S. Rodríguez de Córdoba

Translational Mini-Review Series on Complement Factor H: Genetics and disease associations of human complement factor H

Factor H is an abundant plasma glycoprotein that plays a critical role in the regulation of the complement system in plasma and in the protection of host cells and tissues from damage by complement activation. Several recent studies have described the association of genetic variations of the complement factor H gene (CFH) with atypical haemolytic uraemic syndrome (aHUS), age‐related macular degeneration (AMD) and membranoproliferative glomerulonephritis (MPGN). This review summarizes our current knowledge of CFH genetics and examines the CFH genotype–phenotype correlations that are helping to understand the molecular basis underlying these renal and ocular pathologies.

Lafora disease due to EPM2B mutations A clinical and genetic study

Objective: To study EPM2B gene mutations and genotype-phenotype correlations in patients with Lafora disease. Methods: The authors performed a clinical and mutational analysis of 25 patients, from 23 families, diagnosed with Lafora disease who had not shown mutations in the EPM2A gene. Results: The authors identified 18 mutations in EPM2B, including 12 novel mutations: 4 nonsense mutations (R265X, C26X, W219X, and E67X), a 6-base pair (bp) microdeletion resulting in a two amino acid deletion (V294_K295del), a 4-bp insertion resulting in a frameshift mutation (S339fs12), and 6 missense mutations (D308A, I198N, C68Y, E67Q, P264H, and D233A). In our data set of 77 families with Lafora disease, 54 (70.1%) tested probands have mutations in EPM2A, 21 (27.3%) in EPM2B, and 2 (2.6%) have no mutations in either gene. The course of the disease was longer in patients with EPM2B mutations vs patients with EPM2A mutations. Conclusions: Genetic allelic heterogeneity is present in Lafora disease associated with mutations in EPM2B. Patients with mutations in EPM2A and EPM2B express similar clinical manifestation, although patients with EPM2B-associated Lafora disease seem to have a slightly milder clinical course. The lack of mutations in EPM2A and EPM2B in two families could be because of the presence of mutations in noncoding, nontested regions or the existence of an additional gene associated with Lafora disease.

Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack.

Hepatic parenchymal cells respond in many different ways to acute‐phase cytokines. Some responses may protect against damage by liver‐derived inflammatory mediators. Previous investigations have shown that cytokines cause increased secretion by hepatoma cells of soluble complement regulatory proteins, perhaps providing protection from complement attack. More important to cell protection are the membrane complement regulators. Here we examine, using flow cytometry and Northern blotting, the effects of different cytokines, singly or in combination, on expression of membrane‐bound complement regulators by a hepatoma cell line. The combination of tumour necrosis factor‐alpha, IL‐1β, and IL‐6 caused increased expression of CD55 (three‐fold) and CD59 (two‐fold) and decreased expression of CD46 at day 3 post‐exposure. Interferon‐gamma reduced expression of CD59 and strongly antagonized the up‐regulatory effects on CD59 mediated by the other cytokines. Complement attack on antibody‐sensitized hepatoma cells following a 3‐day incubation with the optimum combination of acute‐phase cytokines revealed increased resistance to complement‐mediated lysis and decreased C3b deposition. During the acute‐phase response there is an increased hepatic synthesis of the majority of complement effector proteins. Simultaneous up‐regulation of expression of CD55 and CD59 may serve to protect hepatocytes from high local concentrations of complement generated during the acute‐phase response.

Mutation and Polymorphism Analysis of the Human Homogentisate 1,2- Dioxygenase Gene in Alkaptonuria Patients

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Analysis of the developmental SIX6 homeobox gene in patients with anophthalmia/microphthalmia

SIX6 is a recently characterized member of the SIX/sine oculis family of homeobox genes that is expressed in the developing and adult retina, in the optic nerve and in the hypothalamic and pituitary regions [Gallardo et al., 1999; Rodrı́guez de Córdoba et al., 2001]. SIX6maps to chromosome 14q22.3q23, a region that is deleted in three individuals with bilateralanophthalmiaandpituitaryanomalies [Bennett etal., 1991; Elliott et al., 1993; Lemyre et al., 1998]. In one of these cases [Bennett et al., 1991], the deletion includes the SIX6 gene, supporting the notion that the ocular and pituitary phenotype in this individual could be caused by SIX6 haploinsufficiency and that SIX6 is a candidate gene for anophthalmia [Gallardo et al., 1999]. Moreover, disruption of the Six6 gene in mice causes pituitary gland and retinal anomalies, often with absence of optic chiasm and optic nerve, resembling the human phenotype [Li et al., 2002]. To evaluate whether the homeobox SIX6 gene is a cause of human eye malformations, we have collected European pedigrees with syndromic or non-syndromic sporadic anophthalmia/ microphthalmia (A/M) and tested them for mutations in SIX6. Patientswere included in the studybased on the only criteria of presenting microphthalmia or anophthalmia. Thirty-six patients are from the A/M Registry at Albert Einstein Medical Center (Philadelphia) and are part of a broad survey of genetic eye disease. The remaining 37 were provided by the Department of Genetics at the Hospital Fundación Jiménez Dı́az (Madrid). DNA from these 73 patients and their relatives was obtained with informed consent following protocols approved by the Institucional Review Boards for human subjects at Albert Einstein Medical Center and Fundación Jiménez Dı́az. Each exon of the SIX6 gene was amplified from genomic DNA of patients and controls using specific primers derived from the 50and 30-intronic sequences. The sequence of these primers and the PCR conditions used for the amplifications are describedelsewhere [Gallardo et al., 1999;Rodrı́guezdeCórdobaetal., 2001]. Direct sequencing of PCRproductswas performed semiautomatically inboth strands inanABI3700 sequencerusinga dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). We have identified four single nucleotide changes compared to the reference SIX6 sequence (accession number: NM_007374) [Gallardo et al., 1999]. Analysis of these nucleotide substitution in a sample of 78 chromosomes from normal individuals demonstrated that three of them [IVS1-185A>G, c.21G>A (L7L), and c.421A>C (N141H)] are frequent single nucleotidepolymorphisms (SNPs) in thehumanSIX6geneand shownoassociationwith theA/Mphenotype (Fig. 1, Table I). In addition to these three polymorphic sites, we report the identification of a patient with a heterozygous amino acid substitution (c.493A>G; T165A) in exon 1 of SIX6 (Fig. 2). The alterationwasnot found in a control population includingmore than 160 chromosomes from normal individuals. The patient carrying the T165A alteration is an 18-year-old female. The patient presented with congenital bilateral asymmetric microphthalmia, cataract, nistagmus, and syndactyly of toes 2/3. At 1 year of age, she underwent cataract extraction onher left eye. Subsequently, she developed secondary glaucoma in this eye. At 5 years of age, she had visual acuity of hand movement in her right eye and amaurosis in her left eye. Due to the lens opacities and the small size of the globes, eye fundi were very difficult to exam. As glaucoma progressed, the left eye was surgically removed at 7 years of age and replaced by a prosthetic eye. At 11 years of age, growth and psychomotor development were normal. Neurological examination and brainMRIwere also normal,with no abnormalities of posterior pituitary gland. Ocular examination at this age showed microphthalmia, cataract, and nystagmus in the right eye with 20/400 visual acuity and a prosthetic left eye. Ultrasonographic biomicroscopy demonstrated a large cyst in the right eye. Her karyotype is normal. She was born to non-consanguineous parents. Ophthamological examinations of the parents revealed normal ocular development and visual acuity. Isolated syndactyly of toes 2/3 is a very frequent minor limb anomaly that we consider unrelated to the ocular phenotype of the patient. Mutations in other genes associated with microphthalmia, like CHX10 [Percin et al., 2000], were excluded in this patient (data not shown). The alteration changes a

Analysis of Alkaptonuria (AKU) Mutations and Polymorphisms Reveals that the CCC Sequence Motif Is a Mutational Hot Spot in the Homogentisate 1,2 Dioxygenase Gene (HGO)

We recently showed that alkaptonuria (AKU) is caused by loss-of-function mutations in the homogentisate 1,2 dioxygenase gene (HGO). Herein we describe haplotype and mutational analyses of HGO in seven new AKU pedigrees. These analyses identified two novel single-nucleotide polymorphisms (INV4+31A-->G and INV11+18A-->G) and six novel AKU mutations (INV1-1G-->A, W60G, Y62C, A122D, P230T, and D291E), which further illustrates the remarkable allelic heterogeneity found in AKU. Reexamination of all 29 mutations and polymorphisms thus far described in HGO shows that these nucleotide changes are not randomly distributed; the CCC sequence motif and its inverted complement, GGG, are preferentially mutated. These analyses also demonstrated that the nucleotide substitutions in HGO do not involve CpG dinucleotides, which illustrates important differences between HGO and other genes for the occurrence of mutation at specific short-sequence motifs. Because the CCC sequence motifs comprise a significant proportion (34.5%) of all mutated bases that have been observed in HGO, we conclude that the CCC triplet is a mutational hot spot in HGO.

Alkaptonuria in the Dominican Republic: identification of the founder AKU mutation and further evidence of mutation hot spots in the HGO gene

Alkaptonuria (AKU, MIM 203500), the first human disease to be recognised as a recessive trait and Archibald Garrods prototype “inborn error of metabolism”,1,2 is a rare disorder of the phenylalanine and tyrosine catabolic pathway caused by the deficiency of homogentisate dioxygenase (HGO, EC 1.13.11.5) activity.3 AKU patients are homozygous, or compound heterozygous, for loss of function mutations in HGO .4 As a consequence of this defect, AKU patients cannot convert homogentisate to maleylacetoacetate, which results in homogentisic aciduria, ochronosis, and arthritis.5 AKU shows remarkable allelic heterogeneity. More than 40 different AKU mutations have been identified in a total of fewer than 100 unrelated patients from many different countries. In addition to the AKU mutations, 19 polymorphisms have been encountered within the human HGO gene (for a complete description of the HGO mutations and polymorphisms see the AKU database (http://www.cib.csic.es/~akudb/index.htm)). The analysis of the haplotype association of polymorphisms in the AKU chromosomes has been very useful for the identification of the different AKU alleles and for tracing their migration during recent human history. In this regard, it has been shown that the three most widespread AKU mutation in Europe, M368V, V300G, and P230S (representing 20%, 5%, and 5% of European AKU chromosomes, respectively) are not recurrent mutations. Instead they are probably old mutations that were introduced into Europe with the founder populations and have spread throughout western Europe with the different migrations.6 Analysis of the HGO mutations and polymorphisms has also shown that the GGG sequence motif (or its reverse complement CCC) is a mutational hot spot in the HGO gene.7 AKU has a very low prevalence (1:100 000-250 000) in most populations. However, in certain areas, such as the Dominican Republic and Slovakia, the incidence of alkaptonuria is unusually high.8,9 In …

Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria

Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.

Molecular analyses of the HGO gene mutations in Turkish alkaptonuria patients suggest that the R58fs mutation originated from Central Asia and was spread throughout Europe and Anatolia by human migrations

Summary: Alkaptonuria (AKU) is a rare metabolic disorder of phenylalanine catabolism that is inherited as an autosomal recessive trait. AKU is caused by loss-of-function mutations in the homogentisate 1,2-dioxygenase (HGO) gene. The deficiency of homogentisate 1,2-dioxygenase activity causes homogentisic aciduria, ochronosis and arthritis. We present the first molecular study of the HGO gene in Turkish AKU patients. Seven unrelated AKU families from different regions in Turkey were analysed. Patients in three families were homozygous for the R58fs mutation; another three families were homozygous for the R225H mutation; and one family was homozygous for the G270R mutation. Analysis of nine intragenic HGO polymorphisms showed that the R58fs, R225H and G270R Turkish AKU mutations are associated with specific HGO haplotypes. The comparison with previously reported haplotypes associated with these mutations from other populations revealed that the R225H is a recurrent mutation in Turkey, whereas G270R most likely has a Slovak origin. Most interestingly, these analyses showed that the Turkish R58fs mutation shares an HGO haplotype with the R58fs mutation found in Finland, Slovakia and India, suggesting that R58fs is an old AKU mutation that probably originated in central Asia and spread throughout Europe and Anatolia during human migrations.

Lack of association between polymorphisms in C4b-binding protein and atypical haemolytic uraemic syndrome in the Spanish population.

Dysregulation of the alternative pathway of complement activation, caused by mutations or polymorphisms in the genes encoding factor H, membrane co‐factor protein, factor I or factor B, is associated strongly with predisposition to atypical haemolytic uraemic syndrome (aHUS). C4b‐binding protein (C4BP), a major regulator of the classical pathway of complement activation, also has capacity to regulate the alternative pathway. Interestingly, the C4BP polymorphism p.Arg240His has been associated recently with predisposition to aHUS and the risk allele His240 showed decreased capacity to regulate the alternative pathway. Identification of novel aHUS predisposition factors has important implications for diagnosis and treatment in a significant number of aHUS patients; thus, we sought to replicate these association studies in an independent cohort of aHUS patients. In this study we show that the C4BP His240 allele corresponds to the C4BP*2 allele identified previously by isoelectric focusing in heterozygosis in 1·9–3·7% of unrelated Caucasians. Crucially, we found no differences between 102 unrelated Spanish aHUS patients and 128 healthy age‐matched Spanish controls for the frequency of carriers of the His240 C4BP allele. This did not support an association between the p.Arg240His C4BP polymorphism and predisposition to aHUS in the Spanish population. In a similar study, we also failed to sustain an association between C4BP polymorphisms and predisposition to age‐related macular degeneration, another disorder which is associated strongly with polymorphisms in factor H, and is thought to involve alternative pathway dysregulation.