Prospect of genetic disorders in Saudi Arabia.

Introduction: Rare diseases (RDs) create a massive burden for governments and families because sufferers of these diseases are required to undergo long-term treatment or rehabilitation to maintain a normal life. In Saudi Arabia (SA), the prevalence of RDs is high as a result of cultural and socio-economic factors. This study, however, aims to shed light on the genetic component of the prevalence of RDs in SA. Methodology: A retrospective study was conducted between September 2020 and December 2021 at King Saud Medical City, a tertiary hospital of the Ministry of Health (MOH), SA. A total of 1080 individuals with 544 potentially relevant variants were included. The index was 738, and the samples were tested in a commercialized laboratory using different molecular techniques, including next-generation sequencing. Result: A total of 867 molecular genetics tests were conducted on 738 probands. These tests included 610 exome sequencing (ES) tests, four genome sequencing (GS) tests, 82 molecular panels, 106 single nucleotide polymorphism (SNP) array, four methylation studies, 58 single-gene studies and three mitochondrial genome sequencing tests. The diagnostic yield among molecular genetics studies was 41.8% in ES, 24% in panels, 12% in SNP array and 24% in single gene studies. The majority of the identified potential variants (68%) were single nucleotide variants (SNV). Other ascertained variants included frameshift (11%), deletion (10%), duplication (5%), splicing (9%), in-frame deletion (3%) and indels (1%). The rate of positive consanguinity was 56%, and the autosomal recessive accounted for 54%. We found a significant correlation between the ES detection rate and positive consanguinity. We illustrated the presence of rare treatable conditions in DNAJC12, SLC19A3, and ALDH7A1, and the presence of the founder effect variant in SKIC2. Neurodevelopmental disorders were the main phenotype for which genetics studies were required (35.7%). Conclusion: This is the sixth-largest local study reporting next-generation sequencing. The results indicate the influence of consanguineous marriages on genetic disease and the burden it causes for the Kingdom of SA. This study highlights the need to enrich our society's knowledge of genetic disorders. We recommend utilising ES as a first-tier test to establish genetic diagnosis in a highly consanguineous population.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): A challenging diagnosis and a rare multiple sclerosis mimic.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an extremely rare hereditary cerebral small vessel disease caused by homozygous or compound heterozygous mutations in the gene coding for high-temperature requirement A serine peptidase 1 (HtrA1). Given the rare nature of the disease, delays in diagnosis and misdiagnosis are not uncommon. In this article, we reported the first case of CARASIL from Saudi Arabia with a novel homozygous variant c.1156C>T in exon 7 of the HTRA1 gene. The patient was initially misdiagnosed with primary progressive multiple sclerosis and treated with rituximab. CARASIL should be considered in the differential diagnosis of patients with suspected atypical progressive multiple sclerosis who have additional signs such as premature scalp alopecia and low back pain with diffuse white matter lesions in brain MRI. Genetic testing is important to confirm the diagnosis.

The diagnostic yield of CGH and WES in neurodevelopmental disorders.

Background: Neurodevelopmental disorders are a group of conditions characterized by developmental delays leading to abnormal brain functions. The methods of diagnosis and treatment of these conditions are complicated, and their treatment involves a combination of various forms of therapy. In recent years, the development of high-resolution technologies has played an important role in revealing the microdeletions, microduplications, and single-nucleotide variants of the chromosomes and how they are linked to the development of neurodevelopmental disorders. The wide implementation and application of molecular methodologies have started to shed light on the functional importance of using the appropriate methods in detecting these genetic variations that are categorized as either pathogenic or benign. The study aimed to compare the diagnostic yield of comparative hybridization (CGH) and whole exome sequencing (WES) in neurodevelopmental disorders among children attending the King Abdullah Specialist Children Hospital, Riyadh, Saudi Arabia. Methods: A retrospective study was conducted between 2015 and 2018 on 105 patients diagnosed with neurodevelopmental disorders through array-based CGH (Array-CGH) and WES. Results: In a sample of 105 patients, 16% was the hit rate of copy number variations (CNVs). WES was requested for CNV-negative patients (n = 79), of which 30% was the hit rate of pathogenic or likely pathogenic single-nucleotide variants. There was a difference in the diagnostic yield between CGH (16%) and WES (30%). Conclusion: WES was a better approach than Array-CGH to detect various DNA mutations or variants. Our findings could guide clinicians, researchers, and testing laboratories select the most cost-effective and appropriate approach for diagnosing their patients.

De Novo Ring Chromosome 15: Molecular Cytogenetic and Clinical Characterization of First Case from Saudi Arabia.

Ring chromosome 15 is a rare chromosomal disorder, which usually occurs during early embryonic development via spontaneous errors and has variable presentation. To date, 89 cases of this condition have been reported. This case report describes a 5-year-old Saudi boy who was diagnosed as having de novo 46,XY,r(15). The patient presented with short stature, speech delay, café au lait spots, and facial dysmorphic features, together with new findings of left crossed fused renal ectopia and 11 ribs. This presentation was compared with the findings of cases reported previously.

HMG-CoA Lyase Deficiency: A Retrospective Study of 62 Saudi Patients.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMG-CoA lyase) is a rare inborn error of leucine degradation and ketone body synthesis, caused by homozygous or compound heterozygous disease-causing variants in HMGCL. To understand the natural history of this disease, we reviewed the biochemical, clinical, and molecular data of 62 patients from 54 different families with confirmed HMG-CoA lyase deficiency (HMGCLD) diagnosis from Saudi Arabia. The majority of the affected individuals were symptomatic. At initial diagnosis, 38 patients (61.29%) presented with hypoglycemia and 49 patients (79.03%) developed metabolic acidosis. In 27 patients (43.54%), the disorder manifested in the neonatal period, mostly within the first days of life, while 35 (56.45%) patients were diagnosed within the first year of life or beyond. All the patients were alive and developed long-term neurological complications during data collection, which may significantly influence their quality of life. Common neurological findings include seizures 17/62 (27.41%), hypotonic 3/62 (4.83%), speech delay 7/62 (11.29%), hyperactivity 4/62 (4.83%), developmental delay 6/62 (9.677%), learning disability 15/62 (24.14%), and ataxic gate 1/62 (1.612%). An MRI of the brain exhibited nonspecific periventricular and deep white matter hyperintense signal changes in 16 patients (25.80%) and cerebral atrophy was found in one (1/62; 1.612%) patient. We identified a founder variant [c.122G>A; p.(Arg41Gln)] in 48 affected individuals (77.41%) in the HMGCL gene. This is the largest cohort of HMGCLD patients reported from Saudi Arabia, signifying this disorder as a likely life-threatening disease, with a high prevalence in the region. Our findings suggest that diagnosis at an early stage with careful dietary management may avoid metabolic crises.

The variant artificial intelligence easy scoring (VARIES) system.

PURPOSE: Medical artificial intelligence (MAI) is artificial intelligence (AI) applied to the healthcare field. AI can be applied to many different aspects of genetics, such as variant classification. With little or no prior experience in AI coding, we share our experience with variant classification using the Variant Artificial Intelligence Easy Scoring (VARIES), an open-access platform, and the Automatic Machine Learning (AutoML) of the Google Cloud Platform. METHODS: We investigated exome sequencing data from a sample of 1410 individuals. The majority (80%) were used for training and 20% for testing. The user-friendly Google Cloud Platform was used to create the VARIES model, and the TRIPOD checklist to develop and validate the prediction model for the development of the VARIES system. RESULTS: The learning rate of the training dataset reached optimal results at an early stage of iteration, with a loss value near zero in approximately 4 min. For the testing dataset, the results for F1 (micro average) was 0.64, F1 (macro average) 0.34, micro-average area under the curve AUC (one-over-rest) 0.81 and the macro-average AUC (one-over-rest) 0.73. The overall performance characteristics of the VARIES model suggest the classifier has a high predictive ability. CONCLUSION: We present a systematic guideline to create a genomic AI prediction tool with high predictive power, using a graphical user interface provided by Google Cloud Platform, with no prior experience in creating the software programs required.

Cut-off values in newborn screening for inborn errors of metabolism in Saudi Arabia.

BACKGROUND: Newborn screening identifies individuals affected by a specific disorder within an apparently healthy population prior to the appearance of symptoms so that appropriate interventions can be initiated in time to minimize the harmful effects. Data on population based cut-off values, disease ranges for true positive cases, false positive rates, true positive rates, cut-off verification and comparisons with international cut-off ranges have not been done for Saudi Arabia. OBJECTIVE: Establish population-based cut-off values and analyte ratios for newborn screening assays and clinically validate the values. DESIGN: Population-based screening. SETTING: Tertiary care hospitals and laboratories. METHODS: After method verification, initial cut-off values were established by analyzing 400-500 dry blood spot (DBS) samples which were further evaluated after one year. About 74 000 patient results were reviewed to establish cut-off ranges from DBS samples received from five different hospitals during 2013-2020. Analysis was performed by tandem mass spectrometry (TMS) and a genetic screening processor. Confirmation of initial positive newborn screening results for different analytes were carried out using gas chromatography-mass spectrometry, high performance liquid chromatography and TMS. MAIN OUTCOME MEASURES: Cut-off values, ratios, positive predictive values, false positive rate, true positive rate and disease range. SAMPLE SIZE: 74 000 samples. RESULTS: Population based cut-off values were calculated at different percentiles. These values were compared with 156 true positive samples and 80 proficiency samples. The false positive rate was less than 0.04 for all the analytes, except for valine, leucine, isovalerylcarnitine (C5), biotinidase (BTD), 17-hydroxyprogesterone and thyroid stimulating hormone. The highest false positive rate was 0.14 for BTD which was due to pre-analytical errors. The analytical positive predictive values were greater than 80% throughout the eight years. CONCLUSION: We have established clinical disease ranges for most of the analytes tested in our lab and several ratios which gives excellent screening specificity and sensitivity for early detection. The samples were representative of the local populations. LIMITATIONS: Need for wider, population-based studies. CONFLICT OF INTEREST: None.

Common disease-associated gene variants in a Saudi Arabian population.

BACKGROUND: Screening programs for the most prevalent conditions occurring in a country is an evidence-based prevention strategy. The burden of autosomal recessive disease variations in Saudi Arabia is high because of the highly consanguineous population. The optimal solution for estimating the carrier frequency of the most prevalent diseases is carrier screening. OBJECTIVES: Identify the most influential recessive alleles associated with disease in the Saudi population. DESIGN: We used clinical whole-exome sequencing data from an in-house familial database to evaluate the most prevalent genetic variations associated with disease in a Saudi population. SETTINGS: King Abdullah International Medical Research Center (KAIMRC) and King Abdulaziz Medical City. METHODS: Whole exome sequencing data obtained from clinical studies of family members, a cohort of 1314 affected and unaffected individuals, were filtered using the in-house pipeline to extract the most prevalent variant in the dataset. MAIN OUTCOME MEASURES: Most prevalent genetic variations associated with disease in the Saudi population. SAMPLE SIZE: 1314 affected and unaffected individuals. RESULTS: We identified 37 autosomal recessive variants and two heterozygous X-linked variants in 35 genes associated with the most prevalent disorders, which included hematologic (32%), endocrine (21%), metabolic (11%) and immunological (10%) diseases. CONCLUSION: This study provides an update of the most frequently occurring alleles, which support future carrier screening programs. LIMITATIONS: Single center that might represent the different regions but may be biased. In addition, most of the families included in the database are part of the proband's genetic identification for specific phenotypes. CONFLICT OF INTEREST: None.

The rate of secondary genomic findings in the Saudi population.

Secondary findings (SF) are defined as genetic conditions discovered unintentionally during an evaluation of raw data for another disease. We aimed to identify the rate of secondary genetic findings in the Saudi population in the 59 genes of the American College of Medical Genetics and Genomics (ACMG) list. In our study, the raw data of 1254 individuals, generated from exome sequencing for clinical purposes, were studied. Variants detected in the 59 genes on the ACMG list of secondary findings were investigated. Pathogenicity classifications were assigned to those variants based on the ACMG scoring system. We identified 2409 variants in the 59 gene list, 45 variants were classified as pathogenic/likely pathogenic variants according to the ACMG classification. The LDLR gene had the greatest number of pathogenic/likely pathogenic variants 12%. Cardiovascular genetic diseases had the highest frequency of disorders detected as secondary findings. In this study, the overall rate of positive cases identified with secondary findings in the Saudi population was 8%. The different in our current study and the previous studies in Saudi Arabia can be explained by the differences between the sequencing method, the criteria used for variant classification, the availability of newer evidence at the time of the publication, and the fact that we identified Saudi novel variants never reported in other populations.

Long-term effectiveness of carglumic acid in patients with propionic acidemia (PA) and methylmalonic acidemia (MMA): a randomized clinical trial.

BACKGROUND: Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare, autosomal recessive inborn errors of metabolism that require life-long medical treatment. The trial aimed to evaluate the effectiveness of the administration of carglumic acid with the standard treatment compared to the standard treatment alone in the management of these organic acidemias. METHODS: The study was a prospective, multicenter, randomized, parallel-group, open-label, controlled clinical trial. Patients aged ≤ 15 years with confirmed PA and MMA were included in the study. Patients were followed up for two years. The primary outcome was the number of emergency room (ER) admissions because of hyperammonemia. Secondary outcomes included plasma ammonia levels over time, time to the first episode of hyperammonemia, biomarkers, and differences in the duration of hospital stay. RESULTS: Thirty-eight patients were included in the study. On the primary efficacy endpoint, a mean of 6.31 ER admissions was observed for the carglumic acid arm, compared with 12.76 for standard treatment, with a significant difference between the groups (p = 0.0095). Of the secondary outcomes, the only significant differences were in glycine and free carnitine levels. CONCLUSION: Using carglumic acid in addition to standard treatment over the long term significantly reduces the number of ER admissions because of hyperammonemia in patients with PA and MMA.

Short stature with low insulin-like growth factor 1 availability due to pregnancy-associated plasma protein A2 deficiency in a Saudi family.

In 2016 a new syndrome with postnatal short stature and low IGF1 bioavailability caused by biallelic loss-of-function mutations in the gene encoding the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) was described in two families. Here we report two siblings of a third family from Saudi Arabia with postnatal growth retardation and decreased IGF1 availability due to a new homozygous nonsense mutation (p.Glu886* in exon 7) in PAPPA2. The two affected males showed progressively severe short stature starting around 8 years of age, moderate microcephaly, decreased bone mineral density, and high circulating levels of total IGF1, IGFBP3, and the IGF acid-labile subunit (IGFALS), with decreased free IGF1 concentrations. Interestingly, circulating IGF2 and IGFBP5 were not increased. An increase in growth velocity and height was seen in the prepuberal patient in response to rhIGF1. These patients contribute to the confirmation of the clinical picture associated with PAPP-A2 deficiency and that the PAPPA2 gene should be studied in all patients with short stature with this characteristic phenotype. Hence, pediatric endocrinologists should measure circulating PAPP-A2 levels in the study of short stature as very low or undetectable levels of this protein can help to focus the diagnosis and treatment.

The Leukodystrophy Spectrum in Saudi Arabia: Epidemiological, Clinical, Radiological, and Genetic Data.

Background: Leukodystrophies (LDs) are inherited heterogeneous conditions that affect the central nervous system with or without peripheral nerve involvement. They are individually rare, but collectively, they are common. Thirty disorders were included by the Global Leukodystrophy Initiative Consortium (GLIA) as LDs. Methods: We conducted a retrospective chart review of a consecutive series of patients diagnosed with different types of LD from four large tertiary referral centers in Riyadh, Saudi Arabia. Only those 30 disorders defined by GLIA as LDs were included. Results: In total, 83 children from 61 families were identified and recruited for this study. The male-to-female ratio was 1.5:1, and a consanguinity rate of 58.5% was observed. An estimated prevalence of 1:48,780 or 2.05/100,000 was observed based on the clinical cohort, whereas a minimum of 1:32,857 or 3.04/100,000 was observed based on the local genetic database. The central region of the country exhibited the highest prevalence of LDs (48.5%). The most common LD was metachromatic leukodystrophy (MLD), and it accounted for 25.3%. The most common disorder based on carrier frequency was AGS. Novel variants were discovered in 51% of the cases, but 49% possessed previously reported variants. Missense variants were high in number and accounted for 73% of all cases. Compared with other disorders, MLD due to saposin b deficiency was more common than expected, Pelizaeus-Merzbacher-like disease was more prevalent than Pelizaeus-Merzbacher disease, and X-linked adrenoleukodystrophy was less common than expected. The mortality rate among our patients with LD was 24%. Conclusion: To the best of our knowledge, this is the largest cohort of patients with LD from Saudi Arabia. We present epidemiological, clinical, radiological, and genetic data. Furthermore, we report 18 variants that have not been reported previously. These findings are of great clinical and molecular utility for diagnosing and managing patients with LD.

Biallelic variants in HPDL cause pure and complicated hereditary spastic paraplegia.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.

Combining exome/genome sequencing with data repository analysis reveals novel gene-disease associations for a wide range of genetic disorders.

PURPOSE: Within this study, we aimed to discover novel gene-disease associations in patients with no genetic diagnosis after exome/genome sequencing (ES/GS). METHODS: We followed two approaches: (1) a patient-centered approach, which after routine diagnostic analysis systematically interrogates variants in genes not yet associated to human diseases; and (2) a gene variant centered approach. For the latter, we focused on de novo variants in patients that presented with neurodevelopmental delay (NDD) and/or intellectual disability (ID), which are the most common reasons for genetic testing referrals. Gene-disease association was assessed using our data repository that combines ES/GS data and Human Phenotype Ontology terms from over 33,000 patients. RESULTS: We propose six novel gene-disease associations based on 38 patients with variants in the BLOC1S1, IPO8, MMP15, PLK1, RAP1GDS1, and ZNF699 genes. Furthermore, our results support causality of 31 additional candidate genes that had little published evidence and no registered OMIM phenotype (56 patients). The phenotypes included syndromic/nonsyndromic NDD/ID, oral-facial-digital syndrome, cardiomyopathies, malformation syndrome, short stature, skeletal dysplasia, and ciliary dyskinesia. CONCLUSION: Our results demonstrate the value of data repositories which combine clinical and genetic data for discovering and confirming gene-disease associations. Genetic laboratories should be encouraged to pursue such analyses for the benefit of undiagnosed patients and their families.

Hypospadias in ring X syndrome.

Ring X is a chromosomal anomaly mainly seen in females with turner syndrome and usually present in mosaic form with 45,X cells (45,X/46,X,r(X)) because of their mitotic instability. In males it is an extremely rare finding because large nullisomy for X chromosome material is likely not compatible with survival. Only two cases of male with ring chromosome X were previously reported. We report here a four-year-old male with ring chromosome X characterized using Karyotype, FISH and array CGH and presenting short stature, microcephaly and hypospadias. Molecular investigations showed 923 Kb terminal deletion on the pseudoautosomal region 1 (PAR1) including SHOX gene followed by a duplication of 2.4 Mb. The absence of functional nullisomy because of a second copy of deleted genes was present in chromosome Y PAR1 region may explain the compatibility with survival in our case of male with ring X. Short stature common with the two previously reported cases is likely related to SHOX gene deletion but also to the effect of "ring syndrome". However, hypospadias was not reported in the previous cases and can be due to the associated duplication outside PAR1 region including in particular PRKX gene coding for a protein involved in urogenital system morphogenesis.

Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.

BACKGROUND: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies". METHODS: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (ß-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation. RESULTS: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between ß-COP and ß'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant ß-COP, with the mutant protein being retarded in the Golgi. CONCLUSIONS: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

Two Novel Homozygous HPS6 Mutations (Double Mutant) Identified by Whole-Exome Sequencing in a Saudi Consanguineous Family Suspected for Oculocutaneous Albinism.

BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive disorder of low or missing pigmentation in the eyes, hair, and skin. Multiple types of OCA, including Hermansky-Pudlak syndrome 6 (HPS6), are distinguished by their genetic cause and pigmentation pattern. HPS6 is characterized by OCA, nose bleeding due to platelet dysfunction, and lysosome storage defect. To date, 25 disease-associated mutations have been reported in the HPS6 gene. METHODS: DNA was extracted from proband, and whole-exome sequencing (WES) was performed using the Illumina NovaSeq platform. Bioinformatic analysis was done with a custom-designed filter pipeline to detect the causative variant. We did Sanger sequencing to confirm the candidate variant and segregation analysis, and protein-based structural analysis to evaluate the functional impact of variants. RESULT: Proband-based WES identified two novel homozygous mutations in HPS6 (double mutation, c.1136C>A and c.1789delG) in an OCA suspect. Sanger sequencing confirmed the WES results. Although no platelet and/or lysosome storage defect was detected in the patient or family, an oculocutaneous albinism diagnosis was established based on the HPS6 mutations. Structural analysis revealed the transformation of abnormalities at protein level for both nonsense and frameshift mutations in HPS6. CONCLUSION: To the best of our knowledge, the double mutation in HPS6 (p.Ser379Ter and p.Ala597GlnfsTer16) represents novel pathogenic variants, not described previously, which we report for the first time in the Saudi family. In silico analyses showed a significant impact on protein structure. WES should be used to identify HPS6 and/or other disease-associated genetic variants in Saudi Arabia, particularly in consanguineous families.

Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort.

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them  could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.