Genome sequencing identifies biallelic variants in SCLT1 in a patient with syndromic nephronophthisis: Reflections on the SCLT1-related ciliopathy spectrum.

Ciliopathies represent a major category of rare multisystem disease. Arriving at a specific diagnosis for a given patient is challenged by the significant genetic and clinical heterogeneity of these conditions. We report the outcome of the diagnostic odyssey of a child with obesity, renal, and retinal disease. Genome sequencing identified biallelic splice site variants in sodium channel and clathrin linker 1 (SCLT1), an emerging ciliopathy gene. We review the literature on all patients reported with biallelic SCLT1 variants highlighting a frequent clinical presentation that overlaps Bardet-Biedl and Senior-Loken syndromes. We also discuss current concepts in syndrome designation in light of these data.

MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

A novel mutation in LAMC3 associated with generalized polymicrogyria of the cortex and epilepsy.

Occipital cortical malformation is a rare neurodevelopmental disorder characterized by pachygyria and polymicrogyria of the occipital lobes as well as global developmental delays and seizures. This condition is due to biallelic, loss-of-function mutations in LAMC3 and has been reported in four unrelated families to date. We report an individual with global delays, seizures, and polymicrogyria that extends beyond the occipital lobes and includes the frontal, parietal, temporal, and occipital lobes. Next-generation sequencing identified a homozygous nonsense mutation in LAMC3: c.3190C>T (p.Gln1064*). This finding extends the cortical phenotype associated with LAMC3 mutations.

Whole-exome sequencing is a valuable diagnostic tool for inherited peripheral neuropathies: Outcomes from a cohort of 50 families.

The inherited peripheral neuropathies (IPNs) are characterized by marked clinical and genetic heterogeneity and include relatively frequent presentations such as Charcot-Marie-Tooth disease and hereditary motor neuropathy, as well as more rare conditions where peripheral neuropathy is associated with additional features. There are over 250 genes known to cause IPN-related disorders but it is estimated that in approximately 50% of affected individuals a molecular diagnosis is not achieved. In this study, we examine the diagnostic utility of whole-exome sequencing (WES) in a cohort of 50 families with 1 or more affected individuals with a molecularly undiagnosed IPN with or without additional features. Pathogenic or likely pathogenic variants in genes known to cause IPN were identified in 24% (12/50) of the families. A further 22% (11/50) of families carried sequence variants in IPN genes in which the significance remains unclear. An additional 12% (6/50) of families had variants in novel IPN candidate genes, 3 of which have been published thus far as novel discoveries (KIF1A, TBCK, and MCM3AP). This study highlights the use of WES in the molecular diagnostic approach of highly heterogeneous disorders, such as IPNs, places it in context of other published neuropathy cohorts, while further highlighting associated benefits for discovery.

Expansion of the GLE1-associated arthrogryposis multiplex congenita clinical spectrum.

Mutations in GLE1 cause two recessive subtypes of arthrogryposis multiplex congenita (AMC), a condition characterized by joint contractures at birth, and all previously reported patients died in the perinatal period. GLE1 related AMC has been almost exclusively reported in the Finnish population and is caused by a relatively common pathogenic splicing mutation in that population. Here, we report two non-Finnish brothers with novel compound heterozygous splicing mutations in GLE1, one of whom has survived to 12 years of age. We also demonstrate low levels of residual wild type transcript in fibroblasts from the surviving brother, suggesting that this residual wild-type transcript may contribute to the relatively longer-term survival in this family. We provide a detailed clinical report on the surviving patient, providing the first insight into the natural history of this rare neuromuscular disease. We also suggest that lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn disease (LAAHD), the two AMC subtypes related to GLE1, do not have sufficient clinical or molecular differentiation to be considered allelic disorders. Rather, GLE1 mutations cause a variable spectrum of AMC severity including a non-lethal variant described herein.

Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.

Whole-exome sequencing broadens the phenotypic spectrum of rare pediatric epilepsy: a retrospective study.

Whole-exome sequencing (WES) has transformed our ability to detect mutations causing rare diseases. FORGE (Finding Of Rare disease GEnes) and Care4Rare Canada are nation-wide projects focused on identifying disease genes using WES and translating this technology to patient care. Rare forms of epilepsy are well-suited for WES and we retrospectively selected FORGE and Care4Rare families with clinical descriptions that included childhood-onset epilepsy or seizures not part of a recognizable syndrome or an early-onset encephalopathy where standard-of-care investigations were unrevealing. Nine families met these criteria and a diagnosis was made in seven, and potentially eight, of the families. In the eight families we identified mutations in genes associated with known neurological and epilepsy disorders: ASAH1, FOLR1, GRIN2A (two families), SCN8A, SYNGAP1 and SYNJ1. A novel and rare mutation was identified in KCNQ2 and was likely responsible for the benign seizures segregating in the family though additional evidence would be required to be definitive. In retrospect, the clinical presentation of four of the patients was considered atypical, thereby broadening the phenotypic spectrum of these conditions. Given the extensive clinical and genetic heterogeneity associated with epilepsy, our findings suggest that WES may be considered when a specific gene is not immediately suspected as causal.

Costs and benefits of non-invasive fetal RhD determination.

OBJECTIVE: Non-invasive fetal Rhesus (Rh) D genotyping, using cell-free fetal DNA (cffDNA) in the maternal blood, allows targeted antenatal anti-RhD prophylaxis in unsensitized RhD-negative pregnant women. The purpose of this study was to determine the cost and benefit of this approach as compared to routine antenatal anti-RhD prophylaxis for all unsensitized RhD-negative pregnant women, as is the current policy in the province of Alberta, Canada. METHODS: This study was a decision analysis based on a theoretical population representing the total number of pregnancies in Alberta over a 1-year period (n = 69 286). A decision tree was created that outlined targeted prophylaxis for unsensitized RhD-negative pregnant women screened for cffDNA (targeted group) vs routine prophylaxis for all unsensitized RhD-negative pregnant women (routine group). Probabilities at each decision point and costs associated with each resource were calculated from local clinical and administrative data. Outcomes measured were cost, number of women sensitized and doses of Rh immunoglobulin (RhIG) administered. RESULTS: The estimated cost per pregnancy for the routine group was 71.43 compared with 67.20 Canadian dollars in the targeted group. The sensitization rates per RhD-negative pregnancy were equal, at 0.0012, for the current and targeted programs. Implementing targeted antenatal anti-RhD prophylaxis would save 4072 doses (20.1%) of RhIG over a 1-year period in Alberta when compared to the current program. CONCLUSIONS: These data support the feasibility of a targeted antenatal anti-RhD prophylaxis program, at a lower cost than that of the existing routine prophylaxis program, with no increased risk of sensitization.

A founder mutation in BBS2 is responsible for Bardet-Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders.

Bardet-Biedl syndrome (BBS) is a multisystem genetically heterogeneous disorder, the clinical features of which are largely the consequence of ciliary dysfunction. BBS is typically inherited in an autosomal recessive fashion, and mutations in at least 14 genes have been identified. Here, we report the identification of a founder mutation in the BBS2 gene as the cause for the increased incidence of this developmental disorder in the Hutterite population. To ascertain the Hutterite BBS locus, we performed a genome-wide single nucleotide polymorphism (SNP) analysis on a single patient and his three unaffected siblings from a Hutterite family. The analysis identified two large SNP blocks that were homozygous in the patient but not in his unaffected siblings, one of these regions contained the BBS2 gene. Sequence analysis and subsequent RNA studies identified and confirmed a novel splice site mutation, c.472-2A>G, in BBS2. This mutation was also found in homozygous form in three subsequently studied Hutterite BBS patients from two different leuts, confirming that this is a founder mutation in the Hutterite population. Further studies are required to determine the frequency of this mutation and its role, if any, in the expression of other ciliopathies in this population.

Mutation of DNAJC19, a human homologue of yeast inner mitochondrial membrane co-chaperones, causes DCMA syndrome, a novel autosomal recessive Barth syndrome-like condition.

BACKGROUND: A novel autosomal recessive condition, dilated cardiomyopathy with ataxia (DCMA) syndrome, has been identified in the Canadian Dariusleut Hutterite population, characterised by early onset dilated cardiomyopathy with conduction defects, non-progressive cerebellar ataxia, testicular dysgenesis, growth failure, and 3-methylglutaconic aciduria. OBJECTIVE: To map DCMA syndrome and identify the mutation underlying this condition. METHODS: A genome wide scan was undertaken on consanguineous Hutterite families using a homozygosity mapping approach in order to identify the DCMA associated chromosomal region. Mutation analysis was carried out on positional candidate genes in this region by sequencing. Reverse transcriptase polymerase chain reaction and bioinformatics analyses were then used to characterise the mutation and determine its effect on the protein product. RESULTS: The association of DCMA syndrome with a 2.2 Mb region of chromosome 3q26.33 was found. A disease associated mutation was identified: IVS3-1 G-->C in the DNAJC19 gene, encoding a DNAJ domain containing protein of previously unknown function (Entrez Gene ID 131118). CONCLUSIONS: The DNAJC19 protein was previously localised to the mitochondria in cardiac myocytes, and shares sequence and organisational similarity with proteins from several species including two yeast mitochondrial inner membrane proteins, Mdj2p and Tim14. Tim14 is a component of the yeast inner mitochondrial membrane presequence translocase, suggesting that the unique phenotype of DCMA may be the result of defective mitochondrial protein import. It is only the second human disorder caused by defects in this pathway that has been identified.

Deletions causing spinal muscular atrophy do not predispose to amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, invariably lethal disease resulting from the premature death of motor neurons of the motor cortex, brainstem, and spinal cord. In approximately 15% of familial ALS cases, the copper/zinc superoxide dismutase gene is mutated; a juvenile form of familial ALS has been linked to chromosome 2. No cause has been identified in the remaining familial ALS cases or in sporadic cases and the selective neurodegenerative mechanism remains unknown. Deletions in 2 genes on chromosome 5q, SMN (survival motor neuron gene) and NAIP (neuronal apoptosis inhibitory protein gene), have been identified in spinal muscular atrophy, a disease also characterized by the loss of motor neurons. These genes are implicated in the regulation of apoptosis, a mechanism that may explain the cell loss found in the brains and spinal cords of patients with ALS. OBJECTIVE: To determine whether the mutations causing neurodegeneration in spinal muscular atrophy are present in patients with ALS in whom the copper/zinc superoxide dismutase gene is not mutated. PATIENTS AND METHODS: Patients in whom ALS was diagnosed were screened for mutations in the SMN and NAIP genes by single strand conformation analysis. RESULTS: We found 1 patient with an exon 7 deletion in the SMN gene; review of clinical status confirmed the molecular diagnosis of spinal muscular atrophy. No mutations were found in the remaining patients. CONCLUSION: The SMN and NAIP gene mutations are specific for spinal muscular atrophy and do not predispose individuals to ALS.

Absence of mutations in superoxide dismutase and catalase genes in patients with Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is an adult-onset, neurodegenerative disorder characterized by a selective loss of the dopaminergic cells of the substantia nigra and by progressive motor decline. Studies have shown aberrant oxidative stress metabolism within the substantia nigra and other dopaminergic regions of the brain in patients with PD. OBJECTIVE: To screen the genes of three free radical detoxifying enzymes--copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase--for mutations in patients with PD. PATIENTS AND METHODS: A total of 107 unrelated patients with PD from two PD populations (familial and sporadic) were screened for mutations in the genes of copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase by single-strand conformation analysis. The diagnosis of PD was based on the clinical observations of resting tremor, rigidity, and bradykinesia. RESULTS: No mutations were identified. However, we did identify an amino acid substitution (glycine to aspartic acid) in exon 9 of the catalase gene in one patient; decreased red blood cell catalase activity was observed in this patient. CONCLUSION: Parkinson's disease is not caused by mutations in the genes of these three detoxifying enzymes. The exon 9 variant in the catalase gene in the one family with PD is most likely a silent mutation and not the genetic cause of PD in this family.

Spinobulbar muscular atrophy can mimic ALS: the importance of genetic testing in male patients with atypical ALS.

The clinical presentation of amyotrophic lateral sclerosis (ALS) is variable and overlaps with that of other motor neuron diseases such as spinobulbar muscular atrophy (SBMA; Kennedy disease). With the identification of disease-specific mutations such as the CAG repeat expansion in the androgen receptor in SBMA, an accurate molecular diagnosis can be made in some patients with motor neuron disease. To determine the extent of misdiagnosis of ALS we screened 147 male ALS patients and 100 unrelated male patients from 100 familial ALS (FALS) kindreds for the presence of the SBMA mutation using polymerase chain reaction methods. We show that ALS was clinically misdiagnosed in 2% of sporadic cases and in two of the 100 FALS kindreds. This study underscores the difficulty in distinguishing SBMA from ALS clinically, particularly in patients who lack the classic signs of each disease.

Absence of mutations in the Mn superoxide dismutase or catalase genes in familial amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is an autosomal dominant, adult onset, neurological disorder caused by the degeneration of motor neurons of the cortex, brainstem and spinal cord. Recently, the defective gene in some FALS families was identified as the Cu/Zn superoxide dismutase (SOD1) gene. However, SOD1 mutations are present in approximately 20% of patients with FALS. We have tested the genes of two more free radical detoxifying enzymes, Mn superoxide dismutase (SOD2) and catalase by single strand conformation analysis (SSCA) for mutations in the remaining FALS cases. No mutations were found in the catalase enzyme in 73 unrelated FALS cases; mutations were not detected in the 66% of the SOD2 gene analyzed. FALS does not appear to be caused by mutations in the SOD2 nor the catalase genes.