Next-generation sequencing and genetic diagnosis of Charcot-Marie-Tooth disease

Over 70 different Charcot-Marie-Tooth disease (CMT)–associated genes have now been discovered and their number is growing. Conventional genetic testing for all CMT genes is cumbersome, expensive, and impractical in an individual patient. Next-generation sequencing (NGS) technology allows cost-effective sequencing of large scale DNA, even entire exome (coding sequences) or whole genome and thus, NGS platform can be employed to effectively target a large number or all CMT-related genes for accurate diagnosis. This overview discusses how NGS can be strategically used for genetic diagnosis in patients with CMT or unexplained neuropathy. A comment is made to combine simple clinical and electrophysiological algorithm to assign patients to major CMT subtypes and then employ NGS to screen for all known mutations in the subtype-specific CMT gene panel.     

Histopathological and immunohistochemical study of the enteric innervations among various types of aganglionoses including isolated and syndromic Hirschsprung disease

We investigated enteric innervations in 15 isolated and five syndromic cases of Hirschsprung disease (HSCR) with immunohistochemistry for the S100 protein (S100), class III α‐tubulin (TUJ1), peripherin, neuronal nitric oxide synthase (nNOS) and CD34. The number of neurites per smooth muscle unit of the circular muscle layer (CML) was counted in the longitudinal sections. TUJ1 was the best marker to detect whole neuritic networks of the enteric nervous system. There were differences in the innervation patterns between isolated rectosigmoid aganglionosis (RS) and long segment aganglionosis (LS) including total colonic aganglionosis and extensive aganglionosis. In the aganglionic bowel (AGB) of LS, no nerve fibers innervated smooth muscle units in the CML in the area from the small bowel to the terminal descending colon. In the rectosigmoid region of every type of isolated HSCR, we observed transmural nerve fibers forming meshworks in the CML with TUJ1 and S100 antibodies. In RS, the neurites running parallel with smooth muscle cells gradually decreased in number in the distal portion. However, in the rectosigmoid AGB in LS, those neurites were absent and most neurites perpendicularly crossed the CML. Hypertrophic nerve trunks (HNT) in the submucous and myenteric plexuses were observed more frequently in the rectosigmoid region than in the rostral portion. Based on these data, it is suggested that the neuritic meshworks in the CML of the rectosigmoid AGB might derive from not only the sacral plexus, via HNT, but also intrinsic neurons in the oligoganglionic bowel. All of the syndromic HSCR were RS. In the AGB of RS with Down syndrome, the distribution of neurite meshworks in the CML is markedly reduced. In the AGB of RS with mental retardation suspected of having Mowat–Wilson syndrome, the density of intramuscular innervation was comparatively higher. In the rostral portion to the AGB of syndromic HSCR, myenteric ganglia were clearly small in size, and more numerous per smooth muscle unit with scarce internodal strands. These dysplastic features fall under neither hyperganglionosis nor hypoganglionosis classifications. We considered that syndromic HSCR might occur on the basis of a dysplastic enteric nervous system caused by genetic alteration.     

Exome sequencing reveals mutations in MFN2 and GDAP1 in severe Charcot–Marie–Tooth disease

The aim of our study was to characterize electrophysiologically and explain the genetic cause of severe Charcot–Marie–Tooth (CMT) in a 3.5‐year‐old with asymptomatic parents and a maternal grandfather with a history of mild adult‐onset axonal neuropathy. Severity of neuropathy was assessed by Charcot–Marie–Tooth neuropathy score (CMTNS). Whole‐exome sequencing was performed using an Illumina TruSeq Exome Enrichment Kit on the HiSeq 1500 with results followed up by Sanger sequencing on an ABI Prism 3500XL (Applied Biosystems, Foster City, CA, USA). Paternity was confirmed using a panel of 15 hypervariable markers. Electrophysiological studies demonstrated severe axonal sensory‐motor neuropathy in the proband, mild motor neuropathy in his mother, and mild sensory‐motor neuropathy in his grandfather. CMTNS in the proband, his mother, and grandfather was 21, 1, and 12, respectively. On genetic analysis, the boy was found to carry a heterozygous dominant MFN2 T236M mutation transmitted via the maternal line and a de novo GDAP1 H123R mutation. Our findings emphasize the need to search for more than one causative mutation when significant intrafamilial variability of CMT phenotype occurs and underline the role of whole‐exome sequencing in the diagnosis of compound forms of CMT disease.     

Hirschsprung disease, unusual face, mental retardation, epilepsy, and congenital heart disease: Goldberg-Shprintzen syndrome

A 5-year-old girl with Hirschsprung disease, unusual facial appearance, psychomotor retardation, epilepsy, and congenital heart disease is reported. Patients with similar clinical features have been reported and they appear to exhibit the recently identified Goldberg-Shprintzen syndrome. It is believed that this girl also exhibits this new syndrome. Cranial computed tomography demonstrated abnormal findings that may suggest defective neuronal migration and/or dysgenesis of the brain. These findings were considered to cause psychomotor retardation and epilepsy in this patient.     

Long-term depression in vivo: effects of sex, stress, diet, and prenatal ethanol exposure

Long-term depression (LTD) of synaptic efficacy has proven a difficult phenomenon to examine in vivo, despite the ease with which it is induced in a variety of in vitro preparations. Prior exposure to an acute stressful episode does however seem to enhance the capacity of the hippocampus to exhibit LTD in vivo in male animals. In the present experiments, we examined the capacity for low-frequency stimuli (low-frequency stimulation (LFS)) to induce LTD in juvenile male and female animals following an acute stress episode. Interestingly, prior exposure to stress was only required for the induction of LTD in male animals, while both control and stressed female animals exhibited equivalent LTD. In animals that were exposed to ethanol in utero, a similar requirement for prior exposure to stress to elicit LTD was found for male, but not female animals. This prenatal ethanol exposure did not in itself alter the capacity for LTD induction in either sex; however, in utero food restriction did enhance LTD induction in both male and female animals, irrespective of whether they were exposed to stress just prior to being administered LFS. These results indicate that in utero dietary restriction more drastically affects CA1 LTD than in utero ethanol exposure. In addition, female animals seem to exhibit LTD in vivo in the absence of stress much more easily than their male counterparts.     

Variable penetrance of COL6A1 null mutations: implications for prenatal diagnosis and genetic counselling in Ullrich congenital muscular dystrophy families

Collagen VI mutations cause mild Bethlem myopathy and severe, progressive Ullrich congenital muscular dystrophy (UCMD). We identified a novel homozygous COL6A1 premature termination mutation in a UCMD patient that causes nonsense-mediated mRNA decay. Collagen VI microfibrils cannot be detected in muscle or fibroblasts. The parents are heterozygous carriers of the mutation and their fibroblasts produce reduced amounts of collagen VI. The molecular findings in the parents are analogous to those reported for a heterozygous COL6A1 premature termination mutation that causes Bethlem myopathy. However, the parents of our UCMD proband are clinically normal. The proband's brother, also a carrier, has clinical features consistent with a mild collagen VI phenotype. Following a request for prenatal diagnosis in a subsequent pregnancy we found the fetus was a heterozygous carrier indicating that it would not be affected with severe UCMD. COL6A1 premature termination mutations exhibit variable penetrance necessitating a cautious approach to genetic counselling.     

Dandy-Walker malformation: prenatal diagnosis and prognosis

Introduction

The difficulty in prognosticating the clinical and intellectual outcome of fetuses presenting with a Dandy-Walker malformation (DWM) comes from the great variety of cystic, median, and retrocerebellar malformations that probably have nothing in common and the variability of the definitions given to these lesions. In addition, many of these lesions can mimic each other. A correct diagnosis cannot be made without a good quality MRI including sagittal views of the vermis and T2-weighted images. We limited the diagnosis of DWM to those malformations with all of the following features: 1) a large median posterior fossa cyst widely communicating with the fourth ventricle, 2) a small, rotated, raised cerebellar vermis, 3) an upwardly displaced tentorium, 4) an enlarged posterior fossa, 5) antero-laterally displaced but apparently normal cerebellar hemispheres, 6) a normal brain stem. If any one of the previous criteria were not met, the malformation was considered distinct from DWM.

Materials and methods

The charts of 26 patients with DWMs (18 females and 8 males; median age 10.5 years) were reviewed retrospectively. The diagnosis of the malformation was made prenatally in 7 children and postnatally in the 19 others. All the patients had both one MRI including axial and sagittal views of the posterior fossa as well as T1- and T2-weighted sequences, and one neuro-psychological investigation. Syndromic DWMs and Dandy Walker variants were excluded from the study. MRIs were reviewed in a blinded manner looking for brain malformation or damage and studying with particular attention the anatomy of the vermis. Systemic malformations were also recorded. Developmental quotient (DQ) and intellectual quotient (IQ) were said to be normal when equal or greater than 85, and low when below this value. Statistical analysis was performed using a Fisher test to analyze the relationship between intellectual performances, vermis anatomy, ventricular size, brain anatomy, and associated malformations.

Results

On scrutiny of sagittal T2 sequences, the vermis, although constantly small, rotated, and pushed towards the tentorium presented as two distinct morphologies, leading us to distinguish two groups of patients. In the first group (n=21), the vermis presented with two fissures, three lobes, and a fastigium as in the normal situation. In this particular group, none of the patients had associated brain malformation and all but 2 were functioning normally. One of the 2 retarded children had a fragile X syndrome. The other had a severe periventricular leukomalacia due to prematurity, which, per se, was sufficient to account for mental delay. In the second group (n=5), the vermis was highly malformed, obviously dysplastic, presenting with only one fissure or no fissure at all. It was constantly associated with major brain anomalies, most often a complete corpus callosum agenesis. All the patients in this group were more or less severely retarded. Vermis anatomy in DWMs was statistically correlated to neurological and intellectual outcome. Is the vermis dysplasia responsible, in itself, for this poor outcome? No answer can be given from this series, because retardation was observed in children who always had both a severely dysplastic vermis and other brain malformations. No other patient-related factor was statistically correlated to the outcome, in particular, hydrocephalus and extracerebral malformations.

Conclusion

We described two types of DWM. The most frequent is characterized by an isolated and partially agenetic vermis. This malformation is compatible with a normal life. The second type consists of a severely abnormally lobulated vermis and associated brain malformation. This malformation is always accompanied by mental retardation.     

Personality,dopamine, and Parkinson's disease: Insights from subthalamic stimulation

Background : Subthalamic stimulation improves the motor and neuropsychiatric symptoms of Parkinson's disease. However, the impact of this treatment on impulse control and personality is the subject of heavy debate. The objective of this study was to investigate personality changes after subthalamic stimulation. Methods : Using Cloninger's biosocial model, we assessed personality in 73 Parkinson's disease patients before and 12 months after subthalamic stimulation accompanied by a drastic reduction in dopaminergic medication. Changes in psychobehavioral symptoms were measured using a battery of validated clinical scales (apathy, depression, anxiety, hyperemotionality, mania, psychosis, punding, and impulse control behaviors). Results : One year after surgery, the harm avoidance personality domain total score increased compared with the baseline (+2.8; 34 patients; P < 0.001), as did 3 of its 4 subdomains: anticipatory worry (+0.7; 10 patients; P = 0.005), shyness (+0.6; 7 patients; P = 0.03), and fatigability (+1.1; 10 patients; P = 0.0014). Evolution of the shyness personality trait correlated with the decrease in dopaminergic medication. Total scores in the other personality domains remained unchanged, except for extravagance, a subdomain of novelty seeking, and persistence, a subdomain of reward dependence, which both decreased following surgery (‐0.3; 7 patients; and ‐0.6; 9 patients; P = 0.03 and P = 0.0019, respectively). Although apathy increased, other psychobehavioral symptoms, including impulse control behaviors and neuropsychiatric nonmotor fluctuations, improved. Depression and anhedonia remained stable. Scores in hypodopaminergia and neuropsychiatric nonmotor OFF correlated with harm avoidance. Scores in hyperdopaminergia and neuropsychiatric nonmotor ON correlated with novelty seeking. Conclusions : When subthalamic stimulation is applied in Parkinson's disease, significant changes in personality traits are observed, which may be related to postoperative tapering of dopaminergic treatment. © 2017 International Parkinson and Movement Disorder Society     

Cervical myelocystocele: prenatal diagnosis and therapeutical considerations

Background  Cervical myelocystocele (CMC) is a very rare congenital malformation and belongs to the spectrum of skin-covered (occult) dysraphisms. Only 15 cases have been so far reported throughout the literature. We report the first case of CMC whose diagnosis was established prenatally by ultrasound imaging (US) followed by fetal magnetic resonance imaging (MR). Case History  A 35-year-old woman was referred for further investigations following prenatal assessment of a fetal cervical mass observed on routine US during pregnancy. Fetal karyotype was normal. Fetal MR confirmed the ultrasonographic findings and led us to strongly suspect the diagnosis of CMC. The newborn was operated on 2 months after birth. The goal of surgical procedure was to remove the malformation and to obtain an untethering of the spinal cord. Twelve months later, the child is still neurologically intact. Discussion  We discuss embryogenesis, different subtypes, associated malformations, and surgical strategy associated with myelocystoceles. Conclusions  This case adds to the existing literature in that it shows for the first time antenatal images of this rare condition and discusses treatment and follow-up implications.     

LRRK2 mutations in Spanish patients with Parkinson disease: frequency, clinical features, and incomplete penetrance

BACKGROUND: Several pathogenic mutations in the LRRK2 gene have been implicated in familial and sporadic cases of Parkinson disease (PD). The R1441G mutation is frequent in Spanish patients of Basque ethnicity with PD, and the G2019S mutation is a common mutation found in several populations worldwide. OBJECTIVES: To determine the frequency of the LRRK2 G2019S and R1441G mutations in PD patients from the non-Basque northeast region of Spain (Catalonia), and to characterize their family history and clinical features. DESIGN: We screened patients for the presence of the LRRK2 R1441G and G2019S mutations. These LRRK2 mutations were detected by restriction endonuclease digestion, and samples with an abnormal electrophoresis pattern were sequenced to identify the exact nucleotide change. The clinical features and family history of patients with LRRK2 mutations were studied in detail. SETTING: The northeast region of Spain.Patients Three hundred two patients with PD. MAIN OUTCOME MEASURES: Onset age, clinical features, and family history of PD and LRRK2 mutations. RESULTS: The R1441G mutation was present in 0.7% of total PD cases. The G2019S mutation was found in 6.4% of familial and 3.4% of sporadic cases. Additionally, we found 1 patient with the R1441C mutation. Age at onset ranged from 33 to 78 years. Clinical features were not different from classic PD, except for 1 patient who presented with monosymptomatic leg rest tremor of 8 years' duration. In addition, a 91-year-old unaffected relative of a patient with the G2019S mutation was found to be a mutation carrier. CONCLUSIONS: The G2019S mutation frequency in PD patients from northeast Spain is similar to that reported in other European regions. The R1441G mutation is very uncommon in Catalonia. The presence of an aged unaffected G2019S mutation carrier supports the previously described occurrence of incomplete penetrance in PD patients with LRRK2 mutations.     

Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study

BACKGROUND: Mutations in LRRK2, the gene that encodes leucine-rich repeat kinase 2, are a cause of Parkinson's disease (PD). The International LRRK2 Consortium was established to answer three key clinical questions: can LRRK2-associated PD be distinguished from idiopathic PD; which mutations in LRRK2 are pathogenic; and what is the age-specific cumulative risk of PD for individuals who inherit or are at risk of inheriting a deleterious mutation in LRRK2? METHODS: Researchers from 21 centres across the world collaborated on this study. The frequency of the common LRRK2 Gly2019Ser mutation was estimated on the basis of data from 24 populations worldwide, and the penetrance of the mutation was defined in 1045 people with mutations in LRRK2 from 133 families. The LRRK2 phenotype was defined on the basis of 59 motor and non-motor symptoms in 356 patients with LRRK2-associated PD and compared with the symptoms of 543 patients with pathologically proven idiopathic PD. FINDINGS: Six mutations met the consortium's criteria for being proven pathogenic. The frequency of the common LRRK2 Gly2019Ser mutation was 1% of patients with sporadic PD and 4% of patients with hereditary PD; the frequency was highest in the middle east and higher in southern Europe than in northern Europe. The risk of PD for a person who inherits the LRRK2 Gly2019Ser mutation was 28% at age 59 years, 51% at 69 years, and 74% at 79 years. The motor symptoms (eg, disease severity, rate of progression, occurrence of falls, and dyskinesia) and non-motor symptoms (eg, cognition and olfaction) of LRRK2-associated PD were more benign than those of idiopathic PD. INTERPRETATION: Mutations in LRRK2 are a clinically relevant cause of PD that merit testing in patients with hereditary PD and in subgroups of patients with PD. However, this knowledge should be applied with caution in the diagnosis and counselling of patients. FUNDING: UK Medical Research Council; UK Parkinson's Disease Society; UK Brain Research Trust; Internationaal Parkinson Fonds; Volkswagen Foundation; National Institutes of Health: National Institute of Neurological Disorders and Stroke and National Institute of Aging; Udall Parkinson's Disease Centre of Excellence; Pacific Alzheimer Research Foundation Centre; Italian Telethon Foundation; Fondazione Grigioni per il Morbo di Parkinson; Michael J Fox Foundation for Parkinson's Research; Safra Global Genetics Consortium; US Department of Veterans Affairs; French Agence Nationale de la Recherche.     

Proline‐rich transmembrane protein 2–negative paroxysmal kinesigenic dyskinesia: Clinical and genetic analyses of 163 patients

Background : Paroxysmal kinesigenic dyskinesia is the most common type of paroxysmal dyskinesia. Approximately half of the cases of paroxysmal kinesigenic dyskinesia worldwide are attributable to proline‐rich transmembrane protein 2 mutations. Objective : The objective of this study was to investigate potential causative genes and clinical characteristics in proline‐rich transmembrane protein 2–negative patients with paroxysmal kinesigenic dyskinesia. Methods : We analyzed clinical manifestations and performed exome sequencing in a cohort of 163 proline‐rich transmembrane protein 2–negative probands, followed by filtering data with a paroxysmal movement disorders gene panel. Sanger sequencing, segregation analysis, and phenotypic reevaluation were used to substantiate the findings. Results : The clinical characteristics of the enrolled 163 probands were summarized. A total of 39 heterozygous variants were identified, of which 33 were classified as benign, likely benign, and uncertain significance. The remaining 6 variants (3 novel, 3 documented) were pathogenic and likely pathogenic. Of these, 3 were de novo (potassium calcium‐activated channel subfamily M alpha 1, c.1534A>G; solute carrier family 2 member 1, c.418G>A; sodium voltage‐gated channel alpha subunit 8, c.3640G>A) in 3 sporadic individuals, respectively. The other 3 (paroxysmal nonkinesiogenic dyskinesia protein, c.956dupA; potassium voltage‐gated channel subfamily A member 1, c.765C>A; Dishevelled, Egl‐10, and Pleckstrin domain containing 5, c.3311C>T) cosegregated in 3 families. All 6 cases presented with typical paroxysmal kinesigenic dyskinesia characteristics, except for the Dishevelled, Egl‐10, and Pleckstrin domain containing 5 family, where the proband's mother had abnormal discharges in her temporal lobes in addition to paroxysmal kinesigenic dyskinesia episodes. Conclusions : Our findings extend the genotypic spectrum of paroxysmal kinesigenic dyskinesia and establish the associations between paroxysmal kinesigenic dyskinesia and genes classically related to other paroxysmal movement disorders. De novo variants might be a cause of sporadic paroxysmal kinesigenic dyskinesia. © 2018 International Parkinson and Movement Disorder Society     

Agenesis of corpus callosum: prenatal diagnosis and prognosis

Introduction Agenesis of corpus callosum (ACC) is commonly diagnosed prenatally. When isolated, it appears to carry a good prognosis but studies are often retrospective and follow-up short. We report a prospective study of 17 children (11 boys, 6 girls) with prenatally diagnosed isolated ACC.Methods Neuropsychological evaluation was performed each year and results at the ages of 2, 4, and 6 years were compared.Results Febrile seizures occurred in 3 patients. Median intellectual quotient (IQ) was within the normal range (80–109) and nonrelated to partial or complete ACC, sex, or febrile seizures. Lower median IQ was significantly related to low cultural status. With age, the number of children with IQ in the lower range (80–89) increased and slowness, attentional troubles, and instability appeared.Conclusion This study demonstrates that if outcome of isolated ACC is favorable, a long follow-up is necessary: with age, IQ in the lower range and behavioral troubles are linked to difficulties in school.     

Congenital hydrocephalus in the northeast of Brazil: epidemiological aspects, prenatal diagnosis, and treatment

Purpose

Congenital hydrocephalus (CH) has higher proportions in developing countries such as Brazil, reaching rates of 3.16:1,000 newborns. Early diagnosis is essential and can be done during the second trimester of pregnancy, highlighting the importance of regular prenatal care. Our objective is to describe the epidemiological profile of children with CH in the state of Bahia.

Methods

Consecutive medical records of patients with CH, aged less than 2years and operated at a pediatric reference hospital between September 2009 and September 2012, were reviewed.

Results

One hundred twenty-one children underwent ventricular peritoneal shunt (VP shunt) due to CH. As for prenatal care, 38 % of pregnant women did it regularly. Males predominated with 56 % of cases with a median age of 3 months, and 68 % were from the countryside area. The most frequent clinical manifestations were bulging fontanelle (37 %) and increased head circumference (30 %). There were 13 (11 %) reports of complications associated with VP shunts. There were nine deaths (7 %), and respiratory complications were the main cause.

Conclusions

Public health measures are the key to increase mothers’ knowledge about the importance of regular prenatal monitoring. There was a predominance of male children, with a median age of 3 months, with the majority coming from the countryside.