[The genetics of hereditary cataract]. / Transmission génétique de la cataracte congénitale.

Congenital cataracts are an important cause of visual impairment in children. Approximately one-third of congenital cataracts are hereditary. The disease, when inherited as an isolated abnormality, is phenotypically and genetically heterogeneous. Autosomal dominant forms with high penetrance appear to be the most common. To date, thirteen genes have been implicated in cataractogenesis. The identification of the genetic mutations causing congenital cataracts will provide a better understanding of cataractogenesis in childhood and provide further insights into normal lens development.

[Genetic deafness:the primary cause of sensorineural hearing loss in children]. / Les surdités génétiques: première cause de surdité de perception de l'enfant.

Genetically-transferred hearing impairments account for more than 50% of cases of pediatric sensorineural hearing defects. Multiple clinical aspects are involved in genetic hearing impairment, including the involvement of other organs, genetic inheritance, and the degree and age at onset of hearing loss. Diagnosis relies on family history, on the systematic investigation of the symptomatology including an associated syndrome, and audiometry testing in parents and siblings. Analysis of the connexin 26 gene is also indicated, as it is frequently involved in this disorder. Further genetic analysis in affected families will aid in detecting other as yet unidentified genes responsible for hearing impairment.

A locus for autosomal dominant colobomatous microphthalmia maps to chromosome 15q12-q15.

Congenital microphthalmia is a common developmental ocular disorder characterized by shortened axial length. Isolated microphthalmia is clinically and genetically heterogeneous and may be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. Here, we studied a five-generation family of Sephardic Jewish origin that included 38 members, of whom 7 have either unilateral or bilateral microphthalmia of variable severity inherited as an autosomal dominant trait with incomplete penetrance. After exclusion of several candidate loci, we performed a genome-scan study and demonstrated linkage to chromosome 15q12-q15. Positive LOD scores were obtained with a maximum at the D15S1007 locus (maximum LOD score 3.77, at recombination fraction 0.00). Haplotype analyses supported the location of the disease-causing gene in a 13.8-cM interval between loci D15S1002 and D15S1040.

Severe hereditary spherocytosis and distal renal tubular acidosis associated with the total absence of band 3.

Absence of band 3, associated with the mutation Coimbra (V488M) in the homozygous state, caused severe hereditary spherocytosis in a young child. Although prenatal testing was made available to the parents, it was declined. Because the fetus stopped moving near term, an emergency cesarean section was performed and a severely anemic, hydropic female baby was delivered. She was resuscitated and initially kept alive with respiratory assistance and hypertransfusion therapy. Cord blood smears revealed erythroblastosis, poikilocytosis, and red cells with stalk-like elongations. Band 3 and protein 4.2 were absent; spectrin, ankyrin, and glycophorin A were significantly reduced. Renal tubular acidosis was detected by the age of 3 months. Nephrocalcinosis appeared soon thereafter. After 3 years of follow-up the child is doing reasonably well on a regimen that includes regular blood transfusions and daily bicarbonate supplements. The long-term prognosis remains uncertain given the potential for hematologic and renal complications. (Blood. 2000;96:1602-1604)

A novel C202F mutation in the connexin26 gene (GJB2) associated with autosomal dominant isolated hearing loss.

Mutations in the GJB2 gene encoding connexin26 (CX26) account for up to 50% of cases of autosomal recessive hearing loss. In contrast, only one GJB2 mutation has been reported to date in an autosomal dominant form of isolated prelingual hearing loss. We report here a novel heterozygous 605G-->T mutation in GJB2 in all affected members of a large family with late childhood onset of autosomal dominant isolated hearing loss. The resulting C202F substitution, which lies in the fourth (M4) transmembrane domain of CX26, may impair connexin oligomerisation. Finally, our study suggests that GJB2 should be screened for heterozygous mutations in patients with autosomal dominant isolated hearing impairment, whatever the severity of the disease.

Heavy transfusions and presence of an anti-protein 4.2 antibody in 4. 2(-) hereditary spherocytosis (949delG).

BACKGROUND AND OBJECTIVE: A patient with hereditary spherocytosis (HS) was found not to have red cell membrane protein 4.2. This rare form of HS, or 4.2 (-) HS, stems from mutations within the ELB42 or the EPB3 genes. The patient had long suffered from a gastric ulcer and impaired liver function. He had had several dramatic episodes of gastrointestinal tract bleeding and had received numerous transfusions. An antibody against a high frequency, undefined antigen was found, creating a transfusional deadlock. We elucidated the responsible mutation and searched for an anti-protein 4.2 antibody. DESIGN AND METHODS: Red cell membranes were analyzed by SDS-PAGE and by Western blotting. Nucleotide sequencing was performed after reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: The not previously described mutation was a single base deletion: 949delG (CGCAECC, exon 7, codon 317) in the homozygous state. It was called protein 4.2 Nancy. The deletion placed a non-sense codon shortly downstream so that no viable polypeptide could be synthesized. The patient carried a strong antibody against protein 4.2 as shown by Western blotting. INTERPRETATION AND CONCLUSIONS: The manifestations resulting from the mutation described were compared with the picture of HS stemming from other ELB42 gene mutations. We discuss the mechanism through which the anti-protein 4.2 antibody developed. There was no way to establish or to rule out whether the antibody participated in the transfusional deadlock found in our patient.

Wagner vitreoretinal degeneration with genetic linkage refinement on chromosome 5q13-q14.

BACKGROUND: It has been previously described that Wagner disease is linked to chromosome 5q13-q14. This study was carried out to describe the ophthalmological aspects and report the results of genetic linkage analysis in a large pedigree affected by Wagner disease. METHODS: Fourty members of one same family agreed to be examined. RESULTS: Twenty patients presented vitreoretinal degeneration in both eyes without any extra-ocular abnormalities. In young patients, visual acuity was usually normal after correction of frequent mild myopia. Presenile cataracts progressed by the third decade and required removal for visual rehabilitation. The primary disorder involved an abnormal vitreous. A few avascular vitreous bands were usually the only optical feature in the mostly empty vitreous cavity. A circumferential vitreous condensation formed in contact with the retina on many spots. Less common retinal findings included retinal detachment, abnormal retinal pigmentation, progressive atrophy of the RPE simulating choroideremia and lattice degeneration. Genetic analysis revealed a highly significant linkage (lod score >5.0) between the disease and 10 markers of the chromosome 5q13-q14 region. Two recombination events allowed us to refine the linked interval to 20 cM between the D5S650 and D5S618 markers. CONCLUSION: Ophthalmological aspects of Wagner's disease appear to progress with age. Regular ophthalmological examination is important for detecting retinal abnormalities. The gene involved in Wagner's disease lies in a 20 cM interval on chromosome 5q13-q14.

Mutation in the zonadhesin-like domain of alpha-tectorin associated with autosomal dominant non-syndromic hearing loss.

A gene responsible for autosomal dominant non-syndromic hearing impairment in two families (DFNA8 and DFNA12) has recently been identified as TECTA encoding alpha-tectorin, a major component of the tectorial membrane. In these families, missense mutations within the zona pellucida domain of alpha-tectorin were associated with stable severe mid-frequency hearing loss. The present study reports linkage to DFNA12 in a new family with autosomal dominant high frequency hearing loss progressing from mild to moderate severity. The candidate region refined to 3.8 cM still contained the TECTA gene. A missense mutation (C1619S) was identified in the zonadhesin-like domain. This mutation abolishes the first of the vicinal cysteines (1619Cys-Gly-Leu- 1622Cys) present in the D4 von Willebrand factor (vWf) type D repeat. These results further support the involvement of TECTA mutations in autosomal dominant hearing impairment, and suggest that vicinal cysteines are involved in tectorial membrane matrix assembly.

Two distinct truncated variants of ankyrin associated with hereditary spherocytosis.

We present two distinct truncated variants of ankyrin associated with mild to moderate hereditary spherocytosis. Ankyrin Saint-Etienne 1 was manifested by an additional band located between bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 39: TGG-->TGA; W1721X. Ankyrin Saint-Etienne 2 appeared as two faint bands underlining bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 41: CGA-->TGA; R1833X. Overall ankyrin was diminished in splenectomized patients. Messenger RNAs Saint-Etienne 1 and 2 amounted to 20 and 37% of the total ankyrin mRNA, respectively. Ankyrin molecules truncated in their C-terminal region retain some ability to bind to the membrane whereas the bulk of nonsense mutations, located in more upstream regions, result in the mere disappearance of one haploid set of ankyrin. In the present cases, it was not possible to apportion the roles of ankyrin reduction and truncation in the pathogenesis of hereditary spherocytosis.

A large deletion within the protein 4.1 gene associated with a stable truncated mRNA and an unaltered tissue-specific alternative splicing.

Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.

Hereditary spherocytosis: from clinical to molecular defects.

Resistance and elastic deformability of red cells are due to a protein network (cytoskeleton) that laminates the lipid bilayer and to proteins that span the latter. All proteins are interconnected. Their structure as well as the structure of the corresponding genes are now well known. Hereditary spherocytosis (HS) is the most common hemolytic anemia due to a red cell membrane defect. It derives from alterations of the following genes: ANK1, EPB3, ELB42, SPTA1 and SPTB. This condition is clinically, biochemically and genetically heterogeneous. The osmotically fragile spherocytes are selectively trapped in the spleen and destroyed. Increased red blood cell destruction causes the three main clinical signs of HS: anemia, jaundice and splenomegaly. In this review we analyze the most recent advances concerning the molecular basis and the clinical course of HS. In particular, we examine the major individual proteins that constitute the skeleton, which are now known to play an essential role in the pathogenesis of HS. This paper also includes a review of the therapeutical approach to HS. Concerning the diagnosis we provide a flow chart from the clinical aspects to the molecular diagnosis.

High frequency of de novo mutations in ankyrin gene (ANK1) in children with hereditary spherocytosis.

OBJECTIVE: To evaluate the frequency of de novo monoallelic expression of the ANK1 gene in hereditary spherocytosis individuals appearing as recessive. STUDY DESIGN: We studied 40 unrelated children with spherocytosis and their normal parents. The genomic distribution of the ankyrin (AC)n dinucleotide repeats was evaluated in the patients showing combined ankyrin and spectrin deficiency. To search for the absence of mRNA derived from one of the two ANK1 genes, cDNA from the heterozygous patients was amplified using polymerase chain reaction. This was analyzed for the (AC)n dinucleotide repeats. RESULTS: Thirty-three hereditary spherocytosis subjects had variable degrees of combined ankyrin and spectrin reduction; 19 were found to be heterozygous for the AC repeat lengths and were further studied. In 12, we found a cDNA polymerase chain reaction product from one ankyrin gene alone. These findings strongly suggested the nonexpression of one of the two ANK1 genes because of the de novo mutational events. CONCLUSION: The de novo loss of an ankyrin allele expression is a frequent cause of hereditary spherocytosis in children with normal parents. Therefore the category of genuinely recessive hereditary spherocytosis cases is further reduced compared with spherocytosis cases because of de novo mutations. The determination of the (AC)n microsatellite polymorphisms appears as a helpful and reliable tool for the discrimination between these two categories.

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis.

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG-->GAG, and P854L, CCG-->CTG) and, additionally, the mutation: G714R, GGG-->AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA-->AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.

Modulation of clinical expression and band 3 deficiency in hereditary spherocytosis.

We present two novel alleles of the anion-exchanger 1 (AE1) gene, allele Coimbra and allele Mondego. Allele Coimbra (V488M, GTG --> ATG) affects a conserved position in the putative second ectoplasmic loop of erythrocyte band 3. In 15 simple heterozygotes, it yielded a mild form of hereditary spherocytosis (HS) with band 3 deficiency (-20% +/- 2%) and a reduced number of 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (H2DIDS) binding sites (-35%). However, two additional heterozygotes presented with an aggravated HS and a more pronounced reduction of band 3 (-40%) and of H2DIDS binding sites (-48%). They carried, in trans to allele Coimbra, allele Mondego, defined by two mutations: E40K, GAG --> AAG, the known mutation Montefiore, and P147S, CCT --> TCT, a novel mutation, both located in the cytoplasmic domain of band 3. Allele Mondego itself resulted in no clinical or hematologic HS signs in the simple heterozygous state. Yet it yielded a slight decrease in band 3 (-6% to -12%) and in the number of H2DIDS binding sites (-19%). Thus, the more pronounced decrease in band 3 in the two compound heterozygotes derived from the additive effects of two unequally expressed AE1 alleles, resulting in a more severe clinical picture.

Ankyrin Bugey: a de novo deletional frameshift variant in exon 6 of the ankyrin gene associated with spherocytosis.

We describe a case of spherocytosis in a French child splenectomized at age 10 years. The parents were devoid of any clinical, hematological, or biochemical abnormalities. Following splenectomy, the proposita exhibited a reduction of red cell membrane ankyrin. The variable number of dinucleotide repeats associated with the erythroid ankyrin gene (ANK1) were studied at the genomic level. The father, the mother, and the proposita had the AC14/AC11, AC14/AC14, and AC14/AC11 genotypes, respectively, although the proposita exhibited a pattern consistent with an AC14,-combination at the cDNA level. We thought there could be a de novo mutation in the ANK1 allele of paternal origin (AC11). A false paternity seemed most unlikely. Based on PCR-amplification of exons, SSCP analysis, and, when appropriate, nucleotide sequencing, we found a one-nucleotide deletion in codon 146 (exon 6): 521delC, ACG-->AG. This placed in phase a TAG triplet normally overlapping codons 150 and 151. Early interruption of translation presumably accounted for the premature degradation of mutant mRNA. Restriction analysis confirmed the presence of the mutation in the proposita and its absence in the parents. The variant was designated ankyrin Bugey.

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio.

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out-of-frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.

Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/ AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC --> AAC; Asp --> Asn in codon 328 of exon 10, and a one-nucleotide deletion : CTG --> CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degadation. The present allele has been designated allele Napoli.

Molecular genetics of hereditary elliptocytosis and hereditary spherocytosis.

Red cells ow their mechanical properties, that is, their resistance and their elastic deformability, to a protein network that laminates the lipid bilayer and to proteins spanning the latter. All proteins are interconnected. Their structure, as well as the structure of the corresponding genes, will be outlined. Numerous mutations have allowed to reclassify hereditary elliptocytosis (HE) and poikilocytosis (HP), and, more recently, hereditary spherocytosis (HS) into well defined subsets of hereditary hemolytic anemias. HE stems from changes in the SPTA1, SPTB, EL1 and (exceptionally) GPYC genes that encode spectrin alpha- and beta- chains, protein 4.1 and glycophorin C/D, respectively. HS derives from altercations in the ANK1, EPB3 and ELB42 genes, encoding ankyrin, band 3 and protein 4.2, respectively, and also in the SPTA1 and SPTB genes. We will present a repertory of the known mutations. Innumerable polymorphisms will not be considered here, except for a few remarkable ones. Some general points must be stressed on. (a) Clinically conspicuous disorders are often the result of two alleles interacting in trans to one another. Whereas one allele causes moderate symptoms by itself, the other one is usually silent in the simple heterozygous (and exceptionally in the homozygous) state. As a result, the number of potentially pathogenic alleles is much more important than had been initially suspected. (b) The reduction or the loss of a protein within multiprotein assemblies are frequently encountered in red cell membrane genetic diseases; it leads to the disruption of the complexes with the possible disappearance of the other proteins than the mutated protein. (c) The above genes being also expressed in nonerythroid tissues, one starts finding multisyndromic conditions adding non-hematological manifestations to hemolysis. It is puzzling, though, that such situations are not more frequent. (d) In practice, the molecular diagnosis of HE and HS has reached a semi-routine stage that helps very much the paediatricians and haematologists.

Spectrin mutations in hereditary elliptocytosis and hereditary spherocytosis.

Hereditary elliptocytosis (HE), its aggravated form hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS) designate a set of congenital hemolytic syndromes. The responsible mutations lie in several genes encoding proteins of the red cell membrane. In particular, they involve the SPTA1 and SPTB genes that encode erythroid spectrin alpha- and beta-chains, respectively. In situ, spectrin is a alpha 2 beta 2 fibrillar tetramer resulting from the head-to-head self-association of two alpha beta dimers. In HE, the 24 known alpha-chain mutations lie in the self-association site or its vicinity, whereas the 17 beta-chain mutations occur in the self-association site itself (record of November 30, 1995). Allele alpha LELY (LELY: Low Expression LYon) is found in ethnic groups remote from one another with a uniform frequency (20-30% of all alpha-alleles). It allows an expanded expression of any HE alpha-allele located in trans and results in severe HE or in HPP. In HS, a number of spectrin mutations have been recorded recently. Allele alpha LEPRA (LEPRA: Low Expression PRAgue) would occur in a recurrent fashion.

A point mutation in the protein 4.2 gene (allele 4.2 Tozeur) associated with hereditary haemolytic anaemia.

A recessively transmitted haemolytic anaemia associated with the lack of protein 4.2 was found in a Tunisian kindred. Trace amounts of this protein (72 kD component) became visible using high-sensitivity Western blots. Band 3 and ankyrin genes were excluded as candidate genes by linkage studies, and nucleotide sequencing of band 3 cytoplasmic domain cDNA revealed no alteration. In contrast, protein 4.2 gene contained in the homozygous state a mutation at position 310: CGA-->CAA (Arg-->Gln). This mutation defining allele 4.2 Tozeur was co-inherited with the disease. The mRNA encoding the variant protein was normal in size and approximately normal in amount. Recombinant protein 4.2 Tozeur bound normally to red cell IOVs but disclosed an increased susceptibility to proteolysis in vitro. We infer that the nearly total absence of protein 4.2 in the patients results from imbalance between destruction and synthesis of mutated protein 4.2 prior to its binding to the membrane.