Alvaro Mesoraca

Identification of five new mutations and three novel polymorphisms in the muscle chloride channel gene (CLCN1) in 20 Italian patients with dominant and recessive myotonia congenita

Autosomal dominant myotonia congenita or Thomsens disease and autosomal recessive myotonia congenita or Beckersare rare nondystrophic disorders due to allelic mutations of the muscle chloride channel gene, CLCN1. We have analysed all 24 exons of the CLCN1 gene, in a panel of 20 unrelated patients (9 with dominant and 11 with recessive myotonia congenita). We have found five novel mutations including two missense (V563I, F708L), one nonsense (C481X), one splicing (IVS19+2T→A), and one frameshift (2264delC), and also detected the recurrent R894X mutation. These account for 10 of the 22 recessive alleles examined, while no mutations were found in the dominant form. We report three novel polymorphisms (‐134 T/G, 898 C/A and 2154T/C). Our results support high molecular heterogeneity of these myotonias in Italian population and provide new insight for the diagnosis and genetic counselling of these diseases. Hum Mutat 11:331, 1998.

Antibiotic Prophylaxis before second-trimester Genetic Amniocentesis (APGA): a single-centre open randomised controlled trial

To compare procedure‐related pregnancy loss after second‐trimester genetic amniocentesis in women given an antibiotic prophylaxis and controls.

Identification of five new mutations and three novel polymorphisms in the muscle chloride channel gene (CLCN1) in 20 Italian patients with dominant and recessive myotonia congenita. Mutations in brief no. 118. Online

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of allsteroids including glucocorticoids, mineralocorticoids and sexsteroids. It has recently been reported that mutations in thesteroidogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel frameshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene. Hum Mutat 11:331, 1998.

Pallister–Killian syndrome: Cytogenetics and molecular investigations of mosaic tetrasomy 12p in prenatal chorionic villus and in amniocytes. Strategy of prenatal diagnosis

OBJECTIVE Pallister-Killian syndrome (PKS) is a rare, sporadic genetic disorder caused by mosaic tetrasomy of the short arm of chromosome 12 (12p). Clinically, PKS is characterized by several systemic abnormalities, such as intellectual impairment, hearing loss, epilepsy, hypotonia, craniofacial dysmorphism, pigmentary skin anomalies, epilepsy, and a variety of congenital malformations. Prenatally, PKS can be suspected in the presence of ultrasound anomalies: diaphragmatic hernia, rhizomelic micromelia, hydrops fetalis, fetal overweight, ventriculomegaly in the central nervous system, congenital heart defects, or absent visualization of the stomach. In all these cases, a detailed genetic study is required. PKS is diagnosed by prenatal genetic analysis through chorionic villus sampling, genetic amniocentesis, and cordocentesis. CASE REPORT We report two cases of PKS with prenatal diagnosis of isochromosome 12p made by cytogenetic studies. The first case is of a 36-year-old pregnant woman who underwent genetic chorionic villus sampling at 13th weeks of gestation after 1st trimester prenatal ultrasound revealed clinical features of PKS: flat nasal bridge and fetal hydrops. The second case is of a 32-year-old pregnant woman with genetic amniocentesis at 17th weeks of gestation that showed mos46,XX[21]/47,XX,+i(12p) associated to PKS. CONCLUSION New molecular cytogenetic techniques array comparative genomic hybridization and fluorescence in-situ hybridization in association with conventional karyotype are pivotal innovative tools to search for chromosomic anomalies and for a complete prenatal diagnosis, especially in cases such as PKS where array comparative genomic hybridization analysis alone could not show mosaicism of i(12p).

Comparative Study of a CGH and Next Generation Sequencing (NGS) forChromosomal Microdeletion and Microduplication Screening

BACKGROUND prenatal genetic diagnosis of rare disorders is undergoing in recent years a significant enhancement through the application of methods of massive parallel sequencing. Despite the quantity and quality of the data produced, just few analytical tools and software have been developed in order to identify structural and numerical chromosomal anomalies through NGS, mostly not compatible with benchtop NGS platform and routine clinical diagnosis. METHODS we developed technical, bioinformatic, interpretive and validation pipelines for Next Generation Sequencing to identify SNPs, indels, aneuploidies, and CNVs (Copy Number Variations). RESULTS we show a new targeted resequencing approach applied to prenatal diagnosis. For sample processing we used an enrichment method for 4,813 genes library preparation; after sequencing our bioinformatic pipelines allowed both SNPs analysis for approximately thirty diseases or diseases family involved in fetus development and numerical chromosomal anomalies screening. CONCLUSIONS results obtained are compatible with those obtained through the gold standard technique, aCGH array, moreover allowing identification of genes involved in chromosome deletions or duplications and exclusion of point mutation on allele not affected by chromosome aberrations.

Next Generation Sequencing Approach in a Prenatal Case of Cardio-Facio-Cutaneus Syndrome

Cardiofaciocutaneous syndrome (CFCS) belongs to a group of developmental disorders due to defects in the Ras/Mitogen-Activated Protein Kinase (RAS/MAPK) signaling pathway named RASophaties. While postnatal presentation of these disorders is well known, the prenatal and neonatal characteristics are less recognized. Noonan syndrome, Costello syndrome, and CFCS diagnosis should be considered in pregnancies with a normal karyotype and in the case of ultrasound findings such as increased nuchal translucency, polyhydramnios, macrosomia and cardiac defect. Because all the RASopathies share similar clinical features, their molecular characterization is complex, time consuming and expensive. Here we report a case of CFCS prenatally diagnosed through Next Generation Prenatal Diagnosis (NGPD), a new targeted approach that allows us to concurrently investigate all the genes involved in the RASophaties.

A new approach for Next Generation Sequencing in prenatal diagnosis applied to a case of Charcot–Marie–Tooth syndrome

In recent years, Next Generation Sequencing (NGS) has become an important tool, not only for gene discovery and research, but also for clinical diagnosis. Genetic diagnosis of rare disorders has developed considerably through the application of massively parallel sequencing methods. To date, a great effort has been made to introduce NGS in noninvasive prenatal testing both for the detection of aneuploidy associated to chromosomes 21, 18 and 13 and, in the latest studies, for single gene disorder analysis. Regarding invasive prenatal diagnosis, NGS has been applied to whole exome or whole genome studies; however, in these cases NGS did not prove useful in routine clinical application, because of many factors, including the necessity of bioinformatics skills during data analysis, the large amount of variants with uncertain clinical significance which were obtained, and the time required for analysis. We have introduced a new target resequencing NGS approach in prenatal diagnosis, called Next Generation Prenatal Diagnosis (NGPD), for the screening of a wide range of genetic disorders in high-risk pregnancies when an ultrasound screening fails to show a definite clinical phenotype, in cases where ultrasound indications must be supported by a molecular genetic diagnosis, or in cases where one or both parents are carriers of a genetic disorder. To the best of our knowledge, this is the first report that describes the use of a multi-gene NGS panel optimized for clinical prenatal diagnosis with a relatively fast turn-around time that can be multiplexed to allow simultaneous analysis of multiple samples. In this report the NGPDmethod was applied for the prenatal genetic diagnosis of Charcot–Marie–Tooth (CMT) disease in a fetus whose mother was affected by early onset CMT. Of all the types of hereditary sensory-motor polyneuropathy, CMT syndrome is the most frequent. From a clinical point of view, the disorder is characterized by distal muscular atrophy associated with malformation of the feet, osteotendinous hyporeflexia and alterations in sensibility. Based on motor conduction velocity, it is possible to identify type 1 (demyelinating form with a particularly reduced conduction speed) and type 2 (axonal form with a normal conduction speed). From a genetic point of view, many genes responsible for CMT have been identified, and transmission can be autosomal dominant, recessive or X linked. At the time of analysis, the 12th week of gestation, the patient was still without a genetic diagnosis. The patient showed postural tremor, sensory impairment and distal muscle atrophy. Weakness and deafness were also present. From the clinical and family history, we could assume a probability of an autosomal dominant-transmitted form of CMT syndrome. At the time of the ultrasound scan no clinical signs emerged. After genetic counseling, the patient underwent a chorionic villous sampling and parents’ blood samples were taken. After exclusion of any chromosomal microdeletions and microduplications associated with CMT disease, through aCGH analysis on CVS and mother’s blood, we used the NGPD method on the same DNA samples. We proceeded directly to trio analysis, followed by confirmation of any mutations identified on the fetus, to reduce time of prenatal diagnosis, which would otherwise have been delayed. Trio analysis was useful not only for identifying variants, but also for studying inheritance patterns. Variants were sorted for autosomal recessive, autosomal dominant, X-linked and de novo mode of inheritance. For the library preparation, we used the Trusight One Sequencing Panel (TSO, Illumina), which enriches for 62 000 exons of 4813 genes. The genes contained in this library were selected by Illumina according to the Human