COASY related pontocerebellar hypoplasia type 12: A common Indian mutation with expansion of the phenotypic spectrum.

Pontocerebellar hypoplasia (PCH) type 12 is a rare, perinatal lethal neurodegenerative genetic disorder caused by biallelic mutations in the COASY gene. Herein, we describe the clinical and neuroradiological profile of nine affected fetuses/neonates from five families identified with a common COASY: c.1486-3C>G biallelic variant. Four of the five families were identified after data reanalysis of unresolved, severe PCH like phenotype and the fifth family through collaboration. The common antenatal phenotype was cerebellar hypoplasia. Microcephaly, arthrogryposis, and intrauterine growth restriction were the shared postnatal findings. The neurological manifestations included seizures, poor sucking, and spasticity. Novel findings of corpus callosum agenesis, simplified gyral pattern, normal sized pons, optic neuropathy, and a small thorax are reported in this series. The allele frequency of the COASY: c.1486-3C>G variant was 0.62% in the available Asian Indian database. We describe this as a possible common Indian origin variant. To the best of our knowledge, this is the largest PCH12 series reported.

Suleiman-El-Hattab syndrome: a histone modification disorder caused by TASP1 deficiency.

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort.

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them  could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.

GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.

Diagnóstico molecular de enfermedades genéticas: del diagnóstico genético al diagnóstico genómico con la secuenciación masiva / Molecular diagnosis of genetic diseases: from genetic to genomic diagnosis using next generation sequencing

En la actualidad se conocen 8.000 enfermedades genéticas monogénicas. La mayoría de ellas son heterogéneas, por lo que el diagnóstico molecular por técnicas convencionales de secuenciación suele ser largo y costoso debido al gran número de genes implicados. El tiempo estimado para el diagnóstico molecular se encuentra entre 1 y 10 años, y este retraso impide que los pacientes reciban medidas terapéuticas y de rehabilitación específicas, que sus familiares entren en programas preventivos y que reciban asesoramiento genético. La secuenciación masiva está cambiando el modelo de diagnóstico molecular de los afectos, sin embargo, los médicos y profesionales de la salud se enfrentan al dilema de la selección del método más eficiente, con el menor coste sanitario y con la mayor precisión de sus resultados. El objetivo de este trabajo es revisar la tecnología de secuenciación masiva y definir las ventajas y los problemas en su utilización.

Currently 8000 monogenic genetic diseases are known. Most of them are heterogeneous, so their molecular diagnosis by conventional sequencing techniques is labour intensive and time consuming due to the large number of genes involved. The estimated time is between 1 and 10 years for molecular diagnosis and this delay prevents patients from receiving therapy and rehabilitation measures, and their families from entering prevention programs and being given genetic counselling. Next generation sequencing (NGS) is changing the model of molecular diagnosis of patients; however, doctors and health professionals are faced with the dilemma of choosing the most efficient method, with lower health care costs and the most accurate results. The aim of this paper is to review the NGS technology and define the advantages and problems in the use of this technology.

Early noninvasive prenatal detection of a fetal CRB1 mutation causing Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. Mutations in the Crumbs homologue 1 (CRB1; OMIM 600105) gene explain 10%-24% of cases with LCA depending on the population. The aim of the present work was to study a fetal mutation associated to LCA in maternal plasma by a new methodology in the noninvasive prenatal diagnosis field: the denaturing High Performance Liquid Chromatography (dHPLC). METHODS: This study presents the case of a compound heterozygous fetus for two mutations in CRB1 (1q3.1-q32.2). dHPLC and automated DNA sequencing were used to detect the paternally inherited fetal mutation in a maternal plasma sample collected at the 12th week of gestation. To test the detection limit of dHPLC, we made serial dilutions of paternal DNA in control DNA. RESULTS: We were able to detect the presence of the paternally inherited fetal CRB1 mutation in maternal plasma by dHPLC. Moreover, by comparing chromatograms of serial dilutions to the plasma sample, we could ascertain that the percentage of fetal DNA in maternal plasma was at least 2%. However, the detection of the fetal mutation was not possible by automated DNA sequencing. CONCLUSIONS: dHPLC seems to be sensitive enough to detect small amounts of fetal DNA in maternal plasma samples. It could be a useful tool for the noninvasive prenatal detection of paternally inherited point mutations associated with retinopathies.

New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma.

BACKGROUND: Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. METHODS: We have used a mini-sequencing based method, the SNaPshot, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. RESULTS: The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. CONCLUSIONS: The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

New type of mutations in three spanish families with choroideremia.

PURPOSE: Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. METHODS: First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. RESULTS: In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. CONCLUSIONS: Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.

MLPA as a screening method of aneuploidy and unbalanced chromosomal rearrangements in spontaneous miscarriages.

OBJECTIVE: The present study aims to validate multiplex ligation-dependent probe amplification (MLPA) technique with subtelomeric probe mixes as a screening method to detect aneuploidy and unbalanced terminal chromosomal rearrangements in spontaneous abortions (SAs). METHODS: MLPA with P036B and P070 probe mixes was performed on 221 miscarriage DNA samples between the 5th and 24th week of gestation. Cytogenetic culture was attempted on 178 miscarriages. Karyotyped miscarriages served as controls in this blinded study. Results were confirmed by quantitative fluorescent-PCR (QF-PCR). RESULTS: Among the karyotyped miscarriages, MLPA was able to detect all the expected aneuploidies, as well as an unbalanced product from a reciprocal translocation, and revealed cryptic deletions and duplications not visible at the 550-band resolution level. In addition, chromosomal anomalies were found in approximately 37% of cases that failed to grow or could not be cultivated. As expected, ploidy changes were not detected. Copy number variation was found for target sequences of P036B (CYFIP1, MRPL41, CAB45) and P070 (DECR2, TNFRSF18) probe mixes. CONCLUSIONS: We propose the use of MLPA with subtelomeric probe mixes as a reliable, rapid and economical first approach to detect aneuploidy and unbalanced terminal chromosomal rearrangements in SAs.

Clinical presentation of a variant of Axenfeld-Rieger syndrome associated with subtelomeric 6p deletion.

We report a 22-year-old female with a variant of the Axenfeld-Rieger Syndrome (ARS) and discuss its relation with the subtelomeric 6p deletion. An ARS variant has been described in two familial cases of Axenfeld-Rieger Anomaly (ARA) featuring specific extra ocular manifestations-hypertelorism, midface hypoplasia, mild sensorial deafness, hydrocephaly, psychomotor delay and flattened femoral epiphyses. We proposed that this set of characteristics represents a separate syndrome within the ARS. On the other hand, there have been reported four cases with cryptic de novo pure 6pter microdeletions detected by specific subtelomeric probes in patients with ARS characteristics. We describe a 6pter deletion detected by SNP genotyping and confirmed by FISH and MLPA involving the FOXC1 gene in a patient with ocular and systemic findings that fit perfectly with the variant mentioned above. We conclude that the ARS variant belongs to the ARS phenotypic spectrum, which includes flattened femoral epiphyses as a feature.

Detection of a paternally inherited fetal mutation in maternal plasma by the use of automated sequencing.

The discovery of circulating fetal DNA in maternal blood has been an encouraging step forward in the prenatal diagnostic field. It has opened up the possibility of development of a noninvasive method for the genetic analysis of the fetus. Many techniques have been applied to the study of this fetal DNA, but automated sequencing has been seldom used. The intention of this study was to use the automated sequencing technique for the detection of a paternally inherited fetal mutation in maternal plasma. Maternal plasma samples from a pregnant woman, whose husband had a mutation (Q134X) in the RP2 gene, which is located in the X-chromosome, were collected at two different gestational ages (10th and 19th week of gestation) in order to determine whether the paternally inherited fetal mutation could be detected by automated sequencing. Restriction analysis was also performed to confirm the results. The fetal mutation was clearly detected in the maternal plasma by the use of automated sequencing. The automated sequencing enables the possibility of analyzing fetal sequences, at a nucleotide level, in order to detect mutations or polymorphisms which are distinguishable from maternal sequences.

Double trisomy in spontaneous miscarriages: cytogenetic and molecular approach.

BACKGROUND: Although single trisomy is the most common chromosomal abnormality observed within first trimester spontaneous abortions (SA) (>50%), double trisomy (DT) ranges from 0.21 to 2.8% in the literature. Since little is known about mechanisms underlying DT, we report the results of our experience with 517 SA, establishing parental origin and cell stage of non-disjunction when possible in DT cases, and making a revision of those previously reported. METHODS: Cytogenetic analysis was performed in all aborted specimens. Quantitative fluorescent PCR (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA) were performed in DT cases in order to assess parental origin and stage of error of aneuploidy in addition to its reliability in detecting aneuploidies. RESULTS: Karyotyping was successful in 321 miscarriages; the rate of DT was 2.18%. Among the seven DT cases reported, three new combinations were found. Maternal origin was established for all DT SA analysed. Meiotic stage of error was presumed meiosis I (MI) for 48,XX+15+22 and 48,XX+8+21, meiosis II (MII) for 48,XXX+18, and MII and MI respectively for 48,XY+18+22. Molecular results agreed with cytogenetic results. CONCLUSIONS: Similar maternal age-related mechanisms could be implicated in both single and double trisomy. Molecular techniques could be useful in diagnosing not only single but multiple aneuploidy and determining its origin. This will improve our knowledge about mechanisms underlying human aneuploidy, and enable appropriate genetic counselling.

Trisomy 2 due to a 3:1 segregation in an abortion studied by QF-PCR and CGH.

Balanced reciprocal translocation is one of the known causes of recurrent spontaneous abortions. Cytogenetic studies of unbalanced miscarriages are difficult due to the growth failure of early loss and usually macerated abortions. We present a molecular study of an abortion in which the father carries a balanced reciprocal translocation t(2;17)(q32.1;q24.3) using QF-PCR and CGH techniques. DNA analysis showed the presence of a trisomy 2 due to a 3:1 interchange segregation. Recombinant events could also be investigated by comparing DNA samples from the family. We propose QF-PCR in addition to CGH as an efficient diagnostic method to improve our knowledge of unbalanced offspring in balanced translocation carriers.

Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploidies in spontaneous miscarriages.

BACKGROUND: Aneuploidies involve approximately 80% of chromosomal anomalies found in spontaneous miscarriages. Since cytogenetic studies show high rates of failure, we have incorporated the quantitative fluorescent polymerase chain reaction (QF-PCR) technique to the study of numerical chromosome anomalies in miscarriages. METHODS: Multiplex and simple QF-PCR assays have been performed on 160 miscarriage and 34 parental DNA samples analysing specific short tandem repeat (STR) markers for chromosomes 2, 7, 13, 15, 16, 18, 21, 22 and X. Cases successfully karyotyped were used as controls in our study. RESULTS: While maternal contamination could be detected in such cases, a molecular result was obtained for 94% of miscarriages without a cytogenetic one. Thirty-six per cent of them were diagnosed with numerical chromosome anomalies. Parental origin of the extra chromosome and the error stage of meiosis could be also determined. CONCLUSIONS: QF-PCR represents a useful and reliable tool to diagnose aneuploidies in spontaneous miscarriages. It provides information about parental and meiotic origin of anomaly, allowing an appropriate genetic counselling.