Unbiased phenotype and genotype matching maximizes gene discovery and diagnostic yield.

PURPOSE: Widespread application of next-generation sequencing, combined with data exchange platforms, has provided molecular diagnoses for countless families. To maximize diagnostic yield, we implemented an unbiased semi-automated genematching algorithm based on genotype and phenotype matching. METHODS: Rare homozygous variants identified in 2 or more affected individuals, but not in healthy individuals, were extracted from our local database of ∼12,000 exomes. Phenotype similarity scores (PSS), based on human phenotype ontology terms, were assigned to each pair of individuals matched at the genotype level using HPOsim. RESULTS: 33,792 genotype-matched pairs were discovered, representing variants in 7567 unique genes. There was an enrichment of PSS ≥0.1 among pathogenic/likely pathogenic variant-level pairs (94.3% in pathogenic/likely pathogenic variant-level matches vs 34.75% in all matches). We highlighted founder or region-specific variants as an internal positive control and proceeded to identify candidate disease genes. Variant-level matches were particularly helpful in cases involving inframe indels and splice region variants beyond the canonical splice sites, which may otherwise have been disregarded, allowing for detection of candidate disease genes, such as KAT2A, RPAIN, and LAMP3. CONCLUSION: Semi-automated genotype matching combined with PSS is a powerful tool to resolve variants of uncertain significance and to identify candidate disease genes.

Pontocerebellar hypoplasia associated with p.Arg183Trp homozygous variant in EXOSC1 gene: A case report.

Pontocerebellar hypoplasia (PCH) is a heterogeneous group of rare neurodegenerative disorders characterized by a wide phenotypic range including severe motor and cognitive impairments, microcephaly, distinctive facial features, and other features according to the type. Several classes of PCH1 have been linked to mutations in the evolutionarily conserved RNA exosome complex that consists of nine subunits (EXOSC1 to EXOSC9) and facilitates the degradation and processing of cytoplasmic and nuclear RNA from the 3' end. Only a single individual with an EXOSC1 mutation was reported with clinical features of PCH type 1 (PCH1F). Here, we report a 3-month-old female with PCH and additional clinical features not previously reported to be associated with PCH1, including dilated cardiomyopathy. On assessment, failure to thrive, microcephaly, distinctive facial features, and bluish sclera, were noted. Whole-exome sequencing was performed and revealed a novel homozygous missense variant c.547C > T (p.Arg183Trp) in the EXOSC1 gene. Functional studies in a budding yeast model that expresses the human EXOSC1 variant Arg183Trp show a slow-growth phenotype, whereas the previously identified PCH1F allele EXOSC1-Ser35Leu is lethal, indicating impaired exosome function for both of these variants. The protein levels of both EXOSC1 variants are reduced compared with wild-type when expressed in budding yeast. Herein, we ascertain the second case of PCH associated with a EXOSC1 variant that causes defects in RNA exosome function and provide a model organism system to distinguish between benign and pathogenic variants in EXOSC1.

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

Hereditary orotic aciduria identified by newborn screening.

Introduction: Hereditary orotic aciduria is an extremely rare, autosomal recessive disease caused by deficiency of uridine monophosphate synthase. Untreated, affected individuals may develop refractory megaloblastic anemia, neurodevelopmental disabilities, and crystalluria. Newborn screening has the potential to identify and enable treatment of affected individuals before they become significantly ill. Methods: Measuring orotic acid as part of expanded newborn screening using flow injection analysis tandem mass spectrometry. Results: Since the addition of orotic acid measurement to the Israeli routine newborn screening program, 1,492,439 neonates have been screened. The screen has identified ten Muslim Arab newborns that remain asymptomatic so far, with DBS orotic acid elevated up to 10 times the upper reference limit. Urine organic acid testing confirmed the presence of orotic aciduria along with homozygous variations in the UMPS gene. Conclusion: Newborn screening measuring of orotic acid, now integrated into the routine tandem mass spectrometry panel, is capable of identifying neonates with hereditary orotic aciduria.

A novel mutation in TTC19 associated with isolated complex III deficiency, cerebellar hypoplasia, and bilateral basal ganglia lesions.

Isolated complex III (cIII) deficiency is a rare biochemical finding in mitochondrial disorders, mainly associated with mutations in mitochondrial DNA MTCYB gene, encoding cytochrome b, or in assembly factor genes (BCS1L, TTC19, UQCC2, and LYRM7), whereas mutations in nuclear genes encoding cIII structural subunits are extremely infrequent. We report here a patient, a 9 year old female born from first cousin related parents, with normal development till 18 months when she showed unsteady gait with frequent falling down, cognitive, and speech worsening. Her course deteriorated progressively. Brain MRI showed cerebellar vermis hypoplasia and bilateral lentiform nucleus high signal lesions. Now she is bed ridden with tetraparesis and severely impaired cognitive and language functions. Biochemical analysis revealed isolated cIII deficiency in muscle, and impaired respiration in fibroblasts. We identified a novel homozygous rearrangement in TTC19 (c.213_229dup), resulting in frameshift with creation of a premature termination codon (p.Gln77Argfs*30). Western blot analysis demonstrated the absence of TTC19 protein in patient's fibroblasts, while Blue-Native Gel Electrophoresis analysis revealed the presence of cIII-specific assembly intermediates. Mutations in TTC19 have been rarely associated with mitochondrial disease to date, being described in about ten patients with heterogeneous clinical presentations, ranging from early onset encephalomyopathy to adult forms with cerebellar ataxia. Contrariwise, the biochemical defect was a common hallmark in TTC19 mutant patients, confirming the importance of TTC19 in cIII assembly/stability. Therefore, we suggest extending the TTC19 mutational screening to all patients with cIII deficiency, independently from their phenotypes.