1. Disease characteristics

1.1 Name of the disease (synonyms)

See Table 1— column 1 for ‘Name of the disease’.

Table 1 Overview of diseases associated with syndromic microphthalmia.

1.2 OMIM# of the disease

See Table 1—column 2 for ‘OMIM# of the disease’.

1.3 Name of the analysed genes or DNA/chromosome segments and OMIM# of the gene(s)

1.3.1 Core genes (irrespective if being tested by Sanger sequencing or next generation

See Table 1, column 4—‘Associated gene(s)’ and column 5—‘OMIM# of associated gene(s) for all genes and related syndromes.

1.3.2 Additional genes (if tested by next-generation sequencing, including whole exome/genome sequencing and panel sequencing)

See Table 2, column 1—‘Gene’ and column 3—‘OMIM# of gene’.

Table 2 Additional genes associated with syndromic and non-syndromic microphthalmia tested with next-generation sequencing.

1.4 Mutational spectrum

An estimated 33–95% of anophthalmia and microphthalmia cases are observed alongside additional non-ocular systemic malformations, with 20–45% of patients diagnosed with a recognised syndrome [1].

Syndromic microphthalmia may be initially difficult to diagnose from birth dependent on the severity of the phenotype and evolution of other signs and symptoms [2]. Only syndromic microphthalmia will be discussed here, but it is important to note for clarity that severe microphthalmia can be used interchangeably with clinical anophthalmia in the literature (see Clinical Utility Gene Card: Non-Syndromic Microphthalmia [1] and Clinical Utility Gene Card: Anophthalmia [3]). Variants in genes such as ALDH1A3, STRA6, GDF6, and GDF3 may cause either syndromic or apparent non-syndromic microphthalmia and clear distinctions are hard to make when classifying these genes.

The disease has a complex aetiology with chromosomal, monogenic, and environmental causes previously reported [4, 5]. Inheritance patterns include autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, de novo sporadic, and mosaicism. Mitochondrial disease caused by HCCS variants has also been identified as a cause of syndromic microphthalmia, although inheritance is not mitochondrial but rather X-linked dominant [5]. The mutational spectrum spans missense, nonsense, deletions, insertions, splice-site variants and chromosomal deletions, duplications and translocations. The more frequently detected variants are described below as syndromic microphthalmia covers a wide range of diseases [6], some of which are ultra-rare.

SOX2 variants account for 20–40% of autosomal dominant cases and the majority of SOX2 variants are monoallelic loss-of-function de novo sporadic [7,8,9,10,11]. SOX2 is often screened with OTX2 in genetic screening of microphthalmia, anophthalmia and coloboma (MAC) and these variants are jointly causal for 60% of all severe bilateral phenotypes [7]. The deletion (NM_003106.3: c.70_89del, p.(Asp24Argfs*65)) is the most frequently detected variant [7, 12]. The SOX2 polyglycine tract between Gly-19 and Gly-23 is a commonly mutated region found in 20% of SOX2 familial variants [7]. Whole gene deletions have also been found at a rate of 28% in a French patient cohort [12].

Eye-field transcription factors (EFTFs) are essential for early eye development and account for a large proportion of syndromic microphthalmia cases. All identified OTX2 variants are heterozygous, with ~40% of these de novo sporadic [7, 13,14,15,16,17]. The duplication (NM_172337.3:c.106dup, p.(Arg36Profs*52)) and nonsense variants (NM_172337.1:c.289C>T p.(Gln97*) and NM_172377.1:c.295C>T p.(Gln99*)) have been most frequently reported [17]. OTX2 whole gene deletions need to be considered when no point variant is found after screening [2, 17]. It is important to consider the large phenoptypic variability resulting from OTX2 variants as these have also been associated with pattern dystrophy of the retinal pigment epithelium, otocephaly-dysgnathia complex, early onset retinal dystrophy, and pituitary dysfunction [18,19,20]. OTX2 missense variants are also associated with extreme intrafamilial variability, where observed phenotypes ranged from severe multiple congenital defects including microphthalmia to complete non-pentrance [16, 21].

Contiguous gene deletions mapped to the locus 14q22-q23 are variable in size and result in a phenotype comparing with MCOPS5 [22, 23]. These deletions span OTX2 and can include important non-EFTF genes like BMP4 [23]. It is important to also consider the large intrafamilial phenotypical variaiblity linked to 14q22 microdeletions during genetic screening [21]. Whole gene deletion of BMP4, and missense and frameshift variants within the gene causes syndromic microphthalmia with complex phenotypes including hypopituitarism and digital anomalies [5, 14, 15].

PAX6 is a master regulator of ocular development, and variants can result in complex phenotypes. Although most PAX6 variants have been identified in aniridia patients, multiple cases of PAX6 heterozygous variants have been identified in syndromic bilateral microphthalmia [7]. PAX6 variants are primarily missense (NM_000280.4:c.767T>C; p.(Val256Ala); c.474C>T p.(Arg38Trp); c.418G>C p.(Arg19Pro)) or compound heterozygous (NM_000280.4, c.[718C>T]; [112C>T] p.(Arg240*);(Arg38Trp), although biallelic variants are extremely rare and associated with severe microphthalmia, microcephaly, and profound CNS defects [12, 24,25,26,27].

Biallelic heterozygous variants in MITF can cause COMMAD syndrome (coloboma, osteopetrosis, microphthalmia, macrocephaly, albinism, deafness). An autosomal recessive biallelic combination involving at least one dominant-negative variant (NM_000248.3, c.952A>G, p.(Arg318*)) was associated with the disease [28].

Other transcription factor variants are responsible for syndromic microphthalmia phenotypes. Variants in FOXE3 can cause autosomal recessive Anterior Segment Dysgenesis 2 (ASGD2) with bilateral microphthalmia and extraocular manifestations [7, 29]. Associated variants are primarily truncations and biallelic, with the most common variant a homozygous nonsense variant (NM012186.3, c.[720C>A]; p.(Cys240*)) [30].

PITX3 variants associated with CTRCT11 are most commonly heterozygous and homozygous deletions and duplications [31, 32]. Autosomal dominant heterozygous nonsense, frameshift, and missense SALL4 variants have been most frequently identified, while compound heterozygous and de novo variants are less common [33,34,35,36].

Two genes in the retionic acid signalling pathway are associated with syndromic microphthalmia. STRA6 biallelic variants have a higher incidence of syndromic rather than isolated microphthalmia [37]. Most frequently, homozygous or compound heterozygous nonsense and missense variants have been identified in patients with autosomal recessive inheritance [38,39,40,41]. Variants in RARB can cause both autosomal dominant and recessive MCOPS12. Compound heterozygous nonsense (NM_000965.4, c.355C>T, p.(Arg119*)), indel frameshift (NM_000965.4:c.1205_1206dup, p.(Ile403Serfs*15)) and de novo missense variants (NM_000965.4:c.1159C>T, p.(Arg387Cys) and NM_000965.4:c.1159C>A, p.(Arg387Ser)) have been identified [42].

Variants in NAA10 cause MCOPS1, also known as Lenz microphthalmia syndrome, which is an X-linked recessive disorder. An intronic splice-site variant (NG_0.31987.1[NM_003491.3]: c.471 + 2T>A, NC_000023.11[NM_003491.3]: c.471 + 2T>A) has been identified in an affected family, but missense variants are more frequently detected [43, 44]. MCOPS2, also known as oculo-facial-cardio-dental disorder, is an X-linked dominant disorder associated with deletions, insertions, duplications and missense (NM_017745.5:c.254C>T, p.(Pro85Leu)) variants in the BCOR gene [45,46,47]. MCOPS14 is associated with MAB21L2 heterozygous de novo and inherited missense variants [48,49,50].

ASGD7 is associated with homozygous frameshift, missense and nonsense PXDN variants [51]. Heterozygosity for a missense variant (NM_000394.3: c.346C>T, p.(Arg116Cys)) at a highly conserved residue in CRYAA was described in multiple cases of CTRCT9 [52, 53].

Variants in CHD7 and SEMA3E can cause CHARGE syndrome although microphthalmia is only associated with CHD7 variants [54,55,56]. CHD7 de novo nonsense and frameshift variants have been identified [56, 57]. SALL4 variants are linked with Duane-ray radial syndrome.

All data were mined from primary literature or curated genomic and phenotype databases, including GeneReviews (http://www.ncbi.nlm.nih.gov/books/NBK1116/), Online Mendelian Inheritance in Man, OMIM (http://omim.org/), and Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/gene.php?). Novel data should be shared through these databases. They were last accessed on 14th November 2019.

1.5 Analytical validation

The oculome exome gene panel contains a sub panel for microphthalmia, anophthalmia, and ocular coloboma which covers the genes involved in syndromic microphthalmia:


The oculome exome gene panel is important as it compensates for the standard exome capture kits that often miss G-C rich genes including those associated with microphthalmia such as SIX3, PITX3, and SHH.

Sanger sequencing is less frequently used to screen genes but is used for validation of identified variants using genomic DNA from a new extraction. This is because different sample collection and processing methodologies, sequencing chemistries, instruments, enrichment techniques, and data analysis methods between labs can affect NGS assay results [58].

It is important to look for segregation to determine whether the variant is de novo in isolated cases, providing a higher likelihood it affects function. In clinical practice, array comparative genomic hybridisation (aCGH) or multiplex ligation-dependent probe amplification assay may be performed initially to detect copy-number variations (CNVs), such as deletions or duplications. Some molecular service labs also offer fluorescence in situ hybridisation to identify or validate structutral variants such as rearrangements or CNV.

1.6 Estimated frequency of the disease

(Incidence at birth (“birth prevalence”) or population prevalence. If known to be variable between ethnic groups, please report):

A range of studies have estimated the prevalence of microphthalmia between 2 and 23 per 100,000 births [7, 59,60,61,62]. An Israeli study investigating early and late onset foetal microphthalmia in caucasian women, reported a prevalence of 41 per 100,000 pregnancies [63]. Microphthalmia accounts for ~3–11% of all blind children born globally and there is little evidence of higher prevalence in ethinic group populations [64, 65]. However, one prospective study in the UK reported that children of Pakistani descent were at a 3.7 times higher risk of developing a disease on the MAC spectrum than children of white British descent [65, 66].

Between 60 and 80% of cases of microphthalmia are syndromic, however lower incidences were found in a Japanese population with only 31% found with systemic features [61, 67,68,69]. Syndromic involvement is expected at 2.7 times higher in bilateral cases of microphthalmia rather then unilateral cases [68]. Epidemiological data suggest risk factors for microphthalmia are maternal age over 40, multiple births, infants of low birthweight and low gestational age [62, 70, 71].




A. (Differential) diagnostics

B. Predictive Testing

C. Risk assessment in relatives

D. Prenatal

Comment: Because of time constraints such as pregnancy, panel diagnostic, whole-exome sequencing, or whole-genome sequencing (WES/WGS) filtering is preferred if there is a request for prenatal diagnosis (which is rare).

2. Test characteristics


Genotype or disease

A: True positives

C: False negative




B: False positives

D: True negative






Sensitivity: A/(A+C)

Specificity: D/(D+B)




Pos.predict.value: A/(A+B)

Neg.predict.value: D/(C+D)

2.1 Analytical sensitivity

(proportion of positive tests if the genotype is present in the analyte).

2.1.1 If tested by conventional Sanger sequencing

Less than 100%. The proportion is likely ≤100%, because primers may be localised on sequences containing SNVs or rare variants, which results in a preferential amplification of one allele (allele dropout). A supplementary deletion/duplication diagnostic test should be performed for genes with a known proportion of large genomic deletions/duplications as outlined in the section ‘Analytical validation’.

2.1.2 If tested by next-generation sequencing

Less than 100%. The proportion is likely ≤100%, because there might be disease-causing variants in regions that could not be enriched and/ or sequenced owing to suboptimal coverage of some regions of interest depending on enrichment or sequencing strategy. If amplicon-based enrichment strategies are being used, primers may be localised on SNVs or rare variants, which results in preferential amplification of one allele. In patients with a highly suggestive phenotype in whom testing for specific gene alterations proves negative, a supplementary deletion/duplication diagnostic test should be performed for genes with a known proportion of large genomic deletions/duplications as outlined in the section ‘Analytical validation’.

2.2 Analytical specificity

(proportion of negative tests if the genotype is not present).

2.2.1 If tested by conventional Sanger sequencing

Nearly 100%. False positives may at the most arise owing to misinterpretation of rare polymorphic variants.

2.2.2 If tested by next-generation sequencing

Less than 100%. The risk of false positives owing to misinterpretation of rare polymorphic variants may be higher compared with Sanger sequencing because of greater number of analysed genes.

2.3 Clinical sensitivity

(proportion of positive tests if the disease is present).

2.3.1 If tested by conventional Sanger sequencing

Of those patients that undergo genetic testing of known causative genes with Sanger sequencing, those with bilateral severe cases will have a 75% diagnostic rate if aCGH and the coding regions of the following genes are screened; SOX2, OTX2, STRA6, ALDH1A3, PAX6, BMP4 [72].

2.3.2 If tested by next-generation sequencing

Variant detection rates are higher when combined WES with aCGH and high-resolution analysis of intragenic microdeletions and microduplications are performed. WGS may aid in the detection of variants affecting function in the promotor region, introns and other non-coding regulatory elements, and provide better coverage than exome sequencing. Regulatory element disruption in microphthalmia remains largely uncharacterised.

2.4 Clinical specificity

(proportion of negative tests if the disease is not present)

The clinical specificity can be dependent on variable factors such as age or family history. In such cases a general statement should be given, even if a quantification can only be made case by case.

2.4.1 If tested by conventional Sanger sequencing

Unknown, however, if microphthalmia is not present, it is unlikely that a positive test will be detected.

2.4.2 If tested by next-generation sequencing

See section ‘If tested by conventional Sanger sequencing’.

2.5 Positive clinical predictive value

(life time risk to develop the disease if the test is positive).

This is a congenital anomaly of the eye, therefore patients will be born with this defect, therefore nearly 100%, however variable expressivity has been noted and the severity of the phenotype may lead to a delay in clinical diagnosis. Visual acuity may be unaffected, or only slightly affected in patients with less severe forms of disease.

2.6 Negative clinical predictive value

(Probability not to develop the disease if the test is negative).

Assume an increased risk based on family history for a non-affected person. Allelic and locus heterogeneity may need to be considered.

Index case in that family had been tested: Nearly 100%. If the non-affected relative is not a carrier of an identified disease-causing variant, they have no increased risk, except a small risk related to the prevalence in the general population.

Index case in that family had not been tested: Unknown.

3. Clinical utility

3.1 (Differential) diagnostics: the tested person is clinically affected

(To be answered if in 1.9 “A” was marked)

3.1.1 Can a diagnosis be made other than through a genetic test?


□(continue with 3.1.4)














Other (please describe)


3.1.2 Describe the burden of alternative diagnostic methods to the patient

The definition of microphthalmia is heterogenous, however, an axial length (AL) of <21 mm in adults and <19 mm in a 1-year-old is most widely accepted as it represents a reduction of 2 SD or more below normal. Microphthalmia can be detected using ultrasound, or less frequently through fetal MRI, during the second trimester, or after birth in conjunction with clinical examination. Microphthalmia can be associated with microcornea, which is defined as a horizontal diameter <9 mm in a newborn and <10 mm in children 2 years and older.

This diagnosis can depend on the phenotypic severity, but it can be made relatively easily and cost-effectively, confirmed by axial length measures through ultrasound biomicroscopy. MRI brain and orbit imaging is recommended to delineate severe microphthalmia from clinical anophthalmia, determine integrity of the globe, optic nerve, optic chiasm and any associated brain anomalies [64, 73]. If this anomaly is found, children should be investigated within a multidisciplinary team, including paediatricians and clinical geneticists, to ensure it is not syndromic. Further monitoring may be required as systemic manifestations may present later in childhood.

3.1.3 How is the cost effectiveness of alternative diagnostic methods to be judged?

Clinical examination and ultrasound imaging provides a cost-effective diagnosis.

3.1.4 Will disease management be influenced by the result of a genetic test?



Therapy (please describe)

Prognosis (please describe)

Yes, if a variant in a gene is associated with a syndrome, it may lead to a search for systemic involvement to prevent co-morbidity and maximise function, for example, patients with CHARGE syndrome (CHD7) suffer from a range of multisystem abnormalities including heart defects, endocrine deficiencies, and sensorineural deafness, hence early diagnosis will lead to prompt supportive treatment, having longterm health economic benefits.

Management (please describe)

Microphthalmia should be managed by specialists with expertise in this condition. If visual function is present, this must be maximised by correcting refractive error and preventing amblyopia. Those with poor vision must be supported by low visual aids and training. MRI imaging of the brain is required to rule out any associated midline neurological or pituitary defects. Referral to neurology and endocrinology may be indicated. If a child has a non-seeing eye, cosmesis can be addressed by fitting cosmetic shells or contact lenses. Socket expansion in severe microphthalmia may be indicated using enlarging conformers. Although genetic counselling can be challenging owing to the extensive range of disease-associated genes and variable expressivity, appropriate counselling can be applied if the mode of inheritance is identified and should be offered to the family

3.2 Predictive setting: the tested person is clinically unaffected but carries an increased risk based on family history

(To be answered if in 1.9 “B” was marked).

3.2.1 Will the result of a genetic test influence lifestyle and prevention?

If the test result is positive (please describe): Microphthalmia is a congenital eye anomaly, therefore if it is not clinically present at birth then this will not develop later in life. However, if an individual is clinically unaffected but is a carrier, this information will inform family planning if the mode of inheritance can be identified.

If the test result is negative (please describe): If the clinically unaffected person has a negative test result, no further follow-up is required. The result will inform family planning.

3.2.2 Which options in view of lifestyle and prevention does a person at-risk have if no genetic test has been done (please describe)?

Vision can be variably affected in microphthalmic patients depending on the severity of the anomaly and other complex ocular features. This may limit schooling and professions that require perfect vision. Hence, a clinically confirmed diagnosis can help to provide guidance on career choice.

Syndromic microphthalmia is phenotypically heterogenous yet can affect almost all systems in the human body. As such, this may severely impact the quality of life of a patient and their ability to participate in society without the need for both physical and medical assistance. These syndromes can impact on education and career choice, personal relationship development, and participation in many activities, including basic human functions. Infant mortality is also an unfortunate circumstance of many syndromes.

3.3 Genetic risk assessment in family members of a diseased person

(To be answered if in 1.9 “C” was marked).

3.3.1 Does the result of a genetic test resolve the genetic situation in that family?

Yes, although there may be variable expressivity, non-penetrance and germline mosaicism, which will complicate the advice that can be given.

3.3.2 Can a genetic test in the index patient save genetic or other tests in family members?

If a disease-causing variant is identified in the index patient, family members can be tested, but complete clinical examination is also helpful. Test negative family members, who are clinically unaffected, do not need any further investigation or monitoring.

3.3.3 Does a positive genetic test result in the index patient enable a predictive test in a family member?

Yes, if the variant is known.

3.4 Prenatal diagnosis

(To be answered if in 1.9 “D” was marked).

3.4.1 Does a positive genetic test result in the index patient enable a prenatal diagnosis?

Yes. Germline mosaicism and/or variable penetrance render the prediction of recurrence risk difficult in monogenic microphthalmic individuals, however, molecular genetic studies for known variants are possible on amniotic fluid foetal cells withdrawn after 14 weeks of gestation or on chronic villus sampling at 10–12 weeks gestation, and can facilitate the diagnosis of microphthalmia. In addition, transvaginal ultrasonography enables the detection of microphthalmia from 12 weeks gestation [74]; the maximal coronal or axial planes of the orbit are measured, and compared with established eye growth charts [63].

Non-invasive prenatal diagnosis of aneuploidies and some monogenic disorders can be achieved by molecular testing of cell-free foetal DNA (cffDNA) from maternal plasma [75,76,77,78,79,80]. While non-invasive prenatal diagnosis of microphthalmia is not currently available, the reduced risk of non-invasive, early screening (7–9 weeks), makes cffDNA a valuable emerging tool for diagnosis of genetic disorders, particularly for patients with known risk [78, 80].

4. If applicable, further consequences of testing

Please assume that the result of a genetic test has no immediate medical consequences. Is there any evidence that a genetic test is nevertheless useful for the patient or his/her relatives? (Please describe).

Beyond potentially defining recurrence risk information dependent on the cause and mode of inheritance, identifying the genetic aetiology may guide genetic counselling. It also contributes to the classification of syndromic or non-syndromic microphthalmia, thereby guiding any subsequent investigations for affected patients. Preimplantation diagnosis may be an option for bilateral severe microphthalmia.