Wat is de opbrengst van verschillende genetische testen bij patiënten met geïsoleerde lip-, kaak en/of palatumschisis?

Advantages and disadvantages of diagnostic tests

Despite no studies were found in which the yield of different diagnostic tests in isolated clefts of the lip and/or palate has been compared, studies do provide information about the different diagnostic genetic tests.

In general, the diagnostic tests could be divided into two types; array-based methods (identifying minor chromosomal anomalies; microdeletions and microduplications) and sequencing methods (identifying gene variants).

Copy number variant analyses (invasive genetic testing)

Large structural alterations of the genome and deletions and duplications of genomic regions termed copy number variations (CNVs), have been studied in CLA/P patients using classical genetic analyses such as FISH, array CGH or, more recently, SNP arrays (Conte, 2016). Also, some studies mention karyotyping; identifying numerical and large chromosomal anomalies. However, small microdeletions and - duplications will be missed by conventional karyotyping.

Maarse (2012) conducted a systematic review to provide a basis for prenatal invasive diagnostics by investigating the prenatal and postnatal prevalence of associated anomalies and chromosomal defects related to CLA/P. They provided a systematic search and used data from the Dutch Oral Cleft Registry to investigate the prevalence of associated anomalies and chromosomal defects both prenatal and postnatal. This review also provided recommendations for diagnostic genetic testing for different types of CLA/P. This review included 20 studies: 3 prenatal, 13 postnatal and 4 combined. This review concluded that array CGH should be considered in case of isolated oral cleft lip on prenatal ultrasound, because the absence of associated anomalies does not exclude the possibility of the presence of an underlying chromosomal defect. The chance of finding chromosomal deficits seems to depend on the type of CLA/P. For example, in presumed isolated CLAP or CP array-based methods are recommended. However, it should be considered that genetic techniques included in the review of Maarse (2012) are nowadays at least (partially) replaced by newer diagnostic genetics tests.

The study of Szczaluba (2015) assessed the utility of array comparative genomic hybridization in 53 Polish newborns with presumed isolated CLA/P. Szczaluba identified 8 unique CNVs in a total of 8/52 patients (15%) using array CGH, including 3 deletions and 5 duplications. The largest rearrangement could be confirmed by karyotyping.

The study of Cao (2016) highly recommends chromosomal microarray analysis (CMA) in prenatal invasive genetic testing, not only for syndromic oral cleft cases but also for non-syndromic cases with soft markers in ultrasound (such as a single umbilical artery). CMA is a variant of array CGH/ SNP array method. The CMA analysis showed an improved detection rate of 15.3% for pathogenic copy-number variants compared with 10.5% for conventional chromosome analysis. Candidate genes including CRKL, AKAP8, SYDE1, BRD4 are worthy of further investigation regarding their role in human palatogenesis.

In the study of Conte (2016) 45 potential candidate genes for deletions and 27 for duplications were found, including several known causative genes for CLA/P, such as SATB2 and MEIS2, and genes that are associated with CLA/P or development of CLA/P. This study found 34 deletions and 24 duplications in genes that have not previously been associated with CLA/P.

In conclusion, the above discussed studies demonstrate genetic testing (incuding karyotyping, FISH, array CGH, and SNP array) can reveal structural and numerical chromosomal anomalies and CNV’s in CLA/P cases. However, the studied population and the genetic techniques differed between the different studies. Even, the resolution of the SNP array analyses can differ between laboratories. Therefore, the outcome of these studies can not be compared.

Next-generation sequencing methods

Next-generation sequencing methods like whole exome sequencing (WES) in family-based designs offer important advantages for identifying gene variants causing complex and heterogeneous disorders like CLA/P (Bureau, 2014, Basha, 2018).

Bureau (2014) conducted a study with WES to search for variants causing CLA/P using affected relatives from 55 multiplex families. Rare single nucleotide variants (SNVs), detected with WES, shared by affected relatives in 348 recognized candidate genes were examined. These 348 candidate genes consisted of 334 autosomal candidate genes for oral clefts (Jugessur, 2009) plus 14 recently confirmed genes and regions yielding genome-wide significance in a meta-analysis (Ludwig, 2012) and a replication study (Beaty, 2013). Bureau (2014) found five novel and potentially damaging SNVs shared by affected distant relatives (CDH1, FGF8, FGFR4, TRPS1, FTCD). One damaging SNV in CDH1 was shared by three affected second cousins from a single family, indicating that this SNV is very unlikely to occur by chance alone.

The study of Basha (2018) also reports on gene variants causing syndromic CL/P using WES. They tested a cohort of 84 individuals diagnosed with non-syndromic CL/P from multiplex families (n=46) to find rare variants in genes causing syndromic CL/P. Patients included in this study had a normal karyotype, the majority had been tested for a 22q11.2 deletion and anIRF6 mutation (Van der Woude syndrome). Patients were diagnosed with bilateral CL/P (n=7), unilateral CL/P (n=16), unilateral cleft lip (CL)(n=8), cleft palate (CP)(n=33), CP-posterior (n=7), submucous cleft palate (SMCP)(n=2), SMCP with bifid uvula (n=2) and velopharyngeal insufficiency (n=9). Genetic variants were analyzed independently in each family and each subject. The following four mutations were found in four different genes in five patients: TP63 (1 family), TXC1 (one family), LRP6 (one family) and GRHL3 (two families). The four mutations co-segregated with the oral cleft in an autosomal dominant manner in all families. This study shows that patients diagnosed with isolated CL/P still can carry a mutated gene causing the CL/P, whole-exome sequencing (WES) / next generation sequencing is therefore recommended when there is a family history with CL/P (Basha, 2018). In 10% of the patients with non-syndromic CL/P with a positive family history a causative Mendelian mutation could be identified. It could be that in an important percentage of the remaining cases, the same genes are involved due to an indel or copy number alteration (CNVs) not easily detectable by WES. Whole genome sequencing will therefore likely be the best choice for diagnostic screens of families with a history of non-syndromic CL/P (Basha, 2018).

WES is increasingly used in the Netherlands in both isolated as syndromic CLA/P. In the Netherlands, however different strategies are used in the cleft centers arout the country. At first, a cleft gene panel analysis can be done. When multiple congenital anomalies are present, analyses of all OMIM morbid genes (based on WES) or a complete trio WES with analyses of the total exome (open exome) can be performed. In trio WES analyses DNA of the parents is included. These tests are offered in a postnatal, but also in a prenatal setting. Gene panels do have differences regarding content, filtering (single versus trio analysis) and classification of variants can differ among centers.

Recently, a retrospective study was performed to obtain insight in the diagnostic yield of cleft gene panel analyses in UMCU (unpublished data). The yield of the gene panel analysis was evaluated from 2015-2020. In this period the gene panel was updated, and the number of genes included in the gene panel is expanded (156 to 195 cleft related genes) (https://www.umcutrecht.nl/nl/next-generation-sequencing-ngs).

In this study 212 CLA/P patients were included. Most genetic tests were done for CP (103, 48.6%), followed by CLA/P (77, 36,3%) and CL (25, 11,8%). In 7 cases (3.3%) cleft type was not defined. All cases underwent cleft gene panel analyses and, in some cases, additional genetic testing (for example ID gene panel, mendeliome or trio WES) was performed.

Cleft gene panel analyses revealed a genetic (syndromic) diagnose, by identification of a pathogenic variant (P) and clinical confirmation of the diagnosis in 11.3 % (24/212) of the cases. Classification of a gene variant of unkown significance (VUS) often required integrated analyses of genetic and clinical data (including pedigree) and regularly subsequent segregation analyses in the family had to be performed.

In (3.8 % (8/212) a causative genetic diagnosis was confirmed by additional genetic testing. Most genetic diagnoses could be confirmed in CP cases (50 %, n=16); versus CL(A)+P) (40.6 %, n=11) and CL (9.4 %, n=3). In one case the cleft type was not defined.

We realize this retrospective inventory probably reflects the outcome of a biased population, because in general practice genetic testing is probably more often performed in children with additional minor or major anomalies or congenital abnormalities and in cases with a positive family history for clefting.

In addition to genepanel analyses and WES, genome wide methylation tests (Episign ©) can be offered to confirm a suspected diagnosis, associated with a specific methylation profile (Aref-Eshghi, 2019; 2020).

Summary

In conclusion, previous studies and the inventory of a Dutch cleft population (n=212) demonstrated broad genetic testing can reveal underlying genetic diagnosis in syndromic clefting but also in cases with (apparent) nonsyndromic isolated clefting. Additional anomalies and family history of clefting might indicate an underlying genetic cause of the cleft. The genetic diagnoses are most frequently identified in cases with CP and CLA/P.

Unfortunately, the current literature does not provide information about the exact yield of different diagnostic genetic tests in an unbiased well subphenotyped cleft population. More specific, the type of clefting in these studies are not uniformly subclassified according the subclassification proposed by Dixon (2011), Mc Bride (2016), Pool (2021) and Vermeij-Keers (2018). Furthermore, the genetic techniques differed between the various available studies.

Further studies, including broad genotype-phenotype evaluation in well subphenotyped population and treatment outcome studies, are necessary to define those children in which genetic testing will have most benefit and determine the optimal strategy of genetic testing in cleft cases.

On the other hand, literature and the retrospective study conducted by UMCU showed that (complex) genetic testing can prevent a long Diagnostic Odyssey. Furthermore, identification of genetic anomalies will allow tailored medical care.

Values and preferences of patients and their parents or guardians

While genetic testing can reveal a genetic cause of CLA/P requiring specific medical care, the advice is to refer all patients with CLA/P (pre- and postnatally), with consent of patient and/or parents, to a department of genetics for genetic counseling. So, they can be informed about the genetic aspects of CLA/P, genetic testing, their recurrence risk, and prenatal diagnostics.

It is of great importance that genetic testing is based on shared decision making and personalized medicine, where clinicians (clinical geneticist, gynaecologist, and/or plastic surgeon) and patients work together to make individual decisions about invasive prenatal diagnostics and the choice of the genetic test. In this process the outcome is based on clinical evidence and expected outcomes with inclusion of patients and/or parents’ preferences and values.

Parents of children with CLA/P and patients with CLA/P should be informed about all options of genetic testing with the pro and cons, including information on incidental findings, and the possibilities of advanced ultrasound research. After consultation it remains to the parents to decide about (further) diagnostic testing.

Professionals should realize that some parents and/or patients do not prefer invasive prenatal testing when a CLA/P is observed in an unborn child and individual considerations of pregnant couples can differ enormously. Also, religion can play a significant role in the decision-making process. However, referral of parents with a fetus with CLA/P to a clinical geneticist is important to discuss the options for further clinical genetic testing and outcomes of these tests.

Guidance of parents in this process is of great importance. Moreover, some parents might be confronted with a severe (lethal) disorder in their child and must decide on continuation or termination of pregnancy (TOP). However, it is also possible that a clinical genetic diagnosis helps in deciding for optimal peri- and postnatal care.

Parents of a child with CLA/P and patients with CLA/P, should also be informed about their recurrence risk and options for future pregnancies, e.g. preimplantation genetic testing (PGD). Furthermore, it is important that parents and future parents are aware of their right to get a second opinion and they can contact patients organizations for more information and empowerment, for example in the Netherlands: www.schisisnederland.nl, http://www.erfelijkheid.nl/(date: May 2021).

Costs/Finances

With clinical application of whole-exome and whole-genome sequencing in CLA/P cases a diagnosis can be made in an early stage, preventing a Diagnostic Odyssey. Also initially in presumed isolated clefts.

The Dutch Health Council indicates that early diagnosis is extremely important. Knowing the genetic origin will lead to better treatment and timely interference for possible future symptoms related to their genetic disorder. Moreover, early diagnosis prevents unnecessary second opinions and additional diagnostic tests and can thus limit the negative consequences for the well-being of the child and the parents. Futher, parents can be informed about risks of recurrence.

The Dutch Health Council states that early genetic testing should be used instead of introducing heel prick screening for untreatable conditions.

On the other hand, an early diagnosis can also be stressful for the parents, while it may take a long time for symptoms to arise. Also, the clinical spectrum of certain diagnoses can be broad, which can create uncertainty.

DNA testing in the Netherlands is totally reimbursed by the basic insurance, however, the mandatory excess will be paid first. It is important that patients are informed about reimbursement rules.

The actual cost-effectiveness of genetic testing cannot be determined until the yield of genetic testing has been determined in an unbiased population, and follow-up studies have been performed to analyse the effect of a specific diagnosis, considering the quality of life of people with CLA/P and their parents.

Acceptance, feasibility and implementation

Nowadays, genetic counselling and testing is increasingly offered to (future) parents of a child with CLA/P in both prenatal and postnatal settings. We have inquired at our eight genetic centres which policy they currently used. The outcome demonstrated all centres, despite variations variations in genetic strategy, discussed and offered genetic testing.

The clinical geneticist is not always present during consultation at the prenatal or postnatal cleft team. When (future) parents opt for genetic testing, they are referred to the genetics department and receive an invitation for a separate consultation. Depending on personal, cultural, and familial considerations and possible consequences, parents can decide which genetic testing will be performed. This decision should be made on accurate information, therefore shared decision making, and counselling is essential, and even more important than diagnostic outcome.

Rationale

1. Aanbeveling voor de arts/professional die schisis vaststelt

The review of Maarse (2012) and the inventory of the outcome of genetic analyses in a Dutch cleft population (unpublished data) demonstrates genetic testing can identify (extreme) rare genetic disorders. Confirmation of a genetic diagnosis can optimize personalized, timely car and improve medical and genetic counselling.

In prenatlly detected CLA/P, advanced ultrasound (GUO) and genetic testing can lead to an underlying diagnosis.

To inform parents of a child with CLA/P or patients with CLA/P about the pros and cons of genetic testing and to promote personalized genetic testing, they should be offered rererral to a genetic department.

2. Aanbeveling voor de klinisch geneticus/medisch specialist

Most studies showed that CLA/P and CP are more often associated with an underlying genetic diagnosis. The presence of additional anomalies and/or a positive family history for orofacial clefting can indicate an underlying genetic cause Certain diagnoses (e.g.CDH1, IRF6, SIX3) are associated with variable expression and reduced penetrance.

Therefore, genetic counseling should entail an extensive medical and family history, specific physical examination and dysmorphological evaluation of index and their parents (see attachment “intakeformulier”, in Dutch).

The strategy of genetic testing in CLA/P should be personalized. The type of cleft, the presence of possible additional minor and major anomalies or abnormalities and the family history must be considered in selecting the best fitting genetic test(s). Moreover, genetic testing can be different in prenatal or postnatal setting. When a cleft is detected prenatally, DNA can be stored from umbilical cord blood for future genetic testing. After birth, blood samples can be taken during surgery. This prevents invasive bloodsampling in the child after birth.

A suggested strategy for the genetic diagnostics is discussed in “Aanbeveling voor optimale strategie voor genetische diagnostiek bij schisis en organisatie van genetische diagnostiek” in this module.

One should realize sequential DNA testing can be associated with increased costs and extensive DNA testing might increase the chance detecting variants of uncertain (or unknown) significance or unsolicited findings. In addition, an important aspect to keep in mind using next-generation sequencing technologies such as WES trio analysis, is that autosomal dominant gene variants with a reduced penetrance (e.g.CDH1, IRF6, SIX3) segregating in a family, might be filtered out in the data analyses.

It is important that parents of a child with CL(A)/P and patients with CL(A)/P are informed about the different aspects of DNA testing, including yield for different cleft type, broad spectrum of cleft syndromes that might be identified, variablility in severity of those syndromes even within families that and the possible identification of variants of uncertain (or unknown) significance. Parents can be referred to informative websites for more information (e.g. Schisis Nederland and Erfocentrum). Parents of a child with CL(A)/P and patients with CL(A)/P should be informed about their recurrence risk and possible choices concerning future pregnancies.

3. Aanbeveling voor optimale strategie voor genetische diagnostiek bij schisis en organisatie van genetische diagnostiek

Genetic testing should be personalized and the type of clefting, additional anamolies and family history should be considered. The strategy, described below, should be considered.

In CLA/P cases, suspected of an underlying chromosomal abnormality, SNP array is the first choice of testing.

In CLA/P cases, without a suspected syndrome diagnosis or chromosomal abnormality, cleft gene panel analyses, including CNV analyses, (WES targeting cleft related genes) should be considered as the first choice of genetic testing. By performing a trio WES, inherited variants with reduced penetrance and/or variable expression segregating in a family might be filtered out and can be missed.

In CLA/P cases with associated anomalies combined SNP array and WES based cleft gene panel analyses (including CNV analyses) should be considered.

In cases with specific dysmorphic features and/or associated congenital anomalies and/or intellectual disability broader genetic testing (for example for ID and/ or congenital anomalies, mendeliome), specific single gene sequencing and additional trio WES) should be considered.

In case of a prenatally diagnosed CLA/P, prenatal invasive diagnostics is offered (chorionic villus test / amniocentesis). Based on the presence of additional ultrasound anomalies at the moment a QF-PCR, SNP array, and/or gene panel analyses and/or trio WES can be performed.

While prenatal genetic testing can be rather complex, for example by revealing (extremely) rare diagnoses with variable phenotypes or unsolicited findings, prenatal counselling should be performed in specialized centres.

Strategy interpretation genetic test results:

A Dutch inventory shows that DNA testing often reveals variants of uncertain (or unknown) significance (unpublised data).

While classification of a gene variant of unkown significance (VUS) requires integrated analyses of genetic, complete phenotypic and clinical data (including pedigree), it is important VUS –es with uncertain classification are discussed by the laboratory specialist and clinical geneticist together. In addition, clinical imput by other medical experts (e.g. gynaecologist, plastic surgeon and paediatrician) and embryologist can be valuable to interprete a variant of unknown significance (VUS).

It is important to inform the laboratory about the eventual conclusion and genetic diagnosis of the patient after follow-up. These data are of great importance for classifying variants in future patients.

A national cleft working group bringing together a multidisciplinary panel of clinical cleft experts (including clinical geneticists, laboratory specialists, plastic surgeons, paediatricians and embryologists) from different expert centres would promote development of specific cleft expertise. Discussing identified genevariants with uncertain pathogenicity, complex patients and novel research knowledge in a regular meeting (every quarter) would expand current knowledge.

Strategy data sharing:

Development of a biobank containing complete cleft data of the dutch population, including genetic and phenotypic data, will help to identify phenotype-genotype correlations and determine pathogenicity of variants of unknown significance (VUS-ses).

It would be desirable to have a national database containing both phenotypic (subclassification) and genetic data (including pathogenic variants as well as variants of unclear significance (VUS)) of CLA/P patients in the Netherlands. The possibility to connect to current national databases of the NVSCA and ICHOM – International Consortium for Health Outcome Measurement) could be explored.

Although, privacy issues and government rules might be limiting factors in implementation of such a database.

Innovation genepanel analysis:

It is also noted that increasing knowledge urge a regular update of the gene panels. Certain genetic causes of clefting can be missed because novel cleft genes are not yet included in the gene panel.

Cost-effectiveness:

For now, the actual benefit and cost-effectiveness of genetic testing cannot be determined until the yield of genetic testing has been investigated in an unbiased population, and follow-up studies have been performed to analyse the effect of a specific diagnosis, considering the quality of life of people with CLA/P and their parents.