Altered Splicing of LAMP2 in a Multigenerational Family from Latvia Affected by Danon Disease.

Background and Objectives: Danon disease is a multisystemic disorder associated with variants in the LAMP2 gene, mainly affecting the cardiac muscle. Here, we report a multigenerational family from Latvia with two male patients, hemizygous for a novel splice-affecting variant c.928+3A>G. Affected patients exhibit a cardiac phenotype, moderate mental disability, and mild retinal changes. Materials and Methods: Both patients underwent either exome or hypertrophic cardiomyopathy gene panel next-generation sequencing. The pathogenic variant effect was determined using reverse transcription, Sanger sequencing, and high-resolution electrophoresis. Results: Evaluation of the splicing process revealed that approximately 80% of the transcripts exhibited a lack of the entire exon 7. This alteration was predicted to cause a shift of the reading frame, consequently introducing a premature stop codon downstream in the sequence. Conclusions: Based on our data, we propose that c.928+3A>G is a pathogenic variant associated with Danon disease.

Clinical and genetic characterization of Netherton syndrome due to SPINK5 founder variant in Latvian population.

OBJECTIVE: Netherton syndrome (NS) (OMIM:256500) is a very rare autosomal recessive multisystem disorder mostly affecting ectodermal derivatives (skin and hair) and immune system. It is caused by biallelic loss-of-function variants in the SPINK5 gene, encoding the protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI). MATERIAL, METHODS AND RESULTS: Here, we describe NS clinical and genetic features of homogenous patient group: 9 individuals from 7 families with similar ethnic background and who have the same SPINK5 variant (NM_006846.4: c.1048C > T, p.(Arg350*)) in homozygous or compound heterozygous states, suggesting that it is a common founder variant in Latvian population. Indeed, we were able to show that the variant is common in general Latvian population, and it shares the same haplotype among the NS individual. It is estimated that the variant arose >1000 years ago. Clinically, all nine patients exhibited typical NS skin changes (scaly erythroderma, ichthyosis linearis circumflexa, itchy skin), except for one patient who has a different skin manifestation-epidermodysplasia. Additionally, we show that developmental delay, previously underrecognized in NS, is a common feature among these patients. CONCLUSIONS: This study shows that the phenotype of NS individuals with the same genotype is highly homogeneous.

The most common European HINT1 neuropathy variant phenotype and its case studies.

HINT1 is an ubiquitous homodimeric purine phosphoramidase belonging to the histidine-triad superfamily. In neurons, HINT1 stabilizes the interaction of different receptors and regulates the effects of their signaling disturbances. Changes in HINT1 gene are associated with autosomal recessive axonal neuropathy with neuromyotonia. Aim of the study was detailed description of patients' phenotype with HINT1 homozygous NM_005340.7: c.110G>C (p.Arg37Pro) variant. Seven homozygous and three compound heterozygous patients were recruited and evaluated using standardized tests for CMT patients, in four patients' nerve ultrasonography was performed. The median age of symptom onset was 10 years (range 1-20), with initial complaints being distal lower limb weakness with gait impairment, combined with muscle stiffness, more pronounced in the hands than in the legs and worsened by cold. Arm muscles became involved later, presenting with distal weakness and hypotrophy. Neuromyotonia was present in all reported patients and is thus a diagnostic hallmark. Electrophysiological studies demonstrated axonal polyneuropathy. Impaired mental performance was observed in six out of ten cases. In all patients with HINT1 neuropathy, ultrasound examination showed significantly reduced muscle volume as well as spontaneous fasciculations and fibrillations. The nerve cross-sectional areas of the median and ulnar nerves were closer to the lower limits of the normal values. None of the investigated nerves had structural changes. Our findings broaden the phenotype of HINT1-neuropathy and have implications for diagnostics and ultrasonographic evaluation of HINT1-neuropathy patients.

Overview of Neuromuscular Disorder Molecular Diagnostic Experience for the Population of Latvia.

Background and Objectives: Genetic testing has become an integral part of health care, allowing the confirmation of thousands of hereditary diseases, including neuromuscular disorders (NMDs). The reported average prevalence of individual inherited NMDs is 3.7-4.99 per 10,000. This number varies greatly in the selected populations after applying population-wide studies. The aim of this study was to evaluate the effect of genetic analysis as the first-tier test in patients with NMD and to calculate the disease prevalence and allelic frequencies for reoccurring genetic variants. Methods: Patients with NMD from Latvia with molecular tests confirming their diagnosis in 2008-2020 were included in this retrospective study. Results: Diagnosis was confirmed in 153 unique cases of all persons tested. Next-generation sequencing resulted in a detection rate of 37%. Two of the most common childhood-onset NMDs in our population were spinal muscular atrophy and dystrophinopathies, with a birth prevalence of 1.01 per 10,000 newborns and 2.08 per 10,000 (male newborn population), respectively. The calculated point prevalence was 0.079 per 10,000 for facioscapulohumeral muscular dystrophy type 1, 0.078 per 10,000 for limb-girdle muscular dystrophy, 0.073 per 10,000 for nondystrophic congenital myotonia, 0.052 per 10,000 for spinobulbar muscular atrophy, and 0.047 per 10,000 for type 1 myotonic dystrophy. Discussion: DNA diagnostics is a successful approach. The carrier frequencies of the common CAPN3, FKRP, SPG11, and HINT1 gene variants as well as that of the SMN1 gene exon 7 deletion in the population of Latvia are comparable with data from Europe. The carrier frequency of the CLCN1 gene variant c.2680C>T p.(Arg894Ter) is 2.11%, and consequently, congenital myotonia is the most frequent NMD in our population.

CAPN3 c.1746-20C>G variant is hypomorphic for LGMD R1 calpain 3-related.

The investigated intronic CAPN3 variant NM_000070.3:c.1746-20C>G occurs in the Central and Eastern Europe with a frequency of >1% and there are conflicting interpretations on its pathogenicity. We collected data on 14 patients carrying the CAPN3 c.1746-20C>G variant in trans position with another CAPN3 pathogenic/likely pathogenic variant. The patients compound heterozygous for the CAPN3 c.1746-20C>G variant presented a phenotype consistent with calpainopathy of mild/medium severity. This variant is most frequent in the North/West regions of Russia and may originate from that area. Molecular studies revealed that different splicing isoforms are produced in the muscle. We hypothesize that c.1746-20C>G is a hypomorphic variant with a reduction of RNA and protein expression and only individuals having a higher ratio of abnormal isoforms are affected. Reclassification of the CAPN3 variant c.1746-20C>G from variant with a conflicting interpretation of pathogenicity to hypomorphic variant explains many unidentified cases of limb girdle muscular dystrophy R1 calpain 3-related in Eastern and Central Europe.

Expanding the mutation and phenotype spectrum of MYH3-associated skeletal disorders.

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-ß signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-ß signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-ß signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome.

Case Report: Two Families With HPDL Related Neurodegeneration.

There are recent reports of associations of variants in the HPDL gene with a hereditary neurological disease that presents with a wide spectrum of clinical severity, ranging from severe neonatal encephalopathy with no psychomotor development to adolescent-onset uncomplicated spastic paraplegia. Here, we report two probands from unrelated families presenting with severe and intermediate variations of the clinical course. A homozygous variant in the HPDL gene was detected in each proband; however, there was no known parental consanguinity. We also highlight reductions in citrate synthase and mitochondrial complex I activity detected in both probands in different tissues, reflecting the previously proposed mitochondrial nature of disease pathogenesis associated with HPDL mutations. Further, we speculate on the functional consequences of the detected variants, although the function and substrate of the HPDL enzyme are currently unknown.

New-Born Screening for Spinal Muscular Atrophy: Results of a Latvian Pilot Study.

New disease-modifying treatments have recently been approved for 5q spinal muscular atrophy (SMA) and early treatment has been associated with a better clinical outcome. Accordingly, new-born screening (NBS) for SMA should be implemented to ensure early diagnosis of affected individuals. The aim of this study was to determine the feasibility and usefulness of NBS for SMA in Latvia. Between February and November of 2021, 10,411 parents consented to participation in the study. DNA testing for the SMN1 exon 7 homozygous deletion was conducted using qPCR with fluorescent locked nucleic acid primers. In the first month of testing, reporting of results took up to a maximum of 17 days after samples arrived in the laboratory. However, following familiarisation with the procedure, the median report time was reduced to 11 days after birth. Forty cases required samples to be taken again due to poor quality of the isolated DNA transpiring from either the quality of the blood punch or manual mistakes during DNA isolation. The SMN1 exon 7 homozygous deletion was identified in two individuals, which was subsequently confirmed by multiplex ligation-dependent probe amplification. When a NBS sample is taken 48 to 72 h after birth and transported to the laboratory within two working days after collection according to legal requirements, DNA test results can be reported to healthcare professionals before the 12th day of life. Expansion of our SMA 5q NBS procedure to the whole of Latvia is feasible and would facilitate early diagnosis and result in more effective treatment. We strongly advocate that SMA is added to the national Latvia Recommended Uniform Screening Panel.

Implication of Genetic Testing and Pregnancy Outcome in a Woman with Unbalanced Translocation t(1;6).

BACKGROUND Parental chromosomal structural abnormalities can lead to diverse chromosomal imbalances at meiotic segregation during gametogenesis and subsequent early pregnancy loss or birth of a child with a chromosomal abnormality. The incidence of unbalanced translocations is 1 per 1000 newborns versus 3 per 1000 newborns for balanced rearrangements. Here, we present the case of a mother with an unbalanced chromosomal translocation and her offspring. CASE REPORT Our patient had a 1p36.31 duplication of 0.22 Mb and 6qter deletion of 1.2 Mb. She had 5 pregnancies with different outcomes. Her first child died 24 h after birth due to a congenital heart defect. Her second pregnancy resulted in the birth of a girl who was postnatally diagnosed with 1p36 deletion syndrome. The third and fourth pregnancies ended spontaneously in the first trimester. For her last pregnancy, the patient underwent a diagnostic amniocentesis at the 16th week of gestation. A large 5.4-Mb pathogenic duplication of 1p36.33 was detected in the fetus and the woman decided to terminate the pregnancy. CONCLUSIONS In this case report, we detail the different pregnancy outcomes induced by the mother's unbalanced chromosomal translocation and review the prenatal diagnostic genetic testing. Our report clearly demonstrates the complementary nature of chromosomal microarrays and conventional karyotyping.

Kohlschütter-Tönz syndrome: Case report with novel feature and detailed review of features associated with ROGDI variants.

Kohlschütter-Tönz syndrome (KTS) is a rare, autosomal recessive syndrome characterized by a triad of epilepsy, amelogenesis imperfecta and severe global developmental delay. It was first described in a Swiss family in 1974 by Alfried Kohlschütter and Otmar Tönz. It is caused by pathogenic variants in the ROGDI gene. To the best of our knowledge, there are currently 43 patients with a confirmed ROGDI gene pathogenic variant reported. Here, we review in detail the clinical manifestations of KTS, provide an overview of all reported genetically confirmed patients, and document an additional case of KTS-a 6-year-old Latvian girl-with a confirmed ROGDI gene pathogenic variant. In contrast to previous reports, we detected idiopathic bilateral nephrocalcinosis in this newly identified KTS patient. Perampanel proved an effective treatment for our patient with prolonged super-refractory status epilepticus. In order to better characterize this rare syndrome and its clinical course, it is important to report any additional symptoms and also the effectiveness of used therapies. Future research should focus on elucidating the mechanisms by which the absence/insufficiency of ROGDI-encoded protein causes the clinical manifestations of KTS. This knowledge could shape possible ways of influencing the disease's natural history with more effective therapies.

Two Cases of Leigh Syndrome in One Family: Diagnostic Challenges and Clinical Management Experience in Latvia.

Leigh syndrome is a neurodegenerative disorder with an incidence of 1 : 40,000 live births. The clinical presentation of LS is highly variable with heterogeneity in the disease-associated symptoms of cerebellar, motor, and extrapyramidal dysfunction and common infections. There is no effective treatment for this condition; as such, the prognosis of this condition is very poor with death occurring within the first few years of life. In this study, we report the first LS case in Latvia with SURF1 pathogenic variants in two siblings. The difficulties encountered establishing a diagnosis for the first proband and the effective prenatal diagnosis for the second offspring that led to termination of the pregnancy are discussed.

Plasma neurofilament light chain as a potential biomarker in Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth (CMT) disease is a chronic, slowly progressing disorder. The lack of specific disease progression biomarkers limits the execution of clinical trials. However, neurofilament light chain (NfL) has been suggested as a potential biomarker for peripheral nervous system disorders. METHODS: Ninety-six CMT disease patients and 60 healthy controls were enrolled in the study. Disease severity assessment included clinical evaluation with CMT Neuropathy Score version 2 (CMTNSv2). Blood plasma NfL concentrations were measured using the single-molecule array NfL assay. RESULTS: The NfL concentration was significantly higher in the CMT disease patient group than in the controls (p < 0.001). Of the CMT disease patients, those with type CMTX1 had a higher NfL level than those in the two other analysed subgroups (CMT1A and other CMT disease types) (p = 0.0498). The NfL concentration had a significant but weak correlation with the CMTNSv2 (rs  = 0.25, p = 0.012). In one CMT disease patient with an extremely elevated NfL level, overlap with chronic inflammatory demyelinating polyneuropathy was suspected. Receiver operating characteristic analysis showed that an NfL concentration of 8.9 pg/ml could be used to discriminate CMT disease patients from controls, with an area under the curve of 0.881. CONCLUSIONS: Our study confirmed that the plasma NfL concentration is significantly higher in CMT disease patients than in controls. Plasma NfL concentration was found to significantly, albeit weakly, reflect the clinical severity of CMT disease. In the future, NfL may be used, either individually or collaboratively, as a biomarker in the clinical context of suspected CMT disease; however, several issues need to be addressed first.

Collagen VI-related limb-girdle syndrome caused by frequent mutation in COL6A3 gene with conflicting reports of pathogenicity.

Recently the scientific community has started to view Bethlem myopathy 1 and Ullrich congenital muscular dystrophy as two extremes of a collagen VI-related myopathy spectrum rather than two separate entities, as both are caused by mutations in one of the collagen VI genes. Here we report three individuals in two families who are homozygous for a COL6A3 mutation (c.7447A> G; p.Lys2483Glu), and compare their clinical features with seven previously published cases. Individuals carrying homozygous or compound heterozygous c.7447A> G, (p.Lys2483Glu) mutation exhibit mild phenotype without loss of ambulation, similar to the cases described previously as Collagen VI-related limb-girdle syndrome. The phenotype could arise due to an aberrant assembly of Von Willebrand factor A domains. Based on these data, we propose that c.7447A> G, (p.Lys2483Glu) is a common pathogenic mutation.

Genetic variation spectrum in ATP7B gene identified in Latvian patients with Wilson disease.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism caused by allelic variants in ATP7B gene. More than 500 distinct variants have been reported, the most common WD causing allelic variant in the patients from Central, Eastern, and Northern Europe is H1069Q. METHODS: All Latvian patients with clinically confirmed WD were screened for the most common mutation p.H1069Q by PCR Bi-PASA method. Direct DNA sequencing of gene ATP7B (all 21 exons) was performed for the patients with WD symptoms, being either heterozygous for H1069Q or without it on any allele. RESULTS: We identified 15 different allelic variants along with eight non-disease-causing allelic variants. Based on the gene molecular analysis and patients' clinical data variant p.His1069Gln was found in 66.9% of WD alleles. Wide clinical variability was observed among individuals with the same ATP7B genotype. The results of our study confirm that neurological manifestations of WD are typically present later than the liver disease but no significant association between the presence/absence of the most common genetic variant and mode of initial WD presentation or age at presentation was identified. CONCLUSIONS: (1) The most prevalent mutation in Latvian patients with Wilson disease was c.3207C>A (p.His1069Gln); (2) No significant phenotype-genotype correlation was found in Latvian patients with Wilson disease; (3) The estimated prevalence of Wilson disease in Latvia is 1 of 24,000 cases which is higher than frequently quoted prevalence of 1: 30,000.

Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies.

BACKGROUND: Limb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis. METHODS: We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes. RESULTS: Analysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound - c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene - c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present. CONCLUSIONS: Genetic diagnosis was possible in 12 of 60 patients (20%). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Dupuytren's Contracture Cosegregation with Limb-Girdle Muscle Dystrophy.

Limb-girdle muscular dystrophies (LGMDs) is a heterogeneous group of muscular dystrophies that mostly affect the pelvic and shoulder girdle muscle groups. We report here a case of neuromuscular disease associated with Dupuytren's contracture, which has never been described before as cosegregating with an autosomal dominant type of inheritance. Dupuytren's contracture is a common disease, especially in Northern Europe. Comorbid conditions associated with Dupuytren's contracture are repetitive trauma to the hands, diabetes, and seizures, but it has never before been associated with neuromuscular disease. We hypothesize that patients may harbor mutations in genes with functions related to neuromuscular disease and Dupuytren's contracture development.