International reference reagents to standardise blood group genotyping: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. MATERIALS AND METHODS: Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. RESULTS: The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. CONCLUSIONS: The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.

Genetic reference materials and their application to haematology.

Genetic investigations are becoming increasingly useful and widespread in many areas of human health. However, there is a worldwide lack of certified reference materials for use in genetic testing, meaning that tests are being run without well validated controls and new assays are more difficult to develop and validate. We have responded to this challenge by starting a programme of developing genetic reference materials (GRMs) for international accreditation and worldwide distribution. Our approach has been to make materials for disorders where testing is commonplace and genotyping errors have been demonstrated. To ensure a continuing supply of DNA, cell lines are established from consenting, phenotypically well-characterised patients and are then grown up in bulk for genomic DNA extraction to yield up to 100 milligrams of DNA. In most cases the DNA is then formulated, freeze-dried and sealed in glass ampoules to ensure greater stability over time and obviate the need for chilled transportation. In this paper we explore the options and routes available to the production of DNA reference materials and describe the establishment of the first internationally recognised reference materials for human genomic DNA, with particular reference to some genetic tests carried out frequently within haematological and cardiovascular laboratories.

DNA binding activity of recombinant SRY from normal males and XY females.

The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.

Testis-determining factor and Y-linked sex reversal.

A gene named SRY, isolated last year from the sex-determining region of the human Y chromosome, satisfies many of the criteria expected of the testis-determining factor gene. Mutations in SRY have been found in XY females, strongly implicating SRY as the testis-determining gene.

DNA variation and the future of human genetics.

The use of DNA variants in the mapping of the human genome and in the positional cloning of monogenic disease genes is well established. Determining the genetic bases of the more common "multifactorial" diseases, however, presents a major challenge. The genetics of these diseases are complicated by the interplay between many genes and the environment. These investigations will require large numbers of DNA markers and the technology to screen large populations with these markers. The systematic identification of the common DNA polymorphisms in the human genome coupled with the development of high throughput screening methods should allow ultimately the elucidation of the genetic component of most clinical and nonclinical phenotypes.

The SRY gene.

SRY was identified in an attempt to clone the mammalian testisdetermining factor. Evidence in the form of sex-reversing mutations and mouse transgenesis experiments indicates that SRY is the testis-determining factor. The cloning of this gene marks the end of the beginning of our understanding of sex determination. This review covers the structure of the SRY gene and its RNA transcripts, along with evidence equating SRY with the mammalian testis-determining factor, its evolution, and biochemical aspects of the SRY protein.

Tescalcin, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, Col9a3, and renin are expressed in the mouse testis during the early stages of gonadal differentiation.

To identify genes that are differentially expressed in the developing testis we used representational difference analysis of complementary DNA from gonads of mouse embryos at 13.5 days postcoitum (dpc). Three genes were identified. One of them was a novel gene termed tescalcin that encoded a putative EF-hand Ca(2+)-binding protein. The open reading frame consisted of 642 nucleotides encoding a protein with 214 amino acids. Analysis of the predicted amino acid sequence revealed an N:-myristoylation motif and several phosphorylation sites in addition to an EF-hand Ca(2+)-binding domain. TESCALCIN: messenger RNA (mRNA) was present in fetal testis, but not in ovary or mesonephros, and was restricted to the testicular cords. Its expression was first detected in the male gonad at 11.5 dpc and demonstrated a pattern consistent with a role in the testis at the early stages of testis differentiation. Tescalcin is expressed in the testis of Kit(W/W-v) mice, indicating that it is not dependent on the presence of germ cells. The other two genes identified were collagen IX alpha3 (Col9a3) and RENIN: Col9a3 expression was present at low levels in male and female gonads at 11.5 dpc. Thereafter, it was markedly up-regulated in the male, but remained very low in the female. Expression of Col9a3 was restricted to testicular cords and was also detected in testis of Kit(W/W-v) mice. RENIN: mRNA was first detected in testis at 12.5 dpc, increased thereafter, and reached a peak at 16.5 dpc. RENIN: mRNA was localized in cells of the interstitium and cells at the border between the gonad and mesonephros. Expression of RENIN: in the ovary was not detected using standard conditions.

Evidence that longer androgen receptor polyglutamine repeats are a causal factor for genital abnormalities.

Moderate to severe undermasculinized genitalia was recently shown to be associated with longer polyglutamine repeats within the androgen receptor [AR(Gln)n]. However, it was unknown whether this was because longer AR(Gln)n contributed to the: 1) etiology; 2) severity; and/or 3) testicular maldescent. Therefore, AR(Gln)n length in 175 males with abnormal genitalia were analyzed according to etiology (known or unknown), severity (complete, severe, and moderate), or testis position (abdominal, inguinal, or scrotal). Etiology (P = 0.01) and severity (P = 0.02) but not testis position (P = 0.52) were associated with AR(Gln)n length. The association between the severity of the genital abnormalities and AR(Gln)n length was due to the close association of severity with the etiology (P < 0.0001). A highly selected group with moderate to severe genital abnormalities and multiple criteria to exclude known etiological factors had a greater AR(Gln)n length (mean, 25.33) than all other samples (mean, 23.11; P = 0.0004). The results suggest that AR(Gln)n length does not influence the severity of undermasculinization or testis descent but instead contributes to the causation of genital abnormalities in a subset of patients. These findings, together with a demonstrated relationship between severity and multifactorial etiology, are incorporated into a proposed model for the involvement of AR(Gln)n length in genital abnormalities.

Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is the most common single entity that results in male under-masculinization, but large cohort studies of AIS have rarely been performed. Over the last decade, nationwide cooperation between pediatric endocrinologists in the United Kingdom has allowed the creation of a database of cases of intersex and ambiguous genitalia where detailed clinical information on every notified case has been collected via a questionnaire. Among the 816 entries recorded by January 1999, there were 105 clinically diagnosed cases of complete AIS (CAIS) and 173 cases of partial AIS (PAIS). A masculinization score was devised by scoring the external phenotype, and a score of 12 represented normal masculinization. Androgen receptor (AR) binding was determined by studying binding capacity (Bmax) and receptor affinity (K(d)), and cases were classified as either zero, abnormal, or normal binding. Mutation screening of all eight exons of the AR gene was performed by single-strand conformational polymorphism analysis, followed by direct DNA sequencing. All cases of PAIS presented within the first month of birth. The median age at presentation of children with CAIS was 1 yr (P10,P90: 0.1,10.4). The testes were palpable in the labioscrotal folds or the inguinal region in 77% and 41% of cases of CAIS and PAIS, respectively. There was marked overlap between the masculinization score of those children with PAIS reared as girls [2.5(P10,P90:1, 6)] and those reared as boys [3(P10,P90:2, 7.5)]. Gonadectomy was performed prepubertally in 66% and postpubertally in 29% of the cases of CAIS. The median age of the latter group was older at 14 yr (P10,P90:0.1,18). No cases of malignancy or carcinoma in situ were reported in the 121 cases of AIS where histology results were available. Biochemical endocrine investigations were reported to have been performed in a greater number of cases of PAIS than CAIS (98% vs. 48%). AR binding was abnormal in 44 of 51 (86%) and 40 of 113 (35%) cases of CAIS and PAIS, respectively. Zero binding was encountered in 29 of 43 (67%) and 1 of 55 (2%) cases of CAIS and PAIS, respectively. Mutational analysis of the AR gene, performed in 102 index cases was positive in 57 of 69 (83%) cases of CAIS and 12 of 43 (28%) cases of PAIS. In 24 of these cases, the mutation identified was novel. The mutations in PAIS cases were all missense, whereas in CAIS the mutations were more diverse. AR binding was only normal in 3 of 69 mutation-positive cases. In the PAIS group, mutation-positive cases had a significantly higher Kd and Bmax compared to the mutation negative cases. The clinical diagnosis of AIS can be confirmed in a significant number of cases by a combination of androgen-binding studies and mutational analysis. There is some correlation between the phenotypic features and the abnormalities discovered on mutational analysis of the AR gene, but there is a need to improve this further by developing optimal bioassays of AR function. The phenotypic heterogeneity among clinically diagnosed cases of AIS emphasizes the need for appropriate comprehensive evaluation of male under-masculinization.

Clinical and molecular evidence for the role of androgens and WT1 in testis descent.

Testicular maldescent is a common congenital disorder associated with testicular cancer and infertility. In this study, testis position was assessed in subjects with genital abnormalities due to AR mutations, Denys-Drash and WAGR syndromes or an unknown aetiology. Subjects with completely female genitalia and an AR mutation or an unknown aetiology had a greater proportion of maldescended testes (intra-abdominal and inguinal) than those with less severe abnormalities (P=0.00027 and P<0.000001, respectively). Whereas subjects with severe, moderate or mild abnormalities and an unknown aetiology, had similar testis positions. The Denys-Drash and WAGR syndrome group had a greater proportion of maldescended testes than the AR mutation (P=0.013) and unknown aetiology groups (P=0.00019). Androgen production and AR binding were normal in three subjects with Denys-Drash and WAGR syndromes. These findings indicate that the relationship between testis descent and genital abnormalities is a multi-factorial process with greater complexity than previously proposed.

Candidate genes in complete and partial XY sex reversal: mutation analysis of SRY, SRY-related genes and FTZ-F1.

The sexual phenotype is established in three steps: (1) the sex chromosome constitution; (2) the differentiation of the gonads; and (3) the response of the internal and external genitalia to the hormones produced by the differentiated gonads. Errors that occur at any of these stages can result in defective sexual differentiation. Therefore the investigation of patients with abnormalities of testis development will help elucidate the mechanisms of sex determination and gonadal differentiation. It was in this way that SRY. the primary testis determining gene was identified. De novo mutations in SRY, result in gonadal dysgenesis by disrupting the DNA-binding activity of the SRY protein. However, only 20% of cases of gonadal dysgenesis, are explained by mutations in SRY or its flanking sequences. Therefore, there are several pieces to this puzzle yet to be discovered and it is hoped that mutation analysis of other genes implicated in gonadal development and differentiation may shed some light on aetiology of gonadal dysgenesis in the remaining 80% of cases.

Developmental aspects of androgen action.

The formation of a testis from the indifferent gonad is the prelude to sequential steps in male sex differentiation orchestrated by time-dependent androgen biosynthesis and action. Information about the cellular and molecular mechanisms of androgen action can be obtained by the study of disorders of sex differentiation in males. The pivotal role of the androgen receptor as a ligand-induced transcription factor is emphasised and preliminary studies are described which attempt to identify developmentally regulated androgen-responsive genes. That androgen action can be modulated by gene polymorphisms is illustrated by the influence of an androgen receptor polyglutamine repeat in the multi-factorial causation of less severe forms of male under-masculinization.

Genetic analysis of the INSL3 gene in patients with maldescent of the testis.

OBJECTIVE: Testicular maldescent is important because it is a common congenital disorder that is associated with an increased risk of infertility and testicular cancer. Murine studies indicate that testicular maldescent can result from disruption of insulin-like factor 3 (INSL3) activity and that it may be more severe when there is concurrent undermasculinisation. Therefore, the INSL3 gene was screened for mutations and polymorphisms that may contribute to testicular maldescent in patients with undermasculinisation as well as those with isolated testicular maldescent. METHODS AND RESULTS: The patient groups consisted of individuals with isolated testicular maldescent (n=28) and patients with undermasculinised genitalia and intra-abdominal (n=24) or inguinal gonads (n=33). The three control groups were: normal males (n=15), males with undermasculinised genitalia and scrotal gonads (n=29) and females (n=82). SSCP/HA mutation screening detected eight variants, five of which were predicted to alter the protein sequence (A-1G, V19L, P25S, A36T, R78H). Three of the amino acid changes (A-1G, V19L, R78H) each occurred in a single control sample and one was identified in a male with undermasculinised genitalia and intra-abdominal testes (P25S). The A36T amino acid polymorphism was found in both patient and control groups at a similar frequency. CONCLUSIONS: The evidence suggests that INSL3 mutations and polymorphisms are not a major cause of testicular maldescent with or without associated undermasculinisation.

Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs.

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

Factors influencing the amount and type of dental services received by older adults in four municipalities in Ontario, Canada.

OBJECTIVES: The purpose of this study was to investigate the factors influencing the pattern (amount and types) of dental care provided to dental visitors participating in the baseline stage of the Ontario Study of the Oral Health of Older Adults. METHODS: We interviewed and examined older adults from four municipalities in Ontario and obtained dental service records for the previous two years for those reporting a dental visit. Individual services and their corresponding time and values were aggregated into categories. We compared the pattern of care for dentate and edentulous subjects, and by age, municipality of residence, and social, demographic and economic characteristics. RESULTS: The 473 subjects received 5,031 services, more than four hours of dental care per subject over the two years. The 29 edentulous subjects received about one-third the number of services compared with the dentate; however, there were no differences in relative time units (RTUs) or relative value units (RVUs). Among the dentate the amount of care (RVUs) varied by municipality of residence and by visiting behavior, but not by age, income, or other socioeconomic variables. CONCLUSIONS: Both dentate and edentulous visitors received over two hours of care per person per year. Dental care patterns were influenced by dental status, area of residence, and visiting behaviors.

Sexual dimorphism in the neonatal gonad.

The neonatal gonad has two distinct forms (i.e., is sexually dimorphic), as judged by morphological and endocrine characteristics. The dimorphic process begins early in embryogenesis. It is well established by the time of birth, by which time the genital ridge has developed into either a testis or an ovary. The mechanisms involved in sex determination involve the Y chromosome, autosomal genes, transcription factors and possibly other unidentified control networks. This review paper describes the morphological changes that occur and the endocrine functions in the developing gonads. It highlights a number of important differences in fetal and neonatal gonadal function. The testis has early histological definition, several determining genes, delayed germ cell maturation, early autonomous steroid secretion, luteinizing hormone (LH) receptor and steroid enzyme expression, high fetal testicular testosterone content, prominent postnatal Leydig and Sertoli cells and high postnatal serum testosterone levels. The ovary has a prolonged monomorphic state, probably one determining gene, germ cells in early meiotic arrest, delayed expression of LH receptor and aromatase, low ovarian oestradiol content, prominent postnatal follicles and low postnatal serum oestradiol levels.

Sex determination.

The cloning of the 'testis determining gene', SRY, promised a revolution in the understanding of sex determination in humans. The failure to isolate further genes involved in sex determination has been a disappointment. The biology of SRY, however, has kept the field exciting. The discoveries of sex reversing SRY mutations with variable penetrance, but with full expressivity, rapid SRY sequence evolution and circular Sry transcripts could not have been predicted.

Cloning and mutational analysis of SRY.

A candidate for the male sex-determining gene has recently been isolated. This sex-determining gene (SRY) has been found to be mutated in some individuals with failed testis development, and, in mouse transgenesis, the SRY murine homologue (Sry) causes female-to-male sex reversal. The cloning of SRY should facilitate the characterisation of other genes in the testis-determining pathway and provide information on the mechanism of mammalian developmental decisions.