María José Trujillo-Tiebas

C9ORF72 hexanucleotide expansions of 20–22 repeats are associated with frontotemporal deterioration

Objective: Expansions of more than 30 hexanucleotide repetitions in the C9ORF72 gene are a common cause of frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). However, the range of 20–30 repetitions is rarely found and still has unclear significance. A screening of our cohort of cases with FTD (n = 109) revealed 4 mutation carriers (>30 repetitions) but also 5 probands with 20–22 confirmed repetitions. This study explored the possible pathogenic correlation of the 20–22 repeats expansion (short expansion). Methods: Comparison of clinical phenotypes between cases with long vs short expansions; search for segregation in the families of probands with short expansion; analysis of the presence of the common founder haplotype, described for expansions >30 repeats, in the cases having the short expansion; and analysis of the distribution of hexanucleotide repeat alleles in a control population. Results: No different patterns were found in the clinical phenotype or aggressiveness of the disease when comparing cases with long or short expansions. Cases in both groups had psychiatric symptoms during 1–3 decades before evolving insidiously to cognitive deterioration. The study of the families with short expansion showed clear segregation of the 20–22 repeats allele with the disease. Moreover, this 20–22 repeats allele was associated in all cases with the pathogenic founder haplotype. None of 216 controls had alleles with more than 14 repetitions. Conclusions: Description of these families suggests that short C9ORF72 hexanucleotide expansions are also related to frontotemporal cognitive deterioration.

New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma

BACKGROUND Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. METHODS We have used a mini-sequencing based method, the SNaPshot, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. RESULTS The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. CONCLUSIONS The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

Non-invasive prenatal diagnosis of single-gene disorders from maternal blood

Prenatal diagnosis (PD) is available for pregnancies at risk of monogenic disorders. However, PD requires the use of invasive obstetric techniques for fetal-sample collection and therefore, involves a risk of fetal loss. Circulating fetal DNA in the maternal bloodstream is being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a challenging discipline because of the biological features of the maternal blood sample. Maternal blood is an unequal mixture of small (and fragmented) amounts of fetal DNA within a wide background of maternal DNA. For this reason, initial NIPD studies have been based on the analysis of specific paternally inherited fetal tracts not present in the maternal genome so as to ensure their fetal origin. Following this strategy, different NIPD studies have been carried out, such as fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD determination, and analysis of single-gene disorders with a paternal origin. The study of the paternal mutation can be used for fetal diagnosis of dominant disorders or to more accurately assess the risk of an affected child in case of recessive diseases. Huntingtons disease, cystic fibrosis, or achondroplasia are some examples of diseases studied using NIPD. New technologies are opening NIPD to the analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is the latest study derived from the use of next-generation sequencing (NGS).

Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene.

PURPOSE Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD), a few cases of autosomal recessive cone-rod dystrophy (arCRD), and autosomal recessive retinitis pigmentosa (arRP). The purpose of this study was to compare high-resolution melting (HRM) analysis with denaturing high-performance liquid chromatography (dHPLC), to evaluate the efficiency of the different screening methodologies. METHODS Thirty-eight STGD, 15 arCRD, and 5 arRP unrelated Spanish patients who had been analyzed with the ABCR microarray were evaluated. The results were confirmed by direct sequencing. In patients with either no or only one mutant allele, ABCA4 was further analyzed by HRM and dHPLC. Haplotype analysis was also performed. RESULTS In a previous microarray analysis, 37 ABCA4 variants (37/116; 31.9%) were found. dHPLC and HRM scanning identified 18 different genotypes in 20 samples. Of the samples studied, 19/20 were identified correctly by HRM and 16/20 by dHPLC. One homozygous mutation was not detected by dHPLC; however, the p.Cys2137Tyr homozygote was distinguished from the wild-type by HRM technique. In the same way, one novel change in exon 5 (p.Arg187His) was found only by means of the HRM technique. In addition, dHPLC identified the mutation p.Trp1724Cys in one sample; however, HRM detected the mutation in two samples. CONCLUSIONS ABCA4 should be analyzed by an optimal screening technique, to perform further characterization of pathologic alleles. The results seemed to show that HRM had better sensitivity and specificity than did dHPLC, with the advantage that some homozygous sequence alterations were identifiable.

A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Noninvasive prenatal diagnosis of monogenic disorders

Introduction: Since the presence of circulating cell-free fetal DNA (ccffDNA) in maternal peripheral blood was demonstrated in 1997, great efforts have been done in order to use this source of fetal material for noninvasive prenatal diagnosis. The advantage that it represents is avoiding the obstetric invasive procedures required for conventional prenatal diagnosis. Areas covered: Efforts are mainly focused on finding the most accurate way to diagnose the most common fetal aneuploidies, paying special attention to trisomy 21. Recent advances in technology offer new diagnostic tools with high degrees of sensitivity thus generating great expectations for this type of diagnosis. However, there are other reasons why pregnant women undergo conventional prenatal diagnosis. Being at risk of transmitting a monogenic disorder is one of them. And although the percentage of those pregnancies may represent a small percentage of the diagnosis performed in the first trimester, these numbers should not be underestimated. Expert opinion: Management of pregnancies at risk of an X-linked Mendelian disorder has changed thanks to the noninvasive fetal sex assessment. As for other Mendelian disorders, until recently, their study was limited to those cases paternally inherited. Nevertheless, the new emerging technologies are also opening the scope to maternally inherited disorders.

Ellis-van Creveld syndrome in a fetus with rhizomelia and polydactyly. Report of a case diagnosed by genetic analysis, and correlation with pathological andradiologic findings

Ellis-van Creveld syndrome is an autosomal recessive disorder mainly characterized by a disproportionate limb dwarfism, chondroectodermal dysplasia, congenital heart disease, postaxial polydactyly, and dysplastic fingernails and teeth. Only 300 cases have been published worldwide. We report a 21-week fetus with rhizomelia and polydactyly detected. Gross photographs, radiologic studies and pathological study were performed leading to the clinico-pathological suspicion of EvC. DNA from fresh fetal tissue was extracted for sequencing the EVC and EVC2 genes. p.W215X and p.R677X mutations were identified in the EVC2 gene in the fetal sample. Parental sample analysis showed the p.W215X mutation to be inherited from the mother and the p.R677X mutation from the father. The clinical information is essential not only to arrive at a correct diagnosis in fetuses with pathologic ultrasound findings, but also to offer a proper genetic counseling to the parents and their relatives.

Late onset retinitis pigmentosa.

Dear Editor: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous set of progressive retinal disorders. To date, mutations in 53 genes have been described to cause RP (http://www.sph.uth.tmc.edu/Retnet/ accessed June 22, 2011). However, these genes account for disease in a little over half of all patients. Also, it is well known that several genes cause distinct or partially overlapping clinical phenotypes. Mutations in the SPATA7 gene have been associated with a severe congenital retinal dystrophy (Leber congenital amaurosis [LCA]) and a form of Juvenile RP. Therefore, a correlation between the severity of mutant alleles in SPATA7 and the clinical phenotype has been established. We performed a genome-wide linkage analysis, using the Illumina HumanCyto-12 SNP array, followed by homozygosity mapping in the 2 affected individuals from a consanguineous Spanish family that revealed 3 regions with a maximum logarithm of the odds (LOD) score of 2.8. The third largest region (3.7 Mb) in chromosome 14 encompassed SPATA7. Sequence analysis allowed identifying a homozygous nonsense mutation (c.253C T; p.Arg85ter) in SPATA7. The homozygous mutation segregated with the disease in the family. Both affected members of the family were clinically evaluated and were diagnosed as typical autosomal recessive retinitis pigmentosa (arRP). The index case complained of night blindness at the age of 25. At that time, the visual field was normal. At the age of 29 she was re-examined due to loss of peripheral visual field and the night blindness. The fundus showed narrowed retinal vessels and bone-spicule pigmentations in the peripheral retina, and the rod and mixed electroretinogram (ERG) showed subnormal responses of the rods in both eyes. Two years later (at 31 years old), the visual field was limited to central 10° and the ERG showed severe decreased of the a and b components in terms of the photopic and scotopic stimulation. This phenotype fits exactly the signs of typical arRP. To date the affected sibling did not complain of noticeable symptoms, night blindness, or visual field constriction; however, the mixed ERG showed delay in the aand b-waves latency and a moderate reduction in the amplitude of the a-wave in both eyes at the age of the 26 years old. Previous studies have established a correlation between the severity of SPATA7 mutant alleles and the phenotype of the patients. LCA has been associated with frameshift and nonsense mutations, which truncate the last half of the SPATA7 protein, which are predicted to be degraded by nonsense-mediated decay (NMD) mechanism. However, childhood-onset RP has been only associated with mutations that truncate a small part of the protein, which should be protected from the NMD mechanism. We report a previously described nonsense mutation in the middle of the coding region. This mutation will cause a truncated protein, which will be degraded by the NMD echanism. According to this hypothesis, the patients carying this mutation should have a severe retinal dystrophy ith a congenital or childhood presentation as previously eported. However, the affected individuals of the studied amily showed a non-early onset retinal dystrophy with a apid progression. Thus, our results contradict the published ypothesis of correlation between the severity of the PATA7 mutations and the phenotypes. These variable phenotypes could not be explained by the urrent understanding of the genetic mechanism. In addiion, this same mutation was found in 3 families from ifferent origins affected with LCA and severe early onset P, in contrast to the phenotype reported here. This phenotypic variability could be explained by the resence of modifier alleles contributing to the penetrance nd expressivity, or intronic variants influencing the splicng accuracy or efficiency of the different transcripts, could enerate an altered splicing product, encoding a stable rotein with residual function. Thus, we hypothesize that ntronic variants in this family may influence the severity f the phenotype associated with the p.Arg85ter mutation n this family. Further genomic studies of this uncharacterzed part of the noncoding region of SPATA7 combined with unctional analysis at the RNA level could help to better lucidate this hypothesis. In conclusion, our data established the first linkage asociation of a loss-of-function mutation in the SPATA7 gene ith a typical RP phenotype and not with LCA or early nset RP. inancial Support: This study was supported by the folowing research grants: FIS PI09/90047, FIS PI09/00459, IBERER INTRA/07/704.1, FUNDALUCE 2010 and NCE 2011. ALMUDENA ÁVILA-FERNÁNDEZ, PHD MARTA CORTÓN, PHD MARÍA I. LÓPEZ-MOLINA, MD ESTHER MARTÍN-GARRIDO DIEGO CANTALAPIEDRA, PHD RUTH FERNÁNDEZ-SÁNCHEZ, PHD FIONA BLANCO-KELLY, MD ROSA RIVEIRO-ÁLVAREZ, PHD SORINA D. TATU, PHD MARÍA J. TRUJILLO-TIEBAS, PHD BLANCA GARCÍA-SANDOVAL, MD, PHD CARMEN AYUSO, MD, PHD Madrid, Spain

Characterization of an acromesomelic dysplasia, Grebe type case: novel mutation affecting the recognition motif at the processing site of GDF5

Acromesomelic dysplasia, Grebe type is a very rare skeletal dysplasia characterized by severe dwarfism with marked micromelia and deformation of the upper and lower limbs, with a proximodistal gradient of severity. CDMP1 gene mutations have been associated with Grebe syndrome, Hunter–Thompson syndrome, Du Pan syndrome and brachydactyly type C. The proband is a 4-year-old boy, born of consanguineous Pakistani parents. Radiographic imaging revealed features typical of Grebe syndrome: severe shortening of the forearms with an acromesomelic pattern following a proximodistal gradient, with distal parts more severely affected than medial parts; hypoplastic hands, with the phalangeal zone more affected than the metacarpal zone; and severe hypoplastic tibial/femoral zones in both limbs. After molecular analyses, the p.Arg377Trp variant in a homozygous pattern was identified in the CDMP1 gene in the affected child. In silico and structural analyses predicted the p.Arg377Trp amino acid change to be pathogenic. Of the 34 mutations described in the CDMP1 gene, four different missense mutations have been associated with Grebe syndrome. The CDMP1 gene encodes growth differentiation factor 5 (GDF5), which plays a role in regulation of limb patterning, joint formation and distal bone growth. Homozygous mutations in the mature domain of GDF5 result in severe limb malformations such as the Grebe type or the Hunter–Thompson type of acromesomelic chondrodysplasia. The p.Arg377Trp mutation is located within the recognition motif at the processing site of GDF5 where the sequence RRKRR changes to WRKRR. The genotype–phenotype correlation allowed not only confirmation of the clinical diagnosis but also appropriate genetic counselling to be offered to this family.

Non-Invasive Prenatal Diagnosis in the Management of Preimplantation Genetic Diagnosis Pregnancies

Prenatal diagnosis (PD) is recommended in pregnancies after a Preimplantation Genetic Diagnosis (PGD). However, conventional PD entails a risk of fetal loss which makes PGD patients reluctant to undergo obstetric invasive procedures. The presence of circulating fetal DNA in maternal blood allows performing a non-invasive prenatal diagnosis (NIPD) without risk for the pregnancy outcome. This work shows the introduction of NIPD for confirmation of PGD results in eight pregnancies. In those pregnancies referred to PGD for an X-linked disorder (six out of eight), fetal sex determination in maternal blood was performed to confirm fetal sex. One pregnancy referred to PGD for Marfan syndrome and one referred for Huntington disease (HD) were also analyzed. In seven out of eight cases, PGD results were confirmed by NIPD in maternal blood. No results were obtained in the HD pregnancy. NIPD in PGD pregnancies can be a reliable alternative for couples that after a long process feel reluctant to undergo PD due to the risk of pregnancy loss.