Collagen type IV-related nephropathies in Portugal: pathogenic COL4A3 and COL4A4 mutations and clinical characterization of 25 families.

Pathogenic mutations in genes COL4A3/COL4A4 are responsible for autosomal Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). We used Sanger sequencing to analyze all exons and splice site regions of COL4A3/COL4A4, in 40 unrelated Portuguese probands with clinical suspicion of AS/TBMN. To assess genotype-phenotype correlations, we compared clinically relevant phenotypes/outcomes between homozygous/compound heterozygous and apparently heterozygous patients. Seventeen novel and four reportedly pathogenic COL4A3/COL4A4 mutations were identified in 62.5% (25/40) of the probands. Regardless of the mutated gene, all patients with ARAS manifested chronic renal failure (CRF) and hearing loss, whereas a minority of the apparently heterozygous patients had CRF or extrarenal symptoms. CRF was diagnosed at a significantly younger age in patients with ARAS. In our families, the occurrence of COL4A3/COL4A4 mutations was higher, while the prevalence of XLAS was lower than expected. Overall, a pathogenic COL4A3/COL4A4/COL4A5 mutation was identified in >50% of patients with fewer than three of the standard diagnostic criteria of AS. With such a population background, simultaneous next-generation sequencing of all three genes may be recommended as the most expedite approach to diagnose collagen IV-related glomerular basement membrane nephropathies.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.

BACKGROUND: Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype-phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. METHODS: Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. RESULTS: The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype-phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. CONCLUSION: A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.

Meiotic segregation of Robertsonian translocations ascertained in cleavage-stage embryos--implications for preimplantation genetic diagnosis.

BACKGROUND: The aim of this study was to ascertain the prevalence of meiotic segregation products in embryos from carriers of 13/14 and 14/21 Robertsonian translocations and to estimate the predictive value of testing single cells using the fluorescence in situ hybridization (FISH) technique, to provide more information for decision-making about PGD. METHODS: In this prospective cohort study, the copy number of translocation chromosomes in nuclei from lysed blastomeres of cleavage-stage embryos was ascertained using locus-specific FISH probes. Logistic regression analysis, controlling for translocation type, female age and fertility status, was used to calculate the odds ratio (OR) of unbalanced segregation products for female and male heterozygotes. The primary diagnostic measure was the predictive value of the test result. The primary outcome measure was the live birth rate per couple. RESULTS: Female carriers were four times more likely than male carriers to produce embryos with an unbalanced translocation product (OR 3.8, 95% confidence interval 2.0-7.2, P < 0.001). The prevalence of abnormality for the chromosomes tested in embryos from female or male heterozygotes was estimated to be 43 or 28%, respectively, while estimates of the predictive value were 93-100 or 96-100% for a normal test result and 79 or 57% for an abnormal test result. The live birth rate per couple was 58% for female carriers and 50% for male carriers. CONCLUSIONS: For female carriers, PGD using FISH could reduce the risk of miscarriage from either translocation or the risk of Down syndrome from the 14/21 Robertsonian translocation. PGD using FISH for male carriers is unlikely to be indicated given the relatively low prevalence of chromosome imbalance and low predictive value.

Renal chloride channel, CLCN5, mutations in Dent's disease.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dent's disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X-linked renal chloride channel gene, CLCN5, which encodes a 746-amino-acid protein with 12-13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dent's disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single-stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag --> tgaag. The mutations were confirmed either by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dent's disease and the results will help to elucidate further the functional domains of this novel chloride channel.

Two sibs with anophthalmia and pulmonary hypoplasia (the Matthew-Wood syndrome).

We describe two sibs with pulmonary hypoplasia and anophthalmia; one also had a number of other malformations. Only one other broadly similar case could be found in the literature, and it was an isolated occurrence. The condition is named the Matthew-Wood syndrome.

Becker muscular dystrophy presenting with complete heart block in the sixth decade.

Becker muscular dystrophy may be associated with myocardial abnormalities which are usually diagnosed after the onset of weakness. We present a patient who developed complete heart block 6 years before the onset of muscle weakness which occurred unusually late at the age of 62 years.

Phenotypic diversity in the Smith-Lemli-Opitz syndrome.

The phenotype of four cases of Smith-Lemli-Opitz syndrome (SLO) with proven defects in cholesterol biosynthesis are compared, and shown to be markedly disparate even between sibs, and demonstrate the dilemma for the clinician. The advent of a biochemical test for SLO has been enormously valuable in determining which patients are truly affected by the condition, but because of the wide phenotypic variation, a diagnosis on clinical features alone remains problematic.

Joint and skin laxity with Dandy-Walker malformation and contractures: a distinct recessive syndrome?

We report the case of a girl who has joint and skin laxity with atrophic scarring, and was diagnosed at birth with a Dandy-Walker malformation. She subsequently developed joint contractures, hydrocephalus and syringomyelia. This case shows some similarities to Ehlers-Danlos syndrome type VI, but with no evidence of lysyl hydroxylase deficiency or ocular fragility. It is likely that she represents a distinct and recognizable syndrome. There was parental consanguinity and a subsequent pregnancy resulted in a similarly affected fetus, suggesting autosomal recessive inheritance.

Clinical and counselling implications of preimplantation genetic diagnosis for Huntington's disease in the UK.

Huntington's disease is an autosomal dominant neurodegenerative disorder that usually occurs in adult life. Individuals at risk can have a gene test before the onset of symptoms, and prenatal diagnosis is available. Preimplantation genetic diagnosis (PGD) for Huntington's disease is now available for couples in whom one partner has the gene for Huntington's disease. A licence to practise PGD is required from the Human Fertilisation and Embryology Authority, and there are several complex issues relating to PGD for Huntington's disease that require consideration. The partner of the Huntington's disease gene carrier should have a presymptomatic test to ensure accuracy in a PGD cycle. There should be a delay between blood sampling and testing for Huntington's disease to allow time for reflection and withdrawal from testing. All PGD treatment has an associated risk of misdiagnosis. If confirmatory prenatal testing is not undertaken after a successful PGD cycle, no confirmation of diagnosis will be obtained at birth. Guidelines indicate that individuals who are at risk cannot be tested before 18 years. There is concern over the ability of a child or adolescent to make an informed choice about testing before this age. Confirmatory testing at birth after PGD would be in direct contravention of these guidelines. In the UK, the law requires consideration of the welfare of children born after assisted conception treatment. Presenting symptoms of Huntington's disease may affect the parenting abilities of an affected individual. There is a need for an assessment of a patient's current Huntington's disease status and their planned provision of care of children if Huntington's disease affects parenting. It has been necessary to create a detailed working protocol for the management of PGD for Huntington's disease to address these issues.

Three hundred and thirty cycles of preimplantation genetic diagnosis for serious genetic disease: clinical considerations affecting outcome.

OBJECTIVE: To report on our experience with preimplantation genetic diagnosis (PGD) cycles performed for serious genetic disease in relation to the clinical factors affecting outcome. DESIGN: Retrospective review of data from a single centre. SETTING: Tertiary referral PGD centre in a London teaching hospital. METHODS: The PGD cycles included 172 cycles for chromosome rearrangements, 96 cycles for single-gene disorders and 62 cycles for X-linked disorders. In vitro fertilisation was the preferred method in chromosome rearrangement and X-linked cases, while intra cytoplasmic sperm injection was used in all single-gene disorders. Appropriate in situ hybridisation fluorescence probes were used in chromosome rearrangement and X-linked cases and polymerase chain reaction was used in single-gene disorders. All pregnancies were followed till delivery. MAIN OUTCOME MEASURE: Live birth rate per PGD cycle started. RESULTS: Eighty-six percent of cycles started (283) reached oocyte retrieval and 3743 eggs were collected, of which 2086 fertilised normally (55.7%). Two hundred and fifty cycles (76%) had embryos sutiable for biopsy on day 3 of in vitro culture, 1714 embryos were biopsied, and in 205 cycles (62%), there was at least one unaffected embryo available for transfer, resulting in 90 pregnancies, 68 clinical pregnancies and 58 live birth. The live birth rate was 18% per cycle started, 21% per egg retrieval and 28% per embryo transfer which significantly affected the live birth outcome. Woman age, number of eggs collected and achieving cryopreservation of surplus embryos had no statistically significant effect on treatment outcome. CONCLUSIONS: The live birth outcome of PGD cycles for serious genetic disorder is modest and is affected by the number of embryos genetically suitable for transfer.

Alport's syndrome.

Alport's syndrome (AS) is a progressive glomerulonephritis which is associated with high tone sensorineural deafness and characteristic eye signs. It accounts for 0.6% of all patients who start renal replacement therapy in Europe, and is most commonly inherited as an X linked disorder with a gene frequency of 1 in 5000. During the last six years several type IV collagen genes have been implicated in the aetiology of AS, and mutation detection studies are enabling genotype/phenotype correlations to be made, as well as facilitating carrier detection and prenatal diagnosis.

The application of molecular biology to the prenatal diagnosis of renal disease.

The rapid development of new techniques in molecular biology is leading to identification of the genes responsible for a wide variety of diseases. Several renal conditions are caused by gene defects and are amenable to this approach. The process of gene mapping is discussed and the current position regarding prenatal diagnosis and carrier testing for genetic renal disease is reviewed.

Localization of the gene for classic Alport syndrome.

The inheritance of Alport syndrome has been controversial for 30 years because no clear diagnostic criteria were established to define a clinically homogeneous group of patients. In this study, 41 families with "classic" Alport syndrome were identified and studied. All the pedigrees are compatible with X-linked inheritance. A formal genetic study confirmed linkage to probe S21 (DXS17), with a maximum LOD score of 4.72 at a recombination frequency of 0.06.

X inactivation patterns in females with Alport's syndrome: a means of selecting against a deleterious gene?

The patterns of X chromosome inactivation in 43 females from families segregating classic Alport's syndrome (AS) (X linked hereditary nephritis with deafness) have been analysed. AS carrier females have a most variable clinical course. The aim of the study was to establish whether there was any correlation between the X inactivation pattern of a carrier female and the severity of her disease. No correlation was found in DNA derived from peripheral blood lymphocytes. However, it remains possible that differential patterns of X inactivation may occur in the tissues affected by AS, namely the basement membrane of the kidney, eye, and ear.

Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5).

A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.

Long-range mapping of the gene for the human alpha 5(IV) collagen chain at Xq22-q23.

The X-linked kidney disorder known as Alport syndrome (AS) has been shown to be due to mutations in the gene for an alpha 5 chain of type IV collagen that maps to Xq22-23. Using overlapping cDNA clones that represent approximately 90% of this gene and pulsed-field gel electrophoresis, we have constructed a 2.4-Mb long-range restriction map around the locus. All of the cDNA clones lie within a 360-kb segment of DNA bounded by CpG islands that contain sites for the rare-cutting enzymes BssHII, MluI, NotI, NruI, SalI, and SfiI. High-resolution PFGE mapping with XhoI shows that the gene is at least 110 kb in size and is one of the largest collagen genes characterized to date. This map will prove useful in the characterization of mutations in individuals affected with AS and will also provide information as to the location of other genes in the region.

A novel G472R mutation in a Turkish family with X-linked Alport syndrome.

Alport syndrome (AS) is a hereditary disorder of progressive nephritis. Most cases are X-linked, but autosomal forms have been reported. The X-linked form is associated with mutations in the COL4A5 gene that encodes the alpha 5 chain of type IV collagen. More than 200 mutations have been reported in X-linked AS. We report a novel 1616 G > A mutation resulting in glycine substitution to arginine at position 472 in a Turkish family with a severely affected man and several variably affected women. This is the first Turkish family in whom the molecular basis of the disease has been reported.

An interstitial deletion of 6p24-p25 proximal to the FKHL7 locus and including AP-2alpha that affects anterior eye chamber development.

The FKHL7 gene has been implicated in the pathogenesis of glaucoma/autosomal dominant iridogoniodysgenesis (IGDA) (IRID1). This has been supported by mutations in some glaucoma and IGDA patients and the development of anterior eye chamber anomalies in patients with 6p deletions affecting the 6p25 region. We report a case with anterior eye chamber anomalies and an interstitial deletion of 6p24-p25 that does not include the FKHL7 gene, suggesting the possible additional involvement of another locus, within 6p24-6p25, in anterior eye chamber development. A candidate gene is AP-2alpha, which is contained within the deleted segment and plays a role in anterior eye chamber development.