TBX5 pathogenic variant in a patient with congenital heart defect and tracheal stenosis.

Congenital tracheal stenosis is a rare life-threatening disorder caused by narrow O-shaped tracheal ring without smooth muscle. Its underlying genetic cause has not been elucidated. We performed whole exome sequencing in a patient with congenital tracheal stenosis and congenital heart defect, and identified a de novo pathogenic TBX5 variant (NM_181486.4:c.680T>C, p.(Ile227Thr)). The Ile227Thr-TBX5 protein was predicted to have a decreased stability by in silico protein structural analyses, and was shown to have a significantly reduced activity for the NPPA promoter by luciferase assay. The results, together with the expression of mouse Tbx5 in the lung and trachea and the development of tracheal cartilage dysplasia in the lung-specific Tbx5 null mice, imply the relevance of TBX5 pathogenic variants to congenital tracheal stenosis.

Microdeletion at ESR1 Intron 6 (DEL_6_75504) Is a Susceptibility Factor for Cryptorchidism and Hypospadias.

CONTEXT: We have previously reported that a specific "AGATC" haplotype in a >34 kb tight linkage disequilibrium (LD) block within ESR1 is strongly associated with cryptorchidism and hypospadias in Japanese boys. OBJECTIVE: We aimed to determine the true susceptibility factor for cryptorchidism and hypospadias linked to the "AGATC" haplotype. METHODS: We performed various molecular studies in hitherto unreported 230 Italian boys (80 with cryptorchidism and 150 with normal genitalia) and previously reported and newly recruited 415 Japanese boys (149 with cryptorchidism, 141 with hypospadias, and 125 with normal genitalia). We also performed ESR1 expression analyses using breast cancer-derived MCF-7 cells. RESULTS: Haplotype analysis revealed the LD block and positive association of the "AGATC" haplotype with cryptorchidism in Italian boys. Whole genome sequencing identified an identical 2249-bp microdeletion (ΔESR1) generated by a microhomology-mediated replication error in both Japanese and Italian boys with the specific haplotype. ΔESR1 was found to be strongly associated with cryptorchidism and hypospadias by Cochran-Armitage trend test and was revealed to show nearly absolute LD with the "AGATC" haplotype. ESR1 expression was upregulated in MCF-7 cells with a homozygous deletion encompassing ΔESR1 and those with a homozygous deletion involving a CTCF-binding site within ΔESR1. CONCLUSION: The results reveal that ΔESR1, which has been registered as "DEL_6_75504" in gnomAD SVs v2.1, is the true susceptibility factor for cryptorchidism and hypospadias. It appears that ΔESR1 was produced in a single ancestral founder of modern humans and has been maintained within the genome of multiple ethnic groups by selection.

Combined pituitary hormone deficiency in a patient with an FGFR1 missense variant: case report and literature review.

Recent studies have indicated that heterozygous loss-of-function variants in fibroblast growth factor receptor 1 (FGFR1) are involved in the development of congenital hypogonadotropic hypogonadism and combined pituitary hormone deficiency (CPHD). We encountered a Japanese boy with short stature and pubertal failure. Endocrine studies showed GH, TSH, and LH/FSH deficiencies, and brain magnetic resonance imaging delineated hypoplastic anterior pituitary and ectopic posterior pituitary. The patient was treated with GH, l-thyroxine, and hCG/rFSH. Next-generation sequencing panel for pituitary dysfunction identified a probably weak disease-associated heterozygous missense variant in FGFR1 (NM_023110.3:c.176A>T:p.(Asp59Val)), together with a probably non-deleterious heterozygous missense variant in KISS1R (NM_032551.5:c.769G>C:p.(Val257Leu)). We also review six previously reported CHPD patients with probably deleterious FGFR1 variants. The data, in conjunction with the previously reported cases, argue for the relevance of FGFR1 variants to the development of CPHD.

Retrotransposition disrupting EBP in a girl and her mother with X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata (CDPX2) is a rare congenital disorder caused by pathogenic variants in EBP on Xp11.23. We encountered a girl and her mother with CDPX2-compatible phenotypes including punctiform calcification in the neonatal period of the girl, and asymmetric limb shortening and ichthyosis following the Blaschko lines in both subjects. Although Sanger direct sequencing failed to reveal a disease-causing variant in EBP, whole genome sequencing (WGS) followed by Manta analysis identified a ~ 4.5 kb insertion at EBP exon 2 of both subjects. The insertion was associated with the hallmarks of retrotransposition such as an antisense poly(A) tail, a target site duplication, and a consensus endonuclease cleavage site, and the inserted sequence harbored full-length SVA_F1 element with 5'- and 3'-transductions containing the Alu sequence. The results imply the relevance of retrotransposition to the human genetic diseases and the usefulness of WGS in the identification of retrotransposition.

Parthenogenetic mosaicism: generation via second polar body retention and unmasking of a likely causative PER2 variant for hypersomnia.

BACKGROUND: Parthenogenetic mosaicism is an extremely rare condition identified only in five subjects to date. The previous studies indicate that this condition is mediated by parthenogenetic activation and is free from a specific phenotype ascribed to unmaking of a maternally inherited recessive variant in the parthenogenetic cell lineage. RESULTS: We examined a 28-year-old Japanese 46,XX female with Silver-Russell syndrome and idiopathic hypersomnia. The results revealed (1) predominance of maternally derived alleles for all the differentially methylated regions examined; (2) no disease-related copy-number variant; (3) two types of regions for all chromosomes, i.e., four BAF (B-allele frequency) band regions with single major microsatellite peaks of maternal origin and single minor microsatellite peaks of non-maternal (paternal) origin, and six BAF band regions with single major microsatellite peaks of maternal origin and two minor microsatellite peaks of maternal and non-maternal (paternal) origin; (4) an unmasked extremely rare PER2 variant (c.1403G>A:p.(Arg468Gln)) with high predicted pathogenicity; (5) mildly affected local structure with altered hydrogen bonds of the p.Arg468Gln-PER2 protein; and (6) nucleus-dominant subcellular distribution of the p.Arg468Gln-PER2 protein. CONCLUSIONS: The above findings imply that the second polar body retention occurred around fertilization, resulting in the generation of the parthenogenetic cell lineage by endoreplication of a female pronucleus and the normal cell lineage by fusion of male and female pronuclei, and that the homozygous PER2 variant in the parthenogenetic cells is the likely causative factor for idiopathic hypersomnia.

Primary ovarian insufficiency in a female with phosphomannomutase-2 gene (PMM2) mutations for congenital disorder of glycosylation.

Primary ovarian insufficiency (POI) is a highly heterogeneous condition, and its underlying causes remain to be clarified in a large fraction of patients. Congenital disorders of glycosylation (CDG) are multisystem diseases caused by mutations of a number of genes involved in N-glycosylation or O-glycosylation, and the most frequent form is PMM2-CDG (alias, CDG-Ia) resulting from biallelic mutations in PMM2 encoding phosphomannomutase-2 involved in N-glycosylation. Here, we examined a 46,XX Japanese female with syndromic POI accompanied by an undetectable level of serum anti-Müllerian hormone (AMH). Whole exome sequencing identified biallelic pathogenic mutations of PMM2 (a novel c.34G>C:p.(Asp12His) of maternal origin and a recurrent c.310C>G:p.(Leu104Val) of paternal origin) (NM_000303.3), and N-glycosylation studies detected asialotransferrin and disialotransferrin characteristic of PMM2-CDG, in addition to normally glycosylated tetrasialotransferrin. Clinical assessment showed cerebellar hypotrophy, which is a fairly characteristic and highly prevalent feature in PMM2-CDG, together with multiple non-specific features reported in PMM2-CDG such as characteristic face, intellectual disability, skeletal abnormalities, and low blood antithrombin III value. These results including the undetectable level of serum AMH, in conjunction with previously reported findings suggestive of the critical role of glycosylation in oocyte development and function, imply that PMM2-CDG almost invariably leads to POI primarily because of the defective oogenesis and/or oocyte-dependent early folliculogenesis rather than the compromised bioactivity of FSH/LH with defective glycosylation. Thus, it is recommended to examine PMM2 in patients with syndromic POI, especially in those with cerebellar ataxia/hypotrophy.

Kagami-Ogata syndrome in a patient with 46,XX,t(2;14)(q11.2;q32.2)mat disrupting MEG3.

Kagami-Ogata syndrome (KOS14) is a rare imprinting disorder characterized by a unique constellation of phenotypes including bell-shaped small thorax with coat-hanger appearance of the ribs. We encountered an African American female infant with KOS14 phenotype and 46,XX,t(2;14)(q11.2;q32.2)mat. After excluding upd(14)pat and an epimutation (hypermethylation) and a deletion affecting the maternally derived 14q32.2 imprinted region, we performed whole-genome sequencing, revealing that the translocation was generated between noncoding region at 2q11.2 and intron 6 of MEG3 at 14q32.2. Subsequent Sanger sequencing for the fusion points showed that the chromosomal fusion on the der(2) chromosome occurred between Chr2:102,193,994 (bp) and Chr14:101,314,628 (bp) in association with an insertion of 5-bp segment of unknown origin and that on the der(14) chromosome took place between Chr14:101,314,627 (bp) and Chr2:102,193,995 (bp) in association with an insertion of 1-bp segment of unknown origin (according to GRCh37/hg19). The results, together with the previous data in patients with KOS14, imply that the MEG3 disruption by 46,XX,t(2;14)(q11.2;q32.2)mat caused silencing of all MEGs including RTL1as and resultant excessive RTL1 expression, leading to the development of KOS14. To our knowledge, while Robertsonian translocations involving chromosome 14 have been reported in KOS14, this is the first case of KOS14 caused by a chromosomal translocation involving the 14q32.2 imprinted region.

Coffin-Lowry syndrome in a girl with 46,XX,t(X;11)(p22;p15)dn: Identification of RPS6KA3 disruption by whole genome sequencing.

We report a Japanese girl with Coffin-Lowry syndrome phenotype such as hypertelorism, hypodontia, and tapering fingers and 46,XX,t(X;11)(p22;p15)dn. Whole genome sequencing revealed RPS6KA3 disruption by the translocation, and X-inactivation analysis indicated preferential inactivation of the normal X chromosome. The results explain the development of an X-linked disease in this girl.

De novo ZBTB7A variant in a patient with macrocephaly, intellectual disability, and sleep apnea: implications for the phenotypic development in 19p13.3 microdeletions.

Interstitial microdeletions at chromosome 19p13.3 are frequently associated with a constellation of clinical features including macrocephaly, characteristic face, intellectual disability, and sleep apnea. Previous studies in 25 patients with 19p13.3 microdeletions have revealed loss of MAP2K2 in 24 patients and that of PIAS4 and ZBTB7A in 23 patients, suggesting that these three adjacent genes are candidate genes for the phenotypic development in 19p13.3 microdeletions. We identified a de novo likely pathogenic heterozygous missense variant of ZBTB7A (NM_015898.3:c.1152C>G, p.(Cys384Trp)) in a Japanese boy with macrocephaly, intellectual disability, and sleep apnea. This variant affects the conserved cysteine residue forming the coordinate bond with Zn2+ ion at the first zinc finger domain, and is predicted to exert a dominant-negative effect because of the generation of homo- and hetero-dimers with the wild-type and variant ZBTB7A proteins. The results argue for a critical relevance of ZBTB7A to the development of most, but probably not all, of the 19p13.3 microdeletion phenotype.

Comprehensive clinical and molecular studies in split-hand/foot malformation: identification of two plausible candidate genes (LRP6 and UBA2).

Split-hand/foot malformation (SHFM) is a clinically and genetically heterogeneous condition. We sequentially performed screening of the previously identified Japanese founder 17p13.3 duplication/triplication involving BHLHA9, array comparative genomic hybridization, and whole exome sequencing (WES) in newly recruited 41 Japanese families with non-syndromic and syndromic SHFM. We also carried out WES in seven families with nonsyndromic and syndromic SHFM in which underlying genetic causes including pathogenic copy-number variants (CNVs) remained undetected in our previous studies of 56 families. Consequently, we identified not only known pathogenic CNVs (17p13.3 duplications/triplications [n = 21], 2q31 deletion [n = 1], and 10q24 duplications [n = 3]) and rare variants in known causative genes (TP63 [n = 3], DLX5 [n = 1], IGF2 [n = 1], WNT10B [n = 3], WNT10B/PORCN [n = 1], and PORCN [n = 1]), but also a de novo 19q13.11 deletion disrupting UBA2 (n = 1) and variants that probably affect function in LRP6 (n = 1) and UBA2 (n = 1). Thus, together with our previous data based on testing of 56 families, molecular studies for a total of 97 families with SHFM revealed underlying genetic causes in 75 families, and clinical studies for the 75 families indicated a certain degree of correlation between genetic causes and phenotypes. The results imply that SHFM primarily occurs as a genetic disorder with genotype-phenotype correlations. Furthermore, the results together with previous data such as the development of SHFM in Lrp6 knockout mice, the presence of SHFM in two subjects with 19q13 deletions involving UBA2, and strong mouse Uba2 expression in the developing limb buds, imply that LRP6 and UBA2 represent plausible candidate genes for SHFM.

Longitudinal serum and urine steroid metabolite profiling in a 46,XY infant with prenatally identified POR deficiency.

Although POR deficiency (PORD) is assumed to be accompanied by excessive placental androgen accumulation and enhanced adrenal and testicular androgen production via the backdoor pathway as well as compromised testicular androgen production via the frontdoor pathway, there is no direct evidence for the flux of excessive placental androgens into the fetal circulation and for the production of dihydrotestosterone (DHT) via the backdoor pathway. We examined longitudinal serum and urine steroid metabolite profiles in a 46,XY infant with PORD who was prenatally identified because of the progressive fetal masculinization and maternal virilization from the mid-gestation and the presence of fetal radio-humeral synostosis and was confirmed to have compound heterozygous mutations of POR (p.Q201X and p.R457H). The results showed (1) markedly and inappropriately elevated serum androstenedione and testosterone (T) values at birth, (2) a markedly increased serum DHT value with a normal DHT/T ratio at birth, (3) transient elevation of serum T and DHT values accompanied by a normal DHT/T ratio and concomitant elevations of intermediate steroid metabolites on both the frontdoor and backdoor pathways at 30 days of age, and (4) persistent PORD-compatible urine steroid profiles. Although the data obtained from a single infantile patient are too premature to be generalized, they imply: (1) the transfer of excessive placental androgens into the fetal as well as the maternal circulations from the mid-gestation, (2) lack of a clinically discernible amount of DHT production via the adrenal backdoor pathway around birth, and (3) the activation of both the frontdoor and backdoor pathways in the testis around the mini-puberty, with no production of a clinically discernible amount of DHT via the testicular backdoor pathway.

Mutation analysis of FGFR1-3 in 11 Japanese patients with syndromic craniosynostoses.

Syndromic craniosynostoses usually occur as single gene disorders. In this study, we analyzed FGFR1-3 genes in four patients with Crouzon syndrome (CS), four patients with Pfeiffer syndrome type 2 (PS-2), one patient with Jackson-Weiss syndrome (JWS), and two patients (sisters) with Muenke syndrome (MS). FGFR2 and FGFR3 mutations were identified in 10 of the 11 patients. Notably, we found a novel FGFR2 p.Asn549Thr mutation in a patient with CS, and a novel FGFR2 p.Ser347Cys mutation in a patient with JWS (thus, this patient was turned out to have an FGFR2-related PS-variant). We also identified an FGFR2 p.Ser252Leu mutation in a phenotypically normal father of a daughter with CS, and an FGFR3 p.Pro250Arg mutation in a mildly macrocephalic father of sisters with MS. These findings, together with previous data, imply that the same FGFR2 mutations can be associated with a wide range of phenotypes including clinically different forms of syndromic craniosynostosis and apparently normal phenotype, depending on other (epi)genetic and environmental factors. Thus, genetic studies are recommended not only for obviously affected individuals but also for family members with apparently normal phenotype or non-specific subtle abnormal phenotype, to allow for pertinent genetic counseling. © 2016 Wiley Periodicals, Inc.

Combined steroidogenic characters of fetal adrenal and Leydig cells in childhood adrenocortical carcinoma.

Although childhood adrenocortical carcinomas (c-ACCs) with a TP53 mutation are known to produce androgens, detailed steroidogenic characters have not been clarified. Here, we examined steroid metabolite profiles and expression patterns of steroidogenic genes in a c-ACC removed from the left adrenal position of a 2-year-old Brazilian boy with precocious puberty, using an atrophic left adrenal gland removed at the time of tumorectomy as a control. The c-ACC produced not only abundant dehydroepiandrosterone-sulfate but also a large amount of testosterone via the Δ5 pathway with Δ5-androstenediol rather than Δ4-androstenedione as the primary intermediate metabolite. Furthermore, the c-ACC was associated with elevated expressions of CYP11A1, CYP17A1, POR, HSD17B3, and SULT2A1, a low but similar expression of CYB5A, and reduced expressions of AKR1C3 (HSD17B5) and HSD3B2. Notably, a Leydig cell marker INSL3 was expressed at a low but detectable level in the c-ACC. Furthermore, molecular studies revealed a maternally inherited heterozygous germline TP53 mutation, and several post-zygotic genetic aberrations in the c-ACC including loss of paternally derived chromosome 17 with a wildtype TP53 and loss of maternally inherited chromosome 11 and resultant marked hyperexpression of paternally expressed growth promoting gene IGF2 and drastic hypoexpression of maternally expressed growth suppressing gene CDKN1C. These results imply the presence of combined steroidogenic properties of fetal adrenal and Leydig cells in this patient's c-ACC with a germline TP53 mutation and several postzygotic carcinogenic events.

Silver-Russell syndrome without body asymmetry in three patients with duplications of maternally derived chromosome 11p15 involving CDKN1C.

We report duplications of maternally derived chromosome 11p15 involving CDKN1C encoding a negative regulator for cell proliferation in three Japanese patients (cases 1 and 2 from family A and case 3 from family B) with Silver-Russell syndrome (SRS) phenotype lacking hemihypotrophy. Chromosome analysis showed 46,XX,der(16)t(11;16)(p15.3;q24.3)mat in case 1, 46,XY,der(16)t(11;16)(p15.3;q24.3)mat in case 2 and a de novo 46,XX,der(17)t(11;17)(p15.4;q25.3) in case 3. Genomewide oligonucleotide-based array comparative genomic hybridization, microsatellite analysis, pyrosequencing-based methylation analysis and direct sequence analysis revealed the presence of maternally derived extra copies of the distal chromosome 11p involving the wild-type CDKN1C (a ~7.98 Mb region in cases 1 and 2 and a ~4.43 Mb region in case 3). The results, in conjunction with the previous findings in patients with similar duplications encompassing CDKN1C and in those with intragenic mutations of CDKN1C, imply that duplications of CDKN1C, as well as relatively mild gain-of-function mutations of CDKN1C lead to SRS subtype that usually lack hemihypotrophy.

Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex.

BACKGROUND: Limb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients. METHODS: This study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses. RESULTS: We identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome. CONCLUSIONS: These results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.

TBX1 mutation identified by exome sequencing in a Japanese family with 22q11.2 deletion syndrome-like craniofacial features and hypocalcemia.

BACKGROUND: Although TBX1 mutations have been identified in patients with 22q11.2 deletion syndrome (22q11.2DS)-like phenotypes including characteristic craniofacial features, cardiovascular anomalies, hypoparathyroidism, and thymic hypoplasia, the frequency of TBX1 mutations remains rare in deletion-negative patients. Thus, it would be reasonable to perform a comprehensive genetic analysis in deletion-negative patients with 22q11.2DS-like phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We studied three subjects with craniofacial features and hypocalcemia (group 1), two subjects with craniofacial features alone (group 2), and three subjects with normal phenotype within a single Japanese family. Fluorescence in situ hybridization analysis excluded chromosome 22q11.2 deletion, and genomewide array comparative genomic hybridization analysis revealed no copy number change specific to group 1 or groups 1+2. However, exome sequencing identified a heterozygous TBX1 frameshift mutation (c.1253delA, p.Y418fsX459) specific to groups 1+2, as well as six missense variants and two in-frame microdeletions specific to groups 1+2 and two missense variants specific to group 1. The TBX1 mutation resided at exon 9C and was predicted to produce a non-functional truncated protein missing the nuclear localization signal and most of the transactivation domain. CONCLUSIONS/SIGNIFICANCE: Clinical features in groups 1+2 are well explained by the TBX1 mutation, while the clinical effects of the remaining variants are largely unknown. Thus, the results exemplify the usefulness of exome sequencing in the identification of disease-causing mutations in familial disorders. Furthermore, the results, in conjunction with the previous data, imply that TBX1 isoform C is the biologically essential variant and that TBX1 mutations are associated with a wide phenotypic spectrum, including most of 22q11.2DS phenotypes.

IMAGe syndrome: clinical and genetic implications based on investigations in three Japanese patients.

OBJECTIVE: Arboleda et al. have recently shown that IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome is caused by gain-of-function mutations of maternally expressed gene CDKN1C on chromosome 11p15.5. However, there is no other report describing clinical findings in patients with molecularly studied IMAGe syndrome. Here, we report clinical and molecular findings in Japanese patients. PATIENTS: We studied a 46,XX patient aged 8·5 years (case 1) and two 46,XY patients aged 16·5 and 15·0 years (cases 2 and 3). RESULTS: Clinical studies revealed not only IMAGe syndrome-compatible phenotypes in cases 1-3, but also hitherto undescribed findings including relative macrocephaly and apparently normal pituitary-gonadal endocrine function in cases 1-3, familial glucocorticoid deficiency (FGD)-like adrenal phenotype and the history of oligohydramnios in case 2, and arachnodactyly in case 3. Sequence analysis of CDKN1C, pyrosequencing-based methylation analysis of KvDMR1 and high-density oligonucleotide array comparative genome hybridization analysis for chromosome 11p15.5 were performed, showing an identical de novo and maternally inherited CDKN1C gain-of-function mutation (p.Asp274Asn) in cases 1 and 2, respectively, and no demonstrable abnormality in case 3. CONCLUSIONS: The results of cases 1 and 2 with CDKN1C mutation would argue the following: [1] relative macrocephaly is consistent with maternal expression of CDKN1C in most tissues and biparental expression of CDKN1C in the foetal brain; [2] FGD-like phenotype can result from CDKN1C mutation; and [3] genital abnormalities may primarily be ascribed to placental dysfunction. Furthermore, lack of CDKN1C mutation in case 3 implies genetic heterogeneity in IMAGe syndrome.

A 68-year-old phenotypically male patient with 21-hydroxylase deficiency and concomitant adrenocortical neoplasm producing testosterone and cortisol.

The steroidogenic enzyme 21-hydroxylase is necessary for the synthesis of both glucocorticoids and mineralocorticoids. 21-hydroxylase is a cytochrome P-450 enzyme and is encoded by the gene CYP21A2. Here we report a 68-year-old phenotypically 'male' but genetically female patient with 21-hydroxylase deficiency (21OHD) and the concomitant virilizing adrenocortical carcinoma. This patient grew up as a male and has not encountered any episodes of adrenal insufficiency without glucocorticoid replacement in his lifetime. A chromosome test at admission, however, identified the 46, XX karyotype, and serum 17-hydroxyprogesterone and urine pregnanetriolone and 11ß-hydroxyandrostendione were all elevated, consistent with 21OHD. Moreover, serum testosterone was 1.90 ng/ml, much higher than the female standard levels, and serum cortisol was 5.7 µg/ml, slightly lower than standard levels. Genetic analysis identified the patient as a heterozygote of the two pathogenic mutations in the CYP21A2 gene: IVS2-13C(A)>G and R356W. Magnetic resonance imaging (MRI) revealed the presence of left adrenal tumor measuring 6 cm, which was subsequently diagnosed as adrenocortical carcinoma based on the criteria of Weiss. Immunohistochemical analysis of the tumor specimens revealed the expression of various enzymes involved in testosterone production, including 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17ß-hydroxysteroid dehydrogenase. Importantly, the expression of immunoreactive 21-hydroxylase was detected in these tumor cells. The levels of adrenal tumor-derived steroid metabolites were all markedly decreased following the surgery. This is the first report on a virilized 21OHD patient associated with the adrenocortical tumor that produces testosterone. Moreover, the concomitant adrenocortical tumor may ameliorate adrenocortical insufficiency by producing cortisol.