Effect of clinical whole exome sequencing in aetiological investigation and reproductive risk prediction for a couple with monogenic inherited diseases.

Objective: To determine the genetic causes of monogenic inherited diseases in a couple using clinical whole exome sequencing (WES) and advise on their reproductive choices. Methods: WES was applied to a couple seeking reproductive advice, the female with short stature and the male with congenital cataracts. Results: (1) The woman exhibited a 13.8 Kb heterozygous deletion at chrX: 591590-605428 (hg19). This region corresponds to exons 2-6 of the short-stature homeobox-containing (SHOX) gene (NM000451). Associated diseases involving the SHOX gene range from severe Leri-Weill dyschondrosteosis to mild nonspecific short stature. Meanwhile, further validation using a quantitative reverse transcription polymerase chain reaction assay confirmed the heterozygous deletion of the SHOX gene in the proband, as well as other family members with similar clinical characteristics (the proband's mother, aunt, and cousin). Multiple pathogenic reports of this variant have been included in the HGMD database. Per the American College of Medical Genetics and Genomics (ACMG) classification criteria, this deletion is classified as pathogenic. (2) For the male patient, a heterozygous variant was detected in the CRYBB3 gene: NM004076: c.226G>A (p.Gly76R). Variants in the CRYBB3 gene can cause Cataract 22 (OMIM: 609741). At present, this variant locus is not included in databases such as the gnomAD, while both SIFT and PolyPhen2 deem this locus 'damaging'. Moreover, further validation by Sanger sequencing confirmed that the variant was inherited from the male patient's mother, who also had cataracts. According to ACMG standards and guidelines, the c.226G>A (p.Gly76Arg) variant in the CRYBB3 gene is classified as having 'uncertain significance'. Conclusion: WES identified pathogenic variants in both individuals, suggesting a 25% chance of a healthy child naturally. Third-generation assisted reproductive techniques are recommended to minimize the risk of affected offspring.

Whole exome sequencing identified a novel splice donor site variant in interleukin 2 receptor alpha chain.

Interleukin 2 receptor alpha chain (IL-2Rα or CD25) deficiency (OMIM #606367) is an immune dysregulation disorder segregating in autosomal recessive form. The disease is caused by biallelic variants in the IL-2Rα gene encoding IL-2Rα also known as CD25 protein. IL-2Rα combines with γ and ß chains of interleukin 2 receptor to form a functional interleukin 2 receptor (IL-2R). In the present study, we identified a Pakistani family presenting a unique presentation of IL-2Rα deficiency. Clinical whole exome sequencing revealed a novel splice donor site variant (NM_001378789.1 (NP_001365718); c.64 + 1G > A) in the IL-2Rα gene. American College of Medical Genetics (ACMG) guidelines interpreted the identified variant as likely pathogenic. The IL-2Rα gene mutation usually presents with autoimmunity and immunodeficiency but in our patient, it presents with congenital diarrhea, metabolic crisis, and strong family history of death in infancy due to the similar complications. Her congenital diarrhea is attributed to autoimmunity in the form of autoimmune enteropathy and eczema. The laboratory findings revealed severe metabolic acidosis hypokalemia and elevated lactate and ammonia levels. This is a new presentation of IL-2Rα gene mutation. The present study highlights the importance of clinical whole exome sequencing in the correct diagnosis of congenital disorders. The study will also help clinical geneticists for genetic counseling and prevention of the disease in the affected family.

Clinical whole-exome sequencing reveals a common pathogenic variant in patients with CoQ10 deficiency: An underdiagnosed cause of mitochondriopathy.

BACKGROUND: Primary CoQ deficiency occurs because of the defective biosynthesis of coenzyme Q, one of the key components of the mitochondrial electron transport chain. Patients with this disease present with a myriad of non-specific symptoms and signs, posing a diagnostic challenge. Whole-exome sequencing is vital in the diagnosis of these cases. CASE: Three unrelated cases presenting as either encephalopathy or cardiomyopathy have been diagnosed to harbor a common pathogenic variant c.370G > A in COQ4. COQ4 encodes a key structural component for stabilizing the multienzymatic CoQ biosynthesis complex. This variant is detected only among East and South Asian populations. CONCLUSIONS: Based on the population data and our case series, COQ4-related mitochondriopathy is likely an underrecognized condition. We recommend including the COQ4 c.370G > A variant as a part of the screening process for mitochondriopathy in Chinese populations.

Deoxythymidylate kinase, DTYMK, is a novel gene for mitochondrial DNA depletion syndrome.

BACKGROUND: Mitochondrial DNA depletion syndrome is a group of heterogeneous diseases with non-specific presentation. The common feature is the quantitative depletion of mitochondrial DNA without qualitative defects. Diagnosis of these diseases poses a challenge and whole exome sequencing is often needed for their diagnoses. CASE: Two siblings of a quartet family, presenting with hypotonia, microcephaly and severe intellectual disability, have been diagnosed to harbor two heterozygous variants in trans in the DTYMK gene of the thymidine biosynthesis pathway. Mitochondrial DNA depletion has been demonstrated in silico in the more severe sibling. CONCLUSIONS: We suggest the consideration of incorporating DTYMK as one of the associated genes of mitochondrial DNA depletion syndrome (MDDS). DTYMK may be the missing link in the mitochondrial nucleotide salvage pathway but further characterization and additional evidence would be needed.

UPD16 itself is not a cause of intrauterine growth restriction.

BACKGROUND: The clinical relevance of uniparental disomy (UPD16) for chromosome 16 is currently unclear. METHODS AND RESULT: We performed chromosome microarray analysis on two fetus and their placentas, fluorescence in situ hybridization (FISH) to exclude the hidden chr16 trisomy mosaicism in the fetuses, and clinical whole-exome sequencing to assess for homozygosity mutations of autosomal-recessive diseases. RESULTS: Microarray analysis of two fetuses had UPD16. The membranous placenta of the case 1 had confined placental mosaicism (CPM) for trisomy 16. Clinical whole-exome sequencing on chromosome 16 revealed three potentially pathogenic single nucleotide polymorphisms (SNPs). Gap-polymerase chain reaction (PCR) and MLPA for a-thal deletions demonstrated that case 2 was homozygous for the -SEA deletion. CONCLUSIONS: The poor outcome in these fetuses may be attributed to other factors, the membranous placenta and the -SEA deletion, respectively. Fetal UPD16 itself might be not correlated with intrauterine growth restriction (IUGR) and thus is not the basic cause of IUGR.

Diagnostic odyssey in severe neurodevelopmental disorders: toward clinical whole-exome sequencing as a first-line diagnostic test.

The current standard of care for diagnosis of severe intellectual disability (ID) and epileptic encephalopathy (EE) results in a diagnostic yield of ∼50%. Affected individuals nonetheless undergo multiple clinical evaluations and low-yield laboratory tests often referred to as a 'diagnostic odyssey'. This study was aimed at assessing the utility of clinical whole-exome sequencing (WES) in individuals with undiagnosed and severe forms of ID and EE, and the feasibility of its implementation in routine practice by a small regional genetic center. We performed WES in a cohort of 43 unrelated individuals with undiagnosed ID and/or EE. All individuals had undergone multiple clinical evaluations and diagnostic tests over the years, with no definitive diagnosis. Sequencing data analysis and interpretation were carried out at the local molecular genetics laboratory. The diagnostic rate of WES reached 32.5% (14 out of 43 individuals). Genetic diagnosis had a direct impact on clinical management in four families, including a prenatal diagnostic test in one family. Our data emphasize the clinical utility and feasibility of WES in individuals with undiagnosed forms of ID and EE and highlight the necessity of close collaborations between ordering physicians, molecular geneticists, bioinformaticians and researchers for accurate data interpretation.

Clinical whole-exome sequencing reveals a novel missense pathogenic variant of GNAO1 in a patient with infantile-onset epilepsy.

BACKGROUND: The cause of infantile-onset epilepsy is complex and is not easily recognized clinically, particularly in paediatric patients who present with non-specific neurological signs, no radiological abnormalities and no metabolic changes. CASE: We report a case of infantile-onset epilepsy in a 10-month-old Chinese girl who presented with non-specific neurological signs, no radiological abnormalities and no biochemical disturbances. She first presented at birth with twitching movements and convulsions of an unknown aetiology. Ambulatory EEG showed epileptic rhythmic activities, the presence of asynchrony and runs of sharp waves over the right parietal and central areas. Given the non-specific neurological features and negative structural and biochemical findings, we applied clinical whole-exome sequencing (WES) to determine the underlying aetiology. WES revealed a novel heterozygous missense pathogenic variant, GNAO1:NM_020988.2:c.118G>A; NP_066268.1:p.Gly40Arg. A genetic analysis of the family confirmed the variant identified is a de novo mutation. CONCLUSIONS: Clinical WES can streamline genetic analysis and sort out pathogenic genes in an unbiased approach. GNAO1 is a disease-causing gene for the autosomal dominant form of early infantile epileptic encephalopathy. The novel pathogenic variant identified in this case should contribute to our understanding of the expanding spectrum of infantile-onset epilepsy.