Wuyan Chen

Comprehensive Genetic Analysis of 182 Unrelated Families with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

BACKGROUND Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. STUDY OBJECTIVE The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. METHODS Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. RESULTS In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. CONCLUSIONS Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.

Phenotypic profiling of parents with cryptic nonclassic congenital adrenal hyperplasia: findings in 145 unrelated families

OBJECTIVE To comprehensively phenotype parents identified with nonclassic congenital adrenal hyperplasia (NCCAH) by family genetic studies, termed here as cryptic NCCAH and to define the incidence of cryptic NCCAH in the parents of a large cohort of patients with 21-hydroxylase deficiency. DESIGN Genotyping was performed on 249 parents of 145 unrelated congenital adrenal hyperplasia (CAH) patients. Parents with two CYP21A2 mutations underwent extensive evaluation. RESULTS Of the 249 parents, ten (4%; seven females and three males) were identified as having cryptic NCCAH. The majority was of ethnicities previously reported to have a higher incidence of NCCAH. Cosyntropin stimulation performed in eight parents provided biochemical confirmation (17-hydroxyprogesterone range 56-364 nmol/l) and cortisol response was ≤500 nmol/l in three parents (38%). Of the seven women (27-54 years) with cryptic NCCAH, four had prior infertility, two reported irregular menses, two had treatment for hirsutism, one had androgenic alopecia. Men were asymptomatic. All cryptic NCCAH parents reported normal puberty and had normal height. Adrenal hypertrophy and a small adrenal myelolipoma were observed in two parents; testicular adrenal rest tissue was not found. CONCLUSIONS Parents diagnosed with NCCAH by genetic testing are mostly asymptomatic. Temporary female infertility and suboptimal cortisol response were commonly observed. Ongoing glucocorticoid therapy is not indicated in adults with CAH identified by family genotype studies unless symptomatic, but glucocorticoid stress coverage should be considered in select cases. Parents of a child with CAH have a 1:25 risk of having NCCAH; if the mother of a child with CAH has infertility, evaluation for NCCAH is indicated.

Junction Site Analysis of Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency

BACKGROUND Chimeric CYP21A1P/CYP21A2 genes, caused by homologous recombination between CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and its highly homologous pseudogene CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene), are common in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). A comprehensive junction site analysis of chimeric CYP21A1P/CYP21A2 genes is needed for optimizing genetic analysis strategy and determining clinical relevance. METHODS We conducted a comprehensive genetic analysis of chimeric CYP21A1P/CYP21A2 genes in a cohort of 202 unrelated 21-OHD patients. Targeted CYP21A2 mutation analysis was performed, and genotyping of chimeric CYP21A1P/CYP21A2 genes was cross-confirmed with Southern blot, RFLP, and multiplex ligation-dependent probe amplification analyses. Junction sites of chimera genes were determined by sequencing the long-PCR products amplified with primers CYP779f and Tena32F. An updated bioinformatics survey of Chi-like sequences was also performed. RESULTS Of 100 probands with a chimeric allele, 96 had a chimera associated with the severe classic salt-wasting form of CAH, and the remaining 4 carried an uncommon attenuated chimera with junction sites upstream of In2G (c.293-13A/C>G), which is associated with a milder phenotype. In addition to 6 of 7 reported chimeras, we identified a novel classic chimera (CH-8) and a novel attenuated chimera (CH-9). Attenuated chimeras explained prior genotype-phenotype discrepancies in 3 of the patients. Sequencing the CYP779f/Tena32F amplicons accurately differentiated between classic and attenuated chimeras. The bioinformatics survey revealed enrichment of Chi-like sequences within or in the vicinity of intron 2. CONCLUSIONS Junction site analysis can explain some genotype-phenotype discrepancies. Sequencing the well-established CYP779f/Tena32F amplicons is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes, which are clinically relevant.

Tenascin-X Haploinsufficiency Associated with Ehlers-Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia

CONTEXT The gene for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, CYP21A2, is flanked by the gene encoding tenascin-X (TNXB), a connective tissue extracellular matrix protein that has been linked to both autosomal dominant and autosomal recessive Ehlers-Danlos syndrome (EDS). A contiguous deletion of CYP21A2 and TNXB has been described. OBJECTIVE The objective of the study was to determine the frequency and clinical significance of TNXB haploinsufficiency in CAH patients. DESIGN, SETTING, AND PARTICIPANTS A total of 192 consecutive unrelated CAH patients being seen as part of an observational study at the National Institutes of Health Clinical Center (Bethesda, MD) were prospectively studied during 2006-2010. Patients were evaluated for clinical evidence of EDS, including cardiac evaluation. DNA was analyzed by PCR, multiplex ligation-dependent probe amplification, Southern blot, and TNXB sequencing. Tenascin-X expression was evaluated by Western blot analysis of fibroblasts and immunostaining of the skin. CAH patients with TNXB haploinsufficiency were compared with age-matched CAH patients with normal TNXB (controls). Phenotyping of 7 parents with TNXB haploinsufficiency was performed. MAIN OUTCOME MEASURES The frequency of TNXB haploinsufficiency among CAH patients and the frequency of EDS symptomatology among CAH patients with TNXB haploinsufficiency and controls. RESULTS TNXB haploinsufficiency, here termed CAH-X syndrome, was present in 7% of CAH patients. Twelve of 91 patients carrying a CYP21A2 deletion (13%) carried a contiguous deletion that extended into TNXB. One patient carried a TNXB premature stop codon. Twelve of 13 patients with CAH-X had EDS clinical features. Patients with CAH-X were more likely than age-matched controls to have joint hypermobility (P < .001), chronic joint pain (P = .003), multiple joint dislocations (P = .004), a structural cardiac valve abnormality by echocardiography (P = .02), and reduced tenascin-X expression by Western blot and immunostaining. A subset of parents had clinical findings. CONCLUSIONS Clinical evaluation for connective tissue dysplasia should be routinely performed in CAH patients, especially those harboring a CYP21A2 deletion.

The Phenotypic Spectrum of Contiguous Deletion of CYP21A2 and Tenascin XB: Quadricuspid Aortic Valve and Other Midline Defects

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency is an autosomal recessive disorder and is the most common cause of ambiguous genitalia in the newborn. The genes encoding 21‐hydroxylase, CYP21A2, and tenascin‐X (TNX), TNXB, are located within the HLA complex, in a region of high gene density termed the RCCX module. The module has multiple pseudogenes as well as tandem repeat sequences that promote misalignment during meiosis leading to complex gene rearrangements, deletions and gene conversion events. CYP21A2 mutations cause CAH, and TNX deficiency has been identified as a cause of hypermobility type Ehlers–Danlos syndrome (EDS). Here we report on a three‐generation family with a heterozygous deletion encompassing CYP21A2 and TNXB that initially came to medical attention due to the diagnosis of CAH in the proposita. Southern blotting and PCR‐based analysis of the RCCX module revealed a CYP21A2 deletion extending into TNXB in one allele and a CYP21A2 point mutation in the other allele. Family history is notable for joint hypermobility. Additional radiological and clinical investigations showed a quadricuspid aortic valve, single kidney, bicornuate uterus and a bifid uvula in the proposita, and mitral valve prolapse in her mother. These findings further delineate the phenotype of the CAH‐TNX contiguous gene deletion syndrome and point to an intersection of connective tissue dysplasias with a common gene‐mediated endocrine disorder.

Comprehensive Mutation Analysis of the CYP21A2 Gene An Efficient Multistep Approach to the Molecular Diagnosis of Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of adrenal steroidogenesis caused by mutations in the CYP21A2 gene. Direct comparison of established and novel methodologies of CYP21A2 genetic analysis in a large cohort representing a wide range of genotypes has not been previously reported. We genotyped a cohort of 129 unrelated patients with 21-OHD, along with 145 available parents, using Southern blot (SB) analysis, multiplex ligation-dependent probe amplification (MLPA), PCR-based restriction fragment length polymorphism (RFLP) analysis, multiplex minisequencing and conversion-specific PCR, duplication-specific amplification, and DNA sequencing. CYP21A2 genotyping identified four duplicated CYP21A2 genes (1.53%) and 79 chimeric CYP21A1P/CYP21A2 genes (30.15%). Parental SB data were essential for determining the CYP21 haplotype in three cases, whereas PCR-based RFLP analysis was necessary for MLPA results to be accurately interpreted in the majority of cases. The comparison of different methods in detecting deletion and duplication showed that MLPA with PCR-based RFLP was comparable with SB analysis, with parental data of 100% sensitivity and specificity. DNA sequencing was required for the identification of 16 (6.1%) rare point mutations and determination of clinically significant chimera junction sites. MLPA with PCR-based RFLP analysis is an excellent substitute for SB analysis in detecting CYP21A2 deletion and duplication and a combination of MLPA, PCR-based RFLP, duplication-specific amplification, and DNA sequencing is a convenient and comprehensive strategy for mutation analysis of the CYP21A2 gene in patients with 21-OHD.

Broadening the Spectrum of Ehlers Danlos Syndrome in Patients With Congenital Adrenal Hyperplasia

CONTEXT The contiguous gene deletion syndrome (CAH-X) was described in a subset (7%) of congenital adrenal hyperplasia (CAH) patients with a TNXA/TNXB chimera, resulting in deletions of CYP21A2, encoding 21-hydroxylase necessary for cortisol biosynthesis, and TNXB, encoding the extracellular matrix glycoprotein tenascin-X (TNX). This TNXA/TNXB chimera is characterized by a 120-bp deletion in exon 35 and results in TNXB haploinsufficiency, disrupted TGF-β signaling, and an Ehlers Danlos syndrome phenotype. OBJECTIVE The objective of the study was to determine the genetic status of TNXB and resulting protein defects in CAH patients with a CAH-X phenotype but not the previously described TNXA/TNXB chimera. Design, Settings, Participants, and Intervention: A total of 246 unrelated CAH patients were screened for TNXB defects. Genetic defects were investigated by Southern blotting, multiplex ligation-dependent probe amplification, Sanger, and next-generation sequencing. Dermal fibroblasts and tissue were used for immunoblotting, immunohistochemical, and coimmunoprecipitation experiments. MAIN OUTCOME MEASURES The genetic and protein status of tenascin-X in phenotypic CAH-X patients was measured. RESULTS Seven families harbor a novel TNXB missense variant c.12174C>G (p.C4058W) and a clinical phenotype consistent with hypermobility-type Ehlers Danlos syndrome. Fourteen CAH probands carry previously described TNXA/TNXB chimeras, and seven unrelated patients carry the novel TNXB variant, resulting in a CAH-X prevalence of 8.5%. This highly conserved pseudogene-derived variant in the TNX fibrinogen-like domain is predicted to be deleterious and disulfide bonded, results in reduced dermal elastin and fibrillin-1 staining and altered TGF-β1 binding, and represents a novel TNXA/TNXB chimera. Tenascin-X protein expression was normal in dermal fibroblasts, suggesting a dominant-negative effect. CONCLUSIONS CAH-X syndrome is commonly found in CAH due to 21-hydroxylase deficiency and may result from various etiological mechanisms.

Ehlers–Danlos Syndrome Caused by Biallelic TNXB Variants in Patients with Congenital Adrenal Hyperplasia

Some variants that cause autosomal‐recessive congenital adrenal hyperplasia (CAH) also cause hypermobility type Ehlers–Danlos syndrome (EDS) due to the monoallelic presence of a chimera disrupting two flanking genes: CYP21A2, encoding 21‐hydroxylase, necessary for cortisol and aldosterone biosynthesis, and TNXB, encoding tenascin‐X, an extracellular matrix protein. Two types of CAH tenascin‐X (CAH‐X) chimeras have been described with a total deletion of CYP21A2 and characteristic TNXB variants. CAH‐X CH‐1 has a TNXB exon 35 120‐bp deletion resulting in haploinsufficiency, and CAH‐X CH‐2 has a TNXB exon 40 c.12174C>G (p.Cys4058Trp) variant resulting in a dominant‐negative effect. We present here three patients with biallelic CAH‐X and identify a novel dominant‐negative chimera termed CAH‐X CH‐3. Compared with monoallelic CAH‐X, biallelic CAH‐X results in a more severe phenotype with skin features characteristic of classical EDS. We present evidence for disrupted tenascin‐X function and computational data linking the type of TNXB variant to disease severity.

Genetics of Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders due to single-gene defects in the various enzymes required for cortisol biosynthesis. CAH represents a continuous phenotypic spectrum with more than 95% of all cases caused by 21-hydroxylase deficiency. Genotyping is an important tool in confirming the diagnosis or carrier state, provides prognostic information on disease severity, and is essential for genetic counseling. In this article, the authors provide an in-depth discussion on the genetics of CAH, including genetic diagnosis, molecular analysis, genotype-phenotype relationships, and counseling of patients and their families.

Revisiting the prevalence of nonclassic congenital adrenal hyperplasia in US Ashkenazi Jews and Caucasians

PurposeNonclassic 21-hydroxylase deficiency, a mild form of congenital adrenal hyperplasia (CAH), is estimated to be the most common autosomal recessive condition, with an especially high prevalence in Ashkenazi Jews (3.7% affected, 30.9% carriers), based on a 1985 HLA-B linkage study of affected families. Affected individuals, especially women, may suffer from hyperandrogenism and infertility. State-of-the-art genetic studies have not been done to confirm these remarkable rates.MethodsCYP21A2 genotyping was performed in 200 unrelated healthy Ashkenazi Jewish subjects and 200 random US Caucasians who did not self-identify as a specific ethnicity using multiplex minisequencing, real-time polymerase chain reaction and junction site analysis.ResultsNonclassic CAH carriership was found similarly in 15% (95% confidence interval (CI): 10.4–20.7) of Ashkenazi Jews and 9.5% (95% CI: 5.8–14.4) of Caucasians (P=0.13). The proportion of Ashkenazi Jewish nonclassic CAH carriers (0.15 versus 0.309, P<0.0001) and disease affected (0.005 versus 0.037, P=0.009) was not as high as previously reported. The estimated prevalence of nonclassic CAH in Caucasians was 1 in 200 (0.5%, 95% CI: 0.01–2.8).ConclusionNonclassic CAH is a common condition, regardless of ethnicity, and should be considered with preconception and infertility counseling.