ABSTRACT

Background:

Brachydactyly A1 (BDA1) is an autosomal dominant disorder. It manifests as shortness/absence of the middle phalanges in the hands and feet. It is caused by a variation in the Indian hedgehog (IHH) gene. The IHH gene plays an important role in the development of limbs. IHH is expressed in the pre-hypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. A lack of IHH prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. This study aimed to identify IHH gene variants in BDA1 individuals.

Methods:

The present study used Sanger sequencing to analyze IHH variants in three exons and in silico tools for detailed variant analysis.

Results:

A total of 44 individuals were sequenced - 14 BDA1 individuals and 30 healthy controls. Sanger sequencing revealed a novel variant in the IHH gene in exon 1 and exon 2. A variant was found to segregate affected individuals in a family.

Conclusion:

The present study findings further expand the mutation spectrum of the IHH gene, and provide detailed mutant protein changes by in silico methods. The study also provides additional evidence that IHH plays an important role in limb development and short stature.

Keywords:

Brachydactyly, missense, Indian hedgehog, phalanges, metacarpals, metatarsals, Sanger sequencing, short hands.

Introduction

Brachydactyly (BD) is characterized by shortening or missing phalanges and toes and abnormal development of the phalanges, metacarpals, and metatarsals [1-4], BD was the first disorder described in terms of autosomal dominant Mendelian inheritance [5,6]. In 2001, BD was added to the international nosology and classification of genetic skeletal disorders [7]. The International Nosology Classification of Skeletal Disorders has divided different skeletal disorders into 42 groups, one of which is BD with extra-skeletal manifestations, and another is BD without extra-skeletal manifestations [8]. It was initially classified by Julia Bell in 1951 into five types, namely, BD types A, B, C, D, and E. Type A is further divided into types A1, A2, and A3 based on the shortening of phalanges. It can be present in isolated forms and is involved in syndromes such as Temtamy preaxial syndrome, Feingold syndrome, Robinow syndrome, and achondroplasia. Types A3 and D were approximately 2% prevalent among all types of BD. The most common types of BD found are A and E. The mechanism of BD has not been extensively explored. It can be associated with other hand and foot anomalies, such as polydactyly, syndactyly, and clinodactyly. The hands and feet of the affected individual appear short, occurring as an isolated form or as a part of the syndrome [9-11].

Brachydactyly A1 (BDA1) (OMIM: 112,500) is characterized by hypoplasia/aplasia of the middle phalanges of digits 2-5. BDA1 can be found in an isolated form and can also present with additional malformations, such as short stature, scoliosis, craniofacial dysmorphism, developmental delay, or intellectual disability. It exists in an autosomal dominant manner. The genetic basis of BDA1 on chromosome number 2q35 in the Indian Hedgehog (IHH) gene was first described by Gao et al. [11]. It is caused by heterozygous pathogenic variants in a specific region of the N-terminal active fragment of the IHH gene [12-15] IHH plays important roles in meditating skeletal condensation, growth, patterning, morphogenesis, pre-hypertrophic chondrocyte formation, chondrocyte differentiation, joint development, and bone formation. IHH was the first gene associated with BDA1 [1,3,11,16,17,18]. To date, only a few pathogenic variants causing BDA have been identified. The IHH gene encodes a member of the hedgehog family located on chromosome number 2. IHH is a peptide hormone that cleaves proteins to form N-terminal functional signals and C-termini, which further regulate the protein [11,18,19]. Secreted IHH proteins play a role in regulating embryonic development, including growth, patterning, and morphogenesis [18,20,21]. The protein encoded by this gene specifically plays a role in skeletal development and differentiation. It acts with factors including proteins involved in bone morphogenesis and other growth factors to induce differentiation of osteoblasts [22,23,24]. In this study, we investigated the clinical and genetic characteristics of individuals with BDA1. We present the clinical and radiographic findings and novel IHH variations in related and unrelated individuals with BDA1 and short stature.

Subjects and Methods

This is a case cohort study in which 44 individuals, including 14 patients, and 30 controls were enrolled from an outpatient tertiary care center. Patients belonged to Himachal Pradesh, Punjab, Haryana Chandigarh, Uttar Pradesh, and Bihar. Individuals included in this study were phenotyped by clinical geneticists and senior residents undergoing advanced training. Peripheral blood samples (2-3 ml) were obtained from all individuals of related and unrelated families. Sanger sequencing was performed to analyze IHH gene variants of the patients. The patients or family members/caretakers provided written informed consent for the publication of the clinical details. This study was ethically approved by the Ethics Committee of the Institute (Ref No: NK17963/PhD/638).

Genomic DNA was extracted from the peripheral blood of the patients. The three exons of the IHH gene (forward and reverse) in the NM_002181.4 transcript were amplified with primers from the NCBI via primer blast. All three exons and intron-exon boundaries of IHH were amplified via PCR and subjected to Sanger sequencing (Table 1). Blood samples were collected in an EDTA vial, and then DNA was extracted using the Qiagen DNA Extraction Kit. The PCR program was set to a denaturation temperature of 95°C for 5 minutes, an annealing temperature of 580°C for 1 minute, and an extension temperature of 72°C for 7 minutes. This PCR cycle was run 35 times. We used a kit (Promega Corporation) to purify the PCR products. The polymerase chain reaction products were bidirectionally sequenced using PCR primers and an ABI Prism Big Dye Terminator Cycle Sequencing Kit and analyzed on an ABI 3100/3700XL sequencer. After capillary electrophoresis, the Sanger sequencing results were analyzed by Finch TV, and in silico tools, including Mutation Taster, Franklin, Mutalyzer, and Varsome, and the pathogenicity of the variants was classified following the American College of Medical Genetics (ACMG)/ Association for Molecular Pathology (AMP) guidelines [25].

Table 1. IHH gene forward, reverse primers of three exons used for Sanger sequencing.

Results

Clinical phenotypes

A total of 44 individuals were examined and 14 affected BDA1 individuals belonged to families from nearby geographical regions. A total of 30 healthy unrelated controls of age group 7-35 years were enrolled in this study, these individuals did not have any skeletal, neurodevelopmental, other genetic disorders or BD. All the affected individuals had abnormalities, including short hands, short feet, growth retardation, and short stature. Three affected individuals belonged to a single family, whereas the others were unrelated. An anthropometric examination was performed for all individuals in the growth laboratory. The height and weight of the bodies of the affected individuals were measured. X-rays were evaluated for the identification of short, missing phalanges in the hands and feet. All 14 BDA1 individuals were found to have short stature. The interpretation of short stature, underweight status, and overweight status is given in Table 1. The hand phenotypes and X-ray images are shown in Figure 1.

Familial case

A family with an AD inheritance pattern was examined in the outpatient clinic and 3 BDA1 individuals in the family underwent genetic evaluation and genetic counseling was provided. The clinical details of the affected members of the family included short stature, short limbs, and abnormalities according to their hands and feet radiographs. There were 7 individuals in the BDA1-affected family, 3 of whom exhibited BD. We clinically examined 3 of the subjects via physical examination and imaging studies, including radiographs of the hands and feet and family history. The younger girl was 7 years old and presented with short hands and feet with a height of 103 cm and a weight of 18 kg. On clinical evaluation, it was found that the other sibling of the girl also had similar short hands and feet, and the father also had similar short hands and feet (Figure 2). On X-ray examination, the middle phalanges of the right and left hands of the three affected individuals were missing (HP:0010239). X-ray of the father’s feet showed shortened 4^th and 5^th metatarsals. Another member of the family was found to be affected with down syndrome.

Figure 1. (a) Phenotype of short hands and X-ray (b) of adult showing missing middle phalanges and short proximal phalanx of thumb in BDA1.

Genetic analysis

Genetic analysis of 44 individuals was done and 14 affected individuals were identified with three different heterozygous IHH variants. The genetic testing by Sanger sequencing revealed a missense variant in exon 1, NM_002181.4 IHH: c.299A>C; p. Asp100Ala, in 3 affected individuals of the family with BDA1. Another variant was detected in exon 1 in the 4 individuals with NM_002181.4 IHH: c.217C>T; p.Arg73Cys; in addition, 7 individuals had a variant in exon 2 namely, NM_002181.4 IHH: c.418T>C; p.Ser140Pro. They had short hands, short feet, and short stature. The Sanger sequencing chromatograms are shown in Figure 2. The affected individuals did not present any other specific signs of other skeletal dysplasia on limb and spine radiographs. The unaffected family members of the patients were not found with a variance of unknown significance (VUS). The list of variants identified in 14 individuals is provided in Table 2. We also performed the sequencing of all IHH exons in the control group, but no significant variants were identified.

Figure 2. A. diagrammatic representation of IHH gene on chromosome number 2 at 2q25 with three exonic regions. B. Sanger chromatograms for heterozygous variation a. IHH: c.299A>C, b. IHH:c.217C>T, c. IHH: c.418T>C in index case and normal in control.

Figure 3. IHH gene variant and protein change in wild type to mutant type.

Table 2. Detailed characteristics of BDA1 patients with identified IHH variants.

In silico analysis of the ACMG/AMP classification of identified variants

The ACMG and the AMP guidelines were followed to interpret the pathogenicity of the identified variants. The allele frequencies for rare and pathogenic alleles in our patients were compared with those in public databases. To provide additional genetic evidence for the association of IHH with the short-stature phenotype, we performed variant analyses by comparing allele frequencies between our cohorts of patients and public databases. The variant was not found to be homozygous in public databases, such as ExAC, gnomAD (Exome), gnomAD (Genome), ESP 6,500, 1,000 Genomes, Iranome, GenomeAsia, GME Variome, Turkish Variome, Mexican DB, India DB, 4.7KJPN and TOPMed Bravo. The rare nonsynonymous missense variants were predicted to be pathogenic and to be of uncertain significance by the in-silico tools Revel, Alpha Missense, Varity, SIFT, Meta-LR, Primate AI, Meta-RNN, Mutation Assessor, Mutation Taster, PROVEAN, and Aggregated Prediction. The details of the prediction scores of the identified variants are given in Table 3.

In-silico analysis of the protein parameters of the identified variants

Variant c.299A>C, p. Asp100Ala showed a negatively charged amino acid change to a nonpolar, aliphatic amino acid. Despite the variance in the uncertainty of the significance of the protein change, c.217C>T and p.Arg>Cys altered the positively charged amino acid to a highly reactive amino acid. Another variant, c.418 T>C, p.Ser>Pro changes the polar, neutral amino acid to a nonpolar, neutral, and aliphatic imino group amino acid. An overall change in the in-silico structure of the protein was observed. The change in the structure is shown in Figure 3.

To analyze the changes in the proteins of the identified variants, we performed an extensive analysis of the protein parameters by using the tool ExPASy https://web.expasy.org/protparam/. This provides us with the various physical and chemical parameters required for a change in a mutant protein. Here, we compared the changes in wild-type protein parameters with those of the identified likely pathogenic and variants of uncertain significance. The protein parameters included the amino acid number, which was the same for the mutant and wild-type strains. The molecular weight, isoelectric point, instability index, and grand average of hydropathicity (GRAVY) slightly changed in all the mutant strains compared to those in the wild type. All the parameters are given in Table 4.

Individuals with mutant type IHH:c.299A>C; p.Asp100Ala were found with missing middle phalanges in the hands and short middle phalanges and metacarpals in the feet. This patient’s protein parameter isoelectric point is increased by 9.09, molecular weight decreased with respect to wild-type IHH. Patients with p.Asp100Ala have 40 negatively charged amino acids in the IHH gene. Aliphatic index and instability index are also increased in the patients with p.Asp100Ala. These changes in parameters significantly correlate and segregate with the phenotypic changes in the patients and thus p.Asp100Ala is likely to be pathogenic.

In mutant c.217C>T; p.Arg73Cys, patients were found with short hands and feet, short 3^rd, 4^th, and 5^th metacarpals, and short stature. Protein parameters in the patients included decreased isoelectric point 8. 78, and decreased molecular weight with respect to wild-type protein parameters. +vly charged amino acid is also decreased 46 with respect to wild type 47 in IHH. Grand average hydropathicity decreased in the patients from the actual value of −0.200. Aliphatic and instability index remain the same as the wild-type parameters of proteins. These changed parameters may play a role in the phenotypic changes in the patients and interpreted as uncertain significant variants. With variant c.418T>C: p.Ser140Pro; the Molecular weight of IHH amino acids increased. Protein parameters like aliphatic and instability index decreased, but other parameters like isoelectric point, +vly, -vly charged remained the same as wild-type protein parameters. Thus, it was interpreted as an uncertain significance variant.

Table 3. In silico analysis prediction score and ACMG/AMP classification of IHH-Identified variants.

Discussion

BD is mostly seen in isolated form but can manifest as a complex syndrome. The overall prevalence rate in the population is not known because many individuals do not present to clinicians. Our study involved the clinical and molecular analysis of BDA1 in 14 individuals from related and unrelated families. A missense heterozygous novel variation was identified in the family of the three affected individuals with heterozygous novel variation in exon 1 of the IHH gene This finding demonstrated the complete penetrance of the gene in the affected individual’s phenotype. The affected individuals presented with short hands, feet and short stature. The middle phalanges of the hands of the affected individuals were missing on X-ray examination. In this study, we found the novel pathogenic variant, NM_002181.4 (IHH): c.299A>C; p. Asp100Ala, which segregates with BDA1 and the persons also had short stature. A pathogenic variant in the same region with different amino acid changes has been described earlier in a Chinese family [12]. Additionally, since multiple affected members of the family harbored the variant and because in silico tools supported the pathogenicity of the variant, we assumed that the variant was likely the cause of the phenotype in the family with short stature and BD. Two other variants were analyzed by the in-silico tools. The ACMG guidelines were predicted to be VUS. The allele frequencies of the identified variants in the patients in different population databases are not presented. These two variants NM_002181.4 (IHH): c.217C>T; p.Arg73Cys and NM_002181.4 (IHH): c.418T>C; and p.Ser140Pro were not reported in the ClinVar database. Our study demonstrated that IHH gene screening by sequencing should be done for individuals with BDA1 or other hand-foot malformations, especially if associated with short stature, in related and unrelated individuals. Further description of the BDA1 multiplex families and also a long-term follow-up of these individuals, especially children can give further insights into the possible correlation of genotype (variant location) with phenotype. Additionally, functional studies will be important for validating VUSs. To date, a few variants in the N-terminal and C-terminal domains of the IHH gene have been characterized [26,27]. First, it is important to phenotype patients by detailed clinical examination, including anthropometric assessments and radiological assessments. Furthermore, parental segregation based on phenotypic appearance is a necessary part of clinical examination. This approach helps reveal the familial nature of the disorder. Targeted gene sequencing of the IHH gene is the gold standard method for exploring monogenic cases of BD and short stature. The best way to detect the genetics of minor skeletal abnormalities is currently available at various centers. This study is a pilot study, and a greater number of patients could not be enrolled because usually syndromic patients are seen in our clinic and isolated BD patients do not visit the hospital. A few unaffected family members did not agree to give samples for testing. Funding issues are also a reason for a limited number of enrollment of patients and controls.

Table 4. In silico analysis, protein parameters of identified variants in the present study.

Conclusion

This study is the first to report a familial case of a novel variant in the northern region of India. Additionally, two variants of uncertain significance were also identified in patients with BDA1 and short stature. In silico tools are helpful for interpretation according to ACMG guidelines. However, additional research is essential to determine the exact pathogenic processes in the development of short stature and BD with the variants, and further ex vivo studies may be needed for a better understanding of this phenomenon.

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