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.

In vitro effects of ethanol, acetaldehyde and fatty acid ethyl esters on human erythrocytes.

In vitro experiments were performed to determine if ethanol was metabolized by human erythrocytes and to investigate if ethanol or its metabolites, acetaldehyde and fatty acid ethyl esters, affected erythrocyte morphology and stability. No detectable metabolism of ethanol was found in erythrocytes, although ethanol itself caused an elevated rate of spontaneous haemolysis in erythrocyte preparations. Physiologically attainable levels of ethanol were found to stabilize erythrocytes against haemolysis induced by sodium hypochlorite, and the presence of ethanol caused a decrease in erythrocyte reactive oxygen species levels, although the mechanism for such a process is unknown. Both physiologically attainable and higher levels of acetaldehyde had no effects on erythrocyte morphology and stability even after a 16 h exposure. Fatty acid ethyl esters caused structural changes and instability in erythrocytes in vitro, but whether such changes occur in vivo has not been established. The results of these studies suggest that the deleterious effects of ethanol consumption on erythrocytes in vivo may be, at least in part, the result of direct effects of unmetabolized ethanol on erythrocyte components.

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.

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.

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.

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.

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.

Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males.

The androgen receptor (AR) is essential to the normal development of the male internal and external genitalia. Consequently, impairment of AR function can result in undermasculinized genitalia that vary from a completely female appearance to isolated hypospadias. Since in vitro studies demonstrate that AR function is reduced by expansion of the polyglutamine tract within the receptor [AR(Gln)(n)]; this study examined whether longer AR(Gln)(n) repeats are associated with moderate to severe undermasculinization. The average AR(Gln)(n) length of the undermasculinized group (n = 78, median 25, interquartile range 23-26) was significantly greater than that of the control population (n = 850, median 23, inter-quartile range 22-26, P = 0.002). The odds ratio of having >/=23 repeats (as opposed to

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.

Mutation analysis of subjects with 46, XX sex reversal and 46, XY gonadal dysgenesis does not support the involvement of SOX3 in testis determination.

Despite the identification of an increasing number of genes involved in sex determination and differentiation, no cause can be attributed to most cases of 46, XY gonadal dysgenesis, approximately 20% of 46, XX males and the majority of subjects with 46, XX true hermaphroditism. Perhaps the most interesting candidate for involvement in sexual development is SOX3, which belongs to the same family of proteins (SOX) as SRY and SOX9, both of which are involved in testis differentiation. As SOX3 is the most likely evolutionary precursor to SRY, it has been proposed that it has retained a role in testis differentiation. Therefore, we screened the coding region and the 5' and 3' flanking region of the SOX3 gene for mutations by means of single-stranded conformation polymorphism and heteroduplex analysis in eight subjects with 46, XX sex reversal (SRY negative) and 25 subjects with 46, XY gonadal dysgenesis. Although no mutations were identified, a nucleotide polymorphism (1056C/T) and a unique synonymous nucleotide change (1182A/C) were detected in a subject with 46, XY gonadal dysgenesis. The single nucleotide polymorphism had a heterozygosity rate of 5.1% (in a control population) and may prove useful for future X-inactivation studies. The absence of SOX3 mutations in these patients suggests that SOX3 is not a cause of abnormal male sexual development and might not be involved in testis differentiation.

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.

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.

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.

Genetic control of gonadal differentiation.

The study of naturally occurring mutations in humans and induced mutations in mice that cause sex reversal has been instrumental in the cloning and functional analysis of genes involved in gonadal differentiation. Several genes required for this complex developmental process have now been identified. The genes LIM1, WT1 and FTZ-F1 have been demonstrated to be involved in the formation of the gonads prior to their differentiation as testes or ovaries. Subsequent sex-specific gonadal differentiation appears to be mediated by the SRY and SOX9 genes in the testis, and the DAX-1 gene in the ovary.

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.

Hyperferritinaemia in the absence of iron overload.

BACKGROUND: Serum ferritin is normally a marker of iron overload. Ferritin genes are sited at chromosomes 19 and 11. Regulation of ferritin synthesis involves an interaction between an iron regulatory protein (IRP) and part of the ferritin mRNA designated the iron regulatory element (IRE). A disorder of ferritin synthesis resulting in hyperferritinaemia in the absence of iron overload has been described recently. PATIENTS AND METHODS: Hyperferritinaemia in the absence of iron overload was detected in a patient who was investigated for possible haemochromatosis. Serum iron, transferrin saturation, and ferritin concentration were studied in 11 members of this patient's family from three generations. Eight members had DNA samples analysed by direct cycle sequencing of the 5' untranslated region of the L ferritin gene. RESULTS: Six of the family members studied had serum ferritin concentrations greater than 900 micrograms/l. However, serum iron and transferrin saturation were normal in these subjects who all had evidence of cataracts. Three affected family members who had genetic studies of the L ferritin gene on chromosome 19 had an A to G point mutation which was not found in unaffected members. CONCLUSIONS: There was complete concordance between a mutated IRE, cataracts, and hyperferritinaemia in three generations of this family. This family study confirms the finding that hereditary hyperferritinaemia in the absence of iron overload is an autosomal dominant inherited disorder.

Evidence to exclude SOX9 as a candidate gene for XY sex reversal without skeletal malformation.

The skeletal malformation syndrome campomelic dysplasia (CMD1) is caused by mutations within the SOX9 gene or chromosomal rearrangement breakpoints outside SOX9. Approximately three quarters of cases of CMD1 in XY subjects show complete or partial sex reversal. As some mutations cause CMD1 alone and others cause CMD1 and sex reversal, it is conceivable that some mutations might cause sex reversal in the absence of CMD1. In this study, we have investigated this possibility by screening the entire coding region of SOX9 in 30 patients with a spectrum of XY sex reversal phenotypes. No mutations were identified, suggesting that SOX9 should not be considered a candidate gene for XY sex reversal without skeletal malformation.

Mutation analysis of the 2 kb 5' to SRY in XY females and XY intersex subjects.

Mutations in the Y linked testis determining gene SRY cause 46,XY sex reversal. However, only about 15% of cases of 46,XY sex reversal are accounted for by mutations in SRY. In this study we have investigated the possibility that mutations affecting the expression of SRY might cause some of the cases of sex reversal in which the coding sequence of SRY is normal. We have screened 2 kb of DNA immediately 5' to the SRY coding sequence in 49 subjects with varying degrees of 46,XY sex reversal. Two variant bases were identified, one of which was determined to be a polymorphism and the other is unique, but familial.