Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity.

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

Non-collagen genes role in digenic Alport syndrome.

BACKGROUND: Alport syndrome is a clinically heterogeneous nephropathy characterized by severe symptomatology at kidney level due to ultrastructural lesions of the glomerular basement membrane (GBM) as consequence of mutations in COL4 genes. The disease has been linked to COL4A3/COL4A4/COL4A5 mutations, which impair GBM functionality and can be inherited in a dominant, recessive or X-linked transmission. Although a targeted Next Generation Sequencing approach has allowed identifying families with pathogenic mutations in more than one COL4 α3-α4-α5 heterotrimer encoding genes, leading to conclude for a digenic pattern of inheritance, the role of non-collagen genes in digenic Alport syndrome has not yet been established. METHODS: We employed a whole-exome sequencing approach on three families in whom a digenic pattern of transmission could be suspected because of a likely biparental contribution or an unexplained phenotype in the proband. RESULTS: We identified in the three probands hypomorphic LAMA5 mutations co-inherited with pathogenic COL4 α4-α5 chains mutations. Segregation analysis revealed that the combination of LAMA5/COL4 variants co-segregate with a fully penetrant phenotype in line with a digenic inheritance. In one of the three probands an hypomorphic variant in NPHS2 was also found, suggesting that role of other kidney disease related-genes as modifiers. CONCLUSION: These findings validate the impact of LAMA5 mutations in digenic ATS and highlight the role of extracellular matrix's genes, basement membrane, slit diaphragm and podocyte cytoskeleton in ATS. This underline the need for a more extensive panel approach in the presence of a digenic ATS, in order to better define clinical severity and recurrence risk for family members.

Human protective response induced by meningococcus B vaccine is mediated by the synergy of multiple bactericidal epitopes.

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.

Unbiased next generation sequencing analysis confirms the existence of autosomal dominant Alport syndrome in a relevant fraction of cases.

The mode of inheritance of Alport syndrome (ATS) has long been controversial. In 1927, the disease was hypothesized as a dominant condition in which males were more severely affected than females. In 1990, it was considered an X-linked (XL) semidominant condition, due to COL4A5 mutations. Later on, a rare autosomal recessive (AR) form due to COL4A3/COL4A4 mutations was identified. An autosomal dominant (AD) form was testified more recently by the description of some large pedigrees but the real existence of this form is still questioned by many and its exact prevalence is unknown. The introduction of next generation sequencing (NGS) allowed us to perform an unbiased simultaneous COL4A3-COL4A4-COL4A5 analysis in 87 Italian families (273 individuals) with clinical suspicion of ATS. In 48 of them (55%), a mutation in one of the three genes was identified: the inheritance was XL semidominant in 65%, recessive in 4% and most interestingly AD in 31% (15 families). The AD form must therefore be seriously taken into account in all pedigrees with affected individuals in each generation. Furthermore, a high frequency of mutations (>50%) was shown in patients with only 1 or 2 clinical criteria, suggesting NGS as first-level analysis in cases with a clinical suspicion of ATS.

The XLMR gene ACSL4 plays a role in dendritic spine architecture.

ACSL4 is a gene involved in non-syndromic X-linked mental retardation. It encodes for a ubiquitous protein that adds coenzyme A to long-chain fatty acids, with a high substrate preference for arachidonic acid. It presents also a brain-specific isoform deriving from an alternative splicing and containing 41 additional N-terminal amino acids. To start to unravelling the link between ACSL4 and mental retardation, we have performed molecular and cell biological studies. By retro-transcription polymerase chain reaction analyses we identified a new transcript with a shorter 5'-UTR region. By immunofluorescence microscopy in embryonic rat hippocampal neurons we report that ACSL4 is associated preferentially to endoplasmic reticulum tubules. ACSL4 knockdown by siRNAs in hippocampal neurons indicated that this protein is largely dispensable for these cells' gross architectural features (i.e. axonal and dendritic formation and final length) yet it is required for the presence of normal spines. In fact, reduced levels of ACSL4 led to a significant reduction in dendritic spine density and an alteration in spine/filopodia distribution. The possible mechanisms behind this phenotype are discussed.

Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection using array CGH.

We describe three patients with retinoblastoma, dysmorphic features and developmental delay. Patients 1 and 2 have high and broad forehead, deeply grooved philtrum, thick anteverted lobes and thick helix. Patient 1 also has dolicocephaly, sacral pit/dimple and toe crowding; patient 2 shows intrauterine growth retardation and short fifth toe. Both patients have partial agenesis of corpus callosum. Patient 3 has growth retardation, microcephaly, thick lower lip and micrognathia. Using array-comparative genomic hybridization (CGH), we identified a 13q14 de novo deletion in patients 1 and 2, while patient 3 had a 7q11.21 maternally inherited deletion, probably not related to the disease. Our results confirm that a distinct facial phenotype is related to a 13q14 deletion. Patients with retinoblastoma and malformations without a peculiar facial phenotype may have a different deletion syndrome or a casual association of mental retardation and retinoblastoma. Using array-CGH, we defined a critical region for mental retardation and dysmorphic features. We compared this deletion with a smaller one in a patient with retinoblastoma (case 4) and identified two distinct critical regions, containing 30 genes. Four genes appear to be good functional candidates for the neurological phenotype: NUFIP1 (nuclear fragile X mental retardation protein 1), HTR2A (serotonin receptor 2A), PCDH8 (prothocaderin 8) and PCDH17 (prothocaderin 17).

The Italian XLMR bank: a clinical and molecular database.

Mental retardation (MR) is a nonprogressive condition characterized by a significant impairment of intellectual capabilities with deficit of cognitive and adaptive functioning and onset before 18 years. Mental retardation occurs in about 2 to 3% of the general population and it is estimated that 25 to 35% of the cases may be due to genetic causes. Among these "genetic" MR, 25 to 30% are probably due to mutations in a gene on the X chromosome (X-linked mental retardation, XLMR). Given the genetic heterogeneity of XLMR, the availability of a considerable number of patients with accurate phenotypic classification is a crucial factor for research. The X-linked Mental Retardation Italian Network, which has been active since 2003, has collected detailed clinical information and biological samples from a vast number of MR patients. Collected samples and clinical information are inserted within the XLMR bank, a comprehensive molecular and clinical web-based database available at the address http://xlmr.unisi.it. The database is organized in three distinct parts. Part I and II contain several electronic schedules to register information on the family, the phenotypic description, the photographs, and a 20 sec movie of the patient. Part III allows the registration of molecular analyses performed on each case; samples and clinical data are usable via password-restricted access. Clinical and molecular centers interested in joining the network may request a password by simply contacting the Medical Genetics of the University of Siena. The XLMR bank is an innovative biological database that allows the collection of molecular and clinical data, combines descriptive and iconographic resources, and represents a fundamental tool for researchers in the field of mental retardation.

2q24-q31 deletion: report of a case and review of the literature.

We report a patient with a de novo interstitial deletion of the long arm of chromosome 2 involving bands 2q24.3-q31.1. The patient shows postnatal growth retardation, microcephaly, ptosis, down-slanting palpebral fissures, long eyelashes and micrognathia. Halluces are long, broad and medially deviated, while the other toes are laterally deviated and remarkably short with hypoplastic phalanges. She also showed developmental delay, seizures, lack of eye contact, stereotypic and repetitive hand movements and sleep disturbances with breath holding. Prenatal and three independent postnatal karyotypes were normal. Array-CGH analysis allowed us to identify and characterize a "de novo" 2q interstitial deletion of about 10.4Mb, involving segment between cytogenetic bands 2q24.3 and 2q31.1. The deletion was confirmed by quantitative PCR. About 30 children with 2q interstitial deletion have been reported. The deletion described here is overlapping with 15 of these cases. We have attempted to compare the clinical features of our patient with 15 overlapping cases. The emerging phenotypes include low birth weight, postnatal growth retardation, mental retardation and developmental delay, microcephaly, and peculiar facial dysmorphisms. Peculiar long and broad halluces with an increased distance between the first and the second toe are ("sandal gap" sign) present in most of the described patients. The gene content analysis of the deleted region revealed the presence of some genes that may be indicated as good candidates in generating both neurological and dysmorphic phenotype in the patient. In particular, a cluster of SCNA genes is located within the deleted region and it is known that loss of function mutations in SCNA1 gene cause a severe form of epilepsy.

An apparently sporadic case of oculopharyngeal muscular dystrophy: the first Italian report.

Here we report the case of a 73-year-old Italian woman affected by genetically confirmed oculopharyngeal muscular dystrophy (OPMD) with a negative family history. As OPMD is usually transmitted as an autosomal-dominant meiotically stable trait, this case allows us to suggest that putative de novo OPMD mutations might occur more frequently than previously thought; moreover, when compatible with a proper clinical phenotype, OPMD might be included in the differential diagnosis even in the absence of a positive family history.

[Clinical and genetic features of the Alport 'syndromes']. / Le 'sindromi' di Alport: dalla clinica alla genetica.

Alport syndrome (ATS) is a clinically and genetically heterogeneous progressive nephropathy often associated with deafness and/or ocular lesions. The histological aspect is characterized by thinning, thickening and splitting of the glomerular basement membrane (GBM). Alport syndrome is caused by mutations in COL4A3 gene (type IV collagen, alfa-3 chain), or COL4A4 gene (type IV collagen, alfa-4 chain) or COL4A5 gene (type IV collagen, alfa-5 chain) genes. Alport syndrome accounts for 1-2% of renal failure cases in Europe, and for 2-3% of transplanted patients in United States. This review focuses on the three types of Alport syndrome which differ in the clinical progression and in the mode of inheritance. The common X-linked form is caused by mutations in the COL4A5 gene and it accounts for 85% of cases. The autosomal dominant and the autosomal recessive forms are caused by mutations in either COL4A3 or COL4A4 genes. The autosomal recessive form which is responsible for the 10-15% of Alport cases, has been known since several years. On the contrary, the autosomal dominant form has only recently been identified in some families. Furthermore, this review will focus on the difficulties encountered during the genetic counselling related to the differential diagnosis between Alport syndrome and Thin Basement Membrane Disease (TBMD). We will report direct experiences of our group showing the difficulties to give an exact prognosis and a correct recurrence risk to the family.

CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms.

BACKGROUND: Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterised by a wide spectrum of clinical manifestations. Both the classic form and preserved speech variant of Rett syndrome are due to mutations in the MECP2 gene. Several other variants of Rett syndrome have been described. In 1985, Hanefeld described a variant with the early appearance of convulsions. In this variant, the normal perinatal period is soon followed by the appearance of seizures, usually infantile spasms. We have observed two patients with signs of Rett syndrome showing acquired microcephaly and stereotypic midline hand movements. The disease started with generalised convulsions and myoclonic fits at 1.5 months in the first patient and with spasms at 10 days in the other, suggesting a diagnosis of the Hanefeld variant. In these patients, MECP2 point mutations and gross rearrangements were excluded by denaturing high performance liquid chromatography and real time quantitative PCR. The ARX and CDKL5 genes have been associated with West syndrome (infantile spasms, hypsarrhythmia, and mental retardation). METHODS: Based on the clinical overlap between the Hanefeld variant and West syndrome, we analysed ARX and CDKL5 in the two girls. RESULTS: We found frameshift deletions in CDKL5 in both patients; one in exon 5 (c.163_166delGAAA) and the other in exon 18 (c.2635_2636delCT). CDKL5 was then analysed in 19 classic Rett and 15 preserved speech variant patients, all MECP2 negative, but no mutations were found. CONCLUSION: Our results show that CDKL5 is responsible for a rare variant of Rett syndrome characterised by early development of convulsions, usually of the spasm type.

Germline mosaicism in Rett syndrome identified by prenatal diagnosis.

Rett syndrome is an X-linked neurodevelopmental dominant disorder that affects almost exclusively girls. The vast majority of cases are sporadic and are caused by de novo mutations in the MECP2 gene, located in Xq28. Only few familial cases have been reported: in four cases, the mother was an asymptomatic carrier and in other four cases, the germline mosaicism in the mother was postulated. Owing to the above reported cases of germline mosaicism, we decided to offer prenatal diagnosis to all expectant mothers with a Rett daughter despite the absence of the causative mutation in parents' blood. We describe here the outcome of the first nine cases of prenatal diagnosis followed by our center. In eight cases, the fetus did not carry the mutation. In one case, the female fetus did carry the same mutation of the affected sister. The couple decided to interrupt the pregnancy and to devolve fetal tissues for research purposes. Our results indicate that prenatal diagnosis should be proposed to all couples with a Rett daughter, even when the mutation is apparently de novo. Moreover, one positive prenatal test among the first nine cases indicates that germline mosaicism may be seriously considered for the assessment of recurrence risk during genetic counseling.

Mitochondrial abnormalities in genetically assessed oculopharyngeal muscular dystrophy.

We report a family with a clinical diagnosis of oculopharyngeal muscular dystrophy in which muscle biopsy showed mitochondrial changes such as cytochrome-c-oxidase-negative fibers and aggregates of mitochondria containing paracrystalline inclusions. Molecular analysis demonstrated a GCG expansion in the poly(A)-binding protein 2 (PABP2) gene and failed to demonstrate multiple deletions of mtDNA. We hypothesize that mitochondrial abnormalities may be a secondary phenomenon. This observation may suggest that the PABP2 gene could interfere in the posttranscriptional regulation of genes involved in mitochondrial function.

Chromosome 2 deletion encompassing the MAP2 gene in a patient with autism and Rett-like features.

We present here a unique case of a 14-year-old female with autism and some features similar to Rett syndrome (RTT). Genetic analysis demonstrated a large deletion of chromosome 2q instead of a MECP2 mutation. Like a Rett patient, she is dyspraxic and shows frequent hand-washing stereotypic activities, hyperpnea, and bruxism. Like a preserved speech variant (PSV) of RTT, she is obese, able to speak in second and third persons, frequently echolalic, and has final normal head circumference and autistic behavior. In addition, she has dysmorphic features such as down-slanting palpebral fissures, low set ears without lobuli, bilateral flat feet, and bilateral syndactyly of the second and third toes, which do not belong to the Rett spectrum. She has a de novo chromosomal deletion in 2q34 of paternal origin. Gene content analysis of the deleted region showed the presence of 47 genes (14 putative and 33 known genes). This region contains some interesting genes such as ADAM23/MDC3, CREB1, KLF7, and MAP2. Because alteration of neuronal maturation, dendritic anomalies, and a decrease in MAP2 immunoreactivity in white matter neurons are well documented in RTT patients, we propose MAP2 gene as a good candidate for the generation of PSV phenotype in this case.

PAX6 mutation in a family with aniridia, congenital ptosis, and mental retardation.

Congenital aniridia is due to deletions and point mutations in the PAX6 gene. We describe here a case of a mother and her two sons with a syndrome comprising congenital aniridia, ptosis, and slight mental retardation. The sons also show behavioral changes. The possibility of deletion around the PAX6 locus was excluded by polymorphism studies and fluorescence in situ hybridization analysis. Mutation screening of the PAX6 gene revealed the presence of a transversion C719A, resulting in the substitution of arginine for serine at residue 119. We suggest that this missense mutation is responsible both for aniridia and ptosis, and possibly also for the observed cognitive dysfunction in this family.

Pseudoxanthoma elasticum: Point mutations in the ABCC6 gene and a large deletion including also ABCC1 and MYH11.

Pseudoxanthoma elasticum (PXE) is a mendelian disorder characterized by calcification of elastic fibers in skin, arteries, and retina. It results in dermal lesions, arterial insufficiency and retinal hemorrhages, leading to macular degeneration. PXE is transmitted either as an autosomal dominant or recessive trait and several sporadic cases have been observed. Mutations in the ABCC6 gene have been identified very recently in patients. Here, we report on a large Italian family affected by pseudoxanthoma elasticum for which linkage analysis had pointed to a region encompassing markers D16S3069-D16S405-D16S3103; hemizygosity of marker D16S405 allowed us to detect a submicroscopic deletion of at least 900 kb involving ABCC6, ABCC1, and MYH11. Mutation analysis on the other allele of the family, as well as on two additional sporadic cases, revealed nonsense (Y227X, R518X, R1164X) and frame-shift (c.960delC) mutations in ABCC6 (MRP6) further confirming the role of this multi-drug resistance gene in the etiology of pseudoxanthoma elasticum. Furthermore, clinical re-examination of members of the family harboring the deletion led to the detection of additional features, potentially caused by the deletion of the MYH11 gene. In the course of the analysis five nonpathogenic variants were found in ABCC6: 1233T>C, 1245G>A, 1838 T>G (V614A), 1890C>G, and 3506+83C>A. Hum Mutat 18:85, 2001.

Dot-and-fleck retinopathy in Alport syndrome caused by a novel mutation in the COL4A5 gene.

PURPOSE: To describe an unusual form of dot-and-fleck retinopathy in a slower progressive form of X-linked Alport syndrome, caused by a novel missense mutation in the COL4A5 gene. METHOD: Ophthalmic examination, polymerase chain reaction, and single-strand conformational polymorphism analysis of genomic DNA were performed in the proband. RESULTS: Ophthalmoscopy revealed classic dot-and-fleck retinopathy but located in an unusual site. A novel COL4A5 gene mutation changing glycine to cysteine at 177 was identified. CONCLUSIONS: Although there is no correlation between mutation site and the resulting phenotype in Alport syndrome, our findings suggest that further novel mutations and different ocular manifestations may be associated with Alport syndrome.

A mutation in the rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males.

Heterozygous mutations in the X-linked MECP2 gene cause Rett syndrome, a severe neurodevelopmental disorder of young females. Only one male presenting an MECP2 mutation has been reported; he survived only to age 1 year, suggesting that mutations in MECP2 are male lethal. Here we report a three-generation family in which two affected males showed severe mental retardation and progressive spasticity, previously mapped in Xq27.2-qter. Two obligate carrier females showed either normal or borderline intelligence, simulating an X-linked recessive trait. The two males and the two obligate carrier females presented a mutation in the MECP2 gene, demonstrating that, in males, MECP2 can be responsible for severe mental retardation associated with neurological disorders.

Preserved speech variant is allelic of classic Rett syndrome.

Rett syndrome is a neurological disorder affecting predominantly females with regression loss of speech and purposeful hand use, after a few months of almost normal development. Postnatal microcephaly, hand dispraxia, stereotypic 'hand-washing' activities, ataxia, and abnormal breathing are among its most characteristic features. Another aspect of this disorder is growth failure. The preserved speech variant (PSV) shares with Rett syndrome the same course and the stereotypic hand-washing activities but it differs in that patients typically recover some degree of speech and hand use and usually do not show growth failure. Progressive scoliosis, epilepsy and other minor handicaps, usually present in Rett syndrome, are rare in the preserved speech variant. Here we explore the spectrum of mutations affecting the MECP2 gene in a group of 25 classic Rett syndrome girls and in three patients with the preserved speech variant. Among the Rett syndrome group, two novel mutational hot spots (R270X and R294X), four novel mutations, two novel small deletions, as well as the previously reported 806delG, R168X and R255X mutations, were identified in 20/25 patients. Of note, among the preserved speech variants, two patients carry deletions of 41 bp and 44 bp each, which are strikingly similar to those observed in classic Rett syndrome. Our results confirm the presence of mutational hot spots in MECP2, broaden the spectrum of mutations, pinpoint additional mutational hot spots and establish that the preserved speech variant is indeed allelic of the classic form. Phenotype variability is only partially dependent on the kind of MECP2 mutation and other mechanisms such as skewed X-inactivation, and/or modifier gene effects should be investigated to explain the variable recovery in speech and hand use.