Clinical Genetic Correlation Research Articles

P268 Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations

P268 Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations

Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations

Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. The cellular and molecular consequences of the lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression of the disease. Here we have analyzed quadriceps muscle biopsies of seven DMD patients aged 2 to 4 years old and five age and gender matched controls using single nuclei RNA sequencing (snRNAseq) and correlated the results obtained with clinical data. SnRNAseq identified significant differences in the proportion of cell population present in the muscle samples, including an increase in the number of regenerative fibers, satellite cells, and fibro-adipogenic progenitor cells (FAPs) and a decrease in the number of slow fibers and smooth muscle cells. Muscle samples from the younger patients with stable mild weakness were characterized by an increase in regenerative fibers, while older patients with moderate and progressive weakness were characterized by loss of muscle fibers and an increase in FAPs. An analysis of the gene expression profile in muscle fibers identified a strong regenerative signature in DMD samples characterized by the upregulation of genes involved in myogenesis and muscle hypertrophy. In the case of FAPs, we observed upregulation of genes involved in the extracellular matrix regeneration but also several signaling pathways. Indeed, further analysis of the potential intercellular communication profile showed a dysregulation of the communication profile in DMD samples identifying FAPs as a key regulator of cell signaling in DMD muscle samples. In conclusion, our study has identified significant differences at the cellular and molecular levels in the different cell populations present in skeletal muscle samples of patients with DMD compared to controls.

Updates on congenital hereditary endothelial dystrophy.

Congenital hereditary endothelial dystrophy (CHED) is a rare genetic corneal disorder causing progressive cornea clouding and significant visual impairment. CHED remains a leading indication for pediatric corneal transplantation despite its infrequency, particularly in regions with high consanguinity rates like Southeast Asia. Identifying the Solute Carrier Family 4 Member 11 (SLC4A11) gene as the genetic basis of CHED has led to the discovery of it's various genetic variations. However, a comprehensive understanding of its clinical-genetic correlation, pathophysiology, and optimal management is ongoing. This review aims to consolidate current knowledge about CHED, covering its genetic origins, pathophysiological mechanisms, clinical presentation, and management strategies. Surgical intervention, such as penetrating keratoplasty (PK), Descemet stripping automated endothelial keratoplasty (DSAEK), and Descemet membrane endothelial keratoplasty (DMEK), remains the primary treatment. DSAEK and DMEK offer advantages over PK, including quicker visual recovery, reduced complications, and longer graft survival, especially in the pediatric age group. The timing of surgical interventions depends on disease severity, age at presentation, comorbidities, and visual potential. Elevated oxidative stress in CHED corneal tissue suggests potential benefits from anti-inflammatory drugs to rescue mutated endothelial cells. Considering the limitations of corneal graft surgeries, exploring novel gene-based molecular therapies are essential for future management. Early diagnosis, appropriate surgical interventions, amblyopia control, and genetic counseling for predictive analysis are pivotal for optimizing CHED management. A multidisciplinary approach involving ophthalmologists, researchers, and genetic counselors is essential for precise diagnosis and optimal care for CHED patients.

Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review.

Mitochondrial encephalomyopathies (MEMP) are heterogeneous multisystem disorders frequently associated with mitochondrial DNA (mtDNA) mutations. Clinical presentation varies considerably in age of onset, course, and severity up to death in early childhood. In this study, we performed molecular genetic analysis for mtDNA pathogenic mutation detection in Serbian children, preliminary diagnosed clinically, biochemically and by brain imaging for mitochondrial encephalomyopathies disorders. Sanger sequencing analysis in three Serbian probands revealed two known pathogenic mutations. Two probands had a heteroplasmic point mutation m.3243A>G in the MT-TL1 gene, which confirmed mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome (MELAS), while a single case clinically manifested for Leigh syndrome had an almost homoplasmic (close to 100%) m.8993T>G mutation in the MT-ATP6 gene. After full mtDNA MITOMASTER analysis and PhyloTree build 17, we report MELAS’ association with haplogroups U and H (U2e and H15 subclades); likewise, the mtDNA-associated Leigh syndrome proband shows a preference for haplogroup H (H34 subclade). Based on clinical–genetic correlation, we suggest that haplogroup H may contribute to the mitochondrial encephalomyopathies’ phenotypic variability of the patients in our study. We conclude that genetic studies for the distinctive mitochondrial encephalomyopathies should be well-considered for realizing clinical severity and possible outcomes.

Clinical, genetic and neuropathological characterization of spinocerebellar ataxia type 37.

The autosomal dominant spinocerebellar ataxias (SCAs) consist of a highly heterogeneous group of rare movement disorders characterized by progressive cerebellar ataxia variably associated with ophthalmoplegia, pyramidal and extrapyramidal signs, dementia, pigmentary retinopathy, seizures, lower motor neuron signs, or peripheral neuropathy. Over 41 different SCA subtypes have been described evidencing the high clinical and genetic heterogeneity. We previously reported a novel spinocerebellar ataxia type subtype, SCA37, linked to an 11-Mb genomic region on 1p32, in a large Spanish ataxia pedigree characterized by ataxia and a pure cerebellar syndrome distinctively presenting with early-altered vertical eye movements. Here we demonstrate the segregation of an unstable intronic ATTTC pentanucleotide repeat mutation within the 1p32 5' non-coding regulatory region of the gene encoding the reelin adaptor protein DAB1, implicated in neuronal migration, as the causative genetic defect of the disease in four Spanish SCA37 families. We describe the clinical-genetic correlation and the first SCA37 neuropathological findings caused by dysregulation of cerebellar DAB1 expression. Post-mortem neuropathology of two patients with SCA37 revealed severe loss of Purkinje cells with abundant astrogliosis, empty baskets, occasional axonal spheroids, and hypertrophic fibres by phosphorylated neurofilament immunostaining in the cerebellar cortex. The remaining cerebellar Purkinje neurons showed loss of calbindin immunoreactivity, aberrant dendrite arborization, nuclear pathology including lobulation, irregularity, and hyperchromatism, and multiple ubiquitinated perisomatic granules immunostained for DAB1. A subpopulation of Purkinje cells was found ectopically mispositioned within the cerebellar cortex. No significant neuropathological alterations were identified in other brain regions in agreement with a pure cerebellar syndrome. Importantly, we found that the ATTTC repeat mutation dysregulated DAB1 expression and induced an RNA switch resulting in the upregulation of reelin-DAB1 and PI3K/AKT signalling in the SCA37 cerebellum. This study reveals the unstable ATTTC repeat mutation within the DAB1 gene as the underlying genetic cause and provides evidence of reelin-DAB1 signalling dysregulation in the spinocerebellar ataxia type 37.

Brain imaging in myotonic dystrophy type 1: A systematic review.

To systematically review brain imaging studies in myotonic dystrophy type 1 (DM1). We searched Embase (index period 1974-2016) and MEDLINE (index period 1946-2016) for studies in patients with DM1 using MRI, magnetic resonance spectroscopy (MRS), functional MRI (fMRI), CT, ultrasound, PET, or SPECT. From 81 studies, we extracted clinical characteristics, primary outcomes, clinical-genetic correlations, and information on potential risk of bias. Results were summarized and pooled prevalence of imaging abnormalities was calculated, where possible. In DM1, various imaging changes are widely dispersed throughout the brain, with apparently little anatomical specificity. We found general atrophy and widespread gray matter volume reductions in all 4 cortical lobes, the basal ganglia, and cerebellum. The pooled prevalence of white matter hyperintensities is 70% (95% CI 64-77), compared with 6% (95% CI 3-12) in unaffected controls. DTI shows increased mean diffusivity in all 4 lobes and reduced fractional anisotropy in virtually all major association, projection, and commissural white matter tracts. Functional studies demonstrate reduced glucose uptake and cerebral perfusion in frontal, parietal, and temporal lobes, and abnormal fMRI connectivity patterns that correlate with personality traits. There is significant between-study heterogeneity in terms of imaging methods, which together with the established clinical variability of DM1 may explain divergent results. Longitudinal studies are remarkably scarce. DM1 brains show widespread white and gray matter involvement throughout the brain, which is supported by abnormal resting-state network, PET/SPECT, and MRS parameters. Longitudinal studies evaluating spatiotemporal imaging changes are essential.

A Scoring System to Predict the Severity of Hirschsprung Disease at Diagnosis and Its Correlation With Molecular Genetics.

Objectives Hirschsprung disease (HSCR) has a wide range of severity. There are nonsevere forms treated conservatively until surgery and severe forms that require an early stoma and prolonged hospitalization. Our objective was to establish a clinical scoring system to predict the severity of HSCR and to evaluate the possible existence of a clinical-genetic correlation. Methods We carried out a retrospective observational study including all HSCR cases treated in our hospital. The sample was divided into severe and nonsevere disease according to the number of surgical procedures, hospitalization time, and episodes of enterocolitis. The proposed score was applied at diagnosis, and the sensitivity, specificity, and optimal cut-point were determined. We conducted a prospective molecular study of RET, EDNRB, and EDN3 on all patients, as well as SOX10 in Waardenburg Syndrome type 4 forms. Results Among the 42 patients treated between 1983 and 2013, 15 met the severe disease criteria. This group had a higher mean score (13.15 ± 2.36) than the nonsevere group (8.15 ± 2.13; p < 0.001). A score ≥11 had a sensitivity of 87% and a specificity of 81% in detecting the severe cases. Causative mutations were identified in 12 patients, 8 of them in the severe group ( p = 0.015). Most of these mutations (75%) were located in the RET proto-oncogene. Conclusion The proposed scoring system enables the early selection of patients with severe behavior of HSCR. A value ≥11 showed good sensitivity and specificity for this purpose. Causative mutations were identified in more than 50% of patients who met the criteria for severe disease.

Voice Alterations in Patients With Spinocerebellar Ataxia Type 7 (SCA7): Clinical-Genetic Correlations

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine triplet located in the coding region of the ATXN7 gene, which is characterized by cerebellar ataxia, pigmentary macular degeneration, and dysarthria. Although dysarthria is a common feature in various SCA, its clinical characterization has been barely approached. In this study, we report, to our knowledge for the first time, a detailed voice analysis in a large series of patients with SCA7, using different vocal parameters, including jitter, shimmer, and fundamental frequency. Patients were molecularly diagnosed using fluorescent-based polymerase chain reaction and capillary electrophoresis, and clinically characterized using the Scale for the Assessment and Rating of Ataxia and the Inventory of Non-Ataxia Symptoms. We found altered jitter, shimmer, and fundamental frequency measurements in patients with SCA7 compared with control subjects (P < 0.05). However, voice impairment was found unrelated with both age at disease onset and size of the cytosine-adenine-guanine triplet tract. Remarkably, jitter and shimmer measurements of patients were found to correlate with their Inventory of Non-Ataxia Symptoms, but not with their Scale for the Assessment and Rating of Ataxia scores, implying that voice impairment is the result of extra-cerebellar manifestations of the disease. We propose that deficiency of the extra-cerebellar component of SCA7 might lead to sudden changes in laryngeal muscle tone, producing instability in sustained vowel phonation. Clinical characterization of voice will help to discriminate SCA7 from other SCA and to guide vocal therapy treatments.

A Scoring System to Predict the Severity of Hirschsprung Disease at Diagnosis and its Correlation with Molecular Genetics.

Hirschsprung disease (HSCR) has a wide range of severity. There are non-severe forms treated conservatively until surgery and severe forms that require an early stoma and prolonged hospitalization. Our objective was to establish a clinical scoring system to predict the severity of HSCR and to evaluate the possible existence of a clinical-genetic correlation. We carried out a retrospective observational study including all HSCR cases treated in our hospital. The sample was divided into severe and non-severe disease according to the number of surgical procedures, hospitalization time and episodes of enterocolitis. The proposed score was applied at diagnosis, and the sensitivity, specificity and optimal cut-point were determined. We conducted a prospective molecular study of RET, EDNRB and EDN3 on all patients, as well as SOX10 in Waardenburg Syndrome type 4 forms. Among the 42 patients treated between 1983 and 2013, 15 met the severe disease criteria. This group had a higher mean score (13.15±2.36) than the non-severe group (8.15±2.13; p<0.001). A score {greater than or equal to}11 had a sensitivity of 87% and a specificity of 81% in detecting the severe cases. Causative mutations were identified in 12 patients, 8 of them in the severe group (p=0.015). Most of these mutations (75%) were located in the RET proto-oncogene. The proposed scoring system enables the early selection of patients with severe behaviour of HSCR. A value {greater than or equal to}11 showed good sensitivity and specificity for this purpose. Causative mutations were identified in more than 50% of patients who met the criteria for severe disease.

PP04.16 – 3030: Phenotypic variability with the same MECP2 mutations – An analysis of the Romanian Rett database

Objective Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder that is mainly caused by mutations in Methyl-CpGbinding protein 2 (MECP2). The classical RTT picture is characterized by acquired microcephaly and a period of regression with loss of purposeful hand skills and of spoken language, gait abnormalities and stereotypic hand movements. Methods We exemplify the phenotypes of R168X and R255X mutations wich are the second and third most frequent MECP2 mutations found. The patients fom the national Rett database were analysed searching for the same mutation type. As of 2014 the database consists of 40 patients with clinical diagnosis of RTT, 27 of these patients have been genetically tested for MECP2 mutation, 16 of which are positive for a mutation in this gene. Results The patients are described regarding prehension, loss of ambulation, loss of speech, hand sterotypies presence, age at regression onset, presence of breathing anomalies; also association of epilepsy diagnosis and its response to treatment is discussed. Two groups of patients with the same mutation were identified – three girls with R255X and three girls with R168X mutation with ages between 13 months to 10 years. They all have clinical pictures of classical Rett with age at regession onset under 18 months for 2 of the girls in each group. Phenotypic and genotypic variability are frequent in RTT and this is reflected in the two groups. In the R255X group (which is associated with epilepsy in the literature) one girl had early onset drug-resistant epilepsy and severe clinical regression; the other kept prehension and gait, has mild-moderate mental retardation and no epilepsy. Conclusion No significant clinical-genetic correlations could be realised, due to the small number of identical mutations in our group. More factors (genetic or nongenetic) that influence RTT clinical picture and outcome may be involved.

G.P.251: The Italian Registry of Limb Girdle Muscular Dystrophy: Natural history, genotype–phenotype correlations and outcome measures

Limb girdle muscular dystrophies (LGMD) are genetic heterogeneous disorders characterized by slow but progressive muscular impairment. Despite the recent identification of new genes, about 30% of these LGMDs remain without molecular diagnosis. However clinical-genetic correlations are fundamental for genetic counselling, definition of natural history and insight into pathogenesis. We collected detailed clinical, biochemical, histological and molecular data of 467 Italian LGMD patients, belonging to 8 neuromuscular Italian centres. Detailed data about neuromuscular, cardiological, respiratory involvement as far as molecular analysis and muscle biopsy were collected. Molecular analysis identified mutations in LGMD causative genes in 312 patients; 109 patients (23.1%) are still without a molecular diagnosis and 46 subjects (9.8%) carried heterozygous mutations in genes responsible for autosomal recessive forms. The relative frequency was as follows: 5.8% LGMD1B, 11.2% LGMD1C, 24.3% LGMD2A, 25.6% LGMD2B, 9.6% LGMD2I, 9.9% LGMD2D, 6.4% LGMD2E, 4.2% LGMD2C, 1.9% LGMD2L, 0.3% LGMD2F. Interestingly 1 LGMD patient (0.3%) carried mutations in ISPD and one patient in LAMA2 gene. Onset spans from the first decade to adulthood; LGMD2E being the most precocious (6.2 ± 5.3 years) and LGMD2L the latest (36.6 ± 7.1 years). Creatine-kinase values were generally increased, especially in sarcoglycanopathies, LGMD2B, LGMD1C. Cardiomyopathy was more frequent in LGMD1B (100%), LGMD2E (47%) and LGMD2I (50%) and restrictive pulmonary involvement in LGMD2I (53%) and LGMD2E (47%). About 30% of patients were wheelchair bound. The study of undiagnosed patients will potentially lead to identification of new LGMD causative genes. Overall this study defined the relative frequency of Italian LGMD and improved the knowledge about clinical, morphological and molecular spectrum as far as their natural history as far as the parameters that can better define the clinical evolution of these forms.

Correlation of clinical and molecular features in spinal bulbar muscular atrophy

Objectives:To characterize the clinical and genetic features of spinal bulbar muscular atrophy (SBMA), a rare neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the androgen receptor gene, in the United Kingdom.Methods:We created a national register for SBMA in the United Kingdom and recruited 61 patients between 2005 and 2013. In our cross-sectional study, we assessed, by direct questioning, impairment of activities of daily living (ADL) milestones, functional rating, and subjective disease impact, and performed correlations with both CAG repeat size and degree of somatic mosaicism. Ten patients were deceased, 46 patients participated in the study, and 5 declined.Results:Subjects had an average age at onset of 43.4 years, and weakness onset most frequently occurred in the lower limbs (87%). Impaired mobility was the most frequently reported problem by patients, followed by bulbar dysfunction. Age distribution of the impairment of ADL milestones showed remarkable overlap with a Japanese study. We have identified a significant correlation between the number of CAG repeats and both age at onset and ADL milestones. Somatic mosaicism also showed a correlation with CAG expansion size and age at onset.Conclusions:Clinical features in SBMA show a substantial overlap when comparing populations with different genetic backgrounds. This finding has major implications, because multicenter trials will be necessary to obtain sufficient power in future clinical trials. Clinical-genetic correlations are strong in SBMA and should inform any clinical research strategy in this condition.

Clinical-Genetic Correlations in Familial Alzheimer's Disease Caused by Presenilin 1 Mutations

We describe the clinical phenotype of nine kindred with presenile Alzheimer's disease (AD) caused by different presenilin 1 (PS1) point mutations, and compare them with reported families with mutations in the same codons. Mutations were in exon 4 (Phe105Val), exon 5 (Pro117Arg, Glu120Gly), exon 6 (His163Arg), exon 7 (Leu226Phe), exon 8 (Val261Leu, Val272Ala, Leu282Arg), and exon 12 (Ile439Ser). Three of these amino acid changes (Phe105Val, Glu120Gly, and Ile439Ser) had not been previously reported. Distinct clinical features, including age of onset, symptoms and signs associated with the cortical-type dementia and aggressiveness of the disease, characterized the different mutations and were quite homogeneous across family members. Age of onset fell within a consistent range: some mutations caused the disease in the thirties (P117R, L226F, V272A), other in the forties (E120G, H163R, V261L, L282R), and other in the fifties (F105V, I439S). Associated features also segregated with specific mutations: early epileptic activity (E120G), spastic paraparesis (V261L), subcortical dementia and parkinsonism (V272A), early language impairment, frontal signs, and myoclonus (L226F), and late myoclonus and seizures (H163R, L282R). Neurological deterioration was particularly aggressive in PS1 mutations with earlier age of onset such as P117R, L226F, and E120G. With few exceptions, a similar clinical phenotype was found in families reported to have either the same mutation or different amino acid changes in the same codons. This series points to a strong influence of the specific genetic defect in the development of the clinical phenotype.

Genetic analysis (PKD2) of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease is a multi-organic hereditary disorder. It is responsible for 7-10% of cases of end stage renal failure. It is caused by mutations in the genes PKD1 and PKD2. Both polycystic kidney disease's forms have a pathogeny and similar clinic, but in the patients with mutation in PKD2, the clinical manifestations appear later and the progression to end stage renal failure happens 10 years later than in the patients with mutation in PKD1. The diagnosis of this disease can be performed through ultrasounds, but the molecular diagnosis offers some advantages, such as the early detection of asymptomatic individuals who carry this genetic defect, in order to perform a preventive monitoring and genetic counselling. In this work, we present the results of the mutational analysis of the PKD2 gene in 18 patients diagnosed with autosomal dominant polycystic kidney disease. The objectives of this work were to analyze the profitability of the genetic study compared with the radiologic study, and perform an early genetic diagnosis in the descendants of patients who were affected by a mutation in the PKD2 gene, trying to establish a correlation between phenotype and genotype. After the genetic analysis, only one family was diagnosed with a mutation in exon 13 of the PKD2 gene (5.56%), which consists on a substitution of the nucleotide adenosine by cytosine (c.2398A>C), which implies that the amino acid methionine is replaced by leucine (p.800Met>Leu). In our population, contrary to what was published in the literature, the mutation of the gene was clinically significant and did segregate with the disease. All the members with a clinical and ultrasound diagnosis of polycystic renal disease presented the above mentioned mutation. We could not confirm any clinical-genetic correlation. Due to the high prevalence of chronic renal failure and terminal chronic renal failure secondary to polycystic kidney disease in our study, an early genetic diagnosis would involve a better prognosis in connection with a closer clinical monitoring.

762-4 Importance of Microdeletions of Chromosomal Region 22q11 In the Etiology of Congenital Conotruncal Malformations: A 2 Year Prospective Study

Microdeletions of 22q11 are a well recognised cause of DiGeorge Syndrome, Velocardiofacial syndrome and familial conotruncal heart disease. We sought to clarify the importance of 22q11 microdeletions and other identifiable cytogenetic abnormalities in the etiology of conotruncal malformations and to establish clinical-genetic correlations. From 6/92-8/94 we prospectively studied 39 consecutive infants presenting to a regional pediatric cardiac centre with a conotruncal malformation. Diagnoses were: tetralogy (21), pulmonary atresia NSD (8), DORV (3). type B IAA (3), truncus (2). absent pulmonary valve syndrome (1) and isolated right aortic arch (1). 17 (44%) were considered dysmorphic by a clinical geneticist. In 5 cases the dysmorphic features were major. but no firm diagnosis was established on clinical grounds. One of these patients was shown to have an unbalanced translocation involving chromosomes 4 &amp; 16. Twelve patients had subtler dysmorphic features. Nine of these cases were prospectively considered to have phenotypic appearances consistent with microdeletion of 22q11. In 7/9 the diagnosis was confirmed using fluorescence in situ hybridisation (FISH); 7 cases were confirmed using the cosmid EO and in one case with an equivocal result with cEO, hemizygosity of the 22q1 1 region was confirmed with use of a second cosmid for the TUPLE-1 gene region (cH487). FISH on samples from 19 infants considered free of phenotypic appearances of 22q1 1 microdeletions were all negative. Amongst the deleted patients. thymic aplasia was demonstrated at the time of cardiac surgery in 3. In the other 4 deleted patients no thymic shadow was evident on neonatal CXR. T cell studies were perfordeleted patients &amp; were normal in 3 cases. Serious infection arose in only 1 patient with 22q11 deletion but with normal T cell studies. Transient symptomatic hypocalcemia developed in 2 patients. both deleted for 22q1 1. We conclude that 22q11 microdeletions are clinically recognisable in early infancy and constitute one of the commonest identifiable genetic causes of congenital head disease. Thymic aplasia is common in deleted patients, though serious infections are rare. These observations have important implications for transfusion management. long term follow-up and for genetic counselling.