Familial trisomy 6p in mother and daughter.

Several patients with partial trisomy 6p resulting from parental balanced translocations or from a de novo duplication or insertion have already been described. Here, we report on the first case of familial pure trisomy 6p as a result of interstitial tandem duplication. The patient, an 11-year-old female, presented with mild dysmorphic features, moderate intellectual disability with behavioral disturbances, immunodeficiency, and epilepsy. Conventional cytogenetic analysis showed a duplication of the 6p region in the patient and in her mother presenting with a partially overlapping phenotype. The rearrangement was confirmed and defined by molecular cytogenetic analysis, including FISH and array CGH analysis showing a gain of ~13.8 Mb from 6p12.3 to 6p21.31. The phenotype of a pure partial trisomy 6p is extremely heterogeneous depending on the gene contents of the duplicated region. The clinical features of our patients have been compared with overlapping cases from the literature.

Nephroplex: a kidney-focused NGS panel highlights the challenges of PKD1 sequencing and identifies a founder BBS4 mutation.

BACKGROUND: Genetic testing of patients with inherited kidney diseases has emerged as a tool of clinical utility by improving the patients' diagnosis, prognosis, surveillance and therapy. METHODS: The present study applied a Next Generation Sequencing (NGS)-based panel, named NephroPlex, testing 115 genes causing renal diseases, to 119 individuals, including 107 probands and 12 relatives. Thirty-five (poly)cystic and 72 non (poly)cystic individuals were enrolled. The latter subgroup of patients included Bardet-Biedl syndrome (BBS) patients, as major components. RESULTS: Disease-causing mutations were identified in 51.5 and 40% of polycystic and non-polycystic individuals, respectively. Autosomal dominant polycystic kidney disease (ADPKD) patients with truncating PKD1 variants showed a trend towards a greater slope of the age-estimated glomerular filtration rate (eGFR) regression line than patients with (i) missense variants, (ii) any PKD2 mutations and (iii) no detected mutations, according to previous findings. The analysis of BBS individuals showed a similar frequency of BBS4,9,10 and 12 mutations. Of note, all BBS4-mutated patients harbored the novel c.332+1G>GTT variant, which was absent in public databases, however, in our internal database, an additional heterozygote carrier was found. All BBS4-mutated individuals originated from the same geographical area encompassing the coastal provinces of Naples. DISCUSSION: In conclusion, these findings indicate the potential for a genetic panel to provide useful information at both clinical and epidemiological levels.

Urine concentrating defect as presenting sign of progressive renal failure in Bardet-Biedl syndrome patients.

BACKGROUND: Urine concentrating defect is a common dysfunction in ciliopathies, even though its underlying mechanism and its prognostic meaning are largely unknown. This study assesses renal function in a cohort of 54 Bardet-Biedl syndrome (BBS) individuals and analyses whether renal hyposthenuria is the result of specific tubule dysfunction and predicts renal disease progression. METHODS: The estimated glomerular filtration rate (eGFR), urine albumin:creatinine ratio (ACR) and maximum urine osmolality (max-Uosm) were measured in all patients. Genetic analysis was conducted in 43 patients. Annual eGFR decline (ΔeGFR) was measured in patients with a median follow-up period of 6.5 years. Urine aquaporin-2 (uAQP2) excretion was measured and the furosemide test was performed in patients and controls. RESULTS: At baseline, 33 (61.1%), 12 (22.2%) and 9 (16.7%) patients showed an eGFR >90, 60-90 and <60 mL/min/1.73 m2, respectively; 27.3% showed an ACR >30 mg/g and 55.8% of patients showed urine concentrating defect in the absence of renal insufficiency. Baseline eGFR, but not max-Uosm, correlated negatively with age. Conversely, truncating mutations affected max-Uosm and showed a trend towards a reduction in eGFR. Max-Uosm correlated with ΔeGFR (P < 0.005), suggesting that urine concentrating defect may predict disease progression. uAQP2 excretion and Na+ and Cl- fractional excretion after furosemide did not differ between hyposthenuric patients and controls, suggesting that specific collecting duct and thick ascending limb dysfunctions are unlikely to play a central role in the pathogenesis of hyposthenuria. CONCLUSIONS: Hyposthenuria is a warning sign predicting poor renal outcome in BBS. The pathophysiology of this defect is most likely beyond defective tubular function.

Enhancer chip: detecting human copy number variations in regulatory elements.

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung's disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.

One hundred twenty-one dystrophin point mutations detected from stored DNA samples by combinatorial denaturing high-performance liquid chromatography.

Duchenne and Becker muscular dystrophies are caused by a large number of different mutations in the dystrophin gene. Outside of the deletion/duplication "hot spots," small mutations occur at unpredictable positions. These account for about 15 to 20% of cases, with the major group being premature stop codons. When the affected male is deceased, carrier testing for family members and prenatal diagnosis become difficult and expensive. We tailored a cost-effective and reliable strategy to discover point mutations from stored DNA samples in the absence of a muscle biopsy. Samples were amplified in combinatorial pools and tested by denaturing high-performance liquid chromatography analysis. An anomalous elution profile belonging to two different pools univocally addressed the allelic variation to an unambiguous sample. Mutations were then detected by sequencing. We identified 121 mutations of 99 different types. Fifty-six patients show stop codons that represent the 46.3% of all cases. Three non-obvious single amino acid mutations were considered as causative. Our data support combinatorial denaturing high-performance liquid chromatography analysis as a clear-cut strategy for time and cost-effective identification of small mutations when only DNA is available.