Diabetic vascular disease and Maturity-onset diabetes of the young

Aim: Vascular disease such as cardiovascular and cerebrovascular diseases, or retinopathy, nephropathy and neuropathy are common in diabetes. Maturity - onset diabetes of the young (MODY) describes a clinically heterogeneous group of familial diabetes characterized by monogenic, autosomal dominant inheritance that generally results from beta cell dysfunction. This study aims to assess the presence of vascular complications on Portuguese patients with a clinical diagnosis of MODY.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

A progressive autosomal recessive cataract locus maps to chromosome 9q13-q22.

Cataracts are the leading cause of blindness in most countries. Although most hereditary cases appear to follow an autosomal dominant pattern of inheritance, autosomal recessive inheritance has been clearly documented and is probably underrecognized. We studied a large family-from a relatively isolated geographic region-whose members were affected by autosomal recessive adult-onset pulverulent cataracts. We mapped the disease locus to a 14-cM interval at a novel disease locus, 9q13-q22 (between markers D9S1123 and D9S257), with a LOD score of 4.7. The study of this progressive and age-related cataract phenotype may provide insight into the cause of the more common sporadic form of age-related cataracts.

Dominant PRPF31 Mutations Are Hypostatic to a Recessive CNOT3 Polymorphism in Retinitis Pigmentosa: A Novel Phenomenon of "Linked Trans-Acting Epistasis".

Mutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease. It has been suggested that a major modulating factor lies in close proximity to the wildtype PRPF31 gene on Chromosome 19, implying that a cis-acting factor directly alters PRPF31 expression. Variable expression of CNOT3 is one determinant of PRPF31 expression. This study explored the relationship between CNOT3 (a trans-acting factor) and its paradoxical cis-acting nature in relation to RP11. Linkage analysis on Chromosome 19 was performed in mutation-carrying families, and the inheritance of the wildtype PRPF31 allele in symptomatic-asymptomatic sibships was assessed-confirming that differential inheritance of wildtype chromosome 19q13 determines the clinical phenotype (P < 2.6 × 10(-7) ). A theoretical model was constructed that explains the apparent conflict between the linkage data and the recent demonstration that a trans-acting factor (CNOT3) is a major nonpenetrance factor: we propose that this apparently cis-acting effect arises due to the intimate linkage of CNOT3 and PRPF31 on Chromosome 19q13-a novel mechanism that we have termed "linked trans-acting epistasis."

A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance.

We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of hereditary retinal degeneration. Although the inheritance pattern and previous linkage mapping pointed to the involvement of the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultrahigh throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, amounts of PRPF31 mRNA derived from the mutant allele were very reduced, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.

Mixed inheritance of equine recurrent airway obstruction

BACKGROUND: Mode of inheritance of equine recurrent airway obstruction (RAO) is unknown. HYPOTHESIS: Major genes are responsible for RAO. ANIMALS: Direct offspring of 2 RAO-affected Warmblood stallions (n = 197; n = 163) and a representative sample of Swiss Warmbloods (n = 401). METHODS: One environmental and 4 genetic models (general, mixed inheritance, major gene, and polygene) were tested for Horse Owner Assessed Respiratory Signs Index (1-4, unaffected to severely affected) by segregation analyses of the 2 half-sib sire families, both combined and separately, using prevalences estimated in a representative sample. RESULTS: In all data sets the mixed inheritance model was most likely to explain the pattern of inheritance. In all 3 datasets the mixed inheritance model did not differ significantly from the general model (P= .62, P= 1.00, and P= .27) but was always better than the major gene model (P < .01) and the polygene model (P < .01). The frequency of the deleterious allele differed considerably between the 2 sire families (P= .23 and P= .06). In both sire families the displacement was large (t= 17.52 and t= 12.24) and the heritability extremely large (h(2)= 1). CONCLUSIONS AND CLINICAL RELEVANCE: Segregation analyses clearly reveal the presence of a major gene playing a role in RAO. In 1 family, the mode of inheritance was autosomal dominant, whereas in the other family it was autosomal recessive. Although the expression of RAO is influenced by exposure to hay, these findings suggest a strong, complex genetic background for RAO.

Inheritance of proportionate dwarfism in Angus cattle

Objective To determine the mode of inheritance of congenital proportionate dwarfism in Angus and Angus crossbred cattle, initially detected in two commercial beef herds in northern New South Wales. Design Matings of normal carrier sires to unrelated cows of diverse breeds, and of one carrier sire to his unaffected daughters. An unrelated Piedmontese bull was also mated to unaffected daughters of the carrier sires. Procedure Two carrier Angus bulls and nine unaffected daughters, all of whom were completely indistinguishable from normal animals, were purchased for controlled breeding studies under known nutritional and disease conditions. Affected and carrier individuals were examined for the presence of obvious chromosomal abnormalities. Results Angus dwarfism has been successfully reproduced under controlled experimental conditions over successive years using unrelated dams and is undoubtedly heritable. The high frequency of occurrence of affected individuals (23/61 = 0.38 +/- .06) among the progeny of matings of the Angus sires to unrelated females of diverse breeding is not compatible with recessive inheritance, because of the negligible frequency of proportionate dwarfism in the breeds of the dams. Both paternal and maternal transmission of the defect was demonstrated, so that imprinting in the strict sense of a gene that is only expressed when received from the male parent appears not to be involved. Tested individuals showed no evidence of gross chromosomal abnormality. Dominant autosomal inheritance with incomplete penetrance was indicated by the lack of expression of the defective gene in the two Angus sires and in three unaffected daughters who produced dwarf calves from matings to the Piedmontese bull. Conclusions The mode of inheritance is that of a single autosomal dominant gene with a penetrance coefficient of 0.75 +/- 0.12, estimated from the observed incidence of 23/61 affected offspring of the two carrier Angus bulls mated to unrelated dams. Simple genetic models involving either (i) an unstable mutant which changes at high frequency to the expressed dominant dwarfing allele during gametogenesis, or (ii) a dominant allele with penetrance determined by an unlinked modifying locus, are shown to be compatible with the experimental data. Both models indicate that penetrance of the dwarfing gene may possibly be higher in matings involving carrier daughters of the two Angus bulls.

A novel mutation in the L12 domain of keratin 5 in the Kobner variant of epidermolysis bullosa simplex

We have identified a novel mutation within the linker L12 region of keratin 5 (K5) in a family with the Kobner variant of epidermolysis bullosa simplex. The pattern of inheritance of the disorder in this family is consistent with an autosomal dominant mode of transmission. Affected individuals develop extensive and generalized blistering at birth or early infancy but in later years clinical manifestations are largely confined to palmo-plantar surfaces. Direct sequencing of polymerase chain reaction products revealed a T to C transition within codon 323 of K5 in affected individuals, resulting in a valine to alanine substitution of the seventh residue within the L12 linker domain. This mutation was not observed in unaffected family members or in 100 K5 alleles of unrelated individuals with normal skin. The other critical regions of K5 and K14 were unremarkable in this family except for common polymorphisms that have been previously described. The valine at position 7 of the L12 domain is absolutely conserved in all type II keratins, and in other intermediate filament subunits as well, which suggests that this residue makes an important contribution to filament integrity. Secondary structure analysis revealed that alanine at this position markedly reduces both the hydrophobicity and the beta-sheet nature of the L12 domain. This is the first report of a mutation at this position in an intermediate filament subunit and reinforces the importance of this region to filament biology.

Childhood absence epilepsy and febrile seizures: A family with a GABAA receptor mutation

Although several genes for idiopathic epilepsies from families with simple Mendelian inheritance have been found, genes for the common idiopathic generalized epilepsies, where inheritance is complex, presently are elusive. We studied a large family with epilepsy where the two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), which offered a special opportunity to identify epilepsy genes. A total of 35 family members had seizures over four generations. The phenotypes comprised typical CAE (eight individuals); FS alone (15), febrile seizures plus (FS+) (three); myoclonic astatic epilepsy (two); generalized epilepsy with tonic-clonic seizures alone (one); partial epilepsy (one); and unclassified epilepsy despite evaluation (two). In three remaining individuals, no information was available. FS were inherited in an autosomal dominant fashion with 75% penetrance. The inheritance of CAE in this family was not simple Mendelian, but suggestive of complex inheritance with the involvement of at least two genes. A GABA(A) receptor gamma2 subunit gene mutation on chromosome 5 segregated with FS, FS+ and CAE, and also occurred in individuals with the other phenotypes. The clinical and molecular data suggest that the GABA(A) receptor subunit mutation alone can account for the FS phenotype. An interaction of this gene with another gene or genes is required for the CAE phenotype in this family. Linkage analysis for a putative second gene contributing to the CAE phenotype suggested possible loci on chromosomes 10, 13, 14 and 15. Examination of these loci in other absence pedigrees is warranted.

Spondylocostal Dysostosis Associated with Methylmalonic Aciduria

Spondylocostal dysostosis (SCD) is a genetic disorder characterized by vertebral segmentation and formation defects associated with changes of the ribs. Autosomal dominant and recessive modes of inheritance have been reported. Methylmalonic aciduria (MMA) is an inborn error of propionate or cobalamin metabolism. It is an autosomal recessive disorder and one of the most frequent forms of branched-chain organic acidurias. Here we report on a case of a Brazilian boy with both diseases. As we know, it is the first case in the literature with the occurrence of both SCD and MMA-the first a skeletal disease and the latter an inborn error of metabolism.

A Novel Autosomal Recessive GJA1 Missense Mutation Linked to Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a rare sclerosing skeletal disorder with progressive hyperostosis of craniofacial bones. CMD can be inherited in an autosomal dominant (AD) trait or occur after de novo mutations in the pyrophosphate transporter ANKH. Although the autosomal recessive (AR)form of CMD had been mapped to 6q21-22 the mutation has been elusive. In this study, we performed whole-exome sequencing for one subject with AR CMD and identified a novel missense mutation (c.716G>A, p.Arg239Gln) in the C-terminus of the gap junction protein alpha-1 (GJA1) coding for connexin 43 (Cx43). We confirmed this mutation in 6 individuals from 3 additional families. The homozygous mutation cosegregated only with affected family members. Connexin 43 is a major component of gap junctions in osteoblasts, osteocytes, osteoclasts and chondrocytes. Gap junctions are responsible for the diffusion of low molecular weight molecules between cells. Mutations in Cx43 cause several dominant and recessive disorders involving developmental abnormalities of bone such as dominant and recessive oculodentodigital dysplasia (ODDD; MIM #164200, 257850) and isolated syndactyly type III (MIM #186100), the characteristic digital anomaly in ODDD. However, characteristic ocular and dental features of ODDD as well as syndactyly are absent in patients with the recessive Arg239Gln Cx43 mutation. Bone remodeling mechanisms disrupted by this novel Cx43 mutation remain to be elucidated.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G&gt;A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

Molecular genetics of "PRPF31" dominant mutations linked to retinitis pigmentosa

ABSTRACT : The retina is one of the most important human sensory tissues since it detects and transmits all visual information from the outside world to the brain. Retinitis pigmentosa (RP) is the name given to a group of inherited diseases that affect specifically the photoreceptors present in the retina and in many instances lead to blindness. Dominant mutations in PRPF31, a gene that encodes for a pre-mRNA splicing factor, cause retinitis pigmentosa with reduced penetrance. We functionally investigated a novel mutation, identified in a large family with autosomal dominant RP, and 7 other mutations, substitutions and microdeletions, in 12 patients from 7 families with PRPF31-linked RP. Seven mutations lead to PRPF31 mRNA with premature stop codons and one to mRNA lacking the exon containing the initiation codon. Quantification of PRPF31 mRNA and protein levels revealed a significant reduction in cell lines derived from patients, compared to non carriers of mutations in PRPF31. Allelic quantification of PRPF31 mRNA indicated that the level of mutated mRNA is very low compared to wild-type mRNA. No mutant protein was detected and the subnuclear localization of wild-type PRPF31 remains the same in cell lines from patients and controls. Blocking nonsense-mediated mRNA decay in cell lines derived from patients partially restored PRPF31 mutated mRNA but derived proteins were still undetectable, even when protein degradation pathways were inhibited. Our results demonstrated that the vast majority of PRPF31 mutations result in null alleles, since they are subject to surveillance mechanisms that degrade mutated mRNA and possibly block its translation. Altogether, these data indicate that the likely cause of PRPF31-linked RP is haploinsufficiency, rather than a dominant negative effect. Penetrance of PRPF31 mutations has been previously demonstrated to be inversely correlated with the level of PRPF31 mRNA, since high expression of wild-type PRPF31 mRNA protects from the disease. Consequently, we have investigated the genetic modifiers that control the expression of PRPF31 by quantifying PRPF31 mRNA levels in cell lines derived from 200 individuals from 15 families representative of the general population. By linkage analyses we identified a 8.2Mb-region on chromosome 14q21-23 that contains a gene involved in the modulation of PRPF31 expression. We also assessed apreviously-mapped penetrance factor invariably located on the wild-type allele and linked to the PRPF31 locus in asymptomatic patients from different families with RP. We demonstrated that this modifier increases the expression of both PRPF31 alleles already at the pre-mRNA level. Finally, our data suggest that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is modulated by at least 2 diffusible compounds, which act on both PRPF31 alleles during their transcription.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Recurrent dominant mutations affecting two adjacent residues in the motor domain of the monomeric kinesin KIF22 result in skeletal dysplasia and joint laxity.

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated.