Streamlined two-step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.

Clinical and Histopathological Features of Gelsolin Amyloidosis Associated with a Novel GSN Variant p.Glu580Lys.

Gelsolin amyloidosis typically presents with corneal lattice dystrophy and is most frequently associated with pathogenic GSN variant p.Asp214Asn. Here we report clinical and histopathological features of gelsolin amyloidosis associated with a novel GSN variant p.Glu580Lys. We studied DNA samples of seven members of a two-generation family. Exome sequencing was performed in the proband, and targeted Sanger sequencing in the others. The heterozygous GSN variant p.Glu580Lys was identified in six patients. The patients exhibited corneal dystrophy (5/6), loose skin (5/6) and/or heart arrhythmia (3/6) and one presented with bilateral optic neuropathy. The impact of the mutation on the protein structure was evaluated in silico. The substitution is located in the fifth domain of gelsolin protein, homologous to the second domain harboring the most common pathogenic variant p.Asp214Asn. Structural investigation revealed that the mutation might affect protein folding. Histopathological analysis showed amyloid deposits in the skin. The p.Glu580Lys is associated with corneal dystrophy, strengthening the association of the fifth domain of gelsolin protein with the typical amyloidosis phenotype. Furthermore, optic neuropathy may be related to the disease and is essential to identify before discussing corneal transplantation.

Evaluation of Optical Genome Mapping in Clinical Genetic Testing of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, resulting in the faulty expression of the DUX4 gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.

A novel splice-site FHOD3 founder variant is a common cause of hypertrophic cardiomyopathy in the population of the Balkans-A cohort study.

Founder variants in sarcomere protein genes account for a significant proportion of disease-causing variants in patients with hypertrophic cardiomyopathy (HCM). However, information on founder variants in non-sarcomeric protein genes, such as FHOD3, which have only recently been associated with HCM, remains scarce. In this study, we conducted a retrospective analysis of exome sequencing data of 134 probands with HCM for recurrent pathogenic variants. We discovered a novel likely pathogenic variant c.1646+2T>C in FHOD3 in heterozygous state in eight probands with HCM and confirmed its presence in seven additional relatives. Individuals with this variant had a wide range of ages at onset of the disease (4-63 years). No adverse cardiac events were observed. Haplotype analysis revealed that the individuals with this variant shared a genomic region of approximately 5 Mbp surrounding the variant, confirming the founder effect of the variant. FHOD3 c.1646+2T>C is estimated to have arisen 58 generations ago (95% CI: 45-81) in a common ancestor living on the Balkans. A founder FHOD3 c.1646+2T>C variant is the second most common genetic variant in our cohort of patients with HCM, occurring in 16% of probands with a known genetic cause of HCM, which represents a substantially higher proportion than the currently estimated 0.5-2% for causal FHOD3 variants. Our study broadens the understanding of the genetic causes of HCM and may improve the diagnosis of this condition, particularly in patients from the Balkans.

Biallelic ATOH1 Gene Variant in Siblings With Pontocerebellar Hypoplasia, Developmental Delay, and Hearing Loss.

Background and Objectives: To report on the novel association of biallelic variant in atonal basic helix-loop-helix transcription factor 1 (ATOH1) gene and pontocerebellar hypoplasia (PCH), severe global developmental delay, intellectual disability, and hearing loss in a family with 2 affected siblings. Methods: A detailed clinical assessment and exome sequencing of peripheral blood sample were performed. Segregation analysis with Sanger sequencing and structural modeling of the variant was performed to support the pathogenicity of the variant. Results: A homozygous missense variant (NM_005172.1:c.481C>G) in the ATOH1 gene was identified in the proband and his affected sister. The segregation analysis subsequently confirmed its segregation with an apparently recessive PCH in this family. ATOH1 encodes for the atonal basic helix-loop-helix (bHLH) transcription factor 1, a core transcription factor in the developing cerebellum, brainstem, and dorsal spinal cord, and in the ear. The identified variant results in the p.(Arg161Gly) amino acid substitution in the evolutionarily conserved DNA-binding bHLH domain of the ATOH1 protein. Biallelic missense variants in this domain were previously reported to result in disordered cerebellar development and hearing loss in animal models. In silico homology modeling revealed that p.Arg161Gly in ATOH1 protein probably disrupts a salt bridge with DNA backbone phosphate and increases the flexibility of the bHLH helix-both of which together affect the binding capability of the bHLH domain to the DNA. Discussion: Based on the sequencing results and evidence from structural modeling of the identified variant, as well as with previous reports of ATOH1 gene disruption, we conclude that ATOH1 may represent a novel candidate gene associated with the phenotype of PCH, global developmental delay, and hearing loss in humans.

Axonal Polyneuropathy in 2 Brothers With a Homozygous Missense Variant in the First Catalytic Domain of PCYT2.

BACKGROUND AND OBJECTIVES: To expand the phenotype and genotype associated with PCYT2-related disorder. METHODS: Exome sequencing data from a patient with molecularly undiagnosed complex spastic paraplegia and axonal motor and sensory polyneuropathy were analyzed. Clinical data and nerve conduction studies of the patient and his affected brother were collected, and their phenotype and genotype were compared with previously reported patients with PCYT2-related disorder. RESULTS: A novel homozygous missense variant in PCYT2 (NM_001184917.2) c.88T>G; p.(Cys30Gly) was identified. This variant is located in a highly conserved tyrosine kinase site and is predicted damaging by several variant annotation tools. Both patients reported here and the previously published patients share several phenotypic features, including short stature, spastic tetraparesis, cerebellar ataxia, epilepsy, and cognitive decline. Axonal polyneuropathy, diagnosed in both brothers, was not previously reported. DISCUSSION: This family with a novel PCYT2 variant expands the clinical spectrum of PCYT2-related disorder to include axonal motor and sensory polyneuropathy and the genetic spectrum to include the variant located in the first catalytic domain, whereas all previously reported variants are located in the second catalytic domain. Further research is required to disentangle the underlying pathophysiologic mechanisms, leading to the complex phenotype of PCYT2-related disorder.

Association between male infertility and genetic variability at the PON1/2 and GSTM1/T1 gene loci.

Environmental xenobiotics such as organophosphate pesticides are known factors involved in male infertility. Paraoxanase (PON) and glutathione transferase (GST) are involved in biotransformation of organophosphate pesticides. Interindividual genetic variations in biotransformation enzyme activities can lead to differences in the susceptibility to male infertility. This case-control study investigated associations between polymorphisms in the PON and GST genes (PON1-55/192, PON2-311, GSTM1/T1) and infertility. The study group consisted of 187 infertile men (86 with non-obstructive azoospermia (NOA) and 101 with oligoasthenoteratozoospermia (OAT)), whereas the control group comprised of 194 fertile men. Statistically significant differences were found in PON1-55MM genotype (chi-squared=7.37; P=0.02) and PON1-55M allele (chi-squared=5.98; P=0.01) distribution between the infertile and fertile men. A separate analysis revealed that significant differences in genotype frequencies were limited to the OAT group (chi-squared=9.11, P=0.01). However, no significant differences in genotype frequencies of other tested polymorphisms (PON1-192, PON2-311, GSTM1/T1) and male infertility were observed. The PON1-55M allele might represent a risk factor for infertility susceptibility in Slovenian men. Further studies with a larger sample size are needed to confirm these findings.

Combination of QF-PCR and aCGH is an efficient diagnostic strategy for the detection of chromosome aberrations in recurrent miscarriage.

BACKGROUND: Our aim was to conduct a comprehensive genetic evaluation using the combination of QF-PCR (quantitative fluorescence polymerase chain reaction) and aCGH (array comparative genomic hybridization) for the detection of the frequency and type of chromosome aberrations in recurrent miscarriage (RM) in the clinical setting. METHODS: This retrospective study was conducted on 73 first-trimester products of conception (POC) between September 2014 and February 2017. The POCs were collected from 73 women with at least one previous miscarriage and analyzed for chromosomal anomalies using QF-PCR and aCGH as part of the routine clinical evaluation. RESULTS: Chromosome aberrations were detected in 52/73 POCs (71.2%), of which 41 (56.2%) were identified by QF-PCR and an additional 11 (15.1%) by aCGH. Numerical aberrations constituted 92.3% of abnormalities, with trisomies as the most common subtype (72.9%). Causative structural aberrations were found in three samples (5.8%). The frequency of chromosome aberrations was not dependent on the number of previous miscarriages, whereas it significantly increased with advanced maternal age. CONCLUSION: Our results confirm that chromosome aberrations are the most common cause of RM and that QF-PCR and aCGH combination should be included in the routine genetic analysis of POCs of couples with miscarriage.