Name: Master Jobandeep Singh Case ID: MD053550

Age: 2 Months Sample Type: Blood

Sex: Male Sample collection date: 11/07/2023
Referring Clinician: Dr. LT.Col Vinod Dagar Sample collection time: 02:00 PM
Test Requested: Whole Exome Sequencing (WES) Reporting date: 31/07/2023

CLINICAL INFORMATION/HISTORY

Master Jobandeep Singh, a 2-month-old male child, presented with the complaints of neonatal diabetes (on insulin injection), sepsis
and meningitis induced seizures.
Previous Investigations: Blood glucose level constantly fluctuating between 90 to 530 mg/dl, low serum C peptide and high HBA1C
(9.7).
Family history: late onset diabetes (45 years) in his grandparents.

RESULT SUMMARY

A Likely Pathogenic variant causative of the reported phenotype was identified.

*Correlation with clinical profile and family history is required.

VARIANTS RELEVANT TO INDICATION FOR TESTING

One likely pathogenic variant in the KCNJ11 gene was identified in this individual. No other variants of relevance to the indication were
identified. Please see below for more detailed variant information.

Gene & ACMG

Variant Zygosity Location Disorder Inheritance
Transcript Classification
Diabetes, permanent neonatal 2,
with or without neurologic
KCNJ11 c.679G>A features Likely
NM_000525.4 p.Glu227Lys Heterozygous Exon 1 [OMIM ID: 618856] Autosomal pathogenic
Dominant PP3, PM2 and
Diabetes mellitus, transient PP5
neonatal 3
[OMIM ID: 610582]:

DETAILED VARIANT INFORMATION (VARIANTS RELEVANT TO INDICATION FOR TESTING)

KCNJ11 Chr. 11:17408960 – Likely pathogenic:
The missense variant NM_000525.4(KCNJ11):c.679G>A has been reported to ClinVar as conflicting interpretations of pathogenicity
with a status of (1 star) criteria provided, multiple submitters, conflicting interpretations (Variation ID 158682 as of 2023-02-10).
The c.679G>A variant is observed in an allele frequency of 0003988% from individuals of gnomAD All background. The c.679G>A
variant is not reported in any individuals in 1kG All. This missense variant is predicted to be damaging by both SIFT and PolyPhen2
and is predicted to be conserved by GERP++ and PhyloP across 100 vertebrates. The identified variant has been previously identified
in patients affected with transient neonatal diabetes in heterozygous state [PMID: 28925365, 32101525, 32893419, 17021801,
23667671]. For these reasons, this variant has been classified as Likely Pathogenic.

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Patient Name: Master Jobandeep Singh Case ID: MD053550

Diabetes, permanent neonatal 2, with or without neurologic features [OMIM ID: 618856]:
Permanent neonatal diabetes mellitus-2 (PNDM2) is characterized by onset of insulin-requiring hyperglycemia within the first
months of life that requires insulin therapy throughout life. Some patients additionally have marked developmental delay, muscle
weakness, and epilepsy. The triad of developmental delay, epilepsy, and neonatal diabetes is known as DEND.

Diabetes mellitus, transient neonatal 3 [OMIM ID: 610582]:

Neonatal diabetes mellitus, defined as insulin-requiring hyperglycemia within the first month of life, is a rare entity. In about half
of the neonates, diabetes is transient and resolves at a median age of 3 months, whereas the rest have a permanent form of
diabetes. In a significant number of patients with transient neonatal diabetes mellitus, diabetes type 2 appears later in life. The
onset and severity of TNDM3 is variable with childhood-onset diabetes, gestational diabetes or adult-onset diabetes described
[MalaCards].

FINDINGS UNRELATED TO PHENOTYPE

This section provides information on variants identified which are unrelated to the provided phenotype.

ACMG Secondary Findings

No clinically relevant variants associated with the ACMG recommended secondary list of genes were found in the sequence data.

Incidental Findings

No variants were detected as incidental findings in the sequenced data which may not be associated with the diagnostic indication
for which the sequencing test was performed.

Carrier Status in the genes related to disease

No Pathogenic or Likely Pathogenic variants were detected.

RECOMMENDATIONS

Based on the clinical features and the observed genetic findings the following have been recommended:

1. Genetic counseling is recommended to discuss the potential clinical implications of this result.
2. Sanger evaluation of the identified variant(s) in the proband, parents and close relatives is recommended.
3. If the above results do not correlate completely with the proband’s phenotype, additional testing is advised based on
the clinician’s recommendation.
REPORTED VARIANTS STATISTICS:

Alternate Allele
Gene/Transcript Variant Depth Allelic Depth dbSNP rsID
Fraction
KCNJ11 c.679G>A 82X 39X 0.47 rs587783672
NM_000525.4

DATA STATISTICS

Total data generated (Gb) 9.9

Reads that passed alignment (%) 99.38

Data > Q30 (%) 93.15

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Patient Name: Master Jobandeep Singh Case ID: MD053550

METHODOLOGY

Sequencing of the protein coding regions of approximately 30Mb of the human exome (targeting approximately 99% of regions in
CCDS and RefSeq) was performed using Illumina NovaSeq platform at a mean depth of 80-100X and % of bases covered at 20X depth
>90% in the target region. The individual’s DNA was extracted and fragmented, with fragments from the coding regions of the selected
gene panel targeted for amplification and sequencing. Reads from the sequence output were aligned to the human reference genome
(GRCh37) using the Burrows-Wheeler Aligner (BWA). Duplicate reads identification and removal, base quality recalibration and re-
alignment of reads based on indels were done using inbuilt DRAGEN bio-IT pipeline. Variants to the reference were called using the
Genomic Analysis Tool Kit (GATK). The variants were annotated and filtered using the Golden Helix VarSeq and Varsome analysis
workflow implementing the ACMG guidelines for interpretation of sequence variants. This includes comparison against the gnomAD
population catalogue of variants in 123,136 exomes, the 1000 Genomes Project Consortium’s publication of 2,500 genomes, the NCBI
ClinVar database of clinical assertions on variant’s pathogenicity and multiple lines of computational evidence on conservation and
functional impact. All variants with minor allele frequency (MAF) of less than 1% in gnomAD database, and disease-causing variants
reported in HGMD, in ClinVar are considered. The investigation for relevant variants is focused on coding exons and flanking +/-10
intronic nucleotides of genes with clear gene-phenotype evidence (based on OMIM information). All potential modes of inheritance
patterns are considered. In addition, provided family history and clinical information are used to evaluate identified variants with
respect to their pathogenicity and causality. This test has not been cleared or approved by the U.S. Food and Drug Administration
(FDA). The FDA has determined that such clearance or approval is not necessary.

VARIANT ASSESSMENT PROCESS

The following databases and in-silico algorithms are used to annotate and evaluate the impact of the variant in the context of human
disease: 1000 genomes, gnomAD, ClinVar, OMIM, dbSNP, NCBI RefSeq Genes, ExAC Gene Constraints, VS-SIFT, VS-PolyPhen2, PhyloP,
GERP++, GeneSplicer, MaxEntScan, NNSplice, PWM Splice Predictor. Analysis was reported using the HGVS nomenclature
(www.hgvs.org/mutnomen) as implemented by the VarSeq transcript annotation algorithm. The reported transcript matches that
used most frequently by the clinical labs submitting to ClinVar.

LIMITATIONS

It should be noted that this test is limited to a limited number of genes and does not include all intronic and non-coding regions. This
report only includes variants that meet a level of evidence threshold for cause or contribution to disease. Certain classes of genomic
variants are also not covered using the NGS testing technology, including triplet repeat expansions, copy number alterations,
translocations and gene fusions or other complex structural rearrangements. More evidence for disease association of genes and
causal pathogenic variants are discovered every year, and it is recommended that genetic variants are re-interpreted with updated
software and annotations periodically.

VARIANT CLASSIFICATION BASED ON ACMG RECOMMENDATIONS

Genetic test results are reported based on the recommendations of American College of Medical Genetics (ACMG) as described
below [1]

Variant A change in a gene. This could be disease causing (pathogenic) or not disease
causing (benign).

Pathogenic A disease-causing variation in a gene which can explain the patients’ symptoms.

Likely A variant which is very likely to contribute to the development of disease. However, the
pathogenic scientific evidence is currently insufficient to prove this conclusively. Additional
evidence is expected to confirm this assertion of pathogenicity

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Patient Name: Master Jobandeep Singh Case ID: MD053550

Variant of uncertain significance A variant which is difficult to classify either as pathogenic (disease causing) or
benign (non-disease causing) based on current available scientific evidence.

ACMG Criteria for classifying Variants.

Very Strong (PVS1)
Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multi-exon
PVS1
deletion) in a gene where LOF is a known mechanism of disease.
Strong (PS)
PS1 Same amino acid change as a previously established pathogenic variant regardless of nucleotide change

PS2 De novo variant (both maternity and paternity confirmed) in a patient with the disease and no family history.

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene
PS3
product.
PS4 The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in
controls.
Moderate (PM)
Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an
PM1 enzyme) without benign variation

PM2 Absent from controls (or at extremely low frequency if recessive) in reputed databases.

PM3 Variant (one of the compound heterozygous), is segregating with a pathogenic variant with known phase after
testing of parents.
PM4 An in-frame deletions/insertions in non-repeat region or stop-loss can alter the protein length.

PM5 A novel missense change at the same amino acid residue where a pathogenic missense variant has already
been determined.
PM6 De novo, without testing in the family.
Supporting (PP)
PP1 A variant in known gene for a disease which is co-segregating in multiple affected family members
PP2 Missense variants are a common mechanism of disease in a gene which has low benign missense variants.

PP3 A deleterious effect of the variant is predicted by multiple lines of computational evidence (conservation,
evolutionary, splicing impact, etc.)

PP4 Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
Reputable source recently reported the variant as pathogenic, but the evidence is not available to the
PP5
laboratory to perform an independent evaluation.

DISCLAIMER

In accordance with the Pre-Conception and Pre-Natal Diagnostic Testing (PCPNDT) Act, 2003- Govt. of India; Lab does not
disclose the gender of the fetus.

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Patient Name: Master Jobandeep Singh Case ID: MD053550

Interpretation of variants in this report is performed to the best knowledge of the laboratory based on the information
available at the time of reporting. The classification of variants can change over time and the laboratory cannot be held
responsible for this. Re-analysis of variants in previously issued reports considering new evidence is not routinely
performed but may be available upon request.

Negative results do not completely exclude the risk/carrier status for these disorders tested (residual risk)

The sensitivity of this assay to detect large deletions/duplications of more than 10bp or copy number variations (CNV) is
70-75%. The CNVs detected must be confirmed by an alternate method.

Due to inherent technology limitations of the assay, not all bases of the exome can be covered by this test. Accordingly,
variants in regions of insufficient coverage may not be identified and/or interpreted. Therefore, it is possible that
pathogenic variants are present in one or more of the genes analyzed but have not been detected. The variants not
detected by the assay that was performed may impact the phenotype.

It is also possible that a pathogenic variant is present in a gene that was not selected for analysis and/or interpretation in
cases where insufficient phenotypic information is available.

Genes with pseudogenes, paralog genes and genes with low complexity may have decreased sensitivity and specificity of
variant detection and interpretation due to inability of the data and analysis tools to unambiguously determine the origin
of the sequence data in such regions.

The mutations have not been validated/confirmed by Sanger sequencing.

Incidental or secondary findings (if any) that meet the ACMG guidelines [2] can be given upon request.

The report shall be generated within turnaround time (TAT), however, such TAT may vary depending upon the complexity
of test(s) requested. Laboratory under no circumstances will be liable for any delay beyond aforementioned TAT.

It is hereby clarified that the report(s) generated from the test(s) do not provide any diagnosis or opinion or recommend
any cure in any manner. Laboratory hereby recommends the patient and/or the guardians of the patients, as the case may
be, to take assistance of the clinician or a certified physician or doctor, to interpret the report(s) thus generated. Laboratory
hereby disclaims all liability arising in connection with the report(s).

In a very few cases genetic tests may not show the correct results, e.g., because of the quality of the material provided to
the laboratory. In cases where any test provided by the laboratory fails for unforeseeable or unknown reasons that cannot
be influenced by the laboratory in advance, the laboratory shall not be responsible for the incomplete, potentially
misleading, or even wrong result of any testing if such could not be recognized by the laboratory in advance.

This is a laboratory developed test and the development and the performance characteristics of this test was determined by
laboratory.

REFERENCES

1. Hamosh, A., Scott, A. F., Amberger, J. S., Bocchini, C. A., &McKusick, V. A. (2005). Online Mendelian Inheritance in Man (OMIM), a
knowledgebase of human genes and genetic disorders. Nucleic Acids Research, 33(Database Issue), D514–D517.
http://doi.org/10.1093/nar/gki033, https://www.omim.org/
2. Landrum, M. J., Lee, J. M., Riley, G. R., Jang, W., Rubinstein, W. S., Church, D. M., &Maglott, D. R. (2014). ClinVar: public archive of
relationships among sequence variation and human phenotype. Nucleic Acids Research, 42(Database issue), D980–D985.
http://doi.org/10.1093/nar/gkt1113 https://www.ncbi.nlm.nih.gov/clinvar/

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Patient Name: Master Jobandeep Singh Case ID: MD053550

3. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., et al On behalf of the ACMG Laboratory Quality Assurance
Committee, H. L. (2015). Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus
Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
Genetics in Medicine: Official Journal of the American College of Medical Genetics, 17(5), 405–424.
http://doi.org/10.1038/gim.2015.30.
4. Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K. dbSNP: the NCBI database of genetic variation. Nucleic
Acids Res. 2001 Jan 1;29(1):308-11.
5. GnomAD database - https://gnomad.broadinstitute.org/.

PMID CITATION
Lin, Y. W., Li, A., Grasso, V., Battaglia, D., Crinò, A., Colombo, C., Barbetti, F., & Nichols, C. G. (2013). Functional
characterization of a novel KCNJ11 in frame mutation-deletion associated with infancy-onset diabetes and a mild
23667671
form of intermediate DEND: a battle between K(ATP) gain of channel activity and loss of channel expression. PloS
one, 8(5), e63758. https://doi.org/10.1371/journal.pone.0063758.
Girard, C. A., Shimomura, K., Proks, P., Absalom, N., Castano, L., Perez de Nanclares, G., & Ashcroft, F. M. (2006).
17021801 Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes. Pflugers Archiv : European journal of
physiology, 453(3), 323–332. https://doi.org/10.1007/s00424-006-0112-3.
Gopi, S., Kavitha, B., Kanthimathi, S., Kannan, A., Kumar, R., Joshi, R., Kanodia, S., Arya, A. D., Pendsey, S., Pendsey,
S., Raghupathy, P., Mohan, V., & Radha, V. (2021). Genotype-phenotype correlation of KATP channel gene defects
32893419
causing permanent neonatal diabetes in Indian patients. Pediatric diabetes, 22(1), 82–92.
https://doi.org/10.1111/pedi.13109.
Devaraja, J., Elder, C., & Scott, A. (2020). Non classic presentations of a genetic mutation typically associated with
32101525 transient neonatal diabetes. Endocrinology, diabetes & metabolism case reports, 2020, 19-0125. Advance online
publication. https://doi.org/10.1530/EDM-19-0125.
Griscelli, F., Feraud, O., Ernault, T., Oudrihri, N., Turhan, A. G., Bonnefond, A., Froguel, P., & Bennaceur-Griscelli, A.
(2017). Generation of an induced pluripotent stem cell (iPSC) line from a patient with maturity-onset diabetes of
28925365
the young type 13 (MODY13) with a the potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11)
mutation. Stem cell research, 23, 178–181. https://doi.org/10.1016/j.scr.2017.07.023.

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Patient Name: Master Jobandeep Singh Case ID: MD053550

Conditions for Reporting

1. It is presumed that the specimen belongs to the patient named or identified, such verification being carried out at the point
of generation of said specimen.
2. A test might not be performed due to following reasons:
a. Specimen Quantity not sufficient (Inadequate collection/spillage during transit).
b. Specimen Quality not acceptable (Hemolysis/clotted/lipemic).
c. Incorrect sample type.
d. Test canceled either on request of patient or doctor.
3. In any of the above cases a fresh specimen will be required for testing and reporting.
4. The results of the tests may vary from lab to lab, time to time for the same patient.
5. The reported results are dependent on individual assay methods, equipment, method sensitivity, specificity and quality of
the specimen received.
6. Partial representation of the report is not allowed.
7. The reported tests are for the notification of the referring doctor, only to assist him/her in the diagnosis and management of
the patient.
8. Report with status "Preliminary" means one or more tests are yet to be reported.
9. This report is not valid for Medico Legal Purpose.
10. Applicable Jurisdiction will be of "Delhi" for any dispute/claim concerning the test(s) & results of the test(s).

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