A missense mutation in ALDH1A3 causes isolated microphthalmia/anophthalmia in nine individuals from an inbred Muslim kindred

Nine affected individuals with isolated anophthalmia/microphthalmia from a large Muslim-inbred kindred were investigated. Assuming autosomal-recessive mode of inheritance, whole-genome linkage analysis, on DNA samples from four affected individuals, was undertaken. Homozygosity mapping techniques were employed and a 1.5-Mbp region, homozygous in all affected individuals, was delineated. The region contained nine genes, one of which, aldehyde dehydrogenase 1 (ALDH1A3), was a clear candidate. This gene seems to encode a key enzyme in the formation of a retinoic-acid gradient along the dorsoventral axis during an early eye development and the development of the olfactory system. Sanger sequence analysis revealed a missense mutation, causing a substitution of valine (Val) to methionine (Met) at position 71. Analyzing the p.Val71Met missense mutation using standard open access software (MutationTaster online, PolyPhen, SIFT/PROVEAN) predicts this variant to be damaging. Enzymatic activity, studied in vitro, showed no changes between the mutated and the wild-type ALDH1A3 protein.     

Isolated hypogonadotropic hypogonadism with SOX2 mutation and anophthalmia/microphthalmia in offspring

Isolated hypogonadotropic hypogonadism (IHH) is a genetically heterogeneous condition in which patients frequently require assisted reproduction to achieve fertility. In patients with IHH who are otherwise well, no particular increased risk of congenital anomalies in the resultant offspring has been highlighted. Heterozygous mutations in SOX2 are the commonest single-gene cause of anophthalmia/microphthalmia (A/M) and sometimes result in pituitary abnormalities. We report a family with a novel frameshift mutation in the SOX2 transactivation domain, p.Gly280AlafsX91, resulting in bilateral anophthalmia and subtle endocrinological abnormalities in a male sibling, and unilateral microphthalmia in a female sibling. The mutation is present in their mother who has IHH, but has no eye disorders or other anomalies. She underwent assisted reproduction to achieve fertility. This report has important implications for the evaluation of patients with IHH, particularly in the setting of planned infertility treatment.     

Molecular findings and clinical data in a cohort of 150 patients with anophthalmia/microphthalmia

Anophthalmia and microphthalmia (AM) are the most severe malformations of the eye, corresponding respectively to reduced size or absent ocular globe. Wide genetic heterogeneity has been reported and different genes have been demonstrated to be causative of syndromic and non‐syndromic forms of AM. We screened seven AM genes [GDF6 (growth differentiation factor 6), FOXE3 (forkhead box E3), OTX2 (orthodenticle protein homolog 2), PAX6 (paired box 6), RAX (retina and anterior neural fold homeobox), SOX2 (SRY sex determining region Y‐box 2), and VSX2 (visual system homeobox 2 gene)] in a cohort of 150 patients with isolated or syndromic AM. The causative genetic defect was identified in 21% of the patients (32/150). Point mutations were identified by direct sequencing of these genes in 25 patients (13 in SOX2, 4 in RAX, 3 in OTX2, 2 in FOXE3, 1 in VSX2, 1 in PAX6, and 1 in GDF6). In addition eight gene deletions (five SOX2, two OTX2 and one RAX) were identified using a semi‐quantitative multiplex polymerase chain reaction (PCR) [quantitative multiplex PCR amplification of short fluorescent fragments (QMPSF)]. The causative genetic defect was identified in 21% of the patients. This result contributes to our knowledge of the molecular basis of AM, and will facilitate accurate genetic counselling.     

Combination of diaphragmatic eventration and microphthalmia/anophthalmia is probably nonrandom

Two sporadic cases of eventration of the diaphragm are reported; one had bilateral colobomatous microphthalmia and the other had anophthalmia. Absence of polydactyly and presence of eventration rather than diaphragmatic hernia helped to exclude Fryns syndrome. These cases together with published cases with overlapping features support the thesis that this combination of defects is nonrandom and of heterogeneous cause. Some cases are due to a pleiotropic gene defect. In other cases, a polytypic developmental field involving an unknown developmental cascade common to the eye and diaphragm may provide a basis for the combination. © 2002 Wiley‐Liss, Inc.     

Non‐syndromic anophthalmia/microphthalmia can be caused by a PORCN variant inherited in X‐linked recessive manner

Anophthalmia and microphthalmia (A/M) represent severe developmental ocular malformations, corresponding, respectively, to absent eyeball or reduced size of the eye. Both anophthalmia and microphthalmia may occur in isolation or as part of a syndrome. Genetic heterogeneity has been demonstrated, and many genes have been reported to be associated with A/M. The advances in high‐throughput sequencing have proven highly effective in defining the molecular basis of A/M. Nevertheless, there are still many patients with unsolved genetic background of the disease, who pose a significant challenge in the molecular diagnostics of A/M. Here we describe a family, with three males affected with the non‐syndromic A/M. Whole exome‐sequencing performed in Patient 1, revealed the presence of a novel probably pathogenic variant c.734A>G, (p.[Tyr245Cys]) in the PORCN gene. Pedigree analysis and segregation of the identified variant in the family confirmed the X‐linked recessive pattern of inheritance. This is the first report of X‐linked recessive non‐syndromic A/M. Until now, pathogenic variants in the PORCN gene have been identified in the patients with Goltz syndrome, but they were inherited in X‐linked dominant mode. The ocular phenotype is the only finding observed in the patients, which allows to exclude the diagnosis of Goltz syndrome.     

Exome sequencing in developmental eye disease leads to identification of causal variants in GJA8, CRYGC,PAX6 and CYP1B1

Developmental eye diseases, including cataract/microcornea, Peters anomaly and coloboma/microphthalmia/anophthalmia, are caused by mutations encoding many different signalling and structural proteins in the developing eye. All modes of Mendelian inheritance occur and many are sporadic cases, so provision of accurate recurrence risk information for families and affected individuals is highly challenging. Extreme genetic heterogeneity renders testing for all known disease genes clinically unavailable with traditional methods. We used whole-exome sequencing in 11 unrelated developmental eye disease patients, as it provides a strategy for assessment of multiple disease genes simultaneously. We identified five causative variants in four patients in four different disease genes, GJA8, CRYGC, PAX6 and CYP1B1. This detection rate (36%) is high for a group of patients where clinical testing is frequently not undertaken due to lack of availability and cost. The results affected clinical management in all cases. These variants were detected in the cataract/microcornea and Peters anomaly patients. In two patients with coloboma/microphthalmia, variants in ABCB6 and GDF3 were identified with incomplete penetrance, highlighting the complex inheritance pattern associated with this phenotype. In the coloboma/microphthalmia patients, four other variants were identified in CYP1B1, and CYP1B1 emerged as a candidate gene to be considered as a modifier in coloboma/microphthalmia.     

Mutation analysis of the STRA6 gene in isolated and non‐isolated anophthalmia/microphthalmia

PDAC syndrome [Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia/microphthalmia (A/M) and Cardiac Defect] is a condition associated with recessive mutations in the STRA6 gene in some of these patients. Recently, cases with isolated anophthalmia have been associated with STRA6 mutations. To determine the minimal findings associated with STRA6 mutations, we performed mutation analysis of the STRA6 gene in 28 cases with anophthalmia. In 7 of the cases the anophthalmia was isolated, in 14 cases it was associated with one of the major features included in PDAC and 7 had other abnormalities. Mutations were identified in two individuals: one with bilateral anophthalmia and some features included in PDAC, who was a compound heterozygote for a missense mutation and a large intragenic deletion, and the second case with all the major features of PDAC and who had a homozygous splicing mutation. This study suggests that STRA6 mutations are more likely to be identified in individuals with A/M and other abnormalities included in the PDAC spectrum, rather than in isolated A/M cases.     

Exome sequencing identifies novel missense and deletion variants in RTN4IP1 associated with optic atrophy,global developmental delay,epilepsy, ataxia,and choreoathetosis

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extraocular manifestations. Optic atrophy‐10 (OPA10) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1, which encodes reticulon 4 interacting protein 1 (RTN4IP1), a mitochondrial ubiquinol oxydo‐reductase. Here we report novel compound heterozygous mutations in RTN4IP1 in a male proband with developmental delay, epilepsy, optic atrophy, ataxia, and choreoathetosis. Workup was notable for transiently elevated lactate and lactate‐to‐pyruvate ratio, brain magnetic resonance imaging with optic atrophy and T2 signal abnormalities, and a nondiagnostic initial genetic workup, including chromosomal microarray and mitochondrial panel testing. Exome sequencing identified a paternally inherited missense variant (c.263T>G, p.Val88Gly) predicted to be deleterious and a maternally inherited deletion encompassing RTN4IP1. To our knowledge, this is the first report of a non‐single nucleotide pathogenic variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between “syndromic” cases and severe RTN4IP1 mutations, and the importance of nonbiased genetic testing, such as ES, to analyze multiple genes and variants types, in patients suspected of having genetic disease.     

Exome sequencing in patients with microphthalmia,anophthalmia, and coloboma (MAC) from a consanguineous population

Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.     

Exome sequencing of 22 genes using tissue from patients with biliary tract cancer

Biliary tract cancers (BTC) are a rare heterogeneous disease group with a dismal prognosis and limited treatment options. The mutational landscape consists of genetic aberrations both shared by and characteristic for anatomical location. Here, we present exome sequencing data on 22 genes from a phase 2 trial using a clinically validated panel used in patients with colorectal cancer. A total of 56 patients were included in a one-armed phase 2 trial investigating the treatment combination of capecitabine, gemcitabine, oxaliplatin, and cetuximab. Tissue DNA yield and quality allowed analysis of 30 patients on our panel including 22 genes. ARID1A (33%) and TP53 (33%) were found to be most frequently mutated followed by KRAS mutations found in 20% of the patients. Mutational aberrations in ARID1A were found more prevalent than expected, whereas TP53 and KRAS were in concordance with earlier reported data. Mutation in CTNNB1 was significantly associated with poor prognosis. Our panel is clinically validated and suitable for a high volume of samples to detect mutations in patients with BTC. However, it is reasonable to assume that the clinical utility could be optimized in this patient group by extending the panel to include BTC specific mutations with potential therapeutic consequences such as IDH1/2, FGFR fusions, ERBB3, and BRCA1/2.     

一个先天性小眼畸形家系的全外显子组测序分析及产前诊断

目的分析1个先天性小眼畸形家系的临床表型及遗传学病因。方法应用高通量测序技术对先证者及其父母进行全外显子组测序,筛选候选致病位点,对其家系进行Sanger测序验证,并通过羊水穿刺和Sanger测序为先证者母亲提供产前诊断。结果全外显子组测序和Sanger测序发现家系中的3例患者均携带OTX2基因c.289C>T(p.R97*)杂合变异,先证者母亲亦携带该变异,但无小眼畸形。先证者的父亲、舅母和胎儿未携带上述变异。结论OTX2基因c.289C>T(p.R97*)杂合变异很可能是该家系的发病原因。上述诊断将有助于该家系的遗传咨询和产前诊断。     

Mutations in ALDH1A3 Represent a Frequent Cause of Microphthalmia/Anophthalmia in Consanguineous Families

Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2.     

Identification of Two Homozygous Sequence Variants in the COL7A1 Gene Underlying Dystrophic Epidermolysis Bullosa by Whole‐Exome Analysis in a Consanguineous Family

Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder with variable severity and heterogeneous genetic involvement. Diagnostic approaches for this condition include clinical evaluations and electron microscopy of patients’ skin biopsies, followed by Sanger sequencing (SS) of a large gene (118 exons) that encodes the alpha chain of type VII collagen (COL7A1) located on Chromosome 3p21.1. However, the use of SS may hinder diagnostic efficiency and lead to delays because it is costly and time‐consuming. We evaluated a 5‐generation consanguineous family with 3 affected individuals presenting the severe generalised DEB phenotype. Human whole‐exome sequencing (WES) revealed 2 homozygous sequence variants: the previously reported variant p.Arg578* in exon 13 and a novel variant p.Arg2063Gln in exon 74 of the COL7A1 gene. Validation by SS, performed on all family members, confirmed the cosegregation of the 2 variants with the disease phenotype. To the best of our knowledge, 2 homozygous COL7A1 variants have never been simultaneously reported in DEB patients; however, the upstream protein truncation variant is more likely to be disease‐causing than the novel missense variant. WES can be used as an efficient molecular diagnostic tool for evaluating autosomal recessive forms of DEB.     

Exome sequencing identifies homozygous variants in MBOAT7 associated with neurodevelopmental disorder

Intellectual disability (ID) is a large group of neurodevelopmental disorders characterized by a congenital limitation in intellectual functioning (reasoning, learning, and problem solving), adaptive behavior (conceptual, social, and practical skills), originated at birth and manifested before the age of 18. By whole exome sequencing of five consanguineous Pakistani families presenting hallmark features of ID, global developmental delay, aggressive and self-injurious behaviors, microcephaly, febrile seizures and facial dysmorphic features, we identified three novel homozygous missense variants (NM_024298.5: c.588G > T; p.Trp196Cys, c.736 T > C; p.Tyr246His and c.524A > C; p. Asp175Ala) and one rare homozygous in-frame deletion variant (c.758_778del;p.Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7) gene previously associated with autosomal recessive neurodevelopmental disorder. The segregation of the variants was validated by Sanger sequencing in all family members. In silico homology modeling of wild-type and mutated proteins revealed substantial changes in the structure of both proteins, indicating a possible effect on function. The identification and validation of new pathogenic MBOAT7 variants in five cases of autosomal recessive ID further highlight the importance of this genes in proper brain function and development.     

Exome sequencing reveals NAA15 and PUF60 as candidate genes associated with intellectual disability

Intellectual Disability (ID) is a clinically heterogeneous condition that affects 2–3% of population worldwide. In recent years, exome sequencing has been a successful strategy for studies of genetic causes of ID, providing a growing list of both candidate and validated ID genes. In this study, exome sequencing was performed on 28 ID patients in 27 patient‐parent trios with the aim to identify de novo variants (DNVs) in known and novel ID associated genes. We report the identification of 25 DNVs out of which five were classified as pathogenic or likely pathogenic. Among these, a two base pair deletion was identified in the PUF60 gene, which is one of three genes in the critical region of the 8q24.3 microdeletion syndrome (Verheij syndrome). Our result adds to the growing evidence that PUF60 is responsible for the majority of the symptoms reported for carriers of a microdeletion across this region. We also report variants in several genes previously not associated with ID, including a de novo missense variant in NAA15. We highlight NAA15 as a novel candidate ID gene based on the vital role of NAA15 in the generation and differentiation of neurons in neonatal brain, the fact that the gene is highly intolerant to loss of function and coding variation, and previously reported DNVs in neurodevelopmental disorders.