Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.     

Molecular analysis of Japanese patients with steroid 21-hydroxylase deficiency

We have designed a rapid and convenient strategy to determine nine of the most common mutations in the 21-hydroxylase gene (CYP21). The frequency of the mutations was investigated in 34 Japanese patients affected with congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency. We characterized 82% of the CAH chromosomes. The most frequent mutations were a C/A to G substitution in intron 2 in the salt-wasting form of the disease and an I172N in the simple virilizing form. Three de novo mutations were found. Two homozygous mutations (S268T and N493S) were detected by direct sequencing of all exons of CYP21 in two siblings, who had a normal genotype at all positions screened. We successfully applied these methods for prenatal diagnosis in one family. These procedures proved to be sensitive and rapid for the detection of the most common known mutations in the CYP21 gene and may be useful for genetic screening. Received: March 29, 1999 / Accepted: May 11, 1999     

CYP21 mutations and congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder caused mainly by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene on chromosome 6p21.3. The wide range of CAH phenotypes is associated with multiple mutations known to affect 21-hydroxylase enzyme activity. To date, 56 different CYP21 mutations have been reported, mostly point mutations, but small deletions or insertions have been described too, as well as complete gene deletions. Fifteen mutations, constituting 90-95% of alleles, are derived from intergenic recombination of DNA sequences between the CYP21 gene and the highly homologous CYP21P pseudogene, while the remaining are spontaneous mutations. A reliable and accurate detection of CYP21 mutations is not only important for clinical diagnosis, but also for carrier detection as there is a high variability in the basal level of 17-hydroxyprogesterone between normal and heterozygous individuals. Several strategies based on polymerase chain reaction (PCR)-driven amplification with allele-specific oligonucleotides to the CYP21 gene have been developed. It has been demonstrated that one reaction for PCR amplification of the CYP21 gene and the chimeric CYP21P/CYP21 gene using mixed primers in combination with nested PCR and single-strand conformation polymorphism is considered highly efficient and accurate for molecular diagnosis of CAH due to 21-hydroxylase deficiency.     

Screening of CYP21 gene mutations in 129 French patients affected by steroid 21-hydroxylase deficiency

The frequency of 12 different mutations of the steroid 21-hydroxylase gene (CYP21) was investigated in 129 French patients affected by congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency. Eighty-nine percent of the CAH chromosomes were characterized. The most frequent mutations were a C-G substitution in intron 2, the deletion of the CYP21 gene and a T-A substitution in exon 4 in the severe form of the disease, and a G-T substitution in exon 7 in the nonclassic form. The correlation between the genotypes and the clinical forms of the disease showed marked variation in the phenotype from a single genotype, suggesting that individual variation and undetected additional mutations on the same CAH chromosome accounted for the phenotype. In 65 informative meioses of CAH families, no de novo mutation was found. © Wiley-Liss, Inc.     

Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees

Steroid 21-hydroxylase deficiency is among the most common inborn errors of metabolism in man. Characterization of mutations in the 21- hydroxylase gene (CYP21) has permitted genetic diagnosis, facilitated by the polymerase chain reaction (PCR). The most common mutation is conversion of an A or C at nt656 to a G in the second intron causing aberrant splicing of mRNA. Homozygosity for nt656G is associated with profoundly deficient adrenal cortisol and aldosterone synthesis, secondary hypersecretion of adrenal androgens, and a severe form of congenital adrenal hyperplasia (CAH) characterized by ambiguous genitalia and/or sodium wasting in newborns. During the course of genetic analysis of CYP21 mutations in CAH families, we and others have noticed a number of relatives genotyped as nt656G homozygotes, yet showing no clinical signs of disease. A number of lines of evidence have led us to propose that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one haplotype during PCR amplification of CYP21. For prenatal diagnosis, we recommend that microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nt656.      

A novel 13-bp deletion in exon 1 of CYP21 gene causing severe congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase gene (CYP21). In most cases, this defect is the result of gene conversion events between the functional CYP21 gene and the adjacent inactive pseudogene (CYP21P). Previous screening for mutations of 21-hydroxylase gene in 51 unrelated Tunisian CAH patients revealed 4 novel mutations that have not been reported to occur in the CYP21P pseudogene. The present paper describes the fifth new small 13-bp deletion in exon 1 found after sequencing the CYP21 gene of a Tunisian patient suffering from the salt-wasting form of CAH. The patient is a girl born to consanguineous parents; she is homozygous for a novel deletion. The 13-bp deletion causes a stop codon at amino acid 47, which is likely to result in an enzyme with no activity. Both parents are heterozygous for the small deletion as confirmed by nested PCR method. This novel mutation has not been reported to occur in the CYP21P pseudogene, indicating a casual mutagenic event rather than a conversion one.     

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.     

Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia.

The majority of congenital adrenal hyperplasia (CAH) cases arise from mutations in the steroid 21-hydroxylase (CYP21) gene. Without reliance on HLA gene linkage analysis, we have developed primers for differential polymerase chain reaction (PCR) amplification of the CYP21 gene and the non-functional CYP21P gene. Using the amplification created restriction site (ACRS) approach for direct mutational detection, a secondary PCR was then performed using a panel of primers specific for each of the 11 known mutations associated with CAH. Subsequent restriction analysis allowed not only the detection but also the determination of the zygosity of the mutations analysed. Existing deletion of the CYP21 gene could also be detected. In the analysis of 20 independent chromosomes in 11 families of CAH patients in Taiwan, four CYP21 mutation types, besides deletion, were detected. Interestingly, in five different alleles, the CYP21P pseudogene contained some polymorphisms generally associated with the CYP21 gene. These results suggest gene conversion events that are occurring in both CYP21P and CYP21 genes. Our combined differential PCR-ACRS protocol is simple and direct and is applicable for prenatal diagnosis of CAH using chorionic villi or amniotic cells.     

Duplication of 111 bases in exon 1 of the CYP21 gene is combined with deletion of CYP21P-C4B genes in steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. A 9.3-kb fragment generated by NdeI and AseI digestion by Southern blot analysis indicated that a consequence of deletion of the C4-CYP21 repeat module was the production of a distinct chimeric CYP21P/CYP21 molecule. In the present study, we report a novel CYP21 genotype in two CAH families in which the gene appeared as 9.4- and 3.3-kb fragments by TaqI digestion, rather than as a chimeric gene. From the analysis of PCR amplification patterns and DNA sequencing, we found that there was a duplication of 111 bases from codons 21 to 57 inserted at codon 58 in exon 1 of the CYP21 gene. In addition, codon 21 in the repeated sequence changed from TGG to AGG. Furthermore, this novel CYP21 gene present in both CAH families showed no mutations at IVS2-12A/C>G, 707-714delGAGACTAC, and P30L. Interestingly, the 5' end region of these two CYP21 genes showed the sequence of the CYP21P gene at nucleotides (nt) -103, -110, -123, and thereafter. Our data suggest that these two CYP21 genes are caused by deletion of the CYP21P, XA, RP2, and C4B genes. Possibly, the additional 111-base duplicated coding sequence may be generated by multiple intergenic recombinations, while there seems to be no relationship with deletion of the CYP21P-C4B regions.     

A novel frameshift mutation (141delT) in exon 1 of the 21‐hydroxylase gene (CYP21) in a patient with the salt wasting form of congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disease with a wide range of clinical manifestation. In 90‐95% of the cases it is caused by 21‐hydroxylase deficiency (OMIM #201910) due to mutations of the CYP21 gene (GDB Accession #M12792). In most cases the CYP21‐inactivating point mutations are transferred by apparent gene conversions from CYP21P to CYP21. In only a few cases point mutations have been described, which are not present in the pseudogene. Using Southern blot analysis and DNA sequencing we have identified a novel mutation (141delT) of the CYP21 gene in a patient suffering from the salt wasting form of CAH. This results in a premature termination of a truncated protein at amino acid position 51 (L51X), which is likely to result in an enzyme with no activity. This novel mutation has not been reported to occur in the CYP21P alleles and it was not found in the CYP21P alleles in this CAH family. Hum Mutat 14:90–91, 1999. © 1999 Wiley‐Liss, Inc.     

21-羟化酶缺乏导致肾上腺皮质增生症的研究进展

先天性肾上腺皮质增生症（Congenital adrenal hyperplasia,CAH）属于常见常染色体隐性遗传病,有着广泛的临床表现。CYP21A2基因是先天性肾上腺皮质增生症的致病基因,90％-95％的21-羟化酶缺乏症患者在CYP21A2基因上存在有害突变。了解CYP21A2基因编码区的常见突变谱和突变热点,主要包括基因的点突变、小缺失、小插入和完全重组等,同时分析基因型与表现型的关系,有着重要的意义。本研究在阐述2-羟化酶缺乏症的分子基础上,就近几年国内外21-羟化酶缺乏症相关内容进行简要综述。     

Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed "apparent gene conversion," from the neighboring CYP21Ppseudogene. A chimeric CYP21P/CYP21gene with its 5' end corresponding to CYP21P and 3' end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with -300 nucleotides of the 5' head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21Pand CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes.     

Clinical and genetic analysis of patients with X‐linked hyper‐IgM syndrome

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency (21‐OHD) is a common autosomal recessive disorder caused by mutations in the CYP21A2 gene. The carrier frequency of CYP21A2 mutations has been estimated to be 1:25 to 1:10 on the basis of newborn screening. The main objective of this study was to determine the carrier frequency in the Cypriot population of mutations in the CYP21A2 gene. Three hundred unrelated subjects (150 males and 150 females) from the general population of Cyprus were screened for mutations in the CYP21A2 gene and its promoter. The CYP21A2 genotype analysis identified six different mutants and revealed a carrier frequency of 9.83% with the mild p.Val281Leu being the most frequent (4.3%), followed by p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) and p.Met283Val (0.17%). The notable high CYP21A2 carrier frequency of the Cypriot population is one of the highest reported so far by genotype analysis. Knowledge of the mutational spectrum of CYP21A2 will enable to optimize mutation detection strategy for genetic diagnosis of 21‐OHD not only in Cyprus, but also the greater Mediterranean region.     

CYP21A2 mutations in Portuguese patients with congenital adrenal hyperplasia: identification of two novel mutations and characterization of four different partial gene conversions

More than 90% of congenital adrenal hyperplasia (CAH) cases are caused by 21-hydroxylase deficiency. In this study, the CYP21 gene was genotyped in 56 Portuguese unrelated patients with clinical symptoms of 21-hydroxylase deficiency, in a total of 112 independent alleles. CYP21A2 mutations were identified in 99.1% of the alleles. The most common point mutation was 1688G>T (25.9%). A previously unreported partial gene conversion, extending from exon 1 to 7, was found in 16.1% of the alleles, in most cases associated to the mutation 1688G>T in the other chromosome, and in patients with nonclassical CAH. Other three distinct partial gene conversions were also identified, with lower frequencies: one extends from exon 1 to 3 and the others from exons 3 to 7 and 3 to 8. Two novel mutations were identified in two salt-wasting patients: a putative splicing mutation, IVS2+5G>A, and the transition 2557C>T, that gives rise to the nonsense mutation R445X. Seven point mutations and a partial gene conversion were responsible for 88 of the studied disease causing alleles, and the overall concordance between genotype and phenotype was 92.9%. With this study the molecular basis of CAH was characterized, for the first time, in Portuguese patients, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling.     

Genotype of congenital adrenal hyperplasia patients with testicular adrenal rest tumor

Testicular adrenal rest tumor (TART) is one of the important complications that can cause infertility in male patients with congenital adrenal hyperplasia (CAH) and should therefore be diagnosed and treated at an early age. The factors that result in TART in CAH have not been completely understood. The aim of this study is to evaluate the genotype-phenotype correlation in CAH patients with TART.MethodAmong 230 malepatients with CAH who were followed upwith regular scrotal ultrasonography in 11 different centers in Turkey, 40 patients who developed TARTand whose CAH diagnosis was confirmed by genetic testing were included in this study. Different approaches and methods were used for genotype analysis in this multicenter study. A few centers first screened the patients for the ten most common mutations in CYP21A2 and performed Sanger sequencing for the remaining regions only if these prior results were inconclusive while the majority of the departments adopted Sanger sequencing for the whole coding regions and exon-intron boundaries as the primary molecular diagnostic approach for patients with either CYP21A2 orCYP11B1 deficiency. The age of CAH diagnosis and TART diagnosis, type of CAH, and identified mutations were recorded.ResultsTART was detected in 17.4% of the cohort [24 patients with salt-wasting (SW) type, four simple virilizing type, and one with nonclassical type with 21-hydroxylase (CYP21A2) deficiency and 11 patients with 11-beta hydroxylase (CYP11B1) deficiency]. The youngest patients with TART presenting with CYP11B1 and CYP21A2 deficiency were of 2 and 4 years, respectively. Eight different pathogenic variants in CYP21A2were identified. The most common genotypes were c.293-13C>G/c.293-13C>G (31%) followed by c.955C>T/c.955C>T(27.6%) and c.1069C>T/c.1069C>T (17.2%). Seven different pathogenic variants were identified in CYP11B1. The most common mutation in CYP11B1 in our study was c.896T>C (p.Leu299Pro).ConclusionWe found that 83% TART patients were affected with SW typeCYP21A2 deficiency,and the frequent mutations detected were c.955C>T (p.Gln319Ter), c.293-13C>G in CYP21A2 and c.896T>C (p.Leu299Pro) inCYP11B1. Patients with CYP11B1 deficiency may develop TART at an earlier age. This study that examined the genotype–phenotype correlation in TART may benefit further investigations in larger series.     

Mutations in 21-hydroxylase gene caused by gene conversion-like events

Two steroid 21-hydroxylase genes (CYP21A and CYP21B) alternate in tandem with two genes for the fourth component of complement (C4A and C4B) on the short arm of chromosome 6 between the loci of HLA-B and HLA-DR. The CYP21B gene encodes an adrenal microsomal cytochrome P-450, which is specific for steroid 21-hydroxylation (P450c21). A defect of this protein would cause 21-hydroxylase deficiency, which is an autosomal recessive disease and is the most common cause of congenital adrenal hyperplasia (CAH). On the other hand, the CYP21A gene, which is homologous to the CYP21B gene up to 98% in the nucleotide sequences, is a pseudogene due to several mutations in the coding region. One of the mutations is a C----T change leading a termination codon, TAG, in the 8th exon. 1) I cloned a CYP21B gene from a patient homozygous for HLA-Bw75-DRw9 by descent. I found a C----T change in the 8th exon of the CYP21B gene. This mutation would prevent a synthesis of 21-hydroxylase and was thought to be a crucial change to cause CAH in this patient. Because there was no apparent gross change in the organization of the C4-CYP21 region and this mutation is usually found in the CYP21A pseudogene, it seemed that a gene conversion-like event transferred the mutation from the CYP21A gene to the CYP21B gene. 2) A population study on the organization of C4-CYP21 region revealed that a reciprocal change, i.e. a T----C change in the 8th exon of the CYP21A gene, was observed in two HLA haplotypes, HLA-B44-DRw13 and HLA-Bw46-DRw8 haplotypes in Japanese population. The reciprocal changes also may be considered as a result of gene conversion-like events.     

A rapid screening for steroid 21‐hydroxylase mutations in patients with congenital adrenal hyperplasia

Steroid 21‐hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH). CAH due to 21‐hydroxylase deficiency is divided into three classes: salt‐wasting (classical), non‐classical and simple virilizing, reflecting different degrees of clinical severity. Using polymerase chain reaction (PCR) and allele‐specific oligonucleotide hybridisation (ASO), we screened the DNA of 62 Caucasian CAH families (heterozygous parents and children) for 14 different and frequently‐found CYP21‐mutations (HGMD). Of the 62 patients (21 males, 41 females), 26 females and 11 males had the classical or salt‐wasting form, 3 females and 1 male had the non‐classical form and 14 females and 7 males had simple virilizing CAH. More than 60% of the patients were compound‐heterozygous. We found the mutations on 110 alleles (out of 124 alleles). There were 30 CYP21 gene deletions/conversions, 3 substitutions (P30L) in exon 1, 30 splice mutations (c.93‐13A/C>G) in intron 2, 26 point mutations (I172N) in exon 4, one cluster of mutations (I236N, V237E, M239K) in exon 6, 8 mutations (V281L and 1760‐1761insT) in exon 7, and 8 nonsense (Q318X) and 4 missense (R356W) mutations in exon 8. Our study supports the case for using this rapid technique for CAH‐family screening as long as alleles from both affected patients and parents are screened in parallel. Hum Mutat 13:505, 1999. © 1999 Wiley‐Liss, Inc.     

Genetic diagnosis of 21‐hydroxylase deficiency: DGGE‐based mutation scanning of CYP21

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21‐hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported, and still novel mutations are detected. This makes genetic diagnosis as well as carrier detection of 21‐hydroxylase deficiency a complicated matter. We developed a simple nonradioactive assay based on the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the CYP21 gene. DGGE allows a fast scanning of PCR‐amplified segments of genes for the presence or absence of any single base pair alterations. We have performed this technique on the coding sequence and intron‐exon junctions of CYP21. Our results emphasize that this procedure constitutes a fast and reliable approach when performing diagnosis of 21‐hydroxylase deficiency. Hum Mutat 13:385–389, 1999. © 1999 Wiley‐Liss, Inc.     

21-Hydroxylase deficiency: Mutational spectrum and Genotype–Phenotype relations analyses by next-generation sequencing and multiplex ligation-dependent probe amplification

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is autosomal recessive disorder of cortisol biosynthesis. Genetic defects in CYP21A2 cause 21OHD. The aim of this study was to determine spectrum of mutations in CYP21A2 in a large cohort and analyze the genotype-phenotype correlation to assess predictive characteristics of genotype. We investigated a total of 113 patients with 21OHD. Next-generation sequencing and Multiplex ligation-dependent probe amplification of the CYP21A2 gene were performed in patients and their parents. The genotypes were categorized into Groups 0, A, B, and C according to the residual 21-hydroxylase activities. In this study, the group A was divided into two subgroups as A1 and A2. Three novel variants were found. The genotype–phenotype correlation of the mutation classification was 91.5%. Positive predictivity of subgroups A1 was higher than groups A and subgroups A2. Our study reports genotype–phenotype correlations in the largest 21OHD cohort in Turkey. This correlation sustained when we analyzed our data in combination with metadata from other published studies. This study confirms that CYP21A2 genotyping with next-generation sequencing and MLPA can accurately and reliably confirm the diagnosis of 21OHD. We propose a new classification by dividing group A into two new subgroups to better predict the phenotype. In light of this very high genotype-phenotype correlation, with their ever-increasing availability, declining cost, and turnaround time, we propose that molecular genetic studies can be more economical and practical alternative to the current initial diagnostic laboratory studies based on assays of intermediary steroid metabolites.     

Detection and assignment of TP53 mutations in tumor DNA using peptide mass signature genotyping

This report describes the application of a new approach to tumor genotyping called peptide mass signature genotyping (PMSG) that is particularly suited to detecting minority sequences in a DNA sample. Detecting minority sequences is essential for accurate tumor genotyping because tumor resections are generally a mixture of malignant and non-malignant cells, with the mutations of interest often outnumbered by the corresponding wild-type alleles. To explore the suitability of PMSG for tumor genotyping, 25 human squamous cell carcinomas of the head and neck, as well as a set of cell lines derived from those tumors, were analyzed for mutations in exons 5 to 8 of the TP53 gene, the exons that encode the DNA-binding domains of the p53 protein. PMSG identified mutations in 11 tumor DNA samples, whereas dideoxy sequencing of the same samples detected mutations in only four. Currently, PMSG can be used to detect mutations that are present in only 20% of the sample DNA, and we expect that this threshold will be lowered significantly as the PMSG process is improved. Hum Mutat 22:158-165, 2003.