Case Studies of Two Classical Imprinting Growth Disorders: Silver–Russell and Beckwith–Wiedemann Syndromes

Abstract

The genetic influences on human growth are being increasingly deciphered. Silver–Russell and Beckwith–Wiedemann syndromes (SRS; BWS) are two relatively common genetic syndromes with under- and overgrowth-related issues being the reason for referral. Aberration in genomic imprinting is the underlying genetic pathomechanism behind these syndromes. Herein, we described a series of children with these two growth disorders and give an orientation to the reader of the concept of imprinting as well as the genetic testing strategy and counseling to be offered in these syndromes.

Introduction

Epigenetics has been defined as “the heritable changes in gene activity which does not involve changes in the DNA sequence.” ¹ The prefix “epi” means above and literally implies over and above the genome. Genomic imprinting is the type of epigenetic change that causes a gene to be expressed depending on its parental origin. In human beings, the majority of genes show expression of both the paternal and the maternal allele (copy). Only a small number of human genes (around 228 genes or approximately 1% of the genome) ² are imprinted with either only the maternal or the paternal allele of those genes being expressed. Imprinted genes have been found to be critical in regulating growth and development, and a germline alteration of their normal monoallelic expression leads to congenital imprinting disorders. ³ Silver–Russell and Beckwith–Wiedemann syndromes (SRS; BWS) are two classical imprinting disorders characterized by growth restriction and overgrowth, respectively.

Clinical Manifestations

The incidence of SRS has been estimated to range from 1:30,000 to 1:1,00,000. SRS is characterized by prenatal and postnatal growth retardation. As the etiology of growth retardation is very heterogenous, it is useful to remember the features that point toward SRS. These are relative macrocephaly (defined as head circumference at birth ≥1.5 standard deviation [SD] score [SDS] above birth weight and/or length SDS), body asymmetry, prominent forehead, and feeding difficulties. Body asymmetry has been defined as limb length discrepancy (LLD) of ≥0.5 cm or arm asymmetry, or LLD <0.5 cm with at least two other asymmetrical body parts (one non-face). Protruding forehead should be seen in a profile view, and if the forehead is projecting beyond the facial plane, then it can be considered as a protruding/prominent forehead. Although this is a subjective criterion, it along with relative macrocephaly are the two best utility diagnostic criteria of the Netchine–Harbison clinical scoring system (NH-CSS) that distinguish SRS from other causes of growth retardation. The NH-CSS is considered to be the most sensitive clinical scoring system for SRS with the sensitivity of 98% and the negative predictive value of 89%. ⁴ The presence of four out of six criteria (small for gestational age, postnatal growth failure, relative macrocephaly at birth, prominent forehead, body asymmetry, feeding difficulties, and/or low body mass index [BMI]) is required for clinical diagnosis of SRS. ⁴

BWS is also a relatively common growth disorder with a prevalence of around 1 in 10,000. ⁵ Contrary to SRS, it is characterized by overgrowth. Recently in 2018, an international consensus group has introduced the concept of BWS spectrum (BWSp) ranging from classical BWS to isolated lateralized overgrowth. ⁶ The group has suggested cardinal and suggestive features of BWSp and the indications of molecular testing based on a scoring system using these features ( Table 1 ). ⁶

Table 1. Scoring and clinical features of Beckwith–Wiedemann spectrum ⁶ .

Cardinal features (2 points each)	Suggestive features (1 point each)
Macroglossia	Birth weight >2 SDS above the mean
Exomphalos	Facial nevus simplex
Lateralized overgrowth	Ear creases and or pits
Multifocal and/or bilateral Wilms tumor or nephroblastomatosis	Transient hypoglycemia (lasting less than 1 wk)
Hyperinsulinism (lasting beyond one week and requiring escalated treatment)	Typical BWSp tumors (neuroblastoma, rhabdomyosarcoma, unilateral Wilms tumor, hepatoblastoma, adrenocortical carcinoma or phaeochromocytoma)
Pathology findings: adrenal cortex cytomegaly, placental mesenchymal dysplasia, or pancreatic adenomatosis	Nephromegaly and/or hepatomegaly
	Umbilical hernia and/or diastasis recti
	Polyhydramnios and/or placentomegaly

Abbreviations: BWSp, BWS spectrum; SDS, standard deviation score.

Note: If total score is ≥4 clinical diagnosis of BWSp would be considered, even if an 11p15 anomaly is not identified. A score between 2 and 4 needs genetic testing for confirmation of diagnosis. A score less than 2 need not go ahead with genetic evaluation.

Molecular Basis of SRS and BWS

There are four known molecular mechanisms causing imprinting disorders: aberrant methylation (epimutations), uniparental disomy, chromosomal imbalances, and genomic mutations in the imprinted genes. Most of SRS and BWS cases are associated with molecular alterations at a cluster of imprinted genes at the chromosomal region 11p15.5. The epigenetic alteration (epimutation) involves aberrant DNA methylation at a locus-specific region and affects the normal gene expression in cis (or nearby genes). To elaborate, the chromosome 11p15 region is understood to have a telomeric domain with imprinting control region 1(ICR1 ideally to be named as H19/IGF2: IG DMR (H19/IGF2: intergenic differentially methylated region), and a centromeric domain with imprinting control region 2 (ICR2) or KCNQ1OT1: TSS DMR ( KCNQ1OT1: transcriptional start site DMR). The ICRs regulate the expression of genes in cis. Several of these genes are growth controlling and tumor-suppressing genes. For example, the genes controlled by the ICR1 are the insulin-like growth factor 2 encoding gene IGF2 and H19 encoding a non-translated long non-coding RNA (lncRNA). In normal individuals, the paternal allele of the ICR1 is methylated, and there occurs expression of IGF2 solely from the paternal allele, whereas H19 , which is thought to play a tumor suppressor role, is maternally expressed. The ICR2 controls the cell cycle inhibitor gene CDKN1C and the gene encoding the regulatory long non-coding RNA- KCNQ1OT1 . Normally, the maternal allele of the ICR2 is methylated, and the CDKN1C and KCNQ1 are expressed from the maternal allele ( Fig. 1A ).

Fig. 1.

Showing possible molecular mechanisms for BWS: ( A ) A normal methylation pattern at chromosome11p15 with maternal allele being methylated at imprinting control region 2 (ICR2) and paternal allele methylated at imprinting control region 1 (ICR1). ( B ) Gain of methylation of ICR1 on maternal allele is seen in 5% to 10% cases of BWS. ( C ) Loss of methylation of ICR2 on maternal allele seen in 50% cases of BWS. ( D ) Gain of methylation at ICR1 along with loss of methylation at ICR2 suggests a paternal uniparental disomy as underlying mechanism of BWS. BWS, Beckwith–Wiedemann syndromes; M, maternal allele; P, paternal allele.

In 80% cases of BWS, there occurs aberrant methylation at the chromosomal region 11p15.5. Fifty percent of patients show loss of methylation (LOM) at ICR2 on the maternal allele (ICR2 LOM) ( Fig. 1C ), and 5 to 10% show gain of methylation (GOM) at ICR1 on the maternal allele (ICR1 GOM) ( Fig. 1B ). The ICR2 LOM at the maternal allele leads to the loss of CDKN1C activity, and the ICR1 GOM at the maternal allele leads to biallelic or overexpression of IGF2 , thus explaining the overgrowth. Another molecular defect seen in approximately 20% cases of BWS is segmental upd(11)pat, leading to overexpression of IGF2 . Intragenic maternal CDKN1C mutations leading to the loss of its function are seen in approximately 5% of sporadic and 40% of familial cases of BWS, and chromosomal abnormalities (deletions/duplications) in 11p15 causing BWS are seen in <5% of patients.

In SRS, up to 50% of the patients can be expected to show LOM at the ICR1 on chromosome 11p15 on the paternal allele (SRS1 # 180860). ⁷ However, SRS shows molecular heterogeneity with up to 10% patients having maternal UPD for chromosome 7 (upd(7)mat) or segmental upd(7q)mat (SRS 2# 618905). ⁸ SRS3 (#616489) is caused by mutation in the IGF2 gene on chromosome 11p15. ⁹ SRS4 (#618907) is caused by mutation in the PLAG1 on chromosome 8q12. SRS5 (#618908) is caused by mutation in the HMGA2 on chromosome 12q14. ¹⁰ Besides chromosome 7 and 11 deletion duplications and MEST gene mutations on 7q32, other molecular aberrations can be rarely observed in patients with SRS. ^{11 12}

Diagnosis

Methylation abnormalities are likely to be seen in the majority of the cases of SRS and BWS, so the testing strategy involves first testing the methylation pattern of the chromosomal region 11p11.5 in patients with SRS or BWS. Although there are several methylation testing techniques (such as bisulfite sequencing and polymerase chain reaction studies), methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is the most widely used genetic test as it gives information on both the methylation status of the target region and the copy number of the targeted region. If a normal methylation pattern is seen with a clinical diagnosis as per the scoring systems suggested, one will have to consider point mutation in one of the imprinted region genes. This requires sequencing (Sanger or next-generation sequencing), e.g., CDKN1C sequencing for BWS or IGF2 for SRS. Also, although a rare cause, approximately 30 different pathogenic copy number variations have been described in SRS ⁴ for which chromosomal microarray would be the test of choice.

It is to be remembered that 30 to 40% patients of clinically diagnosed SRS may fail to show any of the known genetic mechanisms for their disease. Similarly, in up to 20% of patients with a BWS phenotype, a molecular diagnosis remains unknown. One of the reasons for this is the presence of tissue mosaicism in imprinting disorders that requires testing other tissues/multiple tissues such as the excised hyperplastic tissues or skin, or others, besides the blood leucocytes.

Management

The major management issue in treating patients with SRS is growth. Children <2 years presented failure to thrive (FTT), while adults with untreated SRS can be predicted to have height <  − 3 SDS. FTT can be multifactorial due to poor appetite, oromotor difficulties leading to poor feeding and caloric intake, as well as functional and structural gastrointestinal problems. These need to be addressed appropriately in early life. Growth hormone (GH) therapy can be expected to improve the adult height to approximately −2 SDS. Recommendations on the use of GH in SRS can be found in the international consensus statement published in 2017 ⁴

Hypoglycemia is seen in approximately 30 to 60% of patients with BWS. It is common in the neonatal period and generally subsides in a few days with the decrease in the insulin levels. ¹³ Overgrowth (generalized and lateralized) occurs in approximately 43 to 65% of patients. ¹⁴ With the concept of BWSp, macrosomia now is not considered as a cardinal feature of BWS. Children remain in the upper centiles of growth during early childhood, and growth slows down in late childhood. ¹⁵ Other issues to be addressed in managing children with BWS include tumor surveillance and management of macroglossia and omphalocele/other abdominal wall defects.

Genetic Counseling

Most of the cases of SRS occur as sporadic de novo epigenetic alteration with either loss of paternal methylation at the ICR1 or maternal uniparental disomy for chromosome 7 and have a low risk of recurrence of about <1%. However, the risk may be as high as 50% in cases of SRS with genetic alterations as a copy number variant on chromosome 7 or 11. Similarly, for BWS, the risk of recurrence may be low in cases with LOM at ICR2 and GOM at ICR1; however, the risk may be as high as 50% in genetic alterations as CDKN1C pathogenic variant and 11p15.5 microdeletion/microduplication.

Prognosis

In general, the prognosis of BWS is good and children achieve the expected height; however, there is an increased risk of cancers that can be treated successfully if screened early. Also, in cases of SRS, patients tend to show improvement in growth parameters and feeding difficulties with age.

Case Presentations

Patient 1: A male baby was born with a birth weight of 900 g (−6 0.13 z -scores) at 36 weeks to non-consanguineously married couple. He had recurrent episodes of loose stools and vomiting since infancy that gradually resolved by 4 years of age. He had shown a normal gain of developmental milestones. At 10 years of age, parents sought medical attention as he appeared to be not gaining weight as much as his peers. Examination showed weight as 11.4 kg (−10.6 SD as per Indian Academy of Pediatrics' charts), height 108 cm (−5.12 SD), and head circumference 48.2 cm (−4.16 SD), and upper segment-to-lower segment ratio was 0.95:1. Asymmetry of upper limbs was present with right upper limb length measuring 47 cm and left upper limb 44 cm. Lower limb length was equal on both sides. On dysmorphology assessment, he appeared to be thin built with triangular facies and had frontal prominence ( Fig. 2B ), micrognathia, low set ears, and bilateral index finger clinodactyly ( Fig. 2D ). Investigations showed a normal hemogram and liver, renal, thyroid function tests. A clinical diagnosis of SRS was made, and methylation testing (MS-MLPA) for chromosomal region 11p15 revealed hypomethylation of the ICR1 region ( Supplementary Material , available in the online version only). No copy number variations were detected. The family was counseled regarding the presence of an underlying genetic syndrome and the need to follow-up the child for growth monitoring.

Fig. 2.

( A ) Patient 2 at the age of 8 months with face in lateral view showing prominent forehead. ( B ) Patient 1 face-in-front view showing triangular face. ( C ) Patient 2 showing asymmetry of lower limbs. ( D ) Hands of patient 1 showing bilateral index finger clinodactyly. ( E ) Foot of patient 2 showing cutaneous syndactyly of second and third toe.

Patient 2: A male baby was born at term with a birth weight of 1,500 g (−1.73 SD) to non-consanguineously married couple. At 4 year 4 months of age, he was brought with a complaint of not gaining weight and height appropriately. At the age of presentation, his weight was 7 kg (−3 SD), height 91.7 cm (−3 SD), and head circumference 49 cm (−1.67 SD), as per WHO growth charts. His facies resembled that described for previous child. Examination revealed further findings of right-sided single palmer crease, fifth finger clinodactyly, proximal placement of bilateral thumbs, syndactyly of second and third toes ( Fig. 2E ) along with hypogenitalism with the testicular volume of 1 mL. There was a limb girth difference of 2 cm ( Fig. 2C ). The radiographs of the upper limbs likewise demonstrated hypoplasia of left-sided bones with bone age corresponding to 1 year. DNA diagnosis by MS-MLPA showed hypomethylation of the ICR1 region at chromosome 11p15, corroborating with the clinical diagnosis of SRS. The child during follow-up at 8 years had a weight of 10 kg (−5.1 SD) and height 92 cm (−5.9 SD).

Patient 3: A male baby was born at term and had a birth weight of 4,200 g (1.94 SD). Examination showed lax abdomen, bilateral inguinal hernia, bilaterally palpable kidneys, and left hand postaxial polydactyly. He appeared to have coarse facies with protruding tongue ( Fig. 3B ). Bilateral cryptorchidism was present ( Fig. 3C ). Ultrasonogram of abdomen showed bilateral nephromegaly. The scoring for the diagnosis of BWSp ( Table 1 ) revealed a total score of 4. The molecular testing using MS-MLPA showed hypermethylation of ICR1 region at chr 11p15 with normal methylation of ICR2 region providing the molecular diagnosis of BWS ( Supplementary Material , available in the online version only).

Fig. 3.

( A ) Patient 4 in newborn period showing protruding tongue. ( B ) Patient 3 at 3 months of age showing coarse face and macroglossia. ( C ) Patient 3 at 1 year note the coarse face, lax abdomen, cryptorchidism, and macroglossia. ( D ) Note the vertical ear crease in patient 4.

The child in the follow-up visit at 1 year 10 months of age had a weight of 18 kg (3.62 SD), height 94 cm (2.67 SD), BMI 20.3 Kg/m ² (2.99 z-score), and weight for length 3.08 z -score. Macroglossia was more obvious in follow-up. The child was gaining normal developmental milestones.

Patient 4: A baby girl firstborn to a non-consanguineously married couple at 36 weeks of gestation was referred to our center for evaluation of hypoglycemia. There was the history of polyhydramnios in the mother during the antenatal period. The baby's birth weight was 2,800 g (−0.99 SD). She had slight coarseness of the face along with macroglossia ( Fig. 3A ). The ears were low set ears with vertical ear creases ( Fig. 3D ). Her thyroid profile, skeletal survey, and urine for glycosaminoglycans were normal. Ultrasonography of the abdomen was unremarkable. MS-MLPA did not indicate methylation abnormality at the BWS locus. Due to financial constraints, further genetic evaluation could not be performed. According to the BWSp scoring system, she had a score of 5. Therefore, a clinical diagnosis of classical BWS was made. The child was advised regular follow-up and tumor surveillance.

Discussion

Human growth is a complex process being influenced by genetic and environmental factors. The role of genetic factors in growth is being increasingly deciphered. ¹⁶ SRS and BWS are classical imprinting disorders showing dysregulation of locus-specific epigenetic marks or DNA methylation signature. A dysregulation of imprinting tips the normal state of parent-specific gene expression leading to a gene dosage effect. Thus, the majority of the BWS cases result from an increased expression of paternal genes due to the LOM at the ICR2 GOM at ICR1 or paternal uniparental disomy of the entire 11p15 region. On the contrary, the majority of SRS cases result from increased maternal gene expression secondary to paternal hypomethylation at ICR1, with no paternal contribution leading to growth restriction.

The two patients of SRS described above fitted into the clinical diagnosis of SRS as per NH-CSS and, in addition, also showed features of SRS such as clinodactyly and syndactyly of toes found in around 70 and 30%, respectively, of patients with SRS, hypogonadism seen in approximately 40% patients, and mild motor delay seen in approximately 37% patients of SRS. Both our patients of SRS were confirmed by molecular diagnosis with hypomethylation of the ICR1 (H19DR) region. However, as discussed above, SRS exhibits significant molecular heterogeneity. In a recent study to clarify the frequency and the clinical features of patients with etiology-unknown SRS, out of 92 patients who qualified the clinical criteria of SRS but did not show any methylation abnormality in differentially methylated regions known to be associated with SRS, pathogenic variations were seen in CDKN1C, PLAG1, and IGF2 in 4.3% patients. Besides, the study revealed the diagnosis IGF1R abnormality, SHORT syndrome ( PIK3R1 ), Floating–Harbor syndrome ( SRCAP ), Pitt–Hopkins syndrome ( TCF4 ), and Noonan syndrome ( PTPN11 ) in 5.4% of their cohort of patients, highlighting the phenotypic overlap of these syndromes with SRS. ¹⁷

In the two patients with BWS presented here, the score using the BWSp scoring system ( Table1 ) was 4 and 5 for patients 3 and 4, respectively. A child with a total score of 4 or more can be given a clinical diagnosis of classical BWS. Moreover, the group suggested that children whose score is ≥4 would be considered to have BWSp, even if an 11p15 anomaly is not identified (applies to patient 4 presented above). However, patients with a score between 2 and 4 need genetic testing for confirmation of diagnosis, while patients with a score less than 2 need not go ahead with genetic evaluation. ⁶ Patient 3 described above showed the less common aberration of methylation pattern, i.e., hypermethylation of the ICR1 region. Patient 4 failed to show any methylation abnormality at the 11p15 locus, although still qualifying for the clinical diagnosis of BWSp disorder. With the availability of genome-wide methylation testing array technology, patients with clinical BWSp without any detectable methylation defect at 11p15 locus or CDKN1C mutation are being diagnosed with methylation aberrations in other imprinted loci or multi-locus imprinting defects. ¹⁸

It is interesting to note that although SRS and BWS grossly appear to have an opposite phenotypic expression in terms of growth, asymmetry and glycemic metabolic disturbance are common to both these disorders. In fact, it is now being recognized that the classical congenital imprinting disorders (currently there are nine such disorders ¹⁹ ) have overlapping clinical features affecting growth, development, and metabolism.

Conclusion

RSS and BWS are the disorders of growth characterized by dysregulation in imprinting mechanism and other complex genetic alterations. The clinical approach to these disorders is unique from other genetic disorders with methylation studies being the first-line investigation. This knowledge is important for the treating physician to prevent delays in diagnosis and management.