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Outline

• Duchenne muscular dystrophy (DMD)

• Diagnosis of DMD

• Newborn screening for DMD

• Goals of the NYS pilot study

• Screening strategy

• Results of the pilot study

• Factors to consider for the first-tier screen for DMD

• Future screening strategy

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Duchenne Muscular Dystrophy

• Progressive, lethal, X-linked neuromuscular disorder

• Incidence is approx. 1 in 5,000 in live male births

• Symptoms are generally seen by age 2-3

• Mean age of diagnosis is 5 years

• Life expectancy is shortened due to respiratory insufficiency and cardiomyopathy – survival to thirties is becoming more common

• Rare in females – depends on patterns of X chromosome inactivation. Some females may exhibit musculoskeletal symptoms and cardiac disease

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Diagnosis

• Creatine kinase (CK) levels are elevated in people with DMD

• Measure CK activity

• Measure CK-MM concentration (skeletal muscle damage)

• Genetic Testing to detect variants in the DMD gene

• Deletion/duplication analysis

• Sequencing/NGS

• Muscle Biopsy

Immunohistochemistry detecting quantity and location of dystrophin protein

DMD, Duchenne muscular dystrophy; NGS, next generation sequencing

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Newborn Screening for DMD

• 1975 2 midwestern hospitals in the US; Rhone-Alpes region of France; Germany

• 1976 Scotland

• 1979 New Zealand, Belgium

• 1986 Manitoba, Canada; Western Pennsylvania and Brazil

• 1990 Wales

• 1992 Cyprus

• 2007 Columbus and Cincinnati, Ohio pilot study

• 2016 China (Zhejiang province)

• 2017 Australia

• 2019 New York pilot study, China (Guangzhou)

• 2020 Research Triangle Institute International (North Carolina)

• 2021 Taiwan, Boston (Brigham and Women’s Hospital)

• 2024 Ohio [mandated in New York, approved in Massachusetts, Minnesota]

https://www.wadsworth.org/programs/newborn/screening

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DMD Pilot Consortium

• NYS Newborn Screening Program

• Parent Project Muscular Dystrophy (PPMD)

• Northwell Health Hospitals

• Columbia Presbyterian Hospitals

• American College of Medical Genetics and Genomics (ACMG) & Newborn Screening Translational Research Network (NBSTRN)

• PerkinElmer/Revvity

• Funders (Sarepta Therapeutics, PTC Therapeutics, Solid Biosciences, Wave Life Sciences, Pfizer, Inc., PPMD)

- NYS-permitted laboratory performing second-tier testing

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Goals of DMD Consented Pilot Study (2019-2021)

Validate a high-throughput first-tier immunoassay screen for DMD in a high-birth number state
Optimize a second-tier molecular testing strategy for detecting DMD and other muscular dystrophies
Identify infants who will develop DMD before clinically detectable symptom onset

Provide parents the opportunity to discuss the diagnosis with a sub-specialist
Determine treatment course
Participate in clinical trials

Use the results of the pilot testing to provide evidence required for state and federal assessments of the benefits and risks of NBS for DMD
Develop the infrastructure to educate parents and health care providers about DMD NBS
Nominate DMD to the Recommended Uniform Screening Panel (RUSP)

NBS: newborn screening

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Recruitment

In-person recruitment

In-person & remote recruitment (Northwell)

In-person & remote recruitment (Columbia)

Remote recruitment

Study Uptake: 87%

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Screening Strategy

DBS was submitted for genetic testing in March 2021

NYS, New York State; NBS, newborn screening; CK-MM, creatine kinase-MM; SCC, specialty care center; NGS, next generation sequencing; NM, neuromuscular; DBS, dried blood spot; BMD, Becker muscular dystrophy

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Population Screened (1 Oct 2019 – 30 Sep 2021)

	Total	% of total
No. specimens screened	39,646	-
No. Babies Screened	36,781	-
Male	18,654	50.7%
Female	17,993	48.9%
Specimens collected <24 hours	1,429	3.6%
Specimens collected 24-47 hours	35,747	90.2%
Specimens collected 48-71 hours	793	2%
Specimens collected ≥72 hours	1,675	4.2%
Low birth weight (<2500 g)	2,465	6.7%
Normal birth weight (2500-3999 g)	32,048	87.1%
High birth weight (≥4000 g)	2,264	6.2%
Quantity not sufficient (not tested)	8	0.02%
Sub-optimal (tested)	331	0.8%

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CK-MM (ng/ml)

Specimens

CK-MM Concentration Scatter Plot

<72 hrs

168+ hrs

72-167 hrs

Referral cut-off

● DMD/BMD

● Carrier DMD/BMD

CK-MM, creatine kinase-MM; DMD, Duchenne muscular dystrophy; BMD, Becker muscular dystrophy

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Reported as Borderline

296 babies had borderline results (1 in 124 babies)

Characteristics of Borderline Babies
Sex	Male	178 (60%)
	Female	118 (40%)
Age at specimen collection (hours)		1 – 101
Birth weight (g)	< 2,500 2,500 – 3,999 4,000	4 (1.4%) 262 (88.5%) 32 (10.8%)
CK-MM value (ng/ml)		644-3,944

CK-MM, creatine kinase-MM

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Reported as Borderline

• 296 babies had borderline results:

• Repeats were received for 277 babies

• 2 babies had an initial borderline result but were subsequently referred:

• A female DMD carrier

• A male baby with DMD

• 17 families declined repeat testing:

• Mother of a female baby was a known DMD carrier

• 2 families did not return for specimen collection

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Referred Infants

42 infants were referred (1 in 876 babies)

Characteristics of Referred Babies
Sex	Male Female	25 (60%) 17 (40%)
Age at specimen collection (hours)		6-3,360
Birth weight (g)	< 2,500 2,500 – 3,999 4,000	3 (9%) 27 (79%) 4 (12%)
CK-MM value (ng/ml)		993-18,547

CK-MM, creatine kinase-MM

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Turn-around Time for Referrals

Median: 8 days of life

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Follow-up of Referred Infants

Outcome of Follow-up/Second-tier Testing
Confirmed with DMD/BMD	Male with duplication of exon 18 (out-of-frame) Male with deletion of exons 48-49 (in-frame: BMD) Male with deletion of exons 3-43 (out-of-frame) Male with deletion of exon 51 (out-of-frame)
Carrier of DMD	Female with stop codon in exon 63
Carrier of MD	Two babies (1 female, 1 male) are carriers of Limb Girdle MD Two male babies are carriers of LAMA2 MD One male baby is carrier of MD dystroglycanopathy
Other disorders	One female baby was diagnosed with Alagille syndrome (also elevated CK-MM of unknown etiology) One male baby with possible inborn error of metabolism One male baby diagnosed with cerebral palsy and NM respiratory weakness
VUS in genes in NM panel	22 newborns

DMD, Duchenne muscular dystrophy; BMD, Becker muscular dystrophy; MD, muscular dystrophy; NM, neuromuscular;

VUS, variants of uncertain significance; CK-MM, creatine kinase-MM

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Follow-up of Referred Infants cont.

Outcome of Follow-up/Second-tier Testing
CK-MM remained elevated	4 newborns
Birth trauma	10 newborns with shoulder dystocia 3 newborn with nuchal cord complication 2 newborns with hypoxic-ischemic encephalopathy and seizures 8 newborns had breech deliveries
Declined	2 families could not be reached 6 families declined genetic testing 4 families declined NM panel after no pathogenic variants were detected in DMD gene

• Trauma at birth has been shown to lead to elevated CK results [Rudolph and Gross, 1966;38(6):1039-46]

• There are markedly elevated levels of CK-MM following vaginal delivery especially if complicated by forceps, vacuum and breech presentation [Amato et al., Klin Padiatr. 1991;203(5):389-94]

NM, neuromuscular; CK-MM, creatine kinase-MM

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Select Publications from NYS Pilot

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Factors Influencing CK-MM Levels in Newborns

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CK-MM Distribution

CK-MM: creatine kinase-MM

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Age at Collection and CK-MM

(Park et al., Muscle and Nerve 2022;65:652-658)

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Effect of Age at Collection on CK-MM

Park et al., Muscle and Nerve, 2022;65(6):652-658

Blue: 0-47 hrs

Orange: 48-71 hrs

Gray: 72-167 hrs

Yellow: 168+ hrs

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Effect of Gender on CK-MM

CK-MM (ng/ml)

Mean CK-MM was 6% higher in males compared to females.

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Effect of Gestational Age on CK-MM

CK-MM (ng/ml)

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Effect of Birthweight on CK-MM

CK-MM (ng/ml)

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Seasonal Variation

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Multiple Linear Regression of CK-MM

N = 19,914

Variables: race, sex, gestational age, age at collection

Assumption checking verified that a logarithmic transformation of CK-MM is needed before applying MLR

Model: LN(CK-MM) = 2.784 - 0.00061*N_LATINX

- 0.014*N_AA_BLACK + 0.16*N_ASIAN +0.00996*N_WHITE

- 0.059*N_FEMALE + 0.015*GESTAGE_DAYS

- 0.032*AGECOLL

Equivalent Model: CK-MM = 16.2 * 0.9994^N_LATINX * 0.986^N_AA_BLACK * 1.177^N_ASIAN * 1.010^N_WHITE * 0.942^N_FEMALE * 1.015^GESTAGE_DAYS * 0.969^AGECOLL

r² = 0.1053

MLR: multiple linear regression

LN: natural log

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Interpretation of Multiple Linear Regression

(Indicator Variables)

Variable	P-value	Significance	Model Interpretation
Latinx	0.965	Not Significant	N/A
Black	0.473	Not Significant	N/A
Asian	<10^-15	Significant	Asians have ~ 18% higher CK-MM than non-Asians
White	0.565	Not Significant	N/A
Female	<10^-12	Significant	Females have ~ 6% lower CK-MM than males

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Interpretation of Multiple Linear Regression

(Continuous Variables)

Variable	P-value	Significance	Model Interpretation
Gestational Age	<10^-5	Significant	Each day increase in gestational age causes ~ 1.5% increase in CK-MM (compounded)
Age at Collection	<10^-15	Significant	Each hour increase in age at collection causes ~ 3% decrease in CK-MM (compounded)

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NYS Cut-offs were Based on AAC

AAC: age at collection

If the cut-offs were not based on AAC, 99.5% for the entire population would have been: 2,348 ng/ml
If we had used 2,348 ng/ml as the cut-off for every baby, we would have missed:

1 male baby with DMD (CK-MM = 993 ng/ml, specimen collected at 331 hrs)(25% of DMD cases would be missed)
1 female DMD carrier (CK-MM = 1,958 ng/ml, specimen collected at 3360 hrs)(100% of carriers would be missed)

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Premature/NICU/LBW Newborns

Potential for missing LBW/premature babies because these babies have lower CK-MM at birth
NYS NICU protocol for specimen collection:

Admission to NICU (<24 hrs)
Between 48-72 hrs
At 28 days of life or discharge

Consider:

Lowering CK-MM cut-off or
Repeat testing at older age

NICU: neonatal intensive care unit

LBW: low birth weight

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Publications

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Future Screening Strategy

*Cutoffs based on AOC and BW adjustments:

Negative screen reported

Repeat specimen (AOC >1 week, preferably) requested

Referral made to SCC for follow-up

Saliva or blood collection for diagnostic NGS panel for DMD, Del/Dup analysis, CK measurement

DMD/BMD diagnosis

Alternative diagnosis

*Normal CK-MM

*Referral level

CK-MM

Investigating the use of collaborative laboratory integrated report (CLIR) tool, a statistical modeling tool, to reduce false positive CK-MM results.

CK-MM, creatine kinase-MM; AOC, age of collection; BW, birth weight; SCC, specialty care center; NGS, next generation sequencing; Del/Dup, deletion/duplication; DMD/BMD, Duchenne/Becker muscular dystrophy

Normal CK-MM

Elevated

CK-MM

Measurement of CK-MM in DBS at the NYS NBS Program

Projections

99.5% Normal

0.5% Elevated

<0.1 % Referral level

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Conclusion

• During the pilot study, 36,781 newborns were screened for DMD

• Forty-two babies were referred for follow-up:

4 consistent with diagnosis of DMD/BMD (1 in 6,218 males)

6 carriers of various forms of MD

10 (24%) had shoulder dystocia

• We determined that the following factors influence CK-MM levels in newborns:

Age at collection

Birth weight/gestational age

Gender

Seasonality

Race (Asian)

• The data from the pilot was used for nomination of DMD to the RUSP

• In NYS, in October 2023 a bill (S6814/A5042) was signed to perform NBS for DMD

DMD/BMD, Duchenne/Becker muscular dystrophy; MD, muscular dystrophy; CK-MM; creatine kinase-MM; RUSP, recommended uniform screening panel; NBS, newborn screening

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Acknowledgments

• Dr. Michele Caggana (Director, NYS NBS Program)

• Ms. Sunju Park, Ms. Breanne Maloney, Ms. Melissa Pearce (NYS NBS Program)

• DMD Steering Committee

• Ms. Niki Armstrong (PPMD)

• Dr. Dorota Gruber, Dr. David Tegay, Ms. Lorraine Verdade (Northwell Health)

• Dr. Wendy Chung, Ms. Julia Wynn (Columbia Presbyterian)

• Ms. Annie Kennedy (EveryLife Foundation for Rare Diseases)

• Dr. Michele Puryear, Dr. Amy Brower (ACMG/NBSTRN)

• Dr. Mycroft Sowizral (Wadsworth Center), Dr. Roxana Moslehi, Isa Bracket (SUNY, Albany)

• Ms. Hanna Polari (Revvity)

• Funders (Sarepta Therapeutics, PTC Therapeutics, Solid Biosciences, Wave Life Sciences, Pfizer, Inc., PPMD)

• Recruiters and NBS staff at hospitals & NBS Program

• Families participating in the pilot study

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