NGS and phenotypic ontology-based approaches increase the diagnostic yield in syndromic retinal diseases.

Syndromic retinal diseases (SRDs) are a group of complex inherited systemic disorders, with challenging molecular underpinnings and clinical management. Our main goal is to improve clinical and molecular SRDs diagnosis, by applying a structured phenotypic ontology and next-generation sequencing (NGS)-based pipelines. A prospective and retrospective cohort study was performed on 100 probands with an a priori diagnosis of non-Usher SRDs, using available clinical data, including Human Phenotype Ontology annotation, and further classification into seven clinical categories (ciliopathies, specific syndromes and five others). Retrospective molecular diagnosis was assessed using different molecular and bioinformatic methods depending on availability. Subsequently, uncharacterized probands were prospectively screened using other NGS approaches to extend the number of analyzed genes. After phenotypic classification, ciliopathies were the most common SRD (35%). A global characterization rate of 52% was obtained, with six cases incompletely characterized for a gene that partially explained the phenotype. An improved characterization rate was achieved addressing prospective cases (83%) and well-recognizable syndrome (62%) subgroups. The 27% of the fully characterized cases were reclassified into a different clinical category after identification of the disease-causing gene. Clinical-exome sequencing is the most appropriate first-tier approach for prospective cases, whereas whole-exome sequencing and bioinformatic reanalysis increases the diagnosis of uncharacterized retrospective cases to 45%, mostly those with unspecific symptoms. Our study describes a comprehensive approach to SRDs in daily clinical practice and the importance of thorough clinical assessment and selection of the most appropriate molecular test to be used to solve these complex cases and elucidate novel associations.

Could a patient with SMC1A duplication be classified as a human cohesinopathy?

The disorders caused by mutations in genes encoding subunits and accessory proteins of cohesin complex are collectively termed as cohesinopathies. The best known cohesinopathy is Cornelia de Lange Syndrome (CdLS), which is a multisystem developmental disorder characterized by facial dysmorphism, limb malformations, growth and cognitive impairment. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), are responsible for ∼ 70% of CdLS cases. We describe a 16-year-old boy with facial dysmorphism, growth retardation, intellectual disability, hirsutism and small hands, who has a small Supernumerary Marker Chromosome (sSMC) present in mosaic form. sSMC is composed of two duplicated segments encompassing 17 genes including SMC1A gene, at the regions Xp11.22 and Xp11.21q11.1. Clinical comparison between our patient with a previously reported individual with a SMC1A duplication and four male carriers of similar sSMC reported in databases, suggest that they all share clinical features related to cohesinopathies. Although our patient does not have the classical CdLS craniofacial phenotype, he has pre and postnatal growth retardation, intellectual disability and mild musculoskeletal anomalies, features commonly seen in patients with cohesinopathies.

Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers.

The existence of foetal DNA in maternal blood, discovered in 1997, opened new possibilities for noninvasive prenatal diagnosis. This includes foetal sex assessment by the detection of specific Y chromosome sequences in maternal blood, particularly important when a foetus may be affected by an X-linked disorder such as haemophilia. This study aims to validate this sex assessment method and to test its clinical utility in the diagnosis of 15 potentially affected pregnancies in female carriers of haemophilia. In the validation study, 316 maternal blood samples from 196 pregnant women at gestations ranging from 5 weeks to 12 weeks were analysed. In the clinical study, 15 pregnancies at risk of having a haemophilic foetus were tested. All pregnancies in the validation study were correctly diagnosed. The accuracy and specificity of the methodology from the seventh week of gestation was 100%. The sex of all 15 pregnancies identified as being at risk of bearing a haemophilic foetus was correctly diagnosed. Foetal sex assessment by detecting specific Y chromosome sequences in maternal blood is now routinely used in our hospital because of its high accuracy from the seventh week of gestation. Reliable foetal gender determination from maternal blood of pregnant women carriers of haemophilia in the first trimester of gestation can avoid more conventional, invasive methods of prenatal diagnosis.

Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study.

BACKGROUND AND PURPOSE: The presence of cell-free fetal DNA in maternal plasma could allow performing a non-invasive prenatal diagnosis of Huntington disease (HD). The great advantage of this diagnosis is the absence of risk of fetal loss that it entails. METHODS: Maternal plasma from four pregnant women in their first trimester of gestation with a fetus at-risk was studied. In all the four cases, the father was affected. RESULTS: The diagnosis was performed both by a direct study of the mutation and an indirect haplotype study. By the direct analysis, three out of the four fetuses could be correctly diagnosed whilst the indirect analysis was only conclusive in one case. CONCLUSIONS: Non-invasive prenatal diagnosis of HD is possible by the analysis of fetal DNA in maternal plasma. Direct analysis of the mutation has shown higher accuracy than the haplotype analysis except for long expansions. Haplotype analysis would need to be improved for the study of Juvenile-onset HD. This diagnostic method would be limited to those couples with an affected male however this situation represents 80-90% of the pregnancies at-risk of HD. Moreover, it could be used as a confirmation test of healthy embryos transferred on pre-implantation genetic studies of HD.

Cryptic 6q subtelomeric deletion associated with a paracentric inversion in a mildly retarded child.

We report on a girl with minor anomalies and developmental delay carrying an apparently balanced paracentric inversion of chromosome 6q (q22qter). Fluorescent in situ hybridization analysis demonstrated a deletion of the subtelomeric region of 6q. This illustrates the use of specific subtelomeric fluorescent in situ hybridization probes to detect cryptic deletions as an important cause of mental retardation in seemingly balanced chromosome rearrangements.

Retinitis pigmentosa, mental retardation, marked short stature, and brachydactyly in two sibs.

We present two siblings with retinitis pigmentosa, mental retardation, markedly short stature, and brachydactyly. This association of clinical findings appears to be distinct from previously described syndromes and seems to represent the pleiotropic effects of a single autosomal recessive gene.

Choroideremia, sensorineural deafness, and primary ovarian failure in a woman with a balanced X-4 translocation.

We present clinical and cytogenetic studies of a female patient affected with choroideremia, mild sensorineural deafness, and primary amenorrhea showing a balanced translocation between chromosomes X and 4. The breakpoint was precisely defined applying FISH techniques: 46,X,t(X;4)(q21.2;p16.3).ish t(X;4)(D4S96+, D4F26+; wcpX+). The X-chromosomal breakpoint was located within a region where both the choroideremia locus and a deafness locus (DFN3/POU3F4) have been mapped. The presence of X-linked disorders in this balanced carrier of X-autosomal translocations (XAT) can be explained either by the disruption of the structural coding or regulatory sequences of the gene(s) or by the submicroscopic deletion of this region leading to a contiguous gene deletion syndrome. The primary ovarian failure (POF) found in the present case has been already observed in XAT when the breakpoint is within a previously defined critical region (Xq13-26). A position effect is postulated as a possible explanation.

A maternal inherited translocation t(1;22)(q11;p11) in two infertile brothers.

We report two infertile brothers presenting with azoospermia and oligozoospermia. Cytogenetic studies using G-banding and FISH analysis on lymphocyte cultures revealed an autosomal balanced reciprocal translocation t(1;22)(q11;p11) in both males. The same translocation was found in their mother, but not in a third fertile brother and maternal uncle suggesting that this translocation might compromise the male but not the female gametogenesis in this family.

A MELAS phenotype and a paternal inherited inversion of chromosome 10 in a female patient.

A MELAS phenotype and a paternal inherited inversion of chromosome 10 in a female patient: We describe a patient suffering from encephalomyopathy with overlapping symptoms, including MELAS and Kearn-Sayre syndrome features. Mutations in tRNA LEU (UUR) were not found in mtDNA of blood cells, suggesting a different genetic defect. Cytogenetic studies revealed a paternal inherited pericentric inversion of chromosome 10 (p13;q22) pat. Although the presence of the same inversion in the father and in the apparently asymptomatic sister does rather suggest that the concurrence of the mitochondrial disease in the patient was due to chance, some alternative explanations to associate both events might be proposed.

Aniridia as part of a WAGR syndrome in a girl whose brother presented hypospadias.

Aniridia can arise as part of the WAGR syndrome (Wilms tumour. aniridia, genitourinary anomalies, and mental retardation), due to a deletion or chromosomal region 11p13. We report a girl with a complete WAGR syndrome, whose brother presented hypospadias. Cytogenetic, FISH and molecular studies showed a deletion in one chromosome 11 of the patient. No cytogenetic rearrangement or deletion affecting the genes included in this region (PAX6 and WT1) were observed in her brother and parents. This excludes a higher risk than that of the general population for developing Wilms tumour in the brother and supports that the presence of WAGR syndrome in the patient and hypospadias in her brother is a chance association. We conclude that the identification and definition of the deletions in the WAGR region, which include the WT1 locus are important in order to identify a high tumour risk in infant patients with aniridia including those without other WAGR anomalies.

Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease.

PURPOSE: We focused on the improvements of prenatal diagnosis by the analysis of DNA from maternal plasma, using Huntington disease as a model of disease. METHODS: We studied plasma from a pregnancy at risk of having a fetus affected with Huntington disease by the use of two direct analysis of the mutation and polymorphic STRs. RESULTS: Direct methods were not informative. Analysis with STRs revealed the presence of the allele that does not co-segregate with the disease, thus the fetus was healthy. CONCLUSIONS: This strategy is very useful to face complex cases when the direct study is not informative not only for Huntington disease but also for many other disorders.

Huntington disease-unaffected fetus diagnosed from maternal plasma using QF-PCR.

The discovery of fetal DNA in maternal plasma from early pregnancies has led to new opportunities for clinical application. In the last few years there have been numerous reported applications, mainly fetal gender and RhD genotyping. The prenatal diagnosis of some inherited genetic diseases such as Huntington disease is also very frequently required in the prenatal diagnosis routine. We have successfully diagnosed, with a non-invasive procedure, an unaffected HD fetus at the 13th week of gestation using fetal DNA from maternal plasma and the quantitative fluorescent PCR method, which is one of the most sensitive ways to detect fetal DNA in maternal plasma at such an early time of gestation.

Rapid identification of a small dicentric supernumerary marker derived from chromosome 16 with a modified FISH technique on amniotic fluid.

Small supernumerary marker chromosomes are seldom found in prenatal diagnosis and the majority of them are difficult to identify. The only possibility to give a more precise prognosis is by establishing its origin. FISH is the best technique to identify the chromosomal origin, but in the majority of cases large amounts of chromosomal material are needed and this is time consuming. We have used a modification of the FISH technique that allows the hybridization of several probes on one slide. Using this method, we have identified the first de novo mosaic dicentric supernumerary marker derived from chromosome 16 (smaller than chromosome 21) in amniotic fluid. The gestation and the follow-up of the baby were normal.

Sonographic, cytogenetic and DNA analysis in four 69,XXX fetuses diagnosed in the second trimester.

OBJECTIVE: To describe the ultrasound findings and its relationship with the cytogenetic study and the origin of the extra haploid chromosome set in four 69,XXX cases. METHODS: Four pregnant women were referred because of abnormal 2nd trimester ultrasound. Karytoypes, FISH and DNA analysis were performed. RESULTS: All cases presented asymmetrical intrauterine growth retardation, marked oligohydramnios and placental alterations and showed a 69,XXX karyotype. In three cases, DNA analysis allowed to establish the origin of the extra haploid chromosome set. CONCLUSIONS: At least three fetuses had a maternal extra haploid chromosome set. Thus, it has been possible to establish the main ultrasonographic markers and to observe the survival of the fetus until the second trimester when they have a maternal origin.

Segregation of digital number with partial monosomy or trisomy of 13q in familial 5;13 translocation.

It has been postulated that the deletion of band 13q22 may be associated with digital malformations, especially thumb and big toe anomalies. We report a family where the mother is carrying a balanced translocation between chromosomes 5p15 and 13q22. The offspring have a specific and well-defined phenotype depending on which is the unbalanced chromosome in the karyotype. When a partial trisomy of 13q22-->qter is present, the fetuses have polydactyly in the four limbs, and when the fetus is carrying a partial monosomy of this portion, an oligodactyly in all members can be observed.

First mutation (S340X) in choroideremia gene in a Spanish family. Mutations in brief no. 173. Online.

A study of choroideremia gene was performed in Spanish families affected with this disorder. One abnormal pattern was detected in exon eight corresponding to a new mutation not described before. The mutation was identified as a nonsense mutation S340X.

Identification of two rare variants (G-->A at nucleotide 721; C-->T at nucleotide 5200) in the rhodopsin gene. Mutations in brief no. 187. Online.

The authors report two new rare DNA sequence variants in the Rhodopsin gene. This gene is involved in the pathogenesis of some retinal hereditary disorders as Retinitis Pigmentosa. These rare variants are G-->A at nucleotide 721 of the non-coding region and C-->T at nucleotide 5200 within codon 323 which does not alter the aminoacid cysteine. Therefore, they are not implicated in the development of the Retinitis Pigmentosa disease.

Chromosomal mosaicism for isochromosome 11q confined to CVS direct preparations.

OBJECTIVE: To analyse the discrepancy between the karyotype in direct preparations of chorionic villus sampling (CVS) and the fetal karyotype and its possible fetal phenotypic repercussion. METHODS: The karyotype was obtained from direct and cultured preparations of CVS. FISH was performed in direct CVS preparations and in four different areas of term placenta. RESULTS: Karyotype and FISH analysis in CVS revealed a 46,XX/47,XX,+i(11q) cell line. Cultured CVS preparations showed a 46,XX karyotype. Cytogenetic studies in term placenta did not reveal the abnormal cell line. Molecular studies did not detect uniparental disomy for chromosome 11 in the fetus. CONCLUSION: The fetus, at birth, had no phenotypic abnormalities. IUGR was not present during gestation, in accordance with the low proportion of aneuploid cells in term placenta, and UPD for chromosome 11 was not observed.

Prenatal diagnosis on fetal cells obtained from maternal peripheral blood: report of 66 cases.

The potential use of fetal cells circulating in maternal blood for a non-invasive prenatal diagnosis has been widely described. Several authors have developed different methods for the enrichment of fetal cells from maternal peripheral blood. The aim of this study was to make a practical valuation of this new prenatal diagnosis technique, using those methods described as efficient and easy to carry out in a prenatal diagnosis unit. These methods consist of the double-density gradient and the positive selection by magnetic activated cell sorting (MACS) of the fetal erythroblasts, and the posterior study of the cells applying the FISH interphasic technique. Once the technique was ready, we obtained results from the study of 66 venous blood samples from women coming for prenatal diagnosis. Using a specific staining for fetal haemoglobin, fetal cells were identified in 63 cases. Fetal sex was well determined in 56 cases, 23 females and 33 males; in 7 cases the sex determination failed. All the aneuploidies found in a previous prenatal diagnosis were confirmed.