Optical Coherence Tomography (OCT), OCT Angiography and Adaptive Optics (AO) imaging. Central macular thickness (CMT), inner nuclear layer (INL), vessel density in superficial capillary plexus were the main variables evaluated with OCT technology. Cone density, cone spacing, cone regularity, cone dispersion and angular density were the parameters evaluated with AO. Genetic evaluation and trio exome sequencing were performed in all affected individuals. Eight patients (3 males and 5 females) with a mean age of 12.62 years (range 8-18) were enrolled. The mean best corrected visual acuity (BCVA) was 0.18 ± 0.13 logMAR, mean CMT was 291.9 ± 16.6 µm and INL was 26.2 ± 4.6 µm. The absence of a foveal avascular zone (FAZ) was documented by examination of OCT-A in seven patients in the superficial capillary plexus. However, there was a partial FAZ in the deep plexus in patients P5 and P8. Of note, all the patients presented with major retinal vessels clearly crossing the foveal center. All individuals exhibited a grade I or II of foveal hypoplasia. In 5 patients molecular analyses showed an extremely mild form of albinism caused by compound heterozygosity of a TYR pathogenic variant and the hypomorphic p.[Ser192Tyr;Arg402Gln] haplotype. One patient had Waardenburg syndrome type 2A caused by a de novo variant in MITF. Two patients had inconclusive molecular analyses. All the patients displayed abnormalities on OCT-A. Photoreceptor count did not differ from normal subjects according to the current literature, but qualitative analysis of AO imaging showed distinctive features likely related to an abnormal pigment distribution in this subset of individuals. In patients with foveal hypoplasia, genetic and multimodal imaging data, including AO findings, can help understand the physiopathology of the foveal hypoplasia phenotype. This study confirms that cone density and visual function can both be preserved despite the absence of a pit.

Disruption of the podosome adaptor protein TKS4 (SH3PXD2B) causes the skeletal dysplasia, eye, and cardiac abnormalities of Frank-Ter Haar Syndrome.

Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.

Pseudo-rheumatic manifestations of limping: Camptodactyly-arthropathy-coxa vara-pericarditis syndrome: Single case report and review of the literature.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome is a rare genetic disease characterized by tetrad camptodactyly, noninflammatory arthropathy, coxa vara deformity, and pericardial effusion. Arthropathy typically affects large joints and presents with joint swelling in the absence of other signs of inflammation. We described the case of a girl affected by CACP syndrome caused by a novel compound heterozygous variant in proteoglycan 4 gene (c.2831_2832insT; c.3892C > T) and associated with temporomandibular involvement. The patient received treatment with intra-articular hyaluronic acid injections, which presented rapid but transient improvements of pain and range of motion. A literature review of previously reported CACP patients has been performed. Of the patients. 69.2% (101 out of 146) were Middle Eastern, and 65.7% (96) were consanguineous. The median age of onset was 24 months (interquartile range of 12-36 months), and median age of diagnosis was 96 months (interquartile range of 48-156 months). Arthropathy was always present, mainly involving hips (95.2%), knees (92.4%), wrists (87.7%), elbows (79.5%), and ankles (57.5%). Camptodactyly and pericardial effusion were described, respectively, in 97.3% (142) and 15.1% (22) of patients. The main radiological findings were coxa vara (95.2%), femoral changes (64.4%), intraosseus cysts (14.4%), and bone erosion (5%). Of the patients, 32.9% (48) had received a previous juvenile idiopathic arthritis diagnosis. CACP syndrome can be easily misdiagnosed with juvenile idiopathic arthritis. A prolonged lack of response to immunosuppressive therapy associated with typical clinical and radiological features should prompt consideration of this rare syndrome.

FOXG1 is responsible for the congenital variant of Rett syndrome.

Rett syndrome is a severe neurodevelopmental disease caused by mutations in the X-linked gene encoding for the methyl-CpG-binding protein MeCP2. Here, we report the identification of FOXG1-truncating mutations in two patients affected by the congenital variant of Rett syndrome. FOXG1 encodes a brain-specific transcriptional repressor that is essential for early development of the telencephalon. Molecular analysis revealed that Foxg1 might also share common molecular mechanisms with MeCP2 during neuronal development, exhibiting partially overlapping expression domain in postnatal cortex and neuronal subnuclear localization.

Investigation of modifier genes within copy number variations in Rett syndrome.

MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether copy number variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by chromatin immunopreceipitation microarray (ChIP-chip) analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target, in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination, and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.

Mutations in FN1 cause glomerulopathy with fibronectin deposits.

Glomerulopathy with fibronectin (FN) deposits (GFND) is an autosomal dominant disease with age-related penetrance, characterized by proteinuria, microscopic hematuria, hypertension, and massive glomerular deposits of FN that lead to end-stage renal failure. The genetic abnormality underlying GFND was still unknown. We hypothesized that mutations in FN1, which encodes FN, were the cause of GFND. In a large Italian pedigree with eight affected subjects, we found linkage with GFND at the FN1 locus at 2q32. We sequenced the FN1 in 15 unrelated pedigrees and found three heterozygous missense mutations, the W1925R, L1974R, and Y973C, that cosegregated with the disease in six pedigrees. The mutations affected two domains of FN (Hep-II domain for the W1925R and the L1974R, and Hep-III domain for the Y973C) that play key roles in FN-cell interaction and in FN fibrillogenesis. Mutant recombinant Hep-II fragments were expressed, and functional studies revealed a lower binding to heparin and to endothelial cells and podocytes compared with wild-type Hep-II and an impaired capability to induce endothelial cell spreading and cytoskeletal reorganization. Overall dominant mutations in FN1 accounted for 40% of cases of GFND in our study group. These findings may help understanding the pathogenesis of proteinuria and glomerular FN deposits in GFND and possibly in more common renal diseases such as diabetic nephropathy, IgA nephropathy, and lupus nephritis. To our knowledge no FN1 mutation causing a human disease was previously reported.

The Immunomodulatory Effect and Clinical Efficacy of Daratumumab in a Patient With Cold Agglutinin Disease.

Daratumumab is a monoclonal antibody directed against the transmembrane glycoprotein CD38 expressed on plasma cells and lymphoplasmocytes, with a proven efficacy in multiple myeloma. Here we show its clinical efficacy in a patient with cold agglutinin disease (CAD) relapsed after multiple lines of therapy. CAD is caused by cold reactive autoantibodies that induce complement mediated hemolysis and peripheral circulatory symptoms. The disease is also characterized by the presence of monoclonal IgM gammopathy and of a lymphoid bone marrow infiltration that benefits from B-cell targeting therapies (i.e., rituximab) but also from plasma cell directed therapies, such as proteasome inhibitors. In the patient described, we also show that daratumumab therapy influenced the dynamics of several immunoregulatory cytokine levels (IL-6, IL-10, IL-17, IFN-γ, TNF-α, TGF-ß) indicating an immunomodulatory effect of the drug beyond plasma cell depletion. In addition, we provide a literature review on the use of daratumumab in autoimmune conditions, including multi-treated and refractory patients with autoimmune hemolytic anemia (both CAD and warm forms), Evans syndrome (association of autoimmune hemolytic anemia and immune thrombocytopenia) and non-hematologic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.

Aberrant sialylation in a patient with a HNF1α variant and liver adenomatosis.

Glycosylation is a fundamental post-translational modification of proteins that boosts their structural diversity providing subtle and specialized biological properties and functions. All those genetic diseases due to a defective glycan biosynthesis and attachment to the nascent glycoproteins fall within the wide area of congenital disorders of glycosylation (CDG), mostly causing multisystem involvement. In the present paper, we detailed the unique serum N-glycosylation of a CDG-candidate patient with an unexplained neurological phenotype and liver adenomatosis harboring a recurrent pathogenic HNF1α variant. Serum transferrin isoelectric focusing showed a surprising N-glycosylation pattern consisting on hyposialylation, as well as remarkable hypersialylation. Mass spectrometry-based glycomic analyses of individual serum glycoproteins enabled to unveil hypersialylated complex N-glycans comprising up to two sialic acids per antenna. Further advanced MS analysis showed the additional sialic acid is bonded through an α2-6 linkage to the peripheral N-acetylglucosamine residue.

Reverse Phenotyping after Whole-Exome Sequencing in Steroid-Resistant Nephrotic Syndrome.

BACKGROUND AND OBJECTIVES: Nephrotic syndrome is a typical presentation of genetic podocytopathies but occasionally other genetic nephropathies can present as clinically indistinguishable phenocopies. We hypothesized that extended genetic testing followed by reverse phenotyping would increase the diagnostic rate for these patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: All patients diagnosed with nephrotic syndrome and referred to our center between 2000 and 2018 were assessed in this retrospective study. When indicated, whole-exome sequencing and in silico filtering of 298 genes related to CKD were combined with subsequent reverse phenotyping in patients and families. Pathogenic variants were defined according to current guidelines of the American College of Medical Genetics. RESULTS: A total of 111 patients (64 steroid-resistant and 47 steroid-sensitive) were included in the study. Not a single pathogenic variant was detected in the steroid-sensitive group. Overall, 30% (19 out of 64) of steroid-resistant patients had pathogenic variants in podocytopathy genes, whereas a substantial number of variants were identified in other genes, not commonly associated with isolated nephrotic syndrome. Reverse phenotyping, on the basis of a personalized diagnostic workflow, permitted to identify previously unrecognized clinical signs of an unexpected underlying genetic nephropathy in a further 28% (18 out of 64) of patients. These patients showed similar multidrug resistance, but different long-term outcome, when compared with genetic podocytopathies. CONCLUSIONS: Reverse phenotyping increased the diagnostic accuracy in patients referred with the diagnosis of steroid-resistant nephrotic syndrome.

Autosomal dominant Alport syndrome: molecular analysis of the COL4A4 gene and clinical outcome.

BACKGROUND: Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by glomerular basement membrane lesions often associated with hearing loss and ocular anomalies. While the X-linked and the autosomal recessive forms are well known, the autosomal dominant form is not well acknowledged. METHODS: We have clinically investigated 38 patients with a diagnosis of autosomal dominant Alport syndrome belonging to eight different families. The analysis of the COL4A4 gene was performed by denaturing high performance liquid chromatography and automated DNA sequencing. RESULTS: In our cohort of patients, only 24.3% (9/37) reached end-stage renal disease, at the mean age of 51.2 years. Four patients had hearing loss (13.3%) and none ocular changes. Molecular analysis revealed eight novel private COL4A4 gene mutations: three frameshift, three missense and two splice-site mutations. CONCLUSIONS: These data indicate autosomal dominant Alport syndrome as a disease with a low risk of ocular and hearing anomalies but with a significant risk to develop renal failure although at an older age than the X-linked form. We were unable to demonstrate a genotype-phenotype correlation. Altogether, these data make difficult the differential diagnosis with the benign familial haematuria due to heterozygous mutations of COL4A4 and COL4A3, especially in young patients, and with the X-linked form of Alport syndrome in families where only females are affected. A correct diagnosis and prognosis is based on a comprehensive clinical investigation in as many family members as possible associated with a broadly formal genetic analysis of the pedigree.

Italian Rett database and biobank.

Rett syndrome is the second most common cause of severe mental retardation in females, with an incidence of approximately 1 out of 10,000 live female births. In addition to the classic form, a number of Rett variants have been described. MECP2 gene mutations are responsible for about 90% of classic cases and for a lower percentage of variant cases. Recently, CDKL5 mutations have been identified in the early onset seizures variant and other atypical Rett patients. While the high percentage of MECP2 mutations in classic patients supports the hypothesis of a single disease gene, the low frequency of mutated variant cases suggests genetic heterogeneity. Since 1998, we have performed clinical evaluation and molecular analysis of a large number of Italian Rett patients. The Italian Rett Syndrome (RTT) database has been developed to share data and samples of our RTT collection with the scientific community (http://www.biobank.unisi.it). This is the first RTT database that has been connected with a biobank. It allows the user to immediately visualize the list of available RTT samples and, using the "Search by" tool, to rapidly select those with specific clinical and molecular features. By contacting bank curators, users can request the samples of interest for their studies. This database encourages collaboration projects with clinicians and researchers from around the world and provides important resources that will help to better define the pathogenic mechanisms underlying Rett syndrome.

MECP2 deletions and genotype-phenotype correlation in Rett syndrome.

Rett syndrome is a neurodevelopmental disorder that represents one of the most common genetic causes of mental retardation in girls. MECP2 point mutations in exons 2-4 account for about 80% of classic Rett cases and for a lower percentage of variant patients. We investigated the genetic cause in 77 mutation-negative Rett patients (33 classic, 31 variant, and 13 Rett-like cases) by searching missed MECP2 defects. DHPLC analysis of exon 1 and MLPA analysis allowed us to identify the defect in 17 Rett patients: one exon 1 point mutation (c.47_57del) in a classic case and 16 MECP2 large deletions (15/33 classic and 1/31 variant cases). One identical intragenic MECP2 deletion, probably due to gonadal mosaicism, was found in two sisters with discordant phenotype: one classic and one "highly functioning" preserved speech variant. This result indicates that other epigenetic or genetic factors, beside MECP2, may contribute to phenotype modulation. Three out of 16 MECP2 deletions extend to the adjacent centromeric IRAK1 gene. A putative involvement of the hemizygosity of this gene in the ossification process is discussed. Finally, results reported here clearly indicate that MECP2 large deletions are a common cause of classic Rett, and MLPA analysis is mandatory in MECP2-negative patients, especially in those more severely affected (P = 0.044).

Clinical and molecular characterization of a novel INS mutation identified in patients with MODY phenotype.

Correct diagnosis of Maturity-Onset Diabetes of the Young (MODY) is based on genetic tests requiring an appropriate subject selection by clinicians. Mutations in the insulin (INS) gene rarely occur in patients with MODY. This study is aimed at determining the genetic background and clinical phenotype in patients with suspected MODY. 34 patients with suspected MODY, negative for mutations in the GCK, HNF1α, HNF4α, HNF1ß and PDX1 genes, were screened by next generation sequencing (NGS). A heterozygous INS mutation was identified in 4 members of the same family. First genetic tests performed identified two heterozygous silent nucleotide substitutions in MODY3/HNF1α gene. An ineffective attempt to suspend insulin therapy, administering repaglinide and sulphonylureas, was made. DNA was re-sequenced by NGS investigating a set of 102 genes. Genes implicated in the pathway of pancreatic ß-cells, candidate genes for type 2 diabetes mellitus and genes causative of diabetes in mice were selected. A novel heterozygous variant in human preproinsulin INS gene (c.125T > C) was found in the affected family members. The new INS mutation broadens the spectrum of possible INS phenotypes. Screening for INS mutations is warranted not only in neonatal diabetes but also in MODYx patients and in selected patients with type 1 diabetes mellitus negative for autoantibodies. Subjects with complex diseases without a specific phenotype should be studied by NGS because Sanger sequencing is ineffective and time consuming in detecting rare variants.