Genetic spectrum of prenatally diagnosed skeletal dysplasias in a Finnish patient cohort

Abstract Objective This retrospective cohort study aims to describe the genetic spectrum of fetal skeletal dysplasias detected in a Finnish patient cohort and the diagnostic yield of various analysis methods used. Method A total of 121 pregnancies with prenatally suspected or diagnosed skeletal dysplasia were analyzed between 2013 and 2020. Clinical details and findings from genetic testing were collected. Results Abnormal ultrasound triggered further testing in most cases. However, there were several cases with increased nuchal translucency and/or abnormal risk ratio in the first trimester combined screening as the initial finding. Further genetic testing was performed in 84/121 (69.4%) cases. A genetic diagnosis was confirmed in 36/84 (42.9%) cases. Half of the identified cases could be attributed to a founder mutation specific to the Finnish Disease Heritage, whereas the other half consisted of a variety of other genetic defects. Conclusion In our patient cohort, the overall genetic spectrum of prenatally diagnosed skeletal dysplasias was wide. However, the impact of Finnish founder mutations was considerable, suggesting that the genetic spectrum of skeletal dysplasias may differ significantly between populations. This should be taken into consideration during the diagnostic process especially as initial ultrasound findings may be unspecific and the interpretation of ultrasound features is usually difficult.


| INTRODUCTION
Skeletal dysplasias are rare inherited disorders that disrupt the normal bone formation, growth, density, or mineralization occurring in 1/5000 births. 1 The FDH contains 36 monogenic diseases including skeletal dysplasias such as diastrophic dysplasia and cartilage-hair hypoplasia. Albeit overrepresented, the disorders are still rare even in the Finnish population. [4][5][6][7][8][9][10][11] On the other hand, increasing immigration diversifies the spectrum of skeletal dysplasias observed in the Finnish population.
The most severe forms of skeletal dysplasias are associated with significant perinatal morbidity and mortality. 12 According to the Finnish law, the termination of pregnancy (TOP) is allowed before 20 pregnancy weeks if there is considerable suspicion of a severe fetal structural or/and genetic abnormality and before 24 weeks of pregnancy if such an abnormality is diagnosed with a reliable method.
Permission of the National Supervisory Authority for Welfare and Health (Valvira) is required for termination. Diagnosis of prenatalonset skeletal dysplasias mostly relies on ultrasound findings and is supported by MRI and/or genetic analysis from a chorionic villus biopsy or an amniotic fluid sample. Postnatally, additional evidence may be achieved from radiographs and in case of termination or fetal demise, autopsy findings. [13][14][15] Diagnostic accuracy of prenatal US alone is only 40%-68% 14,16,17 and other supportive methods are therefore needed.
When fetal skeletal dysplasia is suspected, the limited time window for genetic diagnostics has been a challenge. Before the emergence of NGS-based methods, the diagnostic possibilities were limited to analyses of some common disease-causing variants such as the recurrent FGFR3 mutations in achondroplasia and thanatophoric dysplasia or the Finnish founder mutations for diastrophic dysplasia and cartilage-hair hypoplasia. Next generation sequencing (NGS) allows simultaneous analysis of several genes in a shorter timeline, considerably expanding the prenatal diagnostic options. This is especially important as other genetic diseases such as arthrogryposis, some connective tissue disorders, and neurological diseases limiting fetal movements may present with findings closely resembling skeletal dysplasias during the fetal period. 18 Most genetic analyses are time-consuming and costly. In addition, ultrasound findings suggesting skeletal dysplasia may not be detected early enough for the specific diagnosis to be available within the legal time window for the termination of pregnancy. Interpretation of the genetic analysis may also be a challenge. Genome-wide analyses such as whole exome sequencing or comparative genomic hybridization (array-CGH) may also result in secondary findings raising ethical issues. 19 The aim of this study was to analyze the genetic spectrum of skeletal dysplasias detected prenatally in a tertiary Finnish referral university hospital and the diagnostic yield of various analysis methods used in obtaining the diagnoses. decision-making. Non-invasive prenatal testing for common trisomies is offered as an alternative to invasive testing in cases with a positive combined screening result and/or slightly increased NT (up to 3.4 mm). In case of fetal demise or termination of pregnancy without prior molecular genetic diagnosis, post-mortem investigations (including fetal autopsy, imaging, and genetic analyses) may be performed to confirm the diagnosis and estimate the recurrence risk in future pregnancies. Parental carrier screening for Finnish disease heritage is not offered at the moment but targeted testing can be offered for close relatives of known carriers. The diagnostic path is depicted in Figure 1.

| MATERIALS AND METHODS
Laboratory methods included trisomy PCR, chromosomal analysis, and array-CGH, which were performed in our own laboratory (HUSLAB). Trisomy PCR with Aneufast Multiplex QF-PCR Kit was used to detect trisomies, 13,18,21 sex chromosome abnormalities, and triploidies. Array-CGH was performed using the Agilent Human there were previous pregnancies with skeletal disease, and in 2/26 cases (1.6%), a parent was known to be a carrier of an autosomal dominantly inherited skeletal disorder.
In 22/121 (18.2%) pregnancies, no invasive fetal testing was offered. These cases included either normal findings in repeated ultrasounds or fetuses with isolated or likely nongenetic abnormalities, such as clubfoot or amniotic band syndrome. In 15 pregnancies, the risk for a previously known familial mutation was ruled out by parental carrier testing and no further analyses were needed.
Analysis for aneuploidies either by karyotyping or rapid testing for common trisomies with real-time quantitative PCR followed by array-CGH was performed as a first-tier test for all fetuses as aneuploidy has been reported to be associated with abnormal skeletal development. 20 In this cohort, three cases of trisomy 18, one case of trisomy 13, and one case of mosaic trisomy 16 combined with a balanced translocation (X; 7) were diagnosed. No cases with a copy number variant explaining a skeletal abnormality were detected. Of the 36 genetically confirmed cases, seven had abnormal first trimester screening results (≥1:250). Nuchal translucency (NT ≥ 3.0 mm) was increased in seven cases. In three cases, both NT and screening results were abnormal.
In the cases associated with the FDH, the diagnosis was reached in 72.2% (n = 13) by targeted testing of the Finnish founder mutation, in two cases (11.1%) with a comprehensive gene panel, in two cases (11.1%) with sequencing of a single gene, and in one case (5.6%) with WES. In the remaining cases (n = 18), gene panels yielded a diagnosis in 55.6% (n = 10). In three cases (16.7%), the diagnoses were obtained with a single gene test, two cases (11.1%) with familial variant testing, and two cases (11.1%) with WES. In one case (5.6%), the result was obtained in a research project. This result was verified in our own laboratory. Figure 3 presents genetic testing methods leading to a confirmed diagnosis.
Of the pregnancies with a genetic diagnosis, nine (25%) led to a live birth while the rest were terminated. At the time of termination, a genetically confirmed diagnosis was available only in five cases (13.9%). Regarding the nine nonterminated pregnancies, in two cases, the diagnosis was reached after the legal time limit for termination had passed at 27 +5 and 30 weeks. In one case, the OI diagnosis of the expectant mother was confirmed during pregnancy, but further fetal testing was not performed until birth. In the remaining 6 cases, the abnormalities were observed in the second trimester ultrasound. In a case of type 1 brachydactyly, termination was not indicated based on the diagnosis. In one case of CHH, one case of spondyloepiphyseal dysplasia, two severe cases of OI, and one case of GRACILE, the family decided to continue with the pregnancy despite the diagnosis.
The course of pregnancies is depicted in Figure 4. The phenotype of the live-born children was in all nine cases in line with the prenatal genetic diagnosis.
Furthermore, there were 25 cases with suspected skeletal disease in which a genetic diagnosis was not obtained during the study period. This group was very heterogeneous. In most of these cases (12/25), fetuses had multiple anomalies with skeletal involvement.
In 7/25 cases, only one skeletal abnormality such as fibular hemimelia was observed. Four fetuses were suspected of having arthrogryposis. Two cases had ultrasound findings consistent with a particular type of skeletal dysplasia, but a genetic diagnosis could not be confirmed.
In two cases, a variant of unknown significance (VUS) was found with genetic testing. In the first case, the fetus had radius aplasia and an absent thumb. The gene panel found a heterozygous variant in the CENPJ gene classified as VUS. The pathogenic CENPJ gene variants have mainly been reported in individuals with microcephaly and its possible association with the fetal findings in this case remains unclear. In the second case, short long bones (humeri, femuri, tibiae, and fibulae) were observed in the second trimester US. A heterozygous variant classified as VUS in the FLNB gene was found with a gene panel. The same variant was also present in the asymptomatic mother. Increased NT was one of the primary findings in eight cases with genetically confirmed skeletal dysplasia. Interestingly, in one of these cases, the primary finding was increased nuchal translucency only. In addition, there were three cases with increased NT combined with abnormal US findings. Two cases had elevated NT and positive family history for skeletal abnormalities. Our observations were therefore consistent with previous reports describing increased NT in association with skeletal dysplasias. 21,22 In one case, with diastrophic dysplasia, with very typical US findings, the specific diagnosis could be obtained as early as at 13 pregnancy weeks. In most cases, however, the diagnostic journey was slow and distinguishing between lethal and nonlethal conditions proved difficult. We have not received any information about misdiagnoses of skeletal dysplasias. However, ultrasound findings of some skeletal diseases, such as hypochondroplasia, may appear later in pregnancy (or after birth) and prenatal diagnosis might therefore not be possible.

| DISCUSSION
Termination of the pregnancy due to skeletal dysplasia (n = 27) took place between 12 +5 and 24 +0 gestational weeks. Almost half of the terminations (n = 13) were performed between weeks 20 +4 and 24 +0 , which marks the legal limit for abortion in Finland. This highlights the fact that in many cases, the suspicion of skeletal dysplasia arises rather late in pregnancy and depending on the local protocol, it may be challenging to obtain the exact diagnosis within the time limit The other 50% of those with a genetically confirmed diagnosis of skeletal dysplasia included relatively common diagnoses such as osteogenesis imperfecta and thanatophoric dysplasia as well as very rare entities that are challenging to diagnose with ultrasound alone.
In the majority of these cases, the diagnosis was obtained either with a comprehensive gene panel or WES. It is also meaningful to observe the relationship between actual skeletal dysplasias and other conditions with a phenotype mimicking them such as lethal congenital contracture syndrome (LCCS) and Freeman-Sheldon syndrome. [28][29][30] The strength of the study was the fact that it was performed in a large tertiary center. Furthermore, the diagnostic process is free of charge and therefore, financial factors do not restrict participation in further prenatal testing and leading to an overall high participation in diagnostic evaluations. We therefore have reason to believe that the results are representative and reliably portray the genetic spectrum of the population. We can conclude that the spectrum of skeletal dysplasias in Finland is largely shaped by the FDH but also globally appearing dysplasias are seen along with rare diagnoses. The spec- It is also noteworthy to examine the cases in which a genetic diagnosis was not obtained. It is undetermined whether these cases represent genetic syndromes yet to be discovered or if the phenotypes are the result of various genetic and nongenetic factors. It is also possible that the evolved testing methods could have led to accurate diagnoses of the unresolved cases in the earlier years of the study in which more limited testing was performed. Hence, further research in this field is required.

| CONCLUSION
In this study, we have reported a representative cohort of prenatally presenting skeletal dysplasias in the Finnish population. There were many primary findings that triggered the diagnostic cascade. In addition to characteristic ultrasound findings, abnormal first trimester screening and increased NT could also be the triggers for further testing. The diagnoses observed in our study population included a considerable number of rare skeletal dysplasias enriched in the Finnish population. There may be considerable populationspecific variation in the mutation spectrum of skeletal dysplasias, which should be considered in the diagnostic process. Hence, it is important that these common founder variants are covered in the diagnostic tests, for example, in gene panels, when ethnic Finnish couples are involved. Suspicion of skeletal dysplasia rose late in pregnancy, so there is a need for prompt and accurate diagnostic methods.
Although abnormal ultrasound findings of the skeletal system were the trigger for further investigations in the majority of cases in our cohort, we advocate that in pregnancies with abnormal combined first trimester screening or/and increased NT thickness as the only abnormal finding(s), the awareness of the possible bone dysplasia should be always kept in mind and meticulous assessment of fetal skeletal system performed.