Helsmoortel-Van der Aa syndrome in a 13-year-old girl with autistic spectrum disorder, dysmorphism, a right solitary kidney, and polycystic ovaries: a case report.

BACKGROUND: Helsmoortel-Van der Aa syndrome was officially documented in 2014. Helsmoortel-Van der Aa syndrome is an extremely rare complex neurodegenerative disorder characterized by reduced intellectual capacity, motor dysfunction, facial dysmorphism, impaired development, and an increased predisposition to autism spectrum disorder. In addition, many patients also present with neuropsychiatric disorders, including attention deficit hyperactivity disorder, anxiety disorders, and various behavioral abnormalities. Helsmoortel-Van der Aa syndrome is challenging to identify solely on the basis of symptoms, and genetic investigations, including exome sequencing, may facilitate diagnosis. CASE PRESENTATION: We report a case of 13-year-old Saudi patient who presented with dysmorphic features as illustrated in Fig. 1, severe mental retardation, autism spectrum disorder, and attention deficit hyperactivity disorder. Initial genetic testing was unremarkable; thus, a clinical exome analysis was performed to identify the genetic basis of the condition. CONCLUSIONS: Clinical exome analysis indicated an autosomal dominant Helsmoortel-Van der Aa syndrome with a likely pathogenic de novo variant within the activity-dependent neuroprotector homeobox (ADNP) gene not previously reported in Helsmoortel-Van der Aa syndrome. The patient had a right-sided solitary kidney and polycystic ovaries, conditions that were not previously associated with HVDAS.

Targeted next-generation sequencing of genes involved in Warfarin Pharmacodynamics and pharmacokinetics pathways using the Saudi Warfarin Pharmacogenetic study (SWAP).

BACKGROUND: Warfarin is an oral anticoagulant commonly used for treatment and prophylaxis against thromboembolic events. Warfarins's narrow therapeutic index window is one of the main challenges in clinical practice; thus, it requires frequent monitoring and dose adjustment to maintain patients' therapeutic range. Warfarin dose variation and response are attributed to several inter-and intra-individuals factors, including genetic variants in enzymes involved in warfarin pharmacokinetics (PK) and pharmacodynamics (PD) pathways. Thus, we aim to utilize the next-generation sequencing (NGS) approach to identify rare and common genetic variants that might be associated with warfarin responsiveness. METHOD AND RESULTS: A predesigned NGS panel that included 16 genes involved in Warfarin PK/PD pathways was used to sequence 786 patients from the Saudi Warfarin Pharmacogenetic Cohort (SWAP). Identified variants were annotated using several annotation tools to identify the pathogenicity and allele frequencies of these variants. We conducted variants-level association tests with warfarin dose. We identified 710 variants within the sequenced genes; 19% were novel variants, with the vast majority being scarce variants. The genetic association tests showed that VKORC1 (rs9923231, and rs61742245), CYP2C9 (rs98332238, rs9332172, rs1057910, rs9332230, rs1799853, rs1057911, and rs9332119), CYP2C19 (rs28399511, and rs3758581), and CYP2C8 (rs11572080 and rs10509681) were significantly associated with warfarin weekly dose. Our model included genetics, and non-genetic factors explained 40.1% of warfarin dose variation. CONCLUSION: The study identifies novel variants associated with warfarin dose in the Saudi population. These variants are more likely to be population-specific variants, suggesting that population-specific studies should be conducted before adopting a universal warfarin genotype-guided dosing algorithm.

Pro106Leu MPL mutation is associated with thrombocytosis and a low risk of thrombosis, splenomegaly and marrow fibrosis.

The P106L mutation in the human myeloproliferative leukemia virus oncogene (MPL) was shown to be associated with hereditary thrombocythemia in Arabs. The clinical and bone marrow (BM) features of P106L mutation are unknown. Genetic databases at two tertiary hospitals in Saudi Arabia were searched to identify patients with the MPL P106L mutation. Clinical data were collected retrospectively and the BM aspirates and biopsies were independently reviewed by two hematopathologists. In total, 115 patients were included. Median age was 33 years of which 31 patients were pediatric and 65 were female. The mutation was homozygous in 87 patients. Thrombocytosis was documented in 107 patients, with a median platelet count of 667 × 109/L. The homozygous genotype was associated with a higher platelet count. Thirty-three patients had an evaluable BM and clustering of megakaryocytes was observed in 30/33 patients. At the time of last follow-up, 114 patients were alive. The median follow-up was 7.8 years from the time of thrombocytosis. No patients developed disease progression to myelofibrosis. The P106L mutation was associated with marked thrombocytosis at a younger age and with a low risk of thrombosis, splenomegaly, and marrow fibrosis. The BM demonstrated normal or hypocellular marrow with megakaryocyte clusters.

Ichthyosis Prematurity Syndrome: A Rare Form but Easily Recognizable Ichthyosis.

Ichthyosis prematurity syndrome is a rare autosomal recessive genodermatosis that is associated with mutations in the SLC27A4 gene. Its onset occurs in early childhood and presents with the clinical triad of premature birth, thick caseous desquamating epidermis, and neonatal asphyxia. Here, we describe a prematurely born baby patient (33 weeks of gestation) with a homozygous variant at the initiation codon site (c.1 A> G, p.Met1Val) in the SLC27A4 gene to raise awareness of this rare syndrome despite its distinctive features as we believe it is still underdiagnosed.

Generation of induced pluripotent stem cell Line KAIMRCi001-A by reprogramming erythroid progenitors from peripheral blood of a healthy Saudi donor.

In this study we isolated and enriched erythroid progenitor cells (EPCs) from a 10 ml peripheral blood sample from a 37-year old healthy Saudi donor. After expansion, these EPCs were reprogrammed using episomal plasmids to generate an induced pluripotent stem (iPS) cell line, KAIMRCi001-A. The pluripotency of this line was confirmed by measuring the expression of typical pluripotency markers and assessing differentiation potential in vitro.

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.

Genetic Profile of Epidermolysis Bullosa Cases in King Abdulaziz Medical City, Riyadh, Saudi Arabia.

Epidermolysis bullosa (EB) is a rare heterogeneous genetic mechanobullous skin disorder that is characterized by increased skin fragility leading to blistering following minor trauma. EB may be inherited as an autosomal dominant or an autosomal recessive disorder and can be classified into dystrophic EB (DEB), junctional EB (JEB), and EB simplex (EBS). A total of 28 Saudi patients with EB were included in this observational, retrospective chart-review study. A consecutive non-probability sampling technique was used to approach all affected patients. Molecular analysis was done to test the patients' genomic DNA using a custom-designed AmpliSeq panel of suspected genes. All disease-causing variants were checked against available public databases. Twelve patients (42.9%) were found to have DEB, 6 patients (21.4%) with JEB, and 10 patients (35.7%) with EBS. The molecular genetic results revealed detections of 24 various homozygous genetic variations in the genes associated with EB, of which 14 were novel mutations. The most frequent variations were detected in COL7A1 in 12 cases (42.9%), followed by LAMB3 in 5 cases (17.9%), TGM5 in 4 cases (14.3%), and other genes. Furthermore, the majority (87.5%) of EB cases were confirmed to have homozygous mutations, and few were documented with positive consanguinity history. Only 3 cases (12.5%) were found to be autosomal dominant displaying heterozygous mutations. This is the first study to establish the EB genetic profile in Saudi Arabia where DEB is the most frequent type. A total of 14 novel mutations were identified that had not been previously reported. Consanguineous marriage is clearly recognized in the Saudi population; therefore, we propose a nationwide EB program that would help extend the spectrum of the genetic profile and help in the diagnosis and better understanding of this disease.

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.

Evolving sequence mutations in the Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) has continued to cause sporadic outbreaks of severe respiratory tract infection over the last 8 years. METHODS: Complete genome sequencing using next-generation sequencing was performed for MERS-CoV isolates from cases that occurred in Riyadh between 2015 and 2019. Phylogenetic analysis and molecular mutational analysis were carried out to investigate disease severity. RESULTS: A total of eight MERS-CoV isolates were subjected to complete genome sequencing. Phylogenetic analysis resulted in the assembly of 7/8 sequences within lineage 3 and one sequence within lineage 4 showing complex genomic recombination. The isolates contained a variety of unique amino acid substitutions in ORF1ab (41), the N protein (10), the S protein (9) and ORF4b (5). CONCLUSION: Our study shows that MERS-CoV is evolving. The emergence of new variants carries the potential for increased virulence and could impose a challenge to the global health system. We recommend the sequencing every new MERS-CoV isolate to observe the changes in the virus and relate them to clinical outcomes.

What is the right sequencing approach? Solo VS extended family analysis in consanguineous populations.

BACKGROUND: Testing strategies is crucial for genetics clinics and testing laboratories. In this study, we tried to compare the hit rate between solo and trio and trio plus testing and between trio and sibship testing. Finally, we studied the impact of extended family analysis, mainly in complex and unsolved cases. METHODS: Three cohorts were used for this analysis: one cohort to assess the hit rate between solo, trio and trio plus testing, another cohort to examine the impact of the testing strategy of sibship genome vs trio-based analysis, and a third cohort to test the impact of an extended family analysis of up to eight family members to lower the number of candidate variants. RESULTS: The hit rates in solo, trio and trio plus testing were 39, 40, and 41%, respectively. The total number of candidate variants in the sibship testing strategy was 117 variants compared to 59 variants in the trio-based analysis. We noticed that the average number of coding candidate variants in trio-based analysis was 1192 variants and 26,454 noncoding variants, and this number was lowered by 50-75% after adding additional family members, with up to two coding and 66 noncoding homozygous variants only, in families with eight family members. CONCLUSION: There was no difference in the hit rate between solo and extended family members. Trio-based analysis was a better approach than sibship testing, even in a consanguineous population. Finally, each additional family member helped to narrow down the number of variants by 50-75%. Our findings could help clinicians, researchers and testing laboratories select the most cost-effective and appropriate sequencing approach for their patients. Furthermore, using extended family analysis is a very useful tool for complex cases with novel genes.

The effect of the VKORC1 promoter variant on warfarin responsiveness in the Saudi WArfarin Pharmacogenetic (SWAP) cohort.

Warfarin is a frequently prescribed oral anticoagulant with a narrow therapeutic index, requiring careful dosing and monitoring. However, patients respond with significant inter-individual variability in terms of the dose and responsiveness of warfarin, attributed to genetic polymorphisms within the genes responsible for the pharmacokinetics and pharmacodynamics of warfarin. Extensive warfarin pharmacogenetic studies have been conducted, including studies resulting in genotype-guided dosing guidelines, but few large scale studies have been conducted with the Saudi population. In this study, we report the study design and baseline characteristics of the Saudi WArfarin Pharmacogenomics (SWAP) cohort, as well as the association of the VKORC1 promoter variants with the warfarin dose and the time to a stable INR. In the 936 Saudi patients recruited in the SWAP study, the minor allele C of rs9923231 was significantly associated with a 8.45 mg higher weekly warfarin dose (p value = 4.0 × 10-46), as well as with a significant delay in achieving a stable INR level. The addition of the rs9923231 status to the model, containing all the significant clinical variables, doubled the warfarin dose explained variance to 31%. The SWAP cohort represents a valuable resource for future research with the objective of identifying rare and prevalent genetic variants, which can be incorporated in personalized anticoagulation therapy for the Saudi population.

A classification system for split-hand/ foot malformation (SHFM): A proposal based on 3 pedigrees with WNT10B mutations.

SHFM6 (OMIM 225300) is caused by WNT10B pathogenic variants (12q13.12). It is one of the rarest forms of SHFM; with only seven pathogenic variants described in the world literature. Furthermore, it has not been determined if SHFM6 has specific phenotypic characteristics. In this paper, we present a case series of three unrelated families with SHFM6 caused by three novel WNT10B pathogenic variants. The index patient of the first family was homozygous for the nonsense variant c.676C > T (p.Arg226*) in the WNT10B gene. The index case of the second family had a homozygous splice variant c.338-1G > C in the WNT10B gene. Finally, the index case of the third family carried two different variants in the WNT10B gene: A nonsense variant (p.Arg226*), and a missense variant (p.Gln86Pro). The latter represents the first compound heterozygous pathogenic variant related to SHFM6. We also offer a classification system for the hand/foot defects to illustrate the specific phenotypic characteristics of SHFM6. Based on this classification and a review of all previously reported cases, we demonstrate that SHFM6 caused by WNT10B pathogenic variants have the following characteristics: more severe feet defects (compared to the hand defects), polydactyly, severe flexion digital contractures, and phalangeal dysplasia.

Targeted SLC19A3 gene sequencing of 3000 Saudi newborn: a pilot study toward newborn screening.

BACKGROUND: Biotin-thiamine-responsive basal ganglia disease (BTBGD) is an autosomal recessive neurometabolic disorder mostly presented in children. The disorder is described as having subacute encephalopathy with confusion, dystonia, and dysarthria triggered by febrile illness that leads to neuroregression and death if untreated. Using biotin and thiamine at an early stage of the disease can lead to significant improvement. METHODS: BTBGD is a treatable disease if diagnosed at an early age and has been frequently reported in Saudi population. Keeping this in mind, the current study screened 3000 Saudi newborns for the SLC19A3 gene mutations using target sequencing, aiming to determine the carrier frequency in Saudi Population and whether BTBGD is a good candidate to be included in the newborn-screened disorders. RESULTS: Using targeted gene sequencing, DNA from 3000 newborns Saudi was screened for the SLC19A3 gene mutations using standard methods. Screening of the SLC19A3 gene revealed a previously reported heterozygous missense mutation (c.1264A>G (p.Thr422Ala) in six unrelated newborns. No probands having homozygous pathogenic mutations were found in the studied cohort. The variant has been frequently reported previously in homozygous state in Saudi population, making it a hot spot mutation. The current study showed that the carrier frequency of SLC19A3 gene mutation is 1 of 500 in Saudi newborns. CONCLUSION: For the first time in the literature, we determined the carrier frequency of SLC19A3 gene mutation in Saudi population. The estimated prevalence is too rare in Saudi population (at least one in million); therefore, the data are not in favor of including such very rare disorders in newborn screening program at population level. However, a larger cohort is needed for a more accurate estimate.

Atypical influenza A(H1N1)pdm09 strains caused an influenza virus outbreak in Saudi Arabia during the 2009-2011 pandemic season.

BACKGROUND: The triple assortment influenza A(H1N1) virus emerged in spring 2009 and disseminated worldwide, including Saudi Arabia. This study was carried out to characterize Saudi influenza isolates in relation to the global strains and to evaluate the potential role of mutated residues in transmission, adaptation, and the pathogenicity of the virus. METHODS: Nasopharyngeal samples (n = 6492) collected between September 2009 to March 2011 from patients with influenza-like illness were screened by PCR for influenza A(H1N1). Phylogenetic and Molecular evolutionary analysis were carried out to place the Saudi strains in relation to the global strains followed by Mutation analysis of surface and internal proteins. RESULTS: Concatenated whole-genome phylogenetic analysis along with hemagglutinin (HA) signature changes, that is, Aspartic Acid (D) at position 187, P83S, S203T, and R223Q confirmed that the Saudi strains belong to the antigenic category of A/California/07/2009. However, phylogenetic analysis revealed unusual strains of A(H1N1) circulating in Saudi Arabia, not belonging to any of known clades, appearing in five distinct groups well supported by group-specific mutations and novel mutation complexes. These cases had characteristic inter- and intragroup substitution patterns while few of their closest matches showed up as sporadic cases the world over. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, and M2 having a putative role in viral fitness and virulence. Bayesian coalescent MCMC analysis revealed that Saudi strains belonged to cluster 2 of A(H1N1)pdm09 and spread a month later as compared to other strains of this cluster. CONCLUSION: Influenza outbreak in Saudi Arabia during 2009-2011 was caused by atypical strains of influenza A(H1N1)pdm09, probably introduced in this community on multiple occasions. To understand the antigenic significance of these novel point mutations and mutation complexes require functional studies, which will be crucial for risk assessment of emergent strains and defining infection control measures.

Upper limb muscle overgrowth with hypoplasia of the index finger: a new over-growth syndrome caused by the somatic PIK3CA mutation c.3140A>G.

BACKGROUND: Scientists have previously described an overgrowth syndrome in Saudi patients and named it 'Upper limb muscle overgrowth with hypoplasia of the index finger' syndrome. CASE PRESENTATION: We describe a new case and document that the syndrome is caused by the somatic PIK3CA mutation c.3140A>G, p.His1047Arg. We also recruited one of the previously reported cases and found the same somatic mutation in the affected muscles. A wider classification of 'PIK3CA-related pathology spectrum' is presented which includes cancer, benign skin lesions/tumors, Cowden syndrome, isolated vascular malformations and various overgrowth syndromes. The latter entity is sub-divided into 3 sub-groups: overgrowth with brain involvement, overgrowth with multiple lipomatosis, and overgrowth without brain involvement/multiple lipomatosis. CONCLUSION: Our literature review indicated that "upper limb muscle overgrowth with hypoplasia of the index finger" is not as rare as previously thought to be. This overgrowth syndrome is unique and is caused by the somatic PIK3CA mutation c.3140A>G.

Whole-genome sequencing offers additional but limited clinical utility compared with reanalysis of whole-exome sequencing.

PURPOSE: Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are used to diagnose genetic and inherited disorders. However, few studies comparing the detection rates of WES and WGS in clinical settings have been performed. METHODS: Variant call format files were generated and raw data analysis was performed in cases in which the final molecular results showed discrepancies. We classified the possible explanations for the discrepancies into three categories: the time interval between the two tests, the technical limitations of WES, and the impact of the sequencing system type. RESULTS: This cohort comprised 108 patients with negative array comparative genomic hybridization and negative or inconclusive WES results before WGS was performed. Ten (9%) patients had positive WGS results. However, after reanalysis the WGS hit rate decreased to 7% (7 cases). In four cases the variants were identified by WES but missed for different reasons. Only 3 cases (3%) were positive by WGS but completely unidentified by WES. CONCLUSION: In this study, we showed that 30% of the positive cases identified by WGS could be identified by reanalyzing the WES raw data, and WGS achieved an only 7% higher detection rate. Therefore, until the cost of WGS approximates that of WES, reanalyzing WES raw data is recommended before performing WGS.

Tetrasomy 18p: case report and review of literature.

Tetrasomy 18p syndrome (Online Mendelian Inheritance in Man 614290) is a very rare chromosomal disorder that is caused by the presence of isochromosome 18p, which is a supernumerary marker composed of two copies of the p arm of chromosome 18. Most tetrasomy 18p cases are de novo cases; however, familial cases have also been reported. It is characterized mainly by developmental delays, cognitive impairment, hypotonia, typical dysmorphic features, and other anomalies. Herein, we report de novo tetrasomy 18p in a 9-month-old boy with dysmorphic features, microcephaly, growth delay, hypotonia, and cerebellar and renal malformations. We compared our case with previously reported ones in the literature. Clinicians should consider tetrasomy 18p in any individual with dysmorphic features and cardiac, skeletal, and renal abnormalities. To the best of our knowledge, we report for the first time an association of this syndrome with partial agenesis of cerebellar vermis.