Prenatal sonographic findings in confirmed cases of Wolf-Hirschhorn syndrome

Wolf-Hirschhorn syndrome (WHS, OMIM #194190), sometimes also referred to as 4p- syndrome or 4p deletion syndrome, is a congenital malformation syndrome characterized by pre- and postnatal growth deficiency, developmental delay, seizures as well as a characteristic craniofacial phenotype ('Greek warrior helmet' appearance). The syndrome is caused by partial loss of material from the distal portion of the short arm of chromosome 4 (4p16.3) and is considered a contiguous gene syndrome involving the two critical regions WHSCR1 and WHSCR2 [1‐3]. Birth incidence of WHS is estimated to be at least 1 in 50,000 with a female predilection of 2:1 [4].

The prenatal diagnosis is difficult due to a large diversity of expression of this syndrome and rather non-specific ultrasound findings like intrauterine growth restriction (IUGR) or increased nuchal translucency (NT) [5]. Compared to the postnatal situation, distinct facial anomalies such as hypertelorism, micrognathia, short philtrum, highly arched eyebrows and protruding eyes can be rather subtle prenatally. However, the composition of different ultrasound signs is the key to guide appropriate genetic testing for achieving a diagnosis. Detection rate by routine karyotyping is around 50–60% [1, 4, 6]. With Fluorescent in situ hybridization (FISH) the reported sensitivity is 95%. Nowadays, chromosomal microarray analysis (CMA) is the method of choice and even Noninvasive Prenatal Testing (NIPT) is increasingly used in microdeletion/duplication screening [7].

In this study, we report on the spectrum of prenatal sonographic features and the outcome of 18 cases with proven WHS. We review the intrauterine phenotypic abnormalities of our and previous small case series from the literature compared to data on postnatal frequencies and abnormalities in affected patients.

This was a retrospective observational study covering the period between 2002 and 2021. Patients were seen at the tertiary level referral centers of Obstetrics and Prenatal Medicine in Bonn (n = 11), Tuebingen (n = 4) and Nuernberg (n = 3), Germany. Referrals to our centers represent a mixed low- and high-risk population and are sent for targeted ultrasound examination or evaluation of suspected fetal anomalies. Most of the cases presented here were sent for a second opinion because of IUGR with or without other sonographic anomalies. All women received a detailed fetal anomaly scan including fetal echocardiography using high-resolution ultrasound equipment. Based on the abnormal ultrasound findings, genetic testing was offered to the patients and involved routine karyotyping as well as fluorescence in situ hybridization (FISH) and/or chromosomal microarray analysis (CMA), if routine karyotyping was unrewarding. Prenatal genetic confirmation of WHS was obtained by cytogenetic (n = 15) and molecular genetic analysis (n = 3. FISH and CMA were performed according to local standards: For metaphase FISH analysis on cultured amniocytes, specific FISH probes for the WHS critical region (WHSCR) were used (Aquarius® FISH Probes, Cytocell, UK; ToTelvysion multicolor FISH, Abbott Molecular, USA; Kreatech™ FISH probes, Kreatech Biotechnology B.V., The Netherlands). CMA was performed on uncultured amniocytes by oligonucleotide aCGH (CytoScan™ Optima Array, Thermo Fisher Scientific, USA). Extensive multidisciplinary counseling included information on the course and outcome of the disease. Pregnancy outcomes were obtained from our perinatal database, neonatal records or autopsy findings. All patients have given written informed consent for data collection, analysis and their use for research. According to the Ethics Committee of the University of Bonn, ethical approval for anonymous retrospective data analysis is not required according to the national guidelines. All methods performed were according to relevant guidelines.

Between January of 2002 and March of 2021, 18 fetuses were diagnosed with Wolf-Hirschhorn syndrome. There was a female-to-male ratio of  > 2.5:1 (72.2% females, 27.8% males). At presentation, the average gestational age was 23 + 1 weeks (range: 13 + 4 to 29 + 1 weeks’, Table 1).The suspected diagnosis of WHS was made based on prenatal ultrasound diagnosis of IUGR (defined as an estimated fetal weight below the 3^rd percentile using the Hadlock formula [8]) together with typical ultrasound findings (Fig. 1). The presumed diagnoses were confirmed prenatally by genetic testing in all 18 cases, and individual genetic results can be seen in Table 2.

To the best of our knowledge, we present the largest prenatal series of confirmed WHS cases so far. Prenatal diagnosis of Wolf-Hirschhorn syndrome is challenging due to unspecific prenatal ultrasound findings associated with this genetic disease. Usually, family history is unremarkable and parental ages are similar to those found in the general population. Most children with WHS are born at term, in about one third of cases with some degree of perinatal distress. Decreased fetal movements in almost all pregnancies affected by WHS have been reported [8].

Together with a total of 65 previously reported prenatal cases of WHS in the English literature [5, 9, 10], we compared prenatal findings in fetuses with WHS to postnatal findings [8] (Table 3). IUGR with an “abnormal facial appearance” are the leading ultrasound findings previously reported in WHS and are seen in > 75% of all cases. Progression of IUGR in the course of pregnancies complicated by WHS had not received specific attention by previous studies, however, evaluation remains difficult since high rates of TOP cause a significant lack of follow-up biometric data. A continuous growth pattern (with all growth parameters below the 3^rd percentile) was seen in 62.5% of WHS fetuses with more than one biometric assessment in our study cohort and could therefore be a typical finding in fetuses with WHS. Unlike previous studies and in an attempt to make ultrasound findings more objective with regards to craniofacial abnormalities, we specifically reviewed our data on the prevalence of microcephaly (HC < 5^th centile for gestational age), which presented in 72.2% (n = 13/18) of fetuses. Microcephaly in WHS had not found any specific consideration in previous reviews, except for the series by Sifakis et al. (2013) with a prevalence of only 8.3%. “Abnormal facial appearance” was present in 85.1% (n = 40/47) fetuses studied by Xing et al., however, craniofacial dysmorphic features were not specifically defined. In the case series by Zhen et al., no craniofacial dysmorphism was noted in a total of 10 fetuses. As a possible explanation the authors stated, that “structural defects rather than subtle morphological changes were targeted by the investigating sonographers”. The assessment of the fetal nasal bone and profile were essential aspects of our study, and hypoplastic or absent nasal bone was observed in 72.2% (n = 13/18, according to reference charts by Sonek et al., 2003) [11]. This is in contrast to 9.2% (n = 6/65) of previously reviewed prenatal cases (Table 3).

A somewhat beaked and triangular shape of the tip of the fetal nose came to our attention (even in the first trimester, Fig. 1c), which, if present, might further raise the suspicion of WHS. Not least, assessment of the fetal ears by prenatal ultrasound should take place in suspected WHS cases, since deformities of the ears and/or preauricular tags are quite common [8] and could also be seen in the one survivor of our cohort.

We found cardiac defects in 50% of our cases, which is consistent with postnatal findings [8], but has not been reported before in such a high frequency in prenatal series (24.6%, n = 16/65). The same applies to the presence of SUA, which was seen in 44.4% in our cases, compared to 4.6% (n = 3/65) [5, 9]. Novel ultrasound findings in our cohort include aberrant right subclavian artery (ARSA, n = 3/18), right aortic arch (RAA, n = 2/18) and overlapping fingers (n = 2/18). One fetus presented with persistent hyperplastic primary vitreous (Fig. 1d).

Abnormal first trimester ultrasound (increased NT or cystic hygroma) has been reported in WHS [10, 12‐14] and some authors suggest FISH and/or CMA for WHS testing if routine karyotyping shows normal results [13]. Most cases in our study presented during second trimester, which is consistent with the literature [5]. The average gestational age at which intrauterine growth retardation typically manifest has not been specifically determined yet, but was not reported before 16 weeks’, so far [5].

Minimal diagnostic criteria for WHS, characterizing the "core" phenotype, are the typical facial appearance (“greek warrior helmet”), intellectual disability, growth delay and seizures (or EEG anomalies) [15]. Phenotypes of WHS can range from mild to severe and are related to the size of the deletion (genotypic-phenotypic correlation), with more severe phenotypic expressions associated with larger deletions [16]. Approximately 20% of WHS cases show deletions restricted to 4p16.3, but a substantial part is caused by larger deletions that can extend as far as 4p14 [17]. Affected patients with deletions less than 3.5 Mb usually express a mild phenotype. Deletion between 5 and 18 Mb cause the classic WHS phenotype and individuals with deletions > 22 Mb typically present major malformations, seizures and severe cognitive impairment [5, 15, 18]. However, the complexity of phenotype-genotype correlation in WHS becomes clear when, for instance, looking at congenital heart defects (CHD) in WHS patients. In the study by Maas et al. (2008), four of eight patients from both large and small deletions presented with CHD, whereas Zollino et al. (2000) and Wieczorek et al. (2000) found CHD in only 13 of 19 patients with large deletions [4, 16, 19]. Both fetuses with complex heart defects in our study cohort showed large and more complex deletions of chromosome 4 and 7 in one case and unbalanced translocation with derivative chromosome 4 (size of deletion unknown) in the other case. Rates of 40 – 45% of unbalanced translocations (both inherited and de-novo) in WHS but also genetic haploinsufficiency and interaction with surrounding genes as well as mutations in modifier genes located outside the WHSCR regions all pose an challenge to genotype–phenotype correlation [15, 20]. Besides extent of the deletion, all these other aspects should be considered when counselling parents [21].

Karyotyping and fluorescent in situ hybridization (FISH, Fig. 2) for WHSCR1 and 2 are the common methods of genetic testing and also diagnosis of WHS by use of NIPT has been reported before. However, smaller deletions (in particular those < 3 Mb) or complex genomic rearrangements can set limits to these techniques and make CMA necessary [7, 22, 23]. About 55% of cases of WHS are caused by de novo terminal or interstitial 4p deletions with low recurrence risk, approximately 40 – 45% result from an unbalanced translocation involving 4p and are either de novo or inherited from a parent with a balanced translocation (n = 5 in our study, Table 2). This should be kept in mind, especially if there is a family history of miscarriage, stillbirth or an affected individual, and should prompt parental testing to exclude balanced translocation in one or both parents. The remaining 5% are complex chromosome rearrangements including ring chromosome 4 or inverted duplications with terminal deletion at 4p [24, 25]. This furthermore highlights the necessity of using CMA as the diagnostic tool.