Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination.

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Optical genome mapping for prenatal diagnosis: A prospective study.

PURPOSE: Cytogenetic analysis provides important information for prenatal decision-making and genetic counseling. Optical genome mapping (OGM) has demonstrated its performances in retrospective studies. In our prospective study, we assessed the quality of DNA obtained from cultures of amniotic fluid (AF) and chorionic villi (CV) and evaluated the ability of OGM to detect all clinically relevant aberrations identified by standard methods. METHODS: A total of 37 prenatal samples from pregnancies with a fetal anomaly on ultrasound were analyzed prospectively by OGM between January 1, 2021 and June 31, 2022. OGM results were interpreted blindly and compared to the results obtained by standard techniques. RESULTS: OGM results were interpretable in 92% of samples. We observed 100% concordance between OGM and karyotype and/or chromosomal microarray results. In addition, OGM identified a median of 30 small (<100 kb) structural variations per case with the involvement of 12 OMIM genes, of which 3 were OMIM morbid genes. CONCLUSION: This prospective study showed OGM performed well in detecting genomic alterations in cell cultures from prenatal samples. The place of OGM in relation to CMA or exome sequencing remains to be defined in order to optimize the prenatal diagnostic procedure.

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Analysis of the cost effectiveness of different strategies for the antenatal diagnosis of chromosomal aberrations in cases of ultrasound-identified fetal abnormalities.

OBJECTIVE: Principal objective of this work was to analyse the cost effectiveness of different sequences of cytogenetic techniques from the hospital's point of view, after prenatal ultrasound has identified fetal malformations. METHODS: Cytogenetic tests were performed for each case in 3 strategies, and their results are reported and compared to one reference strategy. Two new simulated strategies were considered: chromosomal microarrays alone and a direct test + CMA. MAIN OUTCOMES MEASURES: cost-effectiveness ratio. RESULTS: A single test result was positive in 234 of the 835 pregnancies studied (28%). CMA alone would have identified 239 abnormalities. In the simulated direct test + CMA sequence, the direct test alone would have been positive for 66.1% of the abnormalities identified. When testing was indicated for NT, reference strategy (Direct + karyotyping) costs 1 084.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively 992.7 and 550.0 euros by positive test results. For OUM indications, reference strategy costs 2 937.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively, 2 118.4 and 1 304.7 euros by positive test results. CONCLUSIONS: CMA appears to be the most effective test for prenatal cytogenetic diagnosis of fetal abnormalities identified by ultrasound.

Deleterious mutations in ALDH1L2 suggest a novel cause for neuro-ichthyotic syndrome.

Neuro-ichthyotic syndromes are a group of rare genetic diseases mainly associated with perturbations in lipid metabolism, intracellular vesicle trafficking, or glycoprotein synthesis. Here, we report a patient with a neuro-ichthyotic syndrome associated with deleterious mutations in the ALDH1L2 (aldehyde dehydrogenase 1 family member L2) gene encoding for mitochondrial 10-formyltetrahydrofolate dehydrogenase. Using fibroblast culture established from the ALDH1L2-deficient patient, we demonstrated that the enzyme loss impaired mitochondrial function affecting both mitochondrial morphology and the pool of metabolites relevant to ß-oxidation of fatty acids. Cells lacking the enzyme had distorted mitochondria, accumulated acylcarnitine derivatives and Krebs cycle intermediates, and had lower ATP and increased ADP/AMP indicative of a low energy index. Re-expression of functional ALDH1L2 enzyme in deficient cells restored the mitochondrial morphology and the metabolic profile of fibroblasts from healthy individuals. Our study underscores the role of ALDH1L2 in the maintenance of mitochondrial integrity and energy balance of the cell, and suggests the loss of the enzyme as the cause of neuro-cutaneous disease.

Mutation in POLR3K causes hypomyelinating leukodystrophy and abnormal ribosomal RNA regulation.

OBJECTIVE: To identify the genetic cause of hypomyelinating leukodystrophy in 2 consanguineous families. METHODS: Homozygosity mapping combined with whole-exome sequencing of consanguineous families was performed. Mutation consequences were determined by studying the structural change of the protein and by the RNA analysis of patients' fibroblasts. RESULTS: We identified a biallelic mutation in a gene coding for a Pol III-specific subunit, POLR3K (c.121C>T/p.Arg41Trp), that cosegregates with the disease in 2 unrelated patients. Patients expressed neurologic and extraneurologic signs found in POLR3A- and POLR3B-related leukodystrophies with a peculiar severe digestive dysfunction. The mutation impaired the POLR3K-POLR3B interactions resulting in zebrafish in abnormal gut development. Functional studies in the 2 patients' fibroblasts revealed a severe decrease (60%-80%) in the expression of 5S and 7S ribosomal RNAs in comparison with control. CONCLUSIONS: These analyses underlined the key role of ribosomal RNA regulation in the development and maintenance of the white matter and the cerebellum as already reported for diseases related to genes involved in transfer RNA or translation initiation factors.

Sperm meiotic segregation of a balanced interchromosomal reciprocal insertion resulting in recurrent spontaneous miscarriage.

RESEARCH QUESTION: Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? DESIGN: This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. RESULTS: A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. CONCLUSIONS: This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.

TERT promoter status and gene copy number gains: effect on TERT expression and association with prognosis in breast cancer.

Upregulation of the telomerase reverse transcriptase (TERT) gene in human cancers leads to telomerase activation, which contributes to the growth advantage and survival of tumor cells. Molecular mechanisms of TERT upregulation are complex, tumor-specific and can be clinically relevant. To investigate these mechanisms in breast cancer, we sequenced the TERT promoter, evaluated TERT copy number changes and assessed the expression of the MYC oncogene, a known transcriptional TERT regulator, in two breast cancer cohorts comprising a total of 122 patients. No activating TERT promoter mutations were found, suggesting that this mutational mechanism is not likely to be involved in TERT upregulation in breast cancer. The T349C promoter polymorphism found in up to 50% of cases was not correlated with TERT expression, but T349C carriers had significantly shorter disease-free survival. TERT gains (15-25% of cases) were strongly correlated with increased TERT mRNA expression and worse patient prognosis in terms of disease-free and overall survival. Particularly aggressive breast cancers were characterized by an association of TERT gains with MYC overexpression. These results evidence a significant effect of gene copy number gain on the level of TERT expression and provide a new insight into the clinical significance of TERT and MYC upregulation in breast cancer.

A novel 2q14.1q14.3 deletion involving GLI2 and RNU4ATAC genes associated with partial corpus callosum agenesis and severe intrauterine growth retardation.

BACKGROUND: Microdeletions encompassing chromosome bands 2q14.1q14.3 are rare. To date, eight reports of relatively large deletions of this region (∼20 Mb) but only two small deletions (<6 Mb) have been reported. These deletions can cause a variable phenotype depending on the size and location of the deletion. Cognitive disability, facial dysmorphism, and postnatal growth retardation are the most common phenotypic features. CASE: We report on a novel 5.8 Mb deletion of 2q14.1q14.3 identified by array comparative genomic hybridization in a fetus with severe intrauterine growth retardation and partial agenesis of the corpus callosum. The deletion contained 24 coding genes including STEAP3, GLI2, and RNU4ATAC and was inherited from the mild affected mother. A sibling developmental delay and similar dysmorphic facial features was found to have inherited the same deletion. CONCLUSION: This case emphasizes the variable expressivity of the 2q14 microdeletion and reinforces the hypothesis that agenesis of corpus callosum, microcephaly, developmental delay, and distinctive craniofacial features may be part of the phenotypic spectrum characterizing the affected patients. We suggest that GLI2 is a dosage-sensitive gene that may be responsible for the agenesis of corpus callosum observed in the proband. Birth Defects Research (Part A) 106:793-797, 2016. © 2016 Wiley Periodicals, Inc.

Determination of arylsulfatase A pseudodeficiency allele and haplotype frequency in the Tunisian population.

Arylsulfatase A (ASA) is a lysosomal enzyme involved in the catabolism of cerebroside sulfate. ASA deficiency is associated with metachromatic leukodystrophy (MLD). Low ASA activities have also been reported in a more common condition with no apparent clinical consequences termed ASA pseudo-deficiency (ASA-PD) which is associated with two linked mutations in the ASA gene (c.1049A>G and c.*96A>G). This study aimed to investigate the frequency of the two ASA-PD variants and their linkage disequilibrium (LD) among Tunisians. ASA-PD variants were detected in 129 healthy Tunisians and their frequencies were compared to those described worldwide. The frequency of the PD allele was estimated at 17.4% for the overall sample, with c.1049A>G and c.*96A>G frequencies of 25.6 and 17.4%, respectively. This study also revealed a high LD between the two ASA-PD variants (r(2) = 0.61). Inter-population analysis revealed similarities in the ASA-PD genetic structure between Tunisians and populations from Middle East with c.*96A>G frequencies being the highest in the world. A significant North vs. South genetic differentiation in the ASA-PD frequency was also observed in Tunisian population who seems genetically intermediate between Africans, Middle-Easterners and Europeans. This is the first report on the allele frequency of the ASA-PD in North Africa, revealing a relatively high frequency of the PD allele among Tunisians. This study gives also evidence on the importance of discriminating ASA-PD allele from pathological mutations causing MLD and supporting enzymatic activity testing with both sulfatiduria determination and genetic testing in the differential diagnosis of MLD in the Tunisian population.

Insertion of an extra copy of Xq22.2 into 1p36 results in functional duplication of the PLP1 gene in a girl with classical Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder characterized by nystagmus, hypotonia, ataxia, progressive spasticity, and cognitive decline. PMD classically results from a duplication of a genomic segment encompassing the entire PLP1 gene. Since the PLP1 gene is located in Xq22, PMD affects mostly boys. METHODS AND RESULTS: Here we report the case of a girl with typical PMD. Copy number analysis of the PLP1 locus revealed a duplication of the entire gene and FISH analysis showed that the extra copy of the PLP1 gene was actually inserted in chromosome 1p36. This insertion of an additional copy of PLP1 in an autosome led to a functional duplication irrespective of the X-inactivation pattern. Subsequent overexpression of PLP1 was the cause of the PMD phenotype observed in this girl. Further sequencing of the breakpoint junction revealed a microhomology and thus suggested a replication based mechanism (such as FoSTeS or MMBIR). CONCLUSION: This case emphasizes the susceptibility of the PLP1 locus to complex rearrangement likely driven by the Xq22 local genomic architecture. In addition, careful consideration should be given to girls with classical PMD clinical features since they usually experience complex PLP1 genomic alteration with a distinct risk of inheritance.

Prenatal Screening of 21 Microdeletion/Microduplication Syndromes and Subtelomeric Imbalances by MLPA in Fetuses with Increased Nuchal Translucency and Normal Karyotype.

Fetuses with increased nuchal translucency thickness (NT) are at increased risk for chromosomal abnormalities. In case of a normal karyotype, a minority of them may present with structural abnormalities or genetic syndromes, which may be related to submicroscopic chromosomal imbalances. The objective of this study was to evaluate whether MLPA screening of 21 syndromic and subtelomeric regions could improve the detection rate of small chromosomal aberrations in fetuses with increased NT and a normal karyotype. A total of 106 prenatal samples from fetuses with NT ≥ 99th centile and normal R- and G-banding were analyzed by MLPA for subtelomeric imbalances (SALSA P036 and P070) and 21 syndromic regions (SALSA P245). One sample showed a benign CNV (dup(8)pter, FBXO25 gene), and 1 patient was found to have a loss of 18 qter and a gain of 5 pter as a result of an unbalanced translocation. The incidence of cryptic pathogenic variants was <1% or 2.7% when only fetuses with other ultrasound abnormalities were taken into account. Submicroscopic imbalances in fetuses with increased NT may be individually rare, and genome-wide screening seems more likely to improve the diagnostic yield in these fetuses.

Congenital diaphragmatic hernia may be associated with 17q12 microdeletion syndrome.

Microdeletions of 17q12 encompassing TCF2 are associated with maturity-onset of diabetes of the young type 5, cystic renal disease, pancreatic atrophy, Mullerian aplasia in females and variable cognitive impairment. We report on a patient with a de novo 17q12 microdeletion, 1.8 Mb in size, associated with congenital diaphragmatic hernia (CDH). The 5-year-old male patient presented multicystic renal dysplasia kidneys, minor facial dysmorphic features and skeletal anomalies, but neither developmental delay nor behavioral abnormalities. CDH has been previously associated with the 17q12 microdeletion syndrome only in one prenatal case. The present study reinforces the hypothesis that CDH is part of the phenotype for 17q12 microdeletion and that 17q12 encompasses candidate(s) gene(s) involved in diaphragm development. We suggest that PIGW, a gene involved in an early step of GPI biosynthesis, could be a strong candidate gene for CDH.

Clinical and molecular description of a 17q21.33 microduplication in a girl with severe kyphoscoliosis and developmental delay.

High proportion of disease-associated copy number variant maps to chromosome 17. Genomic studies have provided an insight into its complex genomic structure such as relative abundance of segmental duplication and intercepted repetitive elements. 17q21.31, 17q11.2 and 17q12 loci are well known on this chromosome and are associated with microdeletion and microduplication syndrome. No syndrome associated with 17q21.33 locus have been described. We report clinical, cytogenetic and molecular investigations of a 13 years-old girl admitted for evaluation of microcephaly, scoliosis, skeletal defects and learning difficulties. We carried out detailed analysis of the clinical phenotype of this patient and investigated the genetic basis using Agilent 180K Array Comparative Genomic Hybridization. We identified a ∼0.9 Mb de novo microduplication on chromosome 17q21.33. Four genes, COL1A1, SGCA, PPP1R9B and CHAD located within the duplicated region are possible candidates for clinical features present in our patients. Gene expression studies by real-time RT-PCR assay only showed an overexpression of SGCA (P < 0.01), a component of the dystrophin glycoprotein complex. Defect of SGCA was previously shown to lead to severe childhood autosomal recessive muscular dystrophy (LGMD2D) which result in progressive muscle weakness and can also be associated with hyperlordosis or scoliosis. Further cases with similar duplications are expected to be diagnosed. This will contribute to the delineation of this potential new microduplication syndrome and to improve genetic counseling.

De novo 2q36.1q36.3 interstitial deletion involving the PAX3 and EPHA4 genes in a fetus with spina bifida and cleft palate.

BACKGROUND: Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally. METHODS: We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate. RESULTS: Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes. CONCLUSION: The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally.

An unusual clinical severity of 16p11.2 deletion syndrome caused by unmasked recessive mutation of CLN3.

With the introduction of array comparative genomic hybridization (aCGH) techniques in the diagnostic setting of patients with developmental delay and congenital malformations, many new microdeletion syndromes have been recognized. One of these recently recognized microdeletion syndromes is the 16p11.2 deletion syndrome, associated with variable clinical outcomes including developmental delay, autism spectrum disorder, epilepsy, and obesity, but also apparently normal phenotype. We report on a 16-year-old patient with developmental delay, exhibiting retinis pigmentosa with progressive visual failure from the age of 9 years, ataxia, and peripheral neuropathy. Chromosomal microarray analysis identified a 1.7-Mb 16p11.2 deletion encompassing the 593-kb common deletion (∼29.5 to ∼30.1 Mb; Hg18) and the 220-kb distal deletion (∼28.74 to ∼28.95 Mb; Hg18) that partially included the CLN3 gene. As the patient's clinical findings were different from usual 16p11.2 microdeletion phenotypes and showed some features reminiscent of juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease, OMIM 204200), we suspected and confirmed a mutation of the remaining CLN3 allele. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletion represents one explanation for the phenotypic variability observed in chromosomal deletion disorders.

Relevance of different cellular models in determining the effects of mutations on SLC16A2/MCT8 thyroid hormone transporter function and genotype-phenotype correlation.

SLC 16A2, the gene for the second member of the solute carrier family 16 (monocarboxylic acid transporter), located on chromosome Xq13.2, encodes a very efficient thyroid hormone transporter: monocarboxylate transporter 8, MCT8. Its loss of function is responsible in males for a continuum of psychomotor retardation ranging from severe (no motor acquisition, no speech) to mild (ability to walk with help and a few words of speech). Triiodothyronine uptake measurement in transfected cells and, more recently, patient fibroblasts, has been described to study the functional consequences of MCT8 mutations. Here, we describe three novel MCT8 mutations, including one missense variation not clearly predicted to be damaging but found in a severely affected patient. Functional studies in fibroblasts and JEG3 cells demonstrate the usefulness of both cellular models in validating the deleterious effects of a new MCT8 mutation if there is still a doubt as to its pathogenicity. Moreover, the screening of fibroblasts from a large number of patient fibroblasts and of transfected mutations has allowed us to demonstrate that JEG3 transfected cells are more relevant than fibroblasts in revealing a genotype-phenotype correlation.

A new case of 8q22.1 microdeletion restricts the critical region for Nablus mask-like facial syndrome.

Microdeletions of 8q21.3-8q22.1 have been identified in all patients with Nablus mask-like facial syndrome (NMLFS). A recent report of a patient without this specific phenotype presented a 1.6 Mb deletion in this region that partially overlapped with previously reported 8q21.3 microdeletions, thus restricting critical region for this syndrome. We report on another case of an 8q21.3 deletion revealed by array comparative genome hybridization (aCGH) in a 4-year-old child with global developmental delay, autism, microcephaly, but without Nablus phenotype. The size of the interstitial deletion was estimated to span 5.2 Mb. By combining the data from previous reports on 8q21.3-8q22.1 deletions and our case, we were able to narrow the critical region of Nablus syndrome to 0.5 Mb. The deleted region includes FAM92A1, which seems to be a potential candidate gene in NMLFS.