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• W2949949273 abstract "Many patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes [CAH-X chimera 1 (CH-1) and chimera 2 (CH-2)] on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40; this method is amenable to either real-time quantitative PCR or droplet digital PCR (ddPCR). The assay was validated in a cohort of 278 subjects from 146 unrelated CAH families. Results were confirmed by a validated Sanger sequencing platform. A total of 44 CAH-X–positive calls were made, with 42 (26 CH-1 and 16 CH-2) confirmed. The assay had 100% sensitivity (42 true/42 positives), 99.2% specificity (234 true/236 negatives), and an overall 99.3% accuracy (276/278). Calls made by real-time quantitative PCR and ddPCR were consistent (100%), and ddPCR offered easier data interpretation. The CAH-X prevalence was 15.6% (21/135 probands), higher than the previously estimated 8.5%, and was particularly high (29.2% or 21/72) in those with a 30-Kb deletion. This assay is suitable for high-throughput CAH-X screening, especially in subjects testing positive for CAH in neonatal screening. Many patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes [CAH-X chimera 1 (CH-1) and chimera 2 (CH-2)] on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40; this method is amenable to either real-time quantitative PCR or droplet digital PCR (ddPCR). The assay was validated in a cohort of 278 subjects from 146 unrelated CAH families. Results were confirmed by a validated Sanger sequencing platform. A total of 44 CAH-X–positive calls were made, with 42 (26 CH-1 and 16 CH-2) confirmed. The assay had 100% sensitivity (42 true/42 positives), 99.2% specificity (234 true/236 negatives), and an overall 99.3% accuracy (276/278). Calls made by real-time quantitative PCR and ddPCR were consistent (100%), and ddPCR offered easier data interpretation. The CAH-X prevalence was 15.6% (21/135 probands), higher than the previously estimated 8.5%, and was particularly high (29.2% or 21/72) in those with a 30-Kb deletion. This assay is suitable for high-throughput CAH-X screening, especially in subjects testing positive for CAH in neonatal screening. Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders in steroidogenesis, with 95% of cases due to 21-hydroxylase deficiency (Online Mendelian Inheritance in Man number 201910). CAH is classified into three major subtypes according to clinical severity: two classic subtypes of salt-wasting and simple virilizing combined are estimated to occur in 1 in 15,000 live births,1Speiser P.W. Arlt W. Auchus R.J. Baskin L.S. Conway G.S. Merke D.P. Meyer-Bahlburg H.F. Miller W.L. Murad M.H. Oberfield S.E. White P.C. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society Clinical practice guideline.J Clin Endocrinol Metab. 2018; 103: 4043-4088Crossref PubMed Scopus (466) Google Scholar and a mild or late-onset, nonclassic subtype that is more common and affects 1 in 200 to 1 in 1000 Caucasians.2Hannah-Shmouni F. Morissette R. Sinaii N. Elman M. Prezant T.R. Chen W. Pulver A. Merke D.P. Revisiting the prevalence of nonclassic congenital adrenal hyperplasia in US Ashkenazi Jews and Caucasians.Genet Med. 2017; 19: 1276-1279Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Because severe salt-wasting CAH can be life-threatening if left without prompt treatment, CAH screening by a hormonal assay is part of the mandatory neonatal screening in the United States and approximately 40 other countries.3White P.C. Neonatal screening for congenital adrenal hyperplasia.Nat Rev Endocrinol. 2009; 5: 490-498Crossref PubMed Scopus (159) Google Scholar The CYP21A2 gene (Online Mendelian Inheritance in Man number 613815) encoding 21-hydroxylase is mapped at a locus of low copy repeats termed the RCCX module(s) (RP-C4-CYP21-TNX) in the human histocompatibility complex on chromosome 6 (p21.33). RP signifies RP1 (synonym for STK19) encoding a serine/threonine nuclear protein kinase and pseudogene RP2 (STK19P); C4 signifies C4A and C4B encoding two isotopes of complement component 4; CYP21 signifies CYP21A2 and a pseudogene CYP21A1P; and TNX signifies TNXB encoding tenascin-X and a pseudogene TNXA (Figure 1). These gene pairs are highly homologous; thus, the entire locus is vulnerable to unequal crossovers, leading to commonly existing RCCX copy number variation that is found in 14% to 17% of human alleles.4Blanchong C.A. Zhou B. Rupert K.L. Chung E.K. Jones K.N. Sotos J.F. Zipf W.B. Rennebohm R.M. Yung Yu C. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in Caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease.J Exp Med. 2000; 191: 2183-2196Crossref PubMed Scopus (154) Google Scholar, 5Yang Y. Chung E.K. Wu Y.L. Savelli S.L. Nagaraja H.N. Zhou B. Hebert M. Jones K.N. Shu Y. Kitzmiller K. Blanchong C.A. McBride K.L. Higgins G.C. Rennebohm R.M. Rice R.R. Hackshaw K.V. Roubey R.A. Grossman J.M. Tsao B.P. Birmingham D.J. Rovin B.H. Hebert L.A. Yu C.Y. Gene copy-number variation and associated polymorphisms of complement component C4 in human systemic lupus erythematosus (SLE): low copy number is a risk factor for and high copy number is a protective factor against SLE susceptibility in European Americans.Am J Hum Genet. 2007; 80: 1037-1054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 6Banlaki Z. Doleschall M. Rajczy K. Fust G. Szilagyi A. Fine-tuned characterization of RCCX copy number variants and their relationship with extended MHC haplotypes.Genes Immun. 2012; 13: 530-535Crossref PubMed Scopus (17) Google Scholar Among them, chimeric genes of CYP21A1P/CYP21A2 and CYP21A1P-TNXA/TNXB, due to the recombination between respective CYP21 or TNX genes, are two major CAH-causing genotypes accounting for 30% of CAH-causing alleles.7Finkielstain G.P. Chen W. Mehta S.P. Fujimura F.K. Hanna R.M. Van Ryzin C. McDonnell N.B. Merke D.P. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.J Clin Endocrinol Metab. 2011; 96: E161-E172Crossref PubMed Scopus (132) Google Scholar They are also termed 30-Kb deletions. CYP21A1P-TNXA/TNXB chimeras impair both the CYP21A2 and TNXB genes and are pathogenic for hypermobility type of Ehlers-Danlos syndrome (EDS; Online Mendelian Inheritance in Man number 130020).8Burch G.H. Gong Y. Liu W. Dettman R.W. Curry C.J. Smith L. Miller W.L. Bristow J. Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.Nat Genet. 1997; 17: 104-108Crossref PubMed Scopus (275) Google Scholar, 9Schalkwijk J. Zweers M.C. Steijlen P.M. Dean W.B. Taylor G. van Vlijmen I.M. van Haren B. Miller W.L. Bristow J. A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.N Engl J Med. 2001; 345: 1167-1175Crossref PubMed Scopus (305) Google Scholar The presence of both CAH and hypermobility-type EDS due to the contiguous deletion of CYP21A2 and TNXB is termed CAH-X syndrome. The CAH-X chimeras cause EDS in an autosomal dominant manner regardless of CAH status,10Zweers M.C. Bristow J. Steijlen P.M. Dean W.B. Hamel B.C. Otero M. Kucharekova M. Boezeman J.B. Schalkwijk J. Haploinsufficiency of TNXB is associated with hypermobility type of Ehlers-Danlos syndrome.Am J Hum Genet. 2003; 73: 214-217Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 11Chen W. Kim M.S. Shanbhag S. Arai A. VanRyzin C. McDonnell N.B. Merke D.P. The phenotypic spectrum of contiguous deletion of CYP21A2 and tenascin XB: quadricuspid aortic valve and other midline defects.Am J Med Genet A. 2009; 149a: 2803-2808Crossref PubMed Scopus (18) Google Scholar, 12Merke D.P. Chen W. Morissette R. Xu Z. Van Ryzin C. Sachdev V. Hannoush H. Shanbhag S.M. Acevedo A.T. Nishitani M. Arai A.E. McDonnell N.B. Tenascin-X haploinsufficiency associated with Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2013; 98: E379-E387Crossref PubMed Scopus (49) Google Scholar, 13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar although patients with CAH usually have more severe EDS manifestations than do carriers without CAH. We previously found that 8.5% of patients with CAH due to 21-hydroxylase deficiency are affected by CAH-X.13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar EDS is a group of genetic disorders of the connective tissue that affect approximately 1 in 5000 individuals.14Beighton P. De Paepe A. Steinmann B. Tsipouras P. Wenstrup R.J. Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK).Am J Med Genet. 1998; 77: 31-37Crossref PubMed Scopus (1370) Google Scholar Five of the six major EDS types are associated with genes encoding collagens or collagen-modifying enzymes. Hypermobility-type EDS, the most common type of EDS, has been associated with TNXB defects, but the etiology is mostly unknown.9Schalkwijk J. Zweers M.C. Steijlen P.M. Dean W.B. Taylor G. van Vlijmen I.M. van Haren B. Miller W.L. Bristow J. A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.N Engl J Med. 2001; 345: 1167-1175Crossref PubMed Scopus (305) Google Scholar The TNXB gene encodes tenascin-X, a large extracellular matrix–forming glycoprotein that is a crucial component of connective tissue and is found in the dermis, skeletal muscle, heart, and blood vessels.15Bristow J. Tee M.K. Gitelman S.E. Mellon S.H. Miller W.L. Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B.J Cell Biol. 1993; 122: 265-278Crossref PubMed Scopus (256) Google Scholar Although mutations throughout the TNXB gene have been reported to cause hypermobility-type EDS, CAH-X chimeras are most prevalent in CAH populations.9Schalkwijk J. Zweers M.C. Steijlen P.M. Dean W.B. Taylor G. van Vlijmen I.M. van Haren B. Miller W.L. Bristow J. A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.N Engl J Med. 2001; 345: 1167-1175Crossref PubMed Scopus (305) Google Scholar, 10Zweers M.C. Bristow J. Steijlen P.M. Dean W.B. Hamel B.C. Otero M. Kucharekova M. Boezeman J.B. Schalkwijk J. Haploinsufficiency of TNXB is associated with hypermobility type of Ehlers-Danlos syndrome.Am J Hum Genet. 2003; 73: 214-217Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12Merke D.P. Chen W. Morissette R. Xu Z. Van Ryzin C. Sachdev V. Hannoush H. Shanbhag S.M. Acevedo A.T. Nishitani M. Arai A.E. McDonnell N.B. Tenascin-X haploinsufficiency associated with Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2013; 98: E379-E387Crossref PubMed Scopus (49) Google Scholar, 13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar, 16Zweers M.C. Dean W.B. van Kuppevelt T.H. Bristow J. Schalkwijk J. Elastic fiber abnormalities in hypermobility type Ehlers-Danlos syndrome patients with tenascin-X mutations.Clin Genet. 2005; 67: 330-334Crossref PubMed Scopus (43) Google Scholar, 17Demirdas S. Dulfer E. Robert L. Kempers M. van Beek D. Micha D. van Engelen B.G. Hamel B. Schalkwijk J. Loeys B. Maugeri A. Voermans N.C. Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.Clin Genet. 2017; 91: 411-425Crossref PubMed Scopus (37) Google Scholar, 18Penisson-Besnier I. Allamand V. Beurrier P. Martin L. Schalkwijk J. van Vlijmen-Willems I. Gartioux C. Malfait F. Syx D. Macchi L. Marcorelles P. Arbeille B. Croue A. De Paepe A. Dubas F. Compound heterozygous mutations of the TNXB gene cause primary myopathy.Neuromuscul Disord. 2013; 23: 664-669Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Clinical EDS manifestation caused by CAH-X chimeras include joint hypermobility, frequent joint dislocations, skin laxity, tissue fragility, chronic myalgia, and cardiac malformations. A genetic test for the presence of CAH-X chimeras is mostly not available in clinical practice due to technical obstacles including pseudogene interference and the 70-Kb size of the TNXB gene. Currently, the diagnosis of CAH-X relies on clinical evaluation of joint hypermobility and subluxations; this type of evaluation is not reliably performed during infancy. Thus, infants who test positive for CAH based on neonatal screening are not screened for CAH-X. To date, two major types of pathogenic CYP21A1P-TNXA/TNXB chimera have been identified: CAH-X chimera 1 (CH-1) has TNXB exons 35-44 replaced with TNXA, and CAH-X chimera 2 (CH-2) has TNXB exons 40-44 replaced with TNXA.8Burch G.H. Gong Y. Liu W. Dettman R.W. Curry C.J. Smith L. Miller W.L. Bristow J. Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.Nat Genet. 1997; 17: 104-108Crossref PubMed Scopus (275) Google Scholar, 12Merke D.P. Chen W. Morissette R. Xu Z. Van Ryzin C. Sachdev V. Hannoush H. Shanbhag S.M. Acevedo A.T. Nishitani M. Arai A.E. McDonnell N.B. Tenascin-X haploinsufficiency associated with Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2013; 98: E379-E387Crossref PubMed Scopus (49) Google Scholar, 13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar The substitution of TNXB exon 35 by TNXA features a nonsense 120-bp deletion (c.11435_11524+30del) that is causative of tenascin-X haploinsufficiency in CAH-X CH-1, whereas the substitution of TNXB exon 40 by TNXA features two contiguous mutations of c.12150 C>G (synonymous) and c.12174 C>G (p.C4058W) in CAH-X CH-2, with the latter causing more severe EDS manifestations, likely due to a dominant negative effect.13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar In addition, a third chimera, termed CAH-X CH-3 and having TNXB exons 41-44 substituted by TNXA, has been reported in one patient, and its significance is still under investigation.19Chen W. Perritt A.F. Morissette R. Dreiling J.L. Bohn M.F. Mallappa A. Xu Z. Quezado M. Merke D.P. Ehlers-Danlos syndrome caused by Biallelic TNXB variants in patients with congenital adrenal hyperplasia.Hum Mutat. 2016; 37: 893-897Crossref PubMed Scopus (27) Google Scholar In this study, we focused on CAH-X CH-1 and CH-2 only. Here we present an allele-specific PCR–based assay developed to efficiently screen for CAH-X. The assay determines the copy numbers of TNXB exons 35 and 40; CAH-X CH-1 is expected to have copy number losses in both exons 35 and 40, whereas CAH-X CH-2 is expected to have loss in only exon 40. A total of 278 subjects (145 patients, 118 carriers, and 1 unaffected relative from 135 unrelated families of CAH due to 21-hydroxylase deficiency; and 11 patients and 3 carriers from 11 unrelated families of other CAH types) were evaluated. All subjects were enrolled in an ongoing Natural History Study at the NIH Clinical Center (Bethesda, MD; ClinicalTrials.gov identifier: NCT00250159). All subjects (and parents of subjects aged <18 years) gave written informed consent, and the study protocol was approved by the institutional review board at the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Subjects were selected based on the availability of genomic DNA samples at the time of study. All subjects had previously completed comprehensive genetic analysis related to 21-hydroxylase deficiency at the time of study, including a targeted PCR-based CYP21A2 mutation analysis of 12 common CYP21A2 mutations and screening for the presence of a 30-Kb deletion based on 12 single-nucleotide polymorphic markers (Esoterix Laboratory Services, Calabasas Hills, CA). Samples testing positive for a 30-Kb deletion underwent a validated Sanger sequencing test to identify CYP21 chimeras (Prevention Genetics LLC, Marshfield, WI).7Finkielstain G.P. Chen W. Mehta S.P. Fujimura F.K. Hanna R.M. Van Ryzin C. McDonnell N.B. Merke D.P. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.J Clin Endocrinol Metab. 2011; 96: E161-E172Crossref PubMed Scopus (132) Google Scholar, 20Chen W. Xu Z. Sullivan A. Finkielstain G.P. Van Ryzin C. Merke D.P. McDonnell N.B. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency.Clin Chem. 2012; 58: 421-430Crossref PubMed Scopus (44) Google Scholar Patients with rarer forms of CAH underwent testing using commercially available platforms for other CAH types. Subjects with clinical manifestations of EDS were also subjected to a validated Sanger-based test of TNX chimeras (Prevention Genetics).12Merke D.P. Chen W. Morissette R. Xu Z. Van Ryzin C. Sachdev V. Hannoush H. Shanbhag S.M. Acevedo A.T. Nishitani M. Arai A.E. McDonnell N.B. Tenascin-X haploinsufficiency associated with Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2013; 98: E379-E387Crossref PubMed Scopus (49) Google Scholar, 13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar All genomic DNA samples were extracted from frozen peripheral blood by ReproCELL Inc (Beltsville, MD) and stored at −80°C with an estimated concentration of 100 ng/μL. An aliquot of 10 μL of each DNA sample was diluted with 40 μL of water to make 20 ng/μL of working solution and stored at 4°C. Five samples were randomly selected for repeated assay by real-time quantitative PCR (qPCR) on a monthly basis, and the consistent results suggested that the DNA samples were stable without detectable degradation during the study period of 3 months. Each test included two separate qPCR reactions testing TNXB exons 35 and 40, respectively, with hemoglobin subunit β (HBB) as a reference gene. The primers and hydrolysis probes were as described in Table 1. Each qPCR reaction was composed of 1 μL of DNA, 1 unit of MyTaq HS DNA Polymerase (Bioline, London, UK), 12.5 μL of TaqMan Universal PCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, MA), 400 nmol/L of each primer, and 250 nmol/L of each probe for a TNXB exon (35 or 40) and HBB, and PCR-grade water to make a final volume of 25 μL. An ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) equipped with FAM and VIC channels was used for the assay with ROX as a passive reference. PCR reaction was 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 58°C for 20 seconds, and 60°C for 40 seconds (plate read). Samples with an HBB cycle of quantitation (Cq) value within the range of 20 to 30 were subjected to analysis or otherwise repeated by qPCR assay.Table 1Primers and Probes Used for the CAH-X AssayExon/primerSequence, dye, and quencher∗DNA bases specific to the active gene TNXB are marked in lowercase, and bases shared by both TNXB and pseudogene TNXA are shown in capitals.TNXB exon 35 Forward5′-GAGCCTCAGAGTGTGCAGGT-3′ Reverse5′-GTTTTCTTGgCTCCCAcctc-3′ Probe5′-FAM-ctgggatcagccCCTGGAGT-MGB-3′TNXB exon 40 Forward5′-TCCTCAACGGCAACCGc-3′ Reverse5′-GAACACCTGGGAAGCAAGTG-3′ Probe5′-FAM-CGTGTTTTGcGACATGGAGAC-MGB-3′HBB Forward5′-TATCATGCCTCTTTGCACCA-3′ Reverse5′-AATCCAGCCTTATCCCAACC-3′ Probe35′-VIC-CAGCTACAATCCAGCTACCATTCTGC-MGB-3′CAH-X, connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes.∗ DNA bases specific to the active gene TNXB are marked in lowercase, and bases shared by both TNXB and pseudogene TNXA are shown in capitals. Open table in a new tab CAH-X, connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. The copy numbers of TNXB exons 35 and 40 were also tested separately by droplet digital PCR (ddPCR) using the same primers and probes as used in the qPCR assay. Each 20-μL ddPCR reaction contained 1 μL of DNA, 10 μL of 2× ddPCR Supermix for probes (no deoxyuridine triphosphate; Bio-Rad Laboratories, Hercules, CA), 900 nmol/L of each primer, and 250 nmol/L of each probe for a TNXB exon (35 or 40) and HBB. A QX200 AutoDG Droplet Digital PCR System (Bio-Rad Laboratories) was used for the assay by following the manufacturers' instructions, except the PCR reaction was set to be 95°C for 10 minutes; 40 cycles at 94°C for 30 seconds, 58°C for 20 seconds, and 60°C for 40 seconds; a final cycle at 98°C for 10 minutes before cooling to 4°C; with a 2°C/second ramp. Samples with ≥10,000 total accepted droplets and an HBB concentration ranging from 100 to 2000 were subjected to analysis or otherwise repeated by ddPCR assay. For qPCR, the ΔRn (Rn minus baseline with Rn signifying the reporter signal normalized to ROX signal) thresholds for the Cq values of FAM and VIC channels were set as 0.2 and 0.07, respectively. The ratio of TNXB exons 35 or 40 to HBB was calculated as:R=2Cq(VIC)−Cq(FAM),(1) and RX35 <1.2 and RX40 <1 were the cutoff values used for calling the respective exon loss. For ddPCR, standard CNV2 program in QuantaSoft software version 1.7.4.0917 supplied by the manufacturers (Bio-Rad Laboratories) was used to determine the copy numbers of TNXB exons 35 and 40. In both platforms, samples with exons 35 and 40 losses were called as CAH-X CH-1, whereas samples with only exon 40 loss were called as CAH-X CH-2. A total of 44 positive calls (26 CAH-X CH-1 and 18 CAH-X CH-2) were made from the cohort of 278 subjects. Heterozygous, homozygous CAH-X chimeras, and the negatives clearly separated into different clusters (Figure 2). All positive calls were confirmed by Sanger sequencing. Two false-positives of CAH-X CH-2 (one hetero- and one homozygous) were observed, due to a rare TNXB exon 40 haplotype having a c.12150C>G (synonymous) variant without the pathogenic c.12174C>G (p.C4058W). No false-negatives were observed. The copy number assay had a sensitivity of 100% (42 positives/42 true-positives), a specificity of 99.2% (234 negatives/236 true negatives) and an overall accuracy rate of 99.3% (276/278) in determining a CAH-X genotype, and results were consistent between the qPCR and ddPCR systems (Figure 2). Six samples had one-time borderline results by qPCR but were negative in the follow-up repeats (data not shown). There were no borderline results by ddPCR. Notably, one subject (CAH carrier with one CAH-X CH-1 allele) had an unusual two copies of TNXB exon 40 measured. Further analysis revealed that the other allele (non-CAH) had contiguous single-nucleotide polymorphisms (SNPs) of rs77471377 (C>G) and rs4959086 (C>G) to cause a TNXA locus identical to TNXB exon 40 that masked the latter's loss in the CAH-X CH-1 allele. These TNXA SNPs might be common because a total of 46 subjects had ≥2.5 copy numbers measured in TNXB exon 40 (Figure 2). The ratio of TNXB to HBB determined by qPCR and the copy numbers determined by ddPCR provided similar results (Figure 3). However, given that most copy number results fell close to an integer range from 0 to 3, the ddPCR system was considerably more convenient in terms of data interpretation and making the calls. The CAH cohort of this study had a 15.6% (21/135 21-hydroxylase deficiency CAH probands) prevalence of CAH-X that was higher than the previously estimated 8.5%.13Morissette R. Chen W. Perritt A.F. Dreiling J.L. Arai A.E. Sachdev V. Hannoush H. Mallappa A. Xu Z. McDonnell N.B. Quezado M. Merke D.P. Broadening the spectrum of Ehlers Danlos syndrome in patients with congenital adrenal hyperplasia.J Clin Endocrinol Metab. 2015; 100: E1143-E1152Crossref PubMed Scopus (42) Google Scholar The prevalence was especially high (29.2% or 21/72) in the subjects with a 30-Kb deletion genotype. As expected due to autosomal dominant inheritance, the prevalence of CAH-X was found to be similar among the affected patients, carriers, and families of CAH (Table 2).Table 2Prevalence of the CAH-X Chimeras in a Cohort of Subjects with Congenital Adrenal Hyperplasia due to 21-Hydroxylase DeficiencyGenotypeProbandsPatientsCarriersCohortTotal30-Kb deletion∗Carrier of a 30-Kb deletion.Total30-Kb deletion∗Carrier of a 30-Kb deletion.Total30-Kb deletion∗Carrier of a 30-Kb deletion.Total30-Kb deletion∗Carrier of a 30-Kb deletion.CAH-X CH-1†CAH-X CH-1: CYP21A1P-TNXA/TNXB chimera with TNXB exons 35–44 replaced by TNXA.9.6% (13/135)18.1% (13/72)10.3% (15/145)19.7% (15/76)9.3% (11/118)21.6% (11/51)9.9% (26/263)20.5% (26/127)CAH-X CH-2‡CAH-X CH-2: CYP21A1P-TNXA/TNXB chimera with TNXB exons 40–44 replaced by TNXA.5.9% (8/135)11.1% (8/72)6.9% (10/145)13.2% (10/76)5.1% (6/118)11.8% (6/51)6.1% (16/263)12.6% (16/127)CAH-X15.6% (21/135)29.2% (21/72)17.2% (25/145)32.9% (25/76)14.4% (17/118)33.3% (17/51)16.0% (42/263)33.1% (42/127)Two subjects had biallelic CAH-X chimeras. One subject with a CH-2/CH-2 genotype was counted once as CH-2 whereas the other subject with a CH-1/CH-2 genotype was counted once as CH-1 in calculating the prevalence in probands, patients, and cohort, respectively.CAH-X, connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes.∗ Carrier of a 30-Kb deletion.† CAH-X CH-1: CYP21A1P-TNXA/TNXB chimera with TNXB exons 35–44 replaced by TNXA.‡ CAH-X CH-2: CYP21A1P-TNXA/TNXB chimera with TNXB exons 40–44 replaced by TNXA. Open table in a new tab Two subjects had biallelic CAH-X chimeras. One subject with a CH-2/CH-2 genotype was counted once as CH-2 whereas the other subject with a CH-1/CH-2 genotype was counted once as CH-1 in calculating the prevalence in probands, patients, and cohort, respectively. CAH-X, connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. Forty of the 42 CAH-X–positive subjects had a complete or partial clinical evaluation for EDS, and 39 had at least one finding characteristic of EDS. The lone CAH-X–positive subject without EDS findings was an 11-year–old male with CAH carrying a CAH-X CH-1 chimera; and the 2 subjects not evaluated were CAH carriers carrying a CAH-X CH-1 chimera. In general, patients with monoallelic CAH-X CH-1 had fewer EDS characteristics than did patients with monoallelic CAH-X CH-2, and patients with biallelic CAH-X had the most severe EDS phenotype (Table 3). In addition, carriers of CAH who were heterozygous for a CAH-X mutation tended to have less of an EDS phenotype than did their relatives with CAH who carried the same CAH-X mutation.Table 3Clinical Ehlers-Danlos Syndrome Characteristics of Subjects with CAH-XParameterCAH patientsCAH carriersCAH-X CH-1CAH-X CH-2BiallelicCAH-X CH-1CAH-X CH-2n1410295Age, years17.2 ± 10.7 (4–39)13.1 ± 12.0 (2–44)16.0 ± 14.1 (6–26)46.9 ± 11.1 (30–63)40.8 ± 11.3 (21–49)Females/males6/84/60/25/44/1Musculoskeletal Generalized hypermobility∗Generalized hypermobility defined as a Beighton score of 5 of 9 or greater in children and of 4 of 9 or greater in postpubertal adolescents and adults.7/135/102/24/92/5 Small joint hypermobility5/145/101/24/91/5 Large joint hypermobility3/144/101/21/91/5 Subluxations4/143/101/23/90/5 Chronic arthralgia4/142/102/24/91/5 Chronic tendonitis, bursitis or fasciitis2/142/101/23/90/5 Pes planus3/142/101/22/90/5Dermatologic Skin laxity1/142/102/20/90/5 Wide scars0/142/101/20/90/5 Piezogenic papules3/141/102/20/90/" @default.
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• W2949949273 title "High-Throughput Screening for CYP21A1P-TNXA/TNXB Chimeric Genes Responsible for Ehlers-Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia" @default.
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