- Record: found
- Abstract: found
- Article: found
Prenatal diagnosis of developmental and epileptic encephalopathy 9 with a 10.05‐Mb microdeletion at Xq21.31q22.1 inherited from mother: A case report and literature review
research-article
Publication date (Electronic):
11 December 2023
Publisher:
John Wiley and Sons Inc.
Read this article at
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Background
Developmental and epileptic encephalopathy 9 (DEE9) is characterized by early infantile seizures and mild‐to‐severe neuropsychiatric symptoms. Despite being an X‐linked dominant disorder, DEE9 mainly affects heterozygous females or mosaic males, while hemizygous males are less affected. PCDH19 gene has been documented as the causative gene.
Methods
Karyotyping analysis and copy number variation sequencing (CNV‐seq) were performed on a pregnant woman with epilepsy, together with her husband, son, and fetus.
Results
A disease‐causing microdeletion, seq[GRCh37] del(X)(q21.31q22.1) (90310001–100360000), was identified in the pregnant woman and her female fetus. The microdeletion includes the entire PCDH19 gene and is classified as “pathogenic” according to the American College of Medical Genetics and Genomics guidelines.
Conclusion
In this case study, we have not only identified the epilepsy type of the woman as DEE9 but have also made an unfavorable prognosis for her fetus. Our findings from this prenatal case provide valuable clinical resources for prenatal diagnosis and genetic counseling, while also implying the potential of CNV‐seq as a viable method for uncovering PCDH19‐related epilepsy.
Abstract
We performed prenatal diagnosis on a Chinese pregnant woman with epilepsy. A disease‐causing microdeletion at Xq21.31q22.1 including the PCDH19 gene was discovered by CNV‐seq in both the woman and her female fetus. Our case provides valuable clinical resources for prenatal diagnosis and genetic counseling, while also implying the potential of CNV‐seq as a viable method for uncovering PCDH19‐related epilepsy.
Related collections
Most cited references36
- Record: found
- Abstract: found
- Article: not found
Technical standards for the interpretation and reporting of constitutional copy number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen)
Erin Rooney Riggs, Erica Andersen, Athena Cherry … (2019)
Copy number analysis to detect disease-causing losses and gains across the genome is recommended for the evaluation of individuals with neurodevelopmental disorders and/or multiple congenital anomalies, as well as for fetuses with ultrasound abnormalities. In the decade that this analysis has been in widespread clinical use, tremendous strides have been made in understanding the effects of copy number variants (CNVs) in both affected individuals and the general population. However, continued broad implementation of array- and next-generation sequencing-based technologies will expand the types of CNVs encountered in the clinical setting, as well as our understanding of their impact on human health. To assist clinical laboratories in the classification and reporting of CNVs, irrespective of the technology used to identify them, the American College of Medical Genetics and Genomics has developed the following professional standards in collaboration with the NIH-funded Clinical Genome Resource (ClinGen) project. This update introduces a quantitative, evidence-based scoring framework; encourages the implementation of the 5-tier classification system widely used in sequence variant classification; and recommends “uncoupling” the evidence-based classification of a variant from its potential implications for a particular individual. These professional standards will guide the evaluation of constitutional CNVs and encourage consistency and transparency across clinical laboratories.
- Record: found
- Abstract: found
- Article: not found
Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females
Christel Depienne, Delphine Bouteiller, Boris Keren … (2009)
Introduction Epileptic encephalopathies are a group of rare disorders in which impairment of cognitive, behavioural and other brain functions is caused by the same underlying disease process. This heterogeneous group of disorders has multiple aetiologies such as symptomatic brain lesions, metabolic causes and diverse genetic syndromes. Much progress has been made in the past few years in the identification of genes responsible for genetic infantile epileptic encephalopathies. Among the genetic syndromes that have been characterized are: Dravet syndrome (DS), also called severe myoclonic epilepsy of infancy (SMEI, MIM# 607208) [1], CDKL5/STK9 Rett-like epileptic encephalopathy [2],[3], ARX-related epileptic encephalopathies [4], SRPX2-related rolandic epilepsy associated with oral and speech dyspraxia and mental retardation [5], and very recently, female-limited epilepsy and cognitive impairment (EFMR) associated with mutations in PCDH19, the gene encoding the protocadherin 19 on the X chromosome [6]. Dravet syndrome is characterized by the occurrence of generalized or unilateral clonic or tonic–clonic seizures, usually triggered by fever, in the first year of life of a previously normal infant. Later on, other types of seizures occur, including myoclonus, atypical absences and partial seizures [7]. Development is progressively delayed starting from the second year. Susceptibility to febrile seizures persists over time, and status epilepticus is frequent. Epilepsy generally persists despite appropriate anti-epileptic therapy (polytherapy including sodium valproate, clobazam or topiramate and stiripentol). Children with DS typically have poorly developed language and motor skills, learning disabilities and variable degrees of mental retardation [8]. They are usually sporadic cases; however, sib pairs with SMEI, or patients with a family history of epilepsy, have occasionally been reported [9]. Heterozygous de novo mutations in SCN1A, the gene encoding the voltage-gated neuronal sodium channel alpha 1 subunit (Nav1.1), are a major cause of DS [1]. All types of mutations [10] and rearrangements [11]–[15] in SCN1A have been observed in SMEI patients. However, no point mutations or rearrangements have been found in a fraction of patients, now estimated to 20–25% [15]–[18], strongly suggesting that DS is a genetically heterogeneous disorder. Our aim was to identify the gene(s) involved in SCN1A-negative patients with Dravet syndrome. Most of our patients were isolated, excluding the use of classical genetic approaches. Our hypothesis was that genomic micro-rearrangements, which are increasingly identified as causes of human genetic disorders, might be found in a subset of the SCN1A-negative patients with DS, thus identifying new causal genes. In this study, we have searched for genomic rearrangements in 41 SCN1A-negative patients using high-density SNP microarrays (Illumina, 370K). Genes located in the rearrangements were then considered to be candidate genes and were analysed for point mutations by direct sequencing in the remaining negative patients with DS. Results Microarray-Based Identification of a De Novo Deletion on Chromosome Xq22.1 Encompassing PCDH19 An initial series of 41 probands (18 females and 23 males), referred for genetic analysis of Dravet syndrome but negative for point mutation and intragenic rearrangement of SCN1A [15], was screened for genomic rearrangement using Illumina 370CNV microarrays. A hemizygous deletion on chromosome Xq22.1 was identified in a male patient (patient 1 from family 1). This deletion spanned approximately 1 Mb and encompassed a single gene, PCDH19 (Figure 1A). A duplication of the same region was previously reported in one of 776 healthy controls (506 unrelated healthy individuals from Northern Germany and 270 HapMap subjects) [19], but no deletions in healthy individuals have been recorded in the database of genomic variants. Patient 1 and his mother were then analyzed with high-resolution CGH arrays (Nimblegen). This analysis confirmed that the deletion spans 890 Kb, between genomic positions g.98731380 and g.99618794 on chromosome X, and showed that it has occurred de novo since it was not found in the mother of the patient (Figure 1B). 10.1371/journal.pgen.1000381.g001 Figure 1 Identification of a deletion encompassing PCDH19 in a male patient. A) Identification of a hemizygous Xq22.1 deletion with a 370 K SNP microarray (Illumina): Y-axes represent Log R ratio (above) and B allele frequency (below); the X-axis indicates the position on the X chromosome. The red line (log R ratio profile) corresponds to the median smoothing series (Beadstudio). B) Analysis of the patient and his mother with CGH microarrays (Nimblegen), showing that the deletion occurred de novo. Indicated genomic positions are based upon the Ensembl Genome Browser. Black horizontal bars (below) represent the gene (PCDH19) and pseudogenes comprised in the deleted region. Identification of Additional Patients with Point Mutations in the Coding Sequence of PCDH19 by Direct Sequencing PCDH19 encodes protocadherin 19, a transmembrane protein of the cadherin family of calcium-dependent cell–cell adhesion molecules, which is strongly expressed in the central nervous system. In the postnatal brain, protocadherins might be involved in the modulation of synaptic transmission and the generation of specific synaptic connections [20]. PCDH19 was therefore an attractive candidate gene for epilepsies and mental retardation. To test whether a PCDH19 deficiency might be implicated in some epileptic encephalopathies resembling Dravet syndrome, we sequenced the coding region of this gene in 73 SCN1A-negative probands (the remaining 40 patients of the initial series plus 33 additional patients, for a total of 45 females and 28 males). Ten different variants were identified in 11 unrelated female probands at the heterozygous state (Figure 2A). All but one were located in exon 1: three were nonsense mutations (c.142G>T/p.Glu48X, c.352G>T/p.Glu118X, c.859G>T/p.Glu287X), two were small deletions and insertions creating a frameshift (c.506delC/p.Thr169SerfsX43 and c.1036_1040dup/p.Asn347LysfsX23) and the remaining five were missense mutations (c.361G>A/p.Asp121Asn, c.595 G>C/p.Glu199Gln, c.1019A>G/p.Asn340Ser, c.1628 T>C/p.Leu543Pro and c.3319 C>G/p.Arg1107Gly). Glu48X was present in two affected sisters of family 2; Glu118X was identified in an isolated patient (family 3) and Glu287X was found independently in a patient with family history of epilepsy and mental retardation (family 4) and in an isolated patient (family 5). Interestingly, the c.3319 C>G/p.Arg1107Gly missense variant, located in exon 6, was associated with the p.Glu287X mutation in the proband of family 5. In family 6, cytosine 506 (c.506delC) was deleted in a patient whose parents were unaffected, but whose female cousin also had epilepsy and moderate mental retardation. The 5-bp duplication (c.1036_1040dup) was present in the index case of family 7. The p.Asp121Asn mutation was identified in the index case of family 8, who had a sister with epilepsy and psychotic disturbances. Finally, p.Glu199Gln, p.Asn340Ser and p.Leu543Pro variants were identified in the 4 remaining isolated patients (families 9 to 12); Asn340Ser was found in two independent patients (families 10 and 11). These 4 missense variants (p.Asp121Asn, p.Glu199Gln, p.Asn340Ser and p.Leu543Pro) all affected amino-acids in the extracellular domain of protocadherin 19, which are highly conserved in orthologs and in paralogs of PCDH19 in the delta protocadherin family (Figure 2B). Interestingly, p.Arg1107Gly, associated with the de novo Glu287X mutation in the proband of family 5, affected a residue of the protein that is conserved in mammalian orthologs, but not in other species or in paralogs (Figure 2B). To confirm that the variants are pathogenic, we screened 180 healthy Caucasians. Only Arg1107Gly was found in a healthy female individual and was thus considered to be a rare polymorphism. None of the other variants was found in the control population, confirming that they are causal mutations. 10.1371/journal.pgen.1000381.g002 Figure 2 Detection of 9 different point mutations of PCDH19 in 11 female patients by direct sequencing. A) Sequence electropherograms of the mutations and the missense variant (c.3319C>G/p.Arg1107Gly) identified in association with the c.859G>T/p.Glu287X nonsense mutation. The mutation nomenclature is based on the PCDH19 transcript reference EF676096. Nucleotides are numbered according to the cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to the journal guidelines (www.hgvs.org/mutnomen). B) Alignment of the regions surrounding the mutations (indicated by an arrow) in orthologous and paralogous proteins, showing the high conservation of each affected amino-acid in vertebrates and in the delta protocadherin paralogous genes. The parents and relatives of PCDH19-positive patients were also analysed when possible (Figure 3). The p.Glu48X mutation, found in two affected sibs in family 2, was inherited from their asymptomatic father. Likewise, the c.1036_1040dup5, p.Asp121Asn and p.Leu543Pro mutations were inherited from the healthy fathers of the index cases in families 6, 8 and 12. In family 6, the 5-bp duplication was inherited from the paternal grandmother who also had epilepsy and cognitive impairment, and transmitted to the half-brother of the father and his affected daughter (i.e. the index case's cousin, Figure 3). In family 4, the mother of the proband had mental retardation associated with adult-onset epilepsy, a clinical feature also present in the maternal grandmother and maternal aunt; the proband's father also presented with moderate mental retardation but without epilepsy. The Glu287X mutation in this family was also inherited from the father. In contrast, in families 5, 7, 10 and 11, the mutations (p.Glu287X, c.506delC and p.Asn340Ser, respectively) occurred de novo in the index cases, since they were not found in either parent. Interestingly, Arg1107Gly was inherited from the asymptomatic father in family 5. In family 4, only the mother and sisters of the index case were available for genetic analyses. Both sisters, who were monozygous twins, had mild psychomotor and cognitive impairment but never had seizures. Neither the mother nor the sisters had the p.Glu287X mutation. Analysis of the haplotypes in Xq22.1 (PCDH19 locus) with microsatellite markers confirmed that the three sisters (the two twins and the affected proband) received the same X chromosome from their father, with and without the p.Glu287X mutation, which indicates that the mutation also occurred de novo in this family. Finally, in family 9, the mother did not have the p.Glu199Gln mutation but the father remained unavailable for genetic analyses. 10.1371/journal.pgen.1000381.g003 Figure 3 Pedigrees of the families and segregation analysis of the PCDH19 deletion and point mutations. del/+, m/+ or v/+ denote individuals heterozygous for the deletion, mutation or variant, respectively; +/+ denotes individuals carrying homozygous wild-type alleles. Squares represent males, circles females; filled black symbols: patients diagnosed as having Dravet syndrome; right black half: Cognitive delay or impairment; left grey half: adolescence-onset idiopathic epilepsy. Dots in the middle of the squares indicate unaffected mutation carriers. The arrows indicate the index cases. FISH Shows Somatic Mosaïcism in the Male Patient with the PCDH19 Deletion Recently, PCDH19 mutations were shown to cause epilepsy and mental retardation limited to females (EFMR), a familial disorder associating childhood-onset epilepsy and a variable degree of cognitive impairment with an unusual mode of inheritance: this X-linked disorder is found in females with heterozygous mutations but not in males with hemizygous mutations [6]. How, then, can we explain the affected male in our series with a deletion of the entire PCDH19? Random X-inactivation in mutated females normally leads to tissue mosaicism in which two cell populations, one expressing normal PCDH19 and the other expressing the mutated allele, co-exist. To explain why only females are affected, it might be hypothesized that the co-existence of PCDH19-positive and PCDH19-negative cells would be pathogenic whereas homogeneous cell populations (PCDH19-positive in normal individuals but PCDH19-negative in mutated males) would not [6]. A mechanism of this type was previously termed “cellular interference” [21]. Two cell populations would also be found in mosaic males, who, according to this hypothesis, would be affected like mutated females. To test whether our male patient was mosaic for the PCDH19 gene deletion, we compared peripheral blood lymphocytes (PBL) and cultured fibroblasts from the patient by FISH with a probe specific to the PCDH19 genomic region. Although no signal corresponding to PCDH19 was detectable in PBL, a normal PCDH19 allele was found in 53% of the fibroblasts (Figure 4), confirming that the patient was mosaic, in his skin, for the PCDH19 deletion. This result confirms that mutations in PCDH19 can be responsible, in mosaic males, for epileptic encephalopathy phenotypes that are usually limited to females, and strongly supports the hypothesis that cellular interference is the main pathogenic mechanism of the disease. 10.1371/journal.pgen.1000381.g004 Figure 4 FISH analysis of the PCDH19 deletion in the male patient showing somatic mosaicism in fibroblasts. (A) Absence of the specific Xq22.1 probe site on metaphase chromosomes in peripheral blood lymphocytes (PBL); (B) In fibroblasts, presence of one hybridization spot in 53% of the cells and absence of signal in the remaining 47%; C) and D) FISH analysis on PBL (C) and fibroblasts (D) of a female control. PCDH19-specific signals (red) are indicated by arrowheads. Magnification ×1000. Clinical Features of the Patients with PCDH19 Mutations or Deletions The clinical features of the male patient with the PCDH19 deletion and the female patients with PCDH19 point mutations are summarized in Table 1. These patients fulfil the main criteria for DS (see material and methods' section), with a mean age of seizures onset of 9.5 months (ranging from 7.5 to 12 months). Nevertheless, contrary to SCN1A-positive patients (SCN1A-DS), myoclonic jerks, atypical absences, and photosensitivity were unfrequent in PCDH19-positive patients (PCDH19-DS) (3, 3 and 1 patients out of 13, respectively). Only 6 patients presented status epilepticus. The mental delay was mild in 6 patients, moderate in 4 and only 3 patients presented with severe delay. Although much delayed, the language was present in all patients, with 12 out of 13 able to formulate short sentences. 10.1371/journal.pgen.1000381.t001 Table 1 Clinical characteristics of patients with PCDH19 mutations. Family number 1 2 2* 3 4 5 6 7 8 9 10 11 12 Patient N 07 0168 N 06 1257 N 06 1258 N 06 1358 N 07 0627 N 07 0971 18050_31839 N 06 0730 N 07 0661 N 07 1000 N 06 1016 N 06 0991 N 05 1157 Sex M F F F F F F F F F F F F Present age (years) 7 6 3 7 3 13 10 12 2,5 18 3,5 6 8 PMD previous to seizures onset Nl Nl Nl Nl ? Nl Nl Nl ? Nl Nl Nl Nl Age at seizures onset (months) 12 9 11 11 10 7.5 11 9 9 ? 9 8 8 Type of seizures at onset F, GTC, prolonged, repetitive Focal Unilat F, GTC, prolonged, repetitive GTC F, unilat F, unilat GTC Unilat F,unilat F, GTC Partial F,unilat Presence of febrile seizures (FS) +(>50%) + + +(>50%) +(>50%) +(>50%) +(50%) +(>50%) +(>50%) +(>50%) +(>50%) +(>50%) +(>50%) Unilateral (or hemiclonic) seizures + + − − + + + + + + + + + Other seizure types - Partial + + + − + + + + + + + + + - GTC + + + + + + + + + + + + + - Myoclonic jerks + − − − − − + − − − − − − - Absences ? + + ? − − + − − − − − − Repetitive seizures (in clusters) + + + + + + + + + + − + + Status epilepticus + + − − − + − − + + + − − Photosensitivity − − − − − − − − − − + − Persistance of seizures in spite of treatment +(F) + − + + + + − + + − + + Mental retardation moderate/severe mild mild moderate moderate/severe severe mild moderate mild moderate mild mild moderate Language delay +(W-S) +(W-S) − +(W-S) +(abs) +(W-S) +(W-S) +(W-S) +(W) +(W-S) +(W-S) +(W-S) +(W-S) Behavioral disturbances + + − + + + + ? ? − − + + Autistic features + − − + − − − − − − − − − Motor delay + +(mild) +(mild) + +(Hypotonia) + +(mild) − + − − + + Ataxia + + +(mild) + + + − ? − − − + + Other clinical features − − − Tall stature Hyperlaxity − − − − − − − − Treatment (AED) - Sodium Valproate + + + + + + + + + + + + - Clobazam + + + + + + + + + - Clonazepam + + + + + + - Topiramate + + + + + + + - Stiripentol + + + + - Lamotrigine + + + 2*: patient N 06 1258 is the sister of patient N 06 1257 (index case); PMD = psychomotor development, Nl = normal, F = febrile, unit = unilateral, GTC = generalized tonic-clonic, W-S = words-sentences, abs = absent, AED = anti epileptic drugs. Discussion In this study, we used SNP microarrays to search for microrearrangements in patients with clinical features suggestive of Dravet syndrome but without mutations in SCN1A in order to identify new causative genes. The identification of a de novo hemizygous deletion of PCDH19, encoding protocadherin 19, in a male patient led us to screen the coding region of this gene in the remaining patients. Eleven unrelated probands with point mutations in PCDH19, all females, were found. While this study was ongoing, PCDH19 was reported to be the causative gene for female-limited epilepsy and cognitive impairment (EFMR), a disorder characterized by seizure onset in infancy or early childhood and cognitive impairment, which is found only in females in multi-generational families [6]. Since all of our patients with point mutations in PCDH19 were females as previously reported, we investigated the possibility that the male patient in whom the gene was deleted might be mosaic for the deletion. FISH analysis confirmed this latter hypothesis. The thirteen patients with PCDH19 mutation or deletion (12 probands and one sib, family 2) all fulfilled the main criteria for DS and were all negative for mutation or rearrangement in SCN1A after direct sequencing and multiplex ligation-dependent probe amplification (MLPA) [15]. The proportion of PCDH19-DS probands in our series of SCN1A-negative patients was 16% (12/74), or even 25% (11/45) if only female patients were included in the calculation. Considering that approximately 25% of all patients with DS are SCN1A-negative [15], PCDH19 might overall account for 5% of DS patients. PCDH19-DS patients and SCN1A-DS patients have many features in common including: normal psychomotor development before seizures onset, early onset of seizures (before age one year), association of febrile and afebrile seizures, with a high susceptibility of the seizures to fever for all 13 patients, occurrence of hemiclonic or unilateral seizures (11/13), and association of generalized tonic-clonic and focal seizures (12/13), a high proportion of seizures occurring in clusters (12/13), prolonged seizures, a proportion of which lead to status epilepticus, secondary progressive appearance of mental and motor regression and language delay, accompanied, in some cases, with ataxia (Table 1). However, PCDH19-DS patients slightly differ on average from the classical pattern reported in SCN1A-DS. PCDH19-DS patients were slightly older at onset than SCN1A-DS patients (9.5 months, with a range from 7.5 to 12 months, versus 6.3 months, calculated from our series of SCN1A-positive DS patients, p 15 min, that might evolve to status epilepticus), later occurrence of other types of seizures (febrile and afebrile) and cognitive regression. The presence of myoclonic jerks and/or ataxia was considered to be a highly characteristic, although inconstant, feature of the disease that could reinforce a diagnosis; however, their absence did not exclude the clinical diagnosis of Dravet syndrome, since they were not previously observed in all patients with DS [7],[31]. Informed written consent was obtained from the patients' parents before blood sampling. This study was approved by the ethical committee (CCPPRB of Pitié-Salpêtrière Hospital, Paris, n°69-03, 25/9/2003). Screening for Genomic Rearrangements with High-Density SNP Arrays Patients were screened using Illumina 370CNV-Duo genotyping BeadChip arrays (370 K). The Infinium II Genotyping reaction steps were performed according to the manufacturer's specifications (Illumina, San Diego, CA) on the P3S platform (Pitié-Salpêtrière Hospital). Briefly, 750 ng of genomic DNA were isothermally amplified at 37°C overnight. The amplified products were fragmented by a controlled enzymatic process then precipitated with isopropanol. The dried precipitated pellet was resuspended, hybridized to 370CNV-Duo beadchips in a capillary flow-through chamber and incubated overnight at 48°C. The amplified, fragmented DNA samples anneal to locus-specific 50-mers during the hybridization step. Each bead type corresponds to one allele per SNP locus. After hybridization, allelic specificity was conferred by enzymatic single-base extension and fluorescent staining. Arrays were washed and dried for 1 h before imaging using a BeadArray Reader (Illumina). Image data analysis and automated genotype calling was performed using Beadstudio 3.1 (Illumina). All genomic positions were based on the UCSC and Ensembl Genome Browsers. Each copy number variant (CNV) identified in patients was searched in the database of genomic variants (http://projects.tcag.ca/variation/), which repertories the structural variation in the Human genome, to determine whether this CNV is normally present in a control population. Analysis of the Xq22.1 Deletion with Nimblegen CGH Arrays Genomic DNA from the patients was analysed by microarray-based comparative genomic hybridization with the HG18 WG Tiling 385 K CGH array v2.0 (Roche NimbleGen, Madison, WI), according to the NimbleGen hybridization Kit Protocol. Briefly, DNA samples from patients and controls were labelled by random priming: the DNA (1 µg) was denatured in the presence of 5′Cy3- or Cy5-labeled random nanomers (Trilink Biotehcnologies, San Diego, CA) and incubated with 100 units of exo-klenow fragment (NEB, Beverly, MA) and dNTP mix [6 mM each in TE buffer (10 mMTris/1 mM EDTA, pH 7.4, Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 mM EDTA (pH 8.0), precipitated with isopropanol and resuspended in water. The Cy-labelled test sample (Cy3) and the reference sample (Cy5) were combined in 13 µL of Nimblegen Hybridization solution (Roche Nimblegen). After denaturation, hybridization was carried out on a MAUI Hybridization System (BioMicro Systems, Salt Lake City, NE) for 18 h at 42°C. The array was washed with the NimbleGen Wash System (Roche NimbleGen), dried by centrifugation and scanned with the genePix 4000B scanner (Axon Instrument, Union City, CA). Fluorescence intensity (raw data) was obtained from the scanned images of the oligonucleotide tiling arrays with NIMBLESCAN 2.0 extraction software (Nimblegen Systems). For each spot on the array, log2 ratios of the Cy3-labeled test sample versus Cy5 reference sample were calculated. Regions were considered to be duplicated or deleted when result exceeded the +/−0.25. Sequencing of the PCDH19 Coding Sequence Eleven specific primer pairs were designed to amplify the 6 exons and adjacent intron-exon boundaries (∼100 bp from each side of the exons) of the PCDH19 gene (transcript reference EF676096). Primer sequences are available on request. Forward and reverse sequence reactions were performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) using the same primers. G50-purified sequence products were run on an ABI 3730 automated sequencer (PE Applied Biosystems) and data were analyzed with the Seqscape 2.5 software (Applied Biosystems). Mutations identified in the patients were looked for directly in the DNA of available parents by sequencing the corresponding amplicon. If neither parent had the mutation, the parents were tested with microsatellite markers at the Xq22.1 locus to ensure that the mutation occurred de novo. In addition, 180 European controls (90 males and 90 females) were included to test new variants in the PCDH19 gene. Fluorescence In Situ Hybridization (FISH) FISH experiments were performed on peripheral blood lymphocytes (blood samples) and fibroblasts (skin biopsies). Fibroblasts were grown in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose and 110 µg/ml pyruvate (DMEM) supplemented with 10% fetal calf serum (FCS), 0.03% glutamine, 1000 U/ml penicillin/streptomycin in a 5% CO2 atmosphere for 2 weeks before FISH. Lymphocytes were grown in PB-Max medium (Invitrogen) for 3 days. Metaphase chromosome spreads were obtained by standard hypotonic treatment and methanol/acetate (3/1) fixation. The slides were washed with the cytology FISH accessory kit (Dako). A FISH DNA probe, specific for the Xq22.1 region covering PCDH19, was labeled with rhodamine by nick-translation after amplification of the RP11-99E24 BAC (Invitrogen) and cohybridized with a commercial subtelomeric control probe (Cytocell), specific for the pseudo-autosomal region 1 (chromosomes X/Y) labeled with fluorescein isothiocyanate (FITC). The slides were then washed and counterstained with 4,6-diamino-2-phenylindole (DAPI) for chromosome identification. Metaphase cells were examined under a motorized reflected BX61 Olympus fluorescence microscope with filters for separate detection of DAPI, FITC and rhodamine. One hundred metaphase cells were counted to determine the degree of mosaicism in fibroblasts and lymphocytes. Metaphase chromosomes from a karyotypically normal female were used as a control. Statistical Tests Frequencies were compared with the Chi-Square test or the Fisher exact test when appropriate. Means were compared using Mann-Whitney Rank Sum Test. Statistical analysis was performed using SigmaStat 3.5 software.
- Record: found
- Abstract: found
- Article: not found
XLID-causing mutations and associated genes challenged in light of data from large-scale human exome sequencing.
Claire Redin, Jean-Louis Mandel, Amélie Piton (2013)
Because of the unbalanced sex ratio (1.3-1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Author and article information
Contributors
Qin Yang:
ORCID: https://orcid.org/0009-0009-2531-0869
532005936@qq.com
Journal
Journal ID (nlm-ta): Mol Genet Genomic Med
Journal ID (iso-abbrev): Mol Genet Genomic Med
Journal ID (doi): 10.1002/(ISSN)2324-9269
Journal ID (publisher-id): MGG3
Title:
Molecular Genetics & Genomic Medicine
Publisher:
John Wiley and Sons Inc.
(Hoboken
)
ISSN
(Electronic):
2324-9269
Publication date
(Electronic):
11
December
2023
Publication date Collection:
January
2024
Volume: 12
Issue: 1
(
doiID:
10.1002/mgg3.v12.1
)
Electronic Location Identifier: e2338
Affiliations
[
1
]
Suizhou Prenatal Diagnosis and Prenatal Screening Quality Control Center, Department
of Perinatal Health
Suizhou Maternal and Child Health Hospital
Suizhou
Hubei
China
Author notes
[*]
[*
]
Correspondence
Qin Yang, Suizhou Prenatal Diagnosis and Prenatal Screening Quality Control Center, Department of Perinatal Health, Suizhou Maternal and Child Health Hospital, Suizhou, Hubei, China.
Email: 532005936@ 123456qq.com
Qin Yang, Suizhou Prenatal Diagnosis and Prenatal Screening Quality Control Center, Department of Perinatal Health, Suizhou Maternal and Child Health Hospital, Suizhou, Hubei, China.
Email: 532005936@ 123456qq.com
Author information
Article
Publisher ID: MGG32338 Other ID: MGG3-2023-08-1170.R1DOI: 10.1002/mgg3.2338
PMC ID: 10767682
PubMed ID: 38083988
SO-VID: 50cb33f8-298e-4441-b20e-aa5c8fda99b3
Copyright © © 2023 The Authors.
Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.
License:
This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
History
Date
revision received
: 10
November
2023
Date
received
: 14
August
2023
Date
accepted
: 28
November
2023
Page count
Figures: 3,
Tables: 1,
Pages: 8,
Words: 5187
Categories
Subject:
Clinical Report
Subject:
Clinical Reports
Custom metadata
source-schema-version-number 2.0
cover-date January 2024
details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:6.3.6 mode:remove_FC converted:05.01.2024
Data availability: