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.

Alport syndrome: impact of digenic inheritance in patients management.

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X-linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene-splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X-linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.

9q31.1q31.3 deletion in two patients with similar clinical features: a newly recognized microdeletion syndrome?

Interstitial deletions of the long arm of chromosome 9 are rare and most patients have been detected by conventional cytogenetic techniques. Disparities in size and localization are large and no consistent region of overlap has been delineated. We report two similar de novo deletions of 6.3 Mb involving the 9q31.1q31.3 region, identified in two monozygotic twins and one unrelated patient through array-CGH analysis. By cloning the deletion breakpoints, we could show that these deletions are not mediated by segmental duplications. The patients displayed a distinct clinical phenotype characterized by mild intellectual disability, short stature with high body mass index, thick hair, arched eyebrows, flat profile with broad chin and mild prognathism, broad, and slightly overhanging tip of the nose, short neck with cervical gibbus. The twin patients developed a metabolic syndrome (type 2 diabetes, hypercholesterolemia, vascular hypertension) during the third decade of life. Although long-term follow-up and collection of additional patients will be needed to obtain a better definition of the phenotype, our findings characterize a previously undescribed syndromic disorder associated with haploinsufficiency of the chromosome 9q31.1q31.3 region.

Xq28 duplications including MECP2 in five females: Expanding the phenotype to severe mental retardation.

Duplications leading to functional disomy of chromosome Xq28, including MECP2 as the critical dosage-sensitive gene, are associated with a distinct clinical phenotype in males, characterized by severe mental retardation, infantile hypotonia, progressive neurologic impairment, recurrent infections, bladder dysfunction, and absent speech. Female patients with Xq duplications including MECP2 are rare. Only recently submicroscopic duplications of this region on Xq28 have been recognized in four females, and a triplication in a fifth, all in combination with random X-chromosome inactivation (XCI). Based on this small series, it was concluded that in females with MECP2 duplication and random XCI, the typical symptoms of affected boys are not present. We present clinical and molecular data on a series of five females with an Xq28 duplication including the MECP2 gene, both isolated and as the result of a translocation, and compare them with the previously reported cases of small duplications in females. The collected data indicate that the associated phenotype in females is distinct from males with similar duplications, but the clinical effects may be as severe as seen in males.

Expanding the phenotype associated with FOXG1 mutations and in vivo FoxG1 chromatin-binding dynamics.

Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

Novel FOXG1 mutations associated with the congenital variant of Rett syndrome.

BACKGROUND: Rett syndrome is a severe neurodevelopmental disorder representing one of the most common genetic causes of mental retardation in girls. The classic form is caused by MECP2 mutations. In two patients affected by the congenital variant of Rett we have recently identified mutations in the FOXG1 gene encoding a brain specific transcriptional repressor, essential for early development of the telencephalon. METHODS: 60 MECP2/CDKL5 mutation negative European Rett patients (classic and variants), 43 patients with encephalopathy with early onset seizures, and four atypical Rett patients were analysed for mutations in FOXG1. RESULTS AND CONCLUSIONS: Mutations have been identified in four patients, independently classified as congenital Rett variants from France, Spain and Latvia. Clinical data have been compared with the two previously reported patients with mutations in FOXG1. In all cases hypotonia, irresponsiveness and irritability were present in the neonatal period. At birth, head circumference was normal while a deceleration of growth was recognised soon afterwards, leading to severe microcephaly. Motor development was severely impaired and voluntary hand use was absent. In contrast with classic Rett, patients showed poor eye contact. Typical stereotypic hand movements with hand washing and hand mouthing activities were present continuously. Some patients showed abnormal movements of the tongue and jerky movements of the limbs. Brain magnetic resonance imaging showed corpus callosum hypoplasia in most cases, while epilepsy was a variable sign. Scoliosis was present and severe in the older patients. Neurovegetative symptoms typical of Rett were frequently present.

Early-onset seizure variant of Rett syndrome: definition of the clinical diagnostic criteria.

BACKGROUND: Rett syndrome is a severe neurodevelopmental disorder affecting almost exclusively females. Among Rett clinical variants, the early-onset seizure variant describes girls with early onset epilepsy and it is caused by mutations in CDKL5. METHODS: Four previously reported girls and five new cases with CDKL5 mutation, ranging from 14 months to 13 years, were evaluated by two clinical geneticists, classified using a severity score system based on the evaluation of 22 different clinical signs and compared with 128 classic Rett and 25 Zappella variant MECP2-mutated patients, evaluated by the same clinical geneticists. Clinical features were compared with previously described CDKL5 mutated patients. Both the statistical and the descriptive approach have been used to delineate clinical diagnostic criteria. RESULTS: All girls present epilepsy with onset varying from 10 days to 3 months. Patients may present different type of seizures both at onset and during the whole course of the disease; multiple seizure types may also occur in the same individual. After treatment with antiepileptic drugs patients may experience a short seizure-free period but epilepsy progressively relapses. Typical stereotypic hand movements severely affecting the ability to grasp are present. Psychomotor development is severely impaired. In the majority of cases head circumference is within the normal range both at birth and at the time of clinical examination. CONCLUSION: For the practical clinical approach we propose to use six necessary and eight supportive diagnostic criteria. Epilepsy with onset between the first week and 5 months of life, hand stereotypies, as well as severe hypotonia, are included among the necessary criteria.

A 9.3 Mb microdeletion of 3q27.3q29 associated with psychomotor and growth delay, tricuspid valve dysplasia and bifid thumb.

We describe a de novo 3q27.3q29 deletion in a 2.5-year-old female patient with developmental and growth delay, dysmorphic facial features, mild tricuspid valve dysplasia, bifid thumb, clinodactyly of the 2nd toe bilaterally and scoliosis. The deletion overlaps for about 1Mb with the 1.6Mb region commonly deleted in patients with 3q29 microdeletion syndrome. The phenotype of the two syndromes is not completely overlapping, though the most important clinical features, such as mental retardation and microcephaly, occur in both. This suggests that the deletion in our patient causes a distinct clinical phenotype, not described previously. In the deleted region there are 47 annotated genes. Among them, seven are of particular interest for correlation with clinical features of the patient. Two genes, OPA1 and CCDC50, responsible for autosomal dominant optic atrophy and deafness, respectively, may be important for the correct follow-up of the patient.

Diagnostic criteria for the Zappella variant of Rett syndrome (the preserved speech variant).

The preserved speech variant is the milder form of Rett syndrome: affected girls show the same stages of this condition and by the second half of the first decade are making slow progress in manual and verbal abilities. They walk without help, and may be able to make simple drawings and write a few words. Most of them can speak in sentences. Autistic behavior can often be observed. We previously described several cases in the pre-molecular era and subsequently reported a survey of 12 cases with MECP2 mutations. Seventeen new patients with the preserved speech variant and a proven MECP2 mutation have been clinically evaluated. Additional clinical data of our previously described cases are reported. These 29 preserved speech variant cases were compared with 129 classic Rett patients using a clinical severity score system including 22 different signs. There was both statistical and clinical evidence of the existence of this variant. On the basis of their abilities these girls can be distinguished as low-, intermediate- and high-functioning. Girls of the last two groups show a greater homogeneity: they speak in sentences, use their hands more easily, have normal somatic features, mild neurovegetative abnormalities, with autistic behavior in 76%, epilepsy in 30%, while girls of the first group are closer to classic Rett syndrome. The majority of patients carries either missense mutations (especially the p.R133C change) or late truncating mutations in the MECP2 gene. These results confirm the existence of this variant of Rett syndrome (Zappella variant), a clear example of progress of manual and verbal abilities, and not of a "preserved speech" and suggest corresponding diagnostic criteria.

A 2.6 Mb deletion of 6q24.3-25.1 in a patient with growth failure, cardiac septal defect, thin upperlip and asymmetric dysmorphic ears.

We report a female patient with neurodevelopmental delay and peculiar facial features. She has postnatal growth failure and an atrial septal defect. Patent duct arteriosis and tricuspidal insufficiency were also noted at birth. Characteristic facial features include medial flare eyebrows, dysmorphic helix of the right ear, cupshaped left ear, anteverted nares, long and smooth philtrum, thin upper lip, high vaulted palate. Array-CGH analysis demonstrated the presence of a 2.6 Mb deletion in 6q24.3-25.1. The phenotypic features of this case are very similar to those previously reported in a patient with a 7Mb overlapping deletion, pointing to a specific new syndrome. Twenty-two genes are present in the common critical deleted region. Among them, there is the PPP1R14C gene that encodes for KEPI, a PKC-potentiated inhibitory protein for type-1 Ser/Thr protein phosphatase. Its selective distribution in brain and heart well correlates with developmental delay and cardiac anomalies observed in the patient.

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.

Alterations of ballistic movements in epileptic patients with phenytoin intoxication.

We assessed the effects of phenytoin (PHT) overdosage on ballistic arm abduction movements in nine epileptic patients receiving long-term PHT treatment. During the overdosage period, all but one showed clinical abnormalities referable to impaired cerebellar function; one also had slowness of movement. Ballistic movements showed abnormalities in all of the patients although a great variability was present in the type and severity of abnormalities. In four patients, kinematic and EMG recordings differed least from the normal, in four they resembled those described in patients with cerebellar deficits, and in one those described in patients with Parkinson disease. The type and severity of clinical disturbances of voluntary motor control as well as alterations of ballistic movements were not related to specific PHT plasma concentrations. One month after the adjustment of PHT dosage, the patients who had clinical abnormalities completely recovered or markedly improved. Previously observed kinematic and EMG abnormalities completely disappeared or improved markedly.

Effects of expected perturbations on the velocity control of fast arm abduction movements.

A triphasic electromyographic pattern of sequential activation of agonist, antagonist, and again agonist muscles underlies rapid or ballistic limb movements in humans. The first agonist burst reflects muscular force accelerating the limb, the antagonist burst is mainly related to the braking process of movement, while the second agonist burst is considered a reactive adjustment to the deceleration. The duration of the first agonist burst has been reported to be constant for movements of different amplitudes, thus suggesting that only changes in its amplitude contribute to the velocity control of movement. The present research has been undertaken to investigate the strategy whereby the nervous system increases agonist impulsive force for ballistic performance in response to experimental conditions requiring perceptual and/or provisional processes related to expected changes in load or accuracy constraints. The effects of expected perturbations of different strengths on some kinematic and electromyographic variables of fast arm abduction movements performed in a step-tracking task and in an outer-stop terminated task were analyzed in normal subjects. All motor performances were characterized by triphasic electromyographic patterns. In the absence of expected perturbations, the mean velocity of the movements was markedly higher in the outer-stop terminated task. Correspondingly, greater amplitudes and durations of the first agonist burst were observed. In both types of motor tasks, when expected perturbations were inserted, the velocity of the movement increased as well as the amplitude and the duration of the first agonist burst. These results, in agreement with previous observations, indicate that the normal mechanism, whereby the nervous system increases agonist impulsive force in rapid movements, comprises changes not only in the amplitude but also in the duration of the first agonist burst.