Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

The Spectrum of the Heterozygous Effect in Biallelic Mendelian Diseases-The Symptomatic Heterozygote Issue.

Heterozygous carriers of pathogenic/likely pathogenic variants in autosomal recessive disorders seem to be asymptomatic. However, in recent years, an increasing number of case reports have suggested that mild and unspecific symptoms can occur in some heterozygotes, as symptomatic heterozygotes have been identified across different disease types, including neurological, neuromuscular, hematological, and pulmonary diseases. The symptoms are usually milder in heterozygotes than in biallelic variants and occur "later in life". The status of symptomatic heterozygotes as separate entities is often disputed, and alternative diagnoses are considered. Indeed, often only a thin line exists between dual, dominant, and recessive modes of inheritance and symptomatic heterozygosity. Interestingly, recent population studies have found global disease effects in heterozygous carriers of some genetic variants. What makes the few heterozygotes symptomatic, while the majority show no symptoms? The molecular basis of this phenomenon is still unknown. Possible explanations include undiscovered deep-splicing variants, genetic and environmental modifiers, digenic/oligogenic inheritance, skewed methylation patterns, and mutational burden. Symptomatic heterozygotes are rarely reported in the literature, mainly because most did not undergo the complete diagnostic procedure, so alternative diagnoses could not be conclusively excluded. However, despite the increasing accessibility to high-throughput technologies, there still seems to be a small group of patients with mild symptoms and just one variant of autosomes in biallelic diseases. Here, we present some examples, the current state of knowledge, and possible explanations for this phenomenon, and thus argue against the existing dominant/recessive classification.

Deterioration of visual quality and acuity as the first sign of ceroid lipofuscinosis type 3 (CLN3), a rare neurometabolic disease.

Ceroid lipofuscinosis type 3 (CLN3) is an autosomal recessive, neurodegenerative metabolic disease. Typical clinical symptoms include progressive visual loss, epilepsy of unknown etiology and dementia. Presence of lipofuscin deposits with typical pattern of 'fingerprints' and vacuolized lymphocytes suggest the diagnosis of CLN3. Cause of CLN3 are mutations in the CLN3 gene, among which the most frequently found is the large deletion 1.02 kb spreading on exons 7 and 8. We present 4 patients from 2 families, in whom the deterioration of visual quality and acuity was observed as first clinical sign, when they were a few years old and it was successively accompanied by symptoms of neurologic deterioration (like generalized convulsions with consciousness impairment). In all patients the 1.02 kb deletion in the CLN3 gene was detected in homo- or heterozygosity with other CLN3 pathogenic variant. Ultrastructural studies revealed abnormal structures corresponding to 'fingerprint' profiles (FPPs) in conjunctival endothelial cells. It should be emphasized that in patients with blindness of unknown cause the diagnosis of ceroid lipofuscinosis should be considered and in older children-especially CLN3. The facility of the analysis for the presence of 1.02 kb deletion and economic costs are a solid argument for intensive use of this test in the diagnostic procedure of CLN3.

The role of ultrasound and genetic counsel in prenatal diagnosis of split hand/foot malformation with long bone deficiency.

OBJECTIVE: The aim of the report is to highlight importance of careful ultrasound examination and genetic counsel in case of split hand/foot prenatal diagnosis. Evaluation of the accompanying long bone deficiency indicates that array comparative genomic hybridization (aCGH) should be used. CASE REPORT: We present a prenatal diagnosis of split hand/foot malformation with long bone deficiency type 3 (SHFLD3) in a patient suffering from congenital limb anomalies but without previous molecular diagnosis. Genetic consultation and prenatal testing were offered. While karyotype was normal, aCGH revealed microduplication in locus 17p33.3. Microarray analysis was carried out also in pregnant patient and her asymptomatic mother and gave positive results on both. CONCLUSION: Prenatal diagnosis of SHFLD3 either imaging or cytogenetic is possible. Both methods should be used to make a correct diagnosis. Due to reduced penetrance of SHFLD3 not all individuals carrying microduplication present clinical findings. Therefore, pedigree analysis and genetic counsel is important for whole family and not only for affected members. Advances in genetics may allow to establish exact diagnosis in previously undiagnosed patients.

Genetic polymorphisms and serum concentrations of adiponectin and resistin in anorexia nervosa and healthy controls - pilot study.

BACKGROUND: A possible role of adipokines in the regulation of body weight in patients with anorexia nervosa (AN) has been proposed. Polymorphisms in genes encoding adiponectin and resistin in AN have not been widely assessed, yet. OBJECTIVES: 1) Assessment the frequency of ADIPOQ c.45T>G, ADIPOQ c.276G>T polymorphisms in adiponectin and RETN c.62G>A, RETN c.-180C>G in resistin genes in AN patients and control group (C) 2) Analysis of correlation between these polymorphisms and serum ADP or RETN. METHODS: We examined 67 AN girls and 38 C aged 11-18. Analyses of polymorphisms in ADIPOQ and RETN genes were performed using RFLP method and adiponectin and resistin serum levels - with commercially available ELISA kits. RESULTS: In AN subjects, TT genotype in ADIPOQ c.276 polymorphism as well as GG genotype of RETN c.-180 were significantly more frequent than in CG. In ADIPOQ c.45 polymorphic site, TT alleles were the most frequent in both examined groups. In RETN c.62 GA and GG alleles distribution did not differ between the groups and the most frequently observed genotype was GG. The mean serum adiponectin level in AN was significantly higher and resistin - lower than in controls. There were no statistically significant relationships between serum adiponectin and resistin levels and allele frequency in polymorphisms ADIPOQ c.276 as well as RETN c.-180 in the examined groups. CONCLUSION: Differences in genotype frequencies of ADIPOQ c.276 and RETN c.-180 suggest a need for studies on a larger cohort of patients with AN.